phd_delport+filip (1).pdf

7/26/2019 PhD_Delport+Filip (1).pdf

1/182

Doctoral dissertation nr. 1017 at the faculty of Bioscience Engineering of the K.U.Leuven

CHARACTERISATION

OF DNA FUNCTIONALISED

SILICA NANOPARTICLES

FOR BIOASSAY DEVELOPMENT

Filip Delport

Dissertation presented

in partial fulfillment of therequirements for the degreeof Doctor in BioscienceEngineering

February 2012

Supervisor:Prof. J. Lammertyn, MeBioS, K.U.LeuvenProf. B. Sels, COK, K.U.Leuven

Members of the Examination Committee:Prof. B. Nicola, MeBioS, K.U.LeuvenProf. G. Maes, Dept. of Chemistry, K.U.LeuvenProf. L. Lagae, Dept. of Physics, K.U.Leuven, IMECProf. T. Verbiest, Dept. of Chemistry, K.U.Leuven

Prof. I. Pividori, Unitat de Qumica Analtica,Universitat Autnoma de BarcelonaProf. R. Geers, Division of Livestock-Nutrition-Quality, K.U.Leuven (Chair)


2/182

2012 Katholieke Universiteit Leuven, Groep Wetenschap & Technologie, ArenbergDoctoraatsschool,W. de Croylaan 6, 3001 Heverlee, Belgi

Alle rechten voorbehouden. Niets uit deze uitgave mag worden vermenigvuldigd en/of openbaargemaakt worden door middel van druk, fotokopie, microfilm, elektronisch of op welke andere wijzeook zonder voorafgaandelijke schriftelijke toestemming van de uitgever.

All rights reserved. No part of the publication may be reproduced in any form by print, photoprint,microfilm, electronic or any other means without written permission from the publisher.

ISBN 978-90-8826-231-9D/2012/11.109/8


3/182

Index

i

Index ........................................................................................................................................ i

List of symbols ...................................................................................................................... v

Abstract ...............................................................................................................................viiiSamenvatting ......................................................................................................................... x

Ch 1: General Introduction ............................................................................................... 1

1.1 Nanotechnology ............................................................................................................. 1

1.2 Bionanotechnology ........................................................................................................ 5

1.3 Nanoparticle technology ............................................................................................... 6

1.4 Objectives and outline of this thesis .........................................................................10Ch 2 Literature review: Nanoparticles, from theory to diagnostic applications.......15

2.1 Introduction ..................................................................................................................15

2.1.1 Definition ...................................................................................................................16

2.1.2 Nanoparticle materials and shapes .........................................................................17

2.2 Nanoparticle stability ...................................................................................................18

2.3 Silica nanoparticles .......................................................................................................24

2.3.1 Silica nanoparticle surface charge ...........................................................................25

2.3.2 Silica nanoparticle synthesis ....................................................................................25

2.3.2.1 Reverse micro emulsion method.........................................................................27

2.3.2.2 Stber method ........................................................................................................28

2.3.3 Surface conjugation ..................................................................................................28

2.3.3.1 Linker layer .............................................................................................................29

2.3.3.2 Bioconjugation .......................................................................................................30

2.3.4 Effect of bioconjugation on nanoparticle stability and aggregate formation..32

2.3.4.1 Steric interaction ....................................................................................................33

2.3.4.2 Electrostatic interaction ........................................................................................33

2.3.5 Core and surface modifications of the nanoparticles .........................................34

2.4 Biosensing .....................................................................................................................37

2.4.1 Principle......................................................................................................................37

2.4.3 Theranostic applications of silica nanoparticles ..................................................39

2.5 Conclusion ....................................................................................................................44


4/182

Index

ii

Ch 3 Critical Aspects to Control Conjugating DNA on Silica Nanoparticles... ...... 59

3.1 Introduction .................................................................................................................. 59

3.2 Materials and methods ................................................................................................ 613.2.1 Reagents ..................................................................................................................... 61

3.2.2 Titration, DLS and SEM of silica nanoparticles .................................................. 62

3.2.3 Immobilisation of ssDNA on silica nanoparticles .............................................. 63

3.2.4 Quantification of ssDNA on silica nanoparticles ................................................ 67

3.2.5 Hybridisation on ssDNA functionalised silica nanoparticles ............................ 68

3.3 Results and discussion ................................................................................................. 683.3.1 Characterisation of silica nanoparticles ................................................................. 68

3.3.1.1 Size distribution of silica nanoparticles .............................................................. 68

3.3.1.2 Carboxyl density and Zetapotential measurement ........................................... 70

3.3.2 Quantification of ssDNA in the presence of silica nanoparticles..................... 72

3.3.2.1 Effect of silica nanoparticles on fluorescence calibration curve .................... 73

3.3.2.2 Indirect supernatant or direct on nanoparticle detection................................ 74

3.3.3 Functionalisation of silica nanoparticles with ssDNA........................................ 75

3.3.3.1 ssDNA functionalisation on streptavidin or carboxyl coated silicananoparticles ....................................................................................................................... 76

3.3.3.2 One step versus two step immobilisation protocols ........................................ 77

3.3.3.3 Effect of reaction buffer on ssDNA functionalisation of silica

nanoparticles ....................................................................................................................... 78

3.3.3.4 Effect of the washing buffer on ssDNA functionalisation of silicananoparticles ....................................................................................................................... 79

3.3.3.5 Effect of pH on ssDNA functionalisation of silica nanoparticles ................ 81

3.3.3.6 Kinetics of ssDNA functionalisation of silica nanoparticles .......................... 83

3.3.3.7 Effect of nanoparticle size on ssDNA functionalisation of silicananoparticles ....................................................................................................................... 84

3.3.4 ssDNA hybridisation assays on silica nanoparticles ........................................... 85

3.3.4.1 Denaturation test of DNA hybridised on silica nanoparticles ....................... 85

3.3.4.2 Effect of salt on DNA hybridisation efficiency ............................................... 87


5/182

Index

iii

3.3.4. 3 Specificity of DNA hybridisation on ssDNA functionalised silicananoparticles .......................................................................................................................88

3.3.4.4 Kinetics of DNA hybridisation on ssDNA functionalised silicananoparticles .......................................................................................................................89

3.3.4.5 Effect of nanoparticle size on hybridisation kinetics .......................................90

3.3.4.6 Effect of DNA capture probe length on hybridisation efficiency.................91

3.4 Conclusion ....................................................................................................................92

Ch 4 Single Molecule Study of ssDNA Bioconjugated Nanoparticles .................. 103

4.1 Introduction ............................................................................................................... 103

4.2 Materials and methods ............................................................................................. 105

4.2.1 Single molecule microscopy ................................................................................. 105

4.2.1.1 Confocal fluorescence microscopy setup ....................................................... 106

4.2.1.2 Wide field fluorescence microscopy setup ..................................................... 108

4.2.2 Defocused single molecule spectroscopy .......................................................... 109

4.2.3 Preparation of DNA functionalised nanoparticles ........................................... 111

4.3 Results and discussion .............................................................................................. 1134.3.1 Conjugating fluorescent DNA to silica nanoparticles ..................................... 113

4.3.2 Confocal counting by photobleaching ............................................................... 114

4.3.2.1 Photobleaching ................................................................................................... 114

4.3.2.2 Interpretation of photobleaching data ............................................................ 116

4.3.2.3 Statistical processing........................................................................................... 116

4.3.2.4 Polarisation effect ............................................................................................... 1174.3.3 Modified total internal reflection fluorescence (mTIRF) ................................ 118

4.3.3.1 Principles of mTIRF .......................................................................................... 118

4.3.3.2 mTIRF counting by photobleaching ............................................................... 119

4.3.4 Defocused wide field imaging.............................................................................. 121

4.4 Conclusion ................................................................................................................. 124

Ch 5 Fiber Optic Melting Curve Analysis of DNA Immobilised on SilicaNanoparticles ................................................................................................................... 133

5.1 Introduction ............................................................................................................... 133

5.2 Materials and methods ............................................................................................. 135


6/182

Index

iv

5.2.1 Reagents .................................................................................................................. 135

5.2.2 DNA functionalisation of silica nanoparticles .................................................. 136

5.2.3 Fiber optic surface plasmon resonance sensor ................................................. 1365.2.4 Hybridisation of ssDNA functionalised silica nanoparticles on the fibersurface ............................................................................................................................... 139

5.3 Results and discussion .............................................................................................. 140

5.3.1 Sensitivity of the FO-SPR sensor to the refractive index ............................... 140

5.3.2 Optimisation of silica nanoparticle size and concentration ............................ 141

5.3.2 Optimisation of salt on nanoparticle hybridisation .......................................... 143

5.3.3 Backfilling of nanoparticles and the FO-SPR surface ..................................... 145

5.3.4 Effect of base pair overlap on melting temperature ........................................ 146

5.4 Conclusion ................................................................................................................. 151

Ch 6: General conclusions and future research .......................................................... 157

6.1 General conclusions ................................................................................................. 158

6.2 Further research and perspectives.......................................................................... 160

6.2.1 Fundamental research ........................................................................................... 161

6.2.1.1 Orientation and quantification of conjugated biomolecules on

nanoparticles .................................................................................................................... 161

6.2.1.2 Catalysts on nanoparticles ................................................................................. 161

6.2.1.3 Microfluidic applications of nanoparticles ..................................................... 162

6.2.2 Diagnostic concepts .............................................................................................. 162

6.2.2.1 Multiplexed single molecule diagnostics ......................................................... 162

6.2.2.2 Aptamer nanoparticles based aggregation and dissolution .......................... 162

6.2.2.3 Aptamer coated nanoparticles as secondary label in surface plasmonresonance based biosensing ........................................................................................... 164

Bibliography ..................................................................................................................... 166


7/182

List of symbols

v

Symbols and Abbreviations

APTS3-aminopropyltriethoxysilaneBB2 Binding buffer 2

Bp Basepair

cAMP Cyclic adenosine monophosphate

CCD Charge-coupled device

DETA Diethylenetriamine

DLS Dynamic light scattering

DLVO Derjaguin, Landau, Verwey and Oberbeek

DMF DimethylformamideDNA Deoxyribonucleic acid

DTT Dithiotreitol

E Elementary charge

EC European commission

EDC 1-ethyl-3-[3-dimethylaminopropyl]carbodimide hydrochloride

EDTA Ethylenediaminetetraacetic acid

EM Electromagnetic

FAM Carboxyfluorescein

FIFO First-in, first-out

FO Fiber optic

FO-SPR Fiber optic surface plasmon resonance

FWHM Full width half maximum

(h)IgE (human) Immunoglobuline E

I Ionic strength of the electrolyte

IUPAC The International Union of Pure and Applied ChemistryIVD In vitro diagnostics

kB Boltzmann constant

Kd Dissociation constant

LOD Limit of detection

LSPR Localised surface plasmon resonance

MES 2-(N-morpholino)ethanesulfonic acid

Mn Number average molecular weightMRI Magnetic resonance imaging

mTirf Modified total internal reflection fluorescence

MUA HEG Mercaptoundecanoic acid heptaethylene glycol


8/182

List of symbols

vi

Mw Weight average molecular weight

N.A. Numerical aperture

NA Avogadro numberNHS N-hydroxysuccinimide

NNI National Nanotechnology Initiative

NP Nanoparticle

PB Phosphate buffer

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PDI Polydispersity index

PEG Polyethylene glycol

pKa Acid dissociation constant at logarithmic scale

PMA Poly(methyl acrylate)

RME Reverse micro emulsion

RNA Ribonucleic acid

RPM Rotations per minute

rRNA Ribosomal ribonucleic acid

SAM self-assembling monolayerSDS Sodium dodecyl sulfate

SEM Scanning electron microscopy

SMCM Single molecule confocal microscopy

SMS Single-molecule spectroscopy

SPR Surface plasmon resonance

SSC Sodium citrate sodium chloride

ssDNA Single stranded DNA

T Absolute temperature

T10 10 thymidine bases spacer

TE Tris EDTA

TEOS Tetraethyl orthosilicate

Tg Glass temperature

TGK Tris glycine potassium phosphate

TIRFM Total internal reflection fluorescence microscopy

Tm Melting temperature

Htot Total enthalpy difference

0 Permittivity of free space


9/182

List of symbols

vii

r Dielectric constant

(1/) Debye length

lambda, wavelength phi, orientation expressed as a planar angle

theta, orientation expressed as a perpendicular angle


10/182

Abstract

viii

Characterisation of DNA functionalised silica nanoparticles for bioassay

development

Biosensors are a subgroup of chemical sensors that can detect certain target moleculesby reacting specifically with a biological recognition element, such as antibodies and

aptamers. These biological elements are often conjugated to a transducer which, upon

binding of the target, generates an electrochemical, piezoelectric or optical signal.

Many biosensor applications are found in the food, environmental and medical

industry. Bio-functionalised nanoparticles (NPs) often play an important role in these

biosensing concepts either as means to preconcentrate the target molecules of interest

in a sample, and/or to generate or amplify the signal. In this thesis the focus lies on

nano-sized silica bio-conjugates, which can be exploited as valuable tools fordiagnostic and therapeutic purposes. Often, for these applications, ssDNA is linked to

silica NPs as specific bioreceptor. To ensure a predictable target affinity, the

immobilisation of capture ssDNA has to be precisely quantified and qualified. The

quantification of few biomolecules on NPs is a difficult and labour intensive task.

Consequently, despite the fact that small surface changes on the NP can have a drastic

impact on their functionality, many research groups chose to avoid this optimisation

because of the complexity. Therefore, the specific objective of this thesis is to study

the crucial aspects of creating and characterising ssDNA functionalised silicananoparticles and to describe the behaviour of these bioconjugated nanomaterials in

biosensing.

In a first section of this thesis, DNA bulk fluorescence measurement methodologies

were evaluated to quantify the amount of ssDNA bioconjugated to the NPs. First, the

bare silica NPs were characterised by dynamic light scattering, scanning electron

microscopy and titration to determine the size, charge and carboxyl group density on

the surface. Following, a method for accurate detection of DNA on the NPs by

fluorescence measurements was developed. Measuring the quantity of immobilised

ssDNA directly on the NPs resulted in more reliable results. The conjugation of DNA

to the silica NPs was investigated and optimised. Hereto 5 amine and 3 fluorescently

labeled ssDNA was coupled to carboxyl-functionalised NPs using EDC/NHS

chemistry. The number of ssDNA molecules on the NPs could accurately be

quantified in the range between 20 and 1200 molecules per NP. Next, the DNA NPs

were coated with a PEG layer to minimise the non-specific binding during

hybridisation experiments. Non-specific binding was reduced below 1.5% compared

to specifically hybridised ssDNA. The hybridisation capabilities of silica NPs were

tailored by varying the salt concentration of the buffer, the ssDNA capture probe

density and/or the DNA overlap length.


11/182

Abstract

ix

In a second section, the DNA bulk fluorescence measurement methodologies were

compared to DNA single molecule microscopy concepts. Here, the number of DNA

strands on a single NP was counted by stepwise photobleaching and Poisson

modelling. The method of illumination - confocal (Khler) or wide field total internal

reflection fluorescence (TIRF) mode - determined the ratio of visible DNA strands

on the NP. With the confocal microscopy measurements only about two thirds of the

ssDNA as determined from the bulk fluorescence measurements was retrieved. Wide

field microscopy revealed the missing one third ssDNA which was immobilised

perpendicularly to the sample surface. Defocused imaging of fluorescently labeled

ssDNA on NPs provided info on their orientation. No perpendicular angles below

50 were obtained for either illumination mode. However, the TIRF mode revealed a

broader angular distribution compared to the Khler mode.

Finally, in a last section, the characterised silica NPs were studied as a signal enhancer

on a fiber optic surface plasmon resonance (SPR) setup for detection of DNA. One

of the most accurate, real-time optical technologies to study the dynamics of DNA

hybridisation is SPR. The diameter of the NPs had to be sufficiently small (


12/182

Abstract

x

Karakterisatie van DNA gefunctionaliseerde nanopartikels voor het

ontwikkelen van bioassays

Biosensoren behoren tot een subgroep van chemische sensoren die bepaaldedoelmoleculen kunnen detecteren door specifiek te binden met een

bioherkenningselement, zoals een antilichaam of een aptameer. Deze

bioherkenningselementen worden vaak gekoppeld aan een vertaalsysteem, dat bij

binding met het doelmolecule, een elektrochemisch, pizoelektrisch of optisch signaal

genereert. Er bestaan reeds veel biosensortoepassingen in de voedings-, milieu- en

medische sector. Biologisch gefunctionaliseerde nanopartikels (NPs) spelen vaak een

belangrijke rol in deze biosensorconcepten, enerzijds als middel om selectief

doelmoleculen uit stalen op te concentreren, anderzijds om een signaal te genereren ofte versterken. Voor deze toepassingen wordt vaak ssDNA als specifieke receptor

gekoppeld aan silica NPs. Om de performantie van de biosensor te kunnen

voorspellen, dient de bioconjugatie van het DNA op de silica NPs precies

gekwantificeerd te worden. Het kwantificeren van slechts enkele biomoleculen op een

silica NP, echter, is een uitdagende en werk intensieve taak. Ondanks het feit dat de

kleinste verandering op het oppervlak van het NP een zeer grote impact kan hebben

op hun functionaliteit, gaan vele onderzoeksgroepen dit complexe onderwerp uit de

weg. Dit werk heeft dan ook als doel de cruciale aspecten van het functionaliseren enkarakteriseren van silica DNA NPs in kaart te brengen en het gedrag van deze hybride

nanomaterialen in biosensortoepassingen te beschrijven.

In een eerste deel van dit werk werden DNA bulk fluorescentiemetingen gebruikt om

de immobilisatie van ssDNA op de silica NPs te bestuderen. De lege silica

nanodeeltjes werden eerst gekarakteriseerd met behulp van dynamic light scattering,

scanning electron microscopie en titratie om hun grootte, lading en densiteit aan

carboxylgroepen op hun oppervlak te bepalen. Vervolgens werd een accurate methode

ontwikkeld om DNA op de NPs in bulk te detecteren. Door het meten van het aantal

gemmobiliseerde DNA strengen onmiddellijk op de NPs werd een betrouwbaarder

resultaat bekomen. Het koppelen van DNA aan de silica NPs werd onderzocht en

geoptimaliseerd. Hiervoor werd 5 amine and 3 fluorescent gemerkt DNA

gemmobiliseerd op carboxyl gefunctionaliseerde NPs met behulp van EDC/NHS

chemie. Het aantal moleculen op de NPs kon accuraat bepaald worden tussen 20 en

1200 moleculen per partikel. Vervolgens werd een laag polyethyleenglycol op de DNA

gefunctionaliseerde NPs aangebracht om niet- specifieke binding tijdens hybridisatie

te minimaliseren. De niet-specifiek gebonden fractie was kleiner dan 1,5% in

vergelijking met het specifiek gehybridiseerde DNA. De mate waarin de silica NPs


13/182

Abstract

xi

DNA hybridiseren, kon op maat gestuurd worden door de zoutconcentratie van de

buffer, de gemmobiliseerde DNA densiteit en lengte op de NPs aan te passen.

In een tweede deel werden de DNA bulk fluorescentiemetingen getoetst aan singlemolecule microscopietechnieken. Het aantal DNA strengen op n NP werd geteld

door stap voor stap de fluorescente moleculen te photobleachen. Op de bekomen

aantallen DNA moleculen per NP werd het Poisson model toegepast. De manier

waarop het licht in contact gebracht werd met het staal - confocaal met Khler of

wide field totale interne reflectie microscopie - bepaalde de hoeveelheid zichtbare

DNA strengen op het NP. Met de confocale methode werd slechts twee derde van de

hoeveelheid DNA, zoals bepaald via de bulkmethode, teruggevonden. Wide field

microscopie onthulde het ontbrekende gedeelte dat loodrecht ten opzichte van hetoppervlak was gekoppeld. Door het fluorescente DNA op de NPs net buiten de focus

van de microscoop te brengen, kon de orintatie van het DNA op de NPs achterhaald

worden. Er werden echter geen hoeken loodrecht op het oppervlak gevonden met

beide verlichtingsmethoden, hoewel met de totale interne reflectie een bredere

verdeling van hoeken dan de Khler methode werd teruggevonden.

Tot slot werden de gekarakteriseerde silica NPs bestudeerd als signaalversterker op

een optische vezel surface plasmon resonance (SPR) biosensor voor de detectie van

DNA. SPR is n van de meest accurate real time optische technologien om dedynamiek van DNA hybridisatie te bestuderen. De diameter van de NPs moest

voldoende klein zijn opdat er enige interactie mogelijk zou zijn met het oppervlak van

de optische vezel. Ook de zoutconcentratie had een grote impact op de hybridisatie

performantie van het systeem. De concentratie NaCl was optimaal voor hybridisatie

bij 300 mM. Het aantal basenparen van het ssDNA die complementair waren met het

doel DNA had geen impact op de hybridisatiekinetiek. Het smelten van de DNA

gefunctionaliseerde silica NPs gecomplexeerd op de optische vezel werd eveneens

bestudeerd. De berekende en gemeten smelttemperaturen kwamen goed overeen,maar de gemeten temperaturen lagen wel consistent hoger.

Doorheen de hoofdstukken van dit werk werden instrumentele technieken

geselecteerd en aangewend om fundamentele karakterisaties van de DNA

gefunctionaliseerde silica NPs uit te voeren. De ontwikkelingen die in dit werk

gepresenteerd werden, kunnen gemplementeerd worden in een brede waaier aan

biosensorconcepten. De DNA gefunctionaliseerde NPs kunnen toegepast worden bij

het opzuiveren van stalen, als detectiemechanisme of signaalversterking, maar ook alsstructurele component. Het introduceren van aptameren op de silica NPs laat toe om

een verscheidenheid aan doelmoleculen te detecteren. Aptameren zijn

oligonucleotiden die geselecteerd kunnen worden tegen virtueel elk doelmoelcule (bv.


14/182

Abstract

xii

protenen, toxines, ) Aptameer gefunctionaliseerde silica NPs hebben een groot

potentieel voor diagnostische en therapeutische doeleinden, met toepassingen in de

voedsel-, milieu- en medische sector.


15/182

General introduction

1

1

Chapter 1 General Introduction

1.1 Nanotechnology

The worlds population grows and ages, consuming more energy, material, food

and water each day. Finding new ways of using less to create more is key to a

more durable success of mankind. This worlds big question might be solved by a

small answer, nanotechnology. Nanotechnology encompasses the design,

characterisation, production and application of materials and devices at the

nanometer scale. Efficient use of raw materials and energy to create structures,

devices and systems will allow a sustainable lifestyle. Nanotechnology will not only

add economic value, but also create more complex systems. Such a system will

analyse its environment and decide which optimal action to perform from the

microscopic to the macroscopic level. Recent research efforts in theranostics -


16/182

Chapter 1

2

therapeutics and diagnostics - are aimed at creating concepts that will detect a

malignant entity, such as cancer, at the location where it is situated in the body and

respond by locally applying a drug. Materials can learn, remember and act towards

their environment, enabling a whole chain of events to be performed with a

maximum efficiency and certainty. Nanotechnology is introducing the next

technological revolution. In this chapter the general concepts and applications of

nanotechnology are introduced.

One nanometer (nm) is one billionth, or 109, of a meter. In a descriptive context,

the comparative size of a nanometer to a meter is the same as that of a marble to

the size of the earth. As an example of this size range, typical spacing between

carbon-carbon bond lengths in a molecule, are in the range 0.12 0.15 nm, and aDNA double-helix has a diameter around 2 nm. On the other hand, the smallest

cellular life-forms, the bacteria of the genus Mycoplasma, are around 200 nm in

length. Generally, nanotechnology deals with structures sized between 1 to 100 nm

in at least one dimension and involves developing materials or devices within that

size. There is currently limited scientific evidence to strictly support the arbitrary

100 nm value for all materials. This debate for a general definition is not trivial as it

has important legal and regulatory implications (EC 2010a). For the purposes of

this dissertation, the term nanomaterial will be used as an overarching term todescribe materials, particles, or structures with a size dimension less than a few

hundred nm.

Two main approaches are commonly found in nanotechnology. The "bottom-up"

or self-assembly approach builds materials and devices from molecular

components which assemble themselves chemically by principles of molecular

recognition. In the "top-down" approach, nano-objects are reduced by cutting,

milling or shaving from larger entities (Rodgers 2006). While some disciplinesprincipally work with the top down approach, like engineering and materials

science, others are by definition bottom up, for example biology and chemistry

(Figure 1-1). It is mainly at the intersection of these different approaches and

disciplines that a new technology, i.e., nanotechnology, is created.


17/182


3

Figure 1-1. Representation of manageable structure sizes of different disciplines and

a prospection of nanotechnology conjunction and fields of operation (Sleytr 2006).

The top down approach uses natural or synthetic blocks that are cast, sawed or

machined into precisely shaped products. Methods to produce nanomaterials from

bulk materials include solution based chemistry, mechano-chemical processing,

physical and chemical vapour deposition techniques, sonication and etching.Patterned structures, which are found in silicon integrated chip technology, are

realised by suitable lithographic and ion implantation techniques. Nevertheless,

conventional electronics which are made with the photolithographic method can

not be scaled down infinitely due to the limits of physics including radiation

wavelength. An alternative view based on other principles such as the bottom-up

approach is required to overcome this miniaturisation limit. The top down barrier

can be lowered by creating small scale devices, which in their turn will be able to

create even smaller structures. These devices are called molecular machines, whichare able to switch between two molecular states (shapes) in a controlled manner as

part of a repetitious mechanical cycle (Figure 1-2)(Browne et al.2006). Different

types of molecular machines are molecular rotors, elevators, valves, transporters,

muscles and other motor functions used to develop smart materials. The technique

is demonstrated through recent examples of systems capable of effecting

macroscopic movement through concerted molecular motion (Liu et al. 2005).

More molecular machine concepts are conceived by the David Leigh group from

the University of Edinburgh.


18/182

Chapter 1

4

Figure 1-2. Linear molecular motors are a type of molecular machine. The depicted

examples are so-called rotaxanes in which one (or more) rings can move from one

binding site to another along a shaft. The change in equilibrium position of the ring

is triggered by an external signal A) The millimetre-scale directional transport of a

liquid on a surface is achieved by using the light-responsive rotaxanes to expose or

conceal fluoroalkane residues and thereby modify surface tension (Bernaet al

2005).B) An array of flexible microcantilever beams, each coated on one side with amonolayer of 6 billion of the active bistable rotaxane molecules. It undergoes

controllable and reversible bending up and down when it is exposed to the

synchronous addition of aqueous chemical oxidants and reductants. The beam

bending is correlated with flexing of the surface bound molecular muscles (Liuet

a l

2005).

The bottom-up approach manufactures nanomaterials by moving and organising

single atoms or molecules into specific configurations. Manipulation occurs

similarly to processes of molecular machines in living organisms (Abu-Salahet al.

2010). Organisation is found in the self-assembly of lipid molecules into

nanostructures such as micelles (Michelson 2011; Prasad 2008; Rodgers 2006). An

example of atom by atom growth of nanostructures is the placing of individual


19/182


5

atoms on a surface using a Scanning tunnelling microscopy tip (Binnig et al.1982).

Controlling the placement of individual atoms to build small scale complex

products will enable researching and creating nanotechnology processes. The

advantage of the bottom-up approach is the flexibility to control any object,

machine or material through atom by atom (or molecule by molecule) construction

by linking the right resources at the right time and place. Molecular manufacturing

is envisioned to employ these skills to build entire objects. The machine of this

futuristic fabrication method is called a molecular fabricator, a device that would

build an object as complex as a desktop computer (Phoenix et al.2004).

There is much debate on the future implications of nanotechnology.

Nanotechnology may be able to create many new materials and devices with a vastrange of applications, such as in medicine, electronics, biomaterials and energy

production. On the other hand, nanotechnology raises many of the same issues as

any new technology, including concerns about the toxicity and environmental

impact of nanomaterials, and their potential effects on global economics. The

global market for nanotechnology has increased from 8 180 M in 2007 to an

estimated 8 818 M by the end of 2008. It should reach 18 881 M in 2013, a

compound annual growth rate of 16.3 % (Global Industry Analysts 2011).

1.2 Bionanotechnology

Bionanotechnology and nanobiotechnology are terms that are often alternated and

refer to the intersection of nanotechnology and biology. On the one hand, Dutta

(2006) stated that nanobiotechnology is the integration of physical sciences,

molecular engineering, biology, chemistry and biotechnology. On the other hand,

bionanotechnology seeks to find and modify technological uses of natural

nanocomponents like the nanomotors of ATP synthase (Dutta 2006). Accordingto Niemeyer nanobiotechnology involves the processing and fabrication of organic

or biomaterial devices or assemblies (Niemeyer et al. 2004). Here,

Nanobiotechnology refers to the use of biological principles and molecules to

construct and apply nanodevices, whereas bionanotechnology uses

nanotechnological devices and concepts to study biology. No definite distinction

or definition between the use and the application field of these two terms could be

provided and they are treated accordingly.

This thesis is concerned with bionanotechnology which can be subdivided in

molecular machines, biosensors and bioelectronics (Bruchez 2007). The biological

cell is equipped with a variety of molecular machines that perform complex


20/182

Chapter 1

6

mechanical tasks such as cell division or intracellular transport. One can envision

employing these biological motors in artificial environmentsusing motor proteins

for powering or manipulating nanoscale components. Currently realised

applications are merely proof-of-principle demonstrations. Yet, the sheer

availability of an entire ready-to-use toolbox of nanosized biological motors is a

great opportunity that calls for exploration (van den Heuvel et al.2007). Biosensors

and bioelectronics is another sub-discipline that uses biological molecules in

electronic or photonic devices. It seeks to exploit the growing technical ability to

integrate biomolecules with electronics to develop a broad range of functional

devices. An important research aspect is the development of the communication

interface between the biological materials and electronic components (Jin et al.

2008). Biosensors and bioelectronics research also seeks to use biomolecules to

perform the electronic functions that semiconductor devices currently perform,

thereby offering the potential to increase computing-microchip density sufficiently

to continue Moores law down to the nanometer level.

An ideal challenge that will test every functional nut and bolt of

bionanotechnology is finding a treatment solution for cancer (Global industry

analysts, 2011). This disease is the result of compounded abnormal genetic changes

in specific cells. Bionanotechnology may develop quantitative detection andtherapeutic devices for early diagnosis and treatment of cancer. Bionanotechnology

will help design instruments that permeate biological barriers and deliver highly

concentrated and potent drugs focused directly at the erring cells, thereby

enhancing treatment efficacy and reducing side effects. Cardiovascular diseases will

also benefit from bionanotechnology given the feasibility of developing non-

invasive diagnostics and therapy administration systems that target the

atherosclerotic plaque. Ability to sequentially monitor thrombotic and

haemorrhagic activity will aid in preventing strokes and heart attacks. Developingbiodegradable, intelligent nanoparticles (NPs), which can sense abnormal alveolar

functions will aid in effecting drug release only when needed. This is especially vital

in lung inflammatory diseases.

1.3 Nanoparticle technology

Nanoparticles are a type of nanomaterials with size dimensions between 1 100

nm (defined in section 2.1.1). The number of applications making use of NPtechnology is already vast and keeps growing. Some examples of application

domains are in electronics, environment, energy, space, food, consumer products,

chemical sensors and medicine. NPs in electronics, for example quantum dots and


21/182


7

nanotubes, increase the capabilities of electronics devices while reducing their

weight and power consumption. Typical components that can be constructed with

the aid of NPs are transistors, electrodes, memory chips, nano-emissive display

panels and integrated circuits (Liu et al.2009; Ong et al.2005).

Reduction of the environmental impact is obtained through generating less

pollution during the manufacturing of materials. NP based examples are producing

solar cells that generate electricity at a competitive cost, improving the

performance of batteries and making the production of fuels from raw materials

more efficient. Other applications are retroactive by cleaning up organic chemicals

polluting groundwater, capturing carbon dioxide in power plant exhaust, and

clearing volatile organic compounds from air. Durable energy production anddistribution takes large cuts in the income of companies and households. Energy

recuperation and efficiency can be reached with NP based solutions. Examples

include the generation of electricity from waste heat, clothing that generates

electricity, increasing the electricity generated by windmills and reducing friction to

reduce the energy consumption.

As a technology innovating sector, space research has been on the frontline with

NP based applications. Light weight constructions, high tensile strength materials,nanorobots and networks of nanosensors are or will be used in space crafts. Even

in the food sector advances are made in applying smart packaging, anti-microbial

coatings and agents, functional foods, testing in packaged foods and even

interactive foods that change taste and colour by the consumers wishes. In daily

live fabrics which are lighter, stronger, are intelligent, produce energy and can

change properties can be bought. Sport goods are stronger and lighter. Cleaning

products are more effective and can be applied as films to keep windows and

countertops clean.

Typical applications of NPs in the medical field are drug delivery, therapeutics,

diagnostics, imaging and cell repair. Given the current pace of nanotechnology

infusion in medical sciences, solutions are being developed in the treatment of

cancer, cardiovascular diseases, neurological diseases, diabetes, orthopedic ailments

and other infectious diseases. Branched golden NPs cause hyperthermia to

surrounding cells when absorbing infrared light and are investigated as a

theranostic tool against cancer (Van de Broek et al.2011).

In vitro diagnostics (IVD) is more and more based on advancements in

nanotechnology and nanomaterials. The global IVD market includes reagents,

consumables and analysers that are used to perform testing outside the body on


22/182

Chapter 1

8

obtained specimens, such as blood or urine, to measure analytes of interest for

patient evaluation. This market includes immunoassay, clinical chemistry, clinical

microbiology, tissue diagnostics, hemostasis, hematology, molecular diagnostics,

self monitoring blood glucose and point-of-care specific tests. The global IVD

market reached 27 687 M in 2009 and continues to grow at a compound annual

growth rate of 5.4 % (Frost et al. 2010). The global market for nanotechnology

based medical diagnostic kits has been growing from 7 412 M in 2007 to 9 919

M by 2008 (Global Industry Analysts 2011).

A summary of the markets and growth rate of specific NP applications in the

biomedical, pharmaceutical and cosmetic sector; the energy, catalytic and structural

sector; and the electronics and magnetic sector is represented from the year 2007until 2012 inFigure 1-3.The total market size is smaller compared to the general

market sizes described earlier, but the compound annual growth rate varies

between 17 % to almost 30 % (Global Industry Analysts 2011).

Figure 1-3. NP market sizes and growth rate from 2007 until 2012. Comparison of

global NP markets for the biomedical, pharmaceutical and cosmetic sector; the

energy, catalytic and structural sector; and the electronics and magnetic sector.


23/182


9

Recent developments in the diagnostics market are pushed by both established and

emerging companies like for instance Philips and Biocartis. Their innovative point-

of-care diagnostic tests lead the way to how the future diagnostics market will look

like. The starting point was the need to develop blood and saliva testing that can

be carried out in a more patient friendly way, capable of providing speed, ease-of-

use, and robustness with accuracy as obtained in laboratory tests. Philips has

developed a compact, handheld biosensor that is sensitive enough to measure

substances such as hormones, drugs, proteins and nucleic acids. This new type of

biosensor technology uses magnetic NPs to measure picomolar concentrations of

target substances in blood or saliva in a matter of minutes. Integrated into a

disposable biosensor cartridge that inserts into a handheld analyser, it automatically

fills itself from a single drop of blood or saliva. Biocartis is developing novel

diagnostics technology platforms for molecular diagnostics that fully integrate any

sample preparation of nucleic acids, amplification, detection and the generation of

a result without user intervention. A second platform deals with the multiplex

detection of biomarkers.

The emergence of numerous NP fabricators confirms the growing need for these

types of materials. A small selection of companies producing specifically glass

(silica) NPs are Ssnano, Mknano, micromod, Nanograde, Nanopacific, ABCNanotech, Sigma-aldrich, Kisker Biotech, Spherotech, Invitrogen, Ademtech and

Magnamedics. The offered products range from metallic over oxide to polymer

NPs, magnetic to fluorescent cores, bare to succinimide or protein functionalised

surfaces and sizes from 1 nm to 100 m.

NPs are used more and more in many sectors ranging from basic raw materials to

high-end technological applications. The need for control over fabrication

properties of NPs for either bulk or high-grade production rises. Specifically in thehealthcare sector, where the proper operation of the NPs has to ensure the safety

of human health, the conjugation of biomaterials to the NPs has to be precisely

characterised. Many different chemistry strategies exist for conjugating

biomolecules to solid surfaces. Each type of chemistry has different characteristics

concerning type of target to be immobilised, non-specific binding or ability to post

modify. The quantification of biomolecules on NPs used to be performed on an

order of magnitude determination. A more precise quantification is crucial and can

be performed by fluorescent labeling of the target molecule and a minimalinterference of the solid surface. The characterisation at the single NP level should

be in correspondence to trends witnessed on NP bulk level, but has to be

understood as multiplexing concepts with bulk NPs samples envelop different


24/182

Chapter 1

10

reactions at the single NP level. Furthermore, for developing new bio-conjugated

NP applications and concepts, insights into the functionalisation principles and

manipulations of these NPs has to be gained. Biomolecules, which have a defined

non uniform shape, are immobilised in a black box manner with respect to

orientation. SPR sensing devices generally make use of a flow system to operate

the biosensor. An unbiased interaction between the biological moieties and the

solid supports and/or transducers has to be studied. The biological and physical

interaction of biofunctionalised NPs to an upright surface remains largely

uninvestigated. To this day, there still is insufficient knowledge of the behaviour of

biomolecules on or near NPs.

1.4 Objectives and outline of this thesis

Bio-conjungated NPs will be taking a more prominent place in daily live. In this

thesis the focus is on nano-sized glass bio-conjugates, which can be exploited as

valuable tools for diagnostic and therapeutic purposes. For these applications

ssDNA is often linked to silica NPs as specific binding receptor. To ensure a

predictable target affinity, the immobilisation of capture ssDNA has to be precisely

controlled and quantified. The quantification of few biomolecules on NPs is a

difficult and labour intensive task. Consequently, despite the fact that small surfacechanges on the NP can have a dramatic impact on their functionality, many

research groups chose to avoid this optimisation because of the complexity.

The specific objective of this thesis is to study the crucial aspects of

creating and characterising ssDNA functionalised silica nanoparticles and

to describe the behaviour of these bioconjugated nanomaterials for

biosensing.

A schematic outline of this dissertation is given inFigure 1-4.

In Chapter 2 an introduction to NPs and their properties is presented. The stability

theory, or the study of basic NP behaviour, is described for DNA functionalised

silica NPs. The manufacturing methods of silica NPs are discussed as well. Finally,

an introduction to biosensing with silica NPs is supplied.

In Chapter 3 the functionalisation and characterisation of short ssDNA on silica

NPs and their use in DNA sensing is studied. Starting from the characterisation ofthe bare silica NPs and the means to quantify functionalisation of DNA on the

silica NPs, the influence of different immobilisation protocols and complementary

DNA overlap length on hybridisation will be investigated.


25/182


11

In Chapter 4 the results of Chapter 3 are checked at the single molecule level.

Advanced optical technologies are applied to quantify the number of ssDNA

molecules on a single NP, and to relate this information to bulk measurements. In

order to accurately determine low numbers of immobilised ssDNA molecules at a

single NP surface an integrated approach based on Classic Single Molecule

Confocal Microscopy and modified Total Internal Reflection Fluorescence

Microscopy is proposed. Furthermore the possibilities of defocused single

molecule spectroscopy to determine a 3D distribution of oligonucleotides on a

silica NP are investigated.

In Chapter 5 the factors influencing ssDNA, functionalised on silica NPs, to

hybridise with DNA probes immobilised on the FO-SPR sensors gold surface arestudied. First, the boundaries of NP size and concentrations, specificity and buffer

constitution were investigated to obtain trustworthy results. Next, the FO-SPR

setup was used to study binding kinetics of silica NPs functionalised with DNA

strands of a different length. Finally, discrimination of these different length

ssDNA functionalised NPs was obtained by implementing a melting scheme.

In the final chapter a general conclusion is formulated and some suggestions for

future work are postulated. The combination of advancements in NP synthesis,bioconjugation to NP surfaces and optimised sensor detection schemes will open

up new opportunities.


26/182

Figure 1-4. Schematic representation of the structure of this thesis.


27/182


13

Reference List

Abu-Salah KM, Ansari AA, and Alrokayan SA (2010) DNA-Based

Applications in Nanobiotechnology. Journal of Biomedicine andBiotechnology. 2010:715295.

Berna J, Leigh DA, Lubomska M, Mendoza SM, Perez EM, Rudolf P, Teobaldi

G, and Zerbetto F (2005) Macroscopic transport by synthetic molecular

machines. Nature Materials. 4(9):704-710.

Binnig G, Rohrer H, Gerber C, and Weibel E (1982) Tunneling Through A

Controllable Vacuum Gap. Applied Physics Letters. 40(2):178-180.

Browne WR and Feringa BL (2006) Making molecular machines work. Nature

Nanotechnology. 1(1):25-35.

Bruchez, M. P. (2007). Commercialization and future developments in

bionanotechnology. In: Frontiers of engineering: reports on leading-

edge engineering from the 2006 symposium. National Academy of

Engineering. National academies press, Washington DC, 73 - 81.

Dutta, P. (2006) Understanding of Nano Science and Technology. Gupta, S. andDutta, P. Global Vision Publishing House, New Delhi, 330p.

Frost and Sullivan (2010) Strategic Analysis of the Global In Vitro Diagnostics

Market. 122p.

Global Industry Analysts, Inc. (2011) Nanobiotechnology: A Global Strategic

Business Report. 2423p.

Jin YD, Honig T, Ron I, Friedman N, Sheves M, and Cahen D (2008)Bacteriorhodopsin as an electronic conduction medium for

biomolecular electronics. Chemical Society Reviews. 37(11):2422-

2432.

Liu Y, Flood AH, Bonvallett PA, Vignon SA, Northrop BH, Tseng HR,

Jeppesen JO, Huang TJ, Brough B, Baller M, Magonov S, Solares SD,

Goddard WA, Ho CM, and Stoddart JF (2005) Linear artificial

molecular muscles. Journal of the American Chemical Society.

127(27):9745-9759.


28/182

Chapter 1

14

Liu YT, Zhang ZL, Zhao W, Xie XM, and Ye XY (2009) Selective self-

assembly of surface-functionalized carbon nanotubes in block

copolymer template. Carbon. 47(7):1883-1885.

Michelson ES (2011) What Is Nanotechnology and Why Does It Matter? From

Science to Ethics. Science and Public Policy. 38(4):334-335.

Niemeyer, C. M. and Mirkin, C. A. (2004) Nanobiotechnology: concepts,

applications and perspectives. Wiley-VCH, Weinheim, 469p.

Ong BS, Wu YL, Liu P, and Gardner S (2005) Structurally ordered

polythiophene nanoparticles for high-performance organic thin-film

transistors. Advanced Materials. 17(9):1141.

Phoenix C and Drexler E (2004) Safe exponential manufacturing.

Nanotechnology. 15(8):869-872.

Prasad, S. K. (2008) Modern Concepts in Nanotechnology. Discovery

Publishing House, New Delhi, 32p.

Rodgers P (2006) Nanoelectronics: Single file. Nature Nanotechnology.

5(10):1038.

Sleytr U (2006) Nanobiotechnology: An Interdisciplinary Challenge.

Van de Broek B, Devoogdt N, D'Hollander A, Gijs HL, Jans K, Lagae L,

Muyldermans S, Maes G, and Borghs G (2011) Specific Cell Targeting

with Nanobody Conjugated Branched Gold Nanoparticles for

Photothermal Therapy. Acs Nano. 5(6):4319-4328.

van den Heuvel MGL and Dekker C (2007) Motor proteins at work fornanotechnology. Science. 317(5836):333-336.

EC European Commission (2010a) Draft Commission Recommendation on

the definition of the term nanomaterial[online]. EC DG Environment.

Beschikbaar op

http://ec.europa.eu/environment/consultations/nanomaterials.htm


29/182

Literature review: nanoparticles from theory to diagnostic applications

15

2

Chapter 2 Literature review:

Nanoparticles, from theory to diagnostic

applications

2.1 Introduction

Nanomaterials and nanoproducts are being increasingly manufactured and appliedin many day-to-day aspects of human life, such as in medical applications, thecosmetic sector, the microelectronic industry, and the agricultural sector.Nanotechnology includes a wide range of technologies throughout these differentsectors. Although many products already exist today, there is still great potentialfor further growth and development. But for the time being, producing smaller

materials also increases the uncertainties about the consequences of their use.Many studies adress the effect nanoparticles (NPs) might have on human health orthe environment (Dreher 2004; Gwinn et al.2006).


30/182

Chapter 2

16

In the following literature study the what, how and why of silica NPs innanotechnology is reviewed. First a description of a NP is given. Second, the mainforces between NPs are studied. Next, the methods to synthesise and modify silica

NPs are described along with the methods to biochemically conjugate theirsurface. Finally, the interaction between biological material and the NPs isdescribed and some applications of silica NPs in biosensing are highlighted.

2.1.1 Definition

For many years, the definition of NPs, originally put forward by the NationalNanotechnology Initiative (NNI), was used (Roco 2001): Those particles with

dimensions between approximately 1 and 100 nm where unique phenomenaenable novel applications.

Many scientists now consider this 100 nm cut-off too limiting and arbitrary(Maynard et al.2007). There are nanoscale phenomena that occur when individualfeatures are larger than 100 nm or the particles are in assemblies. There are alsomany materials that are not different below the 100 nm threshold than above it.The absence of a concrete definition for nanomaterials is implicitly linked with thebasic question of whether NPs should be regulated differently than their bulkcounterparts (Marquis et al.2011) .

Recently the European commission (EC) proposed a general legislative referencedefinition for nanomaterials. A nanomaterial meets at least one of the followingcriteria: (i) it consists of particles, with one or more external dimensions in the sizerange between 1 nm - 100 nm for more than 1 % of their number size distribution,(ii) it has internal or surface structures in one or more dimensions in the size rangebetween 1 nm 100 nm, (iii) it has a specific surface area by volume greater than

60 m2cm-3, excluding materials consisting of particles with a size lower than 1 nm.In his context a particle was defined as a minute piece of matter with definedphysical boundaries (ISO 146446:2007).

The regulation of engineered NPs requires a widely agreed definition of suchparticles. It is argued that evidence for novel size-dependent properties, rather thanparticle size, should be the primary criterion, especially when the definition isapplied to make decisions about their regulation for environmental, food, health

and safety issues (Auffan et al.2009).


31/182


17

2.1.2 Nanoparticle materials and shapes

The oldest known application of NPs is the use of gold and silver NPs for glass

staining and ceramics colouring. The bright colours are the result of the NPsabsorbing or quenching, scattering, and resonating incoming light. These NPs wereproduced by pouring AuCl4- liquid into molten glass. To improve the lightprotecting abilities of sunscreen small TiO2 plates were added to the crme, whichorganise themselves into a flat reflecting surface. As the many already existingapplications prove, NPs come in many forms and shapes with a multitude ofcharacteristics. NPs can be synthesised starting from various materials with eachmaterial having its own distinct nano-characteristics. Some materials can even be

combined in a single NP to make use of the different material properties. In manyresearch groups the gold and silver NPs are still used abundantly. Other materialsinclude ferro-oxide NPs, which have superparamagnetic properties, or polystyreneNPs, which are prepared from a polymer and have high surface conjugationfunctionality. Quantum dots are a combination of a heavy metal embedded in azinc sulphide casing, which produces a sharp emitted fluorescence peak while theexcitation profile is very wide. The properties and applications of these materialsare further described in section2.3.4.Different methods are needed to fabricate as

well as functionalise the NPs. In this thesis the focus will be put on silica NPs, asthese are easy to fabricate, conjugate, modify and are biocompatible (Gerion et al.2001). Starting from the same basic molecules, a multitude of shapes can befabricated in order to manipulate or alter the NPs properties (Figure 2-1).


32/182

Chapter 2

18

Figure 2-1. Scanning electron microscopy images of different silica NP shapes. A)

amorphous (Bagwe et al .2006), B) spherical (Rieter et al .2007), C) amorphous core

shell (Yanet al .

2007), D) spherical core shell (Kimet al .

2008), E) brush like

mesoporous (Polshettiwar et a l . 2010), F) cage like (Cao et al . 2007), G)

Redispersable silica nanoparticles Fraunhofer-Gesellschaft, H) hexagonal

(Tomczak et al .2005), I) spherical mesoporous (Nandiyanto et al .2009).

2.2 Nanoparticle stability

Depending on their characteristics, silica NPs will interact differently with theirenvironment. In the following paragraphs the parameters influencing interparticlebehaviour are described by the DLVO theory, named after Derjaguin, Landau,Verwey and Oberbeek. Other physical properties, however beyond the scope ofthis thesis, include the NP porosity and the thermal stability of the NPs. The NPporosity changes the density and available surface area of the silica NPs (Trewyn et

al. 2007a). The internal pore and exterior particle surface can be differentlyfunctionalised. These porous NPs are often applied in drug delivery or catalysisapplications. Silica NPs are also known for their thermal stability and remain intact


33/182


19

up to 1000 C (Ma et al. 2004). The silica NPs are even applied to enhance thethermal stability of metallic NPs (Radloff et al.2001; Xia et al.2004).

As silica NPs are often applied in solvents, it is important to distinguish betweenthe different modes of dispersion. The dispersion of particles in a medium isdivided in solutions, colloids and suspensions. Solutions are atoms and moleculeswhich are completely dissolved in a solvent. Suspensions consist of larger particles,which will settle due to gravity. Colloids are composed of intermediate sizeparticles, which stay homogeneously dispersed without dissolving or settling out.

Colloidal NPs are defined as a dispersion of finely-divided solid NPs in a

continuous liquid phase that will eventually settle (Hiemenzet al.

1997). NPsuspensions are of great practical interest in both the industry and everyday life.Common applications are found in coatings and paints, printing inks, ceramics,food, pharmaceutical and household products. The stability of the suspensions isimportant in many of these technical applications. A thermodynamically stabledispersion remains suspended indefinitely, while a kinetically stable dispersionremains suspended for a limited time. NP-suspensions are usually only kineticallystable, not thermodynamically stable, hence NPs tend to agglomerate and settle

over time. The kinetic stability of colloidal NPs in liquids is determined by abalance between attractive and repulsive forces. The particle interactions incolloidal systems will be discussed in the following sections. Due to the importancein this thesis emphasis lies on silica NPs in aqueous suspensions.

Generally, in aqueous suspensions repulsive forces arise from electrostatic andsteric interactions (Barnes et al. 1991; Holmberg et al. 1996; Tadros 1996). SolidNPs normally acquire charges at their surface when suspended in aqueoussolutions. This can occur either by ion adsorption from the medium and/ordifferential loss of ions from the solid crystal lattice (Morrison et al. 2002). Thissurface confined charge, expressed as the electrostatic potential energy of a surface,is often defined as the surface potential.

When a charged surface is present in an aqueous solution some ions are attractedto or repelled from this surface. Thus, the NPs charges are influenced by the ionicstrength of the solution. Counter ions will distribute around the NPs and form anelectrical double layer consisting of an inner layer and a diffuse layer. The inner

layer or Stern layer is comprised of ions, which are firmly attached to the NPsurface (Figure 2-2A). The attached ions have the opposite charge of the NP. Thepotential drops almost linearly through the Stern layer. The potential beyond theStern layer, or at the shear plane of mobile ions, is called the Zeta-potential (Figure


34/182

Chapter 2

20

2-2B). The Zeta-potential can be measured by the electrophoretic mobility of theNPs using dynamic light scattering (DLS).

Figure 2-2. Schematic representation of the Stern layer and the diffuse layer. The

NP sphere represents the NP with intrinsic negative charges and the small spheresrepresent ions with negative and positive charges. In the underlying graph the

potential energy is drawn as a function of the distance from the NP. The surface

potential, the Zeta-potential at the Stern layer, the diffuse layer and the Debye

length are indicated.

The diffuse double layer consists of a mixture of positive and negative ions that areloosely attached to the particle surface (Delgado et al.2005). The main contributionof counter ions in the diffuse layer have an opposite charge than the surfacecharge. The potential decays exponentially from the surface potential through theZeta-potential almost reaching zero at a certain distance from the colloid. Standardtechniques for determination of the Zeta-potential include micro-electrophoresisand dynamic light scattering. Electrophoresis is the motion of dispersed particlesrelative to a fluid under the influence of a spatially uniform electric field (Gas2011). Micro-electrophoresis is a method of studying electrophoresis of variousdispersed particles using optical microscopy. Dynamic light scattering measures thefrequency shift or phase shift of an incident laser beam on the dispersed particles

which depends on the mobility of the NPs.NPs can either repulse or attract each other. Repulsive forces keep the particlesseparated when two particles approach each other. The high concentration of ionsin the electrical double layer between the particles will result in an osmotic pressure


35/182


21

and a coulomb repulsion. The combination of these effects is called the electro-

osmotic effect. The Debye length (1/) is the distance around the NP where allions are either attracted to or repulsed from the NP as indicated by the arrows in

Figure 2-2. Outside of this sphere the charges are screened. Integration of allelectro-active forces around one NP from the surface to the Debye length resultsin the electro-osmotic energy of the NP.

where:

I is the ionic strength of the electrolyte, and here the unit should be mol m -3,

0is the permittivity of free space (8.85 1012F/m),

ris the relative dielectric constant,

kBis the Boltzmann constant (1.38 10-23m2kg s-2K-1),

T is the absolute temperature in kelvin,NAis the Avogadro number (6.021023mol-1),

e is the elementary charge (1.60 10-19coulombs).

The attractive energy of Van der Waals is the sum of all attraction energies exceptfor electrostatic and covalent interactions. Its origin lies in dipole, induced dipoleand dispersion interactions. Van der Waals forces are relatively weak compared to

normal chemical bonds. The magnitude of the Van der Waals energy is surfacearea dependent. Derjaguin, Landau, Verwey and Oberbeek combined the conceptsof electro-osmotic effect and van der Waals interactions resulting in the DLVOtheory (Figure 2-3) (Derjaguin et al.1941; Verwey et al.1999). At far and at shortinterparticle distances the attracting Van der Waals energy is much larger than therepulsive electro-osmotic energy on particles. By increasing the ion concentrationof the suspension the repulsion forces are reduced.


36/182

Chapter 2

22

Figure 2-3. Potential energy of the repulsive and attractive forces as a function of

the interparticle distance. The summation of these two forces results in the DLVO

potential with the typical potential barrier.

The DLVO theory is not effective in describing ordering processes, such as theevolution of colloidal crystals in dilute dispersions with low salt concentrations orthe relation between the formation of colloidal crystals and salt concentrations (Ise1998). When the DLVO theory fails to explain experimental results an extra termis often added. Extra terms are for example hydration, hydrophobic, oscillatoryand water structure forces. The theory is then renamed to the extended DLVOtheory. For example particles with very high hydrophobicity or very highhydrophilicity cannot be easily described by the DLVO theory. For hydrophilicsilica suspended in water the repulsion force at short range (


37/182


23

Figure 2-4. Comparison of energy barrier between two silica particles calculated by

the DLVO or the extended DLVO theory. Reproduced from Ch en et a l . 2007).

Although the hydration forces are generally accepted, some studies attribute theextra stability to a 10 thick gel-like layer of protruding silanol and silicilic acidgroups in water instead of a fixed water layer on the surface (Vigil et al.1994). This

assumption is based on four measured phenomena: (i) a time dependent change ofthe adhesion ability on silica surfaces, (ii) the friction between two surfaces withconsiderable sticking whose magnitude is water and time dependent, (iii) a stableinstead of oscillatory non-DLVO repulsion, and (iv) dynamic contact anglemeasurements with time dependent effects which are not due to surfaceroughness. These phenomena indicate that the silica surfaces slowly undergostructural and chemical changes with water and each other.

The relative magnitude of the above interactions not only governs the stability ofsuspensions but also their rheological behaviour (Hiemenz et al.1997; Savarmand etal. 2003; Zaman et al. 1996). A suspension is stable when the repulsive forcesdominate, while the presence of strong attractive forces causes particle aggregation.The electrostatic and structural repulsion stabilises the suspension kinetically dueto the formation of the potential barrier preventing particles from coagulating(Dabir et al.1996; Guo et al.2004; Kelland 2006; Sjoblom et al.2003; Venkatesan etal. 2005). pH and ionic strength are factors that will influence the relative

magnitude of attractive and repulsive forces. Electrostatically stabilisedsuspensions can be easily destabilised by a change of pH which reduces the surfacepotential, or by increasing the electrolyte (salt) concentration which decreases thethickness of the electrical double layer. In this case the potential barrier can be


38/182

Chapter 2

24

considerably lowered resulting in slow coagulation with reaction limited kinetics oreven fully removed causing rapid coagulation with diffusion limited kinetics.

The coagulation process is not absolute and usually equilibrates to form clusters ofa definite dimension. The size of this cluster is determined by the balance betweenthe attractive and repulsive forces on the NPs. If a particle joins a cluster then itsattractive interactions with those in the cluster result in a decrease of the systems

energy whereas the electro-osmotic repulsive interactions have the systems energyincreased (Branda et al. 2010). The attractive interactions are short ranged,therefore they are established only within the nearest neighbours in the cluster.Repulsive interactions are long ranged, therefore they can involve more distant

particles in the cluster. Hence, if the cluster is big enough, the increase of thesystems energy due to the repulsive forces exceeds the energy decrease due toattraction (Kovalchuk et al.2009).

For electrostatically stabilised colloids at a fixed Zeta-potential the particle sizeshould exceed a certain critical value to enable coexistence of single particles andclusters (Lai et al.2001; Lai et al.2002; Victor et al.1985). It was estimated by Victorand Hansen (Victor et al.1985) that for aqueous suspensions at room temperature

and a Zeta-potential about 25 mV, the critical diameter of particles should beabout 400 nm. Smaller particles will aggregate fast and to a large extent unless theirZeta-potential is large enough. Similar estimations performed by Lai et al.and Laiand Wu respectively gave a critical diameter of about 500 nm and 300 nm (Lai et al.2001; Lai et al.2002; Victor et al.1985).

2.3 Silica nanoparticles

The element silicon is known to exist in a variety of forms that differ bothphysically and chemically (Vigil et al.1994). Although it is not found in its nascentform, it is always present in combination with oxygen (as in silica) or hydroxides(as in silicic acid). Silicon is found in the environment in different forms. 78% ofearths crust consists of silicon and oxygen compounds like quartz, flint, opal,silicates etc. Even in living organisms like sponges, grasses and algae silica is found(Iler 1979). Silicon is also present in dissolved form in the oceans as silicic acid.

Quartz, for example sand, has a crystalline morphology, and fused silica, or glass,has an amorphous structure. Their surface behaviour originates from the atomicstructure of the silica. This surface can be either hydrophobic with siloxane groups(R2SiO) or hydrophilic when the surface exposes silanol groups (R3SiOH). In this


39/182


25

section the surface charge, synthesis, surface conjugation and modification of silicaNPs is discussed.

2.3.1 Silica nanoparticle surface charge

In the case of silica, the NPs generally possess a high surface potential because ofthe high density of active silanol (-SiOH) groups (around 1.8 per nm) on thesurface (Barthel et al. 2002). The different appearances of silica are describedfurther in section 2.3. When dispersed in aqueous solutions the ionisation ofreactive silanol groups causes the appearance of charges on the particle surface.The surface charge density and potential are influenced by pH and ionic strength

of the surrounding solution. At pH levels above the iso-electric point pKa 9.0, thesurface becomes negatively charged due to the following reaction (Dong et al.1998).

At lower pH values the second type of silanol groups with pKa 5.5, react withprotonated water creating positive groups according to the following reaction(Brinker 1988).

According to Dong et al. there are two types of silanol groups at the silica/waterinterface, with different pKa values and different surface populations. The silanolgroups with the lower values of pKa (5.5) and surface population (19%) arebelieved to be isolated silanol groups, because isolated silanol groups can dissociatemore readily compared to the silanol groups coupled to each other through

hydrogen bonds. The silanol groups with the higher values of pKa (9.0) andsurface population (81%) are believed to be those connected to each other throughhydrogen bonds.

2.3.2 Silica nanoparticle synthesis

Spherical silica NPs are generally made by one of two synthetic routes: reversemicro emulsion (RME) or the Stbers method. Like any other synthesis ofcolloids, the diameter of silica particles is mainly controlled by the relativecontribution of nucleation and growth. The two methods are briefly described anddiscussed with both their advantages and disadvantages in the next paragraphs.


40/182

Chapter 2

26

Porous and branched NPs can also be synthesised, but fall beyond the scope ofthis thesis (Figure 2-5)(Bhattacharyya et al.2006).

Figure 2-5. SEM picture of A) mesoporous silica NPs (Bhattacharyyaet al .

2006)

and B) branched silica NPs (Szczech et al .2010).

Both the RME and Stber synthesis pathways are very comparable as both usehydrolysis and condensation of silicate precursors, tetrahydral conformation ofsilicium and oxygen. The hydrolysis and condensation reactions provide precursorspecies and the necessary supersaturation for the formation of particles. Thereaction mechanism is described for the silicate precursor tetraethyl orthosilicate(TEOS) (Figure 2-6).

Figure 2-6. Structure of tetraethyl orthosilicate (TEOS)

During the hydrolysis reaction, the ethoxy group of TEOS reacts with a watermolecule to form an intermediate (Si (OC2H5)4-x (OH)x) with hydroxyl groupssubstituting the ethoxy groups (Figure 8). Ammonia works as a basic catalyst to

facilitate this reaction. The hydrolysis reaction is initiated by the attacks of hydroxylanions on the TEOS molecules. Following the hydrolysis reaction, thecondensation reaction occurs immediately. The hydroxyl group of the intermediate(Si (OC2H5)4-x (OH)x) reacts either with the ethoxy group of other TEOS or the


41/182


27

hydroxyl group of another hydrolysis intermediate to form Si-O-Si bridges (Greenet al.2003).

Figure 2-7. Reaction scheme of the hydrolysis and condensation of silica precursors

to create a silica NP.

After some growth of the silicate precursor a NP is created presenting a mixture ofsurface groups (Figure 2-8). The hydration of this mixture of surface groups isinfluenced by the pH of the solution.

Figure 2-8. Molecular structure of a silica NP presenting different silicic acid

surface groups (Mannet al .

1983).

2.3.2.1 Reverse micro emulsion method

The reverse micelle or water-in-oil (w/o) micro emulsion system is composed of ahomogeneous mixture of water, oil, and surfactant molecules (Lindberg et al.1995;

Osseoasare et al. 1990; Yamauchi et al. 1989). The single-phase micro emulsionsystem is thermodynamically stable. Water nanodroplets form in the bulk oil phase,which then act as containers or nanoreactors for discrete particle formation. Microemulsions have been used as chemical reactors because of their special interfacial


42/182

Chapter 2

28

properties allowing an intimate contact, at nanoscale level, of hydrophilic andhydrophobic domains. Size of the created NPs is generally between 30 and 70 nmand can be precisely controlled by changing the surfactant to solvent ratio. It is

even possible to create hollow spheres with the micro emulsion method. Adrawback is the need for sometimes toxic surfactants and the limited maximumsize of NPs (90 nm, (Han et al.2008)that can be created. Furthermore, the use ofsurfactants necessitates extensive washing to remove the surfactant moleculesbefore any biological application in order to avoid denaturation by the surfactantmolecules. Advantages of the micro emulsion method are the robustness againstmany reaction conditions and smooth surfaces of the NPs (Darbandi et al.2005). On top of that, the micro emulsion system typically generates monodisperse

and highly uniform NPs (Schmidt 1999).

2.3.2.2 Stber method

Hydrolysis of alkyl silicates (TEOS) and subsequent condensation of silicic acid inalcoholic solutions using ammonia as a catalyst was first published in 1968 byStber. Fairly monodisperse silica NPs with diameters ranging from 30 nm to 2

m are synthesised by the Stber method (Stober et al. 1968). In the Stber

synthesis silica particles are formed in the presence of a mixture of ethanol andammonia which is vigorously stirred. The synthesis method is usually performed asa one pot reaction and there is no need for any surfactants. The attained NPsusually have a wider size distribution and it is difficult to produce small sized NPs.Other drawbacks are the high requirements on purity of the reactants, the difficultyand multiplicity of the preparation steps (Bagwe et al. 2004; Darbandi et al. 2005;Rossi et al.2005).

2.3.3 Surface conjugationOnce NPs have been synthesised, before they can be applied for bio-assaydevelopment, an additional layer of linker molecules with various reactivefunctional groups (e.g., amine, thiol, carboxyl or methacrylate) must be attached.These functional groups usually electrostatically stabilise the NPs and /or act as ascaffold for the coupling of biological moieties like dextran, antibodies, DNA orpeptides. The next paragraphs describe the stabilising linking layer, and thedifferent bioconjugation schemes.


43/182


29

2.3.3.1 Linker layer

The functional groups are typically attached by applying an additional silica coating

(post-coating) that contains the desired functional group. This linker layer is oftenmade up of a silane, which reacts with the silica NP, an alkane spacer and afunctional group for further conjugation. In addition to providing the reaction sitesfor bioconjugation, the functional groups also change the colloidal stability of NPsin solution. At a high density and with orderly arrangement of this layer on the NPsurface a self-assembling monolayer (SAM) is formed. SAMs are a type of covalentlinker layer stabilised by dispersion interactions and were discovered in 1983(Nuzzo et al. 1983). These monolayers are constructed from molecules which

assemble themselves autonomically in patterns and structures without humanintervention (Figure 2-9). SAM structures have a well-known interface, providing adefined immobilisation and sometimes orientation of biomolecules (Lu et al.1995).For example, inert negatively charged organo-silane compounds containingphosphonate groups (R3Si-(CH2)n-PO(OH)2) can be introduced into NPs duringpost-coating (Santra et al. 2004). This step increases the repulsive forces betweenthe particles in solution and thus improves long-term NP stability. In addition,polyethylene glycol (PEG, a neutral polymer) can also be introduced to the NP

surface in order to reduce nonspecific binding by inhibiting the adsorption ofundesired charged biomolecules (Hermanson 2008; Wang et al. 2007). The PEGmolecule is generally linked to the coated functional groups and acts as a secondlayer with a different functional group or a mixture of functional groups. By addingdextran, polystyrene or dendrimers to the NP surface the surface area is greatlyenhanced and a multitude of functional groups are added to the NP. Dextran, forinstance, is a sugar-based polymer, which forms a branched hairy structure on theNP surface. It introduces free carboxyl groups and is biocompatible (Lu et al.

2007).


44/182

Chapter 2

30

Figure 2-9. Self-assembled monolayer (Prof. Hiroyuki, www.mtl.kyoto-u.ac.jp).

2.3.3.2 Bioconjugation

When developing a bioconjugated nanomaterial a commonly arising problem is thelow reproducibility and the limited stability of the bio-nanosurface. Special careshould be taken toward the structure of the linking layer and the orientation of thebio-molecule on this layer. The immobilisation of biological components isperformed in many different ways, with the four most important types summed up

in the next paragraphs (Figure 2-10).

Figure 2-10. Classification of bio-molecule immobilisation techniques reproduced

from (Turner et al .1987)).

Physical adsorption can conjugate bio-molecules with weak Van der Waals forces,electrostatic interactions and hydrogen bridges on clay, aluminium, cellulose,

polystyrene, silica gel or glass. Typically, the bio-molecule is not modified, but theNP will lose its bio-functionality at different pH, ionic buffer strength andtemperature (Eggins 2002). It is also possible to apply charged adapter moleculesor proteins modified to incorporate charged domains (Roy et al. 2005; Zhu et al.


45/182


31

2004). The intrinsic charge of DNA allows adsorption to plain silica NPs (Nguyenet al.2007).

Cross linking or covalent bonding produces a chemical bond between the bio-molecule and the NP, a polymer or itself by means of a bifunctional reagent(Eggins 2002). Cross-linked bio-molecules are either adsorbed or covalently boundto the NP surface. Covalent binding is subdivided between affinity basedinteractions (such as streptavidin biotin), and covalent interactions (such ascarbodimide). Covalent binding of bio-molecules to NPs is preferred over theabove methods, not only to avoid desorption from the NP surface, but also tocontrol the number and orientation of the immobilised bio-recognition entities.

Great care should be taken to avoid that these covalent reactions interfere with thefunctionality of the bio-molecule.

The immobilisation method of choice depends on the nature of the bio-molecule,the type of NP, the physico-chemical properties of the analyte and the operatingbounds. The chosen method greatly influences the bio-nanomateria

phd_delport+filip (1).pdf

Documents