Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 867

Spatially Controlled CovalentImmobilization of Biomolecules

on Silicon Surfaces

BY

ELISABETH PAVLOVIC

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2003

List of papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals:

I E. Pavlovic, A.P. Quist, U. Gelius and S. Oscarsson, “Surface functionalization of silicon oxide at room temperature and atmospheric pressure”, Journal of Colloid and Interface Science 254 (2002) 200-203

II E. Pavlovic, A.P. Quist, U. Gelius, L. Nyholm and S. Oscarsson, “Generation of thiolsulfinates/thiolsulfonates by electrooxidation of thiols on silicon surfaces for reversible immobilization of molecules”, Langmuir 19 (2003) 4217-4221

III E. Pavlovic, S. Oscarsson and A.P. Quist, "Nanoscale site-Specific immobilization of proteins through electroactivated disulfide exchange", Nanoletters 3 (2003) 779-781

IV E. Pavlovic, A.P. Quist, L. Nyholm, U. Gelius and S. Oscarsson, “Patterned generation of reactive thiolsulfinates/thiolsulfonates on silicon oxide by electrooxidation using electro-microcontact printing”, submitted to Langmuir (2003)

My input was essential to decisions taken about the theoretical and experimental development of the research projects described in this thesis. I performed all of the chemistry and analysis using XPS and AFM. I took part in the lithographic and evaporation processes, as well as SEM analysis. I was responsible for the writing of all four papers.

The figures in section 6.2 are reproduced with the publishers permission. Paper I: Elsevier Papers II-IV: American Chemical Society

Papers not included in this thesis:

S. Oscarsson; A.P. Quist; E. Pavlovic; O. Öhman. “Immobilization method and surfaces produced using said method”, PCT Int. Appl. (2003), nr. WO 0323402 A1

Å. Rosengren, E. Pavlovic, S. Oscarsson, A. Krajewski, A. Ravaglioli and A. Piancastelli, “Plasma protein adsorption pattern on characterized ceramic biomaterials”, Biomaterials 23 (2002) 1237-1247

A.P. Quist, E. Pavlovic and S. Oscarsson, “Surface nanobiotechnology I: methods and techniques for activation of surfaces and specific immobilization of macromolecules”, Proceedings of the International School on Advanced Material Science and Technology (2002), submitted

A.P. Quist, A. Mechler, E. Pavlovic and R. Lal, “Probing surface structure and dynamics of water in confined state during (de)hydration with phase imaging AFM”, manuscript in preparation

Y. Coffinier, E. Pavlovic, A.P. Quist, U. Gelius, S. Oscarsson and M.A. Vijayalakshmi, “Adsorption of plain and histidine-derivatized pluronic on thiolated silicon oxide surfaces: an XPS and AFM study”, manuscript in preparation

My conference presentations including parts of the work in this thesis:

E. Pavlovic, A.P. Quist, and S. Oscarsson, “Electrooxidation of thiols on silicon surfaces for reversible immobilization of molecules”, oral presentation at the 225th American Chemical Society National Meeting, New Orleans LA, USA, March 23-27, 2003

E. Pavlovic, A.P. Quist, U. Gelius, L. Nyholm, S. Oscarsson, “A strategy for controlled immobilization of molecules on surfaces”, poster presentation at the Materials Research Society Fall Meeting 2002, Boston MA, USA, December 2-6, 2002

E. Pavlovic, A.P. Quist, L. Nyholm, S. Oscarsson and U. Gelius, “XPS studies of a new silicon oxide silanization technique and electrooxidation of surface thiols for immobilization of molecules”, poster presentation at the 9th

International Conference on Electronic Spectroscopy and Structure, Uppsala, Sweden, June 30-July 4, 2003

Contents

1. Introduction.................................................................................................11.1. General aspects....................................................................................1

1.1.1. Immobilization of biomolecules on surfaces ...............................11.1.2. From the microscale to the molecular scale ................................2

1.2. Nanotechnology ..................................................................................21.3. Spatially controlled immobilization of biomolecules .........................3

1.3.1. Lithographic patterning of surfaces .............................................31.3.2. Chemical patterning of surfaces ..................................................3

1.4. About this work...................................................................................4

2. Chemical derivatization of silicon oxide ....................................................62.1. Silicon and silicon oxide .....................................................................62.2. Silanization of silicon oxide................................................................7

2.2.1. The silanization process...............................................................72.2.2. Silanization methods....................................................................9

Pre-treatments ...................................................................................9Liquid phase......................................................................................9Gas phase ........................................................................................10

3. Chemical modifications of organic monolayers .......................................113.1. Chemical activation of thiolated monolayers....................................11

3.1.1. Generation of reactive disulfides ...............................................113.1.2. Generation of thiolsulfinates and thiolsulfonates ......................13

3.2. Electrochemical modifications of organic monolayers .....................153.2.1. Anodic oxidation of hydroquinones ..........................................153.2.2. Anodic oxidation of methyl groups ...........................................15

4. Lithographic modifications of surfaces ....................................................174.1. Photolithography ...............................................................................174.2. Soft lithography.................................................................................18

5. Experimental techniques...........................................................................205.1. Atomic Force Microscopy.................................................................20

5.1.1. Principle.....................................................................................205.1.2. Instrumentation..........................................................................215.1.3. Imaging modes ..........................................................................22

Contact mode ..................................................................................22Tapping mode .................................................................................23

5.1.4. Lateral force microscopy ...........................................................235.1.5. AFM as a tool for nanoscale surface modification ....................24

5.2. X-ray Photoelectron Spectroscopy....................................................255.2.1. Principle.....................................................................................255.2.2. Instrumentation..........................................................................255.2.3. Chemical shift of core electrons ................................................265.2.4. Spin-orbit interaction.................................................................275.2.5. Surface sensitivity......................................................................28

Electron emission............................................................................28Angle dependent analysis ...............................................................29

6. Papers........................................................................................................306.1. Experimental .....................................................................................30

6.1.1. Surface chemistry and electrochemistry....................................30Silanization .....................................................................................30Electroactivation .............................................................................30

6.1.2. Immobilization and release of biomolecules and particles ........316.1.3. Surface analysis .........................................................................32

XPS .................................................................................................32AFM................................................................................................32

6.1.4. Stamp fabrication.......................................................................326.2. Results and Discussion......................................................................33

6.2.1. Paper I........................................................................................336.2.2. Paper II ......................................................................................356.2.3. Paper III .....................................................................................376.2.4. Paper IV.....................................................................................38

7. Conclusion ................................................................................................41

8. Outlook .....................................................................................................428.1. Improvements....................................................................................42

8.1.1. Reversibility ..............................................................................428.1.2. Surface regeneration ..................................................................428.1.3. Alternative surfaces ...................................................................42

8.2. Biological considerations ..................................................................438.3. Applications ......................................................................................43

Acknowledgements.......................................................................................44

References.....................................................................................................46

Abbreviations

AFM Atomic Force Microscopy -gal -Galactosidase

DNA Deoxyribonucleic Acid DPDS Dipyridyl Disulfide DPL Dip-Pen Lithography DTT Dithiothreitol EBL Electron Beam Lithography EDC 1-Ethyl-3(3-dimethylaminopropyl) carbodiimide ESEM Environmental Scanning Electron Microscope FFIB Finely Focused Ion Beam HSA Human Serum Albumin IMFP Inelastic Mean Free Path LFM Lateral Force Microscopy MED Mean Escape Depth µCP Microcontact Printing MP Mercaptopyridine MPP Monoperoxyphthalate 3-MPTMS 3-mercaptopropyltrimethoxysilane NHS N-hydroxysuccinimide PAA Polyacrylic AcidPDMS Poly(dimethylsiloxane) PL Photolithography PFP Pentafluorophenol R.H. Relative Humidity RMS Root Mean Square SAMs Self-Assembled Monolayers SEM Scanning Electron Microscopy SPDP Succinimidyl3-(2-pyridyldithio)propionate SPM Scanning Probe Microscopy SPR Surface Plasmon Resonance STM Scanning Tunneling Microscopy TEM Transmission Electron Microscopy TFCS N-(Trifluoroacetylcaproyloxy) succinimide ester THF Tetrahydrofurane TOA Take-Off Angle XPS X-ray Photoelectron Spectroscopy

I am enough of an artist to draw freely upon my imagination. Imagination is more important than knowledge.

Knowledge is limited. Imagination encircles the world.Albert Einstein (1879 - 1955)

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1. Introduction

1.1. General aspects

1.1.1. Immobilization of biomolecules on surfaces Immobilization of biomolecules on biological membranes is widespread in nature, and in many cases, is needed for biomolecules to be fully active.

Man-made systems rely on immobilization of biomolecules. Although biomolecules can be immobilized by adsorption to surfaces, immobilization through covalent bonds results in an increased stability of the modified surfaces. Immobilization of antibodies for sensing [1,2], separation [3,4]purposes or catalytic units [5] allows the control of parameters such as measurement resolution and precision, purity and/or yield of the products. Immobilization of molecules on a surface can also be required by some analytical methods such as surface plasmon resonance (SPR) [6,7] and is involved in the design of biocompatible materials [8,9] as well.

Covalent attachment is performed via coupling reactions between chemically modified surfaces and biomolecules. Immobilization of molecules can be achieved on a variety of materials, using a wide range of chemistries depending on the molecule/material couple.

Nevertheless, the lack of control of those processes at a molecular level results in the formation of multilayers of molecules that have partly lost their active conformation or are oriented with their active site to the surface. During the past decade, biomimetics was introduced in order to apply the efficiency of biological systems to artificial systems. This required an increased understanding of coupling techniques at the molecular level and the development of new immobilization techniques to control the positioning, orientation and conformation of immobilized biomolecules.

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1.1.2. From the microscale to the molecular scale Since the 1970’s, technology has undergone a miniaturization trend to better satisfy our modern society’s needs for smaller, faster and more efficient devices.

In the mid 80’s, the invention of scanning probe microscopy (SPM) [10,11], featuring an outstanding lateral as well as vertical resolution, allowed the imaging of surfaces down to the atomic level [11]. This was the opportunity to achieve understanding of surface modification techniques on a molecular level. The quasi-simultaneous discovery of self-assembly of alkylthiol monolayers on gold [12-14], soon followed by silane monolayers on silicon oxide [15], led to improved quality of chemical derivatization of surfaces, and shrank the vertical dimension of organic layers to a few nanometers. Later on, molecular studies such as the analysis of the three-dimensional structure and orientation of surface adsorbed single biomolecules were performed [16-18].

This benefitted directly the area of biodevices and biosensors that was also affected by the miniaturization trend, especially under the pressure of growing fields such as genomics and proteomics [19,20]. Microarrays of covalently bound deoxyribonucleic acid (DNA) and proteins are already used in these fields. Patterned surfaces in the biomaterials area are designed to display specific properties toward cells [21,22].

Considerable research efforts have been made during the past decade to try to push the miniaturization process to the limit of the atom. Nevertheless, nowadays, the smallest applications and devices industrially manufactured are in the microscale.

1.2. Nanotechnology Nanotechnology is a manufacturing technology that will allow for devices with at least one dimension below 100 nm in size [23-25]. The concept of building things atom by atom was first introduced by Richard Feynman in 1959 when he said: "The principles of physics, as far as I can see, do not speak against the possibility of maneuvering things atom by atom." There are two main concepts to that: the “bottom-up”, which consists of building devices atom-by atom or molecule-by-molecule, and the “top-down” approach, which relies on “carving” smaller objects out of materials. While a diversity of “top-down” methods is already available, manufacturing of the “bottom-up” type requires the establishment of new techniques enabling a control of the matter at the atomic or molecular level. The “bottom-up” approach being the essence of a new technological era, it is understandable that nanotechnology is still at the stage of concept.

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Nowadays, few “nanoprocesses” can be transferred to production on an industrial scale, the reason being mainly their slow rate of performance.

1.3. Spatially controlled immobilization of biomolecules Surfaces can either be modified physically by creating topographic defects or chemically by patterning with metals or organic molecules. Existing lithographic and chemical methods allow control of the lateral size, shape and position of surface areas designed to immobilize biomolecules. The majority enables obtaining patterned self-assembled monolayers (SAMs), which most often result in specific non-covalent adsorption of biomolecules. Covalent immobilization requires in general an activation step in order to proceed to the coupling. Those reactions sometimes occur in conditions that are harmful to the three-dimensional structure of proteins. It has to be noted that covalent coupling possibilities are limited and most of them result in irreversible immobilization.

1.3.1. Lithographic patterning of surfaces Recently, techniques enabling the creation of defects or inorganic patterns on surfaces, such as finely focused ion beam lithography (FFIB) [26], electron beam lithography (EBL) [27] and photolithography (PL) [28,29], were developed.

Metal deposition using microcontact printing (µCP) [30] and nanolithographic methods based on SPM [31] basically offer similar chemical possibilities as other lithographic techniques.

Direct patterning of surfaces with organic molecules can be performed using µCP [32] or dip-pen lithography (DPL) [33-36]. These techniques are based on a micro or nanoscale deposition process enabling a direct control of the positioning of molecules on surfaces.

These techniques mostly allow for patterned SAMs, resulting in non-covalent adsorption of macromolecules, unless further chemical activation is performed. Coupling chemistry to directly immobilize biomolecules through covalent bonds is limited to gold-sulfur interactions.

1.3.2. Chemical patterning of surfaces Relying on liquid or gas phase chemistry in order to achieve an activation of a chemically modified surface enables access to a diverse choice of coupling reactions but with poor spatial and dimensional control [37-40], in the best case, at the millimeter scale using nanoliter dispensing [41]. Liquid or gas

4

phase chemistry is more often used for the chemical activation of patterned SAMs, and subsequent immobilization of macromolecules [42].

Chemical patterning can be performed by use of irradiation of light-sensitive compounds, which will trigger a reaction leading to covalent bonds between molecules and surfaces [43]. Masks allow for a spatial control of the activation process [44,45]. The reverse case of patterning such as oxidation of thiol groups using light [46,47] can also be achieved and results in an inactivation of the corresponding areas.

Application of a potential difference can also result in chemical patterning [48,49]. In this situation, spatial control can be achieved by use of counter-electrodes of different sizes [50-53]. Patterns can be created by local desorption of alkylthiols from a gold surface resulting from a reduction to thiolates [54,55].

Nevertheless, the SAMs obtained require chemical activation, either before or after patterning to allow covalent immobilization.

1.4. About this work The objective of this thesis was to develop a series of techniques resulting in a “bottom-up” approach for spatially controlled immobilization of biomolecules. The central point of this project was to develop and investigate a novel method for electroactivation of surface thiols to reactive thiolsulfinates/thiolsulfonates allowing for a thiol-disulfide exchange for covalent coupling of molecules and particles. Methods providing foundations and applications of this chemical activation were developed.

A novel surface derivatization method for silicon oxide (paper I) had to be developed, in order to obtain ultraflat and reproducible thiolated organic monolayers that are the starting point for the building-up of biological structures on surfaces.

Then followed the investigation of the electroactivation process of the surface thiol groups based on electrochemical oxidation (paper II) resulting in a thiol-disulfide exchange. This method enables access to a wide range of spatially controlled electrooxidation from the macroscale to the nanoscale and subsequent covalent binding of molecules/particles to the activated areas, with the unique advantage of combining spatial control with covalence and reversibility of the formed bonds, and subsequent regeneration of the surfaces.

Different set-ups were used to perform the activation at different scales (papers II-IV). One of the driving forces in this research was to achieve the immobilization of biomolecules on surfaces on a nanometer scale, which was achieved in paper III. Paper IV describes a technique allowing for

5

microscale activation of thiolated surfaces, which is of interest for many present applications.

This summary contains a brief review of processes and techniques used to achieve the purpose of this thesis and a description of the research leading to the development of the previously mentioned procedures.

6

2. Chemical derivatization of silicon oxide

Surface chemical modification is the first step of the process leading to immobilization of molecules on surfaces and is therefore crucial. Although it is possible to chemically modify different types of surfaces, silicon surfaces were chosen because they do not interfere with sulfur chemistry as gold does, while allowing the formation of monolayers through a self-assembly process. Silicon surfaces also present a very low root mean square (RMS) roughness, which is essential for single molecule studies. Therefore, this chapter will be focused on the description of silanization of silicon oxide.

2.1. Silicon and silicon oxideThe surface of single crystal silicon consists of a layer of native and amorphous silicon oxide SiOx [56], resulting from oxidation in air, with a thickness around 2 nm. Water molecules strongly bound to the silicon oxide surface form a layer of up to 4 Å thick under ambient conditions [57]. Siloxane bonds linking silicon atoms to oxygen atoms at the silicon oxide surface can react with water and generate silanol groups. Silicon oxide surfaces have similar chemical properties as silica gels. The silanols, in average 5 per nm2, are divided in two groups according to their acidity: pH 4-5 and pH 8-9 [58]. The polished sides of the silicon wafers used in this work, covered with the native oxide, have a low roughness of 1-2 Å (measured over 1 µm2 area), which is essential when performing atomic force microscopy (AFM) imaging of single biomolecules.

Silicon in its non-oxidized state is the most widely used material in electronic circuitry, since it is a semi-conductor. The silicon bulk can be doped with impurities: p-doped silicon, such as boron-doped, contains empty electronic states and is an electron acceptor; n-doped silicon such as phosphorus-doped, contains electrons that are not involved in bonds and is an electron donor.

While the native oxide surface has many implications for the surface derivatization chemistry, doping of silicon mainly impacts on electrochemical reactions occurring at silicon surfaces, as mentioned in paper III.

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2.2. Silanization of silicon oxide

2.2.1. The silanization process Silanization can be performed on any type of surface displaying hydroxyl groups, such as mica [59], glass [60,61] and metal oxides [62]. Silanization involves the covalent binding of a silane reagent molecule, either a chlorosilane or an alkyloxysilane (scheme 2.1), to the surface or to another silane molecule through a siloxane bond. The polymerization of the silane molecules depends on both the number of chloro or alkyloxy groups present and the amount of water present on the silicon oxide surface [63]. In this work, 3-mercaptotrimethoxysilane was used for the silanization, since the presence of the thiol group makes it impossible to have a trichlorosilane from a chemical point of view. On the other hand, cross-polymerisation of the silane on the silicon oxide surface is desirable for a dense and stable monolayer, hence the presence of three methoxy groups.

Si OR''

R''O

R''O

R

Si Cl

Cl

Cl

R

Si R'

R''O

R''O

R

Si R'

R''O

R'

R

Si R'

Cl

Cl

R

Si R'

Cl

R'

R

Alkyloxysilanes

Chlorosilanes

( )n ( )n ( )n

( )n ( )n ( )n

R=alkyl, amino alkyl, thioalkyl, alkene, carboxy alkyl... R'=H, CH3 R''=CH3, CH2CH3

Scheme 2.1. Silane molecules used in silanization

As described in scheme 2.2, if the water layer is kept intact (step A), the diffusion of the silane molecules to the surface silanols is decreased. In this case, alkylsilane groups are hydrolysed by water molecules from the adsorbed water layer (step B to C), followed by a condensation step resulting in the formation of siloxane bonds, which mainly occur between adjacent silane molecules (step D). This last step usually requires high temperatures in a dry atmosphere and is known as “curing”. The silane monolayer will thus be mainly adsorbed to the silicon surface through hydrogen bonds mediated by water molecules.

8

H2O layer

(A)

SiOH

400 deg.C, O2

(C)

Si

OMe

HO OH

(CH2)nR

H2O layer

(D)

H2O layer

Si

OMe

OMeMeO

(CH2)nR

Si OMe

MeO

MeO

(CH2)n

R

(E)

(F)

(G)

SiOMe

(CH2)n

R

O

Si

OMeSi

OMe(CH2)n

R

O

Si

OMe

SiOH

(CH2)n

R

O

Si

Si

(CH2)n

R

O

OH

SiHO

(CH2)nR

O

OH

Si

OMe

OMeMeO

(CH2)nR

Si OMe

MeO

MeO

(CH2)n

R

(B)

H2O layer

alkyltrimethoxysilane

SiOMe

(CH2)n

R

OHOMe

Hydrolysis

Condensation

Direct bonding to the surface

SiOH

SiOH

SiOH

SiOH

SiOH

SiOH

SiO-

SiOH

SiOH

SiOH

SiO-

SiOH

SiOH

SiOH

SiO-

SiOH

SiOH

SiOH

SiO-

SiOH

SiOH

SiOH

SiOH

SiOH

SiO SiO

SiO SiO

SiOSiO

SiO

Scheme 2.2. Influence of silicon oxide surface hydration on the silanization mechanism. R is a functional group such as –CH3, –NH2, –SH, –COOH

Dehydration of a silicon oxide surface (step A to E) will lead to direct reaction of the alkylsilane groups with surface acidic silanols (step G).

Even though water present on the silicon surface is necessary to hydrolyse the alkyloxy groups of the silane molecules, the main cause of trouble in the silanization process is water in the reaction environment, leading to

9

polymerisation of the silane molecules before even reaching the silicon surface and resulting in uneven and clumpy layers.

2.2.2. Silanization methods Many silanization methods were developed to derivatize silicates, metal oxides and glasses. Most of them result nevertheless in high roughness, disordered multilayers of silane molecules. The lack of control of the silanization process on the molecular level affects the efficiency of the resulting layers to perform their role in binding of molecules to the surfaces, due to a wrong orientation.

The roughness resulting from the organic layer itself is often smaller than the roughness of glass or metal oxides but when it comes to ultraflat silicon, most of those methods generate a high surface roughness, which makes the use of derivatized silicon in single molecules studies impossible.

Organic monolayers on silicon oxide are usually much less ordered than SAMs obtained on gold, the main reasons being the uneven and heterogeneous presence of water molecules and hydroxyl groups on the surface, and surface contamination.

Pre-treatmentsIt is essential for surface chemistry to occur to clean the surfaces from their contaminants. In the case of silicon oxide, the use of an oxidizing acid wash called “piranha” solution, a mixture of H2O2 and H2SO4, simultaneously removes carbon contamination and increases the number of reactive hydroxyl groups by breaking siloxane bonds [64].

However, the ultimate surface cleaning process for silicon is the etching of the native silicon oxide by use of dilute hydrogen fluoride, and subsequent regrowth of silicon oxide.

The amount of water bound to the oxide is more difficult to control. One way to control the amount of water present on the surface is to remove it by heating the silicon samples to 420 C in a dry oxygen atmosphere [65]. This results in much less densely packed monolayers but each silane molecule is covalently bound directly to the silicon surface through a siloxane bond.

Liquid phase Surfaces can be immersed in a solution of silane reagent molecules in an organic solvent [66]. In this situation, complete elimination of water in the environment is difficult to achieve, and therefore polymerization of silanes in solution, before reaching the silicon oxide surface, can happen to a certain extent. This results in disordered layers, with a high surface roughness and low reproducibility [67].

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Gas phase Gas phase silanization can be performed in vacuum [68], by distillation of the silane reagent, which allows for silanization of silicon oxide surfaces but needs hours of reaction time and sophisticated equipment. In a variation of this technique, the environment is heated [69] to enhance silane evaporation but heat also promotes polymerization of the silane before adsorbing to the silicon surface and therefore does not result in reproducible quality monolayers.

The development of an alternative way of performing reproducible gas phase silanization using an anhydrous argon flow to evaporate and transport silane molecules to the silicon oxide surface is described in paper I.

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3. Chemical modifications of organic monolayers

This chapter describes some of the activation and coupling chemistry involving thiol-disulfide exchanges, and some examples of electrochemistry used for activation or patterning of organic monolayers, since the main focus of this work is covalent binding of molecules to surfaces through formation of disulfide bonds via electrochemically activated thiolsulfinates/ thiolsulfonates.

3.1. Chemical activation of thiolated monolayers A wide variety of chemical reactions allowing coupling of molecules to solid substrates is available. The thiol-disulfide exchange chemistry displays several advantages compared to other coupling reactions: covalent disulfide bonds form efficiently at neutral pH in aqueous solution and the covalent disulfide bonds created are reversible by reduction with dithiothreitol (DTT) [70].

On the other hand, sulfur compounds are easily chemically as well as electrochemically oxidized. They are prone to oxidation by oxygen in air and therefore need to be protected in a non-oxidizing atmosphere. They also display interesting electrochemical properties such as oxidation of thiols to disulfides and sulfur oxides at relatively low voltages [71]. The reduction of disulfides to thiols has however been impossible to achieve without a metal-bound transition [72].

3.1.1. Generation of reactive disulfides Activation of thiol groups by generation of pyridyl disulfides in a liquid

phase reaction has been extensively used for thiol-disulfide exchange coupling [61,73]. Pyridyl activated disulfides can be introduced into biomolecules via the bifunctional reagent succinimidyl 3-(2-pyridyldithio) propionate (SPDP) [74], which can in turn react with thiol groups on

12

surfaces (scheme 3.1). Surface thiols can be activated to pyridyl disulfides as well using dipyridyl disulfide (DPDS) [39].

Si

SH

Si

SH

Si

SH

Si

SH

SiO

S S

N

R Si

S

S

Si

S

S

NH

SNH

S

(B)

R R

Si

SH

Si

SH

SiO

(A)

DTTred

(C)

Si

SH

Si

SH

Si

SH

Si

SH

+DTToxSH

R

SH

R

SiO

Scheme 3.1. Reaction of surface thiols with SPDP activated biomolecules

N

S S

N

Si

S

S

N

Si

SS

N

NH

S

NHS

SH

Si

SS

Si

SS

NH

SNH

S

(A) (B)

(C)(D)

R

R R

DTTredSi

SH

Si

SH

Si

SH

Si

SH

+DTToxSH

R

SH

R

Si

SH

Si

SH

Si

S

Si

S

Si

S

Si

S

Si

SH

Si

SH

SiO SiO

SiO SiO

+

NH

S

Scheme 3.2. Activation of surface disulfides using MP/DPDS; R is a biomolecule

On the other hand, surface thiol groups can easily be oxidized to low reactivity disulfides by oxygen in air. In this situation, the approach is to

13

generate reactive pyridyl disulfides using a combination of mercaptopyridine (MP) and DPDS [69].

The reaction between disulfides groups from the surface and MP/DPDS is described in scheme 3.2: MP splits the surface disulfides which subsequently react with DPDS, creating reactive pyridyl disulfide groups, with release of a MP molecule (step A to B). Surface pyridyl disulfides can, in turn, react with thiol-containing molecules (step C to D). A complete regeneration of the surface thiols is obtained after DTT treatment (step D to A).

This activation process is a typical liquid phase reaction, and therefore spatial control is not possible below the millimeter scale.

3.1.2. Generation of thiolsulfinates and thiolsulfonates Chemical oxidation of thiols results in the formation of reactive thiolsulfinates and/or thiolsulfonates.

Scheme 3.3 describes the chemical oxidation of thiols using hydrogen peroxide (H2O2) [75], which leads to disulfides (step A to B) followed by thiolsulfonates (step B to C). These activated disulfides can be split by a thiol group from a molecule in solution (step C to D), which results in a thiol-disulfide exchange. The immobilized molecules can then be released using DTT (step D to E).

Si

SOH O

Si

SS

R

Si

SS

R

(C)

(D)

H2O2Si

SH

Si

SH

Si

SH

Si

SH

(A) (B)

H2O2

DTTredSi

SH

Si

SH

SHR

SH

R

(E)

Si

SOOH

Si

SOOH

+DTTox

R SH

Si

S

Si

S

Si

S

Si

S

Si

S

Si

SOO

Si

S

Si

SO

O

Si

SOOH

SiO SiO

SiOSiO

SiO

Scheme 3.3. Chemical oxidation of surface thiols to reactive thiolsulfonates; R is a biomolecule

14

A more controlled way, allowing for generation of thiolsulfinates, described in scheme 3.4, consists of creating disulfides using ferrocyanide (step A to B) and subsequently oxidizing them using a one-oxygen donor, e.g. monoperoxyphthalate (MPP) in solution [76] (step B to C).Similarly to the thiolsulfonates, thiolsulfinates react in a thiol-disulfide exchange with thiol-containing molecules in solution (step C to D), which can be released using DTT (step D to E).

Si

SH

Si

SH

Si

SH

Si

SH

(A)

Fe(CN)6

Si

S

Si

S

Si

S

Si

S

Si

SOH

Si

SOH

Si

S

SR

Si

S

SR

(B) (C)

(D)

DTTredSi

SH

Si

SH

Si

SH

Si

SH

+DTTox+H2O

(E)

OOO-

O

OH

2

Mg2+

Si

S

Si

SO

Si

S

Si

SO

R SH

SiO SiO SiO

SiO SiO

SH

RSH

R

Scheme 3.4. Chemical oxidation of surface thiols to reactive thiolsulfinates; R is a biomolecule

The advantage of thiolsulfinates over thiolsulfonates is the possibility of complete regeneration of the thiol surfaces to their initial form since the sulfenic acid groups formed after reaction with a thiol are very unstable and easily reduced back to thiol groups through reaction with water or nearby thiol groups (scheme 3.4, D to E). In the case of thiolsulfonates, up to 50% of the thiols groups are lost to sulfinic acid (scheme 3.3, D to E), which is very difficult to reduce.

Those sulfur oxide species can easily be electrochemically generated [77]. It was demonstrated in paper II that this type of activation can be performed by means of an electrooxidation of the surface thiols. In paper III, nanoscale patterns of activated thiols are generated in one step by application of a potential difference between an AFM tip and a thiolated surface.

15

3.2. Electrochemical modifications of organic monolayers

3.2.1. Anodic oxidation of hydroquinones Electrochemical oxidation resulting in a chemical activation to perform covalent bonding of molecules was achieved on functionalized alkylthiols on gold [48]. The electrooxidation of hydroquinone to benzoquinone was performed, with subsequent reaction of the benzoquinone in a Diels-Alder addition (scheme 3.5) with a pentadiene. This reaction is limited to covalent immobilization of small organic molecules. It is performed in a mixture of tetrahydrofurane (THF) and H2O, which makes it impossible to use proteins, since it will result in denaturation of their three-dimensional structure.

S

OH

OH

S

OH

OH

S

OH

OH

AuS

O

O

S

O

O

S

O

O

AuS

O

O

S

O

O

S

O

O

R

R R R

+V

-VAu

Scheme 3.5. Electrooxidation of hydroquinones to benzoquinones and subsequent Diels-Alder addition; R is a low molecular weight organomolecule

Deprotection of carboxyl groups was also performed by oxidation of hydroxyquinones to benzoquinones [49]. Nevertheless, further coupling of the carboxyls to amine groups requires an additional activation step using a mixture of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) [78].

There was no spatial control in this situation, although in theory, this would be possible by using adequate counter-electrodes.

3.2.2. Anodic oxidation of methyl groups Electrochemical oxidation of top methyl groups of alkylsilane monolayers on silicon oxide (scheme 3.6) has been reported. Different counter-electrodes were used to apply a positive potential difference to selected areas of the surfaces, allowing for creation of electrooxidized patterns of different sizes. Nanoscale patterning was achieved using an AFM tip [50,51], this set-up

16

being more detailed in section 5.1.5. A µCP-type of patterning method, described in section 4.2, was performed with a copper transmission electron microscope (TEM) grid [53] as conductive “hard stamp”. The generated hydrophilicity offers the possibility of building a second silane layer and can in general promote the interaction with polar molecules. This electrochemical activation was not used to perform patterned covalent immobilization of biomolecules, although a chemical activation of the carboxylic groups is possible using pentafluorophenol (PFP) [79] or EDC/NHS [42], with subsequent reaction with amine groups.

These methods for spatially controlled electrooxidation are similar to the ones used in papers III and IV, respectively.

+VSi

CH3

Si

CH3

Si

CH3

Si

COOH

Si

COOH

Si

COOH

SiOSiO

Scheme 3.6. Electrooxidation of alkylsilane methyl groups to carboxyl groups

17

4. Lithographic modifications of surfaces

A wide variety of lithographic techniques is available to produce micro- to nanostuctured surfaces. The two lithographic techniques described in this chapter play an important part in this work: photolithography (PL) was used in the process leading to the technique developed in paper IV and its set-up was inspired by soft lithography.

4.1. PhotolithographyGeneration of micro to nanoscale patterns on silicon/silicon oxide surfaces or deposition of metallic patterns is done by means of lithographic techniques. Even though electron beam lithography (EBL) [27] allows a much higher resolution, the most extensively used lithographic process is PL [28,29], since it enables the production of devices on an industrial scale.

Figure 4.1. Schematic drawing describing the steps involved in photolithography using a negative resist and silicon oxide etching

PL is based on the property of some polymers to be broken up (positive resist) or cross-linked (negative resist) by exposure to ultra-violet light. In order to pattern the surface, a mask is used. The mask is a glass plate coated with a metal emulsion and a layer of thin resist on one side, in which

silicon oxide

maskphotoresist

silicon

Etching

Development

Cleaning

UV light

18

structures are created using other lithographic techniques, such as electron beam lithography [80] or focused laser lithography.

After irradiation, the development step consists of dissolving the non-irradiated negative resist, leaving only the irradiated areas protected (figure 4.1). The opposite process occurs when a positive resist is used.

The following step is plasma etching of the silicon/silicon oxide or evaporation of metals on the non-protected areas. The remaining resist is then cleaned away from the patterned surfaces.

This technique was used to produce the patterned silicon surface used as a mould for the polymer stamp in paper IV. The mould was imaged using scanning electron microscopy (SEM) (figure 4.2).

Figure 4.2. SEM image of a photolithographically patterned silicon wafer used as a mold for stamp making in paper IV. Top view (A) and tilted view (B)

4.2. Soft lithography Soft lithography [81,82], or micro contact printing (µCP), allows modification of specific areas on surfaces by use of a soft polymer stamp, usually made of poly(dimethylsiloxane) (PDMS), which enables conformal contact with the surfaces to pattern (figure 4.3). The PDMS stamp is usually moulded to acquire the desired pattern and dipped into a solution containing the molecules to imprint on the surface. During the contact, molecules from the stamp are deposited on the surfaces, with which they can eventually create covalent bonds, for instance using alkylthiol molecules and gold surfaces [32].

Reverse patterning can also be performed, if the stamp is used to mask certain areas on the surface. In this case, corrugations in the stamp perform as channels to allow surface contact with a solution containing a derivatization reagent [83].

A B

19

Although widely used to achieve micrometer scale patterns on surfaces, recent optimization of this technique resulted in less than a couple of hundred to a few tens of nanometers wide deposition patterns [84,85]. A major problem of the technique is contamination of the surfaces with low-molecular weight PDMS oligomers, since the surfaces in most cases cannot be washed after printing. The main solution for this drawback is washing of the PDMS stamps to decrease the amount of contamination [32].

Figure 4.3. Microcontact printing process

A similar technique using a PDMS stamp coated with a thin layer of evaporated aluminum allows for patterned electroactivation in paper IV.

Contact

PDMS stamp soaked with molecules to print

Surface

Printing

Pressure

20

5. Experimental techniques

Two main analytical techniques were used to perform the studies described in this thesis: atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). They are both extremely surface-sensitive and complement each other, since AFM gives mostly physical information about the surface, while XPS gives the chemical composition. In addition, AFM is also used as a surface modification tool.

5.1. Atomic Force Microscopy SPM started in the early 80’s with the invention of the scanning tunneling microscopy (STM), soon followed by the AFM [10,11]. The SPM technique is based on the centuries old mechanical profilometer principle, which can be briefly described as a tip raster-scanned across a surface. The revolutionary use of piezoelectric crystals to induce nanoscale precision movements of the tip allowed the unique features of this type of microscopy, among others an exceptional vertical resolution. The possibility to see at the atomic level enabled studies of single molecules adsorbed on surfaces, but also allowed for monitoring of inter-molecular interactions. This was the opening for nanotechnology and manufacturing of nanometer scale devices based on molecular assemblies.

5.1.1. Principle As in all SPM techniques, AFM imaging results from scanning a tip over a surface. It is very well suited for non-conductive organic samples since the detection of the tip movement does not depend on a current between tip and surface, as in STM. In AFM, the tip is positioned on one end of a cantilever, which must be extremely sensitive to small force changes between the tip and the sample surface. The use of a piezocrystal to generate minute movements of either tip or surface allows for accurate measurement of the surface topography.

The interaction between the tip and the surface, while in close proximity or contact, depends on several forces:

21

Van der Waals forces (Eq 1)Non-polar entities, close enough in space, can attractively interact by inducing dipoles in each other. The electronic structure of the two atoms will rearrange in order to create partial opposite charges in the bonds.

FvdW = 26dAR

(Eq 1)

where A is the Hamaker constant, R the radius of the tip and d the distance between the tip and the surface. Born repulsive force (Eq 2)Also known as indentation force, it is a short range repulsive interaction that results from the Pauli exclusion principle.

Fi= 32 RDk (Eq 2) where k depends on the Young’s modulus and Poisson coefficient of the sample and D is the indentation distance of the tip into the surface. Electrostatic forces (Eq 3)A long range attractive interaction exists between two opposite charges, and is also called Coulomb interaction.

Fc= 221

rqqk (Eq 3)

where q1 and q2 are the two charges separated by a distance r and k depends on the electric permeability of the medium. Capillary force (Eq 4)Water from the atmosphere condenses in the gap between the tip and the surface. This force depends on the air relative humidity.

F=)/1( dD

Rk (Eq 4)

where k depends on the hydrophobicity of the tip and the water surface energy, D is the distance tip-sample and d is the height of the section of the tip in contact with water.

5.1.2. Instrumentation In general, the interaction forces between tip and sample are monitored by measuring the deflection and/or torque of the cantilever that holds the tip. The main difference between AFM instruments is the mode of vibration of the tip in tapping mode, which can be achieved either mechanically (MultiMode from Digital Instruments) or magnetically (MacMode from Molecular Imaging).

22

The movements of the cantilever due to surface topography are detected as illustrated in figure 5.1. A setpoint voltage, entered by the user, controls the reference position of the laser on the detector and corresponds to a certain distance between tip and sample surface. A laser beam, reflected by the cantilever toward the detector, is deflected up or down when the cantilever is bent. The feedback system compensates the deflection of the laser by regulating the vertical (Z direction) to keep the distance between the tip and the sample surface constant. This regulation is achieved by applying a z-potential to the piezocrystal that supports the sample.

Figure 5.1. AFM operation principle

5.1.3. Imaging modes Height information can be acquired in two different modes: contact and tapping . In both cases, topographic data is given by the vertical movement of the laser on the detector (figure 5.1, panels (A+B) minus panels (C+D)).

Contact mode In the contact mode (figure 5.2, left), the tip is rastered over the surface while in constant contact with it. For this reason, this mode can be more damaging for soft surfaces. Another disadvantage of this mode is the distortion of the image of the surface topography when areas of different friction coefficients are present on the surface, which induce a lateral torsion of the cantilever. This phenomenon is also at the origin of the main advantage of contact mode, which is the ability to perform lateral force microscopy (LFM, see section 5.1.4).

Setpoint FeedbackA B

C

ADC

Computer Z

X

Y

DAC

Image

Cantilever

Laser

Detector

Piezoelectric crystal

D

23

Tapping mode In the tapping mode (figure 5.2, right), also called “intermittent contact” mode, the cantilever is oscillated by a vibrating piezocrystal. This mode is much less damaging to the sample but is restricted to vertical deflection of the laser. Information about the textural nature of the surface can be acquired through the phase imaging, which results from the phase difference between the piezocrystal and the cantilever vibrations.

Figure 5.2. Contact mode imaging (left); tapping mode imaging (right)

5.1.4. Lateral force microscopy When an AFM tip is moved with a 90 scan angle over a surface, a lateral friction force arises from the load applied on the tip and the adhesion between the tip and the surface, as described by equation 5.

Ff = c1A + c2N (Eq 5)

Where A is the contact area between the tip and the surface and N the normal load. The constants c1 and c2 depend on adhesion, among other factors.

Figure 5.3. Lateral torsion of the cantilever over a high friction spot at 90 scan angle

Piezocrystal

AC

Z feedback regulation

Cantilever

Z feedback regulation

A B

C D

Scan direction

A B

C D

A B

C D

Scan direction

24

This force causes a lateral torsion of the cantilever, which is detected by a horizontal movement of the laser in the detector (figure 5.3, panels (A+C) minus panels (B+D)), directly giving rise to a frictional image.

If a constant force is applied to the tip, the friction difference will mainly result from adhesion. LFM allows for analysis of differences in friction resulting for example from different chemical compositions. This imaging mode is suitable for visualization of the local oxidation of surfaces.

5.1.5. AFM as a tool for nanoscale surface modification As nanotechnology developed, contact mode AFM started to be used as a tool for nanoscale modifications of surfaces. For all the methods mentioned below, the resolution of the drawn patterns depends directly on the size of the apex of the tip used.

The simplest examples are mechanical nanoindentation, performed by pressing the tip into the surface to create defects [86] and the use of the AFM tip as a “nanoarm” to assemble atoms on a surface [87]. In dip-pen lithography (DPL), the AFM tip is used as a nanoscale pen [33,34], coated with chemical “inks”, to draw patterns on surfaces. Finally, an AFM set-up can be used as an electrochemical cell to deposit patterns of reduced metal [31] or simply, to perform surface nanoscale anodic oxidation [88].

Figure 5.4. Schematic representation of the set-up used to perform anodic oxidation

This is achieved by applying a potential difference between the tip and the surface of a (semi)conductive sample. In this process, air relative humidity (R.H.) is a crucial factor, since condensation water around the contact point of the tip with the surface will act as a transfer medium and allow electrochemical reactions to occur (figure 5.4). Nanoscale oxidation

AFM tip

Silicon wafer

Water meniscus

Organic monolayer

25

patterns can be drawn on different types of surfaces. Nanoscale anodic oxidation of passivated or alkyl-derivatized silicon surfaces has also been studied [88]. This technique was also used to oxidize organic monolayers on silicon surfaces without growing silicon oxide [50,51].

A similar set-up is used in paper III to achieve nanometer scale electrochemical activation of thiols.

5.2. X-ray Photoelectron Spectroscopy In 1981, Kai Siegbahn was awarded the Nobel Prize for his pioneering work in electron spectroscopy [89], and more specifically X-ray photoelectron spectroscopy (XPS), which is based on the photoelectric effect. This phenomenon was discovered in the 1880’s by Heinrich Hertz [90] and explained by Albert Einstein, winning him the Nobel Prize in 1921 [91].

5.2.1. Principle XPS is based on the photoelectric effect that is the result of the interaction of photons with matter, which produces electrons. Electrons (or photoelectrons) are emitted if the energy of the absorbed photons is high enough. Excess photon energy is transferred to the electron as kinetic energy. The energy exchange during a photoelectric process is described by equation 6.

h = Eb + Ek + spec (Eq 6)

where h is the energy of the incident photon, Eb is the energy required to remove a certain electron from an atom, i.e. the electron binding energy, Ekis the kinetic energy of the emitted electron, and spec is the work function of the electron spectrometer. If Eb is measured with respect to the Fermi level,

spec can be neglected. Therefore, an absolute calibration of the ESCA instrument to a reference Fermi level is needed.

A relatively high X-ray energy (the usual sources, magnesium and aluminum, emit at 1253.6 and 1486.6 eV respectively) is used in order to enable the study of the core electrons of atoms, i.e. the electrons that are not involved in interatomic bonding.

5.2.2. Instrumentation The essential parts of a modern XPS instrument consist of an X-ray

source, an analysis chamber, an electron lens, an analyzer and a detector,

26

placed in interconnected vacuum chambers. To avoid contamination of the sample, the analysis chamber is kept in high vacuum (10-10 mbar).

Figure 5.5 is a schematic drawing of the ESCA 300 instrument from Gammadata Scienta AB. Al K X-rays produced at the rotating anode are selected to a unique photon energy by the monochromator, which also focuses the monochromized X-rays onto the sample surface. The emitted photoelectrons entering the electron lens are retarded and focused onto the entrance slit of the electron analyzer. The analyzer separates them according to their kinetic energy before they reach the detector.

Figure 5.5. Schematic representation of the ESCA 300 instrument

5.2.3. Chemical shift of core electrons Binding energies of electrons are typical for every element and every core orbital. Electrons with reference energies to a certain element are assigned as coming from similar atoms bonded to each other through single bonds, resulting in non-polar bonds. If the atoms are different in electronegativity, the bond is more or less polarized, and charges are unevenly distributed around the atoms. This effect causes the so-called chemical shift of the binding energy from the reference value and was first clearly demonstrated by Hagström et al. in 1963 [92].

Rotating anode

Electrongun

Monochromator quartz crystals

Sample holder/ manipulator

Sample

Electronlens

Detector

Analyser

+V-V

h

e-

Slits

27

The electronic distribution can also be modified at the result of charged surroundings, but this will result in smaller shifts than in the case of an ionic interatomic bond.

Oxidation of elements by oxygen results in chemical shifts of a few tenths to several electronvolts, depending on the oxidation level (table 5.1). Therefore, XPS is a very useful technique to study oxidation processes as well as oxide chemistry.

Table 5.1. Binding energies for the elements encountered in this work

Orbital Group Eb (eV) Ref. C1s C-H/C-C 285.0-285.5 [93] C-O 286.5-286.7 [93] O-C=O (carboxylate) 289.2-289.3 [93] C-N 285.5-286.4 [93] N-C=O (amide) 288.0-288.6 [93]N1s C-N 399.1-400.2 [93] NH3+ (ammonium) 401.5 [93] N-C=O (amide) 399.8 [93]O1s SiOx 532.0 [94] C-O 532.7-533.8 [94]S2s -SH/-S-S- 228.4 (1)

S=O (sulfinyl) 231.0 (1)

O=S=O (sulfonyl) 232.8 (1)

S2p3/2 -SH/-S-S- 164.0 [94] S=O (sulfinyl) 166.6 (2)

O=S=O (sulfonyl) 168.4 (2)

Si2p Si (bulk) 99.3 [94] SiOx (Silicon oxide) 103.3 [94]

(1) S2s binding energies are calculated by adding 64.38 eV to the S2p3/2 binding energies, corresponding to the energy separation between S2s and S2p3/2 [94](2) Chemical shifts taken from [95]

5.2.4. Spin-orbit interaction Signals resulting from the detection of one element can be either a single peak when the orbital angular momentum has a quantum number l = 0, or double peaks when l 0. If l = 0, the only permitted value of j is ½, giving rise to one single peak. The double peaks are due to the spin-orbital couplingand correspond to the two j quantum numbers, j- = l–s and j+ = l+s, where s

28

is the spin quantum number and is equal to ½. The number of electrons in a subshell with quantum number j is 2j+1. The intensity ratio between the j-and j+ subshells reflects approximately the difference of the electron population of these subshells. The energy separation between two such peaks increases with increasing binding energy.

The intensity ratio between the two peaks in the case of an AlKphotoelectron can be approximated by equation 7.

j

j

II

=1212

jj =

1)(21)(2

slsl

(Eq 7)

The effects of the spin-orbit interaction are well illustrated in the case of the sulfur signal: the S2s level results a single peak while the S2p level results in the S2p1/2 and S2p3/2 peaks. The S2p1/2 level has a 1.19 eV higher binding energy than S2p3/2, and half of its intensity.

5.2.5. Surface sensitivity

Electron emission The amount of electrons detected for a certain element will depend on the mean escape depth (MED), described as the average depth normal to the surface from which electrons are emitted. The MED is related to the path of the electrons through the material, called the inelastic mean free path(IMFP). An electron traveling in a solid phase will be subjected to elastic collisions, which can change the direction of its movement but keep its kinetic energy constant, and inelastic collisions, which will decrease its kinetic energy. Therefore, the higher the electron kinetic energy, the longer it will take to stop it.

Eventually, the electrons will stop after a certain number of inelastic collisions. The probability for the occurrence of those collisions influences directly the IMFP and depends on the density of the material. Therefore, the IMFP varies for different materials, and as a consequence, there is a corresponding variation of the MED. In a dense material, the IMFP is shorter, due to the high probability of collisions, and results in a shorter MED. As a result, electrons originating from below a certain depth will not reach the surface of the sample and therefore will not be detected. This effect is responsible for the surface sensitivity of the ESCA measurements.

Since the MED is an average value, there are always a certain percentage of electrons that will reach the surface. This percentage is high when the depth is smaller than the MED. Consequently, fewer and fewer electrons will be analyzed with increasing depth, which means that signals measured from

29

the top-most layers of the surface will have a higher intensity than those from deeper layers. This phenomenon is called attenuation of the signal, and is described by equation 8.

sin0

d

d eII (Eq 8)

where d is the distance between the signal emitting layer and the surface of the material and is the MED, and is the photoemission angle relative to the surface, also called the take-off angle (TOA).

Angle dependent analysis By changing the TOA, it is possible to change the surface sensitivity of the analysis, since most electrons travel in the same direction through the material toward the detector. When the TOA is 90 , the detected electrons have a path perpendicular to the sample surface and therefore, the analyzed depth will reach a maximum. When the TOA is grazing (e.g. 10 ), the depth at which electrons can still be detected is much shorter and the analysis becomes more surface-sensitive. In reality, a fraction of elastically scattered electrons from deeper layers also contributes to the observed intensity at grazing angle. An angle-dependent analysis procedure using two or more TOA allows, at least qualitatively, the resolving of layered surface structures.

30

6. Papers

6.1. Experimental

6.1.1. Surface chemistry and electrochemistry

Silanization A new silanization method that will be used for all the projects in this thesis is described in paper I. Silicon surfaces were cleaned using a Piranha solution, H2SO4:H2O2 30 % (v/v) 2:1, and rinsed with ultrapure water (18 M , low organic content). The wet surfaces were transferred to a 50 ml polypropylene reaction tube and dried with a dry argon flow. The surfaces were then moved up through an opening in a polypropylene grid placed 3 cm above the tube opening and placed on top of the grid, without having been in contact with the ambient atmosphere. The argon flow, which is entering the tube through a pipette tip with a 2 mm diameter, was then set to approximately 1 L/min. The tube was closed with a lid containing 10 l or 20 l of 3-mercaptopropyltrimethoxysilane (3-MPTMS) reagent. The whole process is performed at room temperature (21 C). After silanization, the surfaces were sonicated, first 10 minutes in 99.5% ethanol followed by 10 minutes in ultrapure water and dried in an argon flow.

Electroactivation In paper II, an electrochemical activation of the thiolated monolayer is investigated. A droplet of 100 l of phosphate buffer (10 mM, pH 7.0) is placed in between the thiolated surface and a platinum electrode. Potential differences were applied for 1 minute using a Keithley 2400 source meter with the silicon surface acting as the anode and the platinum electrode as the cathode. After the electroactivation, the surfaces were rinsed and sonicated for 10 minutes in ultrapure water and dried using an argon flow.

The chronoamperometric experiments were performed with a PAR 273 potentiostat using a three-electrode set-up with a platinum wire inserted in

31

the buffer droplet as a pseudo reference electrode with a stable potential measured versus Ag/AgCl.

In paper III, the electroactivation process was transferred to the nanoscale using an AFM tip as counter-electrode. The experiments were conducted in ambient conditions, with different relative humidity (R.H.) percentages, ranging from 28 to 45%.

In paper IV, an aluminum-coated PDMS stamp, divided into a counter-electrode and a reference electrode, was used as cathode to perform the electroactivation. A 5 l droplet of 10 mM phosphate buffer, pH 7.0, was squeezed between the thiolated silicon oxide and the aluminized stamp. A potential difference of 1.85 V vs. the Al reference electrode was applied for one minute using a LC-4B amperometric detector potentiostat.

6.1.2. Immobilization and release of biomolecules and particles In paper II, a fresh 0.1 mM solution of a short peptide, composed of 2 valines and one cysteine, in phosphate buffer (10 mM, pH 7.0) was prepared for each experiment. To achieve covalent binding of peptides, a surface electroactivated using 1.0 V was incubated in 5 ml of peptide solution under agitation. The surface was then sonicated for 10 minutes in ethanol followed by 10 minutes in ultrapure water before being dried with argon. Human Serum Albumin (HSA) was thiolated with SPDP as described elsewhere [75], yielding an average of four thiols/HSA molecule. Thiolated electroactivated surfaces were exposed overnight to a 1µg/ml solution of thiolated HSA in 10 mM phosphate buffer (pH 7.0).

In paper III, surfaces with patterns electroactivated using potential diffrerences ranging from 2.0 V to 5.0 V were exposed for 2 hours to a 5µg/ml solution of -galactosidase ( -Gal) in ultrapure water.

To release the bound molecules, the surfaces were incubated under agitation in 50 mM dithiothreitol (DTT) solution in phosphate buffer (0.1 M, pH 8.0), for one to three hours followed by sonication for 10 minutes in ultrapure water.

Polystyrene beads, 46 nm in diameter, containing surface amine groups were derivatized with approximately 1000 times excess (molecule/bead) of SPDP in 0.1 M phosphate buffer, pH 8.0, for 2 hours. The separation of the beads from excess SPDP and cleavage of the pyridyl disulfide to generate a thiol group were carried out using PD10 columns.

Immobilization of particles on the patterned surfaces was performed by deposition on the surfaces of a 150µl droplet of 1:500 (v/v) thiol-derivatized beads in 10mM phosphate buffer, pH 7.0, which were allowed to react for 90 minutes. The surfaces were then rinsed under running ultrapure water for 5 minutes and dried with an argon flow.

32

The same procedure was followed for the treatment of the surfaces with a 50 mM DTT solution in phosphate buffer (0.1 M, pH 8.0) for one to three hours.

6.1.3. Surface analysis

XPSIn paper I, XPS spectra at 10 and 90 TOA were acquired in an ESCA 300 instrument from GammaData Scienta AB, using a photon energy of 1487 eV and a pass energy of 300 eV. In paper II, only 10 TOA spectra were acquired.

In paper IV, XPS spectra were acquired in a Quantum 2000 Scanning ESCA Microprobe from Physical Electronics using monochromized Al Kx-ray radiation and a pass energy of 57.8 eV.

AFMFor paper I, atomic force microscopy (AFM) imaging was performed with both a PicoSPM from Molecular Imaging in MacMode using magnetically coated silicon based tips and a Digital Instruments Nanoscope IIIa in Tapping Mode using PointProbe silicon tips. Root mean square (RMS) roughness values were measured using the Nanoscope III software over areas of 1 1 µm2 and 500 500 nm2.

The Digital Instrument microscope was also used in intermittent contact mode in paper II.

For papers III and IV, a Digital Instruments Nanoscope IIIa with Ultrasharp silicon tips was used. Friction images were acquired in contact mode and topographic images in intermittent contact mode.

6.1.4. Stamp fabrication The silicon mould used to fabricate the stamp in Paper IV was produced using a complete photolithographic process, which includes the silicon oxide etching step and washing of photoresist residue. Finally, the silicon oxide was etched using 40% HF in order to minimize interaction of the poly(dimethylsiloxane (PDMS) with the mould. The stamp was prepared as described previously [32]. The PDMS stamp was then attached to a glass slide to obtain a rigid support. Two 100 nm thick layers of aluminum were subsequently evaporated on the patterned face of the stamp. The stamp was exposed with a 60 angle to the horizontal and mounted 25 cm above the source, and the aluminum deposition was carried out at an evaporation rate of about 5 nm/s in an E306 Edwards evaporator.

33

The conductivity of the aluminum layer was measured using a Keithley 2400 source meter. The stamps were also imaged using a FEI environmental scanning electron microscope (ESEM) in the scanning SEM mode before and after the Al coating.

6.2. Results and Discussion

6.2.1. Paper I Silanization of silicon oxide is involved in many technological applications. The silanization process and important parameters influencing the resulting monolayers, such as a dry environment and the access of the silane molecules to the silicon oxide surface are described in section 2.1, as well as the existing methods to achieve silanization of silicon oxide and their major drawbacks. In brief, the monolayers obtained using the existing methods do not satisfy the requirements needed by our projects, which are densely packed monolayers, and sufficiently low roughness to allow for single molecule analysis of surface-bound biomolecules.

Paper I describes a novel method to perform silanization of silicon oxide with 3-MPTMS that is fast, occurs at room temperature and ambient pressure and results in such densely packed, ultraflat, stable and highly reproducible monolayers.

Figure 6.1. Schematic representation of the silanization reactor

An argon flow is used to simultaneously provide a dry environment, enhance the evaporation of the silane reagent and transport 3-MPTMS

gridsample

3MPTMS

dry argon

34

molecules to the silicon oxide surface (figure 6.1). XPS data indicated that the silane molecules are correctly oriented with their thiol groups pointing up by comparing C1s and S2s intensity ratios at 10 and 90 TOA (figure 6.2, A and B).

It was evaluated that the surface coverage is a densely packed monolayer after a reaction time of 30 minutes.

AFM images were taken (figure 6.2, C and D) and the RMS roughness of the silanized surfaces was calculated to be 0.19 nm. In addition, the monolayers displayed high stability to hydrolysis.

The development of this method was essential for the outcome of my other projects, since they are all based on thiolated silicon oxide surfaces (Papers II-IV).

Figure 6.2. XPS data: carbon 1s (A) and sulfur 2s (B) at 10 and 90 TOA; reaction time dependence of the sulfur 2s signal (insert); tapping mode AFM images of the silane monolayer before (C) and after (D) sonication

35

6.2.2. Paper II Immobilization of biomolecules on surfaces is central to many applications ranging from biosensors to separation media. A wide variety of coupling chemistries is available to perform immobilization of biomolecules on practically any type of surface. The major drawback when it comes to micro and nanoscale immobilization is that they are all performed in solution, with no spatial control over the process. Chemical oxidation of thiols to thiolsulfinates/thiolsulfonates, as described in section 3.1.2, is a way of activating thiol disulfide exchange between the surface and thiol-containing biomolecules (figure 6.3).

Si

SH

Si

SH

Si

SH

Si

SH

Si

SH

Si

SH

SiOSi

A) B)

CH3

CH3NH O

NH2

CH3

CH3

NH

O

OH

SH

O

Figure 6.3. A thiolated silicon oxide surface (A), and the Valine –Valine –Cysteine peptide (B)

The possibility of achieving this type of activation using electrochemistry offers the opportunity of a spatially controlled immobilization of biomolecules, since the size of the activated areas corresponds to the size and shape of the counter-electrode.

In paper II, we investigate the generation of thiolsulfinates/ thiolsulfonates by electrooxidation of thiol groups of a 3-MPTMS monolayer on silicon oxide (figure 6.3, A) using a platinum counter-electrode, on a scale allowing XPS. In a two-electrode set-up, XPS data showed that generation of thiolsulfinates/thiolsulfonates depends on the applied potential difference (figure 6.4, top).

Using a three-electrode set-up, it was demonstrated that thiolsulfinates/ thiolsulfonates are generated between 0.6V and 0.8V versus Ag/AgCl, indicating that the reaction process is due to an electrochemical oxidation of the thiol groups.

The binding of a short peptide (figure 6.3, B) to the activated surfaces, its release using DTT and the reoxidation of the surface remaining thiols were investigated as well (figure 6.4, bottom). XPS results indicated that saturation of the activated surface by the peptide molecules occurred after 20 minutes of reaction time but only 40% of them were released from the

36

surface. After DTT treatment, the oxidized sulfur amount decreased by half. Reoxidation of the surface generated 24% thiolsulfinates/thiolsulfonates relative to the total sulfur amount.

Even though the reversibility of the immobilization required further investigation, it was shown that the electroactivation process does occur. The development of this method is the starting point of the projects aiming to spatially control biomolecule immobilization on the micro and the nanoscale (papers III and IV).

Figure 6.4. Top: sulfur 2p signal when no potential difference is applied (a), when 0.5 V (b), 0.8 V (c), 1.0 V (d), 1.2 V (e) and 2.0 V (f) are applied; Bottom, left: sulfur 2p signal, and right: corresponding nitrogen 1s signal, before oxidation (a), after oxidation (b), after 5 minutes (c), 15 more minutes (d) and 30 more minutes of exposure to the peptide (e), after treatment with DTT (f) and reoxidation (g)

37

6.2.3. Paper III Paper III is the application of the electroactivation process to the nanoscale. In this paper, an AFM set-up is used as an electrochemical cell to achieve a nanoscale activation of 3-MPTMS monolayers on silicon oxide, as described in section 5.1.5.

Lateral force microscopy (LFM) was used to investigate the localized oxidation of the surfaces (figure 6.5). It was found that the oxidation increased with the voltage and was optimal at a relative humidity (R.H.) between 40 and 42%. Measured line width ranged from 70 to 200 nm. Broadening of the line width with increasing voltage can also be noticed.

The immobilization of a thiol-rich protein, -gal, on the activated patterns was studied using tapping mode AFM (figure 6.6). The images show that -gal is only immobilized on the patterns electroactivated with 2.0, 3.0 and 4.0 volts. This indicates that the thiol monolayer is most probably destroyed at 5.0 V. Growth of silicon oxide can be evaluated from the background topography of the patterns: no increase in height is observed for 2.0 V, a slight increase at 3.0 V, and more significant increases for 4.0 V and 5.0 V.

It was not possible to release the immobilized -gal molecules by treatment with DTT, and this can be explained by the fact that the thiols involved in the immobilization process are parts of the protein structure.

In this paper, the goal of this thesis, which is the immobilization of biomolecules on a nanometer scale, has been reached. Nevertheless, the question of the reversibility of the process still remains to be solved.

Figure 6.5. Friction images of the electroactivated patterns using 1.0 V (A), 3.0 V (B) and 5.0 V (C), at around 40% R.H.

A

2 µm

B C

38

Figure 6.6. Tapping mode images of the topography of the electroactivated patterns after -gal immobilization (A), and zoom on 2.0V (B, top) and 3.0V (B, bottom),3.0V (C, top) and 4.0V (C, bottom), 4.0V (D, top) and 5.0V (D, bottom). Height scale: 10 nm

6.2.4. Paper IV A stamping method was developed to achieve the electroactivation on a

micrometer scale. PDMS stamps, commonly used in microcontact printing (µCP), were metallized in order to be conductive. PDMS stamps were prepared using photolithographically made passivated silicon molds with micrometer scale patterns. Aluminum was chosen for its non-reactivity to disulfides. The tilt of the stamp during the evaporation and two subsequent evaporations of Al were crucial to obtain a conductive coating. The Al coating was divided into two isolated parts in order to use one of them as reference electrode. The stamps were imaged using SEM (figure 6.7).

The Al-coated stamps were used to electroactivate a thiolated silicon oxide surface (figure 6.8). A droplet of buffer was squeezed in between the

2 V

3 V

4 V

5 V5 µm 1 µm

1 µm1 µm

A B

C D

39

stamp and the surface, and a 12 g weight was used to press the stamp onto the silicon surface.

Figure 6.7. Al-coated non-damaged linear pattern stamp (A) and damaged stamp, with visible cracks (B)

Figure 6.8. Schematic drawing of the electro-µCP set-up

A potential difference of 1.85 V versus the Al reference electrode, corresponding to 0.65V versus Ag/AgCl, was applied to the thiolated silicon surface. XPS data showed no change in the total amount of sulfur, indicating no damage was caused by the electroactivation (figure 6.9). A sulfur

A B

Reference electrode

Buffer droplet

SiO

Aluminizedstamp

Weight (12g)

Glass slide

Counter electrode

3MPTMS

Working electrode

Si

Gap between

electrodes

40

oxidation matching 80% of the maximum activation level was obtained (figure 6.9, B).

LFM measurements showed a clearly visible friction pattern matching the stamp pattern (figure 6.10, A).

Thiolated polystyrene beads were immobilized onto the patterns. Tapping mode AFM showed that the binding was specific to the electroactivated pattern, with little unspecific adsorption (figure 6.10, B).

After incubation of the surfaces with a DTT solution, tapping mode AFM showed that the beads were still present. The solution to the successful removal of the immobilized particles most probably resides in increasing the accessibility of the disulfide bonds to the DTT molecules (see section 8.1.1).

Figure 6.9. XPS spectra of sulfur 2s signal before (A) and after electroactivation (B)

Figure 6.10. Friction image of a thiolated surface electroactivated at a potential difference of 0.65V versus Ag/AgCl (A); tapping mode image of polystyrene beads immobilized on the linear electroactivated pattern, height scale 40 nm (B)

A B

A B

20 µm 10 µm

41

7. Conclusion

Spatially controlled immobilization of biomolecules on surfaces was achieved by developing the different steps composing a well-defined strategy: 1. a novel method to perform silanization of silicon oxide surfaces 2. a chemical process allowing to control activation of the resulting

monolayers 3. the use of set-ups enabling different sizes of activated areas

The chemical derivatization of silicon oxide and monolayer activation methods proved to be highly efficient and easy to perform, resulting in ultraflat and densely packed thiolated monolayers, locally activated to thiolsulfinates/ thiolsulfonates using an electrochemical method, which present the advantage of a simultaneous patterning and activation of the thiolated monolayers. Covalent immobilization of thiol-containing biomolecules was achieved on the activated areas, although the reversibility of the formed disulfides remains an aspect to be improved, since release of the bound biomolecules was only partial. The full regeneration of the surface thiols, which would result from activation to a 100% thiolsulfinates, has not been reached yet.

Activation patterns of different sizes were created on the surface by use of an AFM set-up for a nanometer scale patterning, or an electro-microcontact printing method for a microscale patterning.

Even though complete reversibility and regeneration of the modified surfaces has not been achieved, the electroactivation method still displays the potential of reusing the surfaces, which requires further investigation.

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8. Outlook

8.1. Improvements

8.1.1. Reversibility The incomplete reversibility is most probably due to a poor accessibility of the disulfide bonds between the biomolecules and the surfaces. It should be avoided to use thiol groups that are part of the biomolecule’s structure. Instead, thiol-containing spacers could be introduced on the biomolecules.

A strategy to achieve this on biomolecules could be to extend the lysine side chain amine group using N-(trifluoroacetylcaproyloxy) succinimide ester (TFCS) before reaction with SPDP.

Surface derivatization could also be achieved using a mixture of a long chain thiol-containing silane and a shorter alkylsilane, requiring the readjustment of the silanization method to this new situation.

8.1.2. Surface regenerationThe requirement of an optimum activation to 100% thiolsulfinates could in principle be achieved, but the difficulty resides in the extremely narrow range of potentials at which it occurs. In two-electrode set-ups in particular, the probability of success is low because of the drift in potential.

8.1.3. Alternative surfaces This process can be performed only if the surfaces to pattern are (semi)conductive. Therefore, it could be extended to (semi)conductive organic polymers such as carbon black doped polyethylene [96] or triphenylamine doped polycarbonates [97].

43

8.2. Biological considerations This work was mainly focused on the study of the surface chemistry involved in the immobilization process, but a lot remains to be done concerning the stability and activity of the immobilized biomolecules. Studies performed on chemical oxidation generated thiolsulfinates/ thiolsulfonates on agarose gels indicate a higher activity of immobilized -galactosidase is retained compared to other coupling chemistries [98,99]. Nevertheless, the negatively charged silicon oxide does certainly act on the microenvironment, hence influencing the behaviour of the immobilized molecules.

8.3. Applications The versatile method for surface patterning described in this thesis can find applications in all fields requiring immobilization of molecules. Sequential or simultaneous activation of surface areas is possible to achieve and allow immobilization of different types of biomolecules. Nowadays, this is especially interesting for the fabrication of biosensors and patterning of surfaces. In the future, with further development of nanotechnology, this method can be used to control the nanoscale positioning of different units during manufacturing of nanodevices.

44

Acknowledgements

First, I want to thank the people who supervised my PhD studies and those who have been directly collaborating on my research projects: I am grateful to Prof. Sven Oscarsson, my supervisor, and to Prof. Karin Caldwell, the director of the Dept. of Surface Biotechnology (Biomedical Center, Uppsala University), for accepting me as a PhD student in their research group and for giving me support during all the years I have spent in Sweden. I want to thank Prof. Ulrik Gelius, my “non-official” supervisor, for all the help and priceless advices he gave me throughout my studies. Thanks also to Dr. Leif Nyholm and to Angelo Pallin for being such valuable collaborators. Special thanks to Prof. Jan Carlsson for fruitful discussions and help with chemicals. Finally, I am grateful to Prof. Nadir T. Mrabet, my teacher during my undergraduate studies at Henri Poincaré University (Nancy, France) for being the initiator of my connection to the Dept. of Surface Biotechnology.

Second, my moving to the Ångström Laboratories (Uppsala University) was a decisive turn in my PhD studies and therefore, I would like to thank all the people at the Dept. of Physics, and especially the department director, Prof. Hans Siegbahn, for accepting me as a guest researcher and for being so helpful and friendly. Special thanks to Dr. Carla Puglia and her students for allowing me to participate to their project.

Third, I want to thank the staff of the Ångström Laboratories clean room, and especially Rein Kalm, for always being here to help. I would also like to thank the administration, computer support and library staff of the Depts. of Surface Biotechnology and Physics, and the Dept. of Biology and Chemical Engineering (Mälardalen University). I am grateful to Dr. Per Kårsnäs, the director of the Dept. of Biology and Chemical Engineering, for his unconditional support. Special thanks go to Ulla Widenberg, the secretary of the Dept. of Surface Biotechnology, for her help in solving daily administrative problems.

Finally, I want to thank all the members, and especially my fellow graduate students, of the Depts. of Biology and Chemical Engineering, Surface Biotechnology and Physics, for the time we spent together.

To end on private matters, I want to thank my friends in Sweden for being here, and my family and friends around the world for keeping in touch… A special thank-you to my parents, who made me who I am. And last but not

45

least, I am grateful to my partner at work as in life, Arjan Quist, for the help and support he gave me during the last four years and for the essential part he plays in my life.

Uppsala, August 7, 2003

Roam abroad in the world, and take thy fill of its enjoyments before the day shall come when thou must quit it for good.

Saadi (1184 - 1291)

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Acta Universitatis UpsaliensisComprehensive Summaries of Uppsala Dissertations

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ISSN 1104-232XISBN 91-554-5691-X

A doctoral dissertation from the Faculty of Science and Technology, UppsalaUniversity, is usually a summary of a number of papers. A few copies of thecomplete dissertation are kept at major Swedish research libraries, while thesummary alone is distributed internationally through the series ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Science and Technology.(Prior to October, 1993, the series was published under the title “ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Science”.)