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Polymeric microfilters by interference holography :development and applicationsCitation for published version (APA):Prenen, A. M. (2009). Polymeric microfilters by interference holography : development and applications.Technische Universiteit Eindhoven. https://doi.org/10.6100/IR652646

DOI:10.6100/IR652646

Document status and date:Published: 01/01/2009

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https://doi.org/10.6100/IR652646

https://doi.org/10.6100/IR652646

https://research.tue.nl/en/publications/polymeric-microfilters-by-interference-holography--development-and-applications(3a562f4a-8a38-47a8-aab4-2dbefc3ec1c2).html

Polymeric microfilters by

interference holography:

Development and applications

PROEFSCHRIFT

ter verkrijging van de graad van doctor aan deTechnische Universiteit Eindhoven, op gezag van de

rector magnificus, prof.dr.ir. C.J. van Duijn, voor eencommissie aangewezen door het College voor

Promoties in het openbaar te verdedigenop maandag 12 oktober 2009 om 16.00 uur

door

An Maria Prenen

geboren te Bree, Belgie

Dit proefschrift is goedgekeurd door de promotor:

prof.dr. D.J. Broer

Copromotor:dr.ing. C.W.M. Bastiaansen

A catalogue record is available from the Eindhoven University of TechnologyLibrary

ISBN: 978-90-386-2014-5

Printed by the Eindhoven University Press, the Netherlands.

This research was financially supported by NanoNedproject EPC 7120.

Voor mama en papaVoor Xander

i

Contents

Summary v

1 General introduction 11.1 Miniaturization in medical diagnostics . . . . . . . . . . . . . . 21.2 Microfiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . 41.3 Holography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.4 Scope of the thesis . . . . . . . . . . . . . . . . . . . . . . . . . 81.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2 Flow through holographic membranes 132.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142.2 Flow theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142.3 Flow simulations . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2.3.1 Basics of FEM simulations . . . . . . . . . . . . . . . . 182.3.2 Round vs slit-shaped pores . . . . . . . . . . . . . . . . 212.3.3 Aspect ratio . . . . . . . . . . . . . . . . . . . . . . . . . 222.3.4 Vena Contracta . . . . . . . . . . . . . . . . . . . . . . . 23

2.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

3 Fabrication of holographic membranes 293.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303.2 Holography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

3.2.1 Theory of transmission holography . . . . . . . . . . . . 313.2.2 Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313.2.3 Holographic setup . . . . . . . . . . . . . . . . . . . . . 333.2.4 Recording materials . . . . . . . . . . . . . . . . . . . . 34

3.3 Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343.4 Fabrication of holographic membranes . . . . . . . . . . . . . . 37

ii

3.4.1 Square array of circular pores . . . . . . . . . . . . . . . 373.4.2 Slit-shaped pores . . . . . . . . . . . . . . . . . . . . . . 393.4.3 Tapered cross-section . . . . . . . . . . . . . . . . . . . 413.4.4 Integrated support . . . . . . . . . . . . . . . . . . . . . 43

3.5 Experimental verification FEM simulations . . . . . . . . . . . 453.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

4 In-plane membranes 494.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . 514.3 Fabrication of in-plane membranes . . . . . . . . . . . . . . . . 53

4.3.1 Influence of tilt angle . . . . . . . . . . . . . . . . . . . 534.3.2 Influence of periodicity . . . . . . . . . . . . . . . . . . . 54

4.4 Slanted-angle mask holography . . . . . . . . . . . . . . . . . . 554.5 In situ fabricated filters for microfluidics . . . . . . . . . . . . . 554.6 Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . 57

4.6.1 Selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . 574.6.2 Flow resistance . . . . . . . . . . . . . . . . . . . . . . . 58


5 Filters for biomedical applications 635.1 Flow-through microarray technology . . . . . . . . . . . . . . . 64

5.1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . 645.1.2 Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

5.2 New concept:Lateral Immuno Flow-through Elements sensor . . . . . . . . . 685.2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . 685.2.2 Microvortexers . . . . . . . . . . . . . . . . . . . . . . . 705.2.3 In-plane membranes . . . . . . . . . . . . . . . . . . . . 73

5.3 Filtration of blood . . . . . . . . . . . . . . . . . . . . . . . . . 745.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

6 Surface modifications for immobilization of biomolecules 796.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 806.2 Surface treatment . . . . . . . . . . . . . . . . . . . . . . . . . . 81

6.2.1 Capillary filling theory . . . . . . . . . . . . . . . . . . . 81

iii

6.2.2 Capillary flow experiments in an SU8 microchannel . . . 836.3 SU8 functionalization . . . . . . . . . . . . . . . . . . . . . . . 85

6.3.1 Pendant epoxide groups . . . . . . . . . . . . . . . . . . 866.3.2 Immobilization of antibodies . . . . . . . . . . . . . . . 886.3.3 Functional assay on SU8 . . . . . . . . . . . . . . . . . . 90


7 Glancing angle lithography 977.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 987.2 Reflection exposure theory . . . . . . . . . . . . . . . . . . . . . 997.3 Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1017.4 Process parameters . . . . . . . . . . . . . . . . . . . . . . . . . 102

7.4.1 Exposure dose dependence . . . . . . . . . . . . . . . . 1027.4.2 Acid diffusion . . . . . . . . . . . . . . . . . . . . . . . . 1037.4.3 Angle dependence . . . . . . . . . . . . . . . . . . . . . 1047.4.4 Sealing of microstructures . . . . . . . . . . . . . . . . . 105


Samenvatting 109

List of Publications 113

Acknowledgments 115

Curriculum Vitae 117

v

Polymeric microfilters by interference holography:

Development and applications

Summary

In the past two decades, medical diagnostics have shifted their interest from thelab towards point-of-care testing. Biosensors have led to a tremendous progressin the development of new or strongly improved point-of-care tests. Twoimportant components in a biosensor are microchannels and membranes. Themicrochannels enable the reduction of the sample volume, and a membranecan have various functions. For instance, its large internal surface can serveas substrate for the adsorption of biological species and of course it can alsobe used for the filtration of e.g. blood cells or platelets from a sample.

Numerous different types of membranes exist and usually they have ahighly polydisperse pore size which limits efficiency and throughput. Mi-crosieves, or monodisperse microfiltration membranes, have an almost idealgeometry for efficient separation in combination with a high throughput. Nowa-days, several techniques are being developed for the fabrication of these mi-crosieves. Processes such as silicon micromachining, phase separation micro-molding, track etching all have their specific merits, but none are ideal andthey are often very difficult to implement in biosensors. With interferenceholography, membranes can be produced with well-defined pore shapes, a highefficiency and a low pressure drop with the ease of fabrication and materialproperties of polymers. To achieve these, holography makes use of the in-terference pattern generated by two crossing, parallel polarized laser beams.The periodicity of this pattern can be tuned by changing the angle betweenthe two laser beams. A chemically and thermally resistant photoresist, SU8,is used to digitally record the analogue interference pattern. SU8 allows formultiple exposures to an interference pattern due to its high glass transitiontemperature and the final structure is a simple addition of all exposure steps.A wide range of circular pore sizes (0.1 µm - 5 µm) was fabricated by tuning

vi

the angle between the interfering laser beams.To investigate the geometrical parameters influencing the flux through

a holographic membrane, finite element method simulations were executed.Simulations show that elongated, elliptical pores have a several advantages incomparison to the standard, circular pores. Due to their improved surfaceto volume ratio, the flow resistance induced by the presence of the pore wallis lower, resulting in a larger flux. Membranes with slit-shaped pores havethe additional advantage that, in general, they have a larger porosity. It wasshown that these slit-shaped pores can be produced by changing the angle be-tween the interfering laser beam between the two exposure steps or by rotatingthe sample over an angle less than 90 degrees.

The pore wall reduces the throughput of a membrane and, consequently,membranes with pores with a low aspect ratio are usually preferred. Thinmembranes have a lower flow resistance than thick membranes with the sameselectivity, due to the no-slip condition at the pore wall. The membranethickness is easily tuned by changing the thickness of the applied photoresist,either by adjusting the solid content in solution or by changing the spin coatingconditions. Unfortunately, very thin membranes are mechanically weak and,therefore, a special processing route was developed to produce thin membraneswith a small pore size on a mechanical support which is a thick membrane witha very large pore size.

The so-called Vena Contracta effect decreases the flux through the straightpores by restricting the effective flow diameter after a pore to a diameterslightly smaller than the actual pore diameter. Jet-shaped pores are known todecrease the loss in flux due to a Vena Contracta. A light intensity gradientover the film was induced by the addition of a UV absorber. This resultsin small pore diameters at the high light intensity side of the membrane,and large pore diameters at the low light intensity side, in combination with acontinuous gradient in pore diameter. This effectively results in the desired jet-shaped pores, which reduce the Vena Contracta effect. On the other hand, themaximum obtainable porosity is also reduced and consequently an optimumis obtained for a membrane with a inlet pore diameter of 2 µm and an outletpore diameter of 3 µm.

The above described membranes have several merits in classical filteringapplications such as removal of particulate matter from exhaust streams. Nev-ertheless, incorporation of the membranes in microfluidic channels of a biosen-sor offers new opportunities in the design of e.g. protein microarrays.

In its most simple form, a membrane consists of equally spaced, parallel

vii

lamellae in a microchannel. Such lamellae are easily fabricated using eithermask lithography of holography. In a new design of a protein microarray,groups of lamellae are positioned locally in a microchannel, forming adsorptionsites for capture probes. In this way, several membranes can be put in seriesin a microchannel which results in a high signal-to-noise ratio and an excellentselectivity, while fulfilling the low sample volume requirement.

The flow between lamellae is laminar in nature, implying that the transportof biological agents to the lamellae walls relies on diffusion only, resultingin long assay times. To improve this, grooves are applied on the lamellaeto induce a rotational flow and herewith enhance the transport of agents tothe walls. These grooves are generated by a balanced combination of masklithography and a holographic under-exposure.

So-called slanted-angle holography was developed to create membraneswith in-plane pores that can be incorporated in a microchannel. These mem-branes are mechanically more stable than lamellar membranes and they have alarger surface area. Again, the pore size and shape can be chosen by adjustingthe angle between the laser beams and/or the tilt angle. In combination witha lithographic step, in-plane membranes are fabricated in situ inside a mi-crofluidic channel, providing a leak-free connection to the channel wall. Whentested, these membranes show a low pressure drop and an excellent selectivity.

Immobilization of antibodies is a crucial step in the development of mi-croarrays. The protein conformation needs to remain intact and the biologicalfunctionality of the protein has to be preserved. Physical adsorption is mostfrequently used as immobilization technique for proteins. However, covalentbinding provides a much stronger attachment and is therefore preferable. Co-valent coupling techniques using amines or carboxylic acids on a surface havebeen developed to generate this strong binding. SU8 substrates were thereforeprovided with a coating of polyacrylic acid (PAA) or poly-L-lysine (PLL). Re-covery experiments were performed to test the binding of antibodies to thesesurfaces. Both plain SU8 and PLL showed a good recovery, PAA did not. Thesmall difference between PLL and non-treated SU8 led to the choice for non-treated SU8 as substrate for subsequent biological experiments. A functionalsandwich assay was performed on a flat surface of plain SU8. Preliminary re-sults showed a detection limit of 10 nM on a flat substrate. This is still ratherhigh, but lower detection limits (pM) are expected when membranes will beused as a substrate.

Fluid flow through a biosensor is preferably not induced by external pump-ing devices. Often, capillary flow is used making external pumping redundant.

viii

However, SU8 is known for its hydrophobicity which results in no capillary fluxin a non-treated SU8 microchannel. The flow through microchannels was in-creased significantly by increasing the surface energy and the surface polarityof SU8 by a UV-ozone and/or and oxygen plasma treatment. This enables theuse of capillary pressure as a driving force for fluid flow through a microchan-nel.

Finally, contact between the analyzed body fluids and the outside environ-ment is usually unwanted in biosensors. Therefore, a cover that hermeticallyseals the microchannels is required. Glancing angle lithography was developedto solve this issue. Utilizing the non-discrete nature of a reflection interface, athin area at the top of a photosensitive film (SU8) is activated. This activatedarea was crosslinked to form a cover layer during the postprocessing steps.The thickness of the cover layer is controllable by the angle of incidence, theexposure dose and the diffusion time of the photo-activated species. Com-bining this technique with the aforementioned techniques like slanted-angleholography, interference holography and classical lithography, a hermeticallysealed functional biosensing device can be obtained.

Chapter 1

General introduction

Over the last 10 years, medical diagnostics have been shifting their focus fromlaboratory testing towards point-of-care diagnostics. Biosensors, devices thatcan perform laboratory tests on a chip that is smaller than a credit card, areextremely suitable for this task. The ultimate goal is to selectively detect mul-tiple types of species, preferably every molecule present in the analyte (sen-sitivity). This detection needs to be fast, using a very small sample volume,like the sample volumes obtained with a fingerprick. So, selectivity, sensi-tivity, speed and small sample volumes are key elements for biosensing. Tomeet these requirements, microfluidic channels and membranes are frequentlyused as building blocks for biosensors. A microfluidic device is often definedas a device containing at least one channel with a diameter smaller than 1mm, which is very useful for dealing with small sample volumes. The fluidsthat are to be tested in the biosensor are usually complex mixtures that needpretreatments like filtration, mixing or chemical treatment. A membrane isoften incorporated in a biosensor for microfiltration, turbulence induction orjust for providing a large adsorption surface. The research performed in thisthesis deals with the development of new technologies for the fabrication ofmicrostructured elements and membranes for both standard microfiltration ap-plications as well as in microfluidic devices. A special emphasis is devoted towell-defined monodisperse microstructural elements, produced via interferenceholography, to obtain devices with a strongly enhanced performance in termsof selectivity and sensitivity.

1

2 Chapter 1

1.1 Miniaturization in medical diagnostics

About 2 decades ago, medical diagnostics started to shift its focus from labo-ratory testing towards point-of-care testing (POCT). Two factors led to thisshift in approach of patient care: time to result and technological advances.

Firstly, it is beneficial for a patient if the time needed for a diagnosis in cli-nical situations is reduced[1, 2] e.g. in emergency services, intensive care units,etc. Rapid diagnosing enables a rapid treatment of possibly life-threateningconditions like in the early stages of a heart attack.[3] Secondly, the enormoustechnological developments in miniaturization of systems in the past years havemade POCT feasible and practical. Point-of-care sensors - called biosensors -should be able to compete with standard laboratory tests in terms of specificityand sensitivity, and preferably be faster, cheaper, use smaller sample volumesand be easier to use for non-specialized personnel.[4]

One of the best known examples of a biosensor probably is the glucosemeter (figure 1.1), used by diabetics to measure the glucose concentrationin their blood. By means of a fingerprick, a small amount of blood (∼1 µl)is sampled and applied onto a strip which is inserted into a reader. A fewseconds later, the glucose concentration can be read from the LCD screen,and action (e.g. insuline injection) can be taken accordingly. This exampleillustrates some of the key features of biosensor: speed, selectivity and smallsample volume.

Figure 1.1: The most well-known biosensor on the market: the glucose meter,used to detect the glucose level in blood from a fingerprick.[5]

Glucose is a molecule that is relatively abundant in blood, in concentra-tions of approximately 80-110 mg/dl. However, markers for other conditionslike troponin, an enzyme that is produced during cardiac failure, are only spo-radically present (ng/dl[6]) requiring a very sensitive detection device. These

General introduction 3

low detection limits demand new approaches for the detection of biologicalmarkers.

Microfluidics[7–9] are often used to address the key requirements of a biosen-sor (speed, selectivity, sensitivity and sample volume). A microfluidic deviceis defined as a device containing at least one channel with a diameter smallerthan 1 mm, of which some examples are shown in figure 1.2. Due to theirsmall size, the required sample volume for the device is very limited, and ashort time to result can be reached with this technology. However, the minia-turization of these biosensing systems has one major disadvantage: from thesmall amount of analyte the same amount of information or even more needsto be extracted.

Figure 1.2: Microfluidic chips.[10]

Biosensors realize detection by translating a specific nanoscopic interactionbetween receptors present in the sensor and the species to be detected in theanalyte into a macroscopic signal. This signal can be optical, electrical, mag-netic, etc. and should be proportional to the amount of interactions that oc-cur.[11] Ideally the detection is performed in biological fluids like whole blood,saliva and urine. These fluids are usually complex mixtures that need varioustreatments like filtration/purification, mixing and chemical treatments.[12] Aunit that is therefore often implemented in a biosensor is a membrane. Typ-ically, the membranes are used for the separation of particles from liquids,the induction of turbulence for mixing or providing a large specific area foradsorption of receptors or other chemical species.

Frequently, commercially available membranes are implemented in micro-fluidics.[12] Typically, these membranes are non-woven fabrics and exhibit ahighly polydisperse pore structure and high porosity. Consequently, the appli-cation of these membranes results in a large pressure drop which restricts thethroughput of the fluid in the device. Monodisperse microsieves have a lowpressure drop but the actual implementation in a microfluidic device is compli-cated.[12] Therefore, an efficient way to fabricate monodisperse microfiltrationmembranes and to implement them in microchannels is required.

4 Chapter 1

1.2 Microfiltration

Biological species like cells, pollen, bacteria[13] have dimensions in the orderof micrometers, but also non-biological species like particulate matter (PM2.5,PM10

[14]) are in that size range. To filter out contaminants of this size from afluid, microfiltration is the commonly used filtration process. In microfiltrationthe fluid is passed through a microporous structure, a membrane, driven by anexternally applied pressure. Physical separation takes place by size exclusion.

The first person to develop a microfiltration filter was professor Sigmondyfrom the University of Goettingen in Germany in 1935. A couple of years later,the first membrane filters were commercially available, produced by SartoriusGmbH. Two principles of membrane filtration can be distinguished: dead endfiltration and cross-flow filtration, (figure 1.3).

(a) Dead end

Membrane

Membrane

Retentate

Permeate

Feed

Permeate

(b) Cross flow

Figure 1.3: The two filtration modes: dead end (left) and cross flow (right).

The feed (fluid or gas with suspended species) is divided by the membraneinto a retentate (species that are filtered out) and the permeate (filtered so-lution or gas). One of the problems in filtration is membrane fouling, whichcan be decreased by decreasing the transmembrane pressure drop. However,a reduction in pressure drop also reduces the flux through the membrane[15]

which reduces productivity and throughput of product. To compensate forthis loss of flux, highly efficient membranes with a very low flow resistance arerequired.

Standard microporous membranes (figure 1.4) are usually random-networkdense structures that have a large flow resistance due to the large pore sizedistribution.[13] The largest pores determine the selectivity of a membrane,while pores that are smaller increase the flow resistance.

The flow resistance of a membrane with a certain selectivity (size of thelargest pore) is determined by its thickness, pore size distribution and poro-sity.[16] Microsieves, membranes with a well-defined pore geometry, pore sizeand a very low pore size distribution have an optimum balance between se-


(a) Isotropic (b) Anistropic

Figure 1.4: Schematic representation of isotropic and anisotropic polydis-perse membranes. Polydisperse membranes have a large flow resistance due tothe irregular sizes and shapes of their pores.

lectivity and flow resistance.[15,17] Recently, the production and properties ofsuch monodisperse membranes have attracted considerable attention.[15, 18,19]

Several rather elegant routes were proposed in the past to produce polymericand monodisperse microsieves via, for instance, phase separation micromoul-ding,[20] track etching[21] and silicon micromachining.[22] In the above de-scribed studies, the prime objective is often to combine the ease of processingof polymers with the high performance of monodisperse porous media. Never-theless, the above described polymeric membranes also have limitations withrespect to maximum porosity, material properties (mechanical strength) andfreedom of design in pore geometry or in pore size.[23]

Monodisperse membrane fabrication techniques like phase separation mi-cromolding and micromachining are hard to integrate with biosensor produc-tion or it is even completely impossible. For instance, mounting such mem-branes to the microchannel with a leak-free connection is troublesome. As al-ternative fabrication method, possibly circumventing a number of these issues,we investigated holographic techniques for the in situ fabrication of membranesin microchannels. Therefore interference holography is discussed in some moredetail in the next paragraph.

1.3 Holography

Holography is a photographic technique that has been known for over 60 yearsalready.[24] In essence, it consists of capturing not only the brightness, but alsothe intensity variation between light passing through a photographic objectand light from a reference beam. First, an interference pattern is created usingcoherent polarized light, usually from a laser. Then, this interference patternis recorded in a photosensitive film. And lastly, the hologram is reconstructed

6 Chapter 1

using the diffraction of light by this film.[24]

In this research, the first two steps of the holographic process are used: theregistration of an interference pattern in a photosensitive film. A microstruc-ture is obtained by removing the areas of the film that received a certainexposure dose (intensity × exposure time). Alternatively, a microstructurecan also be generated with a so-called negative photoresist and then de unex-posed areas are removed by dissolving or etching. In fact, the major part ofthe research in this thesis is performed with such a negative photoresist (SU8).

The creation of a regular interference pattern depends highly on the co-herence of the light source used for recording the hologram. Nowadays, lasers(Light Amplification by Stimulated Emission of Radiation, invented by T.Maiman[25]) are used as source for coherent light. During the recording ofa hologram, the photosensitive film is placed on the intersection of the twointerfering light beams.

Two types of recording geometries can be discriminated, depending on theorientation of the photosensitive film in the recording setup (figure 1.5). Ifthe sample is perpendicular to the incoming beams, it is called transmissionholography, from the fact that in a transmission hologram the object is recon-structed by the transmitted beam. If the sample is parallel to the incomingbeams, it is called reflection holography. All structures presented in this thesisare created using transmission holography.

θ/2

k1

k2

ex

ey

k1

k2

θ/2 θ/2

Transmission Reflection

Figure 1.5: Recording geometries for transmission and reflection holography.In transmission mode, the two interfering laser beams are incident on the sameside of the photosensitive film, in reflection mode they reach the sample fromboth sides.

In figure 1.6 an example is shown of the different processing steps for aphotoresist. First, a thin layer of the photoresist is applied on a substrate,


usually glass. After this, the sample is heated to evaporate residual solvents inwhich the resist was dissolved. The substrate is then exposed to an intensitypattern which can be induced by a mask or by the interference of laser beams(figure 1.6(b)). Usually, an additional heating step is needed to polymerize, inthe case of a negative photoresist, or de-polymerize, for a positive photoresist,the material.

(a) Spin coating (b) Exposure

(c) Positive resist (d) Negative resist

Figure 1.6: Steps in the processing of photoresist materials.

SU8 (figure 1.7), a negative photoresist based on the cationic polymer-ization of epoxides, is used as recording material. SU8 is often used as chipmaterial in microfluidics and micro-electromechanical systems (MEMS). It isknown for its high aspect ratio patterning and produces very straight verticalside walls. Also, crosslinked SU8 is chemically and thermally resistant andbiocompatible.[26] This all makes SU8 a very suitable material to fabricatemembranes for biosensor applications.

O

CC2H CH2

O

CH3H3C

OCC2H CH2

O

O

CC2H CH2

O

CH3H3C

OCC2H CH2

O

O

CC2H CH2

O

CH3H3C

OCC2H CH2

O

O

CC2H CH2

O

CH3H3C

OCC2H CH2

O

H2C

H2C

H2C

Figure 1.7: The SU8 molecule.

8 Chapter 1

As discussed previously, the combination of interference holography withsuitable photoresist materials is potentially useful in the integration of mi-crostructured elements in the microfluidic channels of a biosensor. An illus-trative example of this is shown in figure 1.8. The microstructured elementsare produced by interference holography, while the channel is fabricated usingstandard photolithography. The direct injection molding of a channel withsuch microstructural elements is impossible for instance because the aspectratios of the structures are very high and/or because so-called undercuts arepresent. The separate production of the elements and the subsequent in-stallation in a preformed microchannel is or almost impossible or extremelylaborious and expensive or both which illustrates the need for new processingschemes.

Figure 1.8: Example of the application of interference holography in theproduction of microstructured elements in a microfluidic channel.

1.4 Scope of the thesis

In chapter 2 a theoretical framework is established, in order to find the mostideal pore geometry for a microfiltration membrane. Finite Element Method(FEM) simulations are used to predict the pressure drop across monodispersemembranes and the flow through membranes with an emphasis on parameterssuch as pore shape, dimension, packing and cross-section. Chapter 3 is con-cerned with the use of holography for the fabrication of the above describedmembranes with pores perpendicular to the substrate. A wide range of poregeometries (round, slit-shaped) and pore arrays (square, hexagonal packing)are produced using holography, as well as different pore sizes (between 5 µmand 50 nm), according to the findings of chapter 2. Also, the simulation re-sults from chapter 2 are experimentally verified. Chapter 4 deals with thefabrication of in-plane membranes, i.e. membranes which have their poresparallel to the substrate. It will be shown that using this technique produces


well-defined pores with a very small pore size distribution and that the poregeometry, size and packing is easily manipulated. Especially these membranesare extremely suitable for the in situ fabrication inside a microfluidic channel.Moreover, the membranes are evaluated in terms of selectivity and pressuredrop. In chapter 5 holographic membranes are adopted for biosensor ap-plications. The usefulness of these membranes in protein microarrays will beelaborated. Additionally, blood cells will be filtered from a blood sample usingin-plane membranes integrated in a microfluidic channel. Chapter 6 dealswith the application of membranes in another type of filtration, namely usinglarge adsorption area intrinsically provided by membranes for the adsorptionof biomolecules without using the actual sieving properties of the membrane.These molecules would normally be too small to be filtered with a micro-filtration membrane. Different surface modifications, both chemical andphysical i.e. the creation of relief structures on an otherwise flat surface andtheir effect on capture of biomolecules are subject of this chapter. An issuethat is often faced when dealing with microfluidics, namely the sealing of mi-crofluidic channels to create a barrier between the fluids inside the device andthe outside environment is the subject of chapter 7. A new exposure andsealing method, called glancing angle lithography is discussed, and differ-ent parameters affecting the film thickness of the cover layer are investigated.Finally, standard microstructural elements such as channels are sealed usingthis method.

1.5 References

[1] N. Drenck. Point of care testing in critical care medicine: the cliniciansview. Clin. Chim. Acta, 307, (2001) 3–7.

[2] M. Fleisher. Point-of-care testing: Does it really improve patient care?Clin. Biochem., 26, (1993) 6–8.

[3] D. Eccleston, C. Reid, A. Brennan, J. Miels, N. Andrianopoulos andH. Krum. Point of care biomarker assays are superior to clinical assess-ment for diagnosis of heart failure in australian emergency departments:Triaged study. Heart Lung Circ., 17 (Supplement 3), (2008) S155 – S155.

[4] P. St-Louis. Status of point-of-care testing: promise, realities, and possi-bilities. Clin. Biochem., 33 (6), (2000) 427–440.

[5] http://www.abbottdiabetescare.nl.

10 Chapter 1

[6] B. Oosterlynck, J. D. Knock and P. Bouckhout. Dringende medischehulpverlening door verpleegkundigen. Acco Medical (2008).

[7] D. B. Weibel and G. M. Whitesides. Applications of microfluidics inchemical biology. Curr. Opin. Chem. Biol., 10, (2006) 584–591.

[8] C. Hansen and S. Quake. Microfluidics in structural biology: smaller,faster...better. Curr. Op. in Struct. Biology, 13, (2003) 538–544.

[9] C. Situma, M. Hashimoto and S. A. Soper. Review: Merging microfluidicswith microarray-based bioassays. Biomol. Eng., 23, (2006) 213–231.

[10] http://www.micronit.com.

[11] T. Vo-Dinh and B. Cullum. Biosensors and biochips: advances in biolog-ical and medical diagnostics. J. Anal. Chem, 366, (2000) 540–551.

[12] J. D. Jong, R. Lammertink and M. Wessling. Membranes and micro-fluidics: a review. Lab Chip, (6), (2006) 1125–1139.

[13] R. Baker. Membrane technology and applications. Wiley, 2 edition (2004).

[14] Council directive 199/30/ec. Official journal of the European Communi-ties (1999).

[15] S. Kuiper, C. van Rijn, W. Nijdam and M. Elwenspoek. Developmentand applications of very high flux microfiltration membranes. J. Membr.Sci., 150, (1998) 1–8.

[16] S. Kuiper, C. van Rijn, W. Nijdam, O. Raspe, H. van Wolferen, G. Kri-jnen and M. Elwenspoek. Filtration of lager beer with microsieves: flux,permeate haze and in-line microscope observations. J. Membr. Sci., 196,(2002) 159–170.

[17] L. J. Heyderman, B. Ketterer, D. Bchle, F. Glaus, B. Haas, H. Schift,K. Vogelsang, J. Gobrecht, L. Tiefenauer, O. Dubochet, P. Surbled andT. Hessler. High volume fabrication of customised nanopore membranechips. Microelectron. Eng., 67-68, (2003) 208–213.

[18] C. van Rijn, W. Nijdam, S. Kuiper, G. Veldhuis, H. van Wolferen andM. Elwenspoek. Microsieves made with laser interference lithography formicro-filtration applications. J. Micromech. Microeng., 9 (2), (1999) 170–172.


[19] S. Kuiper, H. van Wolferen, C. van Rijn, W. Nijdam, G. Krijnen andM. Elwenspoek. Fabrication of microsieves with sub-micron pore size bylaser interference lithography. J. Micromech. Microeng., 11, (2001) 33–37.

[20] M. Girones, I. Akbarsyah, W. Nijdam, C. van Rijn, H. Jansen,R.G.H.Lammertink and M. Wessling. Polymeric microsieves producedby phase separation micromolding. J. Membr. Sci., 283, (2006) 411–424.

[21] I. M. Yamazaki, R. Paterson and L. P. Geraldo. A new generation of tracketched membranes for microfiltration and ultrafiltration. part i. prepara-tion and characterisation. J. Membr. Sci., 118 (2), (1996) 239–245.

[22] C. van Rijn and M. Elwenspoek. Micro filtration membrane sieve withsilicon micro machining for industrial and biomedical applications. IEEEProc. MEMS 1995, page 83.

[23] M. Ulbricht. Advanced functional polymer membranes. Polymer, 47,(2006) 2217–2262.

[24] G. Saxby. Practical holography. IOP, Bristol, 3 edition (2004).

[25] T. Maiman. Stimulated optical radiation in Ruby. Nature, 187, (1960)493–494.

[26] G. Voskerician, M. S. Shive, R. S. Shawgo, H. von Recum, J. M. Anderson,M. J. Cima and R. Langer. Biocompatibility and biofouling of MEMS drugdelivery devices. Biomaterials, 24 (11), (2003) 1959–1967.

Chapter 2

Flow through holographic

membranes

For the development of efficient microfiltration membranes, the critical designparameters need to be known. From a theoretical viewing point, it is clear thatthe porosity (open surface/total surface) has a major role in the flow resis-tance of a microsieve. Also the pore shape, round or slit-shaped, has a largeinfluence on the flow behavior. Slit-shaped pores have a larger volume to sur-face ratio than round pores and thus less flow resistance induced by the no-slipconditions at the wall of the pore. Another important parameter is the as-pect ratio of the membrane. Ideally, the ratio between the pore diameter andthe membrane thickness is between 0.5 and 1, to decrease the flow resistance.Prevention of the Vena Contracta effect is another issue in the optimizationof membrane performance. This is a well-known effect that occurs after ori-fices which decreases the flux through an opening. It is known that this canbe decreased by using jet-shaped pores. This shape causes the flow to followthe pore walls such that the effective flow through the pore is increased. Thetheoretical models illustrate that flow through membranes can be designed witha very low pressure drop and high throughput provided that the membrane hasa high porosity and the pores are short, slit-shaped and tapered.

13

14 Chapter 2

2.1 Introduction

A membrane acts as a semi-permeable barrier layer between two phases. Somecomponents can pass through the membrane (permeate) and others are re-tained by it (retentate). Membranes are neutral or charged, and the drivingforce for separation can be concentration, chemical or electrical gradients orpressure. However, sieving is the main particle retention method in the mi-crofiltration regime (0.5 - 5 µm). Many techniques are being used to fabri-cate polymer microfiltration membranes,[1] like stretching, track etching[2] andphase separation micromolding.[3]

Microfiltration membranes can be subdivided into membranes with a poly-disperse and monodisperse pore diameter. Polydisperse membranes are oftenmanufactured with phase separation techniques and their properties are oftenfar from ideal i.e. the selectivity is determined by the largest pores and theflow resistance is dictated by the smallest pores.[4] A far more ideal situationis obtained with monodisperse membranes such as those produced via phaseseparation micromolding and by interference holography (see later).

Here, Finite Element Methods are used to model the flow rate and pressuredrop of monodisperse membranes. Obviously, parameters like porosity andpore shape have a large influence on the flow characteristics of the membrane,but also the aspect ratio of the pores and their conicalness are investigated.Especially the ratio between the pore diameter and the membrane thickness(aspect ratio) is expected to have a large effect on the flow. Furthermore,it is well-known that jet-shaped pores can decrease the Vena Contracta ef-fect, which inevitably occurs at the membrane pores. By simulating differenttapered pore shapes, the most ideal conical pore shapes are obtained.

Before moving on to the finite element method simulations, the basic theoryof microsieve fluid dynamics is presented in a short review.

2.2 Flow theory

Four main flow regimes exist: continuum flow, slip flow, transition flow andfree molecule flow. The dimensionless parameter that describes which flowregime is applicable, is the Knudsen number.[5] It is related to the Reynoldsnumber and to the Mach number. The Knudsen number is defined as:

Kn =a

L(2.1)

where a is the fluid mean-free path in [m] and L the macroscopic length

Flow through holographic membranes 15

scale of the physical system, also in [m]. In air, the mean free path is about68 nm, which results in a Knudsen number between 0.34 and 6.8 × 10−3 forpore diameters between 0.2 and 10 µm. In figure 2.1 the flow regimes withthe corresponding Knudsen numbers are schematically depicted.

0 Kn 10-3 10-2 10-1 100 101 Kn ∞

Continuum

Flow

Slip Flow Transition Flow Free molecule

Flow

Navier- Stokes Eqns

Slip ConditionsNo-slip

EulerEqns

Burnet Eqn

Boltzmann Eqn

Figure 2.1: Flow regime classification based on Knudsen number.

From this figure it can be found that for holographic membranes in thesize range of 0.2 to 10 µm, the continuum flow theory with either slip orno-slip conditions will hold.[6, 7] For liquids the mean free path is shorter,therefore also in liquid filtration, the continuum flow model will apply and theuse of Navier-Stokes equations therefore is legitimate. In later paragraphs,the validity of FEM simulations using the Navier-Stokes equations will beexperimentally verified since entrance effects are expected to have an influencewhen considering membranes instead of single pores.

The basic equation in continuum fluid dynamics is the well-known Navier-Stokes equation, in its most basic form:[5]

ρdvdt

= −∇ ·P + F (2.2)

with v the vectorial speed in [m s−1], ρ the density in [kg m−3] and F thecombinatorial force vector of all forces working on the fluid in [N m−3]. P isthe generalized pressure in [Pa] and ∇ is the nabla-operator. Or, in a simpler,vector-form:

Inertia︷︸︸︷ρ(∂v

∂t+ v · ∇v

)=

Pressure︷︸︸︷−∇p +

V iscosity︷︸︸︷µ∇2v +

Other body forces︷︸︸︷f (2.3)

It can be assumed that the flow is incompressible - for a gas stream at pres-

16 Chapter 2

sures below 10 kPa this is usually the case[5] - and that the gravitational forcescan be neglected compared to the applied pressure difference. Finally, the as-sumptions of a steady state and no-slip conditions in a cylindrical channel,lead to following simplified form of the N-S equation:

Q =∫ 2π

0dθ

∫ R

0r dr vz r =

πR4

8µ

∆P

lz(2.4)

also known as Poiseuille’s equation. Here Q is the volumetric flow in[m3s−1] through a cylindrical channel with radius R [m] and length lz [m].µ is the dynamic viscosity of the fluid in [Pa s] and ∆P the transmembranepressure in [Pa]. In membrane applications Q is multiplied by the membraneporosity to obtain the average volumetric flow through the membrane area.This is the simplest approximation, but it is inaccurate for small length scalesand does not take into account entrance or slip effects nor the pore array.Since these effects do play an important role in the flow characterization ofmicrosieves, Poiseuille’s equation is not accurate enough for this application.Aspect ratio (t/d), porosity (β), pore shape and Reynolds number (Re) shouldbe considered when developing a model for membrane fluidics. Dagan[8] sug-gests following expression for the pressure drop over an orifice with finitethickness and an aspect ratio of t/d:

∆P =µQ

(d/2)3[16

π

( t

d

)+ 3

](2.5)

This equation applies for very low porosities β → 0, but since our goal isto fabricate membranes with a high porosity, it is not applicable in this case.Tio & Sadhal[9] on the other hand provide a model for the pressure drop of athin array of pores:

∆P =µQ

(d/2)3[1− f(β)

](2.6)

Q is the throughput per pore in [m3s−1]. P is the transmembrane pressure(TMP) in [Pa], the viscosity in [Pa s], R the pore radius in [m], β the porosityand f(β) a function of the porosity defined as:

f(β) =∞∑

i=0

aiβ( 2i+1

2) (2.7)

In equation 2.7, a1 = 0.344, a2 = 0.111 and a3 = 0.066 for a square array


of circular pores, a1 = 0.3389, a2 = 0.1031, a3 = 0.0558 and a4 = 0.0364 fora hexagonal array of circular pores. Van Rijn combined equations 2.5 and 2.6into:[10]

Q =∆P

ηR3

[1

16tπd + 1

][1

1− f(β)

](2.8)

From equations 2.8 and 2.7 it follows that a low aspect ratio and highporosity are required for a high throughput at a given pressure drop andselectivity. The porosity is defined as:

β =πab

Λ1Λ2 sinα(2.9)

with a, b, Λ1 and Λ2 as in figure 2.2.

a

b

Λ1

Λ2

Figure 2.2: Definition of parameters to determine the porosity. The figurerepresents one unit element of a larger array.

All theories give a good indication for the flux through a membrane witha certain porosity. The theory of van Rijn however, describes best the flowthrough a microsieve, which will be elaborated by comparing the flow theoriesdescribed above with finite element method simulations. FEM simulationshave the advantage that modeling the third dimension (in depth of the mem-brane) is readily done, while incorporation of more advanced pore shapes inan analytical model is laborious.

2.3 Flow simulations

It is generally accepted that FEM simulations are a reliable way to obtaininformation on the behavior of fluids around and inside structures.[11] So asto come to an understanding of the parameters that have a significant influenceon the throughput and pressure drop of a membrane, FEM simulations (usingComsol Multiphysics) will be utilized in this paragraph.

18 Chapter 2

2.3.1 Basics of FEM simulations

The Finite Element Method (FEM) approximates a partial differential equa-tion (PDE) problem with a discretization of that original problem. The FEMmethod starts from a mesh, which is the division of a chosen geometry intosmall units with a simple shape, the mesh elements, with the same dimen-sionality as the geometry itself.

Figure 2.3: FEM mesh generation.

For modeling a physical problem, three classes of parameters need to beknown: material properties (density and viscosity of the fluids), initial condi-tions and boundary conditions. A set of partial differential equations governingthe physical phenomenon that is modeled is solved using these parameters.The initial conditions set a starting point for the calculations. Boundary con-ditions specify the values the solution needs to have on the boundaries of thedomain, e.g. no-slip (flow velocity = 0) at the pore wall. Typically theseconstraints are imposed on dependent variables (velocity, pressure,...).

When modeling a flow through a microfiltration membrane, a momentumbalance needs to be solved. The Navier-Stokes equation (equation 2.3) isthe governing equation. Since normally a membrane will be operated at aconstant flow, steady-state conditions apply. In Comsol Multiphysics, theChemical Engineering module offers the opportunity to model a steady-statemomentum balance using Navier-Stokes equations, which will be the standardmodel used in this study. Before solving the momentum balance, a schematicdrawing of the problem, the geometry, needs to be made.

Due to the symmetric layout of the holographic membranes and the pos-sibility to define symmetry boundaries in Comsol, the flow behavior can bededuced from the simulation of a single membrane pore. An axi-symmetric2D geometry is chosen to simulate the flux through a membrane pore. Thesimulated pore geometry consists of an inlet, the pore itself and an outlet asdepicted in figure 2.4. The width of the inlet is determined by the porosity ofthe membrane.

The boundary conditions that apply to this geometry are defined as follows.In figure 2.4, A is the rotational symmetry axis, over which the geometry needsto be rotated over 360◦ to obtain a three-dimensional pore. As mentioned


A

Outlet, P = 0

Inlet, P = ∆P

Membrane

Sin

Sout

Figure 2.4: Schematic representation of the geometry for flow simulations.An inlet and outlet are added to the pore to simulate entrance and outlet effects.The inlet width is determined by the porosity of the membrane. Sin and Sout

are symmetry boundaries.

earlier, no-slip conditions apply at the pore walls. Sin and Sout are symmetryboundaries that ensure a continous connection between adjacent pores. Thetransmembrane pressure drop ∆P is applied between the inlet and the outlet.

Only the two fluids that most likely will be filtered using holographic mem-branes will be simulated: air and water. The choice for those 2 fluids is ratherself-explanatory: air resembles most gases that are filtered with microfiltra-tion and water is the major component of most biological fluids. These twoapplications are of interest for this research. In principle all membrane flowproblems are three dimensional, however, due to the symmetric layout of themicrosieves, the problem can be simplified to two dimensions, which is bene-ficial for the calculation speed.

To justify whether it is correct to used 2D axi-symmetric simulations in-stead of the time-consuming full 3D simulations, the velocity field of 2 identicalpores is compared for both geometries. A pore with a diameter of 2 µm anda length of 2 µm (aspect ratio 1) is considered. An inlet and outlet are as-sumed to have a length of 2 µm and a diameter of 4 µm corresponding toa porosity of 25%. At the inlet an air flow due to a pressure of 1000 Pa isapplied (at these low pressures, incompressible N-S apply), at the surface ofthe membrane, no-slip conditions apply. On the interface between the poresa symmetry condition is imposed. The resulting velocity fields are shown infigure 2.5.

From this figure it is clear that the results from both simulations are iden-tical, and therefore the axi-symmetric simulation is legitimate to replace theelaborate 3D simulations. In the subsequent simulations, therefore mainlyaxi-symmetric geometries will be simulated. Only for the comparison of pore

20 Chapter 2

0.0 0.5 1.0 1.5 2.00.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

Vel

ocity

fiel

d (m

/s)

Distance from center pore ( m)

2D 3D

Figure 2.5: Velocity fields at the outlet of a pore with a diameter of 2 µm,length of 2 µm and an inlet and outlet of respectively 2 µm length and 4 µmwidth. The solid line represents a 2D axi-symmetric geometry, the dashed lineis the 3D geometry, no significant difference can be observed.

shapes, 3D simulations will be used, since for elliptical pore shapes the rota-tional symmetry argument no longer holds.

The last step before adopting FEM to compare membrane geometries, itneeds to be investigated whether the simulation results correspond with resultsof analytical models. These models have been evaluated experimentally manytimes.[12] To find out whether the FEM simulations match the flow modelsdescribed in section 2.2, the flux at a certain transmembrane pressure for atypical membrane is calculated and simulated.

Let us consider a membrane with a porosity of 40%, a pore diameter of 1µm and a pore length of 5 µm. A porosity of 40% in practice means a peri-odicity of 1.4 µm, for a square array of circular pores. In an axi-symmetricgeometry, this corresponds to an inlet and outlet width of 0.7 µm. Usingequations 2.4, 2.5, 2.8 and Comsol to calculate the throughput at pressuresbetween 0 and 3 kPa, graph 2.6 was obtained. The simulation results approx-imate the theoretical outcome, and therefore give a good description of theflow behavior of microfiltration membranes.

For membranes with straight, circular pores in any array, analytical modelsprovide a direct computational method for the determination of flow throughthe membrane. However, when elongated pores, tapered pores or other shapesneed to be compared, FEM simulations are much simpler and versatile in use.


0 1000 2000 30000.0

0.2

0.4

0.6

0.8 Poiseuille Dagan Van Rijn Comsol

Flux

(10-1

2 m3 /s

)

Pressure (Pa)

Figure 2.6: Comparison of flow theories.

2.3.2 Round vs slit-shaped pores

Several microfiltration applications are used for the separation of rather un-deformable nearly spherical particles, e.g. dust particles[13] or bacteria.[14]

For these applications, it is possible (and feasible) to change the pore shapefrom circular to slit-shaped, without affecting the separation selectivity. Slit-shaped pores have some important advantages over circular pores. The flowresistance of a slit-shaped pore is significantly lower than that of a circularpore.[15] Moreover, slits cannot be completely blocked by a spherical particle,allowing fluid to keep flowing and making the membrane less susceptible tofouling. It also results in a smaller contact area between the particle and themembrane which facilitates cleaning of the membrane.

To investigate the actual decrease in flow resistance R upon stretching acircular pore into an elliptical pore, a three dimensional FEM simulation fora membrane with a porosity of 25% and a selectivity (pore diameter) of 2µm is executed. The pressure drop over the membrane is chosen to be 1 kPa(air), and the membrane thickness is 2 µm giving an aspect ratio of 1. Theporosity is chosen constant to simplify the comparison of the flow resistance,since it is known that elliptical pores can be packed to a larger porosity thancircular pores. The flow resistance is derived from the flow (Q, in m3s−1) andthe pressure drop (∆P in Pa):

R =∆P

Q(2.10)

22 Chapter 2

In figure 2.7, the flow results for elliptical pores with a small diameter of2 µm and a large diameter ranging from 2 µm to 4 µm are shown.

2.0 2.5 3.0 3.5 4.00

2

4

6

8

10

Flow

resi

stan

ce (a

.u.)

Large diameter ( m)

Figure 2.7: Flow simulations for a 1 kPa flow through a membrane witha porosity of 25%, for an increasing large diameter of the elliptical pores.Starting point is a round pore with a diameter of 2 µm.

It is found that a slit-shaped pore with an a/b ratio of 2 (figure 2.2)has a flow resistance that is 3 times lower than circular pores with the samediameter, for a constant porosity. This is a significant improvement in flux,which is expected to become even more meaningful when pores with a largeraspect ratio (diameter/membrane thickness 1) are considered.

2.3.3 Aspect ratio

One of the parameters that is easily accessible and does not influence theselectivity of the membrane, is the aspect ratio of membrane pores. Theaspect ratio of a pore is defined as the ratio between the pore diameter and thethickness of the membrane. Since the thickness of a membrane is easy to tune,as will be shown in the experimental part, the only thing to be determined isaspect ratio that results in the largest flux.

Finite element method simulations are used to show the trend that is ob-served when applying a certain pressure (1000 Pa in all simulations performedin this paragraph) over a pore with a certain geometry (aspect ratio). Thestandard 2D axi-symmetric pore geometry is used, the inlet and outlet are2 µm long, and 2 µm in diameter. On the pore walls a no-slip condition isimposed, and a pressure of 1000 Pa is applied over the pore. Simulations arecarried out for air and water. For a fixed pore diameter, the length of the pore


0 1 2 3 4 50

5

10

15

Air

flux

(10-1

2 m

3 /s)

Aspect ratio

(a) Air

0 1 2 3 4 50.0

0.1

0.2

0.3

Wat

er fl

ux (1

0-12 m

3 /s)

Aspect ratio

(b) Water

Figure 2.8: Flux simulations for different membrane aspect ratios for a porediameter of 1 µm. (a) Air flux, (b) Water flux. The air flux is significantlylarger than the water flow, due to the lower viscosity.

is gradually increased, and the flux is calculated. In figure 2.8 two graphsshowing the flux as a function of the aspect ratio for both air and water aredisplayed. A strongly decreasing trend is observed with increasing aspect ra-tio, which is the same for both materials. From equation 2.8 it can be deducedthat the flux is inversely proportional to the aspect ratio. However, there isa more intuitive explanation for this behavior. A higher aspect ratio impliesthat a pore is longer and therefore that more pore wall is present. Since porewalls only add to the flow resistance of the pore (no-slip conditions apply),it is evident that the longer the pore is, the more flow resistance it has, andtherefore a lower flux will be observed. This behavior is universal for all porediameters in the 50 nm to 10 µm range.

It is therefore concluded that the lower the aspect ratio is, the larger will bethe flux through the membrane. Ideally, a membrane would thus be infinitelythin, which is in practice not achievable. Infinitely thin membranes are alsoinfinitely weak. Keeping in mind both the practical execution and optimizationof the flux, a most ’ideal’ aspect ratio is chosen to be between 0.5 and 1. Thismeans that the fluid only has contact with the membrane over a very shorttime, causing the fluid to leave the membrane very quickly.

2.3.4 Vena Contracta

Streamlines and/or flows are not able to change direction in a discrete step.Therefore, after an orifice or a membrane pore, the streamlines that convergedwhen entering the pore will only gradually diverge after the pore, in a smooth

24 Chapter 2

way. This causes the flow diameter to contract to an effective diameter, smallerthan the actual pore diameter. The place where this flow diameter is minimal iscalled the Vena Contracta (Torricelli 1643, figure 2.9). At very high troughput,flow instabilities can occur at the end of the straight channel. The net resultis that the transmembrane pressure drop increases. It is well-known that thiseffect can be reduced using tapered or jet-shaped pores. In order to get a betterinsight in the parameters that play a role in the Vena Contracta effect and tofind the optimum pore geometry to decrease this effect, FEM simulations areperformed.

Membranes with the same selectivity (same pore diameter at the inlet side)and same pore length are simulated. For a transmembrane pressure drop of 1kPa, the flow through membranes with different conical pores are simulated.Again, one pore is simulated and from this, the membrane characteristics arecalculated.

An important aspect that needs to be taken into account is the fact thatwhen tapered pores are used, the maximum achievable porosity decreases.This is due to the fact that at the outlet side of a membrane with taperedpores, the effective pore diameter is larger than at the inlet side. It is notpossible to pack these larger pores as closely together as the small pores, whicheffectively causes the porosity of the inlet side to be lower. Therefore, for thedifferent pore geometries, always a ’maximum achievable porosity’ needs tobe taken into account. In figure 2.10, a schematic drawing of the geometryused in the FEM simulations is shown. The maximum porosity is calculatedby using the minimum pore periodicity. In figure 2.10, this corresponds withthe case that the pore wall thickness at the outlet side (W) is zero.

The inlet pore diameter (din) is kept constant while the outlet pore di-

Vena Contracta

Figure 2.9: Schematic representation of the Vena Contracta effect. Theeffective flow diameter is decreased after passing an orifice.


Membrane

Outlet

Inlet

Outlet

Inlet

Outlet W

din

dout

Pore

Figure 2.10: Geometry for simulations of tapered pores.

ameter (dout) is varied, for a constant pore length and applied pressure. Infigure 2.11, the uncorrected throughput as a function of outlet pore diameterfor different pore lengths for a selectivity of 2 µm is shown.

5 10 15 20 25 300

100

200

300

400

500

Unc

orre

cted

flow

(a.u

.)

Outlet diameter ( m)

2 m 4 m 6 m 8 m 10 m

Figure 2.11: Flow versus outlet diameter of the pores. Larger outlet diameterof the pores results in a larger flux.

As was expected, the throughput of one pore increases with increasingopening angle of the pore. However, as indicated before, it is not correct toconclude from this that the wider the pore diameter at the outlet side, thehigher the flux through the membrane. Therefore, in figure 2.12, the flowcorrected for the maximum achievable porosity is depicted.

From this graph it is evident that a maximum in the flux through a mem-brane (for a fixed pressure drop) exists at a rather small outlet diameter. Thisoptimal geometry is observed at an outlet pore diameter of 3 µm. In para-graph 3.4.3, a method to produce membranes with this pore geometry willbe presented. Nevertheless, it appears that the actual influence of the VenaContracta effect is rather small if corrections are made for maximum porosity.

26 Chapter 2

2 3 4 5 6 70

10

20

30

40

Cor

rect

ed fl

ow (a

.u.)

Outlet diameter ( m)

2 m 4 m 6 m 8 m 10 m

Figure 2.12: Simulation results for flow through tapered pores, corrected formaximum achievable porosity for increasing outlet diameter. An optimum isobserved at an outlet diameter of 3 µm.

Now, the design requirements for an efficient microfiltration membrane areunderstood. In the next chapter, a new technique using interference hologra-phy combined with a negative photoresist will be introduced, and an attemptwill be made to fabricate membranes meeting these requirements in a practicalway.

2.4 Conclusions

The control over pore geometry and dimensions is essential in the fabricationof high performance polymeric membranes. Finite element method simula-tions based on solving Navier-Stokes equations on discrete elements providean insight in the important parameters for membrane design. The main char-acteristics are pore size distribution, porosity, pore shape, aspect ratio andconicalness of the pores.

Since the largest pores determine the selectivity of the membrane and allsmaller pores only contribute to the flow resistance of the microsieve, thepore size distribution should be very narrow. From the equations derivedby van Rijn (equation 2.8), the requirement for a high porosity follows. Alarge open surface increases the efficiency of the membrane enormously. Slit-shaped pores provide a geometry that opens the opportunity for high densitypacking of the pores, combined with a better surface to volume ratio thantheir round counterparts. Slits also have the advantage that they exhibit thesame selectivity, determined by their small diameter. In addition they have a


lower flow resistance, due to the larger open surface. This pore shape is lesssusceptible to fouling and easier to clean.

Long pores also contribute to the flow resistance, caused by the no-slipconditions at the pore wall. Therefore it is beneficial to produce very shortpores, preferably with an aspect ration between 0.5 and 1. The weakeningof the membrane by decreasing its thickness remains an issue which will beaddressed in the next chapter.

A last important factor that determines the throughput of a pore is theconicalness of the pore. Due to an effect called the Vena Contracta, the fluxis limited by a decrease in effective flow diameter, the Vena Contracta. Jet-shaped pores are known to prevent this from happening. For a model mem-brane with a selectivity of 2 µm, the optimum outlet diameter is determinedto be 3 µm in the ideal case.

2.5 References





[5] P. K. Kundu and I. Cohen. Fluid Mechanics. Academic press (2002).

[6] X. Yang, J. M. Yang, Y.-C. Tai and C.-M. Ho. Micro machined membraneparticle filters. Sensor Actuator, 73, (1999) 184–191.

[7] R. Perry and T. Green. Perry’s chemical engineers handbook. 7 edition(1999).

[8] Z. Dagan, S. Weinbaum and R. Pfeffer. An infinite-series solution for thecreeping motion through an orifice of finite length. J. Fluid Mech., 115,(1982) 505–523.

28 Chapter 2

[9] K.-K. Tio and S. Sadhal. Boundary conditions for stokes flows near aporous membrane. Appl. Sc. Res., 52, (1994) 1–20.


[11] X. Yang and Y.-C. Tai. Micromachined particle filter with low powerdissipation. J. Fluids. Eng., 123, (2001) 899–908.


[13] Council directive 199/30/ec. Official journal of the European Communi-ties (1999).

[14] D. Wild. The Immunoassay Handbook. Elsevier, 3 edition (2005).


Chapter 3

Fabrication of holographic

membranes

Interference lithography is a well-known process for the fabrication of micro-structures. Combining this technique with a negative photoresist (SU8) whichhas an excellent mechanical and chemical resistance provides a method to pro-duce monodisperse membranes. The periodicity of the interference patterninduced by two crossing, parallel polarized beams is tunable by adjusting theangle between the beams. SU8 has a non-linear optical response which enablesdigital recording of an analogue interference pattern. Parameters like porosity,pore diameter, aspect ratio, pore shape and conicalness are easily addressed.The glassy state of the photoresist at room temperature permits the use ofseveral consecutive exposure steps. Exposing a photosensitive layer twice to aline pattern, rotated 90 degrees with respect to each other, generates a grid-likestructure in which the pores are formed. Using holography, a wide variety ofpore shapes (round or slit) and pore sizes (100 nm - 5 µm) in different arrayscan be fabricated. To reduce the Vena Contracta effect, jet-shaped pores are anecessity. Addition of a UV absorber to induce an intensity gradient over thephotosensitive material, results in a pore diameter gradient over the thicknessof the membrane, and thus tapered pores are obtained. The membranes pro-duced are also used for the experimental verification of the theoretical predic-tions in chapter 2. Simulated fluxes correspond well with the measured fluxes,confirming that the assumption that simulating one pore suffices to predict thebehavior of an entire membrane, is correct.

This chapter has been partially published as A.M. Prenen, J.C.A. van der Werf, C.W.M.Bastiaansen and D.J. Broer, Adv. Mater. 21, 2009 pp 1751-1755.

29

30 Chapter 3

3.1 Introduction

The removal of particles from liquids and gasses using membranes is a widelyused separation process. A large variety of membranes is commercially avai-lable[1–3] and the properties of these membranes are extensively tuned to thedesired size-selectivity, throughput and pressure drop.[4] Often, these mem-branes are produced via solvent induced phase separation processes whichinvariably results in membranes with a rather large polydispersity in pore sizeand a tortuosity which is far from ideal.[5]

High performance separation membranes should have a narrow pore sizedistribution, a high porosity, a low tortuosity and a good selectivity and per-meability.[4, 5] Recently, the production and properties of such monodispersemembranes have attracted considerable attention.[6–8] Commercially availablemonodisperse microsieves are usually inorganic and possess an excellent chem-ical and thermal resistance. These membranes are produced using lithographictechniques similar to those used in the semiconductor industry. The produc-tion of these membranes is rather laborious and involves a sequence of process-ing steps such as coating, illumination, developing, etching and stripping.[6–9]

Not surprisingly, the cost of these membranes is very high which limits theirusefulness and this despite their excellent performance.

Several rather elegant routes were proposed in the past to produce poly-meric and monodisperse microsieves via, for instance, phase separation mi-cromolding[10] and track etching.[11] The prime objective is often to combinethe ease of processing of polymers with the high performance of monodis-perse porous media. Nevertheless, the above described polymeric membranesalso have limitations with respect to maximum porosity, material properties(mechanical strength), or freedom of design in pore geometry or in pore size.[5]

In this chapter, a new method for the production of monodisperse mem-branes with a wide range of well-controlled pore sizes, pore geometries andpore packings is described. The main distinction with existing polymeric mi-crosieves is the processing route (interference lithography) and materials used(densely crosslinked thermosets). Interference holography[12] is used to obtaina straightforward process with a few processing steps. Interference lithogra-phy is a well-known process for creating micro- and nanostructures for variousapplications, including nano-pillars,[13] nanostructured substrates[14] and 3Dphotonic crystals.[15] Densely crosslinked thermosets are used to enhance thechemical resistance and high temperature performance of the membranes.

Fabrication of holographic membranes 31

3.2 Holography

Holography is a photographic technique in which an interference pattern isrecorded into a photosensitive film. The interference pattern is generatedby intersecting two laser beams with a well-chosen polarization state. Forintensity holography, where the interference pattern consists of intensity mod-ulations only, the interfering beams need to be linearly polarized, with theirpolarizations parallel to each other.

3.2.1 Theory of transmission holography

Consider two coherent laser beams, represented by the electric field vectors E1

and E2, with propagating wave vectors k1 and k2 and polarization pointingout of plane, incident on a sample at angles θ/2 and −θ/2 (see figure 1.5 left).Then, the k-vectors can be represented as:

k1 =2π

λ

(sin

θ

2−→ex + cos

θ

2−→ey

)(3.1)

k2 =2π

λ

(− sin

θ

2−→ex + cos

θ

2−→ey

)(3.2)

with −→ex and −→ey defined as in figure 1.5. The intensity modulation causedby the interference of these two beams is then:

I(x) = I1 + I2 + 2√

I1I2 cos(2k sin

θ

2)

(3.3)

with k = 2πλ and I1 and I2 the intensities of the two beams. The periodicity

of this intensity pattern can then be found from Bragg’s law:

Λ =λ

2 sin θ2

(3.4)

Besides intensity modulations also polarization modulations can be createdusing different polarization states of the two interfering laser beams.

3.2.2 Lasers

The key to a regular interference pattern, and therefore to regular micro-structures, is the coherence of the light used. Nowadays, lasers are used as asource for coherent light. Photons emitted by a laser have an identical phase,direction and frequency, and therefore make a laser suitable as a coherent lightsource. All holographic exposures in this thesis are performed using an Argon

32 Chapter 3

ion (Ar+) laser (BeamLok 2085-25S, SpectraPhysics) operated at the 351.1nm UV line.

Three different processes take place during lasing: absorption, spontaneousemission and stimulated emission.[16] An atom moves up to a higher energylevel when it absorbs energy provided either by a collision with a free elec-tron, excited atom or by interaction with a photon. When an atom decaysspontaneously to a lower energy state, it emits a photon with energy hν anda frequency:

ν =(Ehigh −Elow)

h(3.5)

with h Planck’s constant. An excited atom can also be stimulated to de-cay by interacting with a photon of frequency ν, thereby releasing a photonwhich is identical to the incident photon in phase, direction and frequency.A laser is designed to provide circumstances where the combination of ab-sorption, spontaneous emission and stimulated emission is beneficial for lightamplification.

Since the rate of stimulated emission is proportional to the populationof the higher energy level, it is favorable to have a larger population in thehigher energy level than in the lower energy level. In figure 3.1, the four-level transition scheme of visible Argon is depicted. In the Argon laser, anelectric discharge is used to excite the Argon gas, pumping a neutral atom bytwo collisions with electrons. The first one ionizes the atom to Ar+, and thesecond one excites it from E1 to E3 or to E4, from which the atom almostimmediately decays to E3 since the transition probability for this transition is

Ionizing

transition

Pumping

transition

E4

E1

E2

E3Visible laser

transition

Ar

Ar+

ground state

Figure 3.1: Energy states of Argon.


much higher than the one to E1. If atoms at E3 have a large lifetime, theirpopulation will increase due to the pumping action. Eventually, an atom willdecay from E3 to E2 emitting - either spontaneously or stimulated - a visiblephoton (or UV in this case). When the E2 is instable, rapid return to theground state will occur, establishing a population inversion between E2 (smallpopulation) and E3 (large population). In this way, light is amplified when itpasses the Argon, which is called an ’active medium’.[16]

A resonant optical cavity, defined by two wavelength-selective mirrors, pro-vides feedback to the active medium. Photons that are emitted parallel to thecavity axis are reflected, returning to interact with other excited ions. Stimu-lated emission produces two photons, two become four, four become eight....until an equilibrium between excitation and emission is reached. One of themirrors - the output coupler - transmits a fraction of the energy stored withinthe cavity, the output beam of the laser.

3.2.3 Holographic setup

The laser beam exiting the laser beam is expanded using a beam expanderconsisting of 2 lenses and a pinhole. Focused on the 5 µm pinhole by thefirst lens, the beam is made parallel by the second lens, filtering out inhomo-geneities in intensity. The laser beam is guided through a half wave plate,where the polarization is rotated to 45 degrees. After this, it passes a po-larizing beam splitter (PBS), where the beam is split into 2 perpendicularlypolarized beam of the same intensity (due to the 45 degree incident polariza-tion). The polarization of one of the beams is rotated by a half wave plate toparallel with the polarization of the second beam. A mirror guides the beamsto the sample with the photosensitive film (figure 3.2).

lenslens lens

pinhole

beam-

splitter

mirror

mirror

mirror

λ/2λ/2

laser

Figure 3.2: Holographic recording setup.

34 Chapter 3

3.2.4 Recording materials

Materials for the recording of a holographic interference pattern are photo-sensitive materials that convert the modulations of the interference patterninto modulations of the material itself. A class of recording materials thatis well-known for its excellent lithographic properties is the photoresists. Inthese photoresists, the interference pattern is recorded as a latent image, afterwhich a few extra processing steps are needed to obtain the final lithographicstructure.

Commercially available photoresists include PMMA (Polymethylmethacry-late), PMGI (Polymethylglutarimide), DNQ-Novolac (Diazonaphthoquinone-phenol formaldehyde derivatives) and SU8,[17, 18] an epoxy-based negative pho-toresist (available from MicroChem). SU8 is very often used for applicationswhere a permanent resist pattern is required, e.g. micro-electromechanicalsystem (MEMS) chips, biochips and optoelectronics.[19] The advantages ofSU8 (figure 1.7) are its very high aspect ratio imaging (>10) and the veryhigh chemical and plasma resistance.[20]

3.3 Experimental

The commercially available photoresist SU8 (MicroChem[17,21]), a mixture ofa multifunctional epoxy resin, a photo-acid generator (triarylsulfonium salt)and a solvent (cyclopentanone) is used as membrane material in this study(figure 3.3). After polymerization this monomer forms a densely crosslinkedchemical network with excellent chemical, mechanical and thermal proper-ties. SU8 is a negative photoresist with an exposure dose threshold (±140 mJcm−2[20]) which facilitates the digital recording of an analogue optical field.Glass substrates are cleaned in ethanol and dried with nitrogen gas. A thinlayer of adhesion promoter (Omnicoat, MicroChem) is spin coated (4000 rpmfor 20 seconds, Karl Zuss RC6 spin coater) and baked at 200◦C for 2 minutes.After cooling, SU8 is spin coated (typically 2000 rpm for 40 seconds - thickness6 µm) and heated for 1 minute at 65◦C followed by 2 minutes at 95◦C ona regular hotplate to evaporate the solvent and bring the SU8 in the glassystate. Film thicknesses are tuned by changing the solid concentration and/orspin speed.

The monomer/initiator mixture is in the glassy state at room temperature,and consequently the application of multiple exposure steps is possible. Thechoice for the two exposure steps, two-beam interference approach instead of a


OOC2HC

O

CH2

CH3H3C

O C2HC CH2

O

OC2HC

O

CH2

CH3H3C

O C2HC CH2

O

OC2HC

O

CH2

CH3H3C

O C2HC CH2

O

OC2HC

O

CH2

CH3H3C

O C2HC CH2

O

H2C

H2C

H2C

SU8 molecule

Cyclopentanone

Triarylsulfonium salts

Figure 3.3: Composition of the SU8-2010 mixture: SU8 molecule: monomer,triarylsulfonium salts: initiator and cyclopentanone: solvent.

single exposure three-beam set-up is mainly based on the fact that a two-beamset-up is much more practical in use e.g. when changing periodicities, withrespect to contrast ratios and that more geometries (square arrays e.g.) canbe fabricated using 2 separate exposure steps instead of one. The resultingstructure after development is a simple addition of the different exposures.[22]

The transmission holography setup depicted in figure 3.2, using the 351nm line of a CW Ar+ ion laser (Beamlok 2085-25S, SpectraPhysics) is used.Transmission holography is used to create a latent image of the desired struc-ture in the SU8 negative photoresist. First, vertical lines are recorded (figure3.4(a)), secondly, after rotating the sample 90 degrees, horizontal lines arerecorded (figure 3.4(b)). Figure 3.4(c) shows the resulting optical field thatis recorded, which is the simple addition of the two exposure steps. The rel-ative orientation of the exposure steps determines the final geometry of thestructure 3.4(d).

The interference pattern generated in the holographic set-up is not perfect,i.e. it shows an offset in intensity, the minimum intensity is not zero butsomewhat higher (∼ 0.5 mW cm−2). Due to the threshold of SU8, this is notan issue when fabricating membranes. However, since the intensity pattern issinusoidal instead of a discrete block function, the exposure dose will have an

36 Chapter 3

(a) (b) (c) (d)

Figure 3.4: Schematic overview of the optical fields recorded during the ex-posure step for the fabrication of a square array of circular pores. White rep-resents high light intensity, black represents low intensity. a: first exposure:vertical lines are recorded, b: second exposure: horizontal lines are recordedafter turning the sample over 90 degrees. c: latent intensity image (analogue)after the two exposure steps, d: final structure of the membrane.

influence on the line width that is recorded. This principle is illustrated in fi-gure 3.5. Consider two SU8 films that are irradiated with the same interferencepattern for different exposure times and thus receive a different exposure dose.1

The film receiving the highest exposure dose will have wider areas (lines) thatreceived a dose higher than the threshold dose. The structures remaining aftercrosslinking and development in the high exposure dose case will be wider thanthe ones in the sample with a lower exposure dose.

Low exposure dose

Narrow lines

High exposure dose

Wide lines

Figure 3.5: Schematic representation of the influence of exposure dose online width/fill factor. A higher exposure dose results in wider lines (higher fillfactor) than a low exposure dose.

During exposure a Lewis acid is formed, which acts as a catalyst for thecationic polymerization. Crosslinking occurs predominantly during a secondheating sequence (1 minute 65◦C, 2 minutes at 95◦C) followed by a slow cooling

1Exposure dose and intensity are always designated as the maximum of the interferencepattern.


to minimize build-up of internal stresses. Remaining monomer is removed byimmersing the sample in SU8 developer (mr-dev 600, MicroChem) followed byrinsing with propanol-2 and drying with nitrogen. The analogue character ofthe interference pattern, together with the threshold value of SU8 results incircular pores instead of square pores which would be expected when using aline pattern.

For analysis after fabrication, samples are covered with a 15 nm layer ofgold (K575 XD Turbo sputter coater, Emitech, Ltd.) and imaged with ascanning electron microscope (XL 30 ESEM-FEG, Philips).

3.4 Fabrication of holographic membranes

3.4.1 Square array of circular pores

The most basic design of a microsieve is the square array of circular pores,fabricated using 2 consecutive holographic exposure steps, rotated 90◦ withrespect to each other. From the grating equation:

Λ =λ

2 sin θ2

(3.6)

with Λ in [nm] the periodicity of the interference pattern, λ the laserwavelength (351 nm) and θ/2 the half angle between interfering beams (figure1.5 left) the required angle θ between the laser beams can be deduced. Fora periodicity of 2 µm an angle of θ = 20◦ between the interfering beams isrequired. In a single exposure step the latent image of periodically spaced linesis expected to be formed (figure 3.4(a)), with their width depending on theexposure dose and interference contrast. Rotating the sample over 90◦ withrespect to the interference pattern and exposing again results in a latent squaregrid 3.4(c). The multifunctional epoxy monomer is a negative photoresist andtherefore, pores are expected to be introduced in the regions that received alow exposure dose (lower than the threshold), between the lines (figure 3.4(d)).In figure 3.6, it is shown that such a regular membrane with a thickness of 6µm and a periodicity of 2 µm is indeed obtained with 2 subsequent holographicexposures with an exposure dose of 76 mJ cm−2.

In a subsequent set of experiments, it was attempted to produce a widerange of pore sizes by varying the exposure conditions with respect to lightintensity pattern and/or exposure dose. In figure 3.7, it is shown that poreswith diameters between 100 nm and 4 µm can be obtained which illustrates

38 Chapter 3

Figure 3.6: Standard holographic membrane with a square array of circularpores, thickness is 6 µm, pore diameter is 1 µm.

the versatility of this holographic technique. It has to be noted that due to thenon-zero off-set intensity of the interference pattern, for smaller periodicitiesa lower exposure dose is needed to obtain a fill factor of 50%.

(a) 4 µm (b) 1 µm (c) 100 nm

Figure 3.7: Scanning Electron Microscope (SEM) images of holographicmembranes with different pore diameters a: 4 µm, b: 1 µm, c: 100 nm.

It might seem surprising that pore sizes (diameter of 100 nm) far belowthe wavelength of recording light (351 nm) can be obtained. However, this isa direct consequence of the use of a negative photoresist and the sinusoidalintensity pattern, i.e. the structures between the pore are actually recorded(and their size and periodicity are restricted by the diffraction limit). Withincreasing exposure dose, the walls grow in width and the pores automaticallybecome smaller (not restricted by the diffraction limit). Of course, a penaltyexists in reducing pore size in terms of porosity. The porosity can be increasedby the application of elongated pores.


3.4.2 Slit-shaped pores

Round pores might seem to have the ultimate pore shape, but they are sur-passed by their slit-shaped peers. Elongated pores exhibit many advantagesover circular pores. Their surface to volume ratio is lower than for a circularpore, effectively decreasing the flow resistance induced by the no-slip conditionat a pore wall (paragraph 2.3.2). Furthermore, slits have the tendency to foulless rapidly than round pores.[23]

Holography is a practical technology that can be utilized for the fabricationof membranes with elongated pores. Firstly, a square array of slit-shapedpores is attempted to construct. For this, the two holographic exposure stepsare executed using two different pitches, one for each exposure step. As anexample, the first exposure step is done with a 2.5 µm periodicity (θ = 8◦)and the second with a Λ of 1 µm (θ = 20◦). After post-baking and developing,the membrane shown in figure 3.8 is obtained.

Figure 3.8: SEM micrograph of holographic membrane fabricated with 2 dif-ferent pitches: horizontal: 2.5 micrometer, vertical 1 micrometer.

From this picture it appears that the walls between the pores are notequally wide in horizontal and vertical directions, which is adverse for theporosity. This can be attributed to the sinusoidal nature of the holographicinterference pattern. Due to the threshold dose of SU8, however, the widthof the walls can be controlled by decreasing the exposure dose such that thefill factor of the walls for the large periodicity exposure decreases. This way,thinner walls could be obtained.

A hexagonal array of slit-shaped pores is to be considered next, because avery high porosity is expected in these membranes. The fabrication principleis straightforward, two holographic exposures using the same periodicity Λ areperformed, but instead of rotating the grating vector over 90◦, it is rotatedover 30◦ with respect to the first exposure. The areas of a high and low light

40 Chapter 3

(a) (b) (c) (d)

Figure 3.9: Schematic overview of the optical fields recorded during the ex-posure step for the fabrication of a hexagonal array of slit-shaped pores. Whiterepresents high light intensity, black represents low intensity. a: first exposure:vertical lines are recorded, b: second exposure: lines are recorded after turningthe sample over 60 degrees. c: latent intensity image (analogue) after the twoexposure steps, d: final structure of the membrane.

intensity and latent images recorded are schematically drawn in figure 3.9.As seen from figure 3.9, a hexagonal packing of slit-shaped pores is ex-

pected to form. A membrane is recorded with a 2 µm spacing (θ = 10◦) and arotation of 30◦ of the grating vector for the second exposure. After the post-processing, the membrane from figure 3.10 is obtained. It is clear that thismembrane indeed possesses a high porosity which is partly related to theirslit-like geometry and partly due to the hexagonal packing of pores.

Figure 3.10: Membrane containing slit-shaped pores in a hexagonal array.The large diameter is 2.5 µm, the small diameter is 1 µm. The porosity ofthis membrane is 30%.

The porosity of the membrane in figure 3.10 is 30%, which is significantlyhigher than the porosity of 20% of a comparable membrane (same pitch andsame selectivity) consisting of a square array of circular pores.


3.4.3 Tapered cross-section

In paragraph 2.3.4 it was shown that the Vena Contracta effect can be over-come by applying pores with a tapered pore diameter. That is, the porediameter increases from small at the inlet side to wide at the outlet side ofthe membrane. Consider a membrane with a pore diameter of 2 µm at theinlet side. From the simulations it was concluded that the pore shape withthe highest gain in flux would be that with an outlet pore diameter of 3 µmfor a thickness of 6 µm.

The diameter of the pores of the previously described membranes is vir-tually constant over the cross-section (figure 3.11) of the membrane. This isalso shown in the cross-sectional SEM image of a membrane with a thicknessof 6 µm, a pore diameter of 1.6 µm and a periodicity of 2.3 µm. The homo-geneity of the pore diameter is a direct consequence of the excellent recordingcharacteristics of the multifunctional epoxy monomer.[24, 25]

Figure 3.11: Cross-section SEM image of a standard holographic membranewith a square array of circular pores.

The approach to obtain the tapered pores consists of the addition of asmall amount of 2-(2H-benzotriazol-2-yl)-4,6-di-tert-pentyl phenol (Tinuvin328, Ciba) as a UV-absorber to the photoresist which induces a UV light in-tensity gradient in the cross-section of the polymeric film. The amount ofUV absorber to be added has to be determined for this purpose. To do this,6 µm thick SU8 films on glass substrates were prepared containing differentconcentrations of UV absorber. A transmission spectrum of each sample wasobtained using a Shimadzu UV-3102PC spectrophotometer (equipped withMPC-3100), using a glass substrate for the baseline measurement. In figure3.12 the transmission at 351 nm for different absorber concentrations is de-picted.

From these measurements, the absorption coefficient of the absorber in

42 Chapter 3

0.0 0.5 1.0 1.5 2.00

25

50

75

100

Tran

smis

sion

(%)

Concentration UV absorber (wt%)

Figure 3.12: Transmission of 6 µm thick SU8 films on glass, containingdifferent concentrations of Tinuvin 328.

the epoxide monomer matrix was determined to be 0.3 wt%−1µm−1, usingthe Lambert-Beer equation for absorption:

I

I0= 10−αlc (3.7)

with α the absorption coefficient [wt%−1µm−1], l the path length of the lightthrough the sample [µm] and c the concentration of absorbing species in [wt%]on the SU8 solid content. The effect of the UV absorber concentration on thepore diameter is simulated, taking into account the intensity profile, exposuretime and threshold dose of SU8.[21] In the simulation the diameter of thepores at the bottom was set at 2 µm and the exposure was performed fromthe bottom side. For every concentration, the exposure dose at the front(non-exposed) side of the SU8 film is calculated, using an intensity pattern(θ = 5◦) and an intensity (10.5 mW cm−2, each beam) that should result inmembrane with a pore diameter of 2 µm. Comparing this exposure dose withthe threshold dose results in the final wall thickness at the un-exposed side ofthe membrane.

From the simulation results shown in figure 3.13, the pore diameter at thetop of the membrane is expected to be 3 µm. An experiment using 0.3 wt% ofabsorber in a 6 µm thick membrane with a periodicity of 4.5 µm and a porediameter (at the bottom) of 2 µm was performed. The SEM image in figure3.14 shows that the pore diameters generated in this experiment correspondto the modeled values.


0.0 0.1 0.2 0.3 0.40.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Outlet

Out

let d

iam

eter

(m

)

Concentration UV absorber (wt%)

Inlet

Figure 3.13: Calculation of the outlet pore diameter as function of the con-centration UV-absorber. The inlet diameter is chosen to be 2 µm.

Figure 3.14: Cross section SEM image of membrane with tapered pores.Inlet diameter is 2 micrometer, outlet diameter is 3 micrometer.

3.4.4 Integrated support

The throughput and transmembrane pressure drop of a membrane are highlydependent on the porosity and on the aspect ratio of the pores, cfr. equa-tion 2.8. In general, a low aspect ratio (<1) is preferred (paragraph 2.3.3)but this automatically imposes restrictions on the mechanical strength of themembrane. For instance, a non-supported membrane with a pore size below1 micrometer and an aspect ratio below unity has a thickness of less than 1µm.

Given the fact that these membranes must be handled and mounted, andthat they need to withstand gas or liquid pressure during their use, measuresfor reinforcement are required. Integrated supports consisting of the same

44 Chapter 3

material as the fragile membrane itself are used to provide the membranewith the strength necessary to withstand the handling forces and operatingpressure without detaching from the membrane. Such a support is obtainedby applying an extra, thick (40 - 50 µm) SU8 layer on the original membraneafter its double exposure but prior to the development step, followed by alithographic mask exposure. The lithographic mask has typical dimensionswhich are one or more orders of magnitude larger than the periodicity of theactual membrane itself.

(a) (b) (c)

(d) (e)

Figure 3.15: Schematic view of the processing steps involved for incorporat-ing a support. (a) starting with a membrane (b) apply thick SU8 layer (c)mask lithography (d) crosslink and development (e) stripping.

The mechanical strength of the membrane is then determined by the di-mensions of the structures created in the latter lithographic step rather thanby the pore dimensions.[26] A membrane provided with a grid-shaped incorpo-rated support with support bars that are 200 µm separated and 20 µm wideis shown in figure 3.16.

Figure 3.16: Membrane (pore diameter = 2 µm) with incorporated supportstructure. The support bars are 50 µm high, 20 µm wide and separated by 200µm.


3.5 Experimental verification FEM simulations

To verify whether FEM simulations using Navier-Stokes equations are validto use in the case of holographic membranes, an experimental verification[27]

of the numerically obtained throughput is performed. A flow of nitrogen gasthrough a typical membrane (pore diameter 1 µm, periodicity 3 µm and thick-ness 5 µm) is measured. For these experiments, the membrane is placedbetween 2 rings inside a pressure chamber. The chamber is split into twoseparate chambers, of which the pressure is measured using a liquid column(Lauda Therm 180, hydrophobic oil). The difference between these two mea-sured pressures is the transmembrane pressure drop. The inlet chamber ispressurized with a gas supply, the outlet chamber is connected to a bubbleflow meter.

Figure 3.17: Comparison of the experimentally determined velocity (dots)and the simulated flow velocity (line).[27]

The results are shown in figure 3.17. The dots represent experimentallyobtained face velocities, while the line represents the calculated face velocity. Itis found that for these dimensions finite element simulations give a reasonablygood prediction for the throughput of holographic membranes.[27]

3.6 Conclusions

The control over pore geometry and dimensions is essential in the fabricationof high performance polymeric membranes. The pore diameter of the largestpore in a membrane determines the selectivity, while all smaller pores onlycontribute to the pressure drop of the membrane. Interference holography isshown to be able to produce monodisperse membranes with a very regular poreshape and very narrow pore size distribution. Elongated pores have two majoradvantages over their circular counterparts: a higher flux due to the better

46 Chapter 3

surface to volume ratio and they are less susceptible to fouling. Either byusing two different periodicities or by rotating the second holographic exposureover an angle < 45◦, slit-shaped pores are obtained, in either a square or ahexagonal packing. Pore diameters between 100 nm and 5 µm were obtainedusing the combination of holography and a well-known commercially availablephotoresist, SU8.

Incorporated supports are produced on the membranes using standardmask lithography to mechanically support very thin and weak membranes..The mechanical strength of the membrane is then determined by the super-structure of the support rather than the microstructure of the membrane. Alast important factor that determines the throughput of a pore is the conical-ness of the pore. Due to an effect called the Vena Contracta, the flux is limitedby a decrease in effective flow diameter, the Vena Contracta. Jet-shaped poresare known to prevent this from happening. To obtain tapered pores, a UV-absorber is added to the photoresist, creating an intensity gradient throughoutthe film. This causes wide pore walls at the high intensity side and thin wallsat the low intensity side, resulting in a pore diameter gradient.

Lastly, holographic membranes were used to experimentally verify the re-sults from the FEM simulations from the previous chapter. It is found thatthe measured flux corresponds well with the simulated values, and thereforethat the assumption to simulate only one pore instead of an entire membraneis valid.

Summarizing, interference holography offers a complete toolbox for gen-erating microfiltration membranes, which is expected to create new opportu-nities in high-end membrane applications. In the next chapter, slanted-angleholography will be introduced as a different way to use interference holographyin membrane manufacturing.

3.7 References

[1] http://www.whatman.com.

[2] http://www.kochmembrane.com.

[3] http://www.fluxxion.com.





[7] C. van Rijn, W. Nijdam, S. Kuiper, G. Veldhuis, H. van Wolferen andM. Elwenspoek. Microsieves made with laser interference lithography formicro-filtration applications. J. Micromech. Microeng., 9 (2), (1999) 170–172.


[9] ”http://www.pamgene.com”.



[12] A. Rodriguez, M. Echeverria, M. Ellman, N. Perez, Y. K. Verevkin, C. S.Peng, T. Berthou, Z. Wang, I. Ayerdi, J. Savall and S. M. Olaizola. Laserinterference lithography for nanoscale structuring of materials: From lab-oratory to industry. Microelectron. Eng., xxx (xxx), (2009) xxx.

[13] R. Murthy, J. Ng, E. Selamat, N. Balasubramanian and W. Liu. Siliconnanopillar substrates for enhancing signal intensity in DNA microarrays.Biosens. Bioelectron., 24, (2008) 723–728.

[14] F. A. Zoller, C. Padeste, Y. Ekinci, H. H. Solak and A. Engel. Nanos-tructured substrates for high density protein arrays. Microelectr. Eng., 85,(2008) 1370–1374.

[15] J. H. Moon, A. Small, G.-R. Yi, S.-K. Lee, W.-S. Chang, D. J. Pine andS.-M. Yang. Patterned polymer photonic crystals using soft lithographyand holographic lithography. Synth. Met., 148, (2005) 99–102.

[16] O. Svelto. Principles of Lasers. Plenum Press, New York, 3 edition(1989).

48 Chapter 3

[17] M. Shaw, J. D. Gelorme, N. C. LaBianca, W. E. Conley and S. J. Holmes.Negative photoresists for optical lithography. IBM J. Res. Dev., 41, (1997)81–94.

[18] M. Shaw, D. Nawrocki, R. Hurditch and D. Johnson. Improving theprocess capability of SU8. Microsyst. Techn., 10, (2003) 1–6.

[19] V. Cadarso, A. Llobera, G. Villanueva, V. Seidemann, S. Bttgenbach andJ. Plaza. Polymer microoptoelectromechanical systems: Accelerometersand variable optical attenuators. Sensor and Actuat. A, 145-146, (2008)147 – 153.

[20] MicroChem Corp. SU8 datasheet.

[21] P. M. Dentinger, K. Krafcik, K. Simison, R. P. Janek and J. Hachman.Removal of su8 photoresist for thick film applications. Microelectron.Eng., 61/62, (2002) 1001–1007.

[22] W. Mao, Y. Zhong, J. Dong, and H. Wang. Crystallography of two-dimensional photonic lattices formed by holography of three noncoplanarbeams. J. Opt. Soc. Am. B, 22 (5), (2005) 1085–1091.



[25] H. Lorenz, M. Despont, P. Vettiger and P. Renaud. Fabrication of photo-plastic high-aspect ratio microparts and micromolds using SU8 uv resist.Microsyst. Technol., 3, (1998) 143–146.

[26] S. C. Kitson, W. Barnes and J. Sambles. The fabrication of submicronhexagonal arrays using multiple-exposure optical interferometry. IEEEPhotonics Technol. Lett., 8, (1996) 1662.

[27] M. Courage. Monodisperse holographic membranes. Performance and ap-plications. Master’s thesis, Eindhoven University of Technology (2005).

Chapter 4

In-plane membranes

Incorporation of a membrane functionality in microfluidic systems in biosen-sors has attracted considerable attention lately. Slanted-angle holography en-ables the fabrication of monodisperse membranes with pores that lie in theplane of the membrane. By tilting a photosensitive film with respect to a holo-graphic interference pattern, slanted walls are created. Exposing the film again,tilted towards the opposite side, walls slanted in the other direction are created.The combination of these two walls generates a honeycomb-like structure con-taining areas that did not receive any UV light. Washing out the non-reactedmonomers from these voids leaves a membrane structure with pores parallel tothe film. The pore shape can be adjusted from square to round to slit-shaped,the most optimum in terms of fouling and flux. Size is tuned by adjustingthe periodicity of the interference pattern and the tilt angle of the sample dur-ing exposure. The pores can stretch over an entire film, which is actually notdesirable because high aspect ratios enormously increase the flow resistance.Therefore a combination of mask lithography and two-step slanted-angle holog-raphy is used to obtain membranes with a finite pore length. This technique ishighly compatible with the standard lithographic processes used in microfluidicchip fabrication. Slanted-angle holography will be used later in this chapter forthe in situ fabrication of monodisperse membranes on well-defined locationsinside microchannels. These membranes are leak-free connected to the chan-nel walls. Upon capillary filling of the microchannel, the pressure drop of theselocalized membranes is determined. The selectivity is tested using polystyrenemicrobeads with different diameters.

This chapter will be partially published as: An M. Prenen, Anja Knopf, Cees W.M.Bastiaansen, Dirk J. Broer, In situ fabrication of polymer microsieves for µTAS by slanted-angle holography, in preparation

49

50 Chapter 4

4.1 Introduction

Microfluidic devices are investigated extensively for the control and mani-pulation of small fluid volumes.[1] Usually, a microfluidic device is defined asa device having at least one channel with a diameter of less than 1 mm. Inbiosensors, these devices are often used to process small quantities of fluidor to detect the presence of chemical or biological substances in a small fluidvolume.[2] Typical fluids in microfluidics are: whole blood, blood plasma,saliva, urine, protein or antibody solutions and various buffers. These arecomplex mixtures that need various treatments such as filtering/purification,mixing and chemical treatment. Often, one or more membranes are thereforeincorporated in a microfluidic device.[3] Its function can be diverse includingthe separation of particles from liquids, the induction of turbulence for mixingor providing a large internal surface area for adsorption.

Frequently, commercially available membranes[4] are implemented in micro-fluidics. Typically, these membranes are non-woven fabrics which exhibit ahighly polydisperse pore structure and high porosity. Mounting these mem-branes inside a microfluidic device often poses serious sealing problems.[3] It istherefore advantageous if these microporous filters could be produced in situ.Emulsion photopolymerization[5] of pre-polymer mixtures can result in a localrandom network membrane. Also the fabrication of microporous structuresby controlled phase separation[6] is possible. However, these random networkmembranes exhibit a large pressure drop and restrict the throughput of thefluid in the device significantly.[7]

Monodisperse microsieves on the other hand, have a low pressure drop[8, 9]

but the actual implementation in a microfluidic device is complicated and la-borious. It is therefore advantageous to apply a method that can producemicrosieve filters in situ. In this chapter a production method for microsievesin a microfluidic channel is presented based on the use of lithographic andholographic processes which are common processes in the production of mi-crofluidic devices.

The material used for this purpose is SU8,[10] a photoresist that is fre-quently used as a chip material for micro-total analysis systems (µTAS).[11–13]

It will be shown that the addition of only a few clever exposure steps to thephotolithographic process is needed to obtain monodisperse microsieves incor-porated in a microfluidic device. The main advantage of this approach is thatsealing at the channel walls is not an issue, since the membrane is made of thesame material and during the same processing step as the chip itself.

In-plane membranes 51

4.2 Materials and methods

SU8, a commercially available negative photoresist (MicroChem) is used aschip material in this study. It has a strong non-linear optical response whichresults in a digital recording of the analogue interference pattern. Moreover,the photoresist mixture is in the glassy state at room temperature, minimizingmobility during irradiation and therefore enabling the utilization of severalconsecutive exposure steps.[14]

Glass slides are cleaned in ethanol and dried with nitrogen gas. A 50 nmthin layer of Omnicoat (MicroChem) is spin coated (4000 rpm, 20 seconds,Karl Zuss RC6 spin coater) and cured (2 minutes at 200◦C) to ensure a goodadhesion of the SU8 to the glass substrate. A layer of SU8 is spin coated ordoctor bladed (Erichsen Coatmaster 509MC-1) on a cleaned glass substrate,depending on the required film thickness, followed by a two step pre-bake toevaporate the solvents and to ensure that the photoresist is in the glassy state.The exact heating times for the two layer thicknesses used in this study, 6 µmand 50 µm, are displayed in table 4.1.[14]

Table 4.1: Processing times for standard SU8. The pre-bake step is intendedto evaporate residual solvents before exposure. The crosslinking takes placeduring the post-bake step, after exposure.

Film Pre-bake Pre-bake Post-bake Post-bake DevelopmentThickness 65◦C 95◦C 65◦C 95◦C Time6 µm 1 min 2 min 1 min 2 min < 1 min50 µm 30 min 60 min 1 min 15 min 5 min

The sinusoidal interference pattern generated by the holographic setup de-picted in figure 3.2 is used for the holographic irradiation. The periodicitycan be tuned by changing the angle between the crossing laser beams (equa-tion 3.4). Usually, a photoresist is illuminated with an interference patternwhere the grating vector is perpendicular to the sample normal. Here, thephotoresist is exposed with the sample tilted over an angle α with respect tothe interference pattern such as used in the fabrication of slanted gratings.[15]

The latent image produced by such an exposure is depicted in figure 4.2.Areas of high and low intensities lie in lamellae-like structures that are tiltedover the same angle α (figure 4.2(a)). Likewise, a second exposure step, thistime with a tilt angle −α results in lamellae tilted over −α (figure 4.2(b)).The resulting latent image of the final structure then shows a honeycomb-likeshape, as can be found in figure 4.2(c). During illumination a Lewis acid is

52 Chapter 4

α

θ/2

sample

Figure 4.1: In contrast to ’standard’ holography, the sample is tilted over anangle α with respect to the interference pattern.

formed in the high intensity areas, which acts as a catalyst for the cationicpolymerization. Crosslinking is induced during a second heating sequence(post exposure bake (PEB), for temperatures and times see: table 4.1) whichis followed by slowly cooling down the sample to minimize build-up of inter-nal stresses due to temperature differences between the glass substrate andthe SU8. The final structure is obtained when unreacted monomer is rinsedout of the non-exposed parts by consecutively immersing the sample in SU8developer (mr-dev 600, MicroChem) and in propanol-2 after which it is driedwith nitrogen gas (figure 4.2(d)).

(a) (b) (c) (d)

Figure 4.2: Schematic representation of the latent image formation duringslanted-angle holography. a: exposure over tilt angle α , b: exposure over tiltangle −α , c: resulting latent image. d: resulting structure after crosslinkingand development.

For scanning electron microscopy (SEM) analysis (XL 30 ESEM-FEG,Philips), a 15 nm layer of gold (K575 XD Turbo sputter coater, Emitech,Ltd.) is sputter coated on the sample after fabrication. The selectivity of themembranes fabricated inside the microchannels is characterized using a mix-ture of fluorescein (Aldrich) in water. To this mixture, dark red polystyrenemicrospheres with a diameter of 1 µm or 3 µm (Aldrich) are added. The fil-tration experiments are monitored with a Leica DM600M optical microscopeequipped with an DFC420 camera.


4.3 Fabrication of in-plane membranes

4.3.1 Influence of tilt angle

Upon exposure at a different tilt angle α, the pore shape and array are influ-enced. Smaller tilt angles are expected to result in slit-shaped pores, whilelarge tilt angles should give more round pores. Slit-shaped pores have manyadvantages over round pores. For example, the open area of a membrane withslit-shaped pores is larger than that of a membrane with round pores, whilemaintaining the same selectivity. Therefore, the pressure drop of such a slit-shaped pore is lower than that of a membrane with round pores.[16] Moreover,slit-shaped pores are less susceptible to fouling,[17] due to the lower surface tovolume ratio of the pores.

(a) 10◦ (b) 20◦

(c) 30◦ (d) 40◦

Figure 4.3: Cross-section SEM images of membranes fabricated using a peri-odicity of 1 µm and tilt angles of 10 ◦, 20 ◦, 30 ◦and 40 ◦. It can be seen fromthe images that the angle influences the pore shape from slit-shaped (small α)to round or even square shaped (large α).

In-plane membranes are fabricated using an exposure dose (measured fromthe maximum of the interference pattern) of 74 mJ cm−2 for a periodicity of 1µm, with tilt angles 10◦, 20◦, 30◦and 40◦. After crosslinking and development,the samples are cut and broken to obtain a cross-section for SEM investigation.

54 Chapter 4

In figure 4.3 the effect of changing the tilt angle is clearly illustrated by cross-section SEM images of membranes where α = 10◦, α = 20◦, α = 30◦, α

= 40◦, for a periodicity of 1 µm. From the SEM images, it can be foundthat the smallest diameter of the pores of all four fabricated membranes isapproximately equal. This indicates that all 4 membranes have the sameselectivity, while the membranes with elongated pores (figure 4.3(a)) have asignificantly higher porosity.

4.3.2 Influence of periodicity

The possibility to produce in-plane membranes with a variety of pore sizesand shapes is also explored. Therefore, samples with a 6 µm film of SU8on a glass substrate coated with Omnicoat (MicroChem) are prepared andconsecutively irradiated with a tilt angle of α and -α with respect to theinterference pattern of the intersecting laser beams, followed by post-exposurebake and development.

By changing the periodicity of the interference pattern, the pore size of theresulting membrane is adaptable to the desired diameter. In figure 4.4 this isillustrated by three cross-sectional SEM images of three in-plane membranes,exposed to interference patterns with periodicities of respectively 1, 2 and 7µm, corresponding to θ between the beams of respectively 20◦, 10◦ and 4◦,all membranes are fabricated with a tilt angle α of 30◦. It is observed thatthe recording of small periodicities (e.g. 1 µm) results in the formation ofmembranes with smaller pore diameters, in multiple layers on top of eachother, as is expected.

(a) 1 µm (b) 2 µm (c) 7 µm

Figure 4.4: Cross-section SEM images of in-plane membranes fabricatedwith different periodicities of the interference pattern: 1 µm, 2 µm and 7 µm.


4.4 Slanted-angle mask holography

Ultimately, the above-described membranes are intended to be fabricated insitu in a microchannel. Therefore it must be possible to choose the locationwhere they are formed. The lithographic character of the slanted-angle holo-graphic fabrication method enables to use dedicated masks for this purpose.The areas that are required to contain a membrane should appear transparenton the mask.

SU8 films of 6 µm thickness are prepared on glass slides, with the conven-tional pre-bake conditions. Thereafter, the films are exposed to a holographicinterference pattern with a periodicity of 2 µm, through a mask with a period-icity of 50 µm (fill factor 50%). The samples are illuminated with an exposuredose of 80 mJ cm−2, and attached to the mask using the capillary force ofcyclohexane which acts as a contact fluid. The microscope image from figure4.5 made with an optical microscope (Zeiss Axioplan 2) shows the final result.The pores are clearly visible as horizontal lines in the image, while the lengthof the pore is determined to be 25 µm, exactly the width of the transparentlines on the mask.

Figure 4.5: Microscope image of in-plane membrane fabricated with slanted-angle mask holography. A mask with a periodicity of 50 µm and a fill factorof 50% was used.

4.5 In situ fabricated filters for microfluidics

It is now shown that slanted-angle mask holography is feasible for the localizedproduction of membranes with a well-defined pore length. The logical nextstep is to use the aforementioned techniques to fabricate a microchannel withincorporated monodisperse microporous filters.

To achieve this, a glass substrate is coated with a thin layer of SU8, ty-pically 5 µm, and pre-baked, which is then flood exposed (100 mJ cm−2)

56 Chapter 4

and post-baked. This film will act as an adhesion layer to which the micro-fluidic channel and the membrane will be chemically bonded in order to forma leak-free connection. A 50 µm thick film is then doctor bladed on top of theadhesion layer and pre-baked (30 minutes at 65◦C, followed by 60 minutes at95◦C).

After cooling down, it is first exposed to UV-light (351 nm) through a linemask containing the negative image of the microfluidic channels with a width of75 µm. In a consecutive exposure step, the slanted-angle holographic exposureis performed through a line mask with a smaller periodicity (10 µm). In bothexposures, the sample is brought into optical contact with the mask usingcyclohexane as a contact fluid, to avoid reflections at the SU8-mask interface.The dimensions of the transparent lines are typically in the micrometer range,in this case 5 µm, and they correspond to the final thickness of the membranesin the microfluidic channel. The holographic exposure step is performed withperiodicity of 2 µm and a tilt angle of 40◦. After exposure, the sample iscrosslinked in the post exposure bake (1 minute at 65◦C, 15 minutes at 95◦C),followed by development with mr-dev 600 (MicroChem) and propanol-2.

(a) (b)

Figure 4.6: SEM images of in situ fabricated microfluidic filters. On the leftan overview, on the right a detailed picture of the membrane: pores are openand have narrow pore size distribution with a pore diameter of 720 nm.

An example of a sample containing equally spaced microfluidic channelscontaining a multitude of in-plane membranes is shown in the SEM imagesof figure 4.6. The resulting pore diameter is 720 nm. In the SEM image offigure 4.6(a) the sharp contours of the well-defined membranes are displayed.From this image it can also be observed that both the microfluidic channelwalls and the membrane itself are physically attached to each other - which isexpected, as they are fabricated in the very same process. Also the connection


of the membrane to the SU8 adhesion layer on the bottom looks impeccable.This gives a good indication that the structures are leak-proof. From subfigure4.6(b) it is clearly visible that the membrane pores are open and the pore sizedistribution is very small.

4.6 Characterization

4.6.1 Selectivity

To determine the selectivity of the in-plane membranes fabricated using slanted-angle holography, filtration experiments are performed. Samples containing 50µm wide channels with each channel containing one in-plane membrane areused. Three types of membranes with different pore sizes (0.5 µm, 2.5 µmand 4 µm) and a width of 15 µm are tested on their selectivity. The microflu-idic channel is capillary filled with a dispersion of red polystyrene microbeads(Aldrich) in water, dyed with fluorescein to improve visualization in the op-tical microscope. During the experiment, both sides of the membrane in thechannel are inspected with a Leica DM6000 optical microscope.

Firstly, a channel containing a membrane with a pore diameter of 0.5 µmis filled with a dispersion of 1 µm microspheres (figure 4.7). The channel isfilled from the right side, and the microspheres are observed to be retained bythe 0.5 µm pores.

Figure 4.7: Microscopy image of capillary filling of microchannel containingmembranes with a pore size of 0.5 µm. 1 µm beads added to the microchannelare retained at the feed side of the membrane.

Secondly, a dispersion of 1 µm beads and 3 µm beads is added to a channelwith 2.5 µm pores. In principle, the 1 µm beads should pass through themembrane, while the 3 µm beads should be retained by the membrane. Figure4.8 shows that on the right (feed) side of the membranes a mix of 1 and 3 µm

58 Chapter 4

beads is present, while on the permeate (left) side of the membrane only 1µm beads are observed. This experiment nicely shows the selectivity of aholographic in-plane membrane.

Figure 4.8: Microscopy image of capillary filling of microchannel containingmembranes with a pore size of 2.5 µm. 1 µm and 3 µm beads are added to themicrochannel. The 3 µm beads are retained by the membrane while the 1 µmbeads are passed on.

In a last experiment (figure 4.9), 3 µm beads are added to a channel con-taining a membrane with 4 µm pores. Here, on both sides of the membranemicrobeads are observed, so no microbeads are retained by the membrane,even though the membrane appears to be partially blocked by a cluster ofbeads.

Figure 4.9: Microscopy image of capillary filling of microchannel containingmembranes with a pore size of 4 µm. 3 µm beads are added to the microchan-nel. The 3 µm beads can pass through the membrane.

From these experiments, it is found that the in-plane membranes fabricatedin situ in a microfluidic channel by slanted-angle holography can filter particlesby size exclusion in a very selective way. However, in order for the membraneto be efficient, it also needs to have a low flow resistance.

4.6.2 Flow resistance

The flow resistance of the membranes is determined by measuring the flowvelocity of water before and after a membrane during capillary filling of a


channel. For this experiment, a 50 µm thick sample is used, in which 75 µmwide channels are fabricated using mask lithography, containing a 25 µm widemembrane with a pore size of approximately 2 µm. Several (15) channels arecapillary filled with water, and the face velocity is determined using opticalmicroscopy.

The average initial face velocity for the water is measured to be 95 ± 10cm/s. The error on this is rather large due to the relatively low frame rate(100 frames per second) of the camera on the microscope. This correspondsto a volumetric flow of 0.0225 mm3s−1. The face velocity of the water afterpassing the membrane is determined to be 16 ± 4 mm/s, or 0.004 mm3s−1.The flux is reduced by almost a factor 6, but still the flow rate is acceptable.It has to be noted that the membrane used for this experiment had an aspectratio of 12.5. As was shown in paragraph 2.3.3, by decreasing the thickness ofthe membrane a significant gain in flux can be achieved.

4.7 Conclusions

The implementation of monodisperse microfiltration membranes into micro-fluidic devices with current techniques is impracticable and almost impossible.Slanted-angle holography provides a solution for this.

Combining SU8, a common chip material, and slanted-angle holographycreates an exceptional opportunity to produce monodisperse microfilters formicro-total analysis systems in situ, using a set of cleverly chosen exposuresteps. By tilting the photosensitive film with respect to the holographic in-terference pattern, tilted walls are created. Tilting the sample again, to theopposite side, a honeycomb-like structure is obtained containing areas thatwere not exposed: the pores.

Membranes with a narrow pore size distribution are easily fabricated. Poredimensions are shown to be controllable between 0.5 µm and 3 µm by changingthe angles α and θ. The shape of the pores can be pre-designed between square,round and slit-shaped.

Furthermore, the monodisperse microsieves are manufactured in situ insidethe microchannels using slanted-angle holography during the chip fabricationprocess itself. The chip material and the membrane material are identical.The final device is produced in the same crosslinking step. This approach,therefore, results in leak-proof connections between the channel walls, thechannel bottom and the membranes.

The selectivity of the in situ fabricated membranes is tested using a com-

60 Chapter 4

bination of a colored fluid and microspheres. The microspheres, having adiameter larger than the pore diameter are shown to be retained well by themembranes. But also the flux through a membrane, even with a high aspectratio, is acceptable. Therefore, the above described approach to the in situ fab-rication of membranes in microchannels is believed to provide an exceptionaldesign freedom for the leak-free implementation of filtration functionalitiesinto µTAS.

4.8 References

[1] P. Gravesen, J. Branebjerg and O. S. Jensen. J. Micromech. Microeng,3, (1993) 168–182.

[2] D. Erickson and D. Li. Integrated microfluidic devices. Anal. Chim. Acta,507, (2004) 1126.

[3] J. D. Jong, R. Lammertink and M. Wessling. Membranes and micro-fluidics: a review. Lab Chip, (6), (2006) 1125–1139.

[4] S. Thorslund, O. Klett, F. Nikolajeff, K.Markides and J.Bergquist. A hy-brid poly(dimethylsiloxane) microsystem for on-chip whole blood filtrationoptimized for steroid screening. Biomed. Microdevices, 8, (2006) 73–79.

[5] J. Moorthy and D. Beebe. In situ fabricated porous filters for microsys-tems. Lab Chip, 3, (2003) 62–66.

[6] R. Kurt, L. Simon, R. Penterman, E. Peeters, H. de Koning and D. Broer.Control over the morphology of porous polymeric membranes for flowthrough biosensors. J. Membr. Sci., 321 (1).



[9] L. J. Heyderman, B. Ketterer, D. Bchle, F. Glaus, B. Haas, H. Schift,K. Vogelsang, J. Gobrecht, L. Tiefenauer, O. Dubochet, P. Surbled andT. Hessler. High volume fabrication of customised nanopore membranechips. Microelectron. Eng., 67-68, (2003) 208–213.


[10] M. Shaw, J. D. Gelorme, N. C. LaBianca, W. E. Conley and S. J. Holmes.Negative photoresists for optical lithography. IBM J. Res. Dev., 41, (1997)81–94.

[11] J. Zhang, K. Tan, G. Hong, L. Yang and H. Gong. Polymerization opti-mization of SU8 photoresist and its applications in microfluidic systemsand MEMS. J. Micromech. Microeng., 11, (2001) 20–26.

[12] K. Mogensen, J. Eli-Ali, A. Wolff and J. Kutter. Integration of poly-mer waveguides for optical detection in microfabricated chemical analysissystems. Appl. Opt., 42 (19), (2003) 4072–4079.

[13] Y.-J. Chuang, F.-G. Tseng, J.-H. Cheng and W.-K. Lin. A novel fabri-cation method of embedded micro-channels by using SU8 thick-film pho-toresists. Sensor Actuat. A, 103, (2003) 64–69.


[15] C. van Heesch, H. Jagt, C. Sanchez, H. Cornelissen, D. Broer and C. Bas-tiaansen. Polarized light out-coupling from lightguides for LCDs. Chemi-cal Record, 5 (2), (2005) 59–69.



Chapter 5

Filters for biomedical

applications

Microfluidic devices and more specifically biosensors often have a membraneincorporated in their systems. Its function can be to provide a large surface foradsorption of molecules or immobilization of nano and microparticles, to fil-tering large components from the feed and the induction of turbulence. In thischapter, examples of the use of holographic membranes for microfluidic devicesand biosensors are illustrated. Holographic membranes are used as a substratefor the immobilization of proteins in a flow-through protein microarray. Theultimate goal for microarray technology is to achieve a high sensitivity, a highdensity packing of spots and a fast screening. A holographic membrane with in-ternal support, having a low pressure drop and a highly symmetric design withwell-defined spots is utilized for the application (by inkjet printing) of captureprobes. The internal support gives increased mechanical strength to the mem-brane and limits the spot size by acting as a barrier upon printing of capturemolecules. In addition to the improvement of the standard flow-through micro-array, a new concept for the protein microarray is proposed where instead ofthe common, parallel detection of proteins, an in-series approach is adopted.Localized membranes, either in the shape of lamellae or in-plane membranesare fabricated inside a microfluidic channel. Each of these membranes canbe provided with one type of capture probes and as such act as a detectionsite for one specific agent. In a last example of the application of holographicmembranes, an attempt will be made to filter blood cells from a fingerpricksample using a microfluidic channel containing an in-plane membrane with apore diameter of 4 micrometer.

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64 Chapter 5

5.1 Flow-through microarray technology

5.1.1 Introduction

Microarrays are devices that consist of a support that contains patterns ofcapture molecules which are capable of binding with bio-molecular agents,such as DNA,[1] RNA, proteins,[2] cells[3] or drugs[4] present within an analyte.Microarray-based assays are capable of multiplexing[5] when the position ofeach type of receptor molecule is known. Each assay (for each type of receptor)is carried out in a similar way, such that highly reproducible and quantitativeinformation is generated. Although the major application area of proteinmicroarrays is basic proteome research,[6] microarrays are also very useful asdiagnostic tool for the detection of antibodies in e.g. blood samples.

Detection of molecular interactions can be achieved by using labeled bind-ing molecules or by direct labeling. Fluorescent dyes are the label of choice[7]

for the detection of molecules, but also magnetic or radioactive labeling isbeing used.

(a) (b) (c) (d) (e)

Figure 5.1: Schematic view of a sandwich assay and detection by a fluores-cent probe. a) the analyte is flushed over the substrate on which receptors areplaced. b) Molecular recognition. c) Washing buffer. d) Labeled capture proberecognizes agent and e) binds to the agent. After another washing step, theremaining labels can be detected.

In a standard optical detection setup of a protein microarray using a sand-wich assay for the detection, a membrane substrate is covered with an arrayof capture targets of different receptor molecules (antibodies), e.g. applied viainkjet printing or contact printing.[8] The large internal surface of the mem-brane allows for a high (projected) density of immobilized capture probes onthe substrate, enhancing the sensitivity of the device. The analyte is broughtinto contact with the substrate (figure 5.1.a) using the flow-through principle,the analyte is forced through the membrane using external pumping. Molec-ular recognition takes place between proteins in the analyte and the capture

Filters for biomedical applications 65

probes, resulting in specific binding of the matching proteins to the captureprobes (figure 5.1.b).[9] This step is often referred to as incubation for pro-teins. After the unbound molecules from the analyte are washed away with abuffer solution (figure 5.1.c), fluorescent labeled antibodies are added to thesystem to determine whether the specific binding took place. These labeledantibodies will in turn bind specifically to the capture probe-protein complex,in a sandwich-like way (figure 5.1.d). The presence of the bound proteins canbe detected by means of a CCD camera[10] if the fluorescent probes are excitedto emit light (figure 5.1.e).

Examples of membranes which are currently used for flow-through pro-cessing are Nytran[11] and Pamgene.[12] The former is a nylon membrane con-sisting of randomly distributed pores. Due to the large capillary forces andthe interconnectivity of the pores the spot size of printed capture molecules isrelatively large. Another disadvantage of this non-uniform distribution of thepores is relatively large pressure drop over the system.[13] These problems canbe overcome by using a monodisperse pore distribution of non-interconnectedpores. The basic concept of this membrane, which is based on aluminum oxide(Pamgene), is given in figure 5.2. A disadvantage of this specific system is thenon-transparency of aluminum oxide reducing the detection sensitivity of thedevice by blocking emitted light.

Figure 5.2: The Pamgene membrane in different enlargements. At the lefta strip is shown, more or less in its real dimensions, showing four areas thatare printed with a microarray. Then at an enlargement of 20x one can see thearray of spots containing the various capture probes. At the right-hand sidethe nanoporous membrane is shown in top view and in a cross-section. Theinner surfaces of the membrane pores are coupled to capture probes.

Although each example has its specific disadvantages there is also a com-mon disadvantage, namely fluorescent crosstalk. In fluorescent crosstalk thefluorescence from neighboring spots interferes with the detection of the signalfrom a single spot. To prevent this, spots may need to be separated widely

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resulting in an increase in non-used volume in the membrane. Holographicmembranes can be employed to overcome some of these issues.

5.1.2 Design

SU8 is well-known for its biocompatibility[14] and therefore very suitable assubstrate material for a protein microarray. The holographic membranes fromchapter 3 have the same advantages as the Pamgene membranes, namely non-interconnected pores and well-defined pore size. However, the SU8 holographicmembranes have some additional advantages: the pore size can be selected atwill to obtain an optimum between internal surface and flow resistance, andSU8 is transparent for visible light.[15] In paragraph 3.4.4, it was describedhow a grid can be applied on an existing holographic membrane. This griddivides the membrane in equally spaced reservoirs containing a monodispersemembrane (figure 5.3). Each of these reservoirs can be used for the applicationof a different capture probe. The dimensions of the grid depend only on themask design.

Figure 5.3: Design of the flow-through microarray, in each compartmentof the membrane separated by the support bars, a different type of capturemolecules can be applied.

The flow-through holographic membranes described here have a low pres-sure drop, high density packing of spots, high sensitivity and a high signal-to-noise ratio. In essence the device consists of a polymeric membrane and aninternal support which are both created by a photo-lithographic process. Thesize and geometry of the pores in the membrane are designed in such way thata high specific surface area is created while maintaining a low pressure dropover the system. The internal support gives increased mechanical strengthto the membrane and limits the spot size by acting as a barrier upon print-ing of capture molecules. Due to the barrier properties, the spot size can bedecreased and the spot density is increased.

The microarray is produced by applying a layer of SU8 (MicroChem) on a


glass substrate that has been provided with an adhesion promoter (Omnicoat).A pre-bake step (1 minute at 65◦C, ramping to 95◦C and leave at 95◦C for2 minutes) ensures that all solvent has evaporated and that the SU8 is inthe glassy state - enabling the use of multiple exposure steps. The cooling isperformed slowly to avoid building up unwanted stresses in the film.

The sample is then exposed to a holographic interference pattern to createthe latent image of the membrane in the photoresist (80 mJ cm−2, 351 nm,SpectraPhysics Beamlok 2085-25S). Subsequently, the post-bake step is per-formed. The sample is heated to 65◦C for 1 minute, 95◦C for 2 minutes toinduce crosslinking of the photoresist, rendering the exposed areas insolubleto the developer liquid. The non crosslinked areas are washed away with de-veloper (mr-Dev 600, MicroChem), followed by rinsing with isopropanol. Thisway, the basic membrane is created.

Another layer of photoresist is applied on top of the membrane by meansof spin coating. The pre-bake step is repeated. Hereafter, the sample isexposed to UV laser light through a contact mask to create the latent imageof the support structure. Again, the exposed areas are crosslinked in a post-bake step and the monomers are washed away in the development step. Thepresence of the adhesion promoter allows to release the microarray from thesubstrate by immersing it in water.

The pore geometry of the basic membrane can be adjusted by changing theholographic interference pattern and the rotation angle between the exposuresteps. The design of the contact mask determines the structure of the support,which determines the final shape of the microarray, which is shown in figure5.4.

Figure 5.4: Flow-through protein microarray consisting of a holographicmembrane with pore size 2 µm provided with a grid consisting of 200 µmsquares separated by lines of 20 µm.

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The membranes produced via above described methods will be used toimmobilize antibodies. In the next chapter it will be investigated whetherthe immobilization can be done on bare SU8, or that another surface chem-istry should be applied for immobilization and preservation of the biologicalfunctionality of the antibodies.

5.2 New concept:

Lateral Immuno Flow-through Elements sensor

5.2.1 Introduction

The two most commonly applied microarray setups differ in the flow directionof the analyte towards the detection sites: flow-through and flow-over. Inflow-over devices the analyte flows parallel to a solid substrate. The substratecontains an array of nanowells[16] in which the capture molecules are located.The flow-over concept[17] exhibits a low pressure drop and small spot size. Ba-sically, capture probes can be inkjet printed in series, such that theoreticallythe analyte comes across every capture area. In practice this often is not thecase as flow in microfluidic devices normally is laminar such that transport ofthe target molecules to the capturing probes should occur by diffusion whichresults in long detection times.[18] In addition, this system has a low cap-ture probe density due to the limitation of the specific area (2D) limiting thenumber of capture probes per projected area.

In a flow-through device[19] the sample flows perpendicular through amicroporous substrate containing spots of immobilized capture molecules. Thehigh specific surface area of the microporous substrate causes a large numberof specifically bonded probe molecules on the detection spots, resulting in ahigh sensitivity for the detection of bonded molecules. A drawback is thatthe capture areas are placed in parallel and therefore a specific targeted areamisses many sample molecules as they pass outside the targeted area. In orderto solve this, the sample is pumped through the patterned membrane severaltimes. An example product that is on the market is the Pamgene biosensor,based on etched pores in an aluminum oxide membrane.

Both approaches to microarray design have advantages over each other.The flow-over device requires only small volumes of analyte, thanks to the useof microchannels. Flow-through devices have higher signal-to-noise ratios dueto the high capture probe density on the microporous substrate.[20] On theother hand, large sample volumes are required and the analyte needs to be


pumped through the membrane to enhance sensitivity for low concentrationsof agents.

In our design we try to combine both approaches into a device that hasboth a low sample volume requirement, a high signal-to-noise ratio and a highsensitivity. Thereto, well-defined membranes are located on specific spots in amicrochannel. This way, a high specific surface for adsorption of receptors isobtained on very well-defined places. In figure 5.5, a schematic representationof the basic idea, the lateral flow-through elements sensor (LIFE sensor) is dis-played. Membranes are regularly spaced inside a microchannel that connectsan inlet with an outlet.[21]

Figure 5.5: Schematic overview of the LIFE sensor design. An inlet andoutlet are connected by a microfluidic channel in which locally membranes arepresent.

A prototype is fabricated using standard lithography on SU8 as chip mate-rial. A glass substrate is coated with a thin layer of SU8 which is flood exposedto act as a primer layer. A second layer of SU8 is doctor bladed (ErichsenCoatmaster 509MC-1), pre-baked and mask exposed (100 mJ cm−2). Afterthe standard postprocessing, a 50 µm wide channel containing localized mem-branes consisting of 2 µm wide lamellae with an interspace of 2 µm is obtained(figure 5.6). The membrane as shown in the figure yields a 17 times largeradsorption surface than a flat surface.

Capture probes such as oligonucleotides, DNA, RNA or antibodies willbe immobilized on the surface of the flow-through membranes. Convenientlythese probes can be applied by inkjet printing, which is helped by the largecapillary action of the membrane pores. This prevents contamination of theopen channels with capture probes.

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Figure 5.6: SEM image of a membrane consisting of 2 µm wide lamellae inthe LIFE sensors’ most basic setup. The lamellae tend to tilt slightly, sincethey have a very high aspect ratio and are only attached at the bottom of thechannel.[21,22]

5.2.2 Microvortexers

The flow between the lamellae of the membranes in the LIFE sensor designis laminar (Re < 0.1). Because of this, the transport of species towards thelamellae is governed by diffusion. Diffusion is a rather slow process and fortypical flow velocities (∼ mm/s) and diffusion constants, large flow distancesare required for biomolecules to reach the lamellae by diffusion only. Therefore,a method is desired that enhances the flow of molecules towards the side walls(lamellae).

In shallow microchannels it is well established that grooved surfaces canpattern flows.[23] The application of square grooves in the bottom wall, posi-tioned under an angle with the flow direction, causes a rotational flow in themicrochannel as schematically shown in figure 5.7.[23]

Figure 5.7: Schematic view of rotational flow induced by square grooves.[23]

Practically, these structures are simple to fabricate by standard photo-lithography. But, in contrast to the above described method where the chan-


nels are wide and shallow, in our case the ’channels’ in between the lamellaeare slim and deep, which diminishes the effect of grooves in the bottom of thechannel. Therefore, it is believed that by placing grooves on the sides of thelamellae, as shown in figure 5.8, the rotational flow profile generated by thegrooves can be induced in the transversal direction.

(a) (b)

Figure 5.8: Schematic drawing of (a) lamellae in a microchannel, in theLIFE sensor design. (b) lamellae on which microvortexers have been applied.

Current lithographic techniques do not allow the fabrication of these sur-face structures, especially the tilt angle of the grooves is problematic. Slanted-angle holography could be a candidate for the production of slanted groovesyet, until now only tilted walls were created with this technique. However,by combining two well-chosen consecutive exposure steps, the wanted resultcould be achieved.

A clean microscope slide is coated with a 5 µm layer of SU8, and heated for5 minutes to 95◦C . After cooling down, the sample is flood exposed (EXFOOmnicure S2000, 100 mJ cm−2), and post-baked for 1 minute at 65◦C and2 minutes at 95◦C . On this primer layer, a 50 µm SU8-2100 (MicroChem)film is doctor bladed with an Erichsen Coatmaster 509MC-1 with a custombuilt blade. The solvent is evaporated during a two step heating: 30 minutesat 65◦C followed by 60 minutes at 95◦C). Then, the latent image of themicrochannel with the lamellae is recorded into the SU8 (100 mJ cm−2, 351nm, SpectraPhysics laser) using mask lithography.

During the lithographic mask exposure, due to diffraction/refraction atthe edges of the pattern on the mask, also the regions next to the lamellaereceive an exposure dose (figure 5.9(a)). This dose is rather low, lower thanthe threshold and therefore if standard postprocessing would be executed, noevidence of this exposure would be found in the final structure.

72 Chapter 5

(a) (b) (c) (d)

Figure 5.9: Formation of the latent image during the fabrication of microvor-texers. (a) mask exposure for microchannels, (b) slanted-angle holography (topview only), (c) combination of the two exposure steps and (d) final structureafter postbaking and development.

In a second exposure step, the sample is exposed to a slanted interferencepattern, with a dose that is lower than the threshold dose of SU8 (20 mJcm−2), figure 5.9(b). If this exposure was to be used alone, no structurewould remain after postprocessing. However, the exposure dose of the slanted-angle holographic exposure step adds up to the lithographic exposure dose,locally (in the regions next to the lamellae) raising the exposure dose abovethe threshold level (figure 5.9(c)). After postprocessing, the regions at the wallwhich received light during both exposure steps, will emerge (figure 5.9(d)).These are exactly the grooves that were looked for. In figure 5.10, a SEM imageview to the side of a lamellae is displayed after the post-bake (5 minutes at95◦C, to prevent diffusion) and development.

Figure 5.10: SEM image of slanted grooves fabricated on the side of alamella, side view.

It is clear that the grooves are positioned at an angle, which is a uniquefeature that cannot be achieved using standard lithographic techniques. Infigure 5.11, a top view of a set of lamellae is shown. From the image, it can be


Figure 5.11: SEM image of slanted grooves fabricated on the side of alamella, top view.

observed that the grooves are actually fabricated on the lamellae, 2 µm wide.Here, we have shown that microgrooves can be fabricated at an angle on

the sides of the lamellae. In addition to their flow patterning function, theridges also increase the surface available for protein immobilization. However,as can be seen from figure 5.11, the lamellae tend to bend slightly due to theirhigh aspect ratio. In view of the reproducibility of later assay results this isunwelcome and a solution needs to be found, e.g. by the in situ formation ofa reinforcing cover layer, which is described in chapter 7 or by the formationof in-plane membranes described in the next section.

5.2.3 In-plane membranes

The lamellae that are currently used to form the localized membranes forantibody deposition have one inconvenience, as can be seen from figure 5.6,these lamellae have the tendency to slightly bend . Mechanically this is notan issue, however, in terms of reproducibility of assay results, this poses a realproblem. The flow between two tilted lamellae can not be related to the flowbetween two straight standing lamellae, which asks for a solution.

In the previous chapter, the concept of localized in-plane membranes wasintroduced. These membranes are extremely suitable to replace the lamellaein the design of the flow-through biosensor. They are connected on three sidesto the channel walls and bottom, and are therefore mechanically much morestable than the freestanding lamellae.

For the fabrication, a mask containing sets of 20 transparent lines of 2µm, spaced by 2 µm non-transparent lines is used. In this way, the in-planemembranes created using slanted-angle mask holography will be grouped in

74 Chapter 5

sets of 20 lamellae, positioned perpendicularly to the direction of the flowchannel.

Glass slides are coated with a thin layer of SU8 (5 µm), pre-baked, floodexposed and post-baked. On top of this adhesion layer, a 50 µm thick layer ofSU8 is doctor bladed and pre-baked (30 minutes at 65◦C and 60 minutes at95◦C). The film is first exposed with UV laser light (351 nm, 100 mJ cm−2)through a mask with transparent lines of 75 µm wide (separated by 75 µm)to form the latent image of the channels. Next, the slanted-angle holographicexposure (periodicity 4 µm, 80 mJ cm−2) is executed using the line mask withthe groups of 20 lines, forming the latent image of the membranes arrangedin lamellae perpendicular to the channel. Finally, the post exposure bake iscarried out, 1 minute at 65◦C and 15 minutes at 95◦C to crosslink the epoxidesin the exposed areas. After developing in mr-dev600 (MicroChem) and rinsingwith propanol-2, the sample is dried with nitrogen gas. A SEM image showingthe resulting structure is presented in figure 5.12.

Figure 5.12: SEM image of in-plane membranes grouped in a set of lamellae,fabricated inside a microfluidic channel.

These membranes placed in groups of twenty provide a large adsorptionsurface for the immobilization of capture probes. An extra advantage is thatthe application of those capture probes via inkjet printing remains possible,due to the open top side, and capillary forces will enhance the distribution evenmore. Additionally, the in-plane membranes can be used for their filtrationcapacities to prevent large species from the analyte from contaminating theinternal structures of the device.

5.3 Filtration of blood

As mentioned earlier, removal of large components from samples by filtra-tion is an important issue in the design of a biosensor. In particular, blood


samples contain many bulky species like red blood cells (erythrocytes, diam-eter 6-8 µm), white blood cells (leukocytes, diameter 10-12 µm) and platelets(thrombocytes, diameter 2-4 µm). These cells could cause blocking of themicrostructures inside the biosensor, like e.g. the lamellae from the LIFEsensor. Therefore, these large parts should be filtered out of the blood be-fore entering the actual biosensor device. In-plane membranes can be used toperform this task.

A sample containing a 50 µm microchannel with a 15 µm wide membranewith 4 µm pores was prepared in the way described in paragraph 4.6.1. Ablood sample was obtained by means of a fingerprick, and diluted with water.A droplet of this mixture was pipetted onto the inlet of the microchannel,and the channel was capillary filled from right to left. The experiment wasmonitored using a Leica DM6000 optical microscope.

In figure 5.13, the result of such a filtration experiment is shown. On theright side, cells are stopped by the membrane, while on the left a clear fluidis visible that has passed the membrane. The filtration was successful.

Figure 5.13: Blood filtration using an in-plane membrane inside a microflu-idic channel. On the right side, bulky cells are visible. On the left, a clear,filtered liquid is shown.

5.4 Conclusions

Biosensor platforms, whether they are microarray-based or not, can profitfrom the extraordinary geometrical properties of holographic membranes. Incurrent flow-through microarray devices, holographic membranes can replacethe commonly used non-woven or ceramic membranes. The tunability of theflow characteristics and adsorption surface by pore shape, diameter and array,

76 Chapter 5

and the incorporated grid structure make them the perfect substitute for thesubstrates used nowadays.

In addition to being a replacement unit for current systems, the applicationholographic membranes led to the development of a new type of microarray-based platform. Combining the advantages of the two existing operatingmodes of microarrays, the LIFE concept was introduced. In-plane membranes,lamellae-like, are implemented at specified positions in a microfluidic channelto increase the adsorption surface for capture probe deposition. However, thelamellar structure of the membranes implies that transport of the moleculesof interest is governed by diffusion, which inevitably is slow.

In order to create a rotational flow between the lamellae, the well-knownapproach to apply grooves on the bottom of a microchannel is adopted andadapted to the high aspect ratio structures of our system. To do this, conven-tional lithography is combined with a slanted-angle holographic underexpo-sure. Taking advantage of the otherwise unwanted residual exposure dose atthe edges of the structures together with the threshold dose of SU8, grooveson the sides of the lamellae are fabricated.

Yet, these lamellae have the tendency to bend slightly due to their high as-pect ratio (> 15). The flow between these lamellae is a significant factor in thefinal result of a biological assay, which makes this intrinsic mechanical insta-bility an uninvited consequence of the applauded high aspect ratio lamellae.The application of in-plane membranes which were described extensively inthe previous chapter overcomes this problem. Sets of 20 in-plane membraneswere fabricated inside a microchannel, in a lamellae-like fashion. Attached onthree sides to the microchannel, these membranes can not lean over, while aleak-free connection is established. Application of proteins by inkjet printingremains possible due to the open top side of the membranes. Additionally, theuse of ’lammellar’ membranes, increases the specific surface for absorption.

Large, bulky elements like leukocytes, erythrocytes and thrombocytes couldcause blocking of a biosensor device. In order to prevent this, in-plane mem-branes inside a microfluidic channels were successfully used to filter blood cellsfrom a fingerprick sample.

This shows the versatility of interference holography to manufacture build-ing blocks for bioMEMS in SU8. Nevertheless, the applicability of SU8 for theimmobilization of biomolecules remains to be proven. In the next chapter, theadaptability of SU8 to bind proteins while preserving their biological functio-nality will be studied.


5.5 References

[1] A. Pease, D. Solas, E. Sullivan, M. Cronin, C. Holmes and S. Fodor.Light-generated oligonucleotide arrays for rapid DNA sequence analysis.Proc. Natl. Acad. Sci. U.S.A., 91 (11), (1994) 50225026.

[2] M. G. Protein microarrays and proteomics. Nat. Genet. Suppl., 32, (2002)526532.

[3] M. McClain, C. Culbertson, S. Jacobson, N. Allbritton, C. Sims andJ. Ramsey. Microfluidic devices for the high-throughput chemical analysisof cells. Anal. Chem., 75 (21), (2003) 5646–5655.

[4] H. Ma and K. Y. Horiuchi. Chemical microarray: a new tool for drugscreening and discovery. Drug Discov. Today, 11 (13-14), (2006) 661 –668.

[5] A. Lueking, M. Horn, H. Eickhoff, K. Bussow, H. Lehrach and G. Walter.Protein microarrays for gene expression and antibody screening. Anal.Biochem., 270, (1999) 103–111.

[6] G. MacBeath and S. Schreiber. Printing proteins as microarrays for high-throughput function determination. Science, 289, (2000) 1760–1763.

[7] J. Glockler and P. Angenendt. Protein and antibody microarry technology.J. Chromatogr. B, 797, (2003) 229–240.

[8] A. Hook, N. Voelcker and H. Thissen. Patterned and switchable surfacesfor biomolecular manipulation. Acta Biomater.


[10] R. Graf and R. Friedl. Charge-coupled device camera-based detectionof fluorescence-labeled proteins immobilized on nitrocellulose membranes.Electrophoresis, 22, (2001) 890–895.

[11] http://www.whatman.com.

[12] ”http://www.pamgene.com”.


[14] G. Voskerician, M. S. Shive, R. S. Shawgo, H. von Recum, J. M. Anderson,M. J. Cima and R. Langer. Biocompatibility and biofouling of MEMS drugdelivery devices. Biomaterials, 24 (11), (2003) 1959–1967.

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[16] Y.-J. Chang, C.-Y. Hu, L.-T. Yin, C.-H. Chang and H.-J. Su. Dividablemembrane with multi-reaction wells for microarray biochips. J. Biosc.Bioeng., 106 (1), (2008) 59–64.

[17] C. B. Cohen, E. Chin-Dixon, S. Jeong and T. T. Nikiforov. A microchip-based enzyme assay for Protein Kinase A. Anal. Biochem., 273, (1999)89–97.


[19] D. Mocanu, A. Kolesnychenko, S. Aarts, A. Troost-Dejong, A. Pierik,E. Vossenaar and H. Stapert. Mass transfer effects on DNA hybridizationin a flow-through microarray. J. Biotechn., 139, (2009) 179–185.

[20] R. van Beuningen, H. van Damme, P. Boender, N. Bastiaensen, A. Chanand T. Kievits. Fast and Specific Hybridization Using Flow-Through Mi-croarrays on Porous Metal Oxide. Clin. Chem., 47 (10), (2001) 1931–1933.

[21] Patent: Flow through biosensor. WO2008139389 (2008).

[22] K. Hermans. Latent structured thermally developed reliefs: principles andapplications of photoembossing. Ph.D. thesis, Eindhoven University ofTechnology, the Netherlands (2009).

[23] A. D. Stroock, S. Dertinger, G. Whitesides and A. Ajdari. Patterningflows using grooved surfaces. Anal. Chem., 74, (2002) 5306–5312.

Chapter 6

Surface modifications for

immobilization of

biomolecules

Substrates used for the immobilization of capture probes need to have surfaceproperties that are favorable for binding of biological species. Surface cha-racteristics like hydrophobicity, chemical groups and surface relief structuresshould be controlled and well-defined in substrates which are used in microsys-tems for biological analyses. SU8, with its many epoxide groups, is easilyaccessible for different surface chemistries and modifications. Due to its hy-drophobicity, no capillary flow is possible in an SU8 microchannel withoutincreasing the surface energy. To control the velocity of a capillary flow in amicrofluidic channel, the surface energy is tuned using a UV ozone or oxy-gen plasma treatment. If antibodies are required for assays, they are inkjetprinted on an SU8 substrate that is functionalized with carboxylic acid groupsor amine groups. These are two of the most abundant groups in proteins forwhich binding protocols like EDC-NHS chemistry have been developed. Re-covery experiments are performed on polyacrylic acid, poly-L-lysine and plainSU8 surfaces to find the best surface for protein immobilization. Lastly, afunctional assay is executed to investigate whether the immobilized proteinsretain their biological functionality.

79

80 Chapter 6

6.1 Introduction

Biosensors, in particular microarrays, require substrates with well-defined sur-face properties to attach capture probes to the substrate.[1] Here, the maingoal is to provide a robust attachment of the capture probes to the substratesin an easy way, which maximizes the probe accessibility for the agent to be de-tected. High capture probe densities are required and non-specific interactionsneed to be avoided.

In literature, glass is a widely studied substrate material, because a goodunderstanding of its physical and chemical properties is available.[2, 3] Silaniza-tion is used to covalently bind silanol groups to the glass, providing an organicfunctionality to the glass surface. Silane molecules with different organic endgroups are available and amino and epoxide functionalities are mostly usedfor DNA microarrays.[4, 5] Additionally, the superior optical characteristics ofglass make it the primary substrate material for biosensors.

Polymers, with their low cost and diversity in chemical structures havelately gained interest for the fabrication of substrates for biological applica-tions. Polymers like poly(dimethylsiloxane) (PDMS),[6, 7] polycarbonate (PC),poly(methyl methacrylate) (PMMA),[8, 9] polystyrene, poly(ethylene tereph-thalate) (PET) and cellulose acetate have been studied for microarray appli-cations.[10] Also, the use of nanoparticles[11] and microbeads[12] as supportfor the immobilization of proteins, oligonucleotides and DNA is being investi-gated.

SU8, the chemically inert photoresist material used in the previous chap-ters has turned out to be an excellent substrate material for the fabrication ofbio-micro-electromechanical systems (bioMEMS).[13, 14] For microfluidic sys-tems used in biological applications it is essential to use aqueous fluids. Un-fortunately, SU8 is highly hydrophobic[15] having a low surface energy whichis a disadvantage for these applications. Plasma activation of polymer sur-faces is well-established in rendering surfaces hydrophilic for the adsorption ofbiomolecules.[16] In the first section of this chapter it will be shown that anoxygen plasma and/or UV-ozone treatment increases the surface energy. It isalso demonstrated that the surface energy can be tuned to obtain the desiredcapillary flow rate.

The binding of proteins to SU8 is also of great importance for the correctlyfunctioning of a bioMEMS system. Epoxide groups are relatively abundanton the surface of SU8 structures and epoxide chemistry is often used[17] forthe functionalization of epoxide surfaces. Many of the aminoacids and DNA

Surface modifications for immobilization of biomolecules 81

bases that form the biomolecules contain amine or carboxyl groups, whicheasily bind with the epoxide groups on the surface. Also protein couplingchemistries like EDC-NHS[18–20] use the presence of amines to covalently bindantibodies to a substrate.

Poly-L-lysine and polyacrylic acid coatings will be applied as a sourceof these reactive groups. To test whether these coatings increase the proteinbinding capacity of a surface, recovery experiments will be carried out. Besidesthe amount of immobilized proteins, also their orientation and conformationare of importance, since these determine the biological functionality of theprotein. A functional assay will show whether the immobilized proteins remainbiologically active.

6.2 Surface treatment

Control over the surface properties in microfluidics and bioMEMS is of greatimportance when control over the flow rate is required. Also, when biologicalspecies like proteins or DNA are deposited, e.g. via inkjet printing, the surfaceenergy determines the spot size of printed droplets. Therefore control oversurface energy of the substrate material is an important issue.

6.2.1 Capillary filling theory

The capillary wetting phenomenon has been studied extensively.[21–23] How-ever, it has been found that the flow behavior in microchannels deviates signifi-cantly from the predictions of conventional fluid mechanics. Here, we considerthe capillary flow through a microchannel with a rectangular cross-section.The geometry is shown in figure 6.1.

A mathematical model for flow through such a channel driven by capillaryforce and gravity is available.[24] To simplify the analytical model, a few as-sumptions are made. The fluid is assumed to be Newtonian, having a constantviscosity regardless of the forces acting on it. Water is considered to be a New-tonian fluid.[25] The flow is fully developed laminar and the gravity inside themicrochannel is neglected. The Navier-Stokes equation then becomes:

∂2u

∂y2=

1η

∂P

∂x(6.1)

The contributions of the two driving forces, capillary force and gravity forthe left term in the Navier-Stokes equations are:[24]

82 Chapter 6

Figure 6.1: Geometry for analytical model of capillary driven flow. H is theheight of the droplet of applied fluid in [cm], L is the length of the microchannelin [mm] and h the width/height of the channel.

−∂P

∂x=

1x

(ρgH +

2σ cos θ

h

)(6.2)

By solving equation 6.1 for the no-slip boundary conditions and by inte-grating the velocity over the cross-section and length of the microchannel, anexpression is obtained for the flow time in a microchannel of length L:

t =6ηL2

ρgHh2 + 2hσ cos θ(6.3)

with η the absolute viscosity of water in [Pa s], ρ the density of waterin [kg m−3], σ the surface tension of water in [N m−1], g the gravity in [ms−2], H the height of the reservoir in [cm], h the width of the microchannelin [mm] L the length of the microchannel in [mm] and θ the contact angle ofwater on SU8. In this equation, all but one parameter are geometrical or fluidproperties characteristic to the setup. The only accessible variable to controlflow rates is cos θ, the contact angle. Especially when no external forces areapplied, the surface properties of the channel material play a crucial role.

Due to the hydrophobic character of SU8 (θc = 90◦), microfluidic deviceswould depend on an external pumping device for fluid transport. However, inview of the simplification and miniaturization of µTAS, it is desirable to dis-pose of these external devices. With a hydrophilic surface, the liquid will flowautomatically through the microchannels without any external force needed.Nowadays, the standard technique for rendering SU8 hydrophilic is oxygen


plasma treatment.[15,26] Besides the increase in surface energy, also the abso-lute oxygen content on the surface of SU8 increases, while the carbon contentdecreases. This extra oxygen content emerges in the form of ether, aldehydeand carboxylic acid functional groups.[26] To the latter, carboxylic acids, bio-logical species are known to have a large affinity, which is an extra advantagefor the deposition of these biological species. However, also the molecules ofinterest for the analysis could bind to the surface of the channel where noantibodies have been deposited due to the presence of carboxylic acid. There-fore the areas of the microchannel where no antibodies are printed shouldbe provided with a blocking agent (e.g. Poly-ethylene glycol, PEG) to avoidunwanted binding of other biomolecules to the channel.

6.2.2 Capillary flow experiments in an SU8 microchannel

Capillary flow experiments are carried out in plain channels to find out whetheroxygen plasma or UV-ozone treatment can be used in the setup of our LIFEsensor in SU8. For this purpose, microscope slides were cleaned with ethanoland coated with a thin (5 µm) layer of SU8 which was pre-baked, flood exposed(100 mJ cm−2, EXFO Omnicure) and post-baked to obtain a primer layer towhich the channel will chemically bind. Secondly, a thick (50 µm) layer ofSU8 was doctor bladed (Erichsen Coatmaster 509MC-1) on top of the primerlayer. This coating was pre-baked (30 minutes at 65◦C and 60 minutes at95◦C) to obtain a solvent-free photoresist layer in the glassy state.

The SU8 film is then mask exposed using 351 nm UV laser light (Spectra-Physics Beamlok 2085-25S) using a mask containing channels (non-transparentlines). After standard postprocessing, the samples were plasma activated usingan oxygen plasma (K1050X Plasma Asher, Emitech Ltd.) with an RF powerof 80W for 2 minutes. Another set of samples was treated in a UV-ozonecleaner (UVP PR-100 UV-ozone photoreactor) for 10 minutes.

To determine the effect of the plasma activation and the UV-ozone treat-ment the channels were capillary filled with water via application of a dropletinto an open reservoir on one end of the channel (figure 6.2). The flow velocitywas registered using a Leica DM6000 equipped with a Redlake Motionpro HS3high-speed camera. To visualize a large area of the microchannel, a 5x ob-jective was used. Since high velocities are anticipated, the high-speed camerawas set to record at 300 frames per second.

First, an untreated sample was provided with a droplet of water at the inlet.Unfortunately, no capillary filling of the channel was observed, emphasizing

84 Chapter 6

Reservoir

1mm x 1mmChannel 50 µm

Reservoir

1mm x 1mm

Droplet

Figure 6.2: Setup for capillary filling of microchannel. A droplet of fluidis applied on the inlet reservoir of the channel, after which the channel fillscapillary.

the need for an effective surface treatment.Then, consider the UV-ozone treated microchannel. In figure 6.3, 3 frames,

each taken with an interval of 50 frames (1/6 second) after application of thedroplet are shown. The distance traveled by the fluid front from right to left,well visible in the figure, between the first and the third frame is 2.68 mm.Therefore the effective fluid velocity in the microchannel upon capillary fillingis 8.04 mm/s.

Figure 6.3: Flux measurement of UV-ozone treated (10 minutes) SU8 chan-nel. A flow velocity of 8.04 mm/s is measured.

The flow time through the microchannel is calculated using equation 6.3,where the reservoir height is estimated to be roughly 50 µm which is the chan-nel height (figure 6.1) and a contact angle of 50 degrees.[15] For a length of 2.68mm, the flow time is calculated to be 0.23 seconds, which corresponds ratherwell with the 0.33 seconds that were measured, considering the rudimentarymethod that was used for the application of the droplet. Only a rough esti-mate of the height of the droplet is possible when pipetting a droplet of wateronto the reservoir.

Secondly, the oxygen plasma treated sample was investigated. From litera-ture, a surface energy of approximately 70 N m−1 was found.[15] In figure 6.4,


the results are shown: note that this time, there were only 10 frames betweeneach displayed image. For a distance of 2.18 mm, traveled in 2/30 seconds,this results in a flow velocity of 32.7 mm/s.

Figure 6.4: Flux measurement of oxygen plasma treated channel (80 Watt,2 minutes). A flow velocity of 32.7 mm/s is observed.

The flow time in the channel calculated from the equation is 0.046 seconds,while the measured time is 0.033 seconds, again rather well corresponding toeach other. It is therefore concluded that by changing the surface energy ofthe SU8, either by UV-ozone treatment or by oxygen plasma, the flow velocityof a capillary flow through an SU8 microchannel can be tuned at will. Theresults from the analytical model describe the actual flow rates rather well.The model can therefore be used to predict the surface energy required for acertain flow rate.

The possibility to use capillary flow in SU8 microchannels increases thechances to use SU8 in a biosensor application significantly. However, it shouldalso be possible to immobilize biomolecules and in particular proteins on SU8.Moreover, if these biomolecules are immobilized, they should retain their bio-logical functionality. These issues will be dealt with in the next paragraph.

6.3 SU8 functionalization

In the development of a protein microarray, a crucial step is the immobiliza-tion of proteins on the substrate material, SU8 in the case of the LIFE sensorthat was introduced in the previous chapter. Passive adsorption[27] based onhydrogen bonding is the most common technique for immobilization onto thesolid phase. However, covalent bonding offers a stronger protein-surface at-tachment, but care needs to be taken that the protein conformation remainsintact upon covalent binding.[27] The conformation intactness and orientationof the capture antibodies deposited on the surface determines their ability to

86 Chapter 6

Figure 6.5: Possible orientations of deposited antibodies on a flat sub-strate. The upright position is the optimized orientation, however, even inthis orientation, the conformation intactness of the antibodies will determinewhether they are still biologically active.

bind to the antigens in an assay (figure 6.5). The substrate material determineswhich type of binding mechanism (physical adsorption, covalent binding) willaccount for the protein immobilization. Modifications to the substrate mate-rials to improve protein immobilization performance are common practice.

In this section, the presence of unreacted epoxides in the bulk of SU8 andat the surface of a flat SU8 substrate are investigated. Secondly, SU8 sub-strates are functionalized with polyacrylic acid and poly-L-lysine, to providethe surface with functional groups (carboxylic acid and amines) to which pro-teins have a high affinity. Lastly, preliminary results from a functional assayon an SU8 flat surface will be elaborated.

6.3.1 Pendant epoxide groups

SU8 has eight epoxide groups per molecule, and it is expected that, for mobilityreasons, after crosslinking not all of these epoxides have reacted. In principle,there should be epoxide groups available at the surface for further reaction -either with proteins or with other molecules that supply other functionalitieslike carboxyl or amine.

To determine the amount of unreacted epoxides, infrared spectroscopy(FT-IR, Excalibur FTS 3000 MX) is performed on an SU8 film before ex-posure, after exposure and after crosslinking. The peak of 1608 cm−1 of thearomatic ring C−C stretch mode which does not participate in the crosslinkingprocess is used as internal standard.[28] In figure 6.6 the normalized spectraare depicted.

The peak intensities at 914 cm−1 before and after exposure are compared.After crosslinking, the epoxide peak has not vanished completely, on the con-trary, still 80% of the unreacted epoxides are present. It needs to be noted thatthis analysis was done on a thick film, so that these results do not imply any-


750 1000 1250 1500 1750 20000

1

2

3

4

Nor

mal

ized

abs

orba

nce

(a.u

.)

Wavenumber (cm-1)

Post baked Exposed Not exposed

700 800 900 1000 11000

2

4

6

8

Nor

mal

ized

abs

orba

nce

(a.u

.)

Wavenumber (cm-1)

Not exposed Exposed Post baked

914 cm-1

Figure 6.6: FT-IR spectra of an SU8 film before exposure, after exposureand after crosslinking. The spectra are normalized at the 1608 cm−1 peak.

thing about the presence of free epoxides at the surface. To obtain informationabout the surface epoxides, a second set of experiments was performed.

Glass slides were first sputter coated with Au to obtain a reflective surface(K575 XD Turbo sputter coater, Emitech Ltd.), after which a thin layer of SU8is applied, flood exposed (100 mJ cm−2, EXFO Omnicure S2000) and post-baked. Specular reflection FT-IR spectroscopy (Seagull, Harrick) is performedat an incident angle of 80◦ in order maximize the path length of the infraredbeam through the thin film. In figure 6.7, IR spectra of 4 different concentra-tions of SU8 in cyclopentanone and hence of 4 different layer thicknesses aredisplayed.

1000 1500 2000

0

5

10

15

20

25

30

Nor

mal

ized

Abs

orba

nce

(a.u

)

Wavenumber (cm-1)

5 wt% 10 wt% 15 wt% 20 wt%

Figure 6.7: IR spectra of cured SU8 films, spin coated from 5, 10, 15 and20 wt% SU8 solutions. The 914 cm−1 stretch is indicative for the presence ofunreacted epoxides.

88 Chapter 6

The peak of 914 cm−1 is analyzed for crosslinked and non-crosslinked SU8films. In figure 6.8, the peak intensities for a set of layer thicknesses are shownin a graph for both cured and non-cured films. The data points exhibit a linearrelationship, and the intercepts with the y-axis are calculated. The interceptwith the y-axis represents the extrapolation of the absorbance for a layer ofzero thickness and thus of the surface. The ratio between the epoxides of acrosslinked and a non-crosslinked surface is determined to be 0.4, so 40% ofthe surface epoxide groups is still present after crosslinking of the film.

0 50 100 150 200 250 3000.0

0.1

0.2

0.3

0.4

0.5

0.6

uncured cured

Abs

orba

nce

(a.u

.)

Film thickness (nm)

Figure 6.8: IR absorbance at 914 cm−1 for film thicknesses between 35 nmand 280 nm, crosslinked and non-crosslinked. Extrapolation of the linear fit tothe y-axis leads to the surface concentration of epoxide groups.

These results show that pendant epoxide groups are present at the surfaceof a crosslinked SU8 film. These groups are available for reaction either directlyto proteins, or to molecules that are used for the functionalization of the SU8substrate e.g. containing COOH or NH2 groups.

6.3.2 Immobilization of antibodies

Carboxylic acids and amines are widely used in the surface modification ofsolid surfaces for the immobilization of proteins. Polyacrylic acid (PAA, figure6.9(a)) and poly-L-lysine (PLL, figure 6.9(a)) are polymers that contain manyof these functional groups and are therefore ideal to apply on a substrate for aprotein microarray. In addition, they covalently bind to the unreacted epoxidegroups at the SU8 interface.

Microscope slides are provided with a 2 µm SU8 film, which is dip coatedwith PAA or PLL solutions. Solutions of 1 wt% polyacrylic acid (Sigma)


HO O

n

(a) PAA

O

NH

NH2 n

(b) PLL

Figure 6.9: Polyacrylic acid and poly-L-lysine monomer.

and 0.1 wt% poly-L-lysine (Sigma) in water are prepared. The samples aredipped in the polyacrylic acid or poly-L-lysine solutions for 15 minutes atroom temperature, during which binding should take place. Afterwards, thesamples are rinsed with distilled water to remove excess PAA or PLL. Theseflat substrates will be used in an antibody immobilization study.

Fluorescent labeled donkey-anti-sheepIgG-AF633 (Invitrogen)1 is used asthe model antibody. The antibodies are inkjet printed (110 pL/droplet) inspots after which they are dried overnight in a Petri dish at room temperature.Binding of the antibody to the surface is checked by attempting to wash theantibodies off the surface. The samples are submerged in a washing bufferconsisting of 1x PBS (150 mM Phosphate Buffered Saline, Sigma) with 0.05%Tween-20 (a soap) shaking them gently for 5 min. The antibody recovery isexamined by comparing the spot signal intensities before and after the wash,measured via a LED-CCD setup.

An example of an image of an SU8 substrate on which a pattern of thelabeled antibody is printed, acquired by the CCD is shown in figure 6.10.As printing buffer a 0.05 molar PBS buffer at pH 7.8 was used. This pH ischosen to be neutral, since at more extreme pH values the proteins presumablydenaturate.

From the CCD measurements before and after the washing step, it was de-duced that on plain SU8 64.3% of the antibodies were recovered after washing.On SU8 coated with PAA 32.6% of the printed antibodies were retained andon SU8 coated with PLL 65.7% were left. This means that antibody bindingon SU8 is very acceptable, while polyacrylic acid scores worse. Poly-L-lysineseems to have a slightly higher recovery than ordinary SU8, but the increase isnot significant. Therefore, it is chosen to perform the subsequent immunoas-

1Donkey-anti-sheep IgG - AF633 is an antibody for sheep Immunoglobulin G, producedin a donkey. AF633 designates the fluorescence wavelength, 633 nm in this case.

90 Chapter 6

Figure 6.10: Example of printed pattern of fluorescent labeled antibody.

say experiments on non-treated SU8 surfaces. It should be mentioned thatfor binding the antibody covalently to PAA or PLL other procedures are re-quired such as EDC-NHS treatment[18–20] where further improvement mightbe expected.

6.3.3 Functional assay on SU8

To test whether the biological function of antibodies that are immobilized onSU8 remain intact after immobilization, a model immunoassay is executed. Asa capture antibody, 8 µM mouse-anti-humanCRP (HyTest) is printed usingPBS as print buffer (pH 7.4). CRP (C-reactive protein) is a protein that iswidely used as a marker for inflammation. A fluorescent labeled 1 M donkey-anti-sheepIgG-AF633 in 1x PBS (pH 7.4) is printed as internal control. Afterovernight drying, the samples are washed with PBS to remove non-immobilizedcapture antibodies and a commercially available reaction chamber (SecureSeals, GRACE Bio-Labs) is attached to the sample. Areas of the sampleon which nothing was printed, are blocked with a 2.5% BSA (Bovine SerumAlbumin, Sigma) in PBS for 4 hours at room temperature, to prevent proteinsused in the next steps from binding non-specifically to the SU8 layer.

The analyte, biotin labeled human CRP, diluted to 10 nM, 1 nM and 10pM in PBS, is incubated for 90 minutes at room temperature. As a blank,only the PBS buffer is incubated. The unbound analyte is washed away usingPBS. Afterwards, fluorescent labeled streptavidin-AF633 (Invitrogen) (1:1000diluted) is incubated for 45 minutes. The biotin-streptavidin binding[27] iswell-known, and under normal conditions (pH, temperature) is expected toalways take place. If the immunoassay is successful, this fluorescent label willbe detected after another washing step with PBS.


B BS

(a) (b) (c)

Figure 6.11: Schematic representation of the sandwich assay performed. (a)First, the antibody (mouse-anti-humanCRP) is printed on the SU8 substrate.(b) Then, the sample is incubated with the analyte (biotin labeled human CRP).(c) Lastly, a fluorescent labeled molecule (streptavidin-AF633) is incubated onthe sample. The biotin-streptavidin binding is well-established. If the fluores-cent label is detected, the immunoassay is successful.

Using the LED-CCD setup, the samples are analyzed. The 10 nM analyteis the only one that shows fluorescence at the detection sites (figure 6.12).

Figure 6.12: Image obtained with the LED-CCD setup, showing 3 fluorescentspots from the positions where the sandwich immunoassay was successful. Thebrighter fluorescent spots are corner markers.

This image shows that an immunoassay can be successfully executed ona flat surface of SU8. However, 10 nM is a quite high analyte concentration,but it needs to be kept in mind that these preliminary studies were performedon flat substrates. Using membranes instead of flat substrates increases thesurface for immobilization and is therefore expected to decrease the detectionlimit. Detection limits in the 10-100 pM region are expected. So, the im-munoassay was successful, but there is room for improvement with respect tothe detection limit.

92 Chapter 6

6.4 Conclusions

In the design of a simple and efficient biosensor chip, three things are of impor-tance: flow through the microchannels on-chip (preferably without externaldevices), immobilization of biomolecules for detection and sample pretreat-ment.

The surface energy of the microfluidic channels plays a major role whencontrol over the flow rate is required. An analytical model is adopted to predictthe capillary flow velocity through a square microchannel with different surfaceenergies. SU8 microchannels are treated with UV-ozone and oxygen plasmato increase their surface energy and hence increase the capillary flow ratethrough the channels. A untreated SU8 channel shows no capillary filling atall, while the flow rate of a UV-ozone treated microchannel already is 8.04mm/s. Oxygen plasma treatment even increases this flow rate with a factor 4,to 32.7 mm/s. So, using these surface treatments the surface tension of SU8can be tuned to the values required for a capillary flow.

Immobilization of proteins can be achieved by passive adsorption or bycovalent binding of the proteins to the substrate. The substrate determineswhich type of binding takes place. The free epoxide groups at the surface ofa crosslinked SU8 substrate can be used to bind proteins, but also to applya functional coating (PAA or PLL) to which proteins have a high affinity.Recovery experiments of labeled proteins on these three substrates (SU8, PAA,PLL) revealed that a PLL coating gives only a slightly higher recovery thannon-treated SU8.

Therefore, in the functional assay to determine whether the immobilizedproteins retained their biological functionality upon immobilization, non-treatedflat SU8 substrates were used. Preliminary results show that indeed the func-tionality is retained and that the detection limit on a flat surface is approxi-mately 10 nM.

Two major issues in the fabrication of a biosensor chip were addressed inthis chapter by applying different surface modifications on SU8. This showsthe exceptional applicability of SU8 as a chip material for bioMEMS, which,combined with the versatility of (slanted-angle) interference holography as astructuring technique leads to an unprecedented design freedom in the fabri-cation of biosensor chips.


6.5 References

[1] J. Glockler and P. Angenendt. Protein and antibody microarry technology.J. Chromatogr. B, 797, (2003) 229–240.

[2] R. L. DeRosa, J. A. Cardinale and A. Cooper. Functionalized glass sub-strate for microarray analysis. Thin Solid Films, 515 (7-8), (2007) 4024– 4031.

[3] S. L. Seurynck-Servoss, A. M. White, C. L. Baird, K. D. Rodland andR. C. Zangar. Evaluation of surface chemistries for antibody microarrays.Anal. Biochem., 371 (1), (2007) 105 – 115.

[4] S. D. Conzone and C. Pantano. Glass slides to DNA microarrays. Mat.Today, 7 (3), (2004) 20 – 26.

[5] N. K. Kamisetty, S. P. Pack, M. Nonogawa, K. Yamada, Y. Yoshida,T. Kodaki and K. Makino. Stabilization of the immobilized linkers andDNA probes for DNA microarray fabrication by end-capping of the re-maining unreacted silanol on the glass. J. Biotechnol., 140 (3-4), (2009)242 – 245.

[6] C. Marquette and L. Blum. Direct immobilization inpoly(dimethylsiloxane)for DNA, protein and enzyme fluidic biochips.Anal. Chim. Acta, 506 (2), (2004) 127–132.

[7] M. Moorcroft, W. Meuleman, S. Latham, T. Nicholls, R. Egeland andE. Southern. In situ oligonucleotide synthesis on poly(-dimethylsiloxane):a flexible substrate for microarray fabrication. Nucleic Acids Res., 33 (8),(2005) e75/1e75/10.

[8] F. Fixe, M. Dufva, P. Telleman and C. Christensen. Functionalization ofpoly(methyl methacrylate) (PMMA) as a substrate for DNA microarrays.Nucleic Acids Res., 32 (1), (2004) e9/1e9/8.

[9] F. Fixe, M. Dufva, P. Telleman and C. Christensen. One-step immo-bilization of aminated and thiolated DNA onto poly(methylmethacrylate)(PMMA) substrates. Lab Chip, 4 (3), (2004) 191–195.


94 Chapter 6

[11] L. R. Hilliard, X. Zhao and W. Tan. Immobilization of oligonucleotidesonto silica nanoparticles for DNA hybridization studies. Anal. Chim.Acta, 470 (1), (2002) 51 – 56.

[12] O. Nord, M. Uhln and P.-. Nygren. Microbead display of proteins bycell-free expression of anchored DNA. J. Biotechnol., 106 (1), (2003) 1 –13.

[13] R. Marie, S. Schmid, A. Johansson, L. Ejsing, M. Nordstrm, D. Hfliger,C. B. Christensen, A. Boisen and M. Dufva. Immobilisation of DNA topolymerised SU8 photoresist. Biosens. Bioelectron., 21 (7), (2006) 1327 –1332.

[14] T. B. Christensen, D. D. Bang and A. Wolff. Multiplex polymerase chainreaction (PCR) on a SU8 chip. Microelectron. Eng., 85 (5-6), (2008) 1278– 1281.

[15] F. Walther, P. Davydovskaya, S. Zurcher, M. Kaiser, H. Herberg, A. M.Gigler and R. W. Stark. Stability of the hydrophilic behavior of oxygenplasma activated SU8. J. Micromech. Microeng., 17, (2007) 524–531.

[16] G. M. Messina, C. Satriano and G. Marletta. A multitechnique study ofpreferential protein adsorption on hydrophobic and hydrophilic plasma-modified polymer surfaces. Coll. Surf. B, 70 (1), (2009) 76 – 83.

[17] M. Joshi, R. Pinto, V. R. Rao and S. Mukherji. Silanization and antibodyimmobilization on SU8. App. Surf. Sci., 253 (6), (2007) 3127 – 3132.

[18] P. Vermette and L. Meagher. Immobilization and characterization ofpoly(acrylic acid) graft layers. Langmuir, 18, (2002) 10137–10145.

[19] P. Tengvall, E. Jansson, A. Askendal, P. Thomsen and C. Gretzer. Prepa-ration of multilayer plasma protein films on silicon by EDC/NHS couplingchemistry. Colloids and Surfaces B: Biointerfaces, 28, (2003) 261–272.

[20] N. Adanyi, M. Varadi, N. Kim and I. Szendro. Development of new im-munosensors for determination of contaminants in food. Current AppliedPhysics, 6, (2006) 279–286.

[21] S. Newman. Kinetics of wetting of surfaces by polymers; capillary flow.J. Colloid Interf Sci., 26 (2), (1968) 209 – 213.


[22] P. Joos, P. V. Remoortere and M. Bracke. The kinetics of wetting in acapillary. J. Colloid Interf Sci., 136 (1), (1990) 189 – 197.

[23] T. D. Blake, R. A. Dobson and K. J. Ruschak. Wetting at high capillarynumbers. J. Colloid Interf Sci., 279 (1), (2004) 198 – 205.

[24] W. Jong, T. Kuo, S. Ho, H. Chiu and S. Peng. Flows in rectangularmicrochannels driven by capillary force and gravity. Int. Comm. HeatMass, 34 (2), (2007) 186 – 196.

[25] P. K. Kundu and I. Cohen. Fluid Mechanics. Academic press (2002).

[26] M. Nordstrom, R. Marie, M. Calleja and A. Boisen. Rendering SU8hydrophilic to facilitate use in micro channel fabrication. J. Micromech.Microeng., 14, (2004) 1614–1617.


[28] D. Wong, T. Tan, P. Lee, R. Rawat and A. Patran. Study of X-raylithographic conditions for SU8 by fourier transform infrared spectroscopy.Microelectron. Eng., 83 (10), (2006) 1912 – 1917.

Chapter 7

Glancing angle lithography

SU8 epoxy resin is a notorious material in microfluidic device fabricationbecause of its ability to form high aspect ratio channel structures. A veryimportant aspect in microfluidics is the effective sealing of microchannels toavoid contact with the outside world. Efficient and simple sealing methodswithout damaging the underlying structures remains a challenging issue. Itis demonstrated that combining classical lithographic procedures with a reflec-tion photopolymerization at glancing angles results in a channel cover thatis inextricably bound to the channel walls. The mechanism of glancing anglelithography (by us given the name GALITH) is based on the localized exceedingof the threshold dose for light induced polymerization. The exposure dose isthe summation of the refracted light intensity with oscillatory coupled reflectedintensity at the layer interface. A reflection interface between two dielectricsis not a discrete transition, but rather a continuous change in refractive index.Utilizing an oblique exposure with s-polarized laser light, the GALITH processtakes advantage of the finite penetration depth of reflected light. SU8 is chosenas model system for a thorough investigation of the parameters that are of im-portance for this process. The shallow penetration depth (80 nm) of an obliqueincident laser beam results in an ’activated’ top layer of the same dimensions.Increasing the exposure dose increases the thickness of the remaining film.However, above a certain dose the film thickness remains constant. Allowingthe photo-activated species (initiator) to diffuse before crosslinking the epoxidealso benefits the remaining film thickness. It is illustrated that microchannelscan indeed be sealed effectively using GALITH.

This chapter will be partially published as A.M. Prenen, C.W.M. Bastiaansen and D.J.Broer, Glancing angle lithography (GALITH) for sealing of microchannels, in preparation

97

98 Chapter 7

7.1 Introduction

In many microfluidic and micro-electromechanical systems (MEMS), hermeticsealing of microstructures is required to control the internal atmosphere or toavoid contact with the outer world. Especially in the area of biosensors, whereusually body fluids are analyzed, contact with the analyte is to be avoided,mainly to prevent cross-contamination. Commonly, these components anddevices are prepared on a single substrate, after which the top cover layer isapplied.

Nowadays, different techniques are being studied like adhesive bonding,[1]

solvent bonding,[2] thermal bonding,[3] photo-embossing,[4] resin-gas injection,[5]

incorporation of fibers[6] and the use of sacrificial layers. These techniques usu-ally cause damage to the underlying structures, like geometrical deformation.A new technique that overcomes this issue has recently been developed whichuses localized microwave radiation, with conductive polymer layers.[7, 8]

Above-mentioned techniques use either sacrificial layers or leave an interfa-cial layer which have a significant influence on the device integrity. Thereforeit is desirable to develop new techniques to provide a cover with a good adhe-sion which is preferable chemically and physically equal to the material usedfor the microfluidic device.

In this chapter, a new sealing method is presented for a wide variety ofmicrostructures and systems. Samples are exposed to UV laser light at anoblique angle of incidence, under which conditions the major part of the lightis reflected. However, the reflection interface between two media is not discretebut a continuous transition between refractive indices.[9] For perpendicular in-cident light, this interface has a thickness of approximately half the wavelengthof the reflected light. This reflection interface is therefore ’activated’ duringthe event of reflection if, for instance, a photoresponsive material is present.

This phenomenon, combined with a photo-initiator material, is used toobtain localized polymerization in the top region of a monomer. The commer-cially available photoresist SU8 (MicroChem) is chosen as a model materialto study the influence of exposure dose, diffusion of photo-activated compo-nents and the angle of incidence of the reflected light. Finally, structuresare prepared by standard lithography, which are sealed successfully using thetechnique of reflection polymerization.

Glancing angle lithography 99

7.2 Reflection exposure theory

Light is reflected at the interface of two dielectric media with a different refrac-tive index. The fraction of light reflected from a surface depends on the angleof incidence (θ) and the two refractive indices (ni and nt) of the media. Thereflection coefficients for respectively s-polarized (Rs) and p-polarized light(Rp) are derived from Fresnel’s equations:

Rs =[sin (θt − θi)sin (θt + θi)

]2

(7.1)

Rp =[tan (θt − θi)tan (θt + θi)

]2

(7.2)

Where θi is the angle of incidence, and θt the transmitted angle that can befound from Snell’s law:

ni sin θi = nt sin θt (7.3)

SU8 (MicroChem), the photosensitive material used in this study, has a re-fractive index of 1.6. The values of Rs and Rp for light travelling from air(ni = 1) into SU8 (nt = 1.6, before polymerization) for all possible angles ofincidence are shown in the graph from figure 7.1. From this graph, it is foundthat no total reflection occurs, and therefore no evanescent wave is expectedin these circumstances.[9]

0 20 40 60 80

0.0

0.2

0.4

0.6

0.8

1.0

Ref

lect

ion

coef

ficie

nt

Angle of incidence (º)

Rp Rs

Figure 7.1: S and P polarized light reflection coefficients for the air-SU8interface. No total reflection is observed.

100 Chapter 7

During the event of reflection, some light reaches an inner region of thematerial at which it is reflected. This happens because in practice, reflectionoccurs due to the coordinated oscillator action of molecules, and light travelsover a distance of half the wavelength (λ/2) through the material before it iscompletely reflected.[9] This is the actual reason for using laser light in thisstudy, due to its monochromatic nature, the penetration depth is uniquely de-termined. For grazing incidence, the path length (λ/2) remains the same, butthe penetration depth of the light decreases with increasing angle of incidence.Assume that for each unit of distance dx travelled through the material, thebeam is deflected by an angle dθ. Then, the penetration depth of the λ/2 longpath of the light through the material can be approximated numerically, andthe results are shown in figure 7.2.

0 20 40 60 800

25

50

75

100

125

150

175

Pen

etra

tion

dept

h (n

m)


Figure 7.2: Penetration depth of reflected light for different angles of inci-dence with respect to the sample normal. For grazing incidence, light of 351nm penetrates 80 nm into the layer.

For grazing incidence (angles > 80◦ with respect to the sample normal) thispenetration depth is approximately 80 nm. During glancing angle exposure,the entire film will receive approximately 40% of the incoming light intensity(figure 7.1), while the 80 nm thick top layer will receive the full intensity.In other words, glancing angle exposure is expected to work if the 40% oftransmitted light is lower than the threshold dose, while the dose received bythe top layer is higher than the exposure dose.


7.3 Experimental

The commercially available photoresist SU8 (MicroChem), a mixture of a mul-tifunctional epoxy resin, a photo-acid generator (triarylsulfonium salt) and asolvent (cyclopentanone) is used as recording material in this study. Glasssubstrates are cleaned in ethanol and dried with nitrogen gas. SU8-2010 (Mi-croChem) is spin coated (2000 rpm for 40 seconds - thickness 6 µm) andafterwards heated for 1 minute at 65◦C and 2 minutes at 95◦C to evaporatethe solvent and bring the SU8 in the glassy state. The 351 nm line of theArgon ion laser (Beamlok 2085-25S, SpectraPhysics) is used to carry out theglancing angle exposure with s-polarized light . During exposure, a Lewis acidis formed, which acts as a catalyst for the polymerization. A second heatingsequence, the post exposure bake (PEB), the actual crosslinking takes place.For standard photolithography, this PEB consists of 1 minute at 65◦C followedby 2 minutes at 95◦C. The complete reaction scheme of SU8 can be found infigure 7.3.

O

R1

O+

R1

H

H+SbF6-

R2 OH O+

R1

H O

R1

H

O+ H

R2

O

R1

H

O+ H

R2

O

R1

H

O

R2

H+SbF6-+

Crosslinking

Lewis acidregeneration

Figure 7.3: Schematic representation of SU8 cationic photopolymerization.During exposure, a Lewis acid is generated. Polymerization occurs by theopening of the 1,2 epoxy ring.[10]

102 Chapter 7

After the PEB, the sample is cooled down and remaining monomer is re-moved by immersing the film in mr-dev600 (MicroChem) followed by rinsingwith propanol-2. Film thicknesses are investigated by scanning electron mi-croscope (XL 30 ESEM-FEG, Philips). Prior to SEM analysis samples arecovered with a 15 nm layer of gold (K575 XD Turbo sputter coater, Emitech,Ltd.).

7.4 Process parameters

In order to get a better insight into the mechanism behind reflection poly-merization, three sets of experiments are performed. Firstly, the influenceof exposure dose on the thickness of a freestanding film obtained by reflec-tion polymerization is investigated. After that, the influence of the angle ofincidence and the diffusion of the Lewis acid on the film thickness are exam-ined. In a third set of experiments, several structures, fabricated using masklithography are sealed using reflection polymerization.

7.4.1 Exposure dose dependence

In a first set of experiments, SU8 films of 6 µm thickness are prepared. Thesefilms are irradiated at an angle of 85◦ with the sample normal, with an intensityof 25 mW cm−2 for times ranging from 1 second to 6 seconds. The film isthen heated for 1 minute at 65◦C followed by 2 minutes at 95◦C and slowlycooled down, after which the unreacted monomer is washed away using SU8developer. The thickness of the remaining freestanding film is investigatedusing SEM, and the results of these measurements are shown in figure 7.4.

For exposure doses lower than 37.5 mJ cm−2 no remaining SU8 film couldbe found and it is therefore concluded that this corresponds to the well-knownthreshold dose, albeit that the observed threshold is significantly lower thanthe one found in SU8 datasheets.[11] For exposure doses between 40 and 90 mJcm−2, a linear increase of film thickness with the exposure dose is observed.Exposure doses higher than 90 mJ cm−2 do not lead to thicker films.

During exposure, a certain amount of Lewis acid is formed, depending onthe exposure dose. This acid acts as a catalyst for the crosslinking reaction,inducing crosslinking wherever a certain concentration of this acid is present.During the first part of the post-exposure bake, this acid can diffuse into thebulk of the SU8 layer,[12, 13] the depth depends on the initial concentrationof acid (a higher concentration generates a larger driving force for diffusion).This explains the linear increase in layer thickness at low exposure doses.


(a)

40 60 80 100 120 1400.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Film

thic

knes

s (

m)

Exposure dose @351 nm (mJ/cm2)

(b)

Figure 7.4: (a) SEM image of freestanding film remaining after development.(b)Thickness of the freestanding film obtained by glancing angle exposure ver-sus the exposure dose. Exposures are carried out at 25 mW cm−2 and at anincident angle of 85 ◦ with respect to the sample normal.

However, the time available for diffusion is limited (1 minute), resulting in afinite distance to which the acid can diffuse, and initiate polymerization. Inaddition to that, the photo-active species (triarylsulfonium salt) is present ina limited amount. The combination of these two facts explains the constantfilm thickness for higher exposure doses, as observed in figure 7.4.

7.4.2 Acid diffusion

Since the photoresist is crosslinked during a two-step heating process, it isassumed that diffusion of species could also be of importance for the GALITHprocess. Therefore a set of experiments is performed with 6 µm thick SU8films exposed with 75 mJ cm−2 at an incident angle of 85◦ with respect tothe sample normal. In the post-exposure bake, the heating time at 65◦Cis varied from 0 to 25 minutes. At 65◦C, no polymerization is expected totake place, however, the mobility is expected to increase significantly since itexceeds the glass transition temperature of uncured SU8. After a period at65◦C, the samples are crosslinked at 95◦C after which the sample is cooleddown slowly. The unreacted monomers are washed away in the developmentstep. The thickness of the remaining films is measured using SEM, and theresults are displayed in figure 7.5.

From the figure it can be seen that the layer thickness increases with in-creasing diffusion time, up to 20 minutes. For longer diffusion times, the layerthickness does not increase any further. The fact that a limited amount ofacid is generated during exposure, which then diffuses into the layer, could be

104 Chapter 7

0 5 10 15 20 25

0

1

2

3

Film

thic

knes

s (

m)

Post bake time @ 65º (min)

Figure 7.5: Freestanding film thickness for different ’diffusion times’, periodat 65 ◦C during the post bake step. For a post-bake time of more than 20minutes at 65 ◦C, no further increase in film thickness is observed.

responsible for this effect. After a certain time, 20 minutes in this case, theacid is distributed in such a way that the driving force for diffusion is too lowto cause any significant diffusion. Combining this with the fact that a certainacid concentration is required for polymerization, it is understood that thefilm thickness is limited.

7.4.3 Angle dependence

A parameter that should clearly have an influence on the achieved film thick-ness, is the angle of incidence during the glancing angle exposure. The pos-sibility to influence the film thickness by changing the angle of incidence isexamined. From figure 7.1 it can be found that for incident angles between 75and 90 degrees, the largest portion of s-polarized light (light polarized in theplane of the air-SU8 interface), is reflected from the surface. By making surethat the transmitted exposure dose is lower than the threshold dose of SU8(the minimal exposure dose and acid concentration at which it polymerizes)the angle of incidence can be varied in this narrow range to obtain differentfilm thicknesses. A smaller angle of incidence results in a larger penetrationdepth (figure 7.2) of the reflected light, resulting in a larger activated layer,which results in a thicker film.

In figure 7.6 the outcome of three experiments performed with an exposuredose of 90 mJ cm−2 at incident angles of 75◦, 80◦ and 85◦ is presented. Thefilm thickness decreases linearly with the incident angle, a trend that is alsoobserved in the penetration depth of the light (figure 7.2).


75 80 85

1.0

1.5

2.0Fi

lm th

ickn

ess

(m

)


Figure 7.6: Measured free-standing film thickness for different angles of in-cidence.

7.4.4 Sealing of microstructures

To investigate whether GALITH is truly suitable for the efficient sealing of mi-crofluidic structures, experiments were performed for covering standard holo-graphic and lithographic structures using reflection photopolymerization. 6µm layers of SU8 are first subjected to a grazing incidence exposure at 85◦

and an exposure dose of 90 mJ cm−2, followed by either a mask exposureor exposure to a holographic interference pattern to obtain microchannels ormembranes[14] (figure 7.7).

(a) (b) (c)

Figure 7.7: Schematic view of the exposure steps in glancing angle sealing ofmicrostructures. (a) Glancing angle exposure (b) Mask or holographic exposure(c) After postprocessing, sealed channels remain.

The reason for this counter-intuitive order of exposure steps is that therefractive index in the exposed areas slightly differs from that of unexposedareas, which could cause unwanted refractive or diffractive effects during thegrazing incidence exposure. An example of an exposure sequence where thisorder of mask and holographic exposure steps was not respected is shownin figure 7.8. First, a mask exposure of a periodic pattern of squares was

106 Chapter 7

Figure 7.8: Interference effects in reflection photopolymerization.

executed, after which the glancing angle exposure was performed.For the fabrication of this sample, first a mask exposure is performed using

an EXFO Omicure S2000 UV lamp (λ = 320-390 nm), with an exposure doseof 70 mJ cm−2, followed by a glancing angle exposure. The mask consistsof squares with a size of 10 µm and a periodicity of 20 µm. In the figure itis shown that an edge effect is observed and square shaped voids resemblingflowers are observed instead of covered squares. The protrusions in the cornersare assumed to result from interference.

After the correct exposure sequence, a normal post exposure bake is carriedout, followed by the development and rinsing with propanol-2. In figure 7.9cross-section SEM images of covered holographic membranes and microchan-nels are shown. An open membrane with a pore size of 1 µm is shown in figure7.9(a), and the same membrane is closed in figure 7.9(b). In the figure at theright, microchannels with a diameter of 5 µm are covered.

(a) (b) (c)

Figure 7.9: Scanning Electron Microscope images of a: open membrane(cross-section), b: sealed membrane (cross-section) and c: sealed microchannel(cross-section).

In this study, SU8 is used as a model system, but one can think of morewidespread applications. Any system that uses the addition of light absorbersto induce intensity gradients in order to limit the depth of exposure (e.g. in


the localized induction of phase separation) could in principle be replaced byGALITH, so no more additives are required. On the other hand, the use oflight absorbers could enhance the GALITH effect even further.

7.5 Conclusions

Glancing angle lithography is used to provide a sealing layer to model micro-structures, such as be used in microfluidic systems or micro-electromechanicalsystems (MEMS). The thickness of the obtained barrier layer is shown todepend on a combination of exposure dose, diffusion allowed and angle of in-cidence of the light. The angle of incidence determines the thickness of theactivated layer, while the exposure dose fixes the amount of acid formed in theactivated layer. In the post-exposure bake, this acid is allowed to diffuse intothe non-exposed regions with the diffusion distance depending on the diffusiontime.

7.6 References

[1] M. Martin, D. Watson, W. Bennett and D. J. Hammerstrom. Fabricationof plastic microfluidic components. SPIE Proc., 3515, (1998) 172–176.

[2] I. Glasgow, D. Beebe and V. White. Design rules for polyimide solventbonding. Sens. Mater., 11 (5), (1999) 269–278.

[3] G. Lee, S. Chen, G. Huang, W. Sung and Y. Lin. Microfabricated plasticchips by hot embossing methods and their applications for DNA separationand detection. Sensor Actuat. B, 75, (2001) 142–148.

[4] K. Hermans. Latent structured thermally developed reliefs: principles andapplications of photoembossing. Ph.D. thesis, Eindhoven University ofTechnology, the Netherlands (2009).

[5] S. Lai, C. Xia and L. Lee. A packaging technique for polymer microfluidicplatforms. Anal. Chem., 76, (2004) 1175–1183.

[6] J. Ruano-Lopez, M. Aguirregabiria, M. Tijero, M. Arroyo, J. Elizalde,J. Berganzo, I. aranburu, F. Blanco and M. Solomona. A new SU8 processto integrate buried waveguides and sealed microchannels for a Lab-on-a-Chip. Sensor Actuat. B, 114, (2006) 542–551.

[7] A. Yussuf, I. Sbarski, M. Solomona, N. Trana and J. Hayes. Sealingof polymeric-microfluidic devices by using high frequency electromagnetic

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field and screen printing technique. J. Mater. Process. Technol., 189,(2007) 401–408.

[8] K. Lei, W. Li, N. Budraa and J. Mai. Microwave bonding of polymer-basedsubstrates for potential encapsulated micro/nanofluidic device fabrication.Sensor Actuat. A, 114, (2004) 340–346.

[9] E. Hecht. Optics. Addison-Wesley, 4 edition (2001).

[10] L. J. Guerin. The SU8 homepage. http://www.geocities.com/guerinlj(2008).


[12] C. Becnel, Y. Desta and K. Kelly1. Ultra-deep x-ray lithography of denselypacked SU8 features: Ii. process performance as a function of dose, featureheight and post exposure bake temperature. J. Micromech. Microeng., 15,(2005) 12491259.

[13] M. D. Stewart, H. V. Tran, G. M. Schmid, T. B. Stachowiak, D. J. Beckerand C. G. Willson. Acid catalyst mobility in resist resins. J. Vac. Sci.Technol. B, 20 (6), (2002) 2946–2952.

[14] E. Koukharenko, M. Kraft, J. Ensell and N. Hollinshead. A comparativestudy of different thick photoresists for mems applications. J. Mater. Sci.- Mater. Electron., 16, (2005) 741–747.

Samenvatting

In de medische diagnostiek groeit de vraag naar kleine, handzame apparaat-jes voor het uitvoeren van snelle tests, bijvoorbeeld voor het opsporen vanhartaandoeningen in het beginstadium. Geminiaturiseerde systemen die aller-hande zogenaamde point-of-care tests kunnen uitvoeren worden biosensorengenoemd. Membranen en microkanalen zijn twee belangrijke componentenvan zo’n biosensor. Een microkanaal zorgt ervoor dat kleine samples (zoals1 druppel bloed) voldoende zijn voor het functioneren van de sensor. Mem-branen kunnen gebruikt worden voor het filteren van het sample, maar ookvoor de adsorptie van biomoleculen die later nodig zijn voor het detecterenvan eiwitten, DNA, etc.

Er bestaan verschillende soorten membranen, maar door hun grote poly-dispersiteit zijn de efficientie en de doorstroming beperkt. Microfilters daar-entegen hebben een erg regelmatige structuur met een lage weerstand en eenzeer efficiente scheiding tot gevolg. Men gebruikt verschillende productietech-nieken zoals silicium technologie en fasescheiding voor het maken van dezemicrofilters, elk met zijn voor- en nadelen. Interferentie holografie maakthet mogelijk om membranen te produceren met een goed gedefinieerde porievorm, een hoge efficientie en een lage stromingsweerstand, gebruikmakend vande goede verwerkbaarheid en uitstekende eigenschappen van polymeren. Eeninterferentiepatroon, gevormd door twee kruisende laserbundels wordt vast-gelegd in SU8, een zogenaamde fotoresist die chemisch en thermisch resistentis. Doordat SU8 bij kamertemperatuur in de glastoestand is, zijn meerderevan deze belichtingsstappen mogelijk. De resulterende structuur is dan de somvan alle belichtingsstappen. Poriegroottes tussen 0.1 en 5 micrometer werdenop deze manier behaald.

Om te onderzoeken welke geometrische parameters de grootste invloedhebben op de flux door deze membranen werden eindige elementen simulatiesgebruikt. Lange, elliptische porien blijken verschillende voordelen te hebbent.o.v. ronde porien. Bijvoorbeeld, de weerstand vanwege de poriewand is

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lager vanwege de betere volume-oppervlakte verhouding, maar ook een hogereporositeit kan behaald worden. Langwerpige porien werden gefabriceerd doorde tweede belichtingsstap uit te voeren met een rotatiehoek kleiner dan 90graden.

De wand van de porien is mede oorzaak van de stromingsweerstand. Daar-om is het wenselijk om de lengte van de porien, en daarmee de aanwezigheidvan de wand, te beperken. Hiervoor werden dunne membranen gefabriceerddoor de concentratie vaste stof of de spin coat condities aan te passen.

Het laatste effect dat een rol speelt bij het ontwerpen van een membraanis het Vena Contracta effect. De effectieve uitstroomdiameter van een poriewordt hierdoor kleiner dan de poriediameter, met een verlaging van de fluxtot gevolg. Tapse porien staan ervoor bekend dat ze dit effect kunnen ver-minderen. Door een intensiteitsgradient te induceren doorheen de fotoresist,worden aan de belichte kant van het membraan kleine porien gevormd en aande andere kant grote porien. Dit, in een continue gradient over de film resul-teert in tapse porien. Tapse porien hebben echter een nadeel. Door de grotereuitgangsdiameter, kunnen de porien minder dicht bij elkaar zitten, waardoorde porositeit van het membraan dan weer afneemt.

Om deze microfilters in een biosensor te kunnen gebruiken, moeten dezemembranen ingebouwd kunnen worden in een microkanaal. In de meest een-voudige vorm bestaat een membraan uit lamellen. Deze zijn makkelijk teproduceren met standaard lithografie of met holografie. Een nieuw ontwerpvoor een microarray voor het detecteren van eiwitten werd gebaseerd op lokaalgeplaatste lamellen voor de adsorptie van antilichamen. Op deze manier isslechts een klein sample volume nodig, maar blijft een hoge selectiviteit en eengoede signaal-ruis verhouding behouden.

De stroming tussen zulke lamellen is laminair, zodat transport van te de-tecteren moleculen enkel door diffusie kan plaatsvinden. Diffusie is traag,waardoor een test erg lang zou duren. Om dit proces te versnellen werdengroeven aangebracht op de zijkanten van de lamellen, dewelke een roterendestroming tussen de lamellen veroorzaken. Deze groeven werden gemaakt dooreens slimme combinatie van masker lithografie en onderbelichting tijdens eenholografische belichtingsstap. Lamellen hebben echter nog een groot nadeel,ze hebben de neiging om te vallen. Hierdoor verschillen de afstanden tussenlamellen, wat de reproduceerbaarheid van diagnostische tests zeker niet tengoed komt.

Slanted-angle holografie werd ontwikkeld om membranen te maken metporien die in het vlak liggen, zodat deze in een microkanaal ingebouwd kunnen

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worden. Door de fotogevoelige laag onder een hoek te plaatsen t.o.v. het in-terferentiepatroon worden schuine lamellen gecreeerd. Een tweede belichting,gedraaid naar de andere kant resulteert in een honingraatstructuur die hetmembraan zal vormen. De poriegrootte en -vorm kunnen opnieuw gekozenworden door de hoek tussen de laserbundels aan te passen. In combinatiemet een lithografische stap werden deze membranen in een microkanaal gefab-riceerd. Deze membranen zijn lekvrij verbonden met de bodem en de wandenvan het kanaal. Uit testen blijkt dat de membranen een lage drukval hebbenen een uitstekende selectiviteit. Ook de mechanische stabiliteit is verbeterdten opzichte van de vrijstaande lamellen door de driezijdige verbinding methet kanaal. Het open karakter van de membraanstructuur zorgt ervoor datbiomoleculen kunnen aangebracht worden met technieken als inkjet printen endergelijke.

De cruciale stap in het ontwikkelen van een microarray is het immobiliz-eren van antilichamen. De conformatie van een antilichaam en de biologischefunctie moet behouden blijven tijdens de immobilizatie. Fysische adsorptiewordt meestal gebruikt voor het vasthechten van eiwitten. Maar, een cova-lente binding is veel sterker, en wordt daarom geprefereerd. Covalente bind-ingsprotocollen maken gebruik van amines of carboxylzuren om deze bindingte bewerkstelligen. SU8 oppervlakken werden behandeld met een coating vanpolyacrylzuur of poly-L-lysine. Om te bepalen op welk oppervlak de meeste ei-witten vasthechten werden recuperatie experimenten uitgevoerd. Hierbij wer-den patronen van fluorescent gelabelde antilichamen door middel van inkjetprinten op SU8, PAA en PLL oppervlakken aangebracht. Na een wasstapbleek dat op SU8 en op PLL veel antilichamen waren blijven zitten, en opPAA slechts de helft hiervan. Omdat het aanbrengen van zo’n coating eenextra productiestap met zich meebrengt, werd geopteerd om de biologischefunctionaliteit van de antilichamen te testen op een onbehandeld SU8 opper-vlak. De eerst resultaten tonen aan dat op een vlak substraat een detectielimietvan 10 nM haalbaar is.

Externe pompen om een vloeistofstroming te creeren zijn ongewenst bij hetontwerpen van een draagbare, kleine biosensor. Capillaire stroming voorkomtdat deze externe componenten nodig zouden zijn. SU8 staat echter bekendom zijn hydrofobiciteit, dewelke een capillaire stroming onmogelijk maakt.De oppervlakte-energie van SU8 werd verhoogd door middel van UV-ozon enzuurstofplasma behandelingen, waardoor een capillaire stroming mogelijk is.Op deze manier wordt het gebruik van een externe pomp vermeden.

Als laatste is ook het contact tussen de vloeistoffen in de biosensor en

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de buitenwereld ongewenst. Daarvoor is een hermetische afdichting van demicrokanalen nodig. Glancing angle lithografie maakt gebruik van het nietdiscrete karakter van een reflectie overgang om in de afdichting van de mi-crokanalen te voorzien. De toplaag van de fotogevoelige laag wordt geactiveerdtijdens de belichtingsstap. Tijdens een verwarmingsstap wordt deze laag ver-net zodat een vaste film wordt bekomen. De dikte van deze film hangt af vande invalshoek van het licht, de belichtingsdosis en de diffusietijd.

Door bovenstaande technieken zoals slanted-angle holografie, lithografieen glancing angle lithografie te combineren kan men een volledig afgesloten,functionele biosensor fabriceren.

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List of publications

Articles

• An M. Prenen, J.C.A van der Werf, C.W.M. Bastiaansen, D.J. Broer,Monodisperse, Polymeric Nano- and Microsieves Produced with Inter-ference Holography, Advanced Materials, 21, (2009) 1751-1755.

• An M. Prenen, C.W.M. Bastiaansen, D.J. Broer, Glancing angle lithog-raphy for sealing of microchannels, in preparation

• An M. Prenen, Anja Knopf, Cees W.M. Bastiaansen, Dirk J. Broer,In situ fabrication of polymer microsieves for µTAS by slanted-angleholography, in preparation

• R. J. Vrancken, H. Kusumaatmaja, K. Hermans, A.M. Prenen, O. PierreLouis, C.W.M. Bastiaansen, D.J. Broer, Fully reversible transition fromWenzel to Cassie-Baxter states on corrugated superhydrophobic sur-faces, Advanced Materials, Submitted.

• M. van den Heuvel, A. Prenen, J. Gielen, P. Christianen, D.J. Broer,D. Lowik, J. Van Hest, Patterns of diacetylene-containing peptide am-phiphiles using polarization holography, Journal of the American Chem-ical Society, Submitted

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Patents

• Ko Hermans, An M. Prenen, Cees Bastiaansen, Dirk Broer, Ron vanLieshout, Chamindie Punyadeera, Flow-Through Biosensor, patent num-ber WO2008139389 (A1)

• An M. Prenen, Ko Hermans, Cees Bastiaansen, Dirk Broer, Ron vanLieshout, Chamindie Punyadeera, Membranes suited for immobilizingbiomolecules, Patent Pending (2009)

• Ko Hermans, An M. Prenen, Cees Bastiaansen, Dirk Broer, Ron vanLieshout, Chamindie Punyadeera, Diagnostic device, Patent Pending(2009)

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Acknowledgments

A physicist doing a PhD at a Chemical Engineering faculty, requires someempathy. However, I got quickly adopted by the PICT group, who guided meinto the magic world of chemistry. Many people came across during the past4 years, and I would like to thank some of them in particular.

First of all, I want to thank my promotor Dick Broer and copromotor KeesBastiaansen for giving me the opportunity to do this PhD in your group, ona very exciting project. Dick, it was a pleasure to work with you, you were agreat inspiration for me with your wild ideas for experiments (of which someactually worked!). Kees, your practical approach helped me enormously to getthe greater picture of this research.

I would also like to express my gratitude to the members of the committeefor their contribution to this thesis: Rint Sijbesma, Mathias Wessling, Jan vanEsch and Martin Moller.

Like many projects in the PICT group, also this one was started with areal application in mind. The biosensor described in chapter 5 was developedin collaboration with Philips Research. I would like to thank Ron van Lieshoutand Chamindie Punyadeera for introducing me into a subject that was com-pletely new to me: biochemistry. The membranes described in this thesis werealso used for the capture of DNA, in the development of another biosensor atPhilips. Roel, Bernadet, Irene, Peter and Arie, thanks a million for showingme how to ’fish’ for DNA.

At the start of my PhD, the work of Hans van der Werf was a stepping stonefor the rest of my research. I had also the opportunity to guide 2 students:Anja Knopf and Martijn van Loon.

During these 4 years, I had a desk in the ’big office’ STO 0.26 with con-tinuously changing inhabitants, but with three constants: Pit, Joost and Ko.Thank you guys for the great atmosphere and help during all this time. Andall the other members of PICT: Anastasia, Blanca, Carlos, Charlotte, Chris,Dick de B., Ivelina, Jeremy, Katherine, Ken, Maud, Michael, Nick, Nico, Paul,

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Robert, Shabnam, Shufen, Teun, Ties, Thijs, Youseli and Xiaoran: these 4years wouldn’t have been so much fun without you.

A special word of gratitude goes to my family. Mama, papa, Leen andKoen, thanks for the support and distraction during these past busy months.Especially, refurbishing (and testing...) the terrace was a good excuse for aday ’not having to write’.

Xander, thank you for your love, care and advice during the past hectictime. With our theses finally finished, I’m looking forward to spending moretime with you.

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Curriculum Vitae

14-08-1982 Born in Bree, Belgium

1994 - 1996 Latijn, Heilig Hartinstituut, Bree, Belgium

1996 - 2000 Latijn-Wiskunde, Sint-Augustinusinstituut, Bree, Belgium

2000 - 2002 Kandidaat Natuurkunde, Limburgs Universitair CentrumDiepenbeek, Belgium

2002 - 2005 M.Sc. Applied Physics, Eindhoven University of TechnologyM.Sc. thesis: Modeling and analysis of light-inducedphase separation in lc-polyacrylate systems

2005 - 2009 Ph.D. research, Eindhoven University of TechnologyDepartment of Chemical Engineering and Chemistry,Research group Polymer technologySupervision: Prof. dr. D.J. Broer anddr. C.W.M. Bastiaansen

Other: RPK courses: Polymer Chemistry, Polymer Physics,Polymer Properties

Paramedic (’Ambulancier’) for the Belgian Red Cross

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polymeric microfilters by interference holography : development and applications · applications...

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