nanotechnology in diagnostics

MICROPATTERNING AND MICROELECTROCHEMICAL

CHARACTERISATION OF BIOLOGICAL RECOGNITION

ELEMENTS

Dissertation

zur Erlangung des Grades eines

Doktors der Naturwissenschaften

der Fakultät für Chemie der

Ruhr-Universität Bochum

Vorgelegt von

Eugen Florin Turcu

Bochum, July 2004

This work was carried out between April 2001 and July 2004 at Lehrstuhl für Analytische

Chemie, AG Elektroanalytik & Sensorik under the supervision of Prof. Dr. W. Schuhmann.

Tag der mündlichen Prüfung 20. Juli 2004

Referent Prof. Dr. W. Schuhmann

Korreferent Prof. Dr. W. S. Sheldrick

Prüfer Prof. Dr. C. Wöll

Contents

1. Introduction: Why going smaller?

2. Methods microstructuring

2.1 Lithography 4

2.1.1. Photolithography 4

2.1.2. Soft-lithography 6

2.1.3. Other methods for microstructuring 8

2.2. Ink-jet printing/lithography – the flow through piezo microdispenser 9

3. Tools for probing microstructures

3.1 Scanning Probe Microscopy (SPM) 12

3.2 Scanning Electrochemical Microscopy (SECM) 14

3.3 Notes 25

4. Electrodes for electrochemistry in small volumes

4.1 Integrated working/reference assembly 27

4.1.1. Preparation of precursor electrode 28

4.1.2. Chemical deposition of silver onto

the body of precursor electrodes

30

4.1.3. Application of the coaxial Pt-µWE/Ag-RE in SECM 34

4.2 Miniaturised Ag/AgCl reference electrode 39

5. Micropatterning and microelectrochemical

characterisation of biological recognition elements

5.1 Enzyme microstructures 42

5.1.1 About enzymes 44

5.1.2 Glucose oxidase (GOD) 51

5.1.3 Patterning of GOD by means of piezo microdispenser 53

5.1.4 Visualisation of GOD microstructures by SECM 62

5.2 Defined adhesion/growth of living cells 71

5.2.1 Introduction 72

5.2.2 What is available so far? 73

5.2.3 Results and discussion 74

5.2.4 Conclusions 79

5.3 DNA microstructures 80

5.3.1 DNA microarrays 82

5.3.2 Detection of DNA hybridisation –

What are the options?

87

5.3.3 The repelling mode of SECM –

A new and promising assay for imaging

DNA microarrays and detecting DNA

hybridisation

97

5.3.4 Detection of DNA hybridisation in

the repelling mode of SECM

118

5.3.5 Conclusions and outlook 124

5.4 Notes 127

6. Experimental 131

7. Conclusions 136

8. Acknowledgment 139

9. References 140

10. Curriculum vitae 157

PUBLICATIONS

• Florin Turcu, Albert Schulte, Gerhard Hartwich, Wolfgang Schuhmann. “Label-free

electrochemical recognition of DNA hybridisation by means of modulation of the

feedback current in SECM”, Angew. Chem. Int. Ed., 2004, 43, 3482-3485.

• Florin Turcu, Karla Tratsk-Nitz, Solon Thanos, Wolfgang Schuhmann, Peter

Heiduschka. “Ink-jet printing for micropattern generation of laminin for neuronal

adhesion” J. Neurosci. Methods, 2003, 131, 141-148.

• Albert Schulte, Mathieu Etienne, Florin Turcu, Wolfgang Schuhmann. “High

resolution constant distance scanning electrochemical microscopy on immobilised

enzyme micropatterns” G.I.T. Imaging and Microscopy, 2003, 5, 46-49.

• Florin Turcu, Albert Schulte, Gerhard Hartwich, Wolfgang Schuhmann. “Imaging

immobilised ss-DNA and detecting hybridisation by means of the repelling mode of

scanning electrochemical microscopy (SECM)”, Biosens. Bioelectron., 2004, accepted.

• Florin Turcu, Albert Schulte, Wolfgang Schuhmann. “Scanning electrochemical

microscopy (SECM) in nanolitre droplets using an integrated working/reference

electrode assembly“, Anal. Bioanal. Chem., 2004, submitted.

• Anh Nguyen, Jane Hübner, Florin Turcu, David Melchior, Hans-Willi Kling, Siegmar

Gäb, Oliver J. Schmitz. “Analysis of alkyl polyglicosides by capillary electrophoresis

with pulsed-amperometric detection”, Electrophoresis, 2004, submitted.

Why going smaller?

1

Micropatterning and microelectrochemical

characterisation of biological recognition elements

1. Introduction: Why going smaller?

A question that often comes in mind when reading the title of a new book or journal article,

is “Why this one? Does it say enough about the content of that writing?” Well, most of the

books have very general titles. Let us consider two of them that are widely spread:

“Analytical Chemistry”, “History of …” and many others that have something in common:

they all speak about something, which somehow can be related to this general topic.

Analytical Chemistry explains what an analysis means and how many types we know, how

to perform precise measurements and how to get rid of different errors that could appear

besides, many theoretical and technical insights. Each book has its main content, which

follows the most important ideas of the subject it is speaking about. Authors have found an

open field to express their own thoughts and views about all the things they are writing

about. This gives the explanation why book’s contents look so different even if they are

dealing with the same topic.

This thesis is entitled “Micropatterning and microelectrochemical characterisation of

biological recognition elements” and may suggest to the reader a survey of all kinds of

biological microscopic structures: how to make and how to use them. What, however,

should be expected from this piece of work? In fact, it is tackling the micropatterning of

biological recognition elements. In particular, these were an enzyme, nucleic acids, and

living cells (in particular neurons). A piezoelectric microdispenser has been used as

microprinting device to create complex enzyme structures on different materials and also

was used to prepare oligonucleotide microarrays. Furthermore, the Scanning

Electrochemical Microscope (SECM) has been chosen as the main tool for exploring the

obtained microstructure and for imaging their local (electro)chemical properties with high

spatial resolution.

Before going into details I would like to raise a question: Why do we need

microstructures? If you answer: because we need miniaturisation, my questionnaire moves

further to the next and most important question: Why do we need miniaturisation? There

are unlimited possibilities to answer. However, a clear example for the need of

Why going smaller?

2

miniaturisation can be found with developing computers, tools that everyone loves when

they are small and very efficient in doing lots of work for us.

The long history of mechanical calculators has its roots in the Antiquity when the “abacus”

was the computing tool. Top leading scientists were later involved in the development of

these sophisticated machines: Leonardo da Vinci, Napier (the inventor of logarithms),

Wilhelm Schickard, Blaise Pascal, Gottfried Wilhelm von Leibniz, just to mention the

most famous of them.

Pascal built several mechanical machines, named “Pascalines” for helping his father in

adding and subtracting large sequences of numbers (he was a tax collector). Instead of

abacus and “Neper’s bones“ all other machines were quite big. In the 20th century the

basics of electronic computing systems were about to be discovered and developed by

Alan Turing and John von Neumann. The first representative electronic computer able to

carry out general-purpose computations was borna

in USA in 1946. Its name is ENIAC (Electronic

Numerical Integrator And Computer) (Figure 1).

Just by judging its dimensions and capability one

can conclude that it had been a real “Dinosaur” as

it occupied a large surface (about 1000 square

feetb) for a low computational power (for instance

5000 additions per second). Nevertheless, I

suppose it had been made in the smallest format

possible at that time. About 50 years later, the

new generation of computers became more

compact (less than 1 square feet) due to the

achievements with the miniaturisation of their

components. Something else changed, too: the

computing speed increased about many hundred thousand times. A laptop with more than 2

GHz clock frequency is a common tool these days.

In conclusion, miniaturisation means smaller and smarter tools for tomorrow! This is a

„sine qua non” condition of all future technological development in order to improve our

lives. As Euclid used to finish his (successful) mathematical demonstration: “Quod Erat

Demonstrandum” (QED)!

a J. Mauchly was the chief consultant and J. P. Eckert the chief engineer of team involved in the construction of ENIAC. b 1m2 = 10.76 square feet; thus, ENIAC required a room of about 10x10 m2!

Fig. 1 ENIAC – an early digital computer. It was characterised by a huge volume and poor computational speed (Image courtesy of the Computer History Museum).

Why going smaller?

3

Confession:

It happened in summer 2000, that I received a phone call from Prof. Dr. Elisabeth Csöregi

(Lund University, Sweden). About a year before, I contacted her and asked for help in

elucidating the structure of a polymer, which I accidentally prepared in 1989. Having this

in my mind, I thought that she would, although a bit late, discuss with me about my

request. Well, I was wrong. Instead, she mentioned that I could go to Wolfgang in

Germany. What a surprise! I am lucky! I can go abroad and study. But where is this place

“Wolfgang”? I though it must be a small town because I never heard about it! This were

my thoughts as E. Csöregi went on explaining me I have to send her a CV (curriculum

vitae and not a cyclic voltammogram!) and the details of the passport, a document that I did

not even have at that time. It was days after this event when I realised Wolfgang is not a

place but the name of my future boss!!! Ups! In the next period of time some e-mails were

exchanged between Wolfgang and me, thus I was informed about what I could do in

Germany, particularly, in the Biosensors laboratory at Ruhr University of Bochum.

Biosensors? I never heard about this, but I knew a little about chemical sensors! Pdf files

of publications describing the topics and research directions in Wolfgang’s group were sent

by e-mail. Unfortunately, I couldn’t read them because the suitable software for opening

such files had never been installed on the only computer of the Chemistry Department at

North University of Baia Mare. However, some bits of information were displayed on the

website of the ELAN-group (Electroanalytik, Biosensors) and gave me a rough idea about

my future colleagues and research topic.

As mentioned above, my PhD work is dedicated to micropatterning of biological

recognition elements. Furthermore, a novel coaxial microelectrode for advancing

microelectrochemical measurements in small electrolyte volumes was developed. The

major part of the dissertation, however, was focusing on the development of a simple

strategy to detect hybridisation of nucleic acids on DNA chips. Working on the technology

of DNA microarrays was enjoyable to me not only because it is a hot-topic these days but

also because I had the opportunity to work on something that was completely new to our

lab. The entire work presented in the following chapters is based on the concept of

miniaturisation.

Methods for microstructuring

4

2. Methods for microstructuring

2.1. Lithography

“As tiny as possible” seems to be the present day trend of science and technology with

respect to any kind of device. I would like to present here, the microfabrication, not as a

dead point of miniaturisation but as a reliable multi-purpose technique for fabrication of

chemical and biological analysis tools. Many ways of microstructuring are derived from

common devices used every day. For instance stamps or printer-like “ink” dispensers

occupy an important place among the microstructuring tools; or one can “write” with a

“nano-pencil”. Scaling down structures originated in the field of electronics where devices

became smaller in size and the individual components of the integrated electric circuitries

had to fit on limited space. Nowadays, microfabrication procedure has a strong impact in

most of the areas of contemporary science and technology and the knowledge and

experimental procedures for miniaturisation were transferred from electronics also to

chemistry and biochemistry for creating sensors with better performances. The ability to

generate patterns of biomolecules on different material surfaces is important for biosensor

technology, tissue engineering, and fundamental studies in cell biology. There are several

well established ways to pattern biomolecules onto substrates, such as photolithography,

soft lithography, nano-pen lithography, and spotting techniques. Although this is the

accepted classification of micro/nano-structuring techniques, to my opinion the key word

“lithography”, that itself means “writing on materials”a, is in fact sufficient to cover all the

existing methods. Thus, no matter how the patterned substance reached the substrate, the

underlying method is a lithographical one! However, the following part that is dedicated to

a succinct presentation of each micropatterning approach; the common categorisation will

be used.

2.1.1. Photolithography

Photolithography is the process of copying geometric shapes from a mask to the surface of

a substrate. The working principle resembles the formation of the “positive image” from

the negatives on to a developed film in photography (Figure 2). The steps involved in the

photolithographic processes are: wafer cleaning, barrier layer formation, photoresist

application, soft baking, mask alignment, exposure and development, hard baking.

a Lithography is a combination of the Greek words „lithos“ and „graphein“ with the meaning of stone and write respectively. It denoted, in earlier times, various items, one of which being “art of engraving on precious stones”.


5

The wafer is initially coated by spin-coating with a photoresist. The desired pattern is then

projected onto for example a wafer in a machine called a stepper. The stepper functions

similarly to a slide projector and creates high-contrast monochromatic images. Light from

the source is focussed through some lenses onto a "mask" (reticle), containing the desired

image in order to produce it on the wafer. Unlike a slide projector, the stepper does not

enlarge the image but actually reduces it in a similar way than sunlight is generating a

shadow of a cloud that is smaller than it actual size. When the image is projected onto the

wafer, the photoresist material undergoes some light-induced chemical reactions, which

cause the regions exposed to light to be either more or less susceptible to chemical etch. If

the exposed regions become more susceptible to the etch, the material is called a positive

photoresist, while it is a negative photoresist if it becomes less susceptible.

The resist is finally "developed" by exposing it to the chemical etchant, which removes

either the exposed (positive photoresist) or the unexposed (negative photoresist)

photoresist. The substrate then has a patterned polymer coating on its surface. This pattern

can then be etched into the underlying wafer by either a wet chemical etch or a plasma

etch. The ability to project a clear image of a very small feature onto the wafer is limited

Fig. 2 Comparison between the photography (left) and photolithography (right). In photography, the negative image on the film is transferred onto the photo-paper in such a way that the final positive image is larger as the picture captured on the photo-film. In contrast, in the photolithographic processes, the image grafted on the mask is reduced in size. For simplicity, no lenses are shown.


6

by the wavelength of the light that is used and the aperture of the lense (Rayleigh

diffraction). With ultraviolet light, features of 100-200 nm can be obtained.

The major disadvantages of photolithographical methods are listed below:

- the smallest achievable dimension is limited by the optical diffraction;

- not simply adopted for patterning non-planar surfaces;

- tolerates only little variations in the material that can be used (there is not a large

selection of available photoresists on one hand, and on the other hand common

wafers such as glass or ceramics are not that suitable as substrates);

- provides very poor control over the chemistry of patterned surfaces (especially

when complex organic functional groups are desired at the patterned surface);

- high cost due to the necessary sophisticated facilities and technologies.

2.1.2. Soft-lithography

Alternatives to photolithography were developed in the last 2-3 decades and showed their

potential for micro- and nano-patterning1,2. There are some procedures resorting to

stamps/moulds made of flexible polymers rather made hard materials. "Soft lithography" is

a new high resolution patterning technique developed at Harvard by Prof. G. M.

Whitesides in which the stamp is made from an elastomeric material namely

polydimethylsiloxane - PDMS. Members of the “soft-lithography” family are microcontact

printing (µCP), replica moulding (REM), microtransfer moulding (µTM), micromoulding

in capillaries (MIMIC) and solvent-assisted micromoulding (SAMIM).

Microcontact printing uses a PDMS stamp (copied from a master that is previously

prepared by photolithography) to transfer molecules onto surfaces. In a first step, the stamp

is covered with the desired molecules (thiols, proteins, nucleic acids, enzymes). The stamp

is allowed to dry and afterwards pressed onto the surface to be patterned. The soft rubber-

like stamp provides a large-area contact on the molecular scale, even on rough or slightly

curved surfaces and molecules are transferred directly from the stamp to the surface.

With replica moulding, a liquid precursor of PDMS is pressed against a patterned surface

with nanometer-sized relief structures. After curing, the cross-linked PDMS is cautiously

peeled off the structure perfectly copying the morphology of what was called the original

master3. The nanostructures present on the PDMS replica are, in turn, re-replicated using a

rigid organic polymer, for example, an photochemically curable polyurethane (PU), to

produce polymeric nanostructures very similar to (or indistinguishable from) those on the

surface of the original master. A great advantage of this approach is that it can produce


7

solid copies with smaller or larger

features as the original master! This is

achievable because the PDMS copy can

be mechanically bent (Figure 3).

In microtransfer moulding4, the mould

must be prepared before being used for

patterning. Accordingly, the recessed

regions of a stamp are filled with a

solution or pre-polymer and the exceeding

liquids are removed away from the crests.

The filled mould is then brought into

contact with the substrate. This technique

allows formation of both interconnected

and isolated microstructures and is also

suitable for building up 3D-structures

layer by layer.

With micromoulding in capillaries, the

dried and clean mould is positioned over

the substrate. In the next step the precursor of the polymer is forced to enter the

microscopic channel formed by capillary forces. Once the polymer is hardened, the mould

is removed to reveal newly prepared 3D-structures. If the mould is filled for instance with

a solution of an alkane thiol in ethanol, thiol microstructure are available by this procedure.

In contrast, to all the above-described soft-lithographic procedures, solvent-assisted

micromoulding does not deposit a particular polymer or self-assembled monolayer at the

surface, but removes material from the substrate in an etching process. The mould is placed

on the substrate and a solvent able to corrode the substrate is filled in the capillaries. When

the solvent is completely evaporated, the mould is removed. Quasi-3D-structures are

possibly created.

An intrinsic problem with any lithography based on an elastomeric mould originates from

the material properties of the mould. The softness of the mould leads to mould deformation

in the process of patterning or mould preparation and, of course, the deformation gets

worse as the pattern size becomes smaller, typically for feature sizes smaller than several

hundred nanometers. To overcome this problem, an amorphous fluoropolymer material

Fig. 3 The soft elastomeric structure can be mechanically bend and thus making possible the preparation of copies with larger (l1) or smaller (l2) features as the original master.


8

was used as a mould material. The unique properties of the mould material made it

possible to pattern densely populated extremely fine features (80 nm line-width)5.

2.1.3. Other methods for microstructuring

A promising alternative is the LIGA process that was developed at the Institute of

Microstructure technology (IMT) in the early eighties under the leadership of Dr. W.

Ehrfeld6. LIGA made possible the mass-production of microcomponents at low-cost. The

steps involved are X-ray lithography, electroformation and plastics moulding.

• Deep X-ray lithography: LIGA requires a highly penetrating, intense, and parallel

X-radiation, typically supplied by a synchrotron. The application of X-radiation

imposes the use of specific materials for the mask with the „transparent” part made

of very thin foil of metals such as titanium or berylliumb, and the absorbers

consisting of a comparatively thick layer of gold. The lateral structural information

is transferred by illumination of the mask with deep X-ray radiation into a plastics

layer, normally polymethylmethacrylate (PMMA, Plexiglas), by „shadowing”.

Exposure to radiation modifies the plastic material in such a way that it can be

removed with a suitable solvent, leaving behind the structure of the unexposed

plastic (the „shadowed areas”) as the primary structure. Of note, high aspect ratio

structures with heights of up to 1 mm and a lateral resolution down to 0.2 µm are

obtainable.

• Electroforming: the microcavities generated by the removal of the irradiated plastic

material can be filled with metal by electroforming processes. In this way, the

negative pattern of the plastics structure is generated as a secondary structure out of

metals, such as nickel, copper and gold, or alloys, such as nickel-cobalt and nickel-

iron.

• Plastics moulding: Plastics moulding is the key to low-cost mass production by the

LIGA process. The metal microstructures produced as mentioned above are used as

moulding tools for the production of reliable replicas of the primary structure in

large quantities and at low cost.

Mask-free methods for micropatterning surfaces were established using the tip of scanning

probe microscopes to locally modify surfaces down to nm range. The way of inducing

b Due to its poor electronic structure, Be is also used at large scale for X-ray transparent windows in nuclear reactors and in radiation detectors. As a matter of fact, its low atomic weight recommends it as moderator for slowing down rapid neutrons in a nuclear reactor and hence promoting the self-sustaining nuclear reaction.


9

controlled alteration of substrates by scanning probe lithography (proximal probe

lithography) relies on the following interactions:

- electrical: a scanning tunnelling microscope (STM) tip generates local electrical

fields that modify the surface underneath;

- electrochemical: a microelectrode, the SECM tip, is used to locally generate a

reagent that is able to etch the substrate;

- mechanical: an atomic force microscope (AFM) with its sharp tip operated in the

contact mode scratches the surface or transfers material from the tip to the surface

(dip-pen nanolithography, DPN7-9; Figure 4);

- optical: the optical fibre probe of a near-field scanning microscope (NSOM)

exposes the photoresist at local areas underneath the tip.

2.2. Ink-jet printing/lithography - the flow-through piezo-microdispenser

Simple but cost-effective lithographic processes that can be applied even to irregular

substrates under ambient conditions have been developed. Ink-jet lithography is a valuable

tool providing a non-destructive and localised surface modification technology in which

small droplets of ink (or other liquids) are jetted from a small aperture (nozzle) directly to a

specified position of the substrate.

Professor Hertz from the Department of Electrical Measurements, University of Lund

developed about 25 years ago continuous ink-jet techniques that had the ability to

modulate the ink-flow characteristics for grey-scale ink-jet printing on different media. The

idea of obtaining grey-scale printing was to control the number of droplets deposited in

each pixel (spot) so that the amount of ink volume in each pixel was adjusted to create the

desired grey tone10. May be inspired by the work of Hertz, Thomas Laurell from the same

Fig. 4 “Writing” with a nanoscopic ink-pen that is the AFM tip. The “ink“ molecules that are dissolved in the solution underneath meniscus bind due to their high affinity to the substrate.


10

Department developed a microdispenser11 capable to shoot and deposit picolitre-sized

droplets of solutions on substrate surfaces.

An obvious advantage of this printing procedure over stamp-based methods is that tiny

droplets can be deposited in small cavities12. In contrast to a deposition from a spotting

needle, a microdispenser can be used with much more viscous solutions because it is

actively forcing the solution out of the reservoir using the piezo-actuator. As shown later,

this was helpful when, for instance, an enzyme–polymer mixture that certainly is more

viscous than pure water had to be micropatterned on a chosen substrate. A schematic

representation of a microdispenser is shown in Figure 5.

In brief, the microdispenser is capable to emit ultra small droplets of solutions of choice

based on rapid movements of a flexible silicon membrane. The membranec is coupled to a

piezoelectric ceramic actor (3x3x2 mm3) and fastened by in a plastic frame (Figure 6),

which expands and contracts upon the excitation with suitable alternating voltages. That

way the system is able to generate a series of short pulses of pressure each of them ejecting

a single droplet with a volume of roughly 100 picolitres.

c Two silicon plates, obtained by anisotropic etching (one for the nozzle and another for inlet/outlet of solution and membrane), are glued and sealed with waterproof epoxy. The inner chamber is then enclosed by the two plates to give a volume of few microlitres down to hundreds of nanolitre.

Fig. 5 Schematic representation of the piezo-microdispenser head. Alternative voltage pulses applied to a thin silicon membrane eject picoliter droplets of solution (yellow) towards the substrate.


11

The droplets are released through the specially designed, pyramidal nozzle with an orifice

size of 40×40 µm and directed perpendicular towards the target surface where the

deposition typically make spots with diameter of about 70 µm. If the droplets are shot in a

horizontal plane, they can travel without any change of the trajectory up to several

centimeters, at least if there is not strong air motion. Certainly, droplets emitted

downwards can be delivered with high precision from the nozzle to a chosen area of the

opposing substrate. A precise movement of the target substrate relative to the

microdispenser nozzle during microdispensing can be used to deposit either rows of

individual spots or lines and grids from overlapping lines.

A pump is connected to the chamber via short silicon tubes. The excess of the filling

solution is drained out through the outlet consequently avoiding an exposure of the

membrane to overpressure inside the piezo chamber. Any time when a voltage pulse is

applied to the piezo, one droplet is hurled through the nozzle.

The flow-through microdispenser has proven to be ideal for applications in biochemical

and analytical chemistry where small sample volumes and rapid sample handling are key

features.

Fig. 6 Two versions of a piezo-microdispenser head: nozzle (1), piezo actuator (2), etched silicon chips (3).

Tools for probing microstructures

12

3. Tools for probing microstructures

3.1. Scanning Probe Microscopy (SPM) Microscopes are representing powerful tools for extracting and mapping detailed

information about the microstructure of an object13 and helped to unveil secrets of nature

that were long time hidden to observers since human eyes are limited to a resolution in the

order of 100 µm, which is just about the thickness of a hair14. First scientific observations

using microscopes to explore the microcosmos were reported by R. Hooke (1667) and A.

Leeuwenhoek (1697)a. The design of the optics of microscopesb changed their look

dramatically over time. At the early stage of development, simply a drop of water, oil,

honey or glass fixed on a small round cut in a metallic plate or a tube containing two lenses

fixed at its extremities were the plain devices to observe things otherwise invisible to the

naked eyes. We are used to think about microscopes as a holder that contains a tube having

two lenses: the eyepiece (ocular) and an objective (H. and Z. Janssen, 1590). This is the

more “classical” picture of a microscope. Over the years, microscopes developed into

sophisticated instruments with improved magnification and resolution (maximum allowed

by Abbe diffraction). These were accomplished by using not only visible light but also

electromagnetic waves with a shorter wavelength such as UV or fascicle of accelerated

electrons. Up till the 80’s of last century, visualisation techniques typically generated

secondary images arising from the interaction of light with structural parts of objects under

study. Then a new set of techniques appeared that provided images of an object without

depending on the diffraction of light. Going along with changing the principles of image

generation, the tubular form of the old-fashioned microscopes turned into new designs.

Scanning-probe microscopy (SPM) refers to a large number of techniques that employ

needle-like probes with physically small tip dimension that are mechanically scanned back

and forth across a surface. The probe tip can be made sensitive to a variety of surface

properties and the measure of distance-dependent interactions between the probe tip and

surface is actually used for imaging the interface. The most widely used high-resolution

imaging tools certainly are the atomic force microscope (AFM) and the scanning

tunnelling microscope (STM)15,16. As a matter of fact, these crucial tools, AFM and STM,

a Robert Hooke (Freshwater, 1635 – London, 1703) British scientist known for his work in physics, astronomy and mathematics; studied various microscopic objects and published the in “Micrographia”; Antony van Leeuwenhoek (Delft, Nederland 1632 – id. 1723,) self-educated, he made lenses with great curvature, built microscopes and was the first to observe yeast, bacteria, and other tiny creatures in a drop of water and the circulation of blood corpuscles in capillaries. b The word microscope comes from Greek language: “micro” = small and “skopein” = look at.


13

were invented by the same scientist, namely G. Binnig (he and H. Rohrer were awarded the

Nobel Prize for Physics in 1986 and shared the prize with E. Ruska who first presented the

electron microscope in 1931 in Berlin)c,17. AFM is taking advantage of force interaction

between the sample surface and a sharp tip that is attached to a flexible cantilever.

Recording the deflection of the cantilever as a function of lateral position provides

topographical images with up to nanometer resolution. With STM, on the other hand, a

voltage is applied between a nanometre-sized metal tip and a conducting surface. With the

STM tip being in extreme proximity to surface and scanned, a distance-dependent

tunnelling current is flowing and may alter laterally with changes in the tip-to-sample

separation. STM gives access to imaging the topography of conductors with atomic

resolution and has been successful for example even in imaging individual atoms laying on

compact surfaces. As it is not restricted to conducting surfaces, AFM is more suitable for

biological imaging. This has been demonstrated by employing an AFM for watching

biological macromolecules in motion or simply biopolymers such as DNA that are coiled

or stretched on a substrate18,19.

Although, the different SPM devices (Table 1) have many technical components in

common (i.e. the systems that are used for precise micropositioning of the probe tip), the

working principles behind the instruments differ quite a lot depending on the specific

interaction between the tips and sample. In general, SPM is applicable for “in-situ”

investigation of samples. Indeed, studying the target in its ambient environment is a great

advantage because no vacuum is needed as for example with a scanning electron

microscope (SEM). This and their high spatial resolution made scanning probe

microscopes so popular in a relatively short time.

Table 1

Probe Microscopy Acronym Appearance year Notes

Scanning Tunnelling Microscopy STM 1982

Scanning Near-Field Optical Microscopy

SNOM 1985

Atomic Force Microscopy AFM 1987

Magnetic Force Microscopy MFM 1987

Scanning Electrochemical Microscopy

SECM 1989

in-situ technique

c Although the first (transmission) electron microscope was presented by M. Knoll and E. Ruska in 1931, the scanning electron microscope was in fact discovered by M. Ardenne in 1938. The first commercial available SEM came up in 1966 (Cambridge Instruments Comp.).


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Scanning Ion Conductance Microscopy

SICM 1989

Scanning Capacitance Microscopy SCM

Scanning Chemiluminescence Microscopy

SCLM

Scanning …. Microscopy … … …

Among the numerous SPM devices, only one is based on an important technological

advancement in modern electrochemistry: the voltammetric and potentiometric

microelectrode. Approximately at the same time, W. Engstrom20 and A. J. Bard21

introduced voltammetric microelectrodes as new type of scanning probes and paved the

way for so-called scanning electrochemical microscopy (SECM). Being attached to high-

precision positioning devices, the micrometre-sized tip of a disk-shaped microelectrode

enabled local electrochemical measurements while being scanned across the sample

surface. As will become clear below, the spatial resolution for SECM imaging is somewhat

limited and much lower than for AFM/STM. SECM, on the other hand, can more easily

provide images of variations in interfacial (electro) chemical activity.

3.2. Scanning Electrochemical Microscopy (SECM)

The development of ultramicroelectrodes was one of the most important contributions to

electroanalytical chemistry. As their name implies, ultramicroelectrodes are extremely

small, with dimensions in the order of micrometers or less. This small size and the

electrode characteristics that come with it (low capacitive currents, low IR drop, enhanced

mass transfer - see note 1 - and high faradaic current densities) can be exploited in a

number of unique applications22. Among them are measurements in highly resistive media,

high-speed voltammetry and electroanalysis in very small volumes. The integration of

ultramicroelectrodes into a scanned probe instrument led to the invention of scanning

electrochemical microscopy which allows observing local electrochemical activity at the

liquid/solid interface or at microscopically small object.

How does SECM work?

A little information about the basic principle of SECM will be introduced because this is

necessary to understand the following parts of this work. With SECM, amperometric or

potentiometric ultramicroelectrodes with radii, r, in the order of a few µm or even less are

employed as electrochemically active scanning probes (SECM tips). In case of an

amperometric SECM tip, an electric current will flow if a redox active compound is


15

present and the tip polarised at proper potential. Tip interaction with a target surface via an

electroactive species results in distance-dependent variation of the tip current.

One tool – multiple operational modes

The SECM23,d as a whole and self-containing instrument helps in different modes to

visualise electrochemically active targets or to modify surfaces exposed to electrolyte

solutions. These are the amperometric feedback, the generation/collection, penetration, and

the surface modification mode24. In general, the amperometric tip current (iT) is plotted as

a function of its horizontal coordinates (X and Y) for imaging superficial electrochemical

activity of a sample. In the following, modes I to IV are briefly described and possible

applications discussed.

I. The amperometric feedback mode of SECM

Let us imagine a disk-shaped microelectrode (active diameter smaller than about 50 µm)

that is immersed in a solution containing a redox species (e.g. ferri- or ferrrocyanide).

Shortly after the potential of the electrode is set to a value suitable to oxidise or reduce the

d The acronym SECM stands for the instrument itself - scanning electrochemical microscope - as well as for the specific field of probe microscopy that is scanning electrochemical microscopy.

Fig. 7 Feedback mode of SECM: for a given redox reaction Ox→Red carried out at the SECM tip, a steady-state current is recorded in bulk of the solution I=Ilim (A); in the vicinity of an electrically conductive surface the Ox species are regenerated with the consequence of local rise of Ox concentration and hence a higher current recorded at the SECM tip (B); thus an insulating substrate obstructs the diffusion of Ox to the tip, thus lowering the measured faradic current (C). Generation-collection mode of SECM: for instance, surface confined species generate an electroactive compound that is detected at the SECM tip (D).


16

electroactive compound, a steady-state faradaic current is observed. The magnitude of this

current depends, besides the rate of electron transfer, on the concentration and diffusion

coefficiente of the electrochemically active substance and on the diameter of the

electroactive disk of the microelectrode. For an unstirred solution, the steady-state current

(i∞) (Figure 7A) is purely controlled by diffusional mass transport of the electroactive

species and given by the well-known equation:

acDFni ⋅⋅⋅⋅⋅=∞ 4 eqn. 1

where n denotes the number of electrons taking part in the electrode process, F the Faraday

number (1F = 96484.6 C⋅mol-1), D the diffusion coefficient (cm2⋅s-1)f, c the concentration

of electroactive species (mol⋅cm-3), a the radius of the disk-shaped electrode (cm); i∞ is

thus measured in ampere (A).

Probing a target with SECM clearly requires the microelectrode tip to be brought close to

the target surface since otherwise the surface-specific information can not be collected. In

the vicinity of a surface, however, the current measured by the SECM tip changes

significantly dependent on the tip-to-sample separation and the nature of the approached

surface25,26. How? and why? are the two questions to be answered in the following. We

should consider two cases:

1. the surface is electrochemically active (electric conductor, enzyme modified).

2. the surface is electrochemically inactive (electric insulator, enzyme-free).

1. An electrochemically active surface: the positive feedback

We are bound to a great degree by our own innately human way of thinking and judging

phenomena around us which is the common sense! We just simply have learned that some

things happen and have a certain course. This helps us to understand new items by

comparing the new facts with old schemes in our brain. Quite often, in the

physics/chemistry (especially the quantum mechanic, relativistic theory, quantum

chemistry…) the common sense deplorably fails! During my PhD, I had the chance to

introduce several students to the basics of SECM, and therefore I noticed that typically the

answer to the question “What should the current be if the SECM microelectrode is brought

close to a conductive surface?” is wrong. The students anticipated that the current will drop

to zero when the electrode is approaching a surface; their common sense suggested them

e As obtained from the Fick’s first equation, the diffusion coefficient is a linear function of temperature: D = k⋅R⋅T, where k is a constant, R is the gas constant and T is the temperature of solution. f D has typical values ranging within 10-5 – 10-6 cm2⋅s-1 that depends strongly upon the composition of the electrolyte.


17

that the current must decrease irrespective of whether the surface is conductive or not, the

surface will decrease the mass flow of the reacting species towards the electrode’s disk.

Against the common belief, the current increases (see Figure 7B and 8). Why? We shall

remember that any electroconductive surface will gain a superficial, negative or positive

net charge when it is in contact with a solution of a redox active species.

In SECM practice, the solution contains typically an electroactive species that can undergo

reversibly redox transformation. A redox compound in solution will electrically charge the

inert metallic interface (Pt or Au) to a potential of which polarity and amplitude are

controlled by the ratio of the Red/Ox concentrations of the redox species. The potential of

a gold surface, for instance, in a supporting electrolyte containing only Fe3+ and no other

redox species (especially no Fe2+) is controlled by the electroactive species present in the

bulk of electrolyte (see note 2). According to the Nernstg equation, the open circuit

potential the gold surface, at least under this experimental conditions, is expected to be

significantly more positive than the E0 of the Fe3+/Fe2+ redox couple (see below), and thus

able to oxidise Fe2+ back to Fe3+ (equation 2): g Walther Nernst (Briesen, West Prussia 1864 - Berlin 1941), German physicist and chemist with contributions in thermodynamics; Nobel Prize for Chemistry in 1920.

Fig. 8 Cyclic voltammograms recorded at a 10 µm Pt micro-electrode in bulk (blue line) and at about 15 µm above a gold surface (dark line). Not only is the steady-state current higher close to the surface but the form of CV changes from hysteresis to a perfect S-shape; 5 mM [Fe(CN)6]

3- in 0.1 M phosphate buffer and 3 M NaCl and 0.05 M NaOH; 500 mV/s scan rate; Ag/AgCl 3 M KCl reference electrode.


18

+

+

++++ +=2

3

2323 lg0

//

Fe

Fe

FeFeFeFea

a

nF

RTEE eqn. 2

where E (V) is the actual electrode potential, E0 is the standard electrode potential

measured against a normal hydrogen electrode (NHE) at 25° C, and 1 M activity, a, of the

two ions; R is the gas constant (8.314 JK-1mol-1), T is the absolute temperature (K); n is the

number of electron transferred in the redox reaction. If T = 298 K, the constant in front of

logarithm is 0.059 V. Here, E0 = +0.356 V.

In SECM, a redox mediator is amperometrically reduced (or oxidised) at the SECM tip

with a potential ensuring diffusional control of the tip current. With the SECM tip far

above a surface, tip- generated species simply diffuses into the bulk without undergoing

any further reaction. When the tip is brought close to a contacting surface, however, tip-

generated species has a chance to interact with the charged interface and, due to its

polarisation (see above) it will be oxidised (or reduced). Consequently, the local

concentration of Ox in the gap between the microelectrode tip and surface increases

compared to bulk concentration and hence the amperometric current measured at the tip

increases, too. This effect is called the positive feedback, and represented schematically in

Figure 1B. Important to obtain the negative feedback is to position the SECM tip into the

nearfield. The variation of the positive feedback SECM tip current with tip-to-sample

distance is given by equation 327:

−++=∞ LLL

i

iT

07.1exp33.0

78.068.0)( eqn. 3

where )( area active of radius

)( sample and tipSECMbetween distanceL

m

m

µµ

= .

A plot of iT versus L is known as approach curve and can be used to calculate and adjust

the tip position of a microelectrode of known dimensions.

Note: Surface-immobilised enzyme can act in the similar way as a bare metallic surface

and regenerate redox species that is consumed by the SECM. This process is known as

“enzyme-mediated positive feedback”28.

2. An electrochemically inactive surface: the negative feedback

The surface of insulating target is inert to the redox species and at small tip-to-sample

separation mechanically hinders the free diffusion of the reacting species towards the

electrode’s tip. Due to the lower flux, the amperometric tip current decreases compared to


19

the value obtained with the tip in bulk (Figure 7C). This observation is regarded as

negative feedback. Obviously, the tip-to-sample distance has a major impact on the

hindrance of diffusion and the extent of negative feedback. The degree of negative

feedback also is influenced by the ratio of the total diameter of the microelectrode and the

diameter of the active area. For a diffusion-controlled electrochemical reaction at an

SECM tip, and a disk-shaped electrode with 1:10 disk to insulation ratio, the relative

faradaic current variation obeys with good accuracy (1.2%) equation 4:

−++=

∞

LL

Li

iT

40.2exp66.0

51.129.0

1)( eqn. 4

General note:

A SECM tip is said to be positioned within the working distance when the measured

current is experiencing the influence of the conductive or insulating surface. This criterion

must be achieved in order to allow SECM measurements.

3. SECM imaging in the negative and positive feedback mode

Negative and positive amperometric feedback are highly distance-dependent, and approach

curves (I/I∞ vs. tip-to-sample separation d) permit to place the SECM tip at appropriate

working distances within the regime of the nearfield (at a height of about a few times the

SECM tip radius or less). Typically, image acquisition is achieved by scanning the SECM

tip laterally at a user-defined fixed height and simultaneously recording the tip current as a

function of position (constant-height feedback mode imaging). Figure 9 is showing in a

schematic the current profiles that one expects when a SECM tip is scanned at a constant

height across samples exposing lateral variations in topography and conductivity.

To give you an idea about SECM imaging in the amperometric feedback mode, I would

like to present a SECM image of a fingerprint that was scanned with a Pt microdisk

electrode in a solution containing 5 mM [Ru(NH3)6]3+ as the active mediator. A sample

with alternating conducting and non-conducting areas was made by firmly pressing a

finger tip against a chemically deposited layer of silver on glass. This contact led to local

removal of silver at areas where the papillae of the skin was touching the silver film. As

can be seen from Figure 10, the variation in conductivity and/or heterogeneities and

variation of the flatness of the sample were nicely revealed.


20

Fig. 9 Schematic representation of SECM tip reduction current profiles expected when scanning the tip in amperometric feedback over (A) electrically conductive, (B) electrically non-conductive surfaces that have topographic heterogeneities; a topographically homogenous but electrically heterogeneous surface can also be image in the feedback mode (C).

Fig. 10 SECM micrograph of a fingerprint. The imaging was performed with a 10 µm Pt disk electrode in the feedback mode of SECM. Dark areas correspond to low currents and indicate where the finger’s papillae touched the thin layer of chemically deposited silver on glass surface. Scanning solution: 5 mM rutheniumhexamine chloride in 0.1 M phosphate buffer(pH 6.7).


21

I I. The generation - collection mode of SECM

As the name suggests, it involves the production of an electrochemically active species at

one place, which after a short diffusion through solution is immediately detected at another

place. If the microelectrode is positioned within the working distance, and operated

amperometrically, it generates a concentration profile by consuming a substance available

in solution, and the properly polarised substrate collects the products. Hence, it is said the

SECM works in the tip-generation/substrate-collection mode (TG/SC).

Opposite to the case above mentioned, the generation process can be carried out by the

substrate and the products gathered at the microelectrode, a situation that is described as

substrate-generation/tip-collection mode of SECM29 (Figure 7D) Typical examples where

the tip is used as a collector are studies of the diffusion of metabolites released from living

cells30, corrosion31 of different materials32-34.

III. Penetration mode of SECM

This mode involves the movement of the SECM tip along a single direction that is

perpendicular to the target (Z-axis). Of course, any medium that allows the microelectrode

to break through without harming it is of interest for this SECM mode. Thus, gels,

biological tissues, solutions with a concentration gradient, or even soft films are

appropriate for being investigated with a tip in the penetration mode of SECM.

IV. Surface modification by means of the SECM

The SECM has been successfully used for modifying numerous surfaces35. This is

achievable, on the one hand due to the possibility of accurately moving microelectrodes

over a substrate and on the other hand, by taking advantage of the SECM tips to generate

reagents for directed chemical transformation of the substrate. For instance, inorganic

microstructures were created by tip-induced reduction of different metallic cations36,37.

Also, a SECM-based fabrication of microstructures of conductive polymers38 such as

polypyrrole39 and polyaniline40 and polythiophene41 or polybithiophene42 derivatives by

oxidative formation of polymerisable radical-cations from their monomers has been

described. Self-assembled monolayers (SAM) of alkanethiolates patterned on gold43 by

means of SECM allowed the local formation of biologically active microstructures

containing for instance glucose-oxidase44. Micromachining is a controlled alteration of

surfaces for creating microstructures. It can also be achieved with SECM tip-generated

etching reagents, for instance bromine45 that act as curving tool and has found application

in wet etching of silicon46, some semiconductors47 or metals like copper48.


22

The item writing mode of SECM is occasionally accounted in papers regarding surface

modification by SECM but it is in fact nothing more than the above mentioned

techniques49,50.

Constant distance mode of SECM

Most often SECM experiments are performed with the SECM tip kept at “constant-height”

above the substrate. However, there are some inherent limitations especially on tilted

samples, on surfaces with larger variations in topography and at surfaces that display both,

variation in topography and conductivity. For instance, a particular substrate has a rough

surface with gaps or heights of sizes not far from the total diameter of the scanning tip. If

the tip is positioned in the working distance, and scanned over the structure, it may collide

with the surface protuberances. In the case of a tilt surface, depending upon the scanning

direction, the electrode tip could either crash on the surface or retreat from the working

distance. In both cases, a clear interpretation of the measurements would be difficult

because changes in the tip current arising from distance variations cannot easily be

differentiated from those originating from alterations in surface activity. Moreover, since

the tip-to-sample distance has to be in the orders of few tip radii of the UME (within the

nearfield) it is obvious that with a decreasing electrode size scanning in this mode becomes

hard51. Constant height mode is only applicable when changes of the sample height or the

overall surface tilt do not exceed the tip-to-sample distance.

The introduction of the “constant-distance” SECM employing an optical detection scheme

for shear forces between the electrode tip and the surface permitted the operation of probe

tips with electroactive diameters below a micrometer and simultaneous imaging52,53. The

integrated shear force-sensitive and computer-controlled feedback loop of this mode forces

the SECM tip to follow the contours of the surface during the entire time of scanning and

imaging. This not only allows simultaneous acquisition of the electrochemical tip response,

the sample topography but also effectively prevents against tip crash even with smallest

SECM tips.

The very strict experimental requirements (perfect alignment of the laser source with the

mirrors, photodiode detector and tapered capillary of the ultramicroelectrode) required

with the optical read out of the damping of shear forces, led to efforts for improving the

“constant-height” methods. A non-optical shear force-based distance control was

established by replacing the laser beam and the light-sensitive diode with a piezo

receiver54.


23

SECM measuring tips

At the beginning, most of the SECM tips were fabricated with noble metals or graphite in

disk-shaped micro- and nano-electrodes with active diameters of 1-25 µm55,56. Glass pulled

capillaries constituted the body and the insulation of the electrodes. In addition to disk-

shaped microelectrodes, ring57,58 or hemispherical59,60 electrodes have gained recognition

and found applications in SECM.

New electrode materials allowed direct measurement of pH61,62, or potentiometric

monitoring of anions (chlorine)63. Besides potentiometric, SECM tips were also

manufactured from pulled capillaries filled with ion-selective ionophors64. Carbon fibre

microelectrodes (CFMEs), insulated by anodic electrophoretic deposition of paint

(EDP)65,66 were used as SECM-tips for low-noise recordings of neurotransmitters release67.

A remarkable electrode for simultaneous electrochemical, optical and topographical

images was obtained from an optical fiber surrounded by a gold ring electrode and an

electrophoretic insulating sheath68.

Drawback

Spatial resolution of SECM is primarily determined by the size of the SECM tip and for

that reason not on atomic scale as for instance with scanning tunnelling microscopy (STM)

and atomic force microscopy (AFM). However, the electrochemical nature of the scanning

probe offers an exceptional chemical selectivity and SECM therefore serves as an excellent

tool for studying interfacial (electro)chemical properties and reactions.


24

Home build SECM set-up

All SECM experiments gathered in this volume were carried out with the following set-up

(Figure 11).

Fig. 11 Scheme of representing the scanning electrochemical microscope (left) and photo of the SECM used in this work (right). The microelectrode is positioned close to the sample with the Z stepper motor. When the working distance is achieved, X and Y motors move the sample according to the given scanning parameters: speed, distance between data points and time for data acquisition. Unlike the Faraday cage that is useful anytime when recording low amperometric currents, the vibration-free platform is useless if the SECM tip’s height is not controlled by shear-forces. Scale bar on the right image represents 35 cm.


25

3.3. Notes

1. Short after being introduced to preparation of microelectrodes by Dr. E. Bonsen, (2001) I

had difficulties in understanding, without resolving mathematical equations, why it is

believed the mass transfer towards a microelectrode is enhanced compared to a

macroelectrode! I felt something is missing to me, something like a qualitative

representation of the diffusional processes that take place around a macro- and micro-

electrode the surface. Quite a while after that moment I have got in mind a clear picture of

a reasonable explanation showing why “the mass transfer is improved”. The mass transfer

is improved because the resource of the fresh material that is carried towards the

electrode, by diffusion, is much higher for a microelectrode as compared to a

macroelectrode (Figure a). Let us name the as unit volume (orange square), the volume of

electrolyte of which content of redox species is fully emptied after the potential step was

applied to the electrode. Once the first species were consumed, fresh material is pushed

towards the electrode by forces derived from the chemical potential of the present species.

In the in the case of the macroelectrode a number of unit-volumes are emptied (9 white

squares in this example) and 13 units are in vicinity to supply the mass transfer (blue

squares), whereas, in the case of the microelectrode one white square has only 5 blue

squares around. I think is possible and didactically useful to compare the ratios of the blue

square to white square, because as big are the resulting numbers as efficient is the mass

transfer! For the above example, where the macroelectrode has 1.44 and the

microelectrode 5, it becomes clear why the last mentioned is better in enhancing the

diffusion of reactive species in direction of electrode surface. This model is valid for

spherical geometries as well.

Fig. a Qualitative model explaining why the mass transfer of active species towards an electrode surface is more efficient for microelectrodes as compared to macroelectrodes.


26

2. Example: a portion of a noble metal is immersed in [Fe(CN)6]3- aqueous solution. Thus, the

potential of the surface is polarised at approximately +400 mV versus the Ag/AgCl 3M KCl

reference electrode (potential measurement at zero current, so-called open circuit potential

(OCP) of noble metal - reference electrode couple). An electrolyte containing [Fe(CN)6]4-

lowers the open circuit potential to about -200 mV. These two compounds are partners of a

redox couple. If an electrochemical in relation is carried out at the tip of a SECM that is

positioned in the working distance, for instance the reduction of ferricyanide, the minute

quantity of ferrocyanide will be quickly consumed at the conductive substrate and this

before being able to affect the perturb the OCP of the metal – solution interface. This

means that the species in high excess (usually in bulk) are controlling the potential of the

substrate while the traces of the partner species are thermodynamically instable (Figure b).

The OCP is hence a critical number for it determines what species can exist at a certain

potential (circa 300 mV in the mentioned case).

Fig. b The stability potential of the redox couple ferro/ferricyanide. These domains are revealed in a cyclic voltammogram performed with a 10 µm Pt disk electrode in a solution of 5 mM ferricyanide, 0.1 M phosphate buffer, 3 M NaCl at a scan rate of 100 mV/s; Ag/AgCl 3M KCl reference electrode.

Electrodes for electrochemistry in small volumes

27

4. Electrodes for electrochemistry in small volumes

4.1. Integrated working/reference assembly

The information about SECM probes given before dealt with conventional type of disk-

shaped microelectrodes. In the following, an electrode will be described that is well

suitable for electrochemical measurements in small volumes of electrolytes and especially

for SECM applications in nanoliter droplets. The motivation to work on the development

of such electrode originated from an observation that a colleague of mine complained

against the difficulty to operate a three-electrode configuration in the tiny vials of a

microtitre plate (Figure 12).

When aiming on applying precisely positionable microelectrodes for measurements in

ultra-small electrolyte volumes it is crucially important that the total diameter of their

sensing tips including the insulating material is sufficiently small. Microelectrodes

fulfilling this prerequisite have been constructed either by sealing carbon fibres or thin

metal wires into tapered tips of pulled glass micropipettes56,69,70 or by coating carbon fibres

with a thin layer of an electrochemically depositable polymer 65,66,72, and chemical vapour-

deposited quartz73, respectively. However, these needle-type electrodes have to be jointly

used with a reference electrode (RE) to accomplish at least a 2-electrode configuration as

required for voltammetry.

Fig. 12 Working electrode small, … but the reference?


28

Clearly, a completion of such a 2-electrode assembly in highly restricted space is a

challenging task. Recently a miniaturised dual electrochemical probe for voltammetry and

SECM in nl droplets was introduced74. A carbon fibre was sealed with epoxy into one of

the two tapered channels of pulled theta glass capillaries while the other kept a

macroscopic Ag/AgCl wire in a chloride solution. The reference system was separated

from the analyte solution with an agar salt bridge that was placed in the tip opening. This

probe design obviously had an advantage for measurements in extremely small volumes

since a combined µWE/RE assembly is more easily to position, needs not as much space,

only a single micro-positioning device and avoids the risk of collision, which can occur

when the tips of two separated mobile electrodes are brought closely together and moved

autonomously from each other. However, the preparation of the proposed electrode system

is difficult and needs a lot of hands-on experience to reach a sufficient success rate.

Furthermore, the tapered tips of the double-barrelled microelectrodes are fragile and can

easily break during operation.

In principle, a technically easier alternative for the construction of dual probe tips is

offered by depositing a metal onto the outer surface of the (glass) insulation of disk-shaped

microelectrodes. Polishing the tip of metal-coated microelectrodes would lead to the

exposure of the active disk that is concentrically surrounded by the insulation and an outer

ring of metal and some used this approach for the fabrication of a coaxial microelectrode

designed for in vivo measurements of nitric oxide75. Pt wires were sealed into the tips of

tapered glass capillaries and then silver was sputtered on the glass surface to form a

Ag/AgCl reference electrode. Concentric layers of metal were deposited on the insulation

of microelectrodes by sputtering76, chemical vapour or electroless deposition (also used for

preparation of substrates in microcontact printing77) were also used to establish an active

electrical shield for reducing the stray capacitances and improving the microelectrode

noise78-81. Note: spatial resolved surface analysis (potentiostatic, galvanostatic techniques)

of non-wetting substrates can be also achieved with the scanning droplet cell method82

where the substrate under investigation must act as the working electrode.

4.1.1. Preparation of precursor electrode

Disk-shaped Pt microelectrodes served as precursors for the fabrication of coaxial Pt/Ag

electrode assemblies. They were constructed from 10 µm-diameter Pt wires that were

tightly sealed into the tapered ends of pulled borosilicate glass capillaries (O.D. 1.5 mm,

I.D. 0.75 mm, L 100 mm). In contrast to the well established method of back-connecting

the electrodic material to external copper wire with bi-component silver epoxy glue, which


29

is awkward an alternative and easy method was developed. The Pt and a Cu wire (Ø 0.5

mm) were attached to each other by filling the capillaries with a small amount of Zn

powder and crushed tin solder, which after placement was melted by careful heating in an

coiled filament at high currents. The melting solder entrapped the Zn particles and the

wires ensuring good electrical contact between all components. Smooth Pt micro disks

were exposed by polishing the tip at 90° on emery paper (grade 320 to 2000) and then on a

polishing cloth wetted with alumina suspension (particle sizes: 3 µm, 1 µm, and 0.3 µm).

The ohmic response does not change over a wide range of scan rates; therefore these

electrodes can successfully replace the epoxy back-connected electrodes. Table 2 outlines

the advantages of zinc-based electrodes over epoxy ones.

This method was applied first for thicker platinum wires without using zinc. In this case,

the wires are stiff enough not to bend when adding the copper wire and there was a good

contact. However, for thin platinum wires this method is not suitable because the delicate

wire can break or turn away as the copper is inserted and hence no electrical contact is

established (see Figure 13 A, B). To overcome this problem, the narrow space between the

copper wire and the capillary tip was filled with zinc powder (Figure 13 C, D).

The ohmic resistivity of the Zn-to-Pt connection was found to be low and the electrode

typically worked reliable.

Tabel 2

Bi-component silver epoxy glue Zinc powder

1. most part of the glue is wasted (it

remains onto the preparation dish, gloves)

2. requires a oven for fastening the two

components (800 C, 1 hour)

3. no efficient control during electrode

preparation

4. glue – platinum/copper wires

connection is fragile (once the glass

capillary breaks, the glue-wires contact is

usually lost)

5. expensive

1. no single zinc particle is wasted

2. no oven necessary (ready made

electrode)

3. every preparation step is under control

4. zinc – tin is strongly binds against glass

(because of the instant wetting resin)

5. inexpensive


30

4.1.2. Chemical deposition of silver onto the body of precursor electrode

Well-adhering layers of Ag were accessible only when the glass insulation of Pt

microelectrodes was rubbed with emery paper (grade 320), wiped with soft tissue paper

and rinsed with a stream of acetone in order to remove the grinding dust. Obviously, this

treatment increased the roughness of the substrate herewith offering a better physical

support for anchoring the Ag deposit to the glass through microscopic fissures in its

surface.

In a small tube, 2 ml of 5% AgNO3 solution were then treated with 10% NaOH solution

until no more dark-coloured precipitate (Ag2O) was formed upon addition. The Ag2O was

converted into soluble [Ag(NH3)2]OH by slowly adding 28% NH4OH until the solution

became clear and colourless. Tollens’ reaction and formation of metallic silver was

induced by adding a few ml of a reducing agent, here, it is 10 % glucose. To enhance the

rate of Ag deposition, the body of the Pt microelectrode was preheated with a heat gun, and

then quickly immersed into the freshly prepared silvering bath for only a few seconds,

taken out and immediately dried with the handheld dryer. For uniform and compact Ag

layers the treatment had to be repeated multiple times. For further Ag deposition, the

microelectrodes were rinsed with distilled water before heating (Figure 14). As can be seen

from the Figure 14, the chemically deposited silver appeared as a uniform coating of white

colour. The deposits typically had a morphology reflecting to some extent the irregularities

of the abraded surface of the underlying glass and were mechanically sufficiently stable

not to be damaged by handling of the electrodes and performing electrochemical

Fig. 13 Only solder alone can not connect the copper lead to the thin Pt wire (A, B).

Zinc microparticles can establish an intimate contact between the external copper lead

and the the glass-embedded platinum wire (C, D).


31

measurements. However, to enhance the stability of the silver layer and extend its life-

time, the electrode surface can be covered with ordinary nail varnish.

Finally, a copper wire was firmly attached to the silver layer using a heat shrinking tube

(Figure 15).

Optionally, thicker layers of silver could be deposited on pre-coated microelectrodes by

galvanic Ag deposition from a thiosulphate-based silvering bath (pH 10) (Figure 16). At

room temperature, a current density of 0.5-0.6 A/dm2 was applied to form well-adhering

and stable Ag deposits. Tips of Ag-coated microelectrodes were carefully polished on

emery paper (grade 2000) and polishing cloth soaked with alumina to re-establish a clean

electroactive Pt disk.

Reproducibly, a flat tip geometry was obtained with the 10-µm-diameter Pt wire well

centred in the glass insulation and the thin layer of chemically deposited Ag, respectively.

In the particular case shown, the total tip diameter was about 200 µm giving a RG value

(dtotal/dPt disk) of about 20. However, tips with diameters down to about 50 µm were

obtained by successively polishing them at an angle in order to reduce the thickness of the

glass insulation at the apex and then upright to expose the Pt disk.

A top view of the tip of a polished Pt/Ag electrode assembly is shown in the scanning

electron micrograph of Figure 17. The following experiments were performed with an

electrode identical to the one in Figure 18.

Fig. 14 The transformation of the precursor electrode (left) into the silver coated electrode

(right) requires three steps: blowing hot air on the precursor (1), dipping into the Ag+ solution

(2), rinsing with pure water (3).


32

Fig. 15 The external connection of the coaxial silver layer.

Fig. 16 The thickness and morphology of the chemically deposited silver layer can be adjusted by electroplating of silver in an electrolysis cell with a silver anode.


33

Fig.17 Scanning electron micrograph of the Ag-chemically coated electrode.

Fig. 18 The completed coaxial working/reference electrode.


34

In order to verify ability of this electrode assembly to perform electrochemical

measurements a series of experiments were designed to address this problem. For instance,

Figure 19 compares two cyclic voltammograms of 5 mM [Ru(NH3)6]Cl3 that were

recorded in a single well of a 384-well microtitre plate (Figure 20) at a coaxial Pt-

µWE/Ag-RE electrode assembly with either the Ag layer of the assembly as internal

pseudo reference (A) or a miniaturised Ag/AgCl 3 M KCl system external reference

electrode (B). In both cases, the CVs displayed the expected sigmoidal shape (steady state)

that is characteristic for microelectrodes. The half-wave potential (E1/2) for the reduction of

Ru3+ measured vs. the internal pseudo reference was shifted by about -60 mV as compared

with the external reference electrode. However, the shape of the CVs did not change

notably over time indicating that the coaxial Ag electrode is sufficiently stable to be used

as an integrated pseudo reference for voltammetric measurements, even without

chloridisation.

4.1.3. Applications of the coaxial Pt-µWE/Ag-RE in SECM

It was thought that this electrode could also be turned to the development of a robust and

easy to use (micro-)electrode arrangement supporting straightforward SECM imaging in

nanoliter volumes of solutions. To demonstrate the suitability of the coaxial Pt-µWE/ Ag-

RE electrode assembly for SECM applications, it has been attached to the Z-positioning

element of the SECM and used as a scanning probe with integrated reference electrode in

electrolytes containing 5 mM [Ru(NH3)6]Cl3.

Fig. 19 Cyclic voltammograms recorded vs. the inner reference (A) and an external Ag/AgCl reference electrode (B); 5 mM [Ru(NH3)6]Cl3 in 0.1 M phosphate buffer; 100 mV/s.


35

The Pt-µWE, was operated in the amperometric feedback mode and scanned at constant

height. To induce diffusion-limited reduction of the dissolved redox mediator, it was kept

at -350 mV vs. the integrated coaxial Ag-RE or, in control measurements, vs. the external

Ag/AgCl reference electrode. In a large-volume electrochemical cell, tip approach curves

were recorded in both configurations on insulating (glass) and conducting (gold) surfaces.

The resulting negative (positive) feedback curves were properly overlapping and in good

accordance with the curves from theoretical calculations (not shown). Furthermore,

approach curves were performed on gold using either an ordinary Pt disk microelectrode or

a Pt-µWE/ Ag-RE electrode assembly with the ring of silver as SECM tips. As can be seen

in Figure 21, the obtained positive feedback curves were almost perfectly lying on top of

each other. This clearly demonstrates that a recycling of tip-induced Fe2+ at the conducting

outer ring of silver does not disturb significantly the positive feedback interaction between

the Pt-µWE and a conductive sample surface.

For the given electrode geometry this was expected, since the distance between the Ag ring

and the Pt-µWE is about 100 µm which is far too large to be within the regime of the

electrochemical feedback of a 10 µm diameter microelectrode. In contrast, an alteration of

the feedback behaviour cannot be excluded when reducing the total tip diameter of Pt-

µWE/Ag-RE assemblies below about 20-30 µm. SECM test trials were then carried out in

small droplets (< 1µl) of the mediator solution that was placed on top of an array of four Pt

band microelectrodes (length: 1 mm; width: 25 µm; spacing: 25 µm) (Figure 22).

Fig. 20 Electrochemistry performed with the integrated Pt working / Ag

reference electrode assembly in a single well of a 384-well microtitre plate.


36

As usual, tip approach curves were used to position the tip of the Pt/Ag electrode assembly

in close proximity to the sample surface. Typically, a working distance was chosen at

Fig. 21 Approach curves that were recorded on a gold surface with (∆) the tip of

a coaxial 10 µm-diameter Pt-µWE/Ag-RE electrode assembly and (O) a 10 µm

diameter Pt disk microelectrode of similar RG value and with no silver deposited

on the insulation; scan speed: 1 µm/s; electrolyte: 5 mM [Ru(NH3)6]Cl3/0.1 M KCl.

Fig. 22 Schematic of the SECM experiment designed to scan with the coaxial electrode in

a 500 nl solution and of the Pt array band used as a substrate.


37

which the amperometric tip current dropped off to about 50 % of the value measured in

bulk solution. Figure 23 shows a representative 3-dimensional feedback image of the Pt

microstructure that was acquired with the coaxial Pt-µWE/Ag-RE electrode assembly in a

droplet of 500 nl volume of mediator solution. The Pt bands are clearly visible with

negative feedback observed over insulating areas in the neighbourhood of the Pt bands and

positive feedback due to the regeneration of consumed mediator molecules above the

electrochemically active Pt bands.

Successfully, imaging the Pt band microstructure provided the first evidence about the

operability of the coaxial SECM tip arrangement for measurements in small volumes.

However, we observed that solution evaporation became a critical parameter for image

acquisition due to rapid changes in the volume of the nl droplets. Accordingly, a raise in

mediator concentration took place, ultimately increasing the SECM tip currents in due

course of imaging. This effect is confirmed by Figure 26A showing a set of fifty line-scans

that were obtained on the Pt band microarray one after the other at scan speeds of about 5

µm/s. Within the one hour of the experiment, the amperometric tip current changed

virtually by a factor of two. Especially, with low scan speeds for SECM imaging, the

experiment will take considerable time and hence, a strategy to prevent solution

Fig. 23 3-D SECM image acquired by operating a coaxial Pt-µWE/Ag-RE as dual

electrode scanning probe in the amperometric feedback mode on a Pt band microarray;

scan speed: 5 µm/s; 5 mM [Ru(NH3)6]Cl3) in 0.1 M phosphate buffer.


38

evaporation is of key importance when aiming for long-term SECM measurements in

microvolumes. Various methods have been proposed previously to stabilise small sample

volumes, among them, regulating the humidity in their environment by maintaining a

water-saturated atmosphere in specially designed closed chambers, adding glycerine to the

electrolyte to lower the water vapour pressure, or covering the solution with mineral oil83-

85. Here, the tiny droplets of ruthenium solution were protected with a reasonably thick

layer of paraffin oil against evaporation. As shown in the schematic representation in

Figure 24 and 25, a large-diameter O-ring of appropriate thickness was used to keep the

paraffin oil in place and hinder it to spread that thin that the droplet would be exposed to

air and hence start to fade away.

Fig. 24 Schematic representation of SECM in nanoliter droplet. A layer of paraffin

oil serves as a protection against solution evaporation and the O-ring keeps the

electrolyte in the proper place.


39

This experimental design is simple, and primarily was chosen to gain maximal spatial

freedom for tip movements in X and Y direction. Certainly, the Pt-µWE was first placed

into the droplet before adding the paraffin oil to the cell to avoid a contamination of the

surface of the tip electrode with the oil.

Figure 26B is a clear proof that paraffin oil indeed offered an effective diffusion barrier for

water molecules and did not permit nl-sized droplets to evaporate and change their volume

significantly throughout SECM imaging. Again, fifty line-scans were taken successively

on the Pt band microarray at a scan speed of 5 µm/s but at this time taking advantage of the

aforementioned strategy of evaporation protection. Although the line scans were not fully

superimposed, the current values of the first and fiftieth scans differed from each other by

only 3-4 %. This was clearly indicating that the droplet volume and concentration of the

mediator could be kept reasonably stable for the duration of the experiment when using

coverage with paraffin oil, a prerequisite for successful carrying out long-term SECM

measurements in nl volumes.

4.2. Miniaturised Ag/AgCl reference electrode

SECM experiments performed in a conventional electrochemical cell required the use of a

pseudo-Ag reference electrode considering the bulky Ag/AgCl 3M KCl reference electrode

available on the market. It turned out that a pseudo Ag/AgCl reference can not be used in

oxidising media (such as ferricyanide solutions), because the electrode potential is greatly

affected. For this reason, a reference electrode had to be made, which is stable and small

enough to fit in the available holder of the SECM tip. To build a trustworthy Ag/AgCl 3M

KCl reference electrode, a Pasteur pipette was used as specified (Figure 27) together with:

500 nl of electrolyte

Pt band array

Fig. 25 Photographs showing the aqueous droplet over the four Pt band, all covered by paraffin oil (left) and the immersed electrode positioned at the working distance over the Pt array (right).


40

a ceramic frit with a diameter about 1mm mm, shrinking tube, 3-4 cm of silver wire. The

short cylindrical frit (about 2 mm length) is sealed into the narrowed part of the Pasteur

pipette with the flame of a portable piezo-torch.

The chloridisation of the silver wire was carried out in a typical solution of 3M KCl and

0.1M HCl in water (1:1 volumes) with a Pt coil as counter electrode. A relative thick and

adherent layer of AgCl is obtained in two potential steps: 1 min at 5 V and then 10 min at

15 V.

The shift of the electrode potential measured versus a commercial Ag/AgCl electrode is

within few mV. The reserve of Cl- (3M KCl) inside the capillary is high enough keep the

potential constant and to sustain low currents for long time in a two electrodes set-up as

usually used. A similar reference electrode was chosen as reference electrode for all

experiments described in the next chapters.

A B

Fig. 26 Droplet SECM carried out on a Pt band microarray in 500 nl droplets of a 5 mM [Ru(NH3)6 ]Cl3 in 0.1 M phosphate buffer solution with a coaxial Pt-µWE/Ag-RE as scanning probe. 50 line scans in (A) were recorded with the droplet exposed to air while the ones in (B) were obtained using a layer of paraffin oil to protect the droplet against solution evaporation.


41

Fig. 27 Simple, four-steps procedure for manufacturing of a Ag/AgCl 3M KCl reference electrode; Pasteur pipette (1), storing tube (2), electrode (3), frit (4), glass cutter and the cutting line (5), complete electrode (6). The syringe shows the way of refilling the capillary with fresh KCl. This operation can be done easily at anytime.

Enzyme microstructures

42

5. Micropatterning and microelectrochemical characterisation

of biological recognition elements

5.1. Enzyme Microstructures A biosensor is intended to be specific to a unique substrate (analyte). However, real

samples (physiological fluids, waste waters, wine etc.) are usually made up of multiple

components and monitoring all of them would require a bundle of biosensors. The size and

large number of different biosensors necessary to perform a complex analysis is a hurdle

for small amounts of samples (Figure 28). The actual technology of micropatterning

Biological Recognition Elements (BRE) such as photolithography, soft-lithography, self-

assembled monolayers, piezo-microdispensing permits the creation of multi-analyte arrays

with densely packed biosensors by immobilising BREs onto different substrates and

preserving the activity of these molecules. Thus, an array is a promising candidate for

becoming an ideal tool for detecting a large spectrum of analytes with a miniscule device.

The successful marriage of biology and microelectrochemistry will result in improved

biosensing devices that can have wide applications. This marriage, however, will require

microfabrication methods that effectively assemble sensitive biological components (e.g.

enzymes, nucleic acids, proteins, and viable cells) onto suitable substrates. The work

described throughout this subchapter was focussing on the application of polymers as

structural materials used to entrap enzymes as recognition element.

One example that illustrates our approach is the use of a copolymer of ethylene and vinyl-

acetate (Vinnapas® EP16 W1) and glucose oxidase (GOD) for the assembly of

micropatterned enzymes. The EP16 offers thin-film properties that allow it to be locally

deposited onto different surfaces in response to an applied voltage on a piezo

microdispenser. Once the enzyme and the polymer are mixed, the enzyme can be locally

assembled onto a given surface. SEM (Scanning Electron Microscope) and SECM

micrographs have demonstrated that micro-deposition allows enzyme assembly to be

performed with high spatial selectivity while the deposition conditions are sufficiently mild

to retain the molecular structure of the enzyme (the activity of trapped enzyme had been

high enough to produce detectable amounts of hydrogen peroxide).

1 Wacker Polymer Systems GmbH & Co. KG, Burghausen, Germany


43

Fig. 28 Schematic showing the power of miniaturisation in reducing the number of individual sensors and laboratory space.

About enzymes

44

5.1.1. About enzymes

Due to its unique 3-dimensional structure, each enzyme is specific to its substrate or to

structurally similar compounds. Enzymes and substrates need proper circumstances to

interact with each other. In the second half of the XIXth century, E. Buchnera had

investigated the alcoholic fermentation of sugars and proved the yeast cells are not

necessary to ferment glucose, fructose or maltose. A juicy extract of brewer’s yeast is able

to perform this task. His belief was that a protein, named by him zymase, must be

responsible for this process. Inspite of XIXth century dynamism, lack of adequate

information and minimal equipments made the separation of enzymes from cells and the

structural analysis complicated and thus explaining why Buchner had doubts in

considering the zymase as a member of enzyme family. It was a fact that “there are

important differences between fermenting action and the action of ordinary enzymes.

The latter is solely hydrolytic and can be imitated by the simplest chemical means86”.

Well, he was wrong but this example intended to briefly give a view of the problems and

ideas that scientists had encountered about 100 years ago working on enzymes.

The biological processes that occur within all living organisms are chemical reactions, and

enzymes regulate most. Without enzymes, many of these reactions would not take place at

a perceptible rate. Enzymes are remarkable catalysts! Not only do they effectively

accelerate the rate of the reaction, but they also limit the potential side reactions so that the

yield of a reaction is essentially 100%. The overall energy of the reactant interaction with

the active site can be determined from the equilibrium constants of the reactant-enzyme

complexes [ES]. The equilibrium constants are usually in the order of 102 – 1010 M which

corresponds to free energies of interaction on the order of -12 to -60 kJ/mol. As can be

noticed, these energies are approximately 10 to 15 times lower than those associated with

covalent bonding.

Enzymes catalyse all aspects of cell metabolism. This includes the digestion of food, in

which large nutrient molecules (such as proteins, carbohydrates, and fats) are broken down

into smaller molecules; the conservation and transformation of chemical energy; and the

construction of cellular macromolecules from smaller precursors. Many inherited human

diseases, such as phenylketonuria87 (PKU - a genetic disorder that results in deficiency of

an organism to convert phenylalanine to tyrosine), albinism (a genetic circumstance which

a Eduard Buchner (Munich, Germany 1860 – 1917, Munich) won the Nobel Prise in 1907 for “his biochemical researches and his discovery of cell-free fermentation” (see: http://www.nobel.se/chemistry/laureates/ 1907/buchner-bio.html)

About enzymes

45

results in a lack of melanin pigment in the eyes, skin and hair of both animals and humans)

and many others, result from a deficiency of a particular enzyme.

Enzymes have valuable industrial applications, the fermenting of wine, leavening of bread,

curdling of cheese, or brewing of beer have been practiced from earliest times, but not until

the 19th century were these reactions understood to be the result of the catalytic activity of

enzymes. Medical applications of enzymes make the metabolism products monitoring

easier and less stressful to the patients (one example to be mentioned is the biosensor for

glucose). In the early stages of the studies on fermentation, the notions “enzyme” and

“ferment” coexisted88. Latin and Greek inspired innumerable scientists over time when

looking for a name to be given to a new substance, living creature or phenomenon. And

this is exactly what happened to enzymes: enzumos is a Greek word meaning “yeast” and

fermentum i.e. a Latin word having the same meaning.

Monitoring health-relevant molecules in body fluids became accomplishable in a simple

manner due to the high (sometimes absolute) specificity of enzymes that make them

perfect tools able to function in complex environment such as blood.

Enzyme classification

A close view on the functional structure of enzymes shows proteins are not sole

components but often accompanied by other groups like FADH2, NADH, porphyrins.

Some enzymes made of pure proteins are able to attach and convert the substrate into

product. However there are a large number of enzymes that use special non-proteic units to

establish a catalytic reaction. This is the basis of an enzyme classification (Figure 29) as

briefly depicted below:

1. Holoproteinsb

- are enzymes entirely built from aminoacids

2. Proteides

- contain beside a protein (apoenzyme) that is inactive but specific to a substrate, a

chemical part that undergoes enzyme specific reaction namely cofactorc. Upon

attachment type, the cofactor is called a prosthetic group, if binds covalently and

is called coenzyme if binds loosely (see note 1).

b From Greek “holos” that means “whole” c The cofactor apart from enzyme is able to catalyse the reaction of numerous substrates.

About enzymes

46

Enzyme coding

“Enzyme Committee” (EC)89 that is a subdivision of “Nomenclature Committee of the

International Union of Biochemistry and Molecular Biology” (NC-IUBMB) established

the enzyme nomenclature. The large variety of enzymes determined EC to assign each

enzyme a code made up of four numbers (say A.B.C.D.). “A” indicates one of the 6 major

classes (see note 2). “B” and “C” codes the affiliation to a sub- and a sub-subclass of

enzymes. “D” is just a numerator that shows the place of enzyme in the list with enzymes

of the same sub-subclass. According to EC nomenclature, one can refer to glucose oxidase

as EC 1.1.3.4. Commonly, the glucose oxidase is known as GOD. This name will be used

in the following parts of this work.

Enzyme activity

Enzyme activity means enzyme ability to transform a substrate into a product. Either a

product or a reagent (even the substrate) is experimentally detected in order to measure,

under controlled conditions, the enzyme activity. It is influenced by many factors

(substrate, solvent, temperature, inhibitors, ionic strength, pH and cofactor). Two kinds of

disturbing factors of enzyme activity: 1) specific to each individual enzyme as substrate,

cofactor, and inhibitor. For most of enzymes, some substrates can react with but at

different rates. This justification is valid for the cofactor and inhibitor too; 2) factors that

control the protein conformation as solvent, ionic strength, pH and temperature affects the

protein backbone shape. Temperature influences both the kinetic and the protein

Fig. 29 Enzyme classification. Enzymes only made of proteins are called holoproteides whereas enzyme containing proteins and other compounds are named as proteides. An apoenzyme is inactive as long as the cofactor is apart. The cofactor is covalently or loosely linked to the apoenzyme and thus it is named prosthetic group or coenzyme respectively.

About enzymes

47

conformation. The enzyme activity has to be measured at the optimal temperature of the

given enzyme (see note 3)! Dissolved oxygen must be taken into account for the enzymes

that use it during the substrate transformation.

Enzyme activity related definitions

The amount of enzyme that is involved in a biochemical reaction is difficult to determine

in terms of grams of enzyme, as its purity is often low and not to mention that parts of it

might be in an inactive state. Parameters that are more relevant are the activity of the

enzyme preparation. These activities are typically measured in terms of the activity unit

(U), which is defined as the amount of substrate (micro-, nano- or pico- moles) converted

by a certain enzyme quantity within a unit time. An international unit (I.U.) corresponds to

a unit time of 1 minute. Another unit, which is used but did not receive widespread

acceptance is the katal (kat) even though recommended by International System of Units

(SI). One katal will catalyse the transformation of 1 mol of substrate per second. The old

unit (U) was generally nameless for approximately 30 years and only occasionally called

“international unit.“ It uses minutes instead of seconds as its unit of time, which is not

keeping with SI or with the usual way of expressing rate constants in chemical kinetics.

Sometimes non-standard activity units are used such as Soxhet, Anson (AU – Anson unit),

Kilo Novo, which are based on physical changes for instance lowering viscosity, and

supposedly better understood by industry. These units are gradually falling into disuse. Of

a practical interest is the specific activity defined as the number of units corresponding to 1

milligram of enzyme preparation (U/mg).

Enzyme kinetics

Enzyme catalysed reactions are basically chemical reactions and a kinetic study is

meaningful when aiming to understand the reaction mechanism or calculating the yields.

“Don’t waste clean thinking on dirty enzymes” said E. Rackerd and he meant that

detailed studies on how enzyme catalyses the conversion of one substance to another is

generally waste of time until the enzyme has been purified from other substances that make

up a crude cell extract. The mixture of thousands of different proteins released from a

disrupted cell that typically contains several that direct other rearrangements of the starting

material and the product of the particular enzyme’s action. Only when one has purified the

enzyme to the point that no other enzymes can be detected can one feel assured that a

single type of enzyme molecule determines the conversion of substrate to product and does

nothing more90. d Efraim Racker (1913 Neu-Sandez, Poland – 1991 Syracuse, New York, USA) American scientist that isolated, from mitochondria, the ATP synthase in 1960.

About enzymes

48

Experiments show that:

a. for low substrate concentrations S, the reaction rate is proportional to S,

v = k⋅⋅⋅⋅S (first order reaction);

b. for high substrate concentrations S, the reaction rate is independent on S,

v = k (zero order reaction);

Herein, E stands for the enzyme and S for the substrate. Conclusion: although the net

reaction is simply S P, it must actually have a mechanism with steps that involve the

enzyme reaction. Enzyme kinetics obeys Michaelis-Menten equation (L. Michaelis and M.

Menten, 1913)e. They proposed the following reaction scheme:

In the conversion of S to product P, the enzyme undergoes no net change.

Symbols have the following meanings: S – substrate; E – enzyme; [SE] – activated

complex; P – product; k 1 – constant of activated complex formation; k -1 constant of

activated complex break-up; k 2 – constant of activated complex consumption. From the

above listed experimental data and assuming that the formation and breakdown of the [SE]

has reached a value that is no more changing during reaction (steady-state approximation),

than the following equation can be derived (equation 5):

[ ] [ ][ ]SK

SEkv

M

P

+= 0

2

Accordingly, the rate of the enzymatic reaction depends linearly on the amount of enzyme

added and on the amount of substrate as well.

Particular cases:

1. [ ] [ ]02maxEkvvKS

PPM==→>>

2. [ ][ ] [ ]

M

PM

K

SEkvKS

0

2=→<<

3. [ ]2

maxP

PM

vvKS =→=

e Nowadays, both more general and more specific models of enzyme kinetic exist for the enzymes that do not follow the mechanism described by Michaelis-Menten equation.

S + E [SE] P + Ek1

k-1

k2S + E [SE] P + Ek1

k-1

k1

k-1

k2k2

Eq. 5

About enzymes

49

The third equation can be used to define KM as substrate concentration at which the

reaction rate is half of the maximum rate. KM (normally expressed in mM) is a short hand

of: 1

21

k

kk +− and therefore has no dimension.

Note:

- the fundamental characteristic of the Michaelis-Menten mechanism is a transition

from first-order to zero-order kinetics near a critical substrate concentration that is

KM;

- KM gives quick information about enzyme affinity to the substrate (the lower the

value of KM, the stronger is the binding between substrate and enzyme and hence

more product is obtainable in a given time);

- it is useful to refer to 1

1

k

k− as KS (the dissociation constant of [ES]);

- KM indicates the required substrate concentration to achieving half of the maximum

reaction rate;

- when determining KM, the optimal pH and temperature must be set;

- for the majority of enzymatic systems k2 is small compared to k-1 and thus KM ≅

KS;

- the steady-state approximation, i.e. [SE] = ct., is true as long as the enzyme

concentration is small compared to the concentration of the substrate.

Artificial enzymes

Surprisingly, it has been possible to design assemblies of inorganic or organic molecules

that have remarkable catalytic properties and high specificity to substrate. Enzymes are

isolated and purified from plants or animals and due to abundance of other biomolecules

this process is complex and time consuming. A great advantage could be given by

possibility of on-demand straightforward synthesis of enzyme-like materials. This issue

already raised lot of attention in the last decades and achievements till date are

encouraging. Briefly, three approaches towards the synthesis of such non-natural catalyst

that mimic real enzymes are presented.

Inorganic

An artificial enzyme analogue of Nafion/lead-ruthenium oxide pyrochlore (Py) chemically

modified electrode (NPyCME) was synthesized by in situ precipitation through blocking of

Nafion’s hydrophilic zones91.

About enzymes

50

Organic

The plausible translation of the principle of enzymatic catalysis to artificial catalysts has

attracted also organic/macromolecular scientists since the 60’. One should not forget that

the exclusive properties of enzyme rely on their polymeric structure! Synthetic polymeric

molecules are attractive chiefly because of their chemical and physical properties (can

withstand heat and chemical attacks)92. Moreover, such polymers are obtainable with little

effort for industrial production. In principle, a cross-liked polymer is structured around a

molecule that acts as a template (for best performances of the artificial enzyme, it should

be a transition state analogue of the aimed enzymatic reaction). The monomers are bearing

functional groups that can later interact with the template through covalent or non-covalent

binding. After removal of the template, an imprint containing the functional groups in a

certain spatial orientation remains. The polymer has to have a high degree of cross-linking

in order to preserve the shape of the template. Otherwise the cavity will shrink and the

substrate will not fit in.

Biochemical

It is well known that antibodies are protein complexes used by the immune system to

identify and neutralise foreign entities (called antigens) that accidentally enter a living

organism. Each antibody is binding only a single type of antigens in a similar way as an

enzyme binds its substrate. This feature lead to the idea of creating an artificial antibody

(also: abzyme, catalytic antibody or catmab – from catalytic monoclonal antibodies). So,

how is this accomplished? We shall imagine one is aiming to generate a catalyst for a

reaction having a transition state [TS]∗. In brief, the following steps are required:

1. a substance resembling the structure of the transition state is synthesised (known as

hapten/haptenef;

2. the hapten is then transformed into an antigen by covalent binding of a polypeptide

(usually bovine serum albumin, BSA);

3. subsequently, the newly formed antigen ([TS]∗-BSA) is injected in small portions

in a mouse;

4. the antibody is isolated from the serum of the mouse and used as a catalyst.

The first abzymes were reported in 1986 and since then various types were described93,94.

f Hapten (from the Greek word “haptein” - to fasten) is, formally, an incomplete antigen that can stimulate the antibody production only in combination with a particular protein.

Glucose oxidase

51

5.1.2 Glucose oxidase (GOD)

It is a flavoenzyme1 that catalyse the following reaction:

β-D-Glucose + GOD-FAD δ-D-gluconolactone + GOD-FADH2

Once a GOD molecule had converted a single glucose molecule into gluconolactone, it is

not any longer able to transform more glucose. The redox moiety of GOD changes its

redox state from oxidised to reduced as glucose (reduced form) turns into gluconolactone

(oxidised form). However, GOD can transform an unlimited number of glucose molecules

by the supportive action of dissolved molecular oxygen. It is present in any solution unless

it is purposely removed (physically or chemically) and re-oxidise GOD back to its active

structure with concomitant formation of hydrogen peroxide:

GOD-FADH2 + O2 GOD-FAD + H2O2

The dissolved molecular oxygen is a co-reagent in this case. A glucose biosensor with

improved electrode kinetics is calling for controllable concentration of the co-reagent and

high diffusion towards the core of GOD. New developments in glucose monitoring of

blood are aiming at finding a replacement for the natural co-reagent with electrochemically

active complexes of transition metals such as osmium or ruthenium.

Since the GOD´s discovery as an “antibiotic”, (shown subsequently to be due to peroxide

formation) there has been a wide interest in GOD chiefly because of its value in glucose

determination. Although specific to β-glucose, it is employed to determine total glucose

because as a result of the consumption of β-glucose, the α- form from the equilibrium is

converted to the β- form by mutarotation. Structurally, glucose oxidase consists of two

identical polypeptide chain subunits each of them containing one Fe atom and one FAD

(Figure a). It works in a wide range of pH, but optimal at pH 5.5. As inhibitors Ag+, Cu2+,

Hg2+ are the most important. The GOD activity is colorimetrically detected by the increase

in absorbance at 460 nm resulting from the oxidation of o-dianisidine (highly toxic

compound which can cause skin irritation and sensitisation) through a peroxidase coupled

system. An orange colour is generated in this two steps reaction. One unit of glucose

oxidase causes the oxidation of 1 µmol o-dianisidine per minute at 25oC, pH 6. The KM is

about 33 mM and the turn-over number is approximately 2.3x104 molsubstrate /second ⋅

molenzyme.

1 FAD is the acronym of Flavine-Adenine Dinucleotide; FAD is a member of the adenilic acid derivates that play a key role in biochemical processes as cofactors.

Glucose oxidase

52

Glucose oxidase is perfect to work with as a trial-enzyme because of its high stability in

solution. Dry preparations having a yellowish colour, are stable for years when stored cold.

However, it is incompatible with strong oxidising agents and is better to keep it away from

moisture. Besides these, the low cost, makes it affordable by many laboratories (104 units

costs 20 € or 106 costs 900 €; from Sigma Product Catalogue for Germany 2002-2003).

Only glucose oxidase isolated from Aspergillus niger2 was used in the present experiments,

described in chapter 5.

About FAD

FAD’s (Flavin Adenine Dinucleotide) molecular structure and redox behaviour are shown

in Figure a. FAD could be seen as being made up of riboflavin residue (dark blue) and an

ADP (adenosine diphosphate, shown in red). The redox active parts are coloured in

magenta and located on the isoalloxazine ring (blue, on the right side) of FAD. R denotes

the ADP-[CH2-(CHOH)3-CH2)].

2 Aspergillus niger is a fungus, non-pathogenic to humans, causing the black mould on certain types of fruit and vegetables.

N N

NN

NH2

O

N

N

N

N

O

O

H

CH3

CH3

CH2

CHOH

CHOH

CHOH

CH2

O

PO

OH

OP

O

OH

O C

H2

OHOH

N

N

N

N

O

O

H

CH3

CH3

R H

H

N

N

N

N

O

O

H

CH3

CH3

R

+ 2H+ / + 2e-- 2H+ / - 2e-

FAD oxidate state

FADH2 reduced state

Riboflavin (Vitamine B2) residue

ADP residue

Fig. 30 The molecular structure of FAD. On the left is shown how the riboflavin residue undergoes redox reaction.

Patterning GOD by means of piezo microdispenser

53

5.1.3 Patterning GOD by means of piezo microdispenser 5.1.3.1. Aim

The main goal of the effort is directed to enzyme micropatterning and to demonstrate the

possibility of constructing a complex self-containing biosensor array for multiple

applications: monitoring of drugs, pollutants, or any other compound of interest in water,

food or physiological fluids. Before proceeding to present specific details of this work, the

reader should be acquainted with the enzyme micropatterning techniques available up to

date and their advantages/disadvantages.

Since biosensors are wished to become simply tools for home-monitoring of glucose, urea

and other substances, increasing attention has been given to the formation of localised

biomolecule microstructures in the last decade. Therefore, simple tools coordinated by

hand (capillaries)95 or sophisticated machinery controlled by computers (laser confocal

microscope96,97, SECM, microdispenser) are used to create basic (spots) or complex (lines

or grids) microstructures. The spatial resolution of such micro/nano structures is

determined by precision in controlling the distance between two neighbouring spots.

Consequently, hands-on devices can not compete with the highly precise micropositioning

stepper motor or piezo elements for creating biosensor architectures with high spatial

resolution. However, two ways of placing biological recognition elements onto a surface

are available so far:

Active placement

Active placement refers to the procedure of delivering small droplets of biomolecule

directly at the point of interest (capillary, microdispensers).

Indirect methods

These methods require a chemical pre-modification of the surface aiming at the localised

immobilisation of biomolecules. The affinity of surface is tuned with an appropriate

functionality to facilitate the specific binding of the biomolecules (for instance, self

assembled monolayers of thiols).

It was already shown (M. Mosbach, S. Gaspar98), by means of the substrate generation –

tip collection mode of SECM, that the enzymes are preserving their activity after being

shot through the microdispenser nuzzle (withstanding the shear forces and elevated

pressure). Excellent 3-dimensional SECM images of different surface confined enzymes

were achieved. Nevertheless, the enzyme concentration was not optimised. Conversely, the

polymer matrix used to entrap the enzymes at a certain surface had not the optimal

concentration.


54

Following their foot steps, I sought to further develop the complexity of the enzymatic

patterns and to study the possibility to increase the dynamic rangea of the microsensor

arrays.

Note An enzyme that is interacting with the substrate in bulk will generate a certain amount of

product. If the same quantity of enzyme is by any way grafted to a surface, then the speed

of the product formation is smaller as in bulk. Now, when one reduces the dimensions of

the catalyst patch, one also decreases the amount of product that can be produced within a

given time, and therefore, one increases the difficulty in analysing the product. Typically, a

spot of GOD preparation pasted at a surface by means of piezo microdispenser contains

6.25 x 10-16 mol of GOD (for 1 mg/ml GOD in the spotting mixture and a droplet of 100

pl). In ideal case, with the enzyme free in a large volume of solution, 6.25 x 10-16 mol of

GOD will generate roughly 40 million product molecules per second (and of course will

consume the same number of glucose molecules). If these molecules diffuse away into a

volume of 2 ml (of the electrochemical cell used in SECM measurements), then there is an

analytical difficulty in measuring the product in the resulting ≅ 2 x 10-10 M solution. As

already mentioned, this minute quantity represents an apex for the given amount of GOD

and reaction time. The available product is even less if the enzyme is immobilised into a

thin film of polymer. Several factors merge their contributions to make the reaction

sluggish:

a. the diffusion of the substrate towards the enzyme molecules is hindered by

the film and the surface on which the enzyme lies;

b. enzyme is not free to accommodate its position with regard to the incoming

substrate (as would happen in bulk where it can rotate and translate in any

direction);

c. as D-glucono-1,5-lactone is formed and is accumulating in the film, the

enzymatic reaction is slowed down because this product is (a weak)

inhibitor of GOD.

Taking into account these facts, it becomes clear why a local electrochemical probe must

be placed close to the enzymatic patches in order to be able to measure the enzyme

activity/substrate concentration through the redox active products. A large disk-shaped

electrode is not appropriate for investigating micrometric structures (because it could

a The dynamic range is the ratio of the maximum value versus the lowest detectable value of a specific parameter. Typically, is measured in B (Bel) or dB (decibel), in a logarithmic scale: B=lg(value1/value2).


55

record signal from multiple enzyme microspots at the same time) and hence

microelectrodes were preferred (active diameter 10 µm). Obviously, the microelectrode is

moved across the enzymatic microarray with a micropositioning system (see chapter 2).

We shall find in chapter 3 and 4 a detailed description of the microelectrodes

properties/manufacturing.

5.1.3.2. General consideration about GOD microdispensing

Home-made microdispensing set-up

The schematic view of a home-made microdispenser control set-up is depicted in Figure

31. The trigger signal generated in the computer was send to the wave generator where the

power output of the power supply was modulated in order to produce on-demand-release

of droplets. The rely card made possible the control of two stepper motors via a single

motor-card. A home-made electronic board combined the carrier signal (from the wave

generator) with the power applied to the piezo actuator.

The surface chosen for patterning glucose oxidase was gold sputtered on silicon wafer.

Improved adherence of the gold layer to the silicon support was typically attained by using

an intermediate layer of titanium between Au and silicon. Such plates were cut in

rectangular pieces with appropriate dimensions, 0.5 x 0.5 cm2 (chip). A plastic holder

allowed the placement of 5 chips in a row, at a distance of 1 cm from the head of the

microdispenser. Once a chip was spotted, the next chip was positioned in the shooting

direction of the microdispenser. In this way, it was possible to overcome the drying of the

enzymatic mixture at the nozzle. This unwanted clogging had to be avoided because

normally it compromised the chips by shooting droplets out of the line. Repairing/cleaning

the dispenser is not easy and successful any time. The mixture containing the enzyme was

prepared by adding 1 mg GOD and 2 mg EP16 in 1 ml tri-distilled water. The yellow-

milky solution did still contain polymer aggregates with an overall size that could block the

Fig. 31 Schematic of the control set-up of the microdispenser.


56

microdispenser. Such big particles have to be removed prior to shooting. It was noticed

that a filter unit of 5 micrometers pore-diameter retained not only large amounts of the

polymer but even enzyme. Alternatively, centrifugation of the enzyme-polymer mixture

was found to be a better choice. Centrifugation for half a minute at a speed of 700

rotation/min (for a normal lab centrifuge) was enough to settle the large aggregates. The

clear supernatant had been carefully sucked into a 1 ml syringe and further used to fill the

chamber of the microdispenser. Although the droplets are visible to naked-eye, while

shooting they have to be well illuminated with a fiber optic light from a side in order to see

if they are ejected from the microdispenser. Once a droplet touches the surface of the gold

it appears as a glittery spot for 1-2 seconds till it gets dried. The polymer matrix is

hydrophilic and thus is capturing a lot of water around. In this case the GOD is rather

mobile (swimming between polymer chains) and could dissolve away from the polymer if

not allowed to cure for 20-24 hours before being further used. This GOD preparation had

enough activity for months even after keeping it at room temperature. However, to avoid a

probable loss of enzyme from the polymeric matrix all enzyme micropatterns herein

presented were cured 24 hours.

4.1.3.3. Simple GOD microstructures

A PC in combination with Windows software programmed in Microsoft Visual Basic 3.0

(Microsoft, Unterschleißheim, Germany) was used for the control of all system parameters

Fig. 32 Snapshot of the software interface used for micropatterning with the piezo microdispenser.


57

(like: number of droplets per spot, distance between and the number of lines; number of

spots per line), and also for data acquisition. A snapshot of the user-friendly interface of

the mentioned software is shown in Figure 32.

With this automated device one could simply create a variety of microstructures of GOD

captured in the Vinnapas EP16® polymer dispersion. The SEM micrographs shows, arrays

of spots (Figure 33A) with different enzyme concentration or even grids (Figure 33B) with

variable load of enzyme were achieved. The polymer film is thinner in the central part than

at the rims (see Figure 33C) where apparent heights of 1-2 µm were measured for with the

electron-beam parallel with the surface of the microstructure (the apparent heights was

used because the enzyme-polymer film was not metalised by sputter-coating prior to

imaging). Figure 33D gives a closer look at the nanoscale structure of the sputter coated

gold. As plainly visible, the gold surface is not smooth but full of cracks and gaps. It is

possible that these features have a certain role in stabilising the polymeric layer.

Fig. 33 SEM micrographs of spots and grid of piezo-dispenser patterned GOD containing polymer. The dispensing mixture contains 1 mg/ml enzyme and 2 mg/ml EP16 W (A, B, C). Number of droplets / spot is increasing from the upper right corner to the lower left corner of the image (A, B). Close-up of a single spot (C). Gold sputtered surface appears as broken up. This feature could explain why EP16 adheres so well to this gilded surface (D). Scale bar represent: 200 µm (A, B), 20 µm (C) and 1 µm (D).


58

Gold is a material that has a certain affinity to many compounds, especially to organic

functional groups. Is it possible to immobilise glucose-oxidase on a gold surface without

resorting to any polymers? To address this question, two aqueous solutions were prepared

one containing only polymer (2 mg/ml) and other only enzyme (1 mg/ml). The idea was to

dispense the droplets from each preparation as spots and then to compare the morphology

of the corresponding films. A well adhering and rather homogenous covering was obtained

with the polymer suspension in water (Figure 33A). Surprisingly, the enzyme alone formed

solitary accumulations and hence indicating low natural affinity for gold (Figure 34B)

A versatile tool for micropatterning biological molecules is the microdispenser, it offers a

straightforward procedure to create complex geometrical structures automatically, or semi-

automatically or to generate as already mentioned, enzyme gradients. For this, multiple

droplets having identical composition are directed towards the same area of the target

surface. Now, precautions must be taken owing the fact that 1) the overall enzyme activity

is not increasingly proportional to the number of droplets/spot and 2) the diameter of the

spots is directly proportional to the number of droplets/spot; these two issues will be

discussed in more detail.

Fig. 34 SEM images corresponding to 2 mg/ml pure polymer spot (A) and pure 1 mg/ml GOD spot (B). Scale bars are identical and represent 20 µm. Both were aqueous solutions.


59

Discussion

Enzyme exposure to the substrate and its relation to diameter/number of droplets

In order to optimise the glucose biosensor array, the enzyme concentration in the spots was

varied. Two approaches could be used to change the biological recognition element spot

concentration; firstly, it can simply be achieved by increasing its concentration in the

shooting solution, but the disadvantage is the necessity to refill the dispenser every time

and for each concentration. Secondly, the target place can be shot multiple times and the

advantage is that the complex arrays can be created without refilling the microdispenser. A

series of SEM micrographs shows the spots with variable diameters obtained by increasing

the number of droplets deposited on each target place. For the same composition of the

enzyme solution (1 mg glucose oxidase and 2 mg polymer per ml of tri-distilled water), the

diameter of the spots increases with the number of droplets (say n) as depicted bellow

(Table 3):

Tab. 3

n 1 2 3

Diameter (µm) 75.7 97.0 133.3

Note: the numbers on this table are valid only for the mixture composition described above.

Is the enzyme activity increases with n? (It is about activity of a spot as active entity not

about the specific enzyme activity, which is not affected by n). A negative answer could

suggest a misuse of the enzyme, or in other words that is useless to try to increase spots

activity in this way. Theoretically, a larger volume will produce a larger diameter spot and

considering a constant film height one can calculate the ratio of the droplet/spot radius. Let

us assign the following parameters for droplet and spot (which is in fact a disk) (Table 4):

Tab. 4

Droplet Spot (Disk)

V volume of a single droplet volume of a single spot

R radius of a single droplet -

R' - radius of a single spot

h - height of the spot


60

The volume is the only parameter that has equal values for both droplet and spot, and

hence the possibility to find a relation between the droplet volume and the corresponding

spot radius (Figure 35).

Similar ideas lead to an equation describing the relation of any two spot radii when their

volume ratio is known.

2/1

1

2

2/1

1

2

'

1

'

2

=

=

n

n

V

V

R

R

The ratio of the volumes is equal to the ratio of the number of droplets and this explains the

second term of the upper equation. For instance, if the volume is doubled then the radius

ratio is 41.12 = . For a triple volume the resultant ratio is 73.13 = . These are theoretical

values and will be used as a reference to calculate the deviation of the real spot diameters

and to explain why the “spots activity” is not proportionally increasing with parameter n

(Table 5). '

nR is the radius of a spot made of n droplets.

Tab. 5

Theoretical Experimental Remarks about film

'

1

'

2RR 1.41 1.28 ticker as single drop

'

1

'

3RR 1.73 1.76 expected thickness

Remarks presented here are roughly valid for bigger droplets. Indeed, a larger droplet will

splash and spread more around the impact area as compared to a small one. However, for a

double sized droplet, the film is a bit thicker as for a single droplet of the same solution.

Consequently the active substance in the mixture is less exposed to the solution in the case

of a double droplet as for single one (Figure 36).

Fig. 35 The droplet and its corresponding spot (disk). Radius of the disk is a function of the droplet volume.


61

This behaviour will decrease the response signal of a biosensor (an amperometric current

in this case) because of the “hidden” enzyme in the thick film. A well permeable polymer

film usually is enough to make the active compound reachable from the bulk of the

substrate solution. Vinnapas EP 16 polymer dispersion had a good behaviour in terms of

permeability for water and glucose.

Fig. 36 The exposure of the enzyme in dependence from polymer film thickness; (A) thin film exposes it better as a tick film (B). Light coloured molecules stand for “hidden” enzyme.

Visualisation of GOD microstructures by SECM

62

5.1.4 Visualisation of GOD microstructures by SECM An indication that surface immobilisation of GOD is possible with the use of a piezo-

microdispenser is only half the way showing that the construction of a biosensor could be

done in an easy and flexible manner by ink-jet printing. It is important to demonstrate that

this approach would preserve the natural and main important feature of an enzyme: its

catalytic properties. Thus, it was proceeded to probe the activity of the surface confined

glucose oxidase by measuring the released hydrogen peroxide from a GOD microstructure

in the presence of glucose. The SECM was the chosen instrument for demonstrating a set

of quantitative and qualitative measurements of micropatterns of glucose oxidase.

Microlines with variable enzyme content

The microdispenser chamber was carefully filled with a mixture of GOD (1 mg/ml) and

Vinnapas® EP16 polymer dispersion (2 mg/ml). To obtain continuous lines, each new

droplet was shot out of the nozzle along the X axis with a displacement of 50 µm (for an

average spot diameter of 75 µm). Typically, two lines were spaced at 500 µm to each

other. The freshly dispensed microstructures were allowed to cure overnight at ambient

temperature, before being studied by SECM. Although such an arrangement of

enzyme/polymer microstructures can still be observed with the naked eye, it is better to

inspect them with the aid of a light-microscope to check for possible errors (such as

unwanted satellite droplets or discontinuities in the pattern). Integrated scale bars along the

length were represented by metallic wires with known diameter (either 500 µm Ag, or 10

µm Pt) (Figure 37).

Fig. 37 Optical photographs of GOD/EP16 polymer microstructured on gold surface. The spotting solution contained 1 mg/ml GOD and 2 mg/ml EP16. Metallic wires (Pt 10 µm and Ag 500 µm) were used as scale bars.


63

To facilitate the SECM measurements these enzyme modified gold chips had to be first

placed into an electrochemical cell (EC cell). For this experiment, it could be a simple Petri

dish, a small cup, or a cell screwed from the top. Once the chip (5x5 mm2) is in place, the

two electrodes (a 10 µm Pt disk-shaped ultramicroelectrode and an Ag/AgCl pseudo-

reference electrode) necessary to perform electrochemical measurements are positioned

inside the EC cell. The ultramicroelectrode (UME) is brought manually to about 1 mm

above a clean area of the gold chip and in the vicinity of the enzyme pattern. In this

position, the working microelectrode is too far from the chip to detect the minute

concentration of anything released from the enzyme preparation. The refinement of the

height of electrode was typically achieved in the feedback mode of SECM. In this

particular case, the approach was done in a 5 mM solution of [Ru(NH3)6]3+ in 0.1 M

phosphate buffer pH 6.7, until the tip-current increased with 30% of the bulk value.

Subsequently, the solution of the mediator was removed and the chip was thoroughly

rinsed with pure water. A solution of glucose in 0.1 M phosphate buffer was then poured

into the cell over the GOD micropattern. As the glucose and GOD are reacting, the

products (gluconolactone and H2O2) start to diffuse away into the bulk of the solution.

Now, if the scanning ultramicroelectrode of the SECM is kept at constant potential, namely

+600 mV vs. the reference electrode, the oxidation of hydrogen peroxide occurring at the

Pt disk can be monitored (Figure 38).

Fig. 38 Schematic showing the underlying idea of the measurement of released hydrogen peroxide in the generation-collection mode of SECM. The incoming glucose is converted at the enzyme microstructure into gluconolactone and hydrogen peroxide, which is subsequently detected at the Pt disk electrode.


64

The obtained amperometric current is proportional to the concentration of the substrate if

the enzyme is not in the saturation regime. Because of the motion of the tip, the entire

microstructure is screened and the currents corresponding to specific locations within the

studied area are recorded by a computer.

Calibration curves obtained in the generation-collection mode of SECM

Optimal concentration of enzyme in a microstructure cannot merely be calculated because

the KM value changes when the enzyme is immobilised onto a film at a surface. For this

reason an uncomplicated enzyme micropattern was designed and investigated to find the

favourable concentration of GOD. The spotting mixture included 1 mg/ml GOD and 2

mg/ml polymer Vinnapas® EP16 in 0.1 M phosphate buffer pH 6.7. The produced

microstructure consisted of three parallel lines with 500 µm spacing. The enzyme

concentration was varied through varying the number of droplets deposited per target spot.

Accordingly, the number increased from line 1 (A) to line 3 (C). A 25 µm Pt disk electrode

as SECM probe was scanned over the three lines of the microstructures at different

substrate concentrations (Figure 39). It turned out that the enzyme was used in excess

compared to the amount of available glucose. Therefore, in the amperometric recording

three current peaks with roughly equal heights can be noticed. Nevertheless, the maximal

current response in each case (A, B, C) is proportional to the glucose concentration in bulk.

Fig. 39 Line-scans over a GOD microstructure obtained in generation-collection mode of SECM. Line A was prepared by dispensing 1 droplet/spot whereas lines B and C were prepared with 2 and respectively 3 droplet/spot. Glucose concentration were (mM) 5 – yellow, 7 – red, 10 – cyan, and 20 – for dark blue. Supporting electrolyte 0.1 M phosphate buffer; 25 µm Pt SECM tip; 30% current increase.


65

These plots are, in fact, calibration curves. Thus, to obtain calibration curves, the enzyme

concentration should not be raised to more than 1 mg/ml because the analytical signal

(amperometric current from the oxidation of H2O2) is not improved.

A GOD concentration of 0.1 mg/ml and 2 mg/ml Vinnapas® EP16 in the dispensing

solution, and high concentration of glucose (100 mM) in the bulk, resulted in a saturation

of the enzyme. In this case, a micro-grid with increasing concentration of GOD from top to

bottom and left to right was scanned again with a 25 µm Pt tip in the generation-collection

mode of SECM. Figure 40 shows the SECM (A – bird view and B – three dimensional)

images of this grid. Note: if the enzyme is working in the saturation range, then the kinetic

of the GOD enzymatic reaction depends upon the diffusional transfer of O2 from outside

gas atmosphere.

High-resolution constant-distance SECM on immobilized enzyme micropatterns

Up to this point, enzymatic microstructures were imaged only with the SECM tip moved at

constant height above the surface. This mode of scanning has the advantage of being fast

and straightforward. Furthermore, it can be performed with standard Pt-glass

microelectrodes. However, the constant-height mode of SECM has certain limits on

heterogeneous surfaces displaying variations in both, conductivity along with topography.

On these samples, changes in the tip current arising from distance variations for principal

reasons cannot easily be differentiated from ones originating from alterations in

conductivity. In addition, with the constant-height mode the tip crash is at high risk on

tilted or rough surfaces, especially when decreasing the size of the SECM tip for imaging

Fig. 40 SECM images of a microgrid with increasing GOD concentration from top to bottom and from left to right. The spotting solution contained 0.1 mg/ml enzyme and 2 mg/ml polymer dispersion. Imaging achieved in the generation-collection mode of SECM. (A) bird-view of the grid; white areas indicate higher concentration of hydrogen peroxide; (B) three-dimensional representation of the grid; elevated areas stand for increased enzyme concentration. Glucose concentration was 100 mM. Supporting electrolyte 0.1 M phosphate buffer; 25 µm Pt SECM tip; 30% current increase.


66

at higher resolution. On the contrary, constant-distance SECM offers the advantage of a

accurate control of the tip-to-sample distance since the integrated shear force-sensitive and

computer-controlled feedback loop of this mode forces the SECM tip to follow the

contours of the surface during the entire time of scanning/imaging. This does not only

allow simultaneous acquisition of the electrochemical tip response and the sample

topography but also effectively prevents against tip crash even with smallest SECM tips.

The foundation of the constant-distance SECM, a mode in which the scanning tip follows

the profile of the sample at constant distance, is an oscillating measuring tip that is forced

to vibrate, in the electrolyte solution, at its resonance frequency. This frequency comes

from superposition of the hydrodynamic friction force and the excitation mechanic force.

Any object that happens to be close to (under or aside) the vibrating tip will increase the

friction because it stops the solvent molecules oscillating around the probe tip. Hence, a

change in the vibration properties of the tip appears and is noticed as a shift in vibration

amplitude and phase. This is the heart of shear-force constant distance mode of SECM.

In the non-optical shear force based distance control54, two piezoelectric plates are glued to

the upper part of the highly flexible, needle-like ultramicroelectrodes just above the region

where the pulled capillary starts to get thinner (Figure 41). The upper plate vibrates the

SECM tip at its resonance frequency while the lower serves as a piezoelectric detector of

the amplitude of tip oscillation and frequency, respectively.

Fig. 41 Schematic representation of the set-up as used for constant-distance mode SECM with an integrated non-optical (piezoelectric) detection of the distance-dependent shear forces.


67

During the approach of the SECM tip towards the sample, shear forces start to appear in

close proximity of the surface, which leads to a damping of the tip vibration and a phase

shift, both effectively registered by connecting the detecting piezoelectric plate to a dual-

phase analogue lock-in amplifier. In this way, the tip-to-sample distance is usually

automatically stopped at a user-defined degree of damping of the vibration amplitude, for

example at 70 - 80 % of the unaffected value with the tip far above the surface. Once the

tip is in the working-distance, the tip can be scanned in X and Y directions. The positive or

negative shift in the vibration amplitude and phase as recorded by the lock-in amplifier

provides an output signal for the Z-axis stepper motor that can move the electrode up or

down unless they reach the user-defined set-point value resulted from the electrode

approach curve. Continuous communication between the lock-in-amplifier, computer and

Z positioning system makes up the computer-assisted feedback loop that accurately

controls the tip-to-sample distance all through the scanning experiment.

A first experiment showed how efficient this technique is in guiding the vibrating

microelectrode across a rough surface without colliding the tip with the protuberances on

the surface. A microscopic spot of an enzyme/polymer mixture was dispensed on a glass

surfaces and visualised in air using the unpolished tips of a pulled Pt-nanoelectrode as

scanning probe. The unpolished tip was used because it offers much smaller total tip

dimension as a polished, disk-shaped nanoelectrode. Figure 42A presents a typical

scanning electron micrograph (SEM) of a 70-µm-diameter glucose oxidase/Vinnapas

EP16 spot on glass. The morphology of the spot appears non-homogeneous and displays

small local variations in the density of the enzyme/polymer film. Obviously, most of the

Fig. 42 Scanning electron micrograph (A) and shear-force topography image (B) showing a non- homogeneous distribution of an enzyme/polymer mixture inside the circular area of a microscopic spot of glucose oxidase/ Vinnapas EP 16. The diameter of the spot is about 76 µm.


68

two compounds deposits upon drying in a thin rim at the edge of the spot. Figure 42B

illustrates the topography of the same spot as imaged using the constant-distance mode of

SECM with an integrated piezoelectric detection for shear forces. As with SEM, local

micrometer-sized topographical variations are clearly visible in the shear-force image

underlining unequivocally the ability of the shear-force based constant-distance mode of

SECM to resolve surface topography at high resolution.

The topographic resolution of constant-distance SECM depends on size the sharpest part at

the very end of the probe tips, which is responsible for the shear force contact while the

current resolution is related to the size of the electro active area of the tip. To visualise the

activity of the enzyme microspot, a quartz glass-insulated Pt-disk nanoelectrode had to be

used for the visualisation of the prepared enzyme-containing polymer microstructures.

Such a nanoelectrode can be made by simultaneously pulling a quartz glass capillary

together with an inserted platinum wire using a laser-based micropipette puller56. Careful

grinding the tips of the pulled capillaries on polishing pads leads to the exposure of active

Pt-disks with diameters of far below one micrometer.

With the encouraging output achieved in air, the application of the non-optical shear-force

constant distance SECM was then supposed to be focused on the simultaneous imaging of

topography and enzymatic activity of a polymer-enzyme micropreparation in aqueous

solution (Figure 43).

In order to fulfil this requisite the following strategy was considered: a number of polymer

(P) and polymer-enzyme (P&E) microstructures (such as lines) patterned onto a surface

should present both topographic and chemical activity features when immersed in a

solution containing the substrate of the given enzyme. Thus it should be possible to scan

with the vibrating tip of the SECM positioned within the near field, over these polymeric

microstructures and to detect variations in the samples topography and the amperometric

current image of the mentioned arrangement of lines.

To practically verify the above mentioned assumptions, a polymer micropattern consisting

of three lines (70 µm in width each) made of Vinnapas® EP16 and with glucose oxidase

was microstructured by means of the piezo microdispenser. The enzyme was entrapped

only in the middle line (2 mg/ml polymer and 1 mg/ml GOD) whereas the outer lines

contained pure polymer (2 mg/ml). With this arrangement, high-resolution constant-

distance SECM imaging of the topography and localized glucose oxidase activity was

performed in solutions containing 50 mM glucose. A disk-shaped Pt nanoelectrode with tip

diameters of about 500 nm was used as scanning probe for the measurements. SECM


69

imaging in the constant-distance mode of operation was carried out at scanning speeds of

0.1 - 1 µm s-1 for X and Y displacements, while the stable feedback loop guaranteed a

constant tip-to-sample distance of about 100 - 200 nm. To allow the local detection of

enzymatically generated H2O2 in the generator/collector mode the SECM tip was poised to

a potential of 600 mV vs. an Ag/AgCl pseudo reference electrode. As clearly visible in

Figure 44A, line scans of topography (blue) and amperometric SECM tip current (red)

were both simultaneously acquired by scanning across the enzyme/polymer line-

microstructure (Figure 44B). Although the topographical resolution with disk-shaped Pt

nanoelectrodes was found to be not as good as with the tapered tips of unpolished

electrodes, small lateral variations in the topography of the enzyme/polymer structure are

still visible. On the other hand, an increase in the amperometric tip current was observed

only just above the middle polymer line. This was expected as only the middle line

contained active enzyme and thus, the local production of H2O2 is limited to the area

covered by that line.

Fig. 43 Trajectory of a SECM tip scanned over a 3-dimensional microstructure made of a polymer (P) in which the central part contains a biological recognition element such an enzyme (E). The shift of the resonance frequency of the vibrating tip due to the shear forces is used to obtain the topographic image of the studied microstructure (blue line). Polarisation of the SECM tip at proper potential could be used to achieve an image of the electrochemical activity of the surface by detecting, for instance, a product of the enzymatic reaction that takes place within the polymer matrix (red line).


70

Conclusion

The combination of nanometre-sized SECM tips with the non-optical shear force

positioning allowed simultaneous imaging of the topography and of the local chemical

activity of, for instance, enzyme-containing polymer microstructures with high spatial

resolution. With such achievements, SECM is widening its application field to objects with

nanoscopic surface features and (electro) chemical inhomogeneities.

Fig. 44 (A) Line scans of the topography (blue) and amperometric SECM tip current (red) simultaneously acquired in solution containing 50 mM glucose by scanning across a polymer microstructure consisting of three lines of Vinnapas® EP16, and with GOD immobilised only in the central line (B). Measurements performed in the constant-distance mode of SECM with a 500 nm Pt-disk nanoelectrode. Scale bar: 100 µm.

Defined adhesion/growth of living cells

71

5.2. Defined adhesion/growth of living cells In the beginning of the year 2004, I had an opportunity to listen to a lecture by Professor P.

Fromherz, at the Ruhr University of Bochum. He is the director of Max Plank Institute for

Biochemistry in Martinsried (near Munich) and world-wide known as the initiator of the

research towards binding the neurons to electronic devices, the so-called neuroelectronic

interfacing. According to his confession, the idea of developing such devices came up in an

unexpected way. Getting irritated while using a personal computer (a Macintosh) about 20

years ago, he dreamt to replace the keyboard with an electronic implant in the brain that

could possibly transfer all the wished commands directly to computera. Hearing all this I

was thinking that if this would have been realised by now, I would have wrote my PhD

thesis faster and enjoyed the springtime! Ok, this was only a minor thought. Actually, I

realised how complicated it is to fulfil this taskb, and that it is a complementary part of the

work directed to the growing of neurons on artificial microstructures – it was done in

cooperation with Dr. P. Heiduschka from the Department of Experimental Ophthalmology,

University Eye Hospital Münster. Indeed, the electrical interfacing of semiconductors and

neurons necessitate micropatterning techniques to control neuronal networks.

Artificial networks of living neurons are supposed to facilitate the understanding of the

properties and functions of neurons. Sooner or later they will be grown in-purpose in

damaged tissues of the human/animal body in order to repair locally the connection of the

nerve cells. In the framework of an ELMINOS project, the task was to prepare

microstructures of laminin on different substrates for growing chicken embryo neurons. All

the following results are shared with Karla Tratsk-Nitz, who prepared the neurons, the

special buffers and also incubated neurons over the laminin microstructures (for details see

chapter 6 “Experimental”). The microdispensing work was mostly done at the Eye

Hospital, Münster. For this, I travelled several times to Münster together with a heap of

equipment (computer, microdispenser, holders, wave generator, power supplies and

obviously lots of connectors, plugs and replacements for the microdispenser) (Figure 45).

A close-up of the microdispenser and positioning system is shown in Figure 46.

a P. Fromherz, “Neuroelectronic Interfacing: semiconductor chip with ion channel, nerve cells, and brain”, Nanoelectronics and Information Technology, Editor: Rainer Waser, Wiley-VCH Berlin, 2003, 781-810. b This is a considerable technical challenge because an ionic current in the cell has to interact with the electronic current in the silicon chip and between these two parts (cell–chip) is gap of several tents of nm that blocks the current flow.


72

The aim of the present study was to check whether laminin patterns can be created by

the microdispensing technique and if such patterns are suitable for patterned adhesion of

neurons.

5.2.1. Introduction

Study of properties and function of neurons belongs to the major research fields in life

sciences. Connections between neurons are established by synapses between axons and

Fig. 45 Microdispenser set-up as mounted and used at the Eye Hospital, Münster. (A) wave generator; (B) rely card; (C) power supplies; (D) piezo microdispenser and positioning system; (E) laminar flow bench.

Fig. 46 Close-up of the active area: (A) the microdispenser head; (B) reservoir of the spotting mixture; (C) cold-light lamp; (D) sample positioning stepper motors; (E) microdispenser leads.


73

dendrites. In order to study the properties of neuronal network (signal propagation, or

neurotransmitters release), they should be grown in vitroc in a defined pattern, where

contacts between neurons can easily be traced99,100. Otherwise, such investigation might be

difficult owing the complicated structure of naturally developed neuronal networks.

5.2.2. What is available so far?

A spectrum of different technologies for the directed growth of living cells is available.

Different approaches have been tried to achieve defined adhesion of neurons on artificial

substrates. They include modifications of surface topography by grooves, wells or similar

structures101-105 or spatially dissolved chemical modification of the surface106,107, which can

be achieved by microcontact printing (stamping)108-112, photolithography99,114-116 or plasma

polymerisation117. An alternative method having the ability to create well defined and

reproducible surface microstructures at the micron level is using an excimer laser beam to

micro-sculpture a polymeric film118. If microstructured electrodes are present on the

substrate, they can be coated selectively as it has been demonstrated for adhesion of

neuroblastoma cells on platinum stripes coated with laminin-derived peptides119 and axonal

outgrowth on laminin-coated platinum stripes120. Electroactive structures were also created

by microcontact printing, and a patterned attachment of two different cell types could be

achieved after coupling an RGD peptide to quinone groups121.

Laminin is a large, multi-domain protein122 with many binding cites for cellular

receptors123,124. It plays a crucial role in the development and maturation of the nervous

system125,126. Laminin has been used for many years as substratum for in vitro cultivation

of neurons127-129, and adhesion and growth promoting properties of laminin could be

achieved by adsorptive deposition of a polylysine layer prior to adsorption of laminin130.

There are also reports of patterned deposition of laminin by photolithography131,132 and

microcontact printing133-135.

Many of the methods of patterned surface modification are restricted to certain material

properties of the substrate and/or require expensive equipment and complicated

procedures. On the search for a simple and inexpensive method, which should be suitable

for a variety of substrate materials, we choose the so-called ink-jet printing11. This method

has been known by commercial printers for several years. For this study, we used a piezo-

ceramic actuated dispenser developed at the Lund Institute of Technology (for details see

chapter 2). Initially developed for the handling of very small volumes of nano- or pico- c „In vitro“ experiments/processes are taking place out of a living organism (in a test tube, for instance). Normally they occur in living organisms that means “in vivo”. From the Latin word “vitreum” – glass / vitreus – made of glass.


74

litres in microscaled analytical devices, e.g. for MALDI-TOF MS136,137, the microdispenser

can also be utilised for the creation of substance patterns on substrates, with a high

variability regarding the geometrical shape of the pattern. This has been demonstrated

already by the creation of microstructures glucose oxidase and other enzymes on gold

substrates98. Moreover, several substrate materials can be used.

5.2.3. Results and discussion

In the further described experiments with neurons, continuous lines with one droplet/spot

were made, with a width of 100 µm and a distance of usually 500 µm. The solution shot

onto the substrates contained 2-4 mg/ml Vinnapas® and 20 µg/ml of laminin diluted in

Hank’s balanced salt solution. Since agglomerates of the polymer could clog the

microdispenser nozzle, they were, prior to use, centrifuged (10·g) for 30 seconds and the

agglomerates settled down at the bottom of an Eppendorf tube.

Note: Special circumstances as handling living cells requires a sterile environment.

Therefore, all instrumentation and materials involved in this project were sterilised prior

use with a 70% aqueous solution of ethanol. To circumvent any contamination of the

equipment, the microdispensing set-up (micropump, positioning system, and glass cover

slips) were placed into a laminar flow bench after being disinfected. A constant stream of

filtered and sterilised air was continuously flowed outwards the hood. Accordingly, no

impurities could enter the space where the microstructures are obtained. In addition, one

has to wear rubber gloves all the time and has to clean them with alcoholic solution as

often as one retracts the hands from the hood. The successful growth of neurons depends

partially on the degree of sterility of gloves and equipment.

At the very beginning of this common project, the idea was to check whether the aqueous

solution of laminin is proper for use in this form or it needs a support. The result is shown

in Figure 46A. It can be seen that the liquid in the lines contracts; as a result, single

irregular dots are formed. This behaviour can be attributed to the properties of laminin that

possesses strong intermolecular interactions. In order to obtain continuous lines on the

glass substrate, a mixture of laminin and Vinnapas® was used. Although the contraction is

still visible, continuous lines could be created now (Figure 46B). Therefore, this mixture of

laminin and Vinnapas® was used in the experiments with the neurons.


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As the next step, it was checked, if the laminin molecules entrapped in the polymer are still

accessible to surface binding molecules so that neurons could bind to them. For this

purpose, Vinnapas® lines with and without laminin were created and stained with an anti-

laminin antibody. It could be seen, by fluorescent microscopy, that the antibody was really

bound to the lines containing laminin in a dot-like manner (Figure 47), whereas no staining

can be seen if the anti-laminin antibody or laminin were omitted.

As depicted in the above images, there are also some fluorescent spots visible

outside the lines. This could mean that some laminin molecules might have been

washed out of the polymer matrix during the long procedure of immunochemical

staining. Nevertheless, it should have no significant effect on the patterned adhesion

of neurons, because there is still enough laminin present on the lines.

Fig. 47 Staining of laminin–Vinnapas® lines using the anti-laminin antibody and Cy2®-conjugated secondary antibody.

Fig. 46 Comparison of lines shot on glass slides with: (A) laminin solution and (B) a mixture of laminin and Vinnapas®. Scale bar: 200 µm.


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Moreover, neurons adhering outside the lines are not able to outgrow neurites, so that they

do not become part of a neural network developing on the lines.

A suspension of neurons was applied onto the substrates with the lines. Examples of the

behaviour of neurons on glass cover slips are shown in Figures 48. It is obvious that

adhesion of neurons follows the laminin-Vinnapas® lines.

Coverage of lines with neurons is not perfect at some places indicating some

inhomogeneities in the composition of the polymer lines. Neuronal adhesion is not

restricted solely to the lines, as several cells were found also outside the lines.

In order to verify that adhered cells are neurons, a new set of experiments were carried out

in Bochum this time. Immunocytochemical staining was performed with an anti-

neurofilament antibody. The vast majority of cells were found to be stained indicating that

neurons were attached to the laminin-Vinnapas® lines (Figure 49).

To obtain this fluorescent image, a Vinnapas®-laminin mixture was patterned onto glass

cover slips. For this experiment a solution containing 4 mg polymer and 14 mg laminin/ml

was used. The microstructures were subjected to the following incubating and rinsing

steps:

a) 1 hour incubation in 1% BSA phosphate buffer;

b) 1 hour incubation with α-laminin antibody that previously dissolved in BSA

phosphate buffer;

c) three-times gently rinsing with phosphate buffer;

d) 1 hour incubation with the fluorescent tagged antibody dissolved in 1% BSA

phosphate buffer;

Fig. 48 Examples of neurons cultured on laminin–Vinnapas® lines on glass cover slips.


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e) rinsing with phosphate buffer;

f) 30 minutes immersion in 5% glutaraldehyde;

g) rinsing with phosphate buffer.

h) drying and preserving in a cold, dark place.

Steps d) and g) have to be carried out in the absence of intense light.

It was also checked whether laminin-Vinnapas® lines were suitable for neuronal adhesion

on gold, silicon and glassy carbon. Figure 50 shows the result of a negative control

performed on glassy carbon substrate, where lines were made only with Vinnapas® i.e.

without laminin. No adhesion of neurons occurs on Vinnapas® lines that are lacking

laminin (Figure 50A). Small and faint fluorescent dots were seen on the polymer lines on

all opaque substrates, possibly by non-specific inclusion of the fluorescent dye DiI into the

polymer matrix. Glassy carbon is a material which possesses a variety of heterogeneities in

its structure. They could provide sites for non-specific cell adhesion. Indeed, it was found

that there were several cells on the glassy carbon surface outside the lines. A good

adhesion of neurons onto the polymer lines on glassy carbon could be observed, if laminin

was present in the Vinnapas® (Figure 50B).

Fig. 49 Neurons on a laminin–Vinnapas® line on a glass cover slip. The cells are visualised by means of immunocytochemically staining with an anti-neurofilament anti-body.


78

More cells adhere non-specifically outside the lines, some of them forming dense clusters.

The reason for this behaviour could be leaching of laminin out of the polymer lines and

subsequent local adsorption of laminin at the glassy carbon surface, thus providing

additional potential adhesion sites for the neurons. The cluster-like gathering of the

neurons indicates that pure glassy carbon surface is not appropriate for neuronal adhesion.

A completely unexpected finding was observed on gold substrates. The whole surface was

populated more or less densely with neurons, whereas no cells could be found on the poly-

mer lines (Figure 51A). The bright dots of fluorescent dye visible on the lines represent

most probably stained cellular debris attached to the lines. At the moment, we cannot

explain this behaviour, and further experiments should be performed.

On silicon substrates, only a very weak adhesion of neurons could be found at all (Figure

51B). Although the lines seemed to be preserved, only few neurons adhered to them.

Nevertheless, they exhibited normal neuronal appearance and a good growth of neurites.

Due to small number of adhering cells, individual cells and their connections can easily be

traced.

Fig. 50 Behaviour of neurons on: (A) Vinnapas® lines and (B) laminin–Vinnapas® lines shot onto glassy carbon. The broken lines indicate position and width of the polymer lines. Both scale bars represent 100 µm.

Fig. 51 Behaviour of neurons on laminin–Vinnapas® lines shot onto: (A) gold and (B) silicon substrates. The broken lines indicate position and width of the polymer lines. Both scale bars represent 50 µm.


79

5.2.4. Conclusions

It could be demonstrated that the adhesion of the neurons followed the prepared

micropatterns. Primary embryonic neurons are able to attach to lines made from a mixture

of laminin and Vinnapas®, though to a different extent depending on the substrate. It is not

yet understood why neurons adhered to the pure gold surface instead of the polymer lines,

which contained the adhesion-supporting laminin. It may be that sub-micrometric features

of the surfaces can promote better the cell adhesion and growth on smooth supports rather

than rough ones. Vinnapas® turned out to be a useful polymer matrix for laminin in order

to achieve adhesion of neurons and even outgrowth of neurites. It swells in an aqueous

environment, exhibiting hydrogel-like properties. These findings open the route for the

generation of complex small neuron arrays and for the electrochemical investigation of the

obtained neuron matrix.

DNA microstructures

80

5.3. DNA microstructures Many metaphoric names have been given to the XXth century. The scientific and

technological revolution have created numerous new research fields and the achievement

rates were so high that the century itself was named after key words specific to the new

found disciplines. The century of speed, of electronics, space era and not to mention the

most recent one: the century of genetic engineering. With every new idea and great

discovery, scientists around the world were seeking to contribute with their best to the new

challenge. Deoxyribonucleic acid (DNA), the “super-molecule” encrypting the secrets of

life within its long chain made of four basic units adenine (A), thymine (T), guanine (G),

and cytosine (C) (Figure 52) could not stay apart of this phenomenon. DNA represents a

cellular library with the complete information required to form complex organisms. The

information storage is achieved by a genetic code made up of sequences of the A, T, G and

C units. The problems to be solved had been different in the last decades and it became

time to take advantage of the enormous amount of information encoded in DNA and used

it to get better tools for detecting and curing genetic diseases, inherited or acquired.

New improvements in life care must pay attention to the detection of genetic vectors as

DNA strands which can give a great deal of information about the incipient phase of a

disease development. Each period of mankind history had its particular implements or

methods to identify and to cure diseases. In this century, the application of highly selective

genetic tools in a parallel manner will play a significant role to perform multiple analyses

at once. In this context, DNA chips or microarrays represent the foundation of the DNA-

based high-throughput analysis. It is a currently developing technology that is reshaping

molecular biology. Some ideas about important aspects of DNA microarray are introduced

in the following.

Fig. 52 The four nitrogenous bases present in the structure of DNA.

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The DNA

Any cell of an organism contains in form of DNA molecules the complete set of genetic

information necessary for building a fully functional new similar organism. The nuclear

DNA that is the physical support of this information is called genome. The size of the

genome is usually measured in terms of base pairs (bp) and largely varies from as little as

50 thousand (Phage λa) to billions (human genome has about 3 billions bp). In a cell

nucleus, the long DNA moleculesb are found within entities called genes that are defined as

the physical and functional units of heredity. The unique arrangement of the four

constituents, the nucleotides (A, T, G, C), in a gene ensures the

distinctive features of individual organisms. To a higher level of

organisation, the genes are coupled in an array of genes that are

known as chromosomes. A chromosome is the self-replicating

genetic material during cell division process.

DNA as biopolymer

The structure of a DNA polymer consists of repeating chain

sugar (β-D-deoxyribose) residues linked by phosphate units.

The four organic bases are attached to the side of the chain

(Figure 53). Condensation of a purine or pyrimidine base with

deoxyribose produces a nucleoside. An ester bound between a

phosphate group and the hydroxyl of deoxyribose generates a

nucleotide. Two single DNA strands are able to bind to each

other forming double stranded DNA obeying the base-pairing

rules of Watson and Crick:

• bases of one strand are bound by H-bonds with bases of the other strand;

• purine bases bind pyrimidine bases; thus A pairs with T, and C with G;

• A and T are hold together by two H-bonds, while G and C are hold by three H-

bonds (Figure 54).

Three secondary structures were identified by X-ray crystallography for double stranded

DNA: A-DNA and B-DNA are two geometries of the standard right-handed double

helices, while Z-DNA is the left-handed double-helical structure that is only stable at high

concentrations of NaCl or MgCl2.

a A phage (or bacteriophage) is a virus that contaminates only bacteria. Phage λ specifically infects E. coli. b Stretching the DNA molecule from a single human cell, leads to a DNA string as long as 1.8 meter.

Fig. 53 A fragment of DNA. B1-B3 are nitrogenous bases.

DNA microstructures

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5.3.1. DNA microarrays

A microarray138 is a spatially ordered and miniaturised arrangement of a large number of

surface immobilised reagents. Typically, the spot sizes are less than 200 µm in diameter.

Roger Ekins was the first who described a “microspot multianalyte immunoassay”139,140 in

1989. His team thought this new tool would be of value for the analysis of complex protein

mixtures deriving from recombinant DNA technologies. Nevertheless, E. Southern had the

idea of using nucleic acids molecules to interrogate other nucleic acid molecules attached

to a solid support141. Since 1995, the term “microarray” got slowly in wide spread use.

A microarray is named according to the reagent that is confined at the surface rather than

to the analytes they aim to detect. Figure 55 is exemplifying different types of microarrays

together with possible applications142.

Fig. 54 Formation of hydrogen bonds (red doted lines) between adenine-thymine (AT) and cytosine-guanine (CG) as postulated by Watson-Crick base-pairing rules.

Fig. 55 Example of microarrays and their possible applications.

DNA microstructures

83

It is common use to call the surface-immobilised reagent the probe and the analyte in the

sample the targetc (Figure 56). This nomenclature will be used throughout the chapter

about DNA microarray which also are frequently called DNA chips. It should be

mentioned that the name GeneChip® is owned by Affymetrix Incorporation and refers to

their high density, oligonucleotide-based DNA array. However, the term “gene chip” is

often used as a general terminology within the microarray technology.

DNA microarrays exploit the preferential binding of complementary single-stranded

nucleic acid sequences. The underlying principle is the same for all microarrays, no matter

how they are made. The unknown sample (target) is hybridised to the ordered array of

immobilised DNA strands whose sequence is known (probes)143-145. Due to the large

number of different probes, the microarray can identify thousands of DNA fragments

simultaneously which offers the chance to perform genetic analysis on a huge scale. This is

a prerequisite to gain profit from the mountain of information resulting from the

completion of the Human Genome Project146-149 (HGP, formally started in 1990) which

otherwise would be of no use but simply a collection of data.

Typical applications of DNA microarrays are150-153:

c In the literature one can find two confusing nomenclature systems for referring to hybridisation partners, but both commonly used "probes" and "targets". According to the nomenclature recommended by Bette Phimister (Nature Genetics Supplements, 1999, 21, 1) a "probe" is the tethered nucleic acid with known sequence, whereas a "target" is the free nucleic acid sample whose identity/abundance is being detected.

Fig. 56 Hybridisation at a DNA microarray (chip). DNA targets, from the bulk of the solution, bind to their complementary DNA probes (match) but do not hybridise to the non-complementary ones (mismatch).

DNA microstructures

84

1. identification of the sequence and gene mutation (such as Single Nucleotide

Polymorphism – SNPd);

2. determination of the expression level (abundance) of a genee;

3. comparison of gene expression in different populations of cells (for instance

healthy versus diseased cells, or the evolution of gene expression during the

embryonic development; disease diagnosis);

4. drug discovery (pharmacogenomics) which is aiming to find correlation between

therapeutic responses to drugs and the genetic profiles of patients;

5. toxicological research (toxicogenomics) which is aiming to find the correlations

between toxic responses to poisons and changes in the genetic profiles of the

organisms exposed to such pollutants.

Hopes

The beginning of the era of molecular detection of cancer started last year, when a team of

cancer specialists recruited a group of women for clinical test for breast cancer

investigations. Researchers are convinced that they understand gene expression patterns

now well enough to use them in life-altering treatment decisions. Thus, the first ever

clinical study to assign patients to a standard or aggressive therapy based on a gene scan is

expected to lead to the implementation of DNA microarrays as routine clinical tools in

hospitals within the next years154.

5.3.1.1. The preparation of DNA microarrays

Various materials can be used for grafting DNA strands, but most used are glass, silicon,

plastic or gold-covered slides. The immobilisation of artificial155 or natural

deoxyribonucleic acid on solid supports is a crucial step for any application in the field of

DNA microarrays. It determines the efficacy of the hybridisation and influences the signal

strength for the detection. Thousands of spots of natural or synthesised156 single-stranded

DNA probes in the form of cDNAf / oligonucleotidesg (see note 5) are fabricated by high-

speed robotics using contact or non-contact printing methods157-159.

Owing to the fact that DNA probes can be easily synthesised “in situ”160,161, nucleic acid

microarrays are currently dominating the field. Unlike DNA, there is no chemical

d SNP is a DNA sequence variation that occurs when a single nucleotide in the genome sequence is changed. About 2 of every 3 SNPs involve the replacement of C with T. e The expression (transcription) level of a gene is the amount of its corresponding mRNA present in the cell. f The term cDNA denotes a complementary DNA that is a single-stranded DNA molecule complementary in base sequence to a RNA strand. g Oligonucleotide is a DNA molecule usually composed of 25 or fewer nucleotides; sometimes even 80-mers are called oligonucleotides maybe because they look small compared to a typical cDNA that has 500-5000 bases.

DNA microstructures

85

possibility yet to build proteins at a microarray surface and thus protein molecules must be

delivered to the surface for the formation of protein chips. “In-situ” synthesis of a library

of oligonucleotides consists of stepwise building a number of different DNA strands from

its particular nucleotides by using ink-jet printing162 or photolithographic methods similar

to those used in the semiconductor industry/silicon technology (Affymetrix, Figure 57)163.

A remark about the DNA spot diameter: photolithographic-based in-situ synthesis can

produce extremely tiny spots with diameters down to 2 µm which is much smaller than the

100 µm obtainable by ink-jet spotting.

CombiMatrix is applying Lab-on-a-Chip technology for in-situ simultaneous synthesis of

thousands of DNA probes. Individual addressable microelectrodes of an array locally

generate, via an electrochemical reaction, reagents that facilitate the in-situ synthesis of

DNA fragments. A great advantages of this approach is the highly parallel synthesis of

hundred of thousands molecules since it saves time and reduces costs.

Thiol-modified DNA was patterned with an atomic force microscope (AFM) on a resist-

covered gold surface with line widths as small as 15 nm164. However, patterning with an

AFM tip is not a largely accepted method for DNA microarrays preparation due to the low

production rates.

Fig. 57 Affymetrix is fabricating the high-density probes GeneChip® through a combination of photolithography and combinatorial chemistry. The protected probes of a DNA film are rendered ready for chemical reaction only in the areas exposed to ultraviolet light (1-2). Deprotected DNA probes react with one of the protected monomers (A, T, G, or C) and increase the length of the addressed probes (3). This process is repeated until the desired DNA chip is ready (4-6).

DNA microstructures

86

The most commonly used technology is based on the localised immobilisation of complete

DNA strands that are prepared by solid phase synthesish,165. In this case, the DNA probes

must be modified at one end with an appropriate functional group (thiol166 or amino167) for

establishing a covalent bond between the probe and chip surface.

Fast and precise delivery of the DNA at the substrate surface is typically achieved by

means of a spotting machine or arrayer (Figure 58). A robotic platform transfers the DNA

sample from the wells of a microplate to the slide in the form of a spot under careful

control of the temperature and humidity. Evidently, an automation of the DNA chip

fabrication gives more reproducibly, accurately and quickly access to high density DNA

microarrays. A key element of an arrayer is the spotting pen that is shown schematically in

Figure 59 (see note 6).

h R. B. Merrifield (FortWorth, Texas, USA 1921), Chemistry Nobel Prize Laureate in 1984, „for his development of methodology for chemical synthesis on a solid matrix“.

Fig. 58 Schematic of a spotting machine (arrayer). The precise delivery of the substance at the chip surface occurs by contacting the spotting pen with the chip in a computer-assisted process. The spotting solutions are kept in microtitre plates; washing and drying stations are cleaning and drying the pens in between spotting steps.

Fig. 59 Examples of split-pins. (1) Normal slot filled with the spotting solution; (2) Slot ended at the upper part with a larger cut used for storing the larger quantities of spotting solution.

DNA microstructures

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5.3.2. Detection of DNA hybridisation – What are the options?

The above discussion on DNA microarrays intended to give a brief description of this

revolutionary tool, but did not show anything about the way a DNA microarray could be

used. As a consequence of the Watson and Crick base pairing rules (see note 7) the kind of

interaction between the DNA probe and target is the formation of the double helix, a

process known as hybridisation of DNA. If the DNA targets to be identified are part of a

complex mixture (such as blood or tissue extract), the compatibility/match of the purines

and pyrimidines would guarantee that preferentially the wished targets will be measured.

Accordingly, the fundamental scheme that researchers are looking for is a method to

discriminate between a certain single stranded DNA (ss-DNA) and its corresponding

double strand (ds-DNA) when both of them are possibly present at the microarray surface.

Detection of DNA hybridisation is performed in multiple ways but optical, mass sensitive

and electrochemical techniques are dominating. A concise review of the established

recognition methods of DNA hybridisation is provided in the following. They are

discussed considering their working principle (optical, mass sensitive or electrochemical

methods) or in concordance with the features of the attached active groups/reporters (label

and label-free methods).

5.3.2.1. Optical detection

Either the DNA targets or the probes are functionalised with a fluorescent dye. If the

targets are tagged then the corresponding spots on the chip are not appearing fluorescent

unless the DNA probes are hybridised. If, however, the probes are labelled one has to take

advantage of a quenching of fluorescence. This is only observed if the labelled probe

strands are flattened on the surface of a metal. Following the hybridisation with the

unlabeled targets, the probes will stretch upwards placing the label far above surface and

thus increasing the detected fluorescence (TIFI168 – target induced fluorescence increase,

Figure 60).

In general, sophisticated instrumentation is required for reading out the weak light signals

emitted by the DNA labelled spots169. Laser scanning fluorescence microscopy using for

example fluorophor-labelled target DNA, is known for its excellent sensitivity and

reliability and turned into the standard for the detection of hybridisation. Nevertheless,

thinking about a widespread application the size, price and complexity of fluorescence-

based DNA detection systems as well as the dependence of the method on the long-term

stability of the fluorescent dyes are major drawbacks.

DNA microstructures

88

5.3.2.2. Mass sensitive detection

A widespread and methodically investigated representative of this sensor type is the quartz

crystal micro balance (QCM). A quartz crystal is forced to oscillate at its own resonance

frequency (5 to 20 MHz). If the power used to vibrate the quartz is kept constant, the

oscillation frequency will changes upon mass addition to its surface. The QCM response is

directly related to mass variations and thus easy to interpret (see note 8). This simple

principle is used to monitor hybridisation of nucleic acids by measuring the frequency shift

that follows the formation of the double strand170-172.

Another integrated biosensor technology based on thin-film bulk acoustic resonators has

been introduced. As the above, the detection principle of these sensors is label-free and

relies purely on a resonance frequency shift caused by mass loading of an acoustic

resonator. The sensor has been proved to be suitable to detect proteins as well as DNA

molecules, with a mass sensitivity being 2500 times higher than for a 20 MHz quartz micro

balance173. Such methods are not only label-free but also able to detect in real-time DNA

hybridisation and hence can provide kinetic information. A limitation of this mechanical

approach to detect hybridisation is the difficulty to use it for large number of samples

(parallelisation) in comparative genome studies with the goal to identify hundred or

thousands DNA fragments in relatively short time.

Fig. 60 Target induced fluorescence increase (TIFI) detection of hybridisation is based on the fact that the fluorescence of the label of a single stranded DNA probe is much lower as compared to the fluorescence signal of the hybridised probe.

DNA microstructures

89

5.3.2.3. Electrochemical detection of hybridisation

The complexity of fluorescence-based detection systems as well as the limited

dissemination of mass sensitive techniques is hindering the development of independent

medical points-of-care devices based on DNA microarrays. In view of that, a remarkable

number of electrochemical (EC) hybridisation assays have been proposed as practical

alternatives to optical readouts. As emphasised in a series of recently published review

articles174-179 electrochemical DNA chips can be much simpler in instrumentation and are

easier to miniaturise, since the fabrication of electrochemical devices actually is well

compatible to micro- and nanofabrication. In fact, the recent appearance of commercially

available systems such as Toshiba’s Genelyzer™ (Toshiba, 2003) and Motorola’s

eSensor™ 180 is a good sign that an EC-based technology indeed has the potential to offer

relatively cheap, easy-to use and portable analytical platforms for high-throughput DNA-

based diagnostics. Such sandwich-like hybridisation assay are used for determining

hybridisation or even point mutations via electrochemical redox signals arising from

ferrocene-tagged signalling probes (AMBERi)181,182. Figure 61 gives a typical example of a

sandwich-like DNA chip where generally three DNA fragments are involved: a capture

probe that is confined at the chip surface and binds the target; another DNA fragment,

signalling probe, is labelled and complementary to a part of the target, other as used to

binding the capture probe. With this complicated stepwise procedure, a redox or

fluorescent label is transferred at the chip surface where is afterwards detected and thus

giving a proof for the hybridisation.

In general, electrochemical detection of base pairing benefits from differences in the

intrinsic electrical properties of single (ss) and double (ds) stranded nucleic acids183 and/or

employs easily oxidisable or reducible hybridisation indicators and redox labels. Stacked

arrays of aromatic heterocyclic base pairs in the core of immobilised ds-DNA, for instance,

are strongly supporting long-range electron transfer184-186 through the duplex (π way)

towards conductive carriers, an effect, however, that is worse in the presence of disruptive

mismatches and not observed with ss-DNA187,188. Barton, Hill and co-workers made use of

this observation for establishing sensitive (see note 9) schemes for the electrocatalytic

detection of hybridisation and mismatch recognition189,190.

i AMBER is the acronym for amperometric bioelectronic reporter.

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There is a category of compounds known as intercalatorsj that are appreciated by the DNA

biosensor community because they opened a new route for the detection of ds-DNA. This

approach is simple on the one hand and on the other hand is highly selective, hence

offering good discrimination rates between ss- and ds-DNA (the binding affinities of metal

complexes with DNA are generally in the following order: intercalation, K > 106 M-1 >

hydrophobic interaction, K >105 M -1 > electrostatic interaction, K > 103 M -1)191,192. It had

been shown that the interaction between DNA and some transition metal complexes

changes from electrostatic to intercalative with increasing ionic strength193. Threading

intercalators are derivatives of polyaromatic systems bearing an electroactive label on the

side arms (naphthalene diimide194,195). They intersperse within the ds-DNA thus allowing

EC detection of DNA on arrays (Figure 62). Methylene blue (MB) has good voltammetric

behaviour and an appropriate structure for binding to ds-DNA. Thus, MB could be

employed as signalling tag for ds-detection196. Natural compounds, such as hemin, were

also used as intercalators197. Osmium198, ruthenium or cobalt199,200 complexes have found

applications as electroactive intercalators too, but they are toxic and their synthesis is time

consuming. Electrode surface modification with functionalised multi-walled nanotubes

(MWNT-COOH) turned to be a suitable intermediate for DNA immobilisation with

j An intercalator is a type of DNA ligand that inserts or intercalate between adjacent base pairs of ds-DNA.

Fig. 61 Sandwich-like assay for detection of hybridisation. A capture DNA probe bind first the non-labelled target (1). The signalling DNA probe is immobilised at the chip surface via the target, and thus allowing the redox label to exchange electrons with the electrically polarise chip. A self assembled monolayer containing molecular wires facilitate the current flow. (Motorola eSensor™).

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improved rates of the electron transfer between the electrode and daunomycin as redox

intercalator201.

Other approaches for transduction of hybridisation take advantage of differences in the

binding affinities of dissolved redox active metal complexes for ss-DNA and

ds-DNA202-204, the oxidation of guanine (Figure 63) and adenine moieties205-208 (only these

two are used because the redox potential is suitable) or amplification strategies with metal

nanoparticles209-212. When guanine bases are oxidised at the surface of the microarray

itself, the high potentials involved could hydrolyse the water. The water oxidation can be

overcome by a suitable modification of the DNA chip surface, for instance with indium tin

oxide (ITO)k,213. It has been reported that modifications of the chip/electrode surface with

polyelectrolytes such as poly(allylamine hydrochloride) and poly(styrenesulfonate) and

above-mentioned ITO, lead to notable improvement of signal-noise (S/N) ratio when gold

nanoparticle probes and silver enhancement are used to detect hybridisation214. One should

mention that target-modified gold nanoparticles have another advantage because they

k Indium tin oxide (ITO) is in fact indium oxide doped with tin oxide (In203:Sn02); it is used to prepare transparent conductive coatings by electron-beam evaporation or sputtering. It has numerous applications in display devices (such as flat panels, field emission), photovoltaic devices and heat reflecting mirrors; high melting point: ~1900° C.

Fig. 62 Schematic showing the application of intercalators for detecting DNA hybridisation. The intercalator has a polyaromatic core with labels linked to its side chains. In this case, the redox label is detectable at the electrically polarised chip surface only if hybridisation took place. Intrinsic electroconductive properties of the DNA double helix allows electrons to be wired from the label towards chip or vice versa.

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induce instability in the duplex. This significantly alters the melting profile of the

hybridised DNA, allowing single base mismatch to be perceived215. Silver nanoparticles

labelled with DNA probes and Anodic Stripping Voltammetry (ASV) of Ag+ were used as

ds-DNA detection assay216. Nevertheless, these approaches require extra amounts of noble

metals and supplementary chemical steps for enhancement of signal.

A technique for detecting DNA hybridisation has been reported that is using “electroactive

beads”. It is sensitive but relies on immobilising both the targets and the probes at the

surface of magnetically and electrochemically active microspheres217.

Instead of using dissolved redox active hybridisation indicators, marker molecules can also

be covalently bound to strands of the DNA probe, the target or oligonucleotides that are

used as autonomous signalling probes. An example is the enzyme-amplified hybridisation

test from Heller’s group, which enables detection of duplex formation between surface-

anchored target strands and enzyme-tagged signalling probes by amperometrically

monitoring the product of the enzymes action as it is exposed to the substrate218-222. A

merge project of several German companies, namely Infineon Technologies, Siemens,

Fraunhofer Gesellschaft, November and Eppendorf Instrumente, is employing enzymes in

Fig. 63 Oxidation of guanine bases of single stranded probe DNA can be achieved with tris(2,2’-bipyridyl) ruthenium(III) electrogenerated at the surface of the DNA chip. Hybridised DNA has the guanine bases hidden inside the core of the double helix.

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detection of DNA hybridisation in an interdigitated array of gold electrodes223. In brief,

two neighbouring microelectrodes E1 and E2 are polarised at opposite potentials. They

cycle the redox active compounds released by the enzyme-labelled target upon

hybridisation enabling the detection and quantitation of hybridisation amperometrically

(Figure 64).

An interesting technique based on light-induced electron transfer from a DNA probe label

to the chip has been developed by FRIZ Biochem (Light Addressable Direct Electrical

Readout, LADER). An electron donor/acceptor complex label of a DNA probe is

selectively illuminated with ultraviolet light. If the probe bearing the label is single

stranded, no current is measured, whereas in the case of hybridisation, the ds-DNA can

wire electrons along its π-way. Even if multiple probes labelled with the same light

sensitive complex are simultaneously present at the chip surface, this method permits the

particular DNA probes to be identified by directing the UV beam in the right spot. It

becomes clear that a microelectrode array is not necessary. In addition, a high sensitivity is

achieved due to the ability of the complex to amplify the current by pushing up to 106

charges/second in the measuring electrical circuit (a mediator in bulk ensures that the

Fig. 64 Microelectrode array for amperometric detection and quantitation of hybridisation. Redox active compounds released by an enzyme-labelled target are consequently cycled between the interdigitated electrodes E1 and E2 while the corresponding current is monitored.

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electron donor/acceptor complex is permanently brought back to initial state subsequent to

UV-label interaction) (Figure 65).

From the same company, FRIZ Biochem, a chronocoulometric procedure, Electrically

Detected Displacement Assay (EDDA), uses short strands (4 bases only) of redox-labelled

DNA as reporter to probe the statue of the surface immobilised DNA. This method

requires two chronocoulometric measurements, one prior and another subsequent to

hybridisation. Firstly, the immobilised DNA probes are hybridised with a number of the

labelled reporter. The redox active labels are then kept relatively close to the conducting

chip surface and detectable by means of a potential step that is applied to the chip (working

electrode). The charge transport through the electrical circuit due to the electrochemical

transformation of the label is recorded as a fast decaying charge-time curve. A similar

experiment is then performed after hybridisation of the probes with their complementary

targets. Displacement of the short labelled reporter DNA by the longer targets is reducing

the concentration of redox labels in the vicinity of the chip surface (Figure 66). Therefore,

the integral of the charge-time curve is smaller as in the first case with the difference being

the indication of hybridisation. This detection scheme does not call for target labelling as

many of the other electrochemical assays but still requires the use of labelled signalling

DNA.

Fig. 65 LADER resorts to the use of light induced electron donors/acceptors to pump charges up/down only along π-way. Thus, ss-DNA generates no current while the ds-DNA can wires an amplified number of charges due to the regeneration of the label by the electrochemically active mediator in the bulk of solution.

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Capacitance measurements224 (dependence of the differential capacitance C of the

electrode double layer on potential E, derived from C-E curves), electrochemical

impedance spectroscopy225-227 (frequency response of the impedance Z of the electrode

double layer-EIS) and constant current chronopotentiometry (dependence of dE/dt on the

potential at constant current) are also used to electrochemically study the interaction of

different redox labels with ss- and ds-DNA228.

Janata and co-workers proposed a new approach for simple and direct electrochemical

detection of a hybridisation, which is based on the electrostatic modulation of the flux of

chloride ions through a polypyrrole film into which the DNA probes are entrapped229.

Interestingly, the transport of Cl- ions is controlled by the status of the DNA probes with

higher values of diffusion observed in case of ss-DNA (Figure 67).

Recently, studies were reported on using electrochemical DNA biosensor as a screening

tool for environmental pollution monitoring230. Lucarelli231 and co-workers, for example,

presented a disposable electrochemical biosensors based on ds-DNA that was immobilised

on the surface of a screen-printed graphite electrode (SPGE). Voltammetry was employed

to investigate the electrode surface and changes in the DNA redox properties (oxidation of

guanine base) were monitored in order to study the interactions between DNA and the

Fig. 66 With the electrically detected displacement assay (EDDA), neither the capture probe nor target is labelled. Contrary, short signalling strands of DNA are tagged with a redox active compound and used as a reporter. The reporter that is hybridised with probe strands is displaced when the chip is exposed to the target. In a potential step experiment the replacement of signalling probes with targets is detected as a significant drop of the charge integral.

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analytes. Although yet not specifically addressed to the detection of hybridisation, the

principles behind the strategy may be helpful as well to discriminate between ss- and ds-

DNA.

5.3.2.4. Other methods

Besides the above mentioned schemes for detecting the hybridisation of DNA, a series of

papers suggest surface plasmon resonance232 (SPR) as a label-free and/or real-time

hybridisation assay. The last decade witnessed a remarkable development of SPR use in

biomedical applications233 and it seems that DNA chip technology is on its way to taking

advantage of this reliable and sensitive technique234.

5.3.2.5 Concluding remarks

A number of schemes for detecting hybridisation events on the surface of DNA

microarrays have been developed ranging from optical readouts using a sophisticated and

expensive instrumentation to rather simple electrochemical assays. However, hybridisation

detection is still an active field of ongoing research and development.

Fig. 67 Modulation of Cl- fluxes by polypyrrole (PPy) film for label-free detection of DNA hybridisation.

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5.3.3. The repelling mode of SECM: A new and promising assay for

imaging DNA microarrays and detecting DNA hybridisation

5.3.3.1. SECM and DNA microarrays

Recently, the visualisation of oligonucleotides and polynucleotides (poly[G], calf thymus

DNA) immobilized onto aldehyde-modified glass substrates was achieved in the

generation/collection mode of SECM through the oxidation of guanine residues by tip-

generated [Ru(bpy)3]3+-molecules235 (Figure 68). This ruthenium complex is a strong

oxidising agent and able to oxidise guanines. Compared to non-hybridised DNA spots, an

increased current is observed over an area with hybridised DNA due to the larger number

of guanine bases present in the double strands.

A silver-enhanced SECM imaging of DNA hybridisation was demonstrated236. Capture

probes attached to insulating glass slides were hybridised with biotinylated targets and only

regions where sequence-specific hybridisation had occurred could be developed by the

adsorption of streptavidin-gold nanoparticles followed by electroless silver particle

deposition. The silver staining procedure formed locally conductive regions at which the

SECM tip current was amplified due to the appearance of positive feedback (Figure 69).

The related increase in the measured tip current made hybridised spots visible. In addition,

DNA duplex regions were successfully visualised by SECM using ferrocenyl naphthalene

diimide as intercalating hybridisation indicator237.

Fig. 68 Detection of DNA hybridisation via SECM tip generated [Ru(BiPy)3] (III). This complex can oxidise guanine bases while itself is reduced to the initial state. The oxidation current of [Ru(BiPy)3] (II) is thus higher above the ds-DNA as compared to areas carrying ss-DNA.

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5.3.3.2. Aim

The abovementioned methods that were designed to address a key issue of present genetic

research, the detection of hybridisation on DNA chips, have inherent drawbacks: they are

either complicated due to a high number of steps necessary for delivering the final result,

or they need of sophisticated machinery. Often miniaturisation that is essential for the

fabrication of individual medical point-of-cares instruments is difficult to achieve.

The idea of using a negatively charged redox compound to probe the status of DNA

strands immobilised at a chip surface appeared very exciting and was thought to offer a

straightforward alternative to the present hybridisation detection assays especially to those

based on electrochemistry. This detection scheme turned out to be not only a tool for

detecting hybridisation, but also could be used as an unconventional approach for

inspecting the quality of spotted nucleic acids microarrays.

5.3.3.3. Imaging and detection principle

At pH values above about 5, the phosphate groups of nucleic acids are likely to be

deprotonated. As illustrated in Figure 70, the diffusion of an anionic species An- towards

DNA-modified regions of a surface will therefore be effectively hindered due to

electrostatic repulsion. This coulomb interaction and modulation of diffusional mass

Fig. 69 Detecting hybridisation with feedback mode SECM. A biotinylated target is hybridised to the DNA probes (1,2). In a further step, gold nanoparticles are bound to the double strand via streptavidin – biotin interactions and than a silver reduction is carried out over the gold nanoparticles layer unless a compact film of silver is obtained (3). A ruthenium complex mediator is recycled only above the hybridised area where the silver layer could be produced.

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transport provided the basis for establishing a novel label-free electrochemical detection of

hybridisation.

At a properly polarised SECM tip, [Fe(CN)6]3- can be reduced to [Fe(CN)6]

4-. Far above a

conducting DNA chip surface (Au), a cathodic tip current arises from the diffusion limited

reduction of ferricyanide ions at the tip electrode, and a steady-state current value is

measured. However, close to the chip surface, the tip-generated [Fe(CN)6]4- is diffusing

towards the Au surface and an oxidised back to [Fe(CN)6]3-. This electrochemical

recycling is increasing the tip current compared to the value in bulk (positive feedback of

SECM). Due to the influence of repulsion, the diffusion of tip-generated [Fe(CN)6]4- is

hindered above surfaces carrying DNA and virtually unaffected in the DNA-free areas.

Additional negative charge is introduced by hybridising the probe strands with a

complementary target. Hybridisation thus becomes detectable thanks to an enhancement of

the effect of coulomb interaction on the diffusional flux of electroactive species.

Because the phosphate groups of nucleic acids and a negatively charged mediator are

presenting a repelling force to each other, a DNA chip with the recognition element spotted

in a regular pattern on a conducting surface is electrochemically highly heterogeneous. For

a given density of DNA probes, number of bases per individual strand, ionic strength of the

Fig. 70 Schematic representation of the influence of coulomb interaction on the diffusion of An- towards a DNA-modified surface. Electrostatic repulsion between An- and the phosphate groups at the backbone of the immobilised DNA strands hinders the diffusion of the anionic species to the underlying surface. For ss-DNA, the flux of An- (Iss) is higher than for the ds-DNA (Ids) due to the increase in negative charges through formation of aggregates between the probe and unlabeled target.

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electrolyte solution and concentration of the mediator, the electrostatic interaction and

corresponding modulation of the diffusional transport of [Fe(CN)6]4- to the surface is

mainly determined by the ss/ds status of DNA strands within the probe spots, with fluxes

of the [Fe(CN)6]4- -species being higher in case of ss-DNA. The determination of the

relevant fluxes of anions can be achieved by subjecting DNA chips to local

electrochemical measurements. As shown in Figure 71, these spatially resolved

measurements can be achieved by performing SECM in the amperometric feedback mode.

Moving the SECM tip at fixed height above an oligonucleotide spot gives reason for a

sudden drop in the SECM tip current since here tip-generated [Fe(CN)6]4- molecules are

repelled, transfer rates for Au-induced recycling diminished in turn leading to a decreased

redox amplification by the positive feedback effect.

5.3.3.4. Oligonucleotides and the substrate

To prove the abovementioned hypothesis right, experiments have been carried out on DNA

microarrays of synthetic 20-mer oligonucleotides. They were supplied by FRIZ Biochem

GmbH, Munich, Germany. Immobilisation of the 20mers was accomplished using the self-

assembly of the 3'-thiol modified strands that were spotted from a 250 mM phosphate

buffer aqueous solution on Au sputtered glass surfaces (2.5 x 7.5 cm2) with a professional

microarrayer (see Materials and Methods). Typically, the DNA chips contained DNA spots

Fig. 71 Schematic of the principle of DNA visualisation in the repelling mode of SECM.

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of two different 20-mer sequences, denoted as Oligo8 and Friz12 (O8 and F12

respectively). The base sequences are shown in Figure 72.

Fluorescent tags were necessary to provide comparative fluorescence measurements of the

hybridisation events. As can be noticed from this figure, the two DNA probe strands

include all the four bases. Freshly prepared DNA chips were post-assembled with 1 mM

propanethiol in deionised water, overnight, and at room temperature. This increased

hybridisation efficiency and helped to avoid unspecific adsorption of target strand. The

layout of the DNA chip is depicted in Figure 73A. A thin film of gold is necessary for the

specific anchoring of the thiol-modified DNA probes to the chip, but its adherence to glass

slides is poor. For this reason, an intermediate layer of chromium or titanium is applied on

the glass before gold is sputtered (Figure 73B).

Fig. 72 The base sequence of the probes and targets used for spotting/hybridisation.

Fig. 73A The layout of the DNA chip.

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DNA spots consist of a monomolecular film of DNA molecules and, hence, are not visible

with naked eyes. In order to facilitate the positioning of an SECM tip near to a DNA

microstructure and to shorten the scanning time of the DNA chip by choosing only a

particular area of the chip, a series of marks were stained or engraved at the surface of the

chip. There are two types of marks: temporary and permanent.

Marking the DNA chip

1. Temporary marks – are polycrystalline deposits of potassium phosphates and NaCl

grown at the hydrophilic DNA modified surface of the chip; they are obtained by

rinsing the chip with some drops of 1M NaCl PBS and air drying. The DNA-free

areas are not noticeably changed by this treatment owing the hydrophobic

properties of the thiol-modified surface. Although they are meaningful for the

positioning of the electrode, the crystals are dissolved when the electrolyte is filled

in the electrochemical cell. However, with the help of these momentary marks, the

chip was marked further with permanent marks.

2. Permanent marks – are fine scratches engraved with a scalpel on the gold surface.

Figure 74 indicates these marks on the chip.

The SECM tip is positioned over the DNA microarray in two steps. First, the tip of the

microelectrode is manually placed close to the “L”-shaped scratch (point 2 in Figure 74)

and roughly 1 mm above the surface. Then, the reference electrode is attached to its holder

and the electrolyte is filled into the cell. Subsequently, a computer-assisted precise

electrochemical approach of the electrode is performed to position the electrode tip well

within the feedback distance. For both, electrochemical approach curve and line scan

Fig. 73B The sandwich structure of the DNA chip.

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measurements, the SECM tip is polarised at 0 mV versus the Ag/AgCl 3 M KCl reference

electrode in order to reduce ferricyanide under diffusion-limited conditions. For the chosen

10 µm diameter platinum disk microelectrode, a tip-to-sample distance of 10-15 µm

corresponded to an increase in the amperometric tip response of about 50%. Though the

gold surface was in most of the cases post-assembled with propane-thiol, the SECM

feedback remained to be positive. Of course, longer alkane-thiols (C6 or C12) would

gradually turn the feedback to negative as the access of the anions to the gold surface is

hindered.

5.3.3.5. The electrochemical cell and set-up for measurements on DNA microarrays

Numerous electrochemical cells were and could be designed for SECM applications, but

here are presented those that have been used in the experiments of this PhD work.

1. Conventional electrochemical cell

The rectangular DNA chips were too large to be fixed in the standard electrochemical cells

available in the laboratory. Accordingly, a cell as shown schematically in Figure 75 was

made of Plexiglas. The width was 28, the length 55 and height 15 mm. The inner

cylindrical hole was 15 mm in diameter but had a larger opening at the bottom to allow an

O-ring to be placed. Four screws pressed down the O-ring onto the chip surface in order to

make a water-tight seal. The electrochemical cell with the DNA microarray fixed to the

bottom was mounted on the two-axis translation stage of the SECM. Thus, the

electrochemical cell and the microarray could be driven by computer-controlled stepper

Fig. 74 Marks on the DNA chip as coordinates for the positioning and imaging; vertical line (1), approximate start point of the scan (2), horizontal line (3), exact start point (4).

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motors in X, Y direction with a nominal resolution of 0.625 µm per half step. Tip approach

(Z-movement) was achieved with a third stepper motor that was mounted perpendicular to

the ones moving the cell and kept the Pt disk microelectrode that was used as SECM tip.

2. Simplified cell

In addition to the conventional electrochemical cell, a simplified cell was constructed with

little efforts and only an O-ring defining the electrolyte volume (see Figure 76).

Vacuum fat was used to keep the O-ring in place and obtain a water-tight seal to the chip

surface. The advantage of this simple arrangement is that it tip placement and positioning

is easier for the user since no walls of a chamber are hindering the optical observation of

the microelectrode and the sample. Furthermore, lower volumes of electrolyte are needed

which could be an advantage when one is aiming on monitoring the hybridisation of the

Fig. 75 Drawing of a typical SECM screwed-from-the-top electrochemical cell; the body of the cell is made of Plexiglas (1); O-ring (2); substrate (3); translation stage (4).

Fig. 76 Simplified electrochemical cell for SECM measurements.

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probe strands with their complementary targets in real time while the duplex formation

takes place. Since the chip has to be exposed to a certain concentration of targets, a lower

volume would help to save on the expensive synthetic target oligonucleotides. The solvent

evaporation is considerably reduced if it is ensured that the surface of the solution is not

having a convex curvature but plain or concave (Figure 77).

Fig. 77 The curvature of the surface of the electrolyte in the simplified electrochemical cell has a high impact on the evaporation rate of the solvent: convex (A) > plain (B) > concave (C).

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5.3.3.6. Imaging ss-DNA in the repelling mode of SECM

The preliminary experiments were performed in a ferrocyanide solution in 1M PBS. The

SECM tip was polarised at +400 mV vs. Ag/AgCl 3 M KCl in order to oxidise the

mediator. For this reason, the DNA spots appeared in the SECM images as areas

displaying a sudden drop of the anodic tip current.

Figure 78 represents a SECM image of a DNA microarray that was recorded at the very

beginning of this project. It is a single line scan carried out in 5 mM ferrocyanide in 1M

PBS with a 10 µm Pt disk electrode scanned at 5 µm/s. Obviously this was a remarkable

result because it demonstrated that the underlying idea of the proposed new detection

protocol was right: the downwards peaks as visible in this SECM image are a visualisation

of the DNA spots. Indeed, repelling forces between the negatively charged mediator and

the phosphate groups of the DNA strands on surface seemed to have a strong impact on the

amperometrically feedback current of the SECM. When larger areas of a DNA chip were

scanned, colour bird-view or 3-D plots could be obtained by converting the raw data into

the desired image files (here not shown, see below).

Proof of principle

In order to prove that electrostatic repulsion is responsible for the creation of contrast

between the DNA-modified and bare chip surface, experiments were carried out in the

presence of a negatively and positively charged mediator. The latter should not experience

a repelling force from the phosphate groups of the immobilised ss-DNA and the feedback

response of the SECM tip thus not be disturbed. In Figure 79, the upper curve displays a

Fig. 78 The first SECM scan over a ss-DNA microarray indicated the position of some DNA spots (lower parts of the curve); 5 mM [Fe(CN)6]

4- in 0.1 M phosphate buffer and 1 M NaCl; scan rate 10 µm/s.

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representative amperometric recording taken in a [Fe(CN)6]3- solution during a complete

move over an individual oligonucleotide spot. The current to the left and right are

indicative of the unbiased positive feedback. As expected, the DNA strands drastically

lowered the current values above the DNA spot. This was as expected and verifies the

reduced rates of redox recycling due to electrostatically hindered diffusion of tip generated

species. The drop of the tip current almost completely disappeared when substituting

[Fe(CN)6]3- by [Ru(NH3)6]

3+. With [Ru(NH3)6]3+, only a negligible decrease in the SECM

tip response was observed (Figure 79, lower curve, and inset) which most probably is due

to a pure steric hindrance of diffusion. The different behaviour of the two mediators gave

evidence that really an electrostatic repelling force is responsible for a DNA-induced

modulation of the tip response.

SECM measurements and the evaporation of solvent

A typical DNA microstructure had 24 spot within a 1200 µm x 1500 µm square. Applying

the typical parameters for imaging in the SECM feedback mode (scan speed: forward scan

5-10 µm/s, backward scan 500 µm/s; distance between two measuring points in X

direction: 5-10 µm; 0.5 s waiting time before data acquisition is performed; 25 µm

distance between two neighbouring X-line scans), 10 to 20 hours are needed to complete a

full image of the DNA array.

Fig. 79 Line scans acquired by scanning the tip of a 10 µm diameter Pt microelectrode at a fixed height of 10 µm across a spot of single stranded oligonucleotide. Negatively charged [Fe(CN)6]

3- (5 mM in 0.1 M potassium phoshate/3 M sodium chloride, pH 6.5) was used as the mediator and the SECM tip was polarised to 0 mV vs. Ag/AgCl/3 M KCl. Positively charged [Ru(NH3)6]

3+ (5 mM in 0.1 M potassium phoshate/3 M sodium chloride, pH 6.5) was the mediator and the SECM tip was polarised to –400 mV vs. Ag/AgCl/3 M KCl.

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At this time scale, evaporation of water from the measuring buffer was expected to lead to

a raise in the concentration of the mediator, and hence to an increase of the faradic current

at the SECM tip. However, cyclic voltammograms recorded at the beginning and the end

of the acquisition of a full SECM image of the 24-spot DNA microarray did not display

significant differences in the diffusion-controlled amperometric currents (see Figure 80).

From this it was assumed that solution evaporation at the given experimental conditions

did not influence the experiments on the DNA chips notably.

Non-specific adsorption

Imperfections in the arraying process may lead to inhomogeneities in the deposition of the

DNA spotting solution and the formation of areas with a certain degree of non-specific

adsorbed DNA strands that surround the desired DNA spots. A practical and

straightforward method for lowering the influence of non-specifically adsorbed ss-DNAs is

to post-assembly the DNA chip with alkane-thiols directly after spotting the DNA probes.

As can be clearly seen in the Figure 81, the gloom accompanying the ss-DNA 20-mer spots

(A) is totally vanished for a chip subjected to a post-assembly with hydroxy-propanethiol

(B) since alkane-thiol molecules are able, in a dynamic equilibrium, to displace loosely

bound (physisorbed) DNA probe strands. On the other hand, the presence of a dense

alkane-thiol monolayer is reducing the contrast between the background signal (DNA free

Fig. 80 CVs measured before (black) and after (red) recording of a complete SECM image of a DNA chip in the imaging solution. Image acquisition took 16 h. Amperometric current increase due to the loss of solvent is ignorable; 5 mM ferricyanide in 0.1 M phosphate buffer and 3 M NaCl; scan rate 100 mV/s.

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surface) and DNA spots and thus decreases the sensitivity of the SECM imaging in the

repelling mode of SECM.

Removal of DNA strands that are physically adsorbed at the chip surface can also be

achieved by soaking the chip in saline buffer for few hours (Figure 82A, B). A strong jet of

buffer directed towards the chip surface can be used as well, without fearing that the shear

forces caused by the liquid flow could damage the self-assembled film!

Furthermore, the so-called edge effect (“doughnut” effect; see note 10) is contributing to a

heterogeneity in the distribution of the DNA strands within an individual spot. The

doughnut effect results from a capillary flow of the bulk of a drying droplet outwards the

edge that can carry any dispersed material to the margin (see Figure 83). That a ring-like

deposition of the biological recognition element takes place when spotting a DNA-

containing solution becomes evident from the SECM image shown in Figure 84. The

higher density of probe strands at the edge of the spot leads to an enhanced repelling which

is visible in the profile of this image.

Fig. 81 The effect of thiol post-assembly on the non-specific ss-DNA adsorption: without post-assembly (A) and with hydroxyl-propanethiol (B); 5 mM ferrocyanide in 0.1 M phosphate buffer and 1 M NaCl. Spot size: 100 µm in length.

Fig. 82 The effect of saline buffer soaking on the non-specific adsorption: no buffer soaking (A) and with soaking (B) in 0.1 M phosphate buffer and 1 M NaCl.

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Certainly, such phenomena have to be considered when aiming at quantitative

measurements of ss- or ds-DNA. The repelling mode of SECM is very sensitive to

variations in the density of probes, and thus could be an excellent tool for inspecting

the quality of the spotting procedure.

The microdispenser as an alternative tool for the preparation of DNA microarrays

Most of the experiments for the evaluation of the repelling mode of SECM as a tool to

detect hybridisation on DNA microarrays were carried out on commercially available chips

that were supplied by FRIZ Biochem, Munich, Germany. These microarrays were prepared

Fig. 83 Formation of a ring-like-deposition (RLD)

Fig. 84 The ring-like deposit (RLD) of a ss-DNA spot (30 µM probe concentration) visualised in the repelling mode of SECM; 5 mM ferricyanide in 0.1 M phosphate buffer and 1 M NaCl.

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with a pin-spotting instrument. Alternatively to this routinely used method of patterning

DNA chips, a piezo microdispenser (GeSIM mbh, Germany, Figure 85) was used to

fabricate in-house DNA microarrays. Although the functioning of this particular piezo-

microdispenser is similar to the one presented in Chapter 2 and 5, there are little

differences between them in the electronic part of the set-ups This commercially available

dispenser is capable to deposit spots about 230 µm in diameter. Typically, the spotting

solutions contained 3’ -thiolated adenine 20-mer in 250 mM phosphate buffer (pH 6.7).

Before spotting, the gold surface has been immersed 5 minutes in Piranha mixture and than

rinsed thoroughly with tri-distilled water. Longer contact between the gold-covered glass

slide and the corrosive mixture could peel off the gold film, especially if the metal layer

underneath is exposed and attacked.

Figure 86 displays a home-made DNA array in which the probe spots are still covered with

the spotting solution. After 4 hours of curing time in which the thiolated DNA strands were

chemisorbed onto the gold surface, the chip was rinsed with 1 M NaCl phosphate buffer

and used for scanning in the repelling mode of SECM. The imaging of the single stranded

DNA probes was carried out in 3 M NaCl in 0.1 M phosphate buffer with 5 mM

ferricyanide as mediator.

Due to the fact that the resolution in X and Y direction was not the same (10 µm distance

between two data point along X-axis and 25 µm along Y-axis), the circular spots of

patterned DNA probes were imaged in the SECM micrograph as squares.

Fig. 85 Ink-jet set-up used for micropatterning spots 230 µm diameter of DNA microarrays on gold surfaces. The piezo-dispenser head is shown in the inset.

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Sequence specificity

Two oligonucleotides with equal number of bases but different sequences should offer the

same repelling force when interacting with negatively charged redox species. Although

only two different DNA strands (O8 and F12) were studied, it appeared in the course of all

experiments involving these entities that one type of DNA (F12) blocked more the

diffusion of ferricyanide ions than the other (O8). Figure 87 undoubtedly visualises this

significant difference between the two oligonucleotides. If would be possible to prove

beyond any doubts that strands having the same number of nucleotides but different

sequence are distinguishable by the repelling mode of SECM, this could lead to a

powerful, cheap and uncomplicated assay for comparative genomic studies.

Fig. 87 Repelling mode of SECM is a method possibly sensitive to the sequence of the oligonucleotides immobilised at the chip. Two 20-mers (F12 and O8) oligonucleotides with different sequence can be clearly distinguishable in these SECM micrographs; 5 mM ferricyanide in 0.1 M phosphate buffer and 3 M NaCl; DNA probe concentrations were 100 (first line) and 30 µM (second line) for both F12 and O8.

Fig. 86 Example of a home-made DNA microarray. The photographic image to the left displays a freshly prepared microarray where the droplets of solution are not yet dried. The image on the right is a SECM micrograph of a small area of the same microarray. White spots represent the DNA modified regions of the Au surface (low feedback current). Note: no shadows around the spots and no heterogeneities within the spots are observed!

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5.3.3.7. Factors affecting the imaging quality in the repelling mode of SECM

Imaging of ss-DNAs in the repelling mode of SECM is influenced by a set of parameters.

The conformation of a single flexible polyelectrolyte molecule with a hydrophobic

backbone in aqueous solution is effected by the interplay of the short-range intramolecular

attraction and the long-range Coulomb repulsion238. Besides, electrostatic attraction

between charged cylindrical polyelectrolytes in aqueous medium can be induced by

multivalent counterions239. Any quantitative model wished to describe completely the

interactions between the DNAs or other polyelectrolytes and ions in solution call for an

accurate description of the potential of the electric field around the charged backbones. The

modified Poisson-Boltzmann (MPB) equations together with the Booth’s theory of water

dielectric saturation and an experimental dependence of water dielectric constant on ionic

concentrations are normally used to calculate the mean electrostatic potential and ionic

distributions around a DNA-like highly charged cylindrical polyion240.

Of course, many factors could be identified as important, but accidentally (effect of tip-to-

sample distance) or in purpose, there were four parameters found during the investigations

that were considered critical and sufficient for the proper study of DNA hybridisation.

These achievements are presented in the following.

Effect of DNA probe concentration

At first, in a set of experiments a number of oligonucleotide spots that were different from

each other only in the density of the immobilised probes were examined. Variations in

surface density of the probes were accomplished by spotting equal volumes of probe

solutions, however, with probe concentrations ranging from 100 µM to 1 µM. Although

not exactly known, the surface density was expected to be correlated to the concentration

of the probe in the spotting solution. For this reason the following results will be discussed

in terms of the concentration of the spotting solutions that were used to produce the studied

DNA chips. Figure 88 illustrates in a series of representative SECM line scans and images

that the modulation of the SECM tip current due to repulsion between the mediator and

probe strands is well correlating with the concentration of spotting solution.

Typically, the contrast (defined as the difference of the signals above a probe spot and the

neighbouring surface) was significantly improved and better SECM images of the single

stranded oligonucleotide were achieved at higher probe concentrations. With the lowest

concentration (1 µM), almost no effect was observed and the spots were almost invisible

for the repelling mode of SECM. This was seen as an indication that the strands in fact

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were so loosely arranged that their anionic phosphate groups could not really hinder

[Fe(CN)6]4- molecules to approach the Au surface and undergo redox recycling. In

contrast, oligonucleotide strands in dots spotted from the highest concentrated solution

(100 µM) effectively blocked the diffusion of [Fe(CN)6]4- towards the gold surface and

offered good contrast. However, the “30-µM” spots nearly equally influenced the tip

current.

This suggested that above a certain density the electrical field produced by closely

arranged probe strands is well protecting the spot region against passage of free-diffusing

anions. This, however, explains why the impact of coulomb repulsion on the diffusion of

the [Fe(CN)6]3-/[Fe(CN)6]

4- couple reaches saturation at a critical probe concentration.

Oligonucleotide spots were detectable and could be successfully visualised with the

repelling mode of SECM in a wide range of concentrations. Nevertheless, from the

observed saturation of the influence of electrostatic interaction on the SECM signals at

higher concentration it became obvious that choosing a suitable superficial density of

probes is a prerequisite when aiming on the imaging of DNA strands and detecting

hybridisation. This is confirmed by a qualitative model, which is schematically depicted in

Figure 89.

Fig. 88 Representative SECM line scans (top) and bird-view SECM images (bottom) obtained by imaging 120 µm diameter spots of a 20 base oligonucleotide at concentration ranging from 1 to 100 µM by means of the repelling mode of SECM. Measuring solutions: 5 mM ferricyanide in 3 M NaCl/0.1 M phosphate buffer, pH 5.7 (for the line scans) or 5 mM [Fe(CN)6]3- in 1 M NaCl/0.1 M phosphate buffer, pH 6.3 (for the bird-views). SECM tip: 10 µm Pt disk microelectrode. Scan speed 10 µm/s.

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Due to the presence of anionic phosphate groups in their backbones, individual probe

strands can be considered as thin negatively charged rods that, in first approximation, form

a cylindrical electric field with a radius r1 around themselves. Within r1, anionic species

will be repelled by coulomb interaction and hence will not find access to the surface.

Obviously, this assumption leads to the consideration that probe strands should not be

arranged too close to each other to avoid overlapping of their electric fields. In this case,

the entry for anions within the probe spot would be fully blocked. The observed feedback

current would then become independent from the number of negative charges localised

within the spot thus preventing to distinguish between ss- and hybridised DNA probes at

higher concentration. On the other hand, the distance between two adjacent strands should

not be too large to allow for a significant modulation of the diffusional flux of the redox

species to the underlying Au surface, even after duplex formation.

Effect of mediator concentration

The influence of the redox mediator concentration on the performance of the repelling

mode of SECM for imaging single-stranded nucleic acids was investigated in electrolyte

solutions containing either 5 mM or 50 mM [Fe(CN)6]3-. A selected spot of probe

oligonucleotide was sequentially imaged in these electrolytes with the electrochemical cell

being rinsed many times with phosphate buffer and the scanning solution of choice to

ensure accuracy of the [Fe(CN)6]3-

concentration. Since, the diffusion-limited current at a

disk-shaped microelectrode is proportional to the bulk concentration of the redox species

according to the equation

crDFni ⋅⋅⋅⋅⋅= 4

Fig. 89 Qualitative model representing schematically the coulomb interaction between ss-DNA and a negatively charged redox species indicated by the arrows. The circles represent the “forbidden” area at which anions do not find access to the chip surface.

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with n = number of transferred electron; F = Faraday constant, D = diffusion coefficient,

c = bulk concentration of the redox active compound, and r = radius of the electroactive

electrode surface, a 10 fold increase in the concentration of the redox mediator is leading to

a 10 fold increase in the amperometric tip current in bulk solution. As a matter of fact, also

the current that is observed due to redox recycling at the same tip-to-sample distances is

proportionally higher in solutions of the higher mediator concentration because the

feedback current is directly related to the number of tip-generated species available. As

shown in Figure 90, however, the ratios of tip currents obtained in the vicinity of an

imaged spot and just above it were apparently not dependent on the concentration of the

chosen mediator. Hence, the contrast for imaging oligonucleotides in the repelling mode

can not be enhanced by varying this parameter.

Effect of ionic strength

It is well-known that two charged particles are only facing electrostatic interaction when

their individual electric double layers start to overlap. Since the extension of the electric

double layer is changing with the ionic strength of a surrounding solution, the repelling

force between phosphate groups at immobilised DNA strands and free-diffusing negatively

charged redox species should be strongly affected by the composition of the electrolyte.

Oligonucleotide spots were therefore subjected to repelling-mode SECM measurements in

solutions with significantly different total ion concentrations. The line scans presented in

Fig. 90 Influence of the mediator concentration on the visualisation of ss-DNA in the repelling mode of SECM; probe concentration: 10 µM; 5 mM (top curve) or 50 mM (bottom curve) [Fe(CN)6]

3- in 3 M NaCl/0.1 M phosphate buffer, pH 5.7; SECM tip: 10 µm Pt disk microelectrode; scan speed 10 µm/s.

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Figure 91 demonstrate that the contrast of SECM imaging indeed was enhanced when

lowering the ionic strength of the measuring solution from 3 to 0.02 M.

In fact, the extension of the electrical double layer expands when lowering the ionic

strength. Regarding the model, this corresponds to an increase of r1 and thus to a reduction

in the surface area available for redox recycling. Worth mentioning that the enhancement

in contrast achieved in solutions of low ionic strength is of little benefit for the detection of

hybridisation since the aggregates of probe and target strands are not stable under this

condition. A compromise must be found to ensure on the one hand good SECM imaging

and on the other hand to provide a suitable environment for the stabilisation of double-

stranded DNA after hybridisation has occurred.

Effect of tip-to-sample distance

One and the same oligonucleotide spot was imaged sequentially in the repelling mode with

the SECM tip positioned at working distances of 6 and 15 µm. As expected, the closer the

SECM tip was scanned across the DNA microstructure, the better was the contrast (Figure

92). The reason for this observation is an improved collection of tip-generated [Fe(CN)6]4-

at the chip surface and recycled [Fe(CN)6]3-

at the tip electrode, respectively. Apparently,

the probability for loosing the redox species through lateral diffusion is lower at a smaller

spacing between the microelectrode tip and chip surface. However, even though it is

meaningful to scan in close proximity, precaution must be taken when applying SECM in

constant-height mode. For large scan lengths, variations of the tip-to-sample distance due

to e.g. a surface tilt could easily disturb the current response or even lead to tip crash.

Fig. 91 Effect of the ionic strength of the electrolyte solution on the visualisation of ss-DNA in the repelling mode of SECM. Line scans were obtained at an ionic strength of 0.02 M (left) or 3 M (right). Probe concentration: 10 µM; 5 mM [Fe(CN)6]

3- in phosphate buffer of the given ionic strength. SECM tip: 10 µm Pt disk microelectrode; scan speed 10 µm/s.

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5.3.4. Detection of DNA hybridisation in the repelling mode of SECM

So far, merely the visualisation of single stranded oligonucleotides in the repelling mode of

SECM was demonstrated. Nevertheless, the major challenge of work was to develop a

straightforward electrochemical approach for detecting the hybridisation of DNA in a truly

label-free manner. Being aware of the multiple factors that are affecting the imaging of

ss-DNA, the investigations were proceeded to study the influence of the probe density on

the recognition of hybridisation.

It was an outcome of the first experiments on detection of DNA hybridisation in the

repelling mode of SECM that the way the hybridisation and imaging were carrying out was

not optimal. For instance, the DNA chip subjected to hybridisation and the one designed to

be the control chip were not one and the same. With this approach, a few µl droplet of the

target solution were placed over all the DNA spots of the microarray and covered with a

glass cover slip which basically meant that hybridisation could not be selectively

performed at a given area of the chip. Several questions can arise from such a situation:

- Are observed differences in the current peaks of the ss-DNA and ds-DNA induced

by the hybridisation or are variations in the probe density responsible for the effect?

- Is the SECM tip positioned at the same height over the hybridised and control

chips? Little variations possibly could have an impact on the obtained results?

Well, the difficulties described above were plainly solved in an extremely easy manner,

namely, the spots of one DNA microarray was divided in two sub-areas by means of the

Fig. 92 Influence of the tip-to-sample distance on the visualisation of ss-DNA in the repelling mode of SECM. Line scans were obtained at a tip to sample distance of 6 µm (left) or 15 µm (right); probe concentration: 10 µM; 5 mM [Fe(CN)6]

3- in 3 M NaCl/0.1 M phosphate buffer, pH 5.7; SECM tip: 10 µm Pt disk microelectrode; scan speed 10 µm/s.

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vertical scratch (see Figure 93). In this way, the control and hybridisation experiments can

be performed on the same chip. This ensures that the probe spots should be of fairly

identical properties and that the tip-to-sample distance above the ss- and ds-DNA spots are

not varying too much since they are only a fraction of 1 mm apart.

The status of individual DNA spots was worked out by measuring and comparing the

current response of the SECM tip (a 10 µm Pt disk polarised at 0 mV vs. Ag/AgCl 3 M

KCl and positioned at about 15 µm above the chip surface) that was scanned across two

otherwise identical microstructures one of which, however, had been exposed to the

hybridisation solution containing the complementary target and the other to target-free

hybridisation solution (control). It had been shown in the previous pages that a

considerable number of DNA probe strands is washed off, while soaking the chip in saline

buffer. To be able to perceive the hybridisation event, the control spots and hybridised

areas must be equal times in contact with buffer solutions. Hence, a “blind” hybridisation

had to be carried out only on the control area, whereas the hybridisation solution should

not touch the control region. The vertical scratch defining the control and hybridisation

regions of the chip was found helpful because it is acting as a barrier stopping the solution

wetting the forbidden area at the opposite side (Figure 94).

Fig. 93 Layout of the DNA chip after hybridisation on the right side with DNA target (A’) complementary only for one type of probe (A).

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A typical hybridisation protocol consists of a series of steps:

1. scratch;

2. 2 hours blind hybridisation on the left side (in the hybridisation cell);

3. 2 hours hybridisation on the right side (in the hybridisation cell);

4. chip rinsing with 3 M PBS;

5. SECM measurement with 5 mM ferricyanide in 3 M PBS.

Hybridisation experiments were performed at room temperature with the following

solutions prepared in phosphate buffer (pH 6.5):

Blind hybridisation Real hybridisation

Target (µM) - 2

Buffer (M) 0.1 0.1

NaCl (M) 1 1

SDS (%) 0.01 0.01

Before and after hybridisation the chips were rinsed with 3 M PBS. Owing to the affinity

of gold for many compounds, it is recommended to filter all buffer solutions through filter

units with a pore size of 5 µm. Otherwise, the gold surface will be gradually covered with

impurities from solutions. This could result in unwanted effects on the amperometric

feedback behaviour of the chip, disturb the electrochemical approach of the electrode and

lead to noisy SECM images.

A Petri dish was used as the hybridisation cell (Figure 94) in which the DNA chip and a

small plate filled with the same buffer as used for hybridisation were placed. This closed

system helped to minimise the evaporation of the 5 µl droplet solution of target DNA, that

otherwise will dry out before the completion of the hybridisation.

A synopsis of the results of a typical hybridisation experiment obtained for 100 µM, 10

µM, 5 µM, 3 µM, and 1 µM probe concentration is shown in Figure 95 and 96. Except for

the lowest probe concentration, the DNA spots could be well visualised but a visible

change in the tip current upon hybridisation was only observed at the 10 µM DNA probe.

In theory, binding of the target strands is increasing the net charge and enhancing the

electric field within the probe spot. Accordingly, the “forbidden” area for free-diffusing

redox-active anions is expanded upon hybridisation (r1→r2; Figure 97). For hybridisation

detection, probe strands should not be too close to each other to avoid that the sphere of

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their electrical fields overlap. In that case, already for single strands the flux of anions

towards the chip surface would be blocked to a high extent, making it difficult for the

hybridisation of target to have an additional effect and become detectable. In principle, the

separation between two single strands needs to be approximately 2⋅r2 (or ε) for optimal

detection of base pairing (see note 11).

By means of proper selection of a set of parameters as can be clearly seen in Figure 95,

which are expected to have a significant impact on the possibility to detect DNA

hybridisation with repelling-mode SECM, a good current contrast between probe spots and

hybridised spots can be obtained.

Fig. 94 The DNA hybridisation cell was a simple Petri dish in which the DNA chip and small water container were placed.

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Fig. 95 Detection of hybridi-sation on an oligonucleotide microarray by means of repelling mode of SECM. Single line scans were carried out over pairs of DNA spots. The current traces to the left correspond to non-hybridised strands (control) whereas the traces to the right were acquired on hybridised spots. Probe concentration in spotting solution: 1 to 100 µM. 5 mM [Fe(CN)6]

3- in 3 M NaCl/0.1 M phosphate buffer, pH 5.7. SECM tip: 10 µm Pt disk microelectrode. Scan speed 10 µm/s.

Fig. 96 A close-up of the 10 µM ss-DNA and ds-DNA amperometric current peaks as measured at the scanning tip in the repelling mode of SECM after hybridisation with the complementary target.

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Non-specific adsorption of target during hybridisation process

DNA has a high affinity to gold and therefore is able to forms stable films non-specifically

adsorbed targets on the substrate. Thus, an exposure of a gold surface to a solution

containing DNA molecules is altering its feedback behaviour that way that it appears less

conductive. This is confirmed by the observations shown in Figure 98 (to the left). The

current observed above the control region (not exposed to any dissolved DNA) was much

higher due to positive feedback than in the region which was exposed to target DNA.

In order to diminish the amount of non-specifically adsorbed targets and enhance the

detection of double helix, it is recommended to use detergent additives such as sodium

dodecylsulphate (SDS). Typical SDS concentrations of 0.01% in the hybridisation solution

are of great help since the target could not be any longer observed by SECM at the gold

plane. It should be mentioned that SDS is not efficient at very high target concentration

(10-20 µM) and it is impossible to overcome unwanted physical adsorption of targets by

increasing the SDS concentration because its solubility in saline buffers is rather low.

Interestingly, 2 M NaOH solutions can, in a few minutes, wash off the excessive target.

Moreover, as a side effect, loosely bound DNA probes are removed as well (Figure 98, to

the right). This observation shows that the surface density of DNA probes will change

when the chip is dehybridised in NaOH, which in principle restrains the number of

hybridisation-dehybridisation cycles of a certain microarray.

Fig. 97 Qualitative model representing schematically the coulomb interaction between ds-DNA and negatively charged redox species An-. Upon duplex formation the forbidden area increases with radii changing from r1 (rss) to r2 (rds).

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5.3.5. Conclusions and outlook

A truly label-free electrochemical method for visualising immobilised nucleic acids and

their hybridisation on microarrays was developed. The method is based on the electrostatic

repulsion between the phosphate groups of DNA and free-diffusing [Fe(CN)6]4- ions and is

straightforward and reliable. The coulomb interaction is modulating the diffusion

properties of the electroactive species in the vicinity of probe strands and thus had a strong

impact on the transport of the negatively charged species towards the conducting chip

surface in DNA-modified regions. Using SECM in the amperometric feedback mode,

significant decreases in positive feedback currents are observed above spots of nucleic

acids due to the local appearance of repulsion between the active components of the assay.

The density of the capture probe, the ionic strength of solution and the tip-to-sample

distance are influencing the capability of so-called repelling mode SECM to visualise DNA

while the concentration of the chosen mediator has not effect on the contrast of imaging.

Miniaturisation of the detection set-up

Verification of the developed detection scheme was performed by local electrochemical

measurements in a sophisticated SECM set-up that obviously is not suitable for common

use in medical diagnostics. However, the obtained results promote, in principle, the design

of a simplified electrochemical device that could be produced using microfabrication

technology. A device could, for example, consist of an array of immobilised probes in a

base plate and an array of individually addressable Pt microelectrodes in a cover plate able

of being revolved upon a lateral hinge as suggested in Figure 99 (similar Pt microelectrode

arrays have already found many applications especially in biosensors).

Fig. 98 Non-specifically adsorbed DNA target on a hybridised DNA chip after hybridisation with solution lacking SDS (left). The same chip, after a short 2 M NaOH treatment (right).

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Using specially designed spacer and alignment elements, the microelectrodes and DNA

spots could be arranged on top of each other and kept at proper working distance. The

difference in the response of an individual Pt microelectrode before and after exposure to a

sample would evaluate the status of the opposite capture probe and indicate hybridisation

in case the complementary target had been present in the sample solution.

Label-free, instrument-free detection of DNA hybridisation

The preparation of the DNA chips for hybridisation and SECM measurements required

rinsing-steps for removing unwanted compounds off the chip surface. For instance, saline

buffers were used before marking the DNA chip. During those operations, it was noticed

that salt deposits are formed on the surface of the chip exactly in the positions of the DNA

spots. Could such phenomena be used for specific imaging of immobilised ss- or ds-

DNAs? Theoretically, special circumstances may lead to crystalline deposits of which

dimensions are correlated with the electrical charge of the polyanions because as many

deprotonated phosphate groups are present at a spot, as many counterions would be

necessary to compensate their electrical charge. The preliminary experiments showed that

the amount of salts trapped at DNA-modified areas is proportional to the concentration of

the saline buffer (Figure 100) and that high density spots grow bigger deposits as small

ones, to a certain extent (Figure 101). Hybridisation could not be yet detected. Apparently,

the relation between the concentration of DNA probes and size of the salt deposit was not

as precise as would be needed for detection of DNA hybridisation. However, preliminary

results in “ imaging” DNA chips by means of the appearance of salt deposits are

Fig. 99 Miniaturised clamping device for reading out DNA hybridisation from a multielectrode array.

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encouraging because the differences between DNA probes of various surface densities are

clearly noticeable within the salt patterns (Figure 100). Future work has to be directed

towards finding the appropriate conditions (substances, solvents) for inducing specific

crystal/deposit formation around DNA spots.

Fig. 100 First step to a label-free instrument-free detection of hybridisation? The concentration of the rinsing buffer determines the thickens of the salt deposit at the ss-DNA spots! The yellow rectangular is pointing out a printing error.

Fig. 101 Another suggestive example of dependence between the size of the salt deposit and concentration of ss-DNA probes for two 20-mers with different sequence (F12 and O8).

Micropatterning and microelectrochemical characterisation of biological recognition elements

127

5.4 Notes


1. The term “holoenzyme” denotes an apoenzyme linked to its corresponding cofactor.

2. The six major classes of enzymes according to EC:

1. EC 1 Oxidoreductases

To this class belong all enzymes catalysing oxido-reductions. The common name

is “dehydrogenase” but “oxidase” is used when oxygen is an electron acceptor.

2. EC 2 Transferases

Are enzymes transferring a group from one compound (generally regarded as

donor) to another (acceptor).

3. EC 3 Hydrolases

These enzymes catalyse the hydrolysis of various bonds.

4. EC 4 Lyases

These enzymes cleave C-C, C-O, C-N and other bonds by other means than by

hydrolysis or oxidation.

5. EC 5 Isomerases

They catalyse changes within one molecule.

6. EC 6 Ligases

Ligases are enzymes that catalyse the joining of two molecules with concomitant

hydrolysis of the diphosphate bond in ATP or a similar triphosphate.

3. Optimal working temperature of an enzyme is to be associated with the conditions the

source of enzyme is living under. Aspergillus niger, the fungus used to produce citric acid

and gluconic acid in industry is a accessible source of glucose oxidase. The optimal growth

temperature of this specific organism is 35-37 °C so that any enzyme obtained from this

fungus best works at 35-37 °C. Special application in biochemistry, one great example

being Polymerase Chain Reactiona (PCR) would not become possible without enzymes

that normally function at elevated temperatures. DNA-polymerase that is isolated from

Thermus aquaticus, a bacteria living in hot springs, needs a hot medium (about 72 °C) to

work properly. Hence, its DNA-polymerase is suitable to using in PCR for mass-copying of

DNA excluding cloning techniques.

Directed growth of living cells

4. Laminin is a major component of basement membranes of living cells. It has numerous

biological activities including promotion of cell adhesion, migration, growth, and

differentiation, including neurite outgrowth. Owing these functions it is used as a thin

coating on tissue/cells-culture surfaces or as a soluble additive to culture medium. Laminin

a Kary B. Mullis (1944, USA) American scientist that received the Nobel Prize for „his invention of the polymerase chain reaction (PCR) method” in 1993. The discovery was first presented in 1985.


128

has been shown in culture to stimulate neurite outgrowth, promote cell attachment,

chemotaxis, and cell differentiation.

DNA microstructures

5. Complementary DNA is synthesized in laboratory from a messenger RNA template (mRNA

– messenger RNA). The cDNA are used as probes in DNA microarrays. To understand

better why this is important for the hybridisation experiments, let us follow the steps of a

virtual experiment for the study of a comparative gene expression.

i. choosing the cell population: two different kind of cells of an organism are

selected;

ii. extraction of mRNA and the reverse transcription: genes which code for

protein are transcribed into messenger RNA’s in the cell nucleus. After

being released in the cytoplasm, the mRNA is translated into proteins by

ribosome. Although, this RNA could be used for grafting it onto a

microarray as probes, this is not practical. mRNA is prone to being

destroyed especially by RNA-digesting enzymes that normally are

everywhere where our fingers touched a surface. To prevent the loss of

mRNA, it is preferred to reverse-transcribe the mRNA into more stable

DNA that is the complementary cDNA.

iii. fluorescent labelling of cDNA’s;

iv. hybridisation at the surface of the DNA microarray;

v. scanning the hybridised microarray.

To obtain cDNAs libraries, vital for the mass production of DNA microarrays, the cDNA is

cloned into a plasmid (an extra-chromosomal circular DNA molecules, distinct from the

normal bacterial genome) and then transferred in Escherichia coli where the plasmid is

replicated many times. At last, the cDNA is extracted and purified from the bacterial

content.

6. A preferred method for deposition of very small (1-2 nanolitre) quantities of DNA-probe

solution onto the slide involves the of use pins. The pins of the spotting machines are

made of stainless steel, chromium, titanium or other metals that have sufficient resistant

against corrosion and certain hardness to minimise the blunting of the sharp tips. These

may be "solid" pins, which dispense just once per sample collection action, or "split" pins,

which pick up much more sample at a time and are capable of a multi-dispensing mode of

operation. In either case, sample is transferred passively by means of surface tension as

the tip gently touches the slide surface. Electrical Discharge Machining (EDM) is normally

used to cut the fine slots in the tip of the pins.

7. Several scientists were at the same time in run for elucidating the structure of nucleic acids

in the early 1950s 241. Simultaneously, ongoing research programmes in the physical,

organic and biological chemistry were intersected in order to elucidate a mysterious

process: DNA replication. In these circumstances, teams of foremost scientist as L. Pauling

and R. B. Corey, Wilkins et all242, and R. E. Franklin and R. S. Gosling understood the DNA


129

molecule is a helix. Pauling build a wrong model of the double helix while R. Franklin did

not bother at all about the possible arrangements of the DNA constituents243,244 even

though she was very close to get it245. It was J. D. Watson and F. H. C. Crick, who

suggested a model explaining that: DNA has two anti-parallel and complementary strands

in a double-helix arrangement246. Furthermore, the novel DNA structure even suggested a

possible copying mechanism for the genetic material247. The two DNA chains are held

together by interactions between the nitrogenous bases on opposite strands. These

interactions are called base pairing. The famous DNA Base Pairing Rules, are :

i. adenine (A) and guanine (G) are purines and cytosine (C) and thymine (T)

are pyrimidines. Purines base pair with pyrimidines;

ii. A pairs with T and vice versa while C pairs with G and vice versa;

iii. there are 2H-bonds holding A and T together and 3H-bond for C and G;

iv. because of the additional hydrogen bond, C and G base pair is stronger as

A and T.

8. The relation between the frequency-shift of a quartz crystal microbalance and the change

in the mass of the film attached to the crystal is given the next equation:

∆×××−=∆

A

m

0

6103.2 νν

where ∆ν denotes the shift of the frequency corresponding to ∆m; ν0 is the resonant

frequency of the bare quartz surface; A is the surface of the exposed quartz crystal. For a

given working-frequency of 10-15 MHz, frequency shifts of 10-2 or even lower can be

detected. Consequently, a mass as small as 10-11 g is measurable by QCM248.

9. It is frequent that scientist are reporting extremely low detection limits of ds-DNA, namely

550 amol (J. J. Gooding, “Electrochemical DNA hybridisation biosensors”, Electroanalysis,

2002, 14, page of interest is 1152). I find this is a misused technical detail because such

limits were achieved only with very long DNA chains. For instance, in the above mentioned

case was used a 1497 bases DNA strand and if one takes into account that there are the

same number of reporters (guanines) on each strand, this is equivalent to 1497 of

individual and simple species. If such ideas are accepted, then one should agree the

human being is also able to see with naked eyes single molecules: a big mono-crystal of

diamond is a single molecule, or?

10. The edge (doughnut) effect is a ring-like deposit (RLD)249 which results from a capillary

flow of the bulk of a drying droplet outwards the edge that can carry any dispersed material

to the margin. Example: a coffee drop falls on the table and by splashing and drying out

creates a brownish disk. After the complete drying of the drop, a ring is observed at the

margins. Note: this ring will not be noticed if the coffee contains sugar. Sugar makes the

liquid viscous and the outwards flow is slowed down. Consequently, the ring is less visible.

For a funny explanation please read the book of Robert L. Wolke “Was Einstein seinem

Friseur erzählte”, Piper Verlag GmbH, München, 2001, 255-257.


130

11. At some point in elaborating this qualitative model of DNA-DNA interaction, I sought to

establish an equation between R1 and the optimal separation distance ε for a given set of

DNAs (target and probes) and negative charged redox mediator. Despite the fact that

describing the overall electrostatic interaction between involved parts is not a challenge,

the problem could not be solved because of turning an theoretical equation valid in vacuum

condition to complex environment where the electrical permittivity of solution differs in bulk

and around the polyelectrolyte strands, or where the DNA’s charges are distributed not

along a straight -road but coiled as function of the ionic strength of the solution are only two

examples of significant obstacles not easy surmountable. Nevertheless, the problem is

solvable, and finding the solution could be of great deal of help for mass production of DNA

microarrays, when knowing the optimal surface density could save expensive probes! It

may worth finding the ε distance only if the repelling-like detection techniques will raise

enough attention and have a market among the already commercialised DNA detection

kits.

Experimental

131

6. Experimental Chemicals and Materials for the preparation of microelectrodes

Solutions were prepared with triply-distilled water. Chemicals were purchased from Sigma

Aldrich (Deisenhofen, Germany). Tollens’ reagent for the electroless deposition of silver

consisted of a mixture of 5% AgNO3, 10% NaOH, 28-30% NH4OH, and 10% Glucose.

The Ag electroplating bath consisted of 40 g/L Ag2SO4, 380 g/L Na2S2O3 ⋅5H2O, 20 g/L

Na2B4O7 ⋅10H2O (borax) and 20 g/L Na2SO4 ⋅10H2O. Cyclic voltammetry and SECM were

performed in solutions containing 5 mM [Ru(NH3)6]Cl3 and either 0.1 M KH2P04/ K2HP04

buffer or 0.1 M KCl as supporting electrolyte. Paraffin (mineral) oil was from Sigma (EC

number 232-455-8).

Pt wire was from Goodfellow/Germany and glass capillaries from glass capillaries

Hilgenberg/Germany. Alumina polishing suspension, 3 µm, 1 µm and 0.3 µm was from

Leco Co., Lakeview Ave., USA. Zinc powder was from Fisher Chemicals and soldering

wire from Conrad/Germany. Polishing cloth (red) was from Heraeus Kulzer, Wehrheim,

Germany.

Chemicals and Materials for the preparation of enzyme microstructures

Glucose oxidase, type X-S from Aspergilus niger activity of 119,000 units/g solid, was

purchased from Sigma Chemical Co., St. Louis, USA. The polymer dispersion Vinnapas®

EP16 W was kindly provided by Wacker Polymer Systems GmbH & Co. KG, Burghausen,

Germany. Solutions of glucose oxidase and Vinnapas® EP16 W were prepared with triply-

distilled water. Filter units FP 30/5,0 CN (black rim) were purchased from Schleicher &

Schuell, Dassel, Germany.

Chemicals and Materials used in the experiments regarding the defined

adhesion/growth of living cells

Laminin from the mouse Engelbreth–Holm–Swarm sarcoma was from Boehringer

Mannheim, Germany. DiI was applied as fluorescent dye (1,1-dioctadecyl-3,3,3,3-tetra-

methylindocarbocyanine perchlorate) was from Molecular Probes Europe, Leiden, The

Netherlands.

Four different substrates were used for the experiments: glass cover slips, pure silicon

samples coated with a 130 nm gold layer (Chemistry Department at the Ruhr University,

Bochum), pure silicon samples provided by the Institute of Thin Film and Ion Technology,

Experimental

132

Research Centre Jülich, Germany, and glassy carbon samples (Hochtemperatur-Werkstoffe

GmbH, Thierhaupten, Germany).

Neurones were prepared from embryonic chicken forebrain under sterile conditions.

Chemicals and Materials for the preparation of DNA microstructures

KH2PO4, K2HPO4⋅3H2O, NaCl, K3[Fe(CN)6] and [Ru(NH3)6]Cl3 were from Sigma,

Deisenhofen, Germany and SDS (sodium dodecylsulphate) from Merck, Darmstadt,

Germany.

FRIZ Biochem, Munich, Germany provided oligonucleotide microstructures (DNA chips)

along with the complementary targets.

Instrumentation

Basic components of the SECM set-up

Experiments were carried out with a home-built SECM in a one-compartment cell in two-

electrode configuration. Faraday cage was home build and the low-noise VA10 amplifier

from npi electronics GmbH, Tamm, Germany. Potentials were measured against the

chemically deposited Ag coating or an independent, miniaturised Ag/AgCl/3M KCl

reference electrode. Stepper motors with a resolution of 0.625 µm per half step were from

Owis, Staufen, Germany. The personal computer used software programmed in Microsoft

Visual Basic 3.0 (Microsoft, Unterschleißheim/Germany). High current power supply (32

V and 24 A) was from Statron Elektronik, Fürstenwalde, Germany.

Microdispenser set-up

The microdispenser was operated together with a wave generator from Hewlett Packard,

type 33120A. Stepper motors for precice movements of the substrate had a resolution of

0.625 µm per half step and were from Owis, Staufen, Germany. The personal computer for

controlling the microdispensing procedure used software programmed in Microsoft Visual

Basic 3.0 (Microsoft, Unterschleißheim/Germany).

Microspotter for DNA microarray fabrication

For spotting a microarrayer from Cartesian Technologies, Inc., USA equipped with a

printhead (ChipMaker™) and microspotting pins (120 µm diameter) from Telechem

International, Inc. was used. Electrochemical measurements were carried out with a low-

Experimental

133

noise VA10 amplifier from npi electronics GmbH, Tamm, Germany. Piezo microdispenser

with 700 nl droplet size, was from GeSIM mbh, Germany.

Other instrumentation

A fluorescence microscope (Axiophot, Zeiss, Germany) was used for the observation of

stained neuronal cells.

Experiment protocols

Preparation of glass Pt-disk microelectrodes

Borosilicate glass capillaries (O.D. 1.5 mm, I.D. 0.75 mm, L 100 mm) were tapered down

to form a closed sharp tip, process carried out with a classical capillary puller. The open

end of the capillary is connected to a water vacuum pump all along the heating process.

This reduces the risk of getting air bubbles between the Pt wire and glass insulation, and

additionally, shortens the embedding time of Pt in the molten glass tip. The particular

coiled filament in this case used a current of 17 A to heat glass tube.

Platinum wires with diameters of 10 µm were tightly sealed into these tapered ends of

pulled capillaries. Smooth Pt micro disks were exposed by carefully polishing the tip at 90°

on emery paper (grade 320 to 2000) and then on a polishing cloth wetted with alumina

suspension (particle sizes: 3 µm, 1 µm, and 0.3 µm). The back connection between the Pt

wire and copper lead was established by filling the capillaries with Zn powder (10 µm

particles) unless the protruding Pt is fully covered and then with crushed tin solder, which

after placement was melted by careful heating with the coiled filament at 9 A. The Cu wire

was fastened at the upper part of the capillary with shrinking tube I order to avoid breaking

the contact between Cu and Pt. Note: in the case of Pt wires with diameter bigger as 50

µm, the Zn powder is non necessary but only the solder.

Silver electroplating

The Ag electroplating bath consisted of 40 g/L Ag2SO4, 380 g/L Na2S2O3 · 5H2O, 20 g/L

Na2B4O7 · 10H2O (borax) and 20 g/L Na2SO4 · 10H2O in tri-distilled water. The compounds

are not all easily dissolved in water (i.e. AgSO4) and for this reason the solution has to be

kept in the ultrasonic bath for few minutes. One should know that the colour of the solution

Experimental

134

is dark green or even black even if all substances used to prepare the mixture are

colourless. So, do not waste it!

At room temperature, a current density of 0.5-0.6 A/dm2 is the ideal. A lower current will

take too long time for preparing a reasonable thick layer of silver whereas a higher current

leads to formation of Ag dendrites. The appearance of dendrites is a sign that the

electrochemical reaction is kinetically limited and thus if, however, high current are

allowed to flow through he electrolysis cell, the solution must be stirred to enhance the

mass transport towards the cathode.


Neurons preparation

For the preparation of prepare the neurons, fertilised chicken eggs were incubated at 37.7 0C and 60% humidity for 8–10 days. Dissociated cells were prepared from the forebrain of

the chicken embryo. After decapitation, the brain was dissociated by incubation in 1 mg/ml

trypsin in HBSS for 10 min. at 37 0C. In order to reduce DNA-mediated aggregation of

cells, 100 µl solution of DNAse were added. The cell suspension was washed by

centrifugation at 550 × g for 6 min in HBSS containing trypsin inhibitor solution (1 mg/ml)

and subsequently incubated with trypsin inhibitor (3 min. at 37 0C). After a second

washing step by centrifugation, the cells were re-suspended in the so-called S4-medium,

and cell suspensions were given onto the substrates. The serum-free S4-medium was

prepared starting from Dulbecco’s modified Eagle’s medium (Gibco BRL Life

Technologies, Scotland) according to the procedure described literature1 with the

modification that a higher glucose concentration was applied. After 2 days of cultivation

in the S4 medium in an incubator at 37 0C and with 5% CO2, the cultured neurones were

washed shortly with phosphate buffer saline (PBS), pH 7.4, and fixed with a solution of

4% paraformaldehyde in PBS overnight at 4 0C. The samples were washed again with

PBS. Cells on glass cover slips could be inspected with usual phase contrast microscopy.

The cells located on opaque substrates were stained for 5 s in a solution of DiI in 70%

ethanol. The lipophilic dye is incorporated very quickly into the membranes of cells and

neurites, and cells and neurites can be visualised with a fluorescence microscope2.

1 L. K. Needham, G. I. Tennekoon, G. M. McKhann, “Selective growth of rat Schwann cells in neuron- and serum-free primary culture”, J. Neurosci., 1987, 7, 1-9. 2 P. Godement, J. Vanselow, S. Thanos, F. Bonhoeffer, “A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue”, Development, 1987, 101, 697-713.

Experimental

135

Immunochemistry

For immunochemistry, neuronal cultures were washed and fixed as described above. Then,

they were washed with PBS three times in order to remove the paraformaldehyde and

blocked with 1% bovine serum albumin (BSA) in PBS for 1 h. Antibody against

neurofilament (Sigma) was added in a dilution of 1:400 in 1% BSA in PBS overnight at

4 0C. The samples were washed again three times with PBS, and a secondary antibody

labelled with Cy2® (Jackson Immuno Research Laboratories, West Grove, PA) was added

in a dilution of 1:200 in 1% BSA in PBS for 1 h. After three washing steps with PBS, the

cell cultures were inspected with a fluorescence microscope. The same procedure was

applied for the immunochemical staining of the polymer lines on glass cover slips. In order

to check whether laminin being entrapped in the polymer could be recognised, an anti-

laminin antibody (Sigma) was used for the detection of laminin.

DNA microstructures

Phosphate buffers

The 0.1 M phosphate buffer was prepared from 0.05M KH2PO4 and 0.05 K2HPO4 · 3H2O in

one litter of tri-distilled water (pH 6.7).

Measuring buffer

With the o.1 M phosphate buffer a solution of 1M NaCl was prepared for hybridisation

purposes (pH 6.5). SECM measurements were performed in a phosphate buffer containing

3 M NaCl and 5 mM ferricyanide (pH 5.7).

Dehybridisation solution

The dehybridisation solution contained only 2M NaOH in NaOH. This solution should be

used only fresh and filtrated through at least 5 µm filter unit prior to use.

Conclusions

136

7. Conclusions The aims of this PhD thesis, some of them being defined from the very beginning, others

coming up during the course of this work, are strongly tuned to lab experiments but have

also had to do with development of theoretical aspects of microstructures. Investigations of

microstructures of various components of living cells, which are key elements of the

chemical/biochemical sensor technologies, and their possible applications, ask first of all,

for manufacturing of the microstructures by means of specific tools, a piezo-

microdispenser in this particular case.

It was shown that with the ink-jet printing technique, complex geometries are

microstructured in a straightforward manner. When it was planned to work on the directed

growth of neurons, it turned out that even patterning laminin is a challenge due to its

structural fragility. Although, the experiments were performed in the laboratory of Eye

Hospital, Münster, that was not that familiar to me, it was of great help to have a modular

spotting set-up that could be adapted to the special conditions (clean rooms, flow benches)

of that laboratory. Special care had been taken to avoid laminin getting damaged and to

minimise contaminations of the instrumentation and chips and to preserve the vitality of

neurons. The output was very encouraging giving that the cells clearly followed our

microstructures. These results open a new route to, for instance, development of neuron

networks useful in studying cell growth or for coupling microelectronics to living

organisms. The microdispenser has found another interesting application in the field of

biosensor microarrays. Multi-analyte sensors will offer a great deal of help in monitoring

metabolites or pollutants, and thus, we sought to add study the conditions in which an

enzyme base microarray could be used to monitor glucose concentration. This study

involved the SECM, owing its abilities to probe electrochemically the course of, in this

case enzymatic reactions. It turned out that calibration curves for glucose could be obtained

in a broad domain of concentrations by varying the content of enzyme within the

microstructures.

The tremendous advance of microarrays, especially of those based on nucleic acids, is

mostly directed towards the invention of a sensitive, reliable, simple, and cost-effective,

miniaturised device for the detection of DNA hybridisation on DNA chips. Along with

long-ago established fluorescence detection of hybridisation, the electrochemical methods,

and especially the label-free approaches are given much attention these days because they

fulfil all the requirements for developing individual medical point-of-care devices. With

background knowledge in SECM, I sought to make use of one of the intrinsic property of

Conclusions

137

DNA, namely the negative electrical charges of the DNA backbones, to establish a method

for a label-free electrochemical procedure to visualise surface confined DNAs strand and

to detect their hybridisation. Investigating a number of DNA microarrays in the newly

introduced repelling mode of SECM, the main factors that have an essential impact on the

quality of this mode of DNA imaging were revealed. Moreover, it was noticed that in order

to improve the sensitivity of this technique, the superficial concentration of DNA probes

must have a special value as resulting from a qualitative model described in this work. Part

of the DNA chapter, dedicated to the detection of hybridisation explains the difficulties

that had been overcome before it could be proved that it is possible to distinguish the

double stranded DNA signal from those belonging to the single stranded DNA.

Microelectrochemistry in small volumes is needed when the amount of available analyte is

reduced or for combinatorial electrochemistry in micro- or nano-titre plates. Two or three

electrodes can be easily fit in a single vial of microtitre plate due to the novel electrode

assembly developed in the framework of this PhD. Its fabrication, possible applications

and advantages as well as disadvantages were described. A great advantage of this coaxial

Pt working/Ag reference electrode comes from its easy manufacture with common

instruments. Thus it can be prepared in-house in laboratories lacking sophisticated

instruments for coating by sputtering or vapour deposition of metals. Additionally, a

miniaturised Ag/AgCl x M reference electrode preparation was presented, and as the above

mentioned electrode system, this can be produced with available material in any

laboratory.

In conclusion, by shortly mentioning some of the significant results of this PhD it was

sought to outline the alternative methods or improvements that could be of use in analytical

chemistry, developed at some points in the course of my PhD time.

Personal note

Thanks to the variety of fields this PhD covered over the last few years, I had the

opportunity to learn/understand concepts, theories related to that part of science regarded

as electrochemistry, and to gain practical experience in manipulating specific

instrumentation. I have found a proper atmosphere, in this group, for developing my own

ideas and checking whether there are valid or not. However, the different research topics I

was involved in matched nicely with each other and thus allowed me to unify the results

within a broad title such as “Micropatterning and microelectrochemical characterisation of

biological recognition elements”.

Conclusions

138

There are several items I would like to continue in one way or another. For instance, I

believe that the micropatterning of biomolecules can still be further improved and

performed with common instrument in a reliable and reproducible way; the detection of

DNA hybridisation should end up with a miniaturised and trustworthy device based either

on electrostatic repulsion between an anion and the deprotonated phosphate groups, or

based on the DNA-induced salt crystallisation (DISC) at the DNA spots that theoretically

could differentiate ss- from ds-DNA. Manufacturing of integrated electrode assemblies is a

funny and relaxing habit that could deliver more useful SECM tips or just simple

miniaturised electrodes.

I hope I will find a chance to go on with these and other scientific plans!

Acknowledgment

139

8. Acknowledgment I would like to thank all those who have been generous with their time and helped me by

any mean over the last three years. I am in particular thankful to:

My esteemed supervisor Prof. Dr. Wolfgang Schuhmann

- for his guidance along electrochemistry, constant encouragement, patience and

understanding;

Dr. Albert Schulte

- for the sleepless nights in the lab and his helping from the very beginning and all

along my PhD time; he also guided me in writing this thesis.

Dr. Thomas Erichsen

- for his useful software and daily computer troubleshooting;

Dr. Gerhard Hartwich

- for providing DNA chips and knowledge about DNA microarrays.

Dr. Peter Heiduschka

- for the successful collaboration on neuron growth on micropatterned surfaces.

Kalathur Ravi

- for making the English readable and for the pleasant teamwork in the lab.

Dr. Rolf Neuser

- for the great SEM images and his friendliness.

Gerhard Hartwich and Wolfgang Schuhmann. Düsseldorf, October 2002.

References

140

9. References


1. Y. Xia, G. M. Whitesides, “Soft-lithography”, Angew. Chem. Int. Ed., 1999, 37, 550-575.

2. Y. Xia, J. A. Rogers, K. E. Paul, G. M. Whitesides, “Unconventional methods for fabricating

and patterning nanostructures”, Chem. Rev., 1999, 99, 1823-1848.

3. Y. Xia, E. Kim, X. M. Zhao, J. A. Rogers, M. Prentiss, G. M. Whitesides, “Complex optical

surfaces formed by replica moulding against elastomeric masters”, Science, 1996, 273,

347-349.

4. J. Feng, C. Gao, J. Shen, „Micropatterning biomacromolecules on aldehyde-enriched

polyester surfaces by a microtransfer technique“, Chem. Mater., 2004, 16, 1319-1322.

5. D.-Y. Khang, H. H. Lee, “Sub-100 nm patterning with an amorphous fluoropolymer mold”,

Langmuir, 2004, 20, 2445-2448.

6. http://www.fzk.de/stellent/groups/imt/

7. L. M. Demers, D. S. Ginger, S.-J. Park, Z. Li, S.-W. Chung, C. A. Mirkin, „Direct patterning

of modified oligonucleotides on metals and insulators by dip-pen nanolithography”,

Science, 2002, 296, 1836-1838.

8. H. Zhang, Z. Li, C. A. Mirkin, „Dip-pen nanolithography-based methodology for preparing

arrays of nanostructures functionalized with oligonucleotides“, Adv. Mater., 2002, 14, 1472-

1474.

9. H. Zhang, C. A. Mirkin, „DPN-generated nanostructures made of gold, silver, and

palladium”, Science, 2004, 16, 1480-1484.

10. http://www.imaging.org/resources/leinkjet/part1.cfm to part5.cfm.

11. T. Laurell, L. Wallman, J. Nilsson, „Design and development of a silicon microfabricated

flow-through dispenser for on-line picolitre sample handling“, J. Micromech. Microeng.,

1999, 9, 369-376.

12. S. Neugebauer, S. R. Evans, Z. P. Aguilar, M. Mosbach, I. Fritsch, W. Schuhmann,

„Analysis in ultrasmall volumes: microdispensing of picoliter droplets and analysis without

protection from evaporation“, Anal. Chem., 2004, 76, 458-463.


13. “The physical basis of biochemistry – the foundation of molecular biophysics”, by Peter R.

Bergheton, Springer Verlag, New York, 1998.

14. http://www.nobel.se/physics/educational/microscopes/

15. http://www.nobel.se/physics/laureates/1986/binnig-lecture.pdf, “Scanning tunnelling micro-

scopy from birth to adolescence”.

References

141

16. G. Attard, C. Barnes, „Surfaces“, Oxford University Press, Great Clarendon Street, Oxford,

OX2 6DP, 2003, pp. 57-62.

17. “Chronologie Chemie – Entdecker und Entdeckungen” by Sieghard Neufeld, Wiley-VCH

Verlag GmbH & Co. KGaA, Weinheim, 2003, 3. Auflage, pp. 193, 215-216.

18. http://homer.hsr.ornl.gov/cbps/AFMImaging.htm

19. H. G. Hansma, “Varieties of imaging with scanning probe microscopes”, Proc. Natl. Acad.

Sci. USA , 1999, 96, 14678-14680.

20. R. C. Engstrom, M. Weber, D. J. Wunder, R. Burgess, S. Winquist, “Measurements within

the diffusion layer using a microelectrode probe”, Anal. Chem., 1986, 58, 844-848.

21. A. J. Bard, F.-R. F. Fan, J. Kwak, O. Lev, “Scanning electrochemical microscopy.

Introduction and principles”, Anal. Chem., 1989, 61, 132-138.

22. J. Heinze, “Ultramicroelectrodes in electrochemistry”, Angew. Chem. Int. Ed., 1993, 32,

1268-1288.

23. “Scanning electrochemical microscopy” edited by A. J. Bard and M. V. Mirkin, Marcel

Dekker Inc., New-York, 2001.

24. G. Nagy, L. Nagy, “Scanning electrochemical microscopy: a new way of making

electrochemical experiments” Fresenius J. Anal. Chem., 2000, 366, 735 – 744.

25. A. J. Bard, F. R. F. Fan, J. Kwak, O. Lev, “Scanning electrochemical microscopy.

Introduction and principles”, Anal. Chem., 1989, 61, 132-138.

26. J. Kwak, A. J. Bard, “Scanning electrochemical microscopy. Theory of the feedback mode”,

Anal. Chem., 1989, 61, 1221-1227.

27. M. V. Mirkin, F. R. F. Fan, A. J. Bard, “Scanning electrochemical microscopy. 13.

Evaluation of tip shapes of nanometer size microelectrodes”, J. Electroanal. Chem., 1992,

328, 47-62.

28. C. Kranz, G. Wittstock, H. Wohlschläger, W. Schuhmann, “Imaging of microstructured

biochemically active surfaces by means of scanning electrochemical microscopy”,

Electrochim. Acta, 1997, 42, 3105-3111.

29. R. D. Martin, P. R. Unwin, “Theory and experiment for the substrate generation/tip

collection mode of the scanning electrochemical microscope: application as an approach

foe measuring the diffusion coefficient ratio of a redox couple”, Anal. Chem., 1998, 70, 276-

284.

30. T. Yasukawa, T. Kaya, T. Matsue, "Characterization and imaging of single cells with

scanning electrochemical microscopy", Electroanalysis, 2000, 12, 653-659.

31. D. O. Wipf, "Initiation and study of localized corrosion by scanning electrochemical

microscopy", Colloid. Surf. A, 1994, 93, 251-261.

References

142

32. N. Casillas, S. J. Charlebois, W. H. Smyrl, H. S. White, "Scanning electrochemical

microscopy of precursor sites for pitting corrosion on titanium", J. Electrochem. Soc.,

1993, 140, L142-L145.

33. Y. Y. Zhu, D. E. Williams, "Scanning electrochemical microscopic observation of a

precursor state to pitting corrosion of stainless-steel", J. Electrochem. Soc., 1997, 144,

L43-L45.

34. A. Schulte, S. Belger, W. Schuhmann, „Corrosion of NiTi shape-memory alloys:

Visualization by means of potentiometric “constant-distance” scanning electrochemical

microscopy“, Material Science Forum, 2002, 394-3, 145-148.

35. A. J. Bard, G. Denuault, C.M. Lee, D. Mandler, D.O. Wipf, “Scanning electrochemical

microscopy - A new technique for the characterization and modification of surfaces”, Acc.

Chem. Res., 1990, 23, 357-363.

36. D. Mandler, A. J. Bard, "A new approach to the high resolution electrodeposition of metals

via the feedback mode of the scanning electrochemical microscope", J. Electrochem. Soc.,

1990, 137, 1079-1086.

37. E. Ammann, D. Mandler, "Local deposition of gold on silicon by the scanning

electrochemical microscope", J. Electrochem. Soc., 2001, 148, C533-C539.

38. K. Borgwarth, N. Rohde, C. Ricken, M. L. Hallensleben, D. Mandler, J. Heinze, "Electroless

deposition of conducting polymers using the scanning electrochemical microscope",

Advan. Mater., 1999, 11, 1221-1226.

39. C. Kranz, H. E. Gaub, W. Schuhmann, "Polypyrrole towers grown with the scanning

electrochemical microscope", Adv. Mater., 1996, 8, 634-637.

40. J. F. Zhou, D. O. Wipf, "Deposition of conducting polyaniline patterns with the scanning

electrochemical microscope", J. Electrochem. Soc., 1997, 144, 1202-1207.

41. C. Marck, K. Borgwarth, J. Heinze, “Generation of polythiophene micropatterns by

scanning electrochemical microscopy”, Chem. Mater., 2001, 13, 747-752.

42. C. Marck, K. Borgwarth, J. Heinze, “Micropatterns of poly(4,4’-dimethoxy-2,2’-bithiophene)

generated by the scanning electrochemical microscope”, Adv. Mater., 2001, 13, 47-51.

43. G. Wittstock, R. Hesse, W. Schuhmann, "Patterned self-assembled alkanethiolate

monolayers on gold. Patterning and imaging by means of scanning electrochemical

microscopy", Electroanalysis, 1997, 9, 746-750.

44. G. Wittstock, W. Schuhmann, "Formation and imaging of microscopic enzymatically active

spots on an alkanethiolate-covered gold electrode by scanning electrochemical

microscopy", Anal. Chem., 1997, 69, 5059-5066.

45. S. Meltzer, D. Mandler, "Study of silicon etching in HBr solutions using a scanning

electrochemical microscope", J. Chem. Soc., Faraday Trans., 1995, 91, 1019-1024.

References

143

46. D. Mandler, A. J. Bard, "High resolution etching of semiconductors by the feedback mode

of the scanning electrochemical microscope", J. Electrochem. Soc., 1990, 137, 2468-2472.

47. D. Mandler, A. J. Bard, "Hole injection and etching studies of GaAs using the scanning

electrochemical microscope" Langmuir, 1990, 6, 489-1494.

48. D. Mandler, A. J. Bard, "Scanning electrochemical microscopy - the application of the

feedback mode for high resolution copper etching", J. Electrochem. Soc., 1989, 136, 3143-

3144.

49. W. B. Nowall, D. O. Wipf, W. G. Kuhr, "Localized avidin/biotin derivatization of glassy-

carbon electrodes using Secm", Anal. Chem., 1998, 70, 2601-2606.

50. I. Turyan, U. O. Krasovec, B. Orel, T. Saraidorov, R. Reisfeld, D. Mandler, "Writing-

reading-erasing on tungsten oxide films using the scanning electrochemical microscope",

Adv. Mater., 2000, 12, 330-333.

51. Y. H. Shao, M. V. Mirkin, G. Fish, S. Kokotov, D. Palanker, A. Lewis, "Nanometer-sized

electrochemical sensors", Anal. Chem., 1997, 69, 1627-1634.

52. M. Ludwig, C. Kranz, W. Schuhmann, H.E. Gaub, „Topography feedback mechanism for

the scanning electrochemical microscope based on hydrodynamic-forces between tip and

sample“, Rev. Sci. Instr., 1995, 66, 2857-2860.

53. A. Hengstenberg, C. Kranz, W. Schuhmann, “Facilitated tip-positioning and applications of

non-electrode tips in scanning electrochemical microscopy using a shear force based

constant-distance mode”, Chem. Eur. J., 2000, 6, 1547-1554.

54. B. Ballesteros Katemann, A. Schulte, W. Schuhmann, „Constant-distance mode scanning

electrochemical microscopy (SECM)-part I: Adaptation of a non-optical shear-force-based

positioning mode for SECM tips”, Chem. Eur. J., 2003, 9, 2025-2033.

55. C. M. Lee, C. J. Miller, A. J. Bard, "Scanning electrochemical microscopy: Preparation of

submicrometer electrodes", Anal. Chem., 1991, 63, 78-83.

56. B. Ballesteros Katemann, W. Schuhmann. „Fabrication and characterization of needle-type

Pt-disk nanoelectrodes“, Electroanalysis, 2002, 14, 22-28.

57. Y. Lee, S. Amemiya, A. J. Bard, “Scanning electrochemical microscopy. 41. Theory and

characterization of ring electrodes”, Anal. Chem., 2001, 73, 2261-2267.

58. P. Liljeroth, C. Johans, C. J. Slevin, B. M.Quinn, K. Kontturi, “Micro ring –disk electrode

probes for scanning electrochemical microscopy”, Electrochem. Commun., 2002, 4, 67 –

71.

59. Y. Selzer, D. Mandler, "Scanning electrochemical microscopy. Theory of the feedback

mode for hemispherical ultramicroelectrodes: steady-state and transient behaviour", Anal.

Chem., 2000, 72, 2383-2390.

References

144

60. J. Mauzeroll, E. A. Hueske, A. J. Bard, „Scanning electrochemical microscopy. 48. Hg/Pt

hemispherical ultramicroelectrodes: fabrication and characterization”, Anal. Chem., 2003,

75, 3880-3889.

61. D. O. Wipf, F. Y. Ge, T. W. Spaine, J. E. Bauer, "Microscopic measurement of pH with

iridium oxide microelectrodes", Anal. Chem., 2000, 72, 4921-4927.

62. C. Wei, A. J. Bard, I. Kapui, G. Nagy, K. Tóth, "Scanning electrochemical microscopy.32.

Gallium ultramicroelectrodes and their application in ion-selective probes", Anal. Chem.,

1996, 68, 2651-2655.

63. N. J. Gray, P. R. Unwin, "Simple procedure for the fabrication of silver/silver chloride

potentiometric electrodes with micrometer and smaller dimensions: application to scanning

electrochemical microscopy", Analyst, 2000, 125, 889-893.

64. B. R. Horrocks, M. V. Mirkin, D. T. Pierce, A. J. Bard, G. Nagy, K. Tóth, "Scanning

electrochemical microscopy .19. Ion-selective potentiometric microscopy", Anal. Chem.,

1993, 65, 1213-1224.

65. A. Schulte, R. H. Chow, “A simple method for insulating carbon fiber microelectrodes using

anodic electrophoretic deposition of paint”, Anal. Chem., 1996, 68, 3054-3058.

66. A. Schulte, R. H. Chow, „Cylindrically etched carbon-fiber microelectrodes for low-noise

amperometric recording of cellular secretion”, Anal. Chem., 1998, 70, 985-990.

67. A. Hengstenberg, A. Blochl, I. D. Dietzel, W. Schuhmann, "Spatially-resolved detection of

neurotransmitter secretion from individual cells by means of scanning electrochemical

microscopy", Angew. Chem. Int. Ed., 2001, 40, 905-908.

68. Y. Lee, Z. Ding, A. J. Bard, “Combined scanning electrochemical/optical microscopy with

shear force and current feedback”, Anal. Chem., 2002, 74, 3634-3643.


69. B. D. Pendley, H. D. Abruna, “Construction of submicrometer voltammetric electrodes”,

Anal. Chem., 1990, 62, 782-784.

70. K. T. Kawagoe, J. A. Jankowski, R. M. Wightman, “Etched carbon-fiber electrodes as

amperometric detectors of catecholamine secretion from isolated biological cells”, Anal.

Chem., 1991, 63, 1589-1594.

71. K. Potje Kamloth, J. Janata, M. Josowicz, “Electrochemically prepared insulations of

carbon fibers microelectrodes”, Ber. Bunsen-Ges. Phys. Chem., 1990, 93, 1480-1485.

72. T. G. Strein, A. G. Ewing, “Characterization of submicron-sized carbon electrodes insulated

with a phenol-allylphenol copolymer”, Anal. Chem., 1992, 64, 1368-1373.

73. G. Zhao, G. M. Giolando, J. R. Kirchhoff, “Chemical vapor deposition fabrication and

characterization of silica-coated carbon fiber ultramicroelectrodes ”, Anal. Chem., 1995, 67,

2592-2598.

References

145

74. T. W. Spaine, J. E. Baur, „A positionable Microcell for electrochemistry and scanning

electrochemical microscopy in subnanoliter volumes“, Anal. Chem., 2001, 73, 930-938.

75. Y. Kitamura, T. Uzawa, K. Oka, Y. Komai, H. Ogawa, N. Takizawa, H. Kobayahi, K.

Tanishita, “Microcoaxial electrode for in vivo nitric oxide measurement”, Anal. Chem., 2000,

72, 2957-2962.

76. P. Liljeroth, C. Johans, C. J. Slevin, B. M. Quinn, K. Kontturi, “Micro ring –disk electrode

probes for scanning electrochemical microscopy”, Electrochem. Comm., 2002, 4, 67-71.

77. Y. Xia, N. Venkateswaran, D. Qin, J. Tien, G. M. Whitesides, „Use of electroless silver as

the substrate in microcontact printing of alkanethiols and ist application in microfabrication“,

Langmuir, 1998, 14, 363-371.

78. F. Sachs, R. McGarrigle, “An almost completely shielded microelectrode”, J. Neurosci.

Methods, 1980, 3, 151-157.

79. G. Kottra, E. Fromter, “A simple method for constructing shielded, low-capacitance glass

microelectrodes”, Pflugers Archiv. Europ. J. Physiol., 1982, 395/2, 156-158.

80. X. Zhang, Y. Kislyak, J. Lin, A. Dickson, L. Cardosa, M. Broderick, H. Fein, “Nanometer

size electrode for nitric oxide and S-nitrosothiols measurement”, Electrochem. Comm.,

2002, 4, 11-16.

81. W. Pernkopf, M. Sagl, G. Fafilek, P. Lindhart, J. O. Besenhard, H. Kronberger, G. E. Nauer,

„Development of a microelectrode for high resolution investigations of redox potentials and

impedance spectroscopy in corrosion studies“, Electrochem. Soc. 2002, 2002, 13, 558-

565.

82. M. M. Lohrengel, A. Moehring, M. Pilaski, „Electrochemical surface analysis with the

scanning droplet cell“, Fresenius J. Anal.Chem., 2000, 367, 334-339.

83. A. C. Clark, P. Beyer-Hietpas, A. G. Ewing, “Electrochemical analysis in picoliter

microvials”, Anal. Chem., 1997, 69, 259-263.

84. J. C. Ball, D. L. Scott, J. K. Lumpp, S. Daunert, J. Wang, L. G. Bachas, “Electrochemistry in

nanovials fabricated by combining screen printing and laser micromachining”, Anal. Chem.,

2000, 72, 497-501.

85. C. D. T. Bratten, P. H. Cobbold, J. M. Cooper, “Micromachining sensors for electrochemical

measurement in subnanoliter volumes”, Anal. Chem., 1997, 69, 253-258.

Micropatterning and microelectrochemical characterisation of biological

recognition elements


86. Berichte des Deutschen chemischer Gesellschaft, 1897, 30, 117.

References

146

87. “Medical Dictionary”, Published by Geddes & Grosset, David Dale House, New Lanark ML

119DJ, Scotland, 2001.

88. Irene Strube, Rüdiger Stolz, Horst Remane “Geschichte der Chemie” VEB, Berlin 1988, p.

170

89. http://www.chem.qmul.ac.uk/iubmb/enzyme/

90. M. P. Deutscher “Methods in Enzymology“ vol. 182 - Guide to protein purification,

Academic Press Inc. 1990

91. J. M. Zen, A. S. Kumar, “A mimicking enzyme analogue for chemical sensors”, Acc. Chem.

Res. 2001, 34, 772-780.

92. G. Wulff, „Enzyme-like catalysis by molecularly imprinted polymers“, Chem. Rev., 2002,

102, 1-27.

93. J. D. Stewart, S. J. Benkovic, „Recent developments in catalytic antibodies“, Int. Rev.

Immunol., 1993, 10, 229-240.

94. R. A. Gibbs, S. Taylor, S. J. Benkovic, “Antibody-catalysed rearrangement of peptide

bond”, Science, 1992, 258, 303-305.

95. H. Shiku, Y. Hara, T. Matsue, I. Uchida, I. Yamauchi, “Dual immunoassay of human

chorionic-gonadotropin and human placental-lactogen at a miocrofabricated substrate by

scanning electrochemical microscopy”, J. Electroanal. Chem. 1997, 438, 187-190.

96. N. Dontha, W. B. Nowall, W. G. Kuhr, “Generation of biotin/avidin/enzyme nanostructures

with maskless photolithography“, Anal. Chem. 1997, 69, 2619-2625.

97. S. A. Brooks, W. P. Ambrose, W. G. Kuhr, “Micrometer dimension derivatization of

biosensor surfaces using confocal dynamic patterning”, Anal. Chem. 1999, 71, 2558-2563.

98. S. Gaspar, M. Mosbach, L. Wallman, T. Laurell, E. Csöregi, W. Schuhmann, „A method for

the design and study of enzyme microstructures formed by means of a flow-through

microdispenser”, Anal. Chem., 2001, 73, 4254-4261.


99. Q. Y. Liu, M. Coulombe, J. Dumm, K. M. Schaffer, A. E. Schaffner, J. L. Barker, et al.

„Synaptic connectivity in hippocampal neuronal networks cultured on micropatterned

surfaces“, Dev. Brain. Res., 2000, 120, 223-231.

100. C. Wyart, C. Ybert, L. Bourdieu, C. Herr, C. Prinz, D. Chatenay, “Constrained synaptic

connectivity in functional mammalian neuronal networks grown on patterned surfaces”, J.

Neurosci. Methods, 2002, 117, 123-131.

101. P. Clark „Cell behaviour on micropatterned surfaces“, Biosens. Bioelectron., 1994, 9, 657-

661.

References

147

102. P. Heiduschka, I. Romann, H. Ecken, M. Schöning, W. Schuhmann, S. Thanos, „Defined

adhesion and growth of neurons on artificial structured substrates“, Electrochim. Acta,

2001, 47, 299-307.

103. M. Merz, P. Fromherz, „Polyester microstructures for topographical control of outgrowth

and synapse formation of snail neurons“, Adv. Mater., 2002, 14, 141-144.

104. M. P. Maher, J. Pine, J. Wright, Y. C. Tai, “The neurochip: a new multielectrode device for

stimulating and recording from cultured neurons”, J. Neurosci. Methods, 1999, 87, 45-46.

105. S. Tatic-Lucic, J. A. Wright, Y. C. Tai, J. Pine, “Silicon cultured-neuron prosthetic devices

for in vivo and in vitro studies”, Sens. Actuators Part B: Chem., 1997, 43, 105-109.

106. A. S. Blawas, W. M. Reichert, „Protein patterning“, Biomaterials, 1998, 19, 595-609.

107. R. S. Kane, S. Takayama, E. Ostuni, D. E. Ingber, G. M. Whitesides, “Patterning proteins

and cells using soft lithography”, Biomaterials, 1999, 20, 2363-2376.

108. Y. Yoshihiro, „Surface micropatterning to regulate cell functions”, Biomaterials, 1999, 20,

2333-2342.

109. L. Kam, W. Shain, J. N. Turner, R. Bizios, “Axonal outgrowth of hippocampal neurons on

micro-scale networks of polylysine-conjugated laminin”, Biomaterials, 2001, 22, 1049-1054.

110. M. Scholl, C. Sprössler, M. Denyer, M Krause, K. Nakajima, A. Maelicke, et.al., „Ordered

networks of rat hippocampal neurons attached to silicon oxide surfaces”, J. Neurosci.

Methods, 2000, 104, 65-75.

111. B. C. Wheeler, J. M. Corey, G. J. Brewer, D. W. Branch, “Microcontact printing for precise

control of nerve cell growth in culture”, J. Biomech. Eng., 1999, 121, 73-78.

112. H. Takano, J.-Y. Sul, M. L. Mozzanti, R. T. Doyle, P. G. Haydon, M. D. Porter,

“Micropatterned substrates: approach to probing intercellular communication pathways”,

Anal. Chem., 2002, 74, 4640-4646.

113. J. Hyun, H. Ma, Z. Zhang, T. P. Beebe Jr., A. Chilkoti, „Universal route to cell

micropatterning using an amphiphilic comb polymer“, Adv. Mater., 2003, 15, 576-579.

114. D. V. Nicolau, T. Taguchi, H. Taniguchi, H. Tanigawa, S. Yoshikawa, “Patterning neuronal

and glia cells on light-assisted functionalised photoresists”, Biosens. Bioelectron., 1999, 14,

317-325.

115. H. Sorribas, C. Padeste, L. Tiefenauer, „Photolithographic generation of protein

micropatterns for neuron culture applications“, Biomaterials, 2002, 23, 893-900.

116. C. Wyart, C. Ybert, L. Bourdieu, C. Herr, C. Prinz, D. Chatenay, „Constrained synaptic

connectivity in functional mammalian neuronal networks grown on patterned surfaces”, J.

Neurosci. Methods, 2002, 117, 123-131.

References

148

117. N. A. Bullett, R. D. Short, T. O’Leary, A. J. Beck, C. W. I. Douglas, M. Cambray-Deakin, et

al., “Direct imaging of plasma-polymerised chemical micropatterns”, Surf. Interface Anal.,

2001, 31, 1074-1076.

118. A. C. Duncan, F. Weisbuch, F. Rouais, S. Lazare, Ch. Baquey, „Laser microfabricated

model surfaces for controlled cell growth”, Biosens. Bioelectron., 2002, 17, 413-426.

119. P. Heiduschka, W. Göpel, W. Beck, W. Kraas, S. Kienle, G. Jung, „Microstructured peptide-

functionalised surfaces by electrochemical polymerisation“, Chem. Eur. J., 1996, 2, 667-

672.

120. P. Heiduschka, I. Romann, H. Ecken, M. Schöning, W. Schuhmann, S. Thanos, „Defined

adhesion and growth of neurons on artificial structured substrates“, Electrochim. Acta,

2001, 47, 299-307.

121. M. N. Yousaf, B. T. Houseman, M. Mrksich, “Using electroactive substrates to pattern the

attachment of two different cell populations”, Proc. Natl. Acad. Sci. USA, 2001, 98, 5992–

5996.

122. K. Beck, I. Hunter, J. Engel, „Structure and function of laminin: anatomy of a multidomain

glycoprotein“, FASEB J., 1990, 4, 148-160.

123. D. Gullberg, P. Ekblom, „Extracellular matrix and its receptors during development“, Int. J.

Dev. Biol., 1995, 39, 845-854.

124. A. M. Mercurio, “Laminin receptors achieving specificity through cooperation”, Trends Cell

Biol., 1995, 5, 419-423.

125. H. K. Kleinman, F. B. Cannon, G. W. Laurie, J. R. Hassel, M. Aumailley, V. P. Teranova, et

al., “Biological activities of laminin“, J. Cell Biochem., 1987, 27, 317-325.

126. G. R. Martin, R. Timpl, „Laminin and other basement membrane components“, Ann. Rev.

Cell Biol., 1987, 3, 57-85.

127. P. Liesi, D. Dahl, A. Vaheri, „Neurons cultured from developing rat brain attach and spread

preferentially to laminin“, J. Neurosci. Res., 1984, 11, 241-251.

128. S. L. Rogers, P. C. Letourneau, S. L. Palm, J. McCarthy, L. T. Furcht, “Neurite extension by

peripheral and central nervous system neurons in response to substratum-bound

fibronectin and laminin“, Dev. Biol., 1983, 98, 212-220.

129. S. Thanos, M. Bähr, Y.-A. Barde, J. Vanselow, “Survival and axonal elongation of adult rat

retinal ganglion cells: in vitro effects of lesioned sciatic nerve and brain derived

neurotrophic factor (BDNF)”, Eur. J. Neurosci., 1989, 1, 19-26.

130. P. Clark, S. Britland, P. Connolly, „Growth cone guidance and neuron morphology on

micropatterned laminin surfaces“, J. Cell Sci., 1993, 105, 203-212.

References

149

131. M. Matsuzawa, K. Kobayashi, K. Sugioka, W. Knoll, “A biocompatible interface for the

geometrical guidance of central neurons in vitro”, J. Colloid Interface Sci., 1998, 202, 213-

221.

132. C. L. Klein, M. Scholl, A. Maelicke, „Neuronal networks in vitro: formation and organisation

on biofunctionalised surfaces”, J. Mater. Sci. Mater. Med., 1999, 10, 721-727.

133. L. Kam, W. Shain, J. N. Turner, R. Bizios, “Axonal outgrowth of hippocampal neuros on

microscale networks of polylysine-conjugated laminin”, Biomaterials, 2001, 22, 1049-1054.

134. L. Lauer, C. Klein, A. Offehäusser, „Spot compliant neronal networks by structure optimised

microcontact printing“, Biomaterials, 2001, 22, 1925-1932.

135. L. Lauer, A. Vogt, C. K. Yeung, W. Knoll, A. Offenhäusser, „Electrophysiological recordings

of patterned rat brain stem slice neurons“, Biomaterials, 2002, 23, 3123-3130.

136. S. Ekström, P. Önnerfjord, J. Nilsson, M. Bengtson, T. Laurell, G. Marko-Varga, “Integrated

microanalytical technology enabling rapid and automated protein identification”,

Anal.Chem., 2000, 72, 286-293.

137. T. Miliotis, S. Kjellström, J. Nilsson, T. Laurell, L.-E. Edholm, G. Marko-Varga, “Capillary

liquid chromatography interfaced to matrix-assisted laser desorption/ionisation time-of-flight

mass spectrometry using an on-line coupled piezoelectric flow-through microdispenser”, J.

Mass. Spectrom., 2000, 35, 369-377.

DNA microstructures

138. “DNA Microarrays – a molecular cloning manual”, edited by D. Bowtell and J. Sambrook,

Cold Springs Harbor Laboratory Press, 2003.

139. R. Ekins, F. Chu, E. Biggart, „Development of microspot multi-analyte ratiometric

immunoassay using dual fluorescent-labelled antibodies“, Anal. Chim. Acta, 1989, 227, 73-

96.

140. R. Ekins, F. W. Chu, “Microarrays: their origins and applications”, Trends in Biotechnology,

1999, 17, 217-218.

141. E. M. Southern, “Detection of specific sequences among DNA fragments separated by gel

electrophoresis”, J. Mol. Biol., 1975, 98, 503-517.

142. M. G. Weller, „Classification of protein microarrays and related techniques“, Ana. Bioanal.

Chem., 2003, 375, 15-17.

143. M. J. Heller, “DNA microarray technology: Devices, systems, and applications”, Annu. Rev.

Biomed. Eng. 2002, 4, 129-153.

144. B. Lemieux, A. Aharoni, M. Schena, “Overview of DNA chip technology”, Mol. Breeding,

1998, 4, 277-289

145. G. Ramsay, “DNA chips: State-of-the-art”, Nat. Biotechnol. 1998, 16, 40-44.

References

150

146. P. S. Lee, K. H. Lee, „Genomic analysis”, Curr. Opin. Biotech., 2000, 11, 171-175.

147. L. M. Steinmetz, R. W. Davis, „High-density arrays and insights into genome function”,

Biotechnol. Genet. Eng. Rew., 2000, 17, 109-146.

148. International Human Genome Sequencing Consortium, “Initial sequencing and analysis of

the human genome”, Nature, 2001, 409, 860-921.

149. D. D. Shoemaker, P. S. Linsley, “Recent developments in DNA microarrays”, Curr. Opin.

Microbiol. 2002, 5, 334-337.

150. K. M. Kurian, J. C. Watson, A. H. Wyllie, “DNA chip technology”, J. Pathol., 1999, 187, 267-

271.

151. J. H. Hacia, “Resequencing and mutational analysis using oligonucleotide microarrays”,

Nature Genetics Supplement, 1999, 21, 42-47.

152. C. Debouck, P. N. Goodfellow, “DNA microarrays in drug discovery and development”

Nature Genetics Supplement, 1999, 21, 48-50.

153. F. S. Collins, E. D. Green, A. E. Guttmacher, M. S. Guyer, “A vision for the future of

genomics research”, Nature, 2003, 422, 835-847.

154. M. Branca, “Putting gene arrays to the test”, Science, 2003, 300, 238.

155. “Oligonucleotide synthesis – a practical approach”, edited by M. J. Gait, IRL Press, Oxford,

1984.

156. „Nucleic acids in chemistry and biology“, edited by G. M. Blackburn, M. G. Gait, Michael J.

Gait, Oxford University Press, 1996.

157. V. G. Cheung, M. Morley, F. Aguilar, A. Massimi, R. Kucherlapati, G. Childs, „Making and

reading microarrays“, Nature Genetics Supplement, 1999, 21, 15-19.

158. M. C. Pirrung, “Die Herstellung von DNA-Chips“, Angew. Chem., 2002, 114, 1326-1341.

159. R. D. Egeland, F. Marken, E. M. Southern, „An Electrochemical Redox Couple Activated by

Microelectrodes for Confined Chemical Patterning of Surfaces”, Anal. Chem., 2002, 74,

1590-1596.

160. R. J. Lipshutz, S. P.A. Fodor, T. R. Gingeras, D. J. Lockhart, „High density synthetic

oligonucleotide array”, Nature Genetics Supplement, 1999, 21, 20-24.

161. A. Jung, “DNA chip technology”, Anal. Bioanal. Chem., 2002, 372, 41-42.

162. A. P. Blanchard, R. J. Kaiser, L. E. Hood, “High-density oligonucleotide arrays”, Biosens.

Bioelectron., 1996, 11, 687-690.

163. http:// www.affymetrix.com

164. P. W. Schwartz, “Meniscus Force Nanografting: Nanoscopic Patterning of DNA“, Langmuir,

2001, 17, 5971-5977.

165. B. Merrifield, http://www.nobel.se/chemistry/laureats/1984/merrifield-lecture.pdf.

References

151

166. R. Lenigk, M. Carles, N. Y. Ip, N. J. Sucher, „Surface Characterization of a Silicon-Chip-

Based DNA Microarray“, Langmuir, 2001, 17, 2497-2501.

167. S. J. Oh, S. J. Cho, C. O. Kim, J. W. Park, “Characteristics of DNA microarrays fabricated

on various aminosilane layers”, Langmuir, 2002, 18, 1764-1769.

168. http://www.frizbiochem.de/

169. F. Perraut, A. Lagrange, P. Pouteau, O. Peyssonneaux, P. Puget, G. McGall, L. Menou, R.

Gonzalez, P. Labeye, F. Ginot “A new generation of scanners for DNA chips”, Biosens.

Bioelectron., 2002, 17, 803-813.

170. F. Caruso, E. Rodda, D. N. Furlong, “Quartz crystal micro-balance study of DNA

immobilisation and hybridisation for nucleic acid sensor development”, Anal. Chem., 1997,

69, 2043–2049.

171. I. Willner, F. Patolsky, Y. Weizmann, B. Willner, „Amplified detection of single-base

mismatches in DNA using microgravimetric quartz-crystal-microbalance transduction”,

Talanta, 2002, 56, 847-856.

172. T. Livache, E. Maillart, N. Lassalle, P. Mailley, B. Corso, P. Guedon, A. Roget, Y. Levy,

“Polypyrrole based DNA hybridisation assays: study of label free detection processes

versus fluorescence on microchips”, Journal of pharmaceutical and biomedical analysis,

2003, 32, 687-696.

173. R. Gabl, H.-D. Feucht, H. Zeininger, G. Eckstein, M. Schreiter, R. Primig, D. Pitzer, W.

Wersing, „First results on label-free detection of DNA and protein molecules using a novel

integrated sensor technology based on gravimetric detection principles”, Biosens.

Bioelectron., 2004, 19, 615-620.

174. P. de-los-Santos-Alvarez, M. J. Lobo-Castanon, A. J. Miranda-Ordieres, P. Tunon-Blanco,

“Current strategies for electrochemical detection of DNA with solid electrodes” Anal.

Bioanal. Chem., 2004, 378, 104-118.

175. K. Kerman, M. Kobayashi, E. Tamiya, “Recent trends in electrochemical DNA biosensor

technology” Meas. Sci. Technol., 2004, 15, R1-R11.

176. T. G. Drummond, M. G. Hill, J. K. Barton, “Electrochemical DNA sensors”, Nat. Biotechnol.

2003, 21, 1192-1199.

177. J. J. Gooding, “Electrochemical DNA hyhridization biosensors”, Electroanalysis, 2002, 14,

1149-1156.

178. E. Paleček, F. Jelen, “Electrochemistry of nucleic acids and development of DNA sensors”,

Crit. Rev. Anal. Chem., 2002, 32, 261-270.

179. J. Wang, “Electrochemical nucleic acid biosensors”, Anal. Chim. Acta, 2002, 469, 63-71.

180. http://www.motorola.com/lifesciences/esensor/tech_system_works.html

References

152

181. C. J. Yu, Y. J. Wan, H. Yowanto, J. Li, C. L. Tao, M. D. James, C. L. Tan, G. F. Blackburn,

T. J. Meade, “Electronic detection of single-base mismatches in DNA with ferrocene-

modified probes”, J. Am. Chem. Soc., 2001,123, 11155-11161.

182. M. Nakayama, T. Ihara, K. Nakamo, M. Maeda, “DNA sensors using a ferrocene-

oligonucleotide coniugate”, Talenta, 2002, 56, 857-866.

183. E. Paleček, “Past, present and future of nucleic acids electrochemistry”, Talanta, 2002, 56,

809-819.

184. J. Jortner, M. Bixon, T. Langenbacher, M. E. Michel-Beyerle, „Charge transfer and

transport in DNA“, Proc. Natl. Acad. Sci. USA, 1998, 95, 12759–12765.

185. P. T. Henderson, D. Jones, G. Hampikian, Y. KAN, G. B. Schuster, “Long-distance charge

transport in duplex DNA: The phonon-assisted polaron-like hopping mechanism”, Proc.

Natl. Acad. Sci. USA, 1999, 96, 8353–8358.

186. H. A. Wagenknecht, “Ladungstransfer durch die DNA”, Chemie in unserer Zeit, 2002, 36,

318-330.

187. R. E. Holmlin, P. J. Dandliker, J. K. Barton, „Charge transfer through the DNA base stack”,

Angew. Chem. Int. Edit., 1997, 36, 2715-2730.

188. S. O. Kelley, R. E. Holmlin, E. D. A. Stemp, J. K. Barton, “Photoinduced electron transfer in

ethidium-modified DNA duplexes: Dependence on distance and base stacking” J. Am.

Chem. Soc., 1997, 119, 9861-9870.

189. E. M. Boon, D. M. Ceres, T. G. Drummond, M. G. Hill, J. K. Barton, “Mutation detection by

electro-catalysis at DNA-modified electrodes”, Nat. Biotechnol., 2000, 18, 1096-1100.

190. S. O. Kelley, E. M. Boon, J. K. Barton, N. M. Jackson, M. G. Hill, “Single-base mismatch

detection based on charge transduction through DNA”, Nucleic Acids Res., 1999, 27, 4830-

4837.

191. M. T. Carter, M. Rodriguez, A. J. Bard, “Voltammetric studies of the interaction of metal

chelates with DNA. 2. Tris-chelated complexes of Co(III) and Iron(II) with 1,10-

phenantroline and 2,2’-bipyridine“, J. Am. Chem. Soc., 1989, 111, 8901-8911.

192. A. E. Friedman, J. C. Chambron, J. P. Sauvage, N. J. Turro, J. K. Barton, “A molecular light

switch for DNA:Ru(bpy)2(dppz)2+“, J. Am. Chem. Soc., 1990, 112, 4960-4962.

193. A. Erdem, B. Meric, K. Kerman, T. Dalbasti, M. Ozsoz, „Detection of interaction between

metal complex indicator and DNA by using, electrochemical biosensor“, Electroanalysis,

1999, 11, 1372-1376.

194. M. Takagi, “Threading intercalation to double-stranded DNA and the application to DNA

sensing. Electrochemical array technique”, Pure and Applied Chemistry, 2001, 73, 1573-

1577.

References

153

195. S. Takenaka, K. Yamashita, M. Takagi, Y. Uto, H. Kondo, “DNA sensing on a DNA probe-

modified electrode using ferrocenylnaphthalene diimide as the electrochemically active

ligand”, Anal. Chem., 2000, 72, 1334-1341.

196. J. Gu, X. Lu, H. Ju, “DNA sensor for recognition of native yeast DNA sequence with

methylene blue as an electrochemical hybridisation indicator”, Electroanalysis, 2002, 14,

949-954.

197. P. Kara, D. Ozkan, K. Kerman, B. Meric, A. Erdem, M. Ozsoz, „DNA sensing on glassy

carbon electrodes by using hemin as the electrochemical hybridisation label“, Anal.

Bioanal. Chem., 2002, 373, 710-716.

198. K. Maruyama, Y. Mishima, K. Minagawa, J. Motonaka, “DNA Sensor with a

dipyridophenazine complex of osmium (II) as an electrochemical probe”, Anal. Chem.,

2002, 74, 3698-3703.

199. G. Song, L. Li, L. Liu, G. Fang, S. Lu, Z. He, Y. Zeng, “Fluorometric determination of DNA

using a new ruthenium complex Ru(bpy)2PIP(V) as nucleic acid probe”, Analytical

Sciences, 2002, 18, 757-759.

200. W. Miao, A. J. Bard, “Electrogenerated chemiluminescence. 72. Determination of

immobilized DNA and C-Reactive protein on Au(111) electrodes using tris(2,2’-bipyridyl)

ruthenium(II) Labels”, Anal. Chem., 2003, 75, 5825-5834.

201. H. Cai, X. Cao, Y. Jiang, P. He, “Carbon nanotube-enhanced electrochemical DNA

biosensor for DNA hybridisation detection”, Anal. Bioanal. Chem., 2003, 375, 287-293.

202. A. Erdem, B. Meric, K. Kerman, T. Dalbasti, M. Ozsoz, “Detection of interaction between

metal complex indicator and DNA by using electrochemical biosensor”, Electroanalysis,

1999, 11, 1372-1376.

203. M. E. Napier, C. R. Loomis, M. F. Sistare, J. Kim, A. E. Eckhardt, H. H. Thorp, “Probing

biomolecule recognition with electron transfer: Electrochemical sensors for DNA

hybridisation”, Bioconjugate Chem. 1997, 8, 906-913.

204. K. M. Millan, S. R. Mikkelsen, “Sequence-selective biosensor for DNA-based on

electroactive hybridization indicators”, Anal. Chem., 1993, 65, 2317-2323.

205. B. Meric, K. Kerman, D. Ozkan, P. Kara, M. Ozsoz, “Indicator-free electrochemical DNA

biosensor based on adenine and guanine signals”, Electroanalysis, 2002, 14, 1245-1250.

206. P. de-los-Santos-Alvarez, M. J. Lobo-Castanon, A. J. Miranda-Ordieres, P. Tunon-Blanco

“Voltammetric determination of underivatized oligonucleotides on graphite electrodes

based on their oxidation products” Anal. Chem., 2002, 74, 3342-3347.

207. F. Lucarelli, A. Kicela, I. Palchetti, G. Marrazza, M. Mascini, “Electrochemical DNA

biosensor for analysis of waste water samples”, Bioelectrochemistry, 2002, 58, 113-118.

208. J. Wang, G. Rivas, J. R. Fernandes, J. L. L. Paz, M. Jiang, R. Waymire, “Indicator-free

electrochemical DNA hybridization biosensor”, Anal. Chim. Acta, 1998, 375, 197-203.

References

154

209. W. Fritzsche, T. A. Taton, “Metal nanoparticles as labels for heterogeneous, chip-based

DNA detection”, Nanotechnology, 2003, 14, R63-R73.

210. M. Ozsoz, A. Erdem, K. Kerman, D. Ozkan, B. Tugrul, N. Topcuoglu, H. Ekren, M. Taylan,

“Electrochemical genosensor based on colloidal gold nanoparticles for the detection of

Factor V Leiden mutation using disposable pencil graphite electrodes”, Anal. Chem., 2003,

75, 2181-2187.

211. S. J. Park, T. A. Taton, C. A. Mirkin, “Array-based electrical detection of DNA with

nanoparticle probes”, Science, 2002, 295, 1503-1506.

212. J. Wang, D. K. Xu, A. N. Kawde, R. Polsky, “Metal nanoparticle-based electro-chemical

stripping potentiometric detection of DNA hybridisation”, Anal. Chem., 2001, 73, 5576-

5581.

213. P. M. Armistead, H. H. Thorp, “Modification of indium tin oxide electrodes with nucleic

acids: detection of attomole quantities of immobilized DNA by electrocatalysis”, Anal.

Chem., 2000, 72, 3764-3770.

214. T. M-H. Lee, L-L. Li, I-M. Hsing, “Enhanced electrochemical detection of DNA hybridisation

based on electrode-surface modification”, Langmuir, 2003, 19, 4338-4343.

215. T. A. Taton, C. A. Mirkin, R. L. Letsinger, “Scanometric DNA array detection with

nanoparticle probes”, Science, 2000, 289, 1757-1760.

216. H. Cai, Y. Xu, N. Zhu, P. He, Y. Fang, “An electrochemical DNA hybridisation detection

assay based on a silver nanoparticle label”, Analyst, 2002, 127, 803-808.

217. J. Wang, R. Polsky, A. Merkoci, K. L. Turner, „Electroactive beads for ultrasensitive DNA

detection”, Langmuir, 2003, 19, 989-991.

218. Y. C. Zhang, H. H. Kim, A. Heller, “Enzyme-amplified amperometric detection of 3000

copies of DNA in a 10-muL droplet at 0.5 fM concentration”, Anal. Chem., 2003, 75, 3267-

3269.

219. C. N. Campbell, D. Gal, N. Cristler, C. Banditrat, A. Heller, “Enzyme-amplified

amperometric sandwich test for RNA and DNA”, Anal. Chem., 2002, 74, 158-162.

220. M. Dequaire, A. Heller, “Screen printing of nucleic acid detecting carbon electrodes”, Anal.

Chem., 2002, 74, 4370-4377.

221. Y. C. Zhang, H. H. Kim, N. Mano, M. Dequaire, A. Heller, “Simple enzyme-amplified

amperometric detection of a 38-base oligonucleotide at 20 pmol L-1 concentration in a 30-

µm L droplet”, Anal. Bioanal. Chem., 2002, 374, 1050-1055.

222. K. Ikebukuro, Y. kohiki, K. Sode, „Amperometric DNA sensor using the pyrroquinoline

quinone glucose dehydrogenase-avidin conjugate“, Biosens. Bioelectron., 2002, 17, 1075-

1080.

References

155

223. M. Gabig-Ciminska, A. Holmgren, H. Andresen, K. Bundvig Barken, M. Wümpelmann, J.

Albers, R. Hintsche, A. Breitenstein, P. Neubauer, M. Los, A. Czyz, G. Wegrzyn, G.

Silfversparre, B. Jürgen, T. Schweder, S. –O. Enfors, „Electric chip for rapid detection and

quantitation of nucleic acids“, Biosens. Bioelectron., 2004, 19, 537-546.

224. F. Wei, B. Sun, Y. Guo, X. S. Zhao, „Monitoring DNA hybridization on alkyl modified silicon

surface through capacitance measurement”, Biosens. Bioelectron., 2003, 18, 1157-1163.

225. F. Patolsky, E. Katz, A. Bardea, I. Willner, „Enzyme-linked amplified electrochemical

sensing of oligonucleotide-DNA interaction by means of the precipitation of an insoluble

product and using impedance spectroscopy“, Langmuir, 1999, 15, 3703-3706.

226. C. A. Marquette, I. Lawrence, C. Polychronakos, M.F. Lawrence, “Impedance based DNA

chip for direct Tm measurement”, Talanta, 2002, 56, 763–768.

227. G. Farace, G. Lillie, T. Hianik, P. Payne, P. Vadgama, “Reagentless biosensing using

electrochemical impedance spectroscopy”, Bioelectrochemistry, 2002, 55, 1-3.

228. S. Hason, J. Dvorak , F. Jelen, V. Vetterl, „Interaction of DNA with echinomycin at the

mercury electrode surface as detected by impedance and chronopotentiometric

measurements”, Talanta, 2002, 56, 905-913.

229. L. A. Thompson, J. Kowalik, M. Josowicz, J. Janata, “Label-free DNA hybridization probe

based on a conducting polymer”, J. Am. Chem. Soc., 2003, 125, 324 -325.

230. J. Wang, G. Rivas, X. Cai, E. Palecek, P. Nielsen, H. Shiraishi, N. Dontha, D. Luo, C.

Parrado, M. Chicharro, P. A. M. Farias, F. S. Valera, D. H. Grant, M. Ozsoz, M. N. Flair,

“DNA electrochemical biosensors for environmental monitoring. A review”, Analytica

Chimica Acta, 1997, 347, 1-8.

231. F. Lucarelli, I. Palchetti, G. Marrazza, M. Mascini, “Electrochemical DNA biosensor as a

screening tool for the detection of toxicants in water and wastewater samples”, Talanta,

2002, 56, 949-957.

232. P. Englebienne, A. Van Hoonacker, M. Verhas, “Surface plasmon resonance: principles,

methods and applications in medical sciences”, Spectroscopy, 2003, 17, 255-273.

233. P. Guedon, T. Livache, F. Martin, F. Lesbre, A. Roget, G. Bidan, Y. Levy, “Characterization

and optimization of a real-time, parallel, labe-free, polypyrrole-based DNA sensor by

surface plasmon resonance imaging”, Anal. Chem., 2000, 72, 6003-6009.

234. B. P. Nelson, T. E. Grimsrud, M. R. Liles, R. M. Goodman, R. M. Corn, “Surface plasmon

resonance imaging measurements of DNA and RNA hybridisation adsorption onto DNA

microarrays”, Anal. Chem., 2001, 73, 1-7.

235. J. Wang, F. Zhou, „Scanning electrochemical microscopic imaging of surface-confined

DNA probes and their hybridisation via guanine oxidation”, J. Electroanal. Chem., 2002,

537, 95-102.

References

156

236. J. Wang, F. Y. Song, F. M. Zhou, „Silver-enhanced imaging of DNA hybridization at DNA

microarrays with scanning electrochemical microscopy”, Langmuir, 2002, 18, 6653-6658.

237. K. Yamashita, M. Takagi, K. Uchida, H. Kondo, S. Takenaga, “Visualization of DNA

microarrays by scanning electrochemical microscopy (SECM)”, Analyst, 2001, 126, 1210-

1211.

238. S. Minko, A. Kiriy, G. Gorodyska, M. Stamm, “Mineralization of single flexible

polyelectrolyte molecules”, J. Am. Chem. Soc., 2002, 124, 10192-10197.

239. P. Lyubartsev, J. X. Tang, P. A. Janmey, L. Nordenskiöld, „Electrostatically Induced

Polyelectrolyte Association of Rodlike Virus Particles”, Phys. Rev. Lett., 1998, 81, 5465-

5468.

240. S. Gavryushov, P. Zielenkiewicz, “ Electrostatics of a DNA-like Polyelectrolyte: Effects of

Solvent Dielectric Saturation and Polarization of Ion Hydration Shells”, J. Phys. Chem. B,

1999, 103, 5860-5868.

Notes chapter 5

241. E. Pennisi, “DNA’s cast of thousands”, Science, 2003, 300, 282-285.

242. M. H. F. Wilkins, A. R. Stokes, H. R. Wilson, “Molecular structure of deoxypentose nucleic

acids”, Nature, 1953, 171, 738-740.

243. R. E. Franklin, R. G. Gosling, „Molecular configuration in sodium thymonucleate“, Nature,

1953, 171, 740-741.

244. R. E. Franklin, R. G. Gosling, „Evidence for 2-chain helix in crystalline structure of sodium

deoxiribonucleate“, Nature, 1953, 172, 156-157.

245. A. Klug, “Rosalind Franklin and the double helix”, Nature, 1974, 248, 787.

246. J. D. Watson, F. H. C. Crick, “A structure for deoxyribose nucleic acid”, Nature, 1953, 171,

737-738.

247. J. D. Watson, F. H. C. Crick, “Genetical implications of the structure of deoxyribonucleic

acid”, Nature, 1953, 171, 964-967.

248. M. Otto, „Analytische Chemie“, VCH Verlagsgesellschaft GmbH, Weinheim, 1995, pp. 586.

249. R. D. Deegan, O. Bakajin, T. F. Dupont, G. Huber, S. R. Nagel, T. A. Witten, “Capillary flow

explains ring stains from dried liquid drops”, Nature, 1997, 389, 827-829.

10. CURRICULUM VITAE

FAMILY NAME

FIRST NAME

ADDRESS

Tel.

E-mail

DATE OF BIRTH

PLACE OF BIRTH

NATIONALITY

MARITAL STATUS

TURCU

EUGEN FLORIN

Markstrasse 329

44801 Bochum, Germany

0049-234-3384897 (private)

0049-234-3226202 (work)

[email protected]

1973.10.18

Baia Mare, Romania

Romanian

Married

Florin Turcu

Bochum, 05.07.2004 158

ACADEMIC DEGREE

• Diploma in Physics and Chemistry, from the Faculty of Science,

North University of Baia Mare, Romania; Diploma thesis: “Active

protection against corrosion“, July 1997.

EDUCATION

• Since 01.04.2001, PhD student with Prof. Dr. Wolfgang Schuhmann,

Lehrstuhl für Analytische Chemie, Abteilung Elektroanalytik und

Sensorik, Ruhr Universität Bochum. The topics of the PhD thesis

include the creation of enzyme microstructures and micropatterns of

living cells, as well as the development of a new, truly label-free

electrochemical detection of DNA hybridisation.

• 1999 – 2000 Research assistant at Department of Chemistry, North

University of Baia Mare; Chairman and organizer of the Chemistry

Club that was designed for participants from the Faculty of Science

and the local high schools. Also, I taught Chemistry at my high

school.

• 1997 - 1999 Technical assistant at Chemistry Department, North

University of Baia Mare.

• 1992 – 1997 Studies in Physics and Chemistry at the North

University of Baia Mare, Romania. Graduation: July 1997.

RESEARCH EXPERIENCE

• Basic knowledge about (micro-) electrochemistry.

• Scanning electrochemical microscopy (SECM) and its application for

the visualisation of variety of samples (enzyme microstructures, DNA

spots, patterns of conductive polymers).

Florin Turcu

Bochum, 05.07.2004 159

• DNA microarrays and electrochemical-based label-free detection of

DNA hybridisation.

• Fabrication and characterisation of enzyme microstructures.

• Fabrication of micropatterns of living (neuronal) cells.

• Microelectrode fabrication, characterisation and application.

• Four-probe conductivity measurements.

ADDITIONAL QUALIFICATIONS

• Soft Skills I & II: “Communicating with Audience & Giving

Presentations” at Graduate School of Chemistry and Biochemistry,

Ruhr Universität Bochum, 11th-17th July 2003.

• Fluent in spoken English and good in written English.

• German (good in understanding)

Florin Turcu

Bochum, 05.07.2004 160

PUBLICATIONS

• Florin Turcu, Albert Schulte, Gerhard Hartwich, Wolfgang

Schuhmann. “Label-free electrochemical recognition of DNA

hybridisation by means of modulation of the feedback current in

SECM”, Angew. Chem. Int. Ed., 2004, 43, 3482-3485; Angew.

Chem., 116, 3564-3567.

• Florin Turcu, Karla Tratsk-Nitz, Solon Thanos, Wolfgang Schuhmann

Peter Heiduschka. “Ink-jet printing for micropattern generation of

laminin for neuronal adhesion” Journal of Neuroscience Methods,

2003, 131, 141-148.

• Albert Schulte, Mathieu Etienne, Florin Turcu, Wolfgang Schuhmann.

“High resolution constant distance scanning electrochemical

microscopy on immobilised enzyme micropatterns” G.I.T. Imaging

and Microscopy, 2003, 5, 46-49.

• Florin Turcu, Albert Schulte, Gerhard Hartwich, Wolfgang

Schuhmann. “Imaging immobilised ss-DNA and detecting

hybridisation by means of the repelling mode of scanning

electrochemical microscopy (SECM)”, Biosens. Bioelectron., 2004, in

press.

• Florin Turcu, Albert Schulte, Wolfgang Schuhmann. “Scanning

electrochemical microscopy (SECM) in nanolitre droplets using an

integrated working/reference electrode assembly“, Anal. Bioanal.

Chem., 2004, submitted.

• Anh Nguyen, Jane Hübner, Florin Turcu, David Melchior, Hans-Willi

Kling, Siegmar Gäb, Oliver J. Schmitz. “Analysis of alkyl

polyglicosides by capillary electrophoresis with pulsed-amperometric

detection”, Electrophoresis, 2004, submitted.

Florin Turcu

Bochum, 05.07.2004 161

CONFERENCES

Florin Turcu, Dominik Schäfer, Albert Schulte, Gerhard Hartwich,

Wolfgang Schuhmann. “Repelling mode of SECM: a new approach for

visualising DNA microarrays and detecting hybridisation” The 3rd

Workshop on Scanning Electrochemical Microscopy (SECM), 11-12th

June 2004, Dublin City University, Dublin, Ireland, Poster presentation.

Anh Nguyen Minh Nguyet, Florin Turcu, Jane Hübner, Oliver J. Schmitz,

Siegmar Gäb. “Optimisation of the analysis of alkyl polyglycosides by

MEKC-PAD” 17th International Symposium on Microscale Separation and

Capillary Electrophoresis, 8-12th February 2004, Salzburg, Austria,

Poster presentation, P166.

Florin Turcu, Albert Schulte, Gerhard Hartwich, Wolfgang Schuhmann.

„Label-free electrochemical detection of hybridisation events on DNA

chips”, 4th EMBL PhD Student International Symposium, 20-22nd

November 2003, Heidelberg, Germany, Poster presentation.

Florin Turcu, Albert Schulte, Wolfgang Schuhmann, Gerhard Hartwich,

Herbert Wieder, Thomas Kratzmüller. „Ink-jet dispensing of DNA

microarrays and label-free electrochemical detection of DNA-

hybridisation events“ Workshop on new trends in nucleic acid based

biosensors, University of Florence, Polo Scientifico di Sesto Fiorentino,

25-28th October 2003, Oral presentation, Book of Abstracts p. 30.

Florin Turcu, Albert Schulte, Wolfgang Schuhmann. „Coaxial reference-

working electrode assembly for electrochemistry in small volumes“

ELACH-6 Conference, Vienna, Austria, 14-17th of September, 2003,

Oral presentation, Book of Abstracts V13.

Wolfgang Schuhmann, Albert Schulte, Mathieu Etienne, Florin Turcu,

Ingrid Fritsch. „High-resolution shear-force dependent constant-distance

Florin Turcu

Bochum, 05.07.2004 162

mode SECM“ 226th ACS National Meeting, New York, USA, 7-11th

September 2003.

Florin Turcu, Herbert Wieder, Thomas Kratzmüller, P. Frischmann, G.

Hartwich, W. Schuhmann. “Label-freie electrochemische Detektion von

Oligonukleotid-Hybridisierung mittels electrochemischer Rastermikros-

kopie (SECM)”, 3. BioSensorSymposion (BSS), 30. März / 1. April 2003

Potsdam, Germany, Oral Präsentation, Tagungsbuch p. 28.

Florin Turcu, Wolfgang Schuhmann. “Lokale Detektion von Oligonukleo-

tidspots mittels elektrochemischer Rastermikroskopie (SECM)”, INCOM-

Sondersymposium / 74. AGEF-Seminar, 26 März 2003, Düsseldorf,

Germany, Oral Präsentation.

Mathieu Etienne, Florin Turcu, Albert Schulte, Wolfgang Schuhmann.

High Resolution SECM imaging of complex enzyme microstructures“,

53rd Annual Meeting of the International Society of Electrochemistry,

ISE 2002, 15-20 September, Düsseldorf, Germany, Poster

presentation, Book of Abstracts p. 76.

Peter Heiduschka, K. Tratsk-Nitz, S. Thanos, Florin Turcu, Wolfgang

Schuhmann. “Defined adhesion and growth of neurones on

microstructured polymer patterns made by ink-jet printing”, 53rd Annual

Meeting of the International Society of Electrochemistry, ISE 2002,

15-20 September, Düsseldorf, Germany, Oral Presentation, Book of

Abstracts p. 187.

Florin Turcu, Mathieu Etienne, Bernardo Ballesteros Katemann, Marcus

Mosbach, Thomas Erichsen, Wolfgang Schuhmann. “Formation of

chemically active microstructures as a basis for novel miniaturised

analytical devices”, 9th International Conference on Electroanalysis,

Florin Turcu

Bochum, 05.07.2004 163

9-13 June 2002, Cracow, Poland, Oral presentation O 46 in the Book of

Abstracts.

Florin Turcu, Mathieu Etienne, Bertrand Ngounou, Thomas Erichsen,

Wolfgang Schuhmann. “Non-manual immobilisation of biological

recognition elements as a prerequisite for the preparation of

miniaturised biosensor systems”, The Seventh World Congress on

Biosensors, Kyoto, Japan, 15-17 May 2002, Poster presentation

P2-3.22 in the Abstracts Book.

Florin Turcu, Karla Tratsk, Peter Heiduschka, Albert Schulte, Wolfgang

Schuhmann. “Polymere Mikrostrukturen als Basis zur lokalisierten

Zelladhäsion“, ELMINOS, 26 April 2002, Düsseldorf, Germany, Poster

Präsentation.

Florin Turcu „Polymer receptor for a methanol chemical sensor“, The

XXVth Chemistry and Chemical Engineering National Conference, 6-8th of

October 1999, Calimanesti-Caciulata, Romania, Poster presentation PS

3, Book of Abstracts pp. 266.

Florin Turcu „Study on the chemical separation of silver from silver-

copper electrotechnical alloy”, Chemistry and Chemical Engineering

National Conference, 16-18th of October 1997, Bucharest, Romania,

Poster presentation, Book of Abstracts vol. 1, pp. 85-86.

Bochum, 05.07.2004

nanotechnology in diagnostics

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