Preparation and Modification of Thin Carbon Films
by
Callie Reynolds Massey-Reed Fairman
A thesis presented in fulfilment Of the requirements for the degree of Doctor of Philosophy
School of Chemistry The University of New South Wales Sydney, Australia
2010
CERTIFICATE OF ORIGINALITY
‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged.’
Signature ……………………………….
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COPYRIGHT AND DAI STATEMENT
‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.
I authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International.
I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.’
SIGNATURE: ……………………………….
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ACKNOWLEDGEMENTS
I am very grateful for the help I have received through out my PhD., not just from my supervisors and group members but from the school of chemistry, my friends, and most importantly my family.
Firstly I would like to thank my supervisors Justin and Brynn, for not only allowing me to undertake a PhD with them, but also for their guidance and support through out my studies. They have given me opportunities that I am grateful for. I especially would like to thank Justin, for his patience, and helping me to grow as a chemist and a researcher.
I would also like to thank the members of the biosensors and biodevices group, both past and present. You have all helped me grow as a researcher, ideas and thought’s are all appreciated. In particular I would like to thank Gouzhen Liu for helping me when I first started with the group, for putting up with constantly asking which wires were which. For Michael Jones, Paul Eggers and Paulo Da Silva for your guidance in all things synthetic. Michael and Paulo, I followed you from honours to my PhD, I enjoyed our coffee breaks, and the endless jokes. In particular from the echem group, Kate, Leo, Pauline, Lyn, Steven Yannolatuos, Josh P., Guozhen, Alicia, Kris, Till, and Erwann, I have had fun working you. The electrochem lab never seemed brighter! Kate, thank you for all your help, fun and guidance in the lab.
Thank you to all the staff in chemistry, you have all made my student life at UNSW such a great experience. From Ian and Paul down in the store and work shop to Anne in the office and all the academic staff. A special thanks goes to SOCS past and present, thank you for the fun nights, the Friday night drinks, the balls, and the general tom foolery had.
For the five weeks that I spent in Paris at ITODYS, Paris 7 university, thank you for letting me work with you, I had so much fun and learnt some very handy new skills. Thank you Michel Delamar, Vincent Noël, and Greg Marche.
I don’t think I wouldn’t have made it so far with all the ups and downs with out my friends. Kate, Leo, Nick P., Kasey, Sam, Sabrina, Paulo, Doug, Maggie, Pauline, Steven R.D.G., Matt, Bin, Oanh, Alisdair, Honman, Erica, Yeng, Sandra, Brad, Jason and Jason, Laurent, Michael, Trung, Lyn, Guozhen, Tom and all the lunch time people. You have listened to endless accounts of broken machinery, and missing parts. It has have been great fun to hang out with both in chemistry and outside as well. My friends out side of chemistry have also been
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a great support, Ken, Jane, Nick, Nep, Heidi, Liti, Simi, Emily, and all my church family. A special thanks goes to Samantha Furfari and Kasey Wood, thank you for the girl’s nights out, the coffees and lunches.
A special thanks goes to Kate, you have been such a great friend. You have put up with my ups and downs, helped me with problems, read my thesis with gigantic sentences. I am glad that I under took my PhD. just to have found such a great friend. One day we will write that book on indoor tents.
My family, you have supported me through out my studies. Mum, thank you for proof reading my thesis, thank you for the dinners and shopping trips, the early morning phone calls. Mum, Dad and Sean your support through out my PhD. is heartfelt, thank you for believing in me, and for your wisdom and guidance through out all my life. Thank you goes to Jeff’s family as well, for their support and love. Jeff, my wonderful, fantastic, husband; you have supported me emotionally and financially. I promise this is it, no more study, well not for a very long time. You have kept me grounded, you have put up with my early morning starts of 5 am in the lab, the late night finishes, the Saturdays in the lab, and the odd Sunday, the running late for dinners, and the lack of a wife for the last two months. You have even been there to help me empty tubs of water, when our lab was flooding on a weekend. Words are not enough.
Finally, I need to thank Teflon tape, rubber bungs and God. My PhD. would not have function with out any of these.
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ABSTRACT
Thin carbon films, in the form of pyrolysed photoresist films (PPF), are being increasingly used for electrochemical devices. This research investigates the preparation and subsequent modification of PPF through both the electrochemical reduction of aryl diazonium salts and through the UV attachment of alkenes to the iodinated surface.
The pyrolysis of photoresist films produces PPF that is a smooth (rms < 0.5 nm), amorphous carbon film that can be used for further electrochemical studies. The pyrolysis method was investigated to see the affect of holding the pyrolysis at three different temperatures during the pyrolysis and the corresponding times for which they were held; the effect of gas flow on the PPF was also addressed. It was seen that the gas flow whilst not affecting the roughness of the surfaces, the electrochemical cleanliness towards ferricyanide was affected, whilst different temperatures during the pyrolysis and the length of time they held was also seen to have some affect on both the electrochemical performance as well.
The modification of PPF was carried out with aryl diazonium derivatives of oligo(ethylene glycols) (OEGs). The protein resistance of these modified surfaces were also investigated and compared to the equivalent gold modified surfaces, both the effect of the chain length of the OEG as well as the change in hydrophilicity of the distal end was investigated. For a comparison OEG thiol modified gold surfaces were used. A Fluorescein isothiocyanate labelled protein, bovine serum albumin, was used and two methods employed to study the protein resistance. These methods were the elution of adsorbed protein from surface and the measurement of the protein whilst on the surface using fluorescence microscopy.
The use of iodine plasma to modify the PPF produces a surface similar to that of both hydrosilysed silicon and hydrogenated diamond surfaces. The use of UV light at 514 nm to activate the surface and attach alkenes was employed. UV addition of alkenes to the surface allows patterning as was observed via the modification of the iodinated carbon film with undec-10-enyl-2,2,2-trifluoroethanethioate on areas exposed
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to light and the deprotection of the thiol which was then exposed to gold nano particles and surface patterning was investigated with SEM. This will allow the production of nanoarray sensors in the future.
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LIST OF PUBLICATIONS
C. Fairman, S.S.C. Yu, G. Liu, A.J. Downard, D.B. Hibbert, and J.J. Gooding, 2008, Exploration of Variables in the Fabrication of Pyrolysed Photoresist, Journal of Solid State Electrochemistry 12: 1357-1365.
C. Fairman, G. Liu, D.B. Hibbert, J.J. Gooding, 2011, Comparing the electrochemical performance of pyrolysed photoresist film electrodes to glassy carbon electrodes for sensing applications, ICONN 2010 proceedings, accepted.
J. S. Quinton, A. Deslandes, A. Barlow, J.G. Shapter, C. Fairman, J.J. Gooding, and D.B. Hibbert, 2008, RF Plasma Functionalized Carbon Surfaces for Supporting Sensor Architectures, Current Applied Physics 8: 376-379.
A. Deslandes, M. Jasieniak, M. Ionescu, J.G. Shapter, C. Fairman, J.J. Gooding, D.B. Hibbert, and J.S. Quinton, ToF-SIMS PCA characterisation of methane and hydrogen plasma modified HOPG, Surface and Interface Analysis, in press.
S. M. Khor, G. Liu, C. Fairman, S. G. Iyengar, J. J. Gooding, 2010, The Importance of Interfacial Design for the Sensitivity of A Label-Free Electrochemical Immuno-Biosensor for Small Organic Molecules, Biosensors and Bioelectronics, in press.
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TABLE OF CONTENTS
Title Page...... i Certificate of Originality ...... ii Copyright and DAI Statement ...... iii Acknowledgements ...... iv Abstract...... vi Publications...... viii Table of Contents ...... ix List of Abbreviations...... xvi
CHAPTER ONE – GENERAL INTRODUCTION
1.1 INTRODUCTION...... 2
1.2 BIOSENSORS ...... 3
1.2.1 TRANSDUCERS...... 4
1.2.1 CATALYTIC BIOSENSORS...... 5
1.2.2 AFFINITY SENSORS...... 8
1.3 FACTORS IMPORTANT FOR AFFINITY BIOSENSORS ...... 12
1.3.1 ELECTRODE SURFACE STRUCTURE...... 12
1.3.2 BIOSENSING INTERFACE ...... 14 1.3.2.1 Protein Resistance ...... 15 1.3.2.2 Binding of the Affinity Biosensor Interface...... 17
1.3 CARBON AS AN ELECTRODE MATERIAL ...... 19
1.3.1 CARBON PASTE ...... 19
1.3.2 HIGHLY ORDERED PYROLYTIC GRAPHITE ...... 19
1.3.3 DIAMOND-LIKE CARBON...... 22
1.3.4 HYDROGENATED AMORPHOUS CARBON...... 23
1.3.5 GLASSY CARBON ...... 24
1.3.5 THIN CARBON FILMS ...... 26
1.4 MODIFICATION OF CARBON ...... 28
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1.4.1 POLYMERS AND MEMBRANES ...... 28
1.4.2 HEATING AND OXIDATION OF CARBON SURFACES ...... 31
1.4.3 AMINES AND ARYLACETATES ...... 32
1.4.4 MODIFICATION OF CARBON SURFACES BY ARYL DIAZONIUM SALTS...... 35
1.4.5 HYDROSILYLATION, HYDROGENATION AND THE ATTACHMENT OF ALKENES...... 39
1.5 WHICH CARBON AND WHICH MODIFICATION METHODS TO USE?...... 42
1.6 REFERENCES ...... 43
CHAPTER TWO – EXPERIMENTAL METHOD
2.1 MATERIALS ...... 64
2.1.1 CHEMICALS AND REAGENTS ...... 64
2.1.2 PREPARATION OF BUFFERS AND STANDARD SOLUTIONS ...... 68
2.2 INSTRUMENTATION ...... 69
2.2.1 ANALYSIS ...... 69 2.2.1.1 Synthetic analysis ...... 69 2.2.1.1.1 NMR...... 69 2.2.1.2 Surface Analysis...... 70 2.2.1.2.1 Electrochemistry...... 70 2.2.1.2.1.1 Cyclic Voltammetry ...... 72 2.2.1.2.1.2 Osteryoung Square Wave Voltammetry...... 73 2.2.1.2.2 Contact Angle ...... 74 2.2.1.2.3 X-ray Photoelectron Spectroscopy...... 76 2.2.1.2.3 Scanning Electron Microscopy...... 76 2.2.1.2.4 Atomic Force Microscopy ...... 77 2.2.1.2.5 Fluorescence Micrpscopy ...... 78
2.2.2 SURFACE PREPARATION ...... 79 2.2.2.1 Spincoater ...... 79 2.2.2.2 Oven...... 79 2.2.2.3 Tube Furnace ...... 79 2.2.2.4 Plasma Chamber ...... 80
2.3 PREPARATION OF ELECTRODES AND ANALYSIS ...... 81
2.3.1 Preparation of Electrodes ...... 81
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2.3.1.1 General Procedure for PPF Preparation ...... 81 2.3.1.2 Glassy Carbon ...... 82
2.3.2 ANALYSIS OF SURFACES...... 82 2.3.2.1 Electrochemical measurements ...... 82
2.4 REFERENCES ...... 84
CHAPTER THREE - THE EXPLORATION OF VARIABLES IN THE
FABRICATION OF PYROLYSED PHOTORESIST
3.1 INTRODUCTION...... 87
3.2 EXPERIMENTAL METHOD ...... 90
3.2.1 PPF PREPARATION ...... 90 3.2.2 EXPERIMENTAL DESIGN ...... 92 3.2.3 PHYSICAL CHARACTERISATION OF FILMS...... 94 3.2.4 ELECTROCHEMICAL CHARACTERISATION...... 95 3.2.4.1 Glassy Carbon ...... 95 3.2.4.2 Electron Transfer Rates ...... 96 3.2.5 SYNTHESIS OF 4-CARBOXYPHENYL DIAZONIUM TETRAFLUOROBORATE...... 97 3.2.6 MODIFICATION OF ELECTRODES...... 98 3.6.2.1 Electrochemical Reduction of 4-Carboxyphenyl Diazonium Tetrafluoroborate...... 98 3.2.6.2 Attachment of Peptide to Surface ...... 98 3.2.6.3 Copper Accumulation ...... 99
3.3 RESULTS AND DISCUSSION ...... 100
3.3.1 CHOICE OF FACTOR VALUES...... 101 3.3.3 UNCERTAINTY IN PREPARATION AND MEASUREMENT ...... 106
3.3.4 PHYSICAL CHARACTERISTICS OF PPF SAMPLES...... 107 3.3.4.1 Resistivity ...... 107 3.3.4.2 Film Thickness ...... 108 3.3.4.3 Roughness ...... 109 3.3.5 ELECTROCHEMICAL CHARACTERISTICS OF PPF SAMPLES ...... 110 3.3.6 ELECTROCHEMICAL USABILITY AND FUNCTIONALITY...... 114 3.3.6.1 Complexation of Copper(II) with the Peptide Gly-Gly-His Bound to a Carbon Substrate...... 116
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3.3.6.2 Complexation of Copper (II) to Gly-Gly-His...... 118
3.4 CONCLUSION ...... 121
3.5 REFERENCES ...... 123
CHAPTER FOUR - PROTEIN RESISTANCE OF OLIGO(ETHYLENE GLYCOL)
ARYL DIAZONIUM DERIVATIVES
4.1 INTRODUCTION...... 128
4.1.1 ZWITTERIONIC SPECIES...... 129 4.1.2 POLYMERS ...... 130 4.1.3 OLIGO ETHYLENE GLYCOLS ...... 132
4.2 EXPERIMENTAL METHOD ...... 137
4.2.1 GENERAL SYNTHESIS OF ARYL DIAZONIUM DERIVATIVES OF ETHYLENE GLYCOL .... 137 4.2.1.1 Synthesis of 2-(2-(2-(4-nitro-phenoxy)-ethoxy)-ethoxy)-ethanol...... 138 4.2.1.2 Tri(ethoxy-(ethoxy(ethoxy))) aniline ...... 138 4.2.1.3 Synthesis of 4-(2-(2-(2-hydroxy-ethoxy)ethoxy)ethoxy) bezenediazonium
(OEG3OH)...... 139 4.2.1.4 Nitrobenzene Decanol ...... 140 4.2.1.5 4-(decyloxy)aniline...... 141 4.2.1.6 1-(decyloxy)-4-Aryl diazonium tetrafluoroborate (Decane derivative)...... 141 4.2.1.7 Monomethylether(ethoxy-(ethoxy(ethoxy))) Nitrobenzene ...... 142 4.2.1.8 Monomethylether(ethoxy-(ethoxy(ethoxy))) Aniline...... 143 4.2.1.9 Mono methylether(ethoxy-(ethoxy(ethoxy))) Aryl Diazonium Tetrafluoroborate
(OEG3OMe) ...... 143 4.2.1.10 Hexaethylene Glycol Nitrobenzene ...... 144 4.2.1.11 Hexaethylene Glycol Aniline...... 145
4.2.1.12 Hexaethylene Glycol Aryl Diazonium Tetrafluoroborate (OEG6OH) ...... 146 4.2.1.13 Monomethylether Hexaethylene Glycol Nitrobenzene...... 146 4.2.1.14 Monomethylether Hexaethylene Glycol Aniline...... 147 4.2.1.15 Monomethylether Hexaethylene Glycol Aryl Diazonium Tetrafluoroborate
(OEG6OMe) ...... 148 4.2.2 MODIFICATION OF SURFACES ...... 149 4.2.2.1 Preparation of Surfaces ...... 149
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4.2.2.1.1 Pyrolysed Photoresist Films...... 149 4.2.2.1.2 Gold ...... 150 4.2.2.2 Thiol attachment to gold surfaces ...... 150 4.2.2.3 Premade Aryl Diazonium Attachment...... 151 4.2.2.4 Acetonitrile In situ Formation of Aryl Diazonium Salts and Attachment ...... 152 4.2.2.5 Aqueous In situ Formation of Aryl Diazonium Salts and Attachment ...... 152 4.2.2.6 Electrochemistry of modified surfaces ...... 153 4.2.3 NON-SPECIFIC PROTEIN ADSORPTION MEASUREMENTS...... 153 4.2.3.1 Elution of Protein from the surface ...... 153 4.2.3.2 Measurement of Protein on the Surface...... 154 4.2.3.3 Non-Specific Protein Adsorption with Relationship to Time...... 156 4.2.4 CONTACT ANGLE ...... 157
4.3 RESULTS AND DISCUSSION ...... 158
4.3.1 ELECTROCHEMISTRY OF MODIFIED SURFACES ...... 160 4.3.1.1 Attachment of Oligo(Ethylene) Glycols...... 161 4.3.1.2 Electrochemical Passivation of Modified Surfaces...... 164 4.3.2 CONTACT ANGLES ...... 166
4.3.3 EFFECT OF MODIFICATION METHOD ON RESISTANCE TO NON-SPECIFIC PROTEIN
ADSORPTION ...... 168 4.3.4 EXPOSURE TIME AND EFFECT ON NON-SPECIFIC PROTEIN ADSORPTION...... 170 4.3.5 RESISTANCE TO NON-SPECIFIC PROTEIN ADSORPTION ON PPF SURFACES ...... 172 4.3.5.1 Elution of Fluorescently Labelled Protein from the Surface ...... 172 4.3.5.2 Fluorescent Microscopy...... 174
4.4 CONCLUSION ...... 180
4.5 REFERENCES ...... 181
CHAPTER FIVE – IODINATION OF PPF AND ALKENE MODIFICATION
5.1 INTRODUCTION...... 190
5.1.1 COMBINING STABILITY OF COVALENT BONDS AND MONOLAYERS...... 190 5.1.1.1 Alkenes and Alkynes on Silicon ...... 190 5.1.1.2 Alkenes on Diamond...... 192
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5.1.2 IODINATED SURFACES ...... 194
5.2 EXPERIMENTAL METHODS...... 195
5.2.1 PREPARATION OF PPF...... 195 5.2.2 IODINATION OF PPF ...... 195 5.2.3 ALKENE AND ALKYNE ADDITION TO AN IODINATED PPF SURFACE ...... 196 5.2.4 ANALYSIS OF THE IODINATED AND UV REACTED SURFACES ...... 198 5.2.4.1 Contact angle...... 198 5.2.4.2 Electrochemical characterisation...... 198 5.2.4.3 X-ray Photoelectron Spectroscopy ...... 198 5.2.4.4 Atomic Force Microscopy ...... 199 5.2.4.5 “Click” Chemistry...... 199 5.2.4.5 Patterning of Iodinated PPF...... 199 5.2.4.5.1 Synthesis of Gold nano particles ...... 199 5.2.4.5.2 Patterning of the Iodinated PPF Surface with S-undec-10-enyl-2,2,2- trifluoroethanethioate...... 200
5.2.4.5.3 Deprotection of C11-S-TFA Modified Surfaces...... 201 5.2.4.5.4 Attaching Gold Nanoparticles to the Modified Surface...... 201 5.2.4.5.5 SEM Imaging ...... 201
5.3 RESULTS AND DISCUSSION ...... 201
5.3.1 IODINATION OF PYROLYSED PHOTORESIST FILMS ...... 202 5.3.1.1 XPS of Iodinated Surface ...... 203 5.3.1.4 Roughness of PPF After Iodination...... 206 5.3.1.2 Electrochemical Behaviour of the Surface...... 208 5.3.1.3 Hydrophobicity of Iodinated PPF ...... 215
5.3.2 MODIFICATION OF IODINATED PPF BY U.V. ACTIVATION ...... 217 5.3.2.1 Reaction of Iodinated PPF withUndecylenic Acid at 254 nm ...... 218 5.3.2.1.1 Electrochemistry of Iodinated PPF Exposed to Undecylenic Acid at U.V. Wavelength 254 nm ...... 219 5.3.2.1.2 Contact Angle of Iodinated PPF Exposed to Undecylenic Acid at U.V. Wavelength 254 nm ...... 220 5.3.2.2 Reaction of Iodinated PPF with Undecylenic Acid at 514 nm ...... 222 5.3.2.2.1 Contact Angle of Iodinated PPF Exposed to Undecylenic Acid at U.V. Wavelength 514 nm ...... 223 5.3.2.2.2 Electrochemistry of Iodinated PPF Exposed to Undecylenic Acid at U.V. Wavelength 514 nm ...... 224 5.3.3 MODIFICATION OF IODINATED PPF WITH ALKYNES ...... 234
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5.3.3.1 Modification with Nonadiyne and Use of “Click” Chemistry...... 234 5.3.4 PATTERNING OF IODINATED PPF ...... 238
5.3.4.1 Modification with C11-S-TFA...... 239 5.3.4.2 Patterning with Trifluoroacetyl Thioldecene...... 241
5.4 CONCLUSION ...... 243
5.5 REFERENCES ...... 244
CHAPTER SIX – CONCLUSION AND FUTURE WORK
6.1 SUMMARY...... 251
6.2 FUTURE WORK...... 255
6.2.1 FURTHER INVESTIGATING THE EFFECTS OF PLASMA IODINATION AND ALKENE/ALKYNE
ADDITION ...... 255 6.2.1.1 Photolithograpy...... 256 6.2.2 BIOLOGICAL APPLICATIONS ...... 257 6.2.2.1 Affinity Sensors ...... 257 6.2.2.2.1 Development of a Mutlianalyte Sensor...... 259
6.3 CONCLUDING REMARKS...... 260
6.4 REFERENCES ...... 261
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LIST OF ABBREVIATIONS
AFM Atomic Force Microsopy BSA-FITC Bovine Ablimun Serum Fluoroscene Isothiocyante Conjugate CV Cyclic Voltammetry DNA Deoxyribonucleic acid EDC 1-Ethyl-3-(3-dimethylamino)propyl) carbodiimide E Electrode potential EDTA Ethylenediaminetetraacetic acid FAD Flavine Adenine Dinucleotide Phosphate GC Glassy Carbon GOx Glucose Oxidase Gly-Gly-His Glycine-Glycine-Histidine MES 2-(N-morpholino)-ethanesulfonic acid mol Mole NHS N-Hydroxysuccinimide NMR Nuclear Magnetic Resonance OEG Oligo(Ethylene Glycol) OSWV Osteryoung Square Wave Voltametry PPF Pyrolysed Photoresist Films PQQ Pyrroloquinolino quinone RMS Root Mean Square SAM Self Assembled Monolayer SEM Scanning Electron Microscopy TEM Transmission Electron Microscopy U.V. Ultra Violet XPS X-ray Photoelectron Spectroscopy
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Chapter One
General Introduction
1.1 INTRODUCTION
The desire for quick results, and easy to use systems, to real time analysis on the body and the environment are very important in today’s society.1 Many systems are already in place that allow us to carry out these tests, but they require trained professionals and time. One such example is the ability to detect heavy metals in a water system. Generally this requires samples to be taken and sent off to a laboratory to be analysed by an inductively coupled plasma mass spectrometer (ICPMS) which requires sample preparation and a qualified trained technician to run the samples.2 This is where a small hand held device that is portable and quick would be ideal.3-4 Samples could be taken and directly analysed at the site without tedious sample preparation. The device would also have to be specific for the target analyte and be resistant to interferences from other species in the sample. These same features are also applicable for many medical tests that require a blood sample to be taken and sent to a pathology laboratory. Typically in medical tests the samples take a couple of days to prepare before the appropriate test can be conducted and the results determined.5 For both of these examples the use of “biosensors” would be applicable.3-4, 6-7
The aims of this thesis are to investigate new carbon surfaces as well as modification for use in affinity biosensors. The following chapter will discuss the back ground to biosensors in particular affinity biosensors, what aspects are important to the sensitivity of the sensors.
Chapter 1 | 2
1.2 BIOSENSORS
The general basis of a “biosensor” is one that has the ability to detect an analyte with a biological recognition element immobilized directly onto a signal transducer.6-9
The biological recognition element includes species such as enzymes, peptides,10 DNA fragments,11-13 or antibodies.14 The analyte reacts with the biological recognition element that is specific to the target molecule. This reaction is then converted to a signal by the transducer that is either physical or physiochemical (Fig. 1.1).6 The most common types of transducer mechanisms used are in the form of optical, acoustic, or electrochemical.3, 6-7
Figure 1.1 Generalised construct of a biosensor.9
Chapter 1 | 3
1.2.1 TRANSDUCERS
The transducer converts the information produced by the analyte reacting with the biorecognition element into an electronic signal that can then be read out by the end user.5, 9, 15
Piezoelectric materials, such as quartz, are frequently used for acoustic wave transducer systems where the frequency of vibration, when an oscillating circuit is applied to the piezoelectric material, is dependent on the amount of substance absorbed onto the surface of the material.9, 16-17 The change in vibrational frequency, as the analyte reacts with the biorecognition element, can be measured. Small changes in the frequency (0.1 Hz) correspond to mass and viscosity changes (10-10 - 10-11 g), though there is no distinction between specific adsorption and non-specific adsorption.15-18
For optical transducers, the reaction of the analyte with the biorecognition element causes a change in the optical characteristics.5, 9, 16, 19 This response can be measured with fluorescence,20 absorbance or reflectance.21-22 For example, optical biosensors using porous silicon, the detection of the analyte is reliant on the change in the wavelength of reflected light at the interface which is caused by the change in refractive index.23-25 The drawback for optical sensors is that the media that the sensor is being measured in, needs to have low turbidity or better still to be transparent to the wavelength of light being employed for the signal generation.
Electrochemical transducers can be used to measure the signal produced in various ways.9, 15, 26 Potentiometric and amperometric are two ways that the signal can
Chapter 1 | 4
be measured from the transducer. These methods require the transducer to be an electrode. In potentiometric methods the ion concentration is measured.27-28 An amperometric transducer measures the currents produced from the change in oxidation state which occurs as the analyte binds with the biorecognition element.29-31
For the rest of this discussion the focus will be on amperometric electrochemical transducers. The use of amperometric electrochemical biosensors allows individual target molecules to be measured directly on the electrode surface using biorecognition species attached the electrode surface. There are two different classifications of biosensors depending on the biorecognition species used. The two classifications are catalytic biosensors, and affinity biosensors.
1.2.1 CATALYTIC BIOSENSORS
A catalytic biosensor relies on the reaction of the analyte with the biorecognition element causing the catalytic conversion of the analyte from a non-detectable species to a detectable species.8 The most common form of a catalytic biosensor is one using enzymes.1, 30, 32-36 The enzyme does not only act as the catalyst but also as the biorecognition element.
There are several ways for immobilising the enzyme on the transducer/electrode surface. In some cases the immobilisation of the enzyme on the surface is by the coating of the surface with the biorecognition element and the catalyst.37-38 The coating of the surface is secured in place with a dialysis membrane, allowing the transfer of species through the membrane but not the other way around.32 Immobilisation of the
Chapter 1 | 5
biorecognition element and catalyst can also occur by inclusion in a polymer. The polymer can be formed through either electrochemical or chemical means.39
Electrochemical redox reactions cause polymer formation and the incorporation of the biorecognition element.26
Not all reactions of the analyte with the biorecognition element are able to produce a signal that is directly detected by the transducer/electrode. A mediator that can be easily oxidised or reduced maybe required. The redox reaction in the mediator occurs from the reaction of the analyte with the biorecognition element. Once the biorecognition element reacts with the analyte, the mediator undergoes subsequent redox reactions that can then be detected by the transducer.9, 32, 40 As the analyte reacts with the enzyme, the enzyme is reduced. This then causes the subsequent reduction of the mediator and the enzyme is oxidised back to its original state. The transducer/electrode detects the change in oxidation state of the mediator.
Perhaps the most successful and well known of these systems is the glucose sensor used by diabetics.1 A glucose sensor consists of an electrode surface, a mediator and glucose oxidase (Fig. 1.2).37, 41 The glucose is oxidised in the blood sample by glucose oxidase that is adsorbed onto the transducer. The reduced glucose oxidase
(GOx) is then in turn re-oxidised by the mediator, which is then oxidised by the electrode and produces a current and thus a measurement of the amount of glucose present is given.37 The mediator used can be either oxygen, as in Nature where hydrogen peroxide is produced, or in commercial glucose sensors the mediator used is ferrocene or ferricyanide.6, 42 As the glucose is oxidised there are electrons produced which in turn cause the reduction of the mediator, ferrocene or ferricyanide.37-38, 41-42
Chapter 1 | 6
The current produced from the oxidation of the mediator by the electrode is then transferred to an electrical signal that can be readout by the end user.
Figure 1.2 The construct of a glucose sensor used by diabetics, where the glucose is adsorbed
onto an electrode surface and the electrical signal is produced by the oxidation of glucose and
subsequent oxidation of GOx.32
This type of sensor, although the biorecognition element is specific for the analyte, is prone to interference. Non-specific oxidation/reduction of unwanted redox active molecules can cause interference. In the case of the glucose sensor, ascorbic acid as well as uric acid and molecular oxygen can cause interference. As the glucose is oxidised by the glucose oxidase, molecular oxygen in the sample can in turn be oxidised instead of the mediator thus not all the glucose is detected.33 By “electrically wiring” into the enzyme this interference can be reduced. Willner and co-workers attached an electrochemical relay, pyrroloquinolino quinone (PQQ), to the aminoethyl flavine adenine dinucleotide phosphate (amino-FAD) in the glucose oxidase (Fig. 1.3).30, 33 By reconstituting the glucose oxidase around the amino-FAD attached to a surface bound
PQQ, the electrons produced via the oxidation of glucose can be transported to the electrode transducer. Similar work has been carried out by Gooding and co-workers
Chapter 1 | 7
where norbornylogous bridges and carbon nanotubes were inserted into glucose oxidase and attached to the electrode surface.43
Figure 1.3 “electrical wiring” of glucose oxidase. The glucose oxidase is reconstituted around
FAD that is bound to the gold surface with pyrroloquinolino quinone. As the glucose is
oxidised, FAD is in turn oxidised and the electrons are passed down the “electrical wire” to the
PQQ and then to the surface.33
These catalytic biosensors though are still not interference free and it is difficult to detect small molecules using this technique.
1.2.2 AFFINITY SENSORS
An affinity biosensor uses species that are specific to the analyte whereby the reaction of the analyte with the biorecognition element causes a signal without further reactions.9, 15-16, 44 The use of an affinity sensor provides a broader range of analytes due to a larger amount of biorecognition elements that can be used. As the name suggests the biorecognition element and analyte can act in a receptor-ligand like fashion.5, 9, 15-16 Affinity sensors can be used to detect various analytes. The
Chapter 1 | 8
biorecognition element can be either antibodies, nucleic acids, DNA, receptor proteins and biomimetic materials.3, 5, 9, 29, 45-46
The use of the peptide Glycine-Glycine-Histidine (Gly-Gly-His) to detect copper in water has previously been documented by Gooding and co-workers.10, 29, 47-49 When the peptide is attached to the surface and not coordinated to copper it produces no electrochemistry, but when the peptide complexes with free copper (II) it brings the copper (II) close enough to the surface of the electrode so that is able to be reduced and oxidised, thus the amount of copper (II) can be detected (Fig 1.4).10, 47 The use of the peptide sequence (Gly-Gly-His) instead of cysteine by itself gives better selectivity towards copper and shows little interference from other heavy metals.47, 49 The ability to attach the peptide to the surface using an alkanethiol species allows free movement of the peptide to be maintained.10 Free movement is achieved by the ability of the gold surface to rearrange on the surface to give a monolayer with lowest energy allowing the monolayer to rearrange to give the peptide enough space to bind to the copper.49-52
O O O O OH OH O NH HN O N N Cu2+ N NH N N + Cu2+ N O N O H H
S S
Figure 1.4 Gly-Gly-His copper sensor construct on a gold substrate, showing no
electrochemical behaviour without copper and once bound to copper electrochemical behaviour
is seen.10, 47
Chapter 1 | 9
The main type of affinity biosensors use antibodies or antigens, due to their affinity for specific proteins and analytes, which also allows for a wider range of analytes to be detected. These affinity biosensors are classified as immunosensors.3, 15,
53 Antibodies are proteins related to the immune system and are produced by B- lymphocytes in animals, in response to a foreign substance otherwise known as an antigen.3 The general structure of an antibody can be represented by the class of antibodies known as IgG or γ-globulin. Antibodies have a molecular weight between
150,000 and 160,000 Daltons. They are Y shaped, with each arm consisting of a light chain and a heavy chain held together with a disulphide bridge (Fig. 1.5).3, 54
Figure 1.5 Basic structure of an antibody showing the different fragments. The Fc fragment is
3, 5, 55 the crystalline fragment. The F(ab) and F(ab’)2 are the antibody binding fragments.
The main problem with immunosensors is the transduction of the binding event.
Many electrochemical immunosensors use competitive binding or co-enzymes that
Chapter 1 | 10
undergo an oxidation change converting the event into a transducable signal. An example of an electrochemical immunosensor has been explored by Liu et al..14, 43, 56
The use of biotin attached to the surface enables the measurement of antibiotin. Unlike other methods though, the use of labels or competitive binding is not needed. Biotin is attached to the redox active molecule ferrocene, which is attached to the surface using a molecular wire (Fig. 1.6).43 The ferrocene is able to be oxidised and reduced, thus producing an electrochemical signal. When the antibiotin binds to the biotin, as it is large in comparison to the sensor construct it not only engulfs the biotin but also the ferrocene. Once the ferrocene is covered with the antibiotin it is no longer able to be oxidised or reduced due to the inability of counter ions to access the ferrocene. Thus a decrease in the electrochemical signal is observed.43
Figure 1.6 Antibiotin immunosensor. As the antibiotin binds to the surface bound biotin, it
blocks access of electrons to the ferrocene thus reducing the electrochemical signal produced.
The concentration of biotin can be correlated to the amount of signal reduction.43, 56
Chapter 1 | 11
The difference between affinity biosensors and catalytic biosensors is the ability to detect small molecules, with specific biorecognition species, and little interference.
1.3 FACTORS IMPORTANT FOR AFFINITY BIOSENSORS
The sensitivity, stability and selectivity of an affinity biosensor are important.
The transducer and its structure, and the biosensing interface can influence these factors. Selectivity relies on the ability of the surface to only bind the target analyte and that species alone.6-7 Sensitivity of the affinity biosensor not only relies on the ability of the biorecognition element to bind to the analyte but also on the ease to which the analyte can bind to the biorecognition element.57 The thermodynamics of the analyte binding with the biorecognition element can also be affected making the biosensor slower in response.58 The stability of the sensor requires the binding of the biorecognition element to be robust as well as the recognition element itself.9, 15, 59-60
1.3.1 ELECTRODE SURFACE STRUCTURE
The surface structure of the electrode is important and has the ability to affect the sensitivity of the affinity biosensor. The environment that the biorecognition element is in will affect the thermodynamics between it and the analyte. It is important that all the biorecognition elements on the surface are in the same environment to ensure similar thermodynamics between each and the analyte.3, 35, 58, 61-62 The loss of movement of the biorecognition element in some cases can affect both the binding kinetics and reaction thermodynamics.35, 57-58, 63-64
Chapter 1 | 12
The surface roughness of the electrode now becomes important to the sensitivity of the affinity biosensor.35, 57, 62 If the surface is rough, the biorecognition elements are not all available to react with the analyte.61 This in comparison to those on a smooth surface where all species are able to react thus allowing more recognition sites and increasing the amount of change that can be recorded and hence the sensitivity (Fig.
1.7). Having an affinity biosensor with a smoother surface will increase the sensitivity.57, 65-66 The ability to control the surface topography is required for both preparation of the surface and for the subsequent modification with the biorecognition element.57
Figure 1.7 a) The affect of a rough surface on the environment of the biorecognition element.
Not all biorecognition elements are able to be accessed by the analyte. b) The affect of a
smooth surface on the environment of the biorecognition element, allowing access to all
biorecognition elements and increasing sensitivity.
Eggers et al. have also investigated the effect of the distance of the redox probe from the electrode surface and its depth into the diffusion layer. It was shown that the distance the redox probe is into the diffusion layer could affect the electron transfer rate,
Chapter 1 | 13
the further it is into the diffusion layer the slower the electron transfer and the lower the formal potential.67-69 If an affinity biosensor similar to the one used by Gooding and co- workers is to be assembled the surface roughness is important as this will affect the sensitivity of the electrochemical signal produced.
The electrode surface that is to be investigated for the use of an affinity biosensor needs to have a surface with near atomic smoothness, this will allow for greater sensor sensitivity. By having a surface with near atomic smoothness will allow for the biorecognition element as well as the redox probe to be in the same environment over the entire electrode surface producing greater sensitivity in the affinity biosensor.
1.3.2 BIOSENSING INTERFACE
As affinity biosensors are to be used in complicated matrixes, the biorecognition layer needs to be bound to the surface to give a reagent less solid-state device. The method used to bind the biorecognition element to the surface needs to be stable towards oxidation in solution. It also needs to be stable for storage. Since many affinity biosensors will be used for biological samples, the interface also needs to resist adsorption of unwanted proteins and allow for fast thermodynamics between the analyte and the biorecognition element. The use of a diluent between the biorecognition elements allows space for freedom of movement for the binding of the biorecognition element and the analyte, thus giving rise to faster thermodynamics.35, 57-58, 63-64
Chapter 1 | 14
1.3.2.1 PROTEIN RESISTANCE
The exposure of the affinity biosensor surface to biological matrixes such as blood, requires the surface to only allow the binding of the wanted analyte to the biorecognition element. The use of a specific biorecognition element for the analyte will partly allow this, but the surface may be prone to non-specific protein adsorption.
There are several ways of combating this problem, and they all involve the diluting of the biorecognition element with a protein resistant diluent.70-75 The use of poly(ethylene glycols) to form a polymer on the surface with the biorecognition element is one such method.76-77 The molecular weight of the polymer can have an effect on the protein resistance.
The use of shorter single chains of poly(ethylene glycol) in the form of oligo(ethylene glycols) (OEG’s), allow for a more controlled formation of the protein resistant diluent.72, 78-82 Grunze and co-workers observed that for alkanethiol OEG’s on gold the length of the chain was an important factor as to the ability of the surface to resist non-specific protein adsorption.82 By changing the chain length from three OEG units (Fig. 1.8 b) to six OEG units (Fig. 1.8b) the amount of protein adsorbed onto the surface was decreased. The hydrophilicity of the distal end was also important. When the distal end was changed from a methoxy (Fig. 1.8c) to a hydroxy (Fig. 1.8a) the amount of non-specifically adsorbed protein was also shown to decrease.82-83
Chapter 1 | 15
HS O OH a O O
HS O O O b O O O OH
c HS O O O O
Figure 1.8 a) Alkanethiol OEG with three OEG units and a hydroxy distal end. b) Alkanethiol
OEG with six OEG units and a hydroxy distal end. c) Alkanethiol OEG with three OEG units
and a methoxy distal end.81, 84
The final common method for the reduction in non-specific protein adsorption is to use zwitterionic species on the surface (Fig 1.9a and 1.9b) or to make the surface zwitterionic like (Fig. 1.9c).71, 80, 85-89 By using a diluent that has both positively and negatively charged species on the surface the amount of non-specific protein adsorption has been observed to decrease.86, 88 The biosensor surface can also be modified to be zwitterionic like. This requires the use of two different diluents, one positively charged and the other negatively charged. This gives the surface an overall neutral charge at an appropriate pH and has been shown to also decrease the amount of protein adsorption.71,
86-87
a b c
+ + + +
- - - - - +
------
+ + + + + + + +
Figure 1.9 a) The modification of a surface with a zwitterionic species that has a negative
distal end and a positive centre. b) The modification of a surface with a zwitterionic species
that has a positive distal end and a negative centre. c) A modified surface with both positive
and negative species giving the surface a zwitterionic like character.
Chapter 1 | 16
By using a diluent that is able to resist non-specific protein the specificity of a affinity biosensor as well as the sensitivity would be increased. By reducing non- specific protein adsorption on the affinity biosensor it would allow the target molecule access to the biorecognition element. The use of zwitterionic surfaces though requires the pH of the matrix being tested to be at the right level so that both elements are positively charged and negatively charged at the same time. It is the hope that affinity biosensors in the future will be used with little to no sample preparation, using oligo(ethylene glycols) are the diluent to reduce non-specific protein adsorption requires no adjustment of the pH and thus will be further investigated.
1.3.2.2 BINDING OF THE AFFINITY BIOSENSOR INTERFACE
The two most common surfaces for electrochemical biosensor transducers are gold and carbon, though the use of silicon surfaces and other metals is an emerging field.90-91 Both gold and carbon surfaces are able to be modified. Gold can be modified with thiols to form a sulphur-carbon bond,59 and carbon can be modified in several ways including polymer layers,26 aryl diazonium salts,92-93 and even alkenes.94 All of these modification methods will be discussed below. The ideal surface for an electrochemical biosensor is one that has reliable electrochemistry; this requires a stable surface.95
The modification of gold is generally carried out by the use of alkanethiols which spontaneously absorb onto the surface of gold to form a gold-thiolate bond.96
The use of an alkanethiol modified gold surface allows the formation of a well understood monolayer, which can then be used for various electrochemical methods
Chapter 1 | 17
including that of electrochemical biosensors.45, 59, 97 It has been shown that longer alkanethiol chains form a more ordered monolayer with the alkyl chains positioned at an angle of 30° than layers formed of shorter chain lengths.98
There are several issues with using thiols for the modification of gold. Firstly they are able rearrange on the surface to give a monolayer with lowest energy.51-52 The gold-thiol bond is also prone to oxidation as it is highly polar, and has a narrow potential window of +0.8 V and -1.4 V.50, 99-100 Packing density, chain length and distal end group can affect the stability of the gold-thiol. Shorter chain lengths can reduce this potential window to -0.4 V to +0.6 V. The monolayers are also liable to desorb at elevated temperatures.96 Alkanethiol modified gold surfaces are also unstable to sulfonates and sulfinates, and are oxidised with UV light.101-102 These instability issues can be overcome for storage by sealing in silver foil to protect it from light. The stability issues still come into play in the modification process as for some sensors there are multiple steps each with its own liquid environment.95, 103 These stability issues in the use of gold for electrochemical biosensors give rise to the search for another viable surface to be used.
The use of carbon, as previously mentioned, is another highly used surface for electrochemical biosensors. There are many carbon surfaces including carbon paste, highly ordered pyrolytic graphite (HOPG), diamond like films, glassy carbon and thin carbon films. Is there a carbon surface that provides better stability and good electrochemical properties?
Chapter 1 | 18
1.3 CARBON AS AN ELECTRODE MATERIAL
1.3.1 CARBON PASTE
Carbon paste electrodes are as their name indicates made of carbon mixed with a liquid that has low water solubility and low volatility.104-105 These are cheap, easy to construct electrodes. They react well electrochemically, but are extremely rough and not stable over long periods of time.105 The media in which the carbon is mixed can affect the ability of the electrode to perform. This can be seen in work by Lindquist,105 where several different carbon paste electrodes were made and their performance was tested. On glassy carbon electrode the reduction of hydroquinone is irreversible.
Lindquist showed that a carbon paste electrode made from 1-bromonaphthalene produced irreversible voltammograms with hydroquinone.105 Carbon paste electrodes made with either paraffin oil, silicone fluid or decahydronapthalene all gave good reversible peaks for hydroquinone.
These electrodes though do not provide a smooth enough surface for an affinity biosensors. The biorecognition element is also only loosely bound, which makes long exposure time of the sensor in media difficult.
1.3.2 HIGHLY ORDERED PYROLYTIC GRAPHITE
Highly ordered pyrolytic graphite (HOPG) can be atomically smooth, but due to its structure of edge and basal planes the electrochemical response towards many redox active complexes can be affected.106-107 Basal plane HOPG is generally used for STM
Chapter 1 | 19
imaging as it is atomically flat carbon with few or no defects.108 HOPG is produced by using high temperature decomposition of gaseous hydrocarbons and then applying pressure at high temperatures. The common gaseous hydrocarbons used are acetylene or methane gas.109-110 The higher the temperature used during formation, the more ordered and parallel the graphitic layers become.111 The use of pressure during the annealing process removes “ripples” in the basal plane and removes impurities in the graphite sheet, thus forming a near ideal graphite.112 HOPG is made of edge and basal planes. The basal plane is atomically smooth with variances due to step edges. The edge planes are rough,113 and functionalised with oxygen. During polishing of the edge planes unsatisfied valances are formed due to bonds breaking and they are able to react with oxygen and water to form oxygen containing functional groups.
It has been seen that edge and basal planes behave differently with regards to electron transfer. Both inner sphere and outer sphere redox systems, such as ferricyanide and ruthenium hexamine respectively, behave the same way on basal plane
HOPG. Slow electron transfer rates of ruthenium hexamine and ferricyanide on basal planes, indicate that it is not surface impurities that hinder the process but the surface itself.114 It has been seen that electron transfer on the basal plane can be improved by both electrochemical and laser activation of the surface. These processes increase the edge defects of the surface.106, 108, 115-116 The surface of basal plane HOPG is low in oxygen and the cleaning of these impurities from this surface does not dramatically increase the surface kinetics. However, increasing the amount of oxygen on edge planes has been shown to slow the electron transfer rate due to the linking of the graphite sheets.117
Chapter 1 | 20
HOPG is a semimetal in nature, from Raman spectroscopy the 1360/1582 cm-1 peak ratio (D/G) can indicate how ordered the carbon structure is. Basal plane HOPG is highly ordered with a ratio of D to G band value close to zero (0.0005), whilst edge plane HOPG is highly disordered (1.1). The capacitance of basal order HOPG is low and linked with the semimetal nature of the material. Edge plane HOPG shows high reactivity towards electron transfer and adsorption in comparison to basal plane HOPG that has low electron transfer rates and minimum adsorption although the bulk structure is the same Fig 1.10.106
Figure 1.10 Bulk configuration of HOPG showing edge and basal planes.106, 108, 115
The use of HOPG as an surface for mass produced electrodes is undesirable due to the difficulty of mass production and cost. This fact combined with both slow electron transport for basal planes and rough surfaces for edge planes makes another carbon alternative sought after.
Chapter 1 | 21
1.3.3 DIAMOND-LIKE CARBON
The use of diamond as a carbon electrode substrate is a new development.
Diamond like carbon film is made from the deposition in plasma of methane and hydrogen gas mix, which forms diamond like films with polycrystalline structure or microcrystals where the carbons are linked in a tetrahedral construct with sp3 hybridisation.118
Diamond by itself though is non-conducting and needs to be doped during the formation process with boron to provide conductance through the substrate. From here boron doped diamond like films (BDD) can be used for electrochemistry by further modification with alkenes after the surface has been modified. BDD provides a wide working potential window and a low background.119 The BDD diamond is comprised of diamond like micro crystalline structures. Hence the structure is not the pure sp3 structure but can contain sp2 as well due to the inclusion of boron.120-121
Diamond surfaces have been used to further investigate non-specific protein resistance of oligo(ethylene glycols) for use in biosensors. The modification of the diamond surface was carried out with alkenes via activation of the surface with UV light, as will be discussed later in chapter 1 section 1.4.5. The modified surfaces produced were able to reduce non-specific protein adsorption similar to that of oligo(ethylene glycol) thiol modified gold surfaces.72, 122 The diamond surfaces were also modified with amine terminated alkenes which were used to further modify the surface with sulfosuccinimidyl-6'-(biotinamido)-6-hexamidohexanoate (NHS-SS-
Biotin). This allowed the diamond to be modified with biotin, by exposing the surface
Chapter 1 | 22
to fluorescently labelled avidin, the binding of the avidin to biotin modified surfaces and oligo(ethylene glycol) modified surfaces was measured using fluorescent microscopy.72, 94, 122 Diamond surfaces have also been used to bind DNA to the surface.90-91
Further study into the use of this surface as a biosensor substrate is still required.
Although BDD is electrochemically conducting the surface roughness though, is not near atomically smooth which is wanted for an affinity biosensor. Is there a smoother electrochemically conducting carbon surface that can be used?
1.3.4 HYDROGENATED AMORPHOUS CARBON
Amorphous carbon is a mixture of both sp2 and sp3 carbon, and it is highly disordered. Hydrogenated amorphous carbon (a-C:H) films can be formed using either microwave plasma chemical vapour deposition (MW-PCVD) or plasma enhanced chemical vapour deposition (PE-CVD) of acetylene and hydrogen gas. The structure of the a-C:H can vary on the flow rate of hydrogen into the CVD chamber. As the ratio of hydrogen to acetylene is increased the amount of sp3 configured carbon also increases in comparison to sp2 configured carbon.123 The surfaces produced are similar to diamond like films as both are non-polar and similar to basal plane HOPG.124 The surfaces produced are stable in air, and are slower to oxidise than that of polished glassy carbon.125 Roughening of the surface occurs during formation of a-C:H and root mean square (rms) values range from 5 nm to 120 nm.123 The roughness depends on the acetylene to hydrogen ratio; ratios of 15:150 and 15:270 (acetylene to hydrogen respectively) give surfaces with low rms values due to the formation of more sp3 bonds
Chapter 1 | 23
and the structure is more dense. Ratios on either side of this have higher rms values for low acetylene to hydrogen ratios more sp2 bonds are formed and at higher ratios there is polymeric carbon due to excess hydrogen.123
Similarly to diamond films and BDD, a-C:H films can be modified through the use of alkenes and UV light. Hamers and co-workers have also functionalised a-C:H films with alkenes and further modified with s-DNA. The modified surfaces were then hybridised with DNA.126
For the improvement of sensitivity of affinity biosensors surface roughness needs to be minimised, as discussed earlier a-C:H have a wide range of surface roughness (rms of 5 nm to 120 nm)123, this makes a-C:H not smooth enough for the use of an affinity sensor if surface sensitivity is to be improved.
1.3.5 GLASSY CARBON
Glassy carbon (GC) is commonly used as an electrode material in electrochemical sensors as it has a wide potential window127-129 and is a very stable material under a wide range of conditions.130 GC is a good electrode material for most redox reactions and can be modified by the reduction of various compounds, as will be discussed later. The behaviour towards outer sphere redox species131 produces good electrochemistry, though the behaviour of inner sphere redox species can vary depending on the preparation methods used.132
Chapter 1 | 24
GC is made from the high heat treatment of phenolic resins in which intermolecular cross linking of the hydroxyl groups on the phenolic groups and methylene groups, with the release of water. This gives the resulting glassy carbon domains of graphite in an amorphous phase consisting of entangled aromatic ribbons that are crossed linked with sp3 bonding (Fig. 1.11).133-135
Figure 1.11 Generalised structural model of GC showing graphitic ribbon stacks.134
The elimination of water occurs between the temperatures of 350 °C and 500 °C forming the aromatic ribbons, though they are not perfect and are randomly orientated.134 At 500 °C to 1500 °C hydrogen is released and the ribbons stack closer together and cross linking occurs.134, 136 At higher than 1500 °C graphitisation occurs at these temperatures the aromatic ribbons start to flatten and the boundaries between the ribbons become more localised.136-137 However true graphitisation cannot occur as the aromatic ribbons are too long and would need to break in order for this to occur.
GC has been used for several biosensors as shown (section 1.2.2) previously the work by Liu et al.43, 56 shows the use of glassy carbon in the construct of an affinity
Chapter 1 | 25
biosensor through the modification technique of the electrochemical reduction of aryl diazonium salts (this will be discussed later in chapter 1 section 1.4.4).14, 56 The affinity biosensor worked through the use of biotin attached to ferrocene which in turn is bound to the GC electrode surface. The biosensor gives and electrochemical signal due to the electrochemical reduction and oxidation of ferrocene. As anitbiotin binds to the biotin it blocks the access of electrons to the ferrocene, thus the signal is reduced depending on the amount of antibiotin in the sample. Glassy carbon has also been modified with oligo(ethylene glycols) and oligo(monomethyl ethers) to investigate non-specific protein adsorption. Both Maeda et al.138 and Downard and co-workers139 have shown that the use of these molecules on the surface has the ability to reduce non-specific protein adsorption in comparison to bare surfaces.138-139
The drawback with glassy carbon is that the surface of the final material is rough on the molecular level. This roughness has implications for modern trends in sensing where molecular level control over the modification of an electrode surface is important. Therefore, if the advantages of GC are to be exploited over as wide a range of potential applications as possible there is a need for smoother surfaces to allow molecular level control of the surface modification, for high resolution patterning, and for AFM investigation into the layers formed by various surface modification techniques.
1.3.5 THIN CARBON FILMS
An alternative to GC is thin carbon films.140-142 Previous studies by McCreery and co-workers143-145 and the Kinoshita group146-149 have shown that thin carbon films
Chapter 1 | 26
can be formed by the pyrolysis of photoresist films. The thin carbon films produced give smoother carbon surfaces than are achievable with conventional methods of fabricating GC (with a rms value less than 0.5 nm compared with 4.5 - 44 nm for
GC).143-147 In common with surfaces of GC, films formed from this process are mainly amorphous with graphitic regions. This can be seen by the presence of the relative D and G bands in the Raman spectra around 1360 cm-1 and 1600 cm-1. These two bands can give an idea as to how much disorder is present in the structure.145
For the formation of thin carbon films a thin film of photoresist is laid down on a substrate and then pyrolysed in a tube furnace in a reducing atmosphere to give a conducting carbon film.143 The attractiveness of pyrolysed photoresist films (PPF) is that it not only provides a much smoother surface but also has similar electrochemistry to that of GC. Furthermore it is compatible with bulk manufacture of a range of conducting patterned carbon structures.144, 146-147 PPF has also been shown to have low oxygen content (the atomic percent ratios from x-ray photoelectron spectroscopy the ratios of oxygen to carbon are approximately 0.05) for films pyrolysed at temperatures greater than 700 ºC and they are slow to be oxidised in ambient atmospheres. The low
O/C ratio is important if these materials are to be used as micro electrodes and micro batteries as the O/C ratio influences the electron transfer rate of some redox probes.140,
144
The use of PPF as an electrode surface for affinity biosensors will be discussed later in chapter 3 section 3.3.6.2.
Chapter 1 | 27
1.4 MODIFICATION OF CARBON
Carbon surfaces, as previously mentioned, can be modified in several ways;
1. Through the use of polymers on the surface.
2. Through the production of radicals on the surface, via heat and vacuum.
3. Making the surface reactive with carboxylic acids and amines, the
surface can be subsequently modified.
4. Using electrochemical modification to produce stable covalently bound
layers.
5. Recent progress into the use of alkenes has been shown to modify
diamond-like surfaces.
1.4.1 POLYMERS AND MEMBRANES
Polymer layers on the surface of carbon use the ability to combine the conducting properties of certain polymers and the ease of incorporating not only recognition elements, but several wanted functionalities such as protein resistance and specification.150-151 Polymers can be used as a membrane either over the recognition element, or in the form of a sandwich arrangement with a polymer membrane on either side of the recognition element, or with the polymer itself being the recognition element as illustrated in Fig. 1.12.26, 152
Chapter 1 | 28
a b c
Figure 1.12 Modification of carbon surfaces with polymer membranes. a) The biorecognition element is sandwiched between to polymer layers. The first is conducting; the second can act as
a membrane allowing only certain species through. b) The polymer layer is a membrane over
the biorecognition element. c) The biorecognition element is incorporated into the polymer.26
The polymers can be formed in several ways, including via electrochemical polymerisation, and chemical polymerisation. Polypyrrole is used as a polymer for biosensing as it is electrochemically conducting, and can be produced from the electrochemical oxidation of pyrrole in aqueous sulphuric acid, reacting through the α,
α'-carbons and some reaction of the β-carbons causing cross linking of the chain. By forming the polymer in a biological media, enzymes can be incorporated into the polymer.153 Several other polymers that are able to be electrochemically formed can also be incorporated with enzymes via the same method.26 This method has the advantage of being able to control the biological loading of the polymer through the concentration of the media. Modifying the pyrrole monomer with a recognition element is another way to incorporate them into the polymer.154-155 As the polymerisation of the
Chapter 1 | 29
pyrrole can still occur and in a similar manner to the enzymes the recognition element is thus incorporated into the polymer.
The use of Nafion, a sulfonated tetrafluoroethylene based fluoropolymer- copolymer, can be used for selectivity. Nafion polymer on the electrode surfaces repels ascorbic acid as it is negatively charged.156-157 The electrodes with a layer of Nafion on top of the electrode showed better resistance to ascorbic acid than electrodes with a mixture of Nafion and GOx.156
The use of membranes can work in a similar manner. The recognition elements are physisorbed onto the surface and a membrane, generally a dialysis membrane or a non-conducting polymer, is then placed over the modified surface.32, 158 The membrane has two purposes, firstly as a method to stabilise the modified surface in solution, and secondly as a way to reduce interferences via size or charge exclusion. This method can be seen in a lactose sensor where the two enzymes galactosidase and glucose oxidase are trapped behind a dialysis membrane that has a low molecular cut off weight at 3.5 ×
103.39, 158
These methods both allow the inclusion of recognition elements and help to reduce interference by competing substances, though neither allow sensitive detection due to mass transfer through either the polymer or membrane.
Chapter 1 | 30
1.4.2 HEATING AND OXIDATION OF CARBON SURFACES
The oxidation of carbon surfaces produces carboxylic acids, quinonic, ketonic or hydroxylic groups on the surface (Fig. 1.13). These groups can then be reacted to further modify the surface. This oxidation can occur through treatment of the surface with either hot, strong acids, or by heating the substrate to around 400 °C and exposing it to oxygen.159 This can be seen in the work by Paxinos et al.160 where the GC was anodically oxidised using nitric acid, sulphuric acid and potassium dichromate and passing a charge through the surface of 2 C cm-2. Cobalt diaminosarcophgine was then attached covalently to the GC surface once the oxidised surface was activated with N- cyclohexyl-N'-[2-(N-methylmorpholino)-ethyl]-carbodiimide-p-toluene sulfonate.160
The oxidation of the surface is carried out under harsh conditions and also increases the surface roughness.161-163
Carboxylic acid Phenol o-quinone Lactone HO O Carbonyl OH O O O O O O O
p-quinone
O
Figure 1.13 Carboxylic acids, quinonic, ketonic or hydroxylic groups on the surface of
oxidised carbon.164
Chapter 1 | 31
Amine groups can be introduced to the surface by the use of ammonia. Amine termination of the surface can be accomplished using either plasma or ammonia or a combination of the two; Firstly by the exposure of the carbon surface with ammonia plasma.165 Secondly, amines can also be introduced onto the surface by polishing GC electrodes in the presence of ammonia.128 Thirdly, using a mixture of plasma exposure and ammonia exposure the carbon can be modified by exposing the carbon surface to argon radio frequency plasma and subsequent exposure to ammonia vapour.166-168 The amine terminated surface can then be reacted with N-hydroxysuccinimide esters. This was shown by Saveant and co-workers through the attachment of a ferrocene N- hydroxysuccinimide ester to an amine terminated surface as well as attaching biotin to the surface.166, 169
The surface preparation required to terminate the carbon surfaces with amines or to oxidise the surface requires harsh conditions, which roughen the surface. Little work has also been carried out on the use of these two methods for making mixed monolayers on the surface, which is a requirement for affinity biosensors.
1.4.3 AMINES AND ARYLACETATES
Primary and secondary amines can be electrochemically oxidised in either ethanol or acetonitrile to produce a radical that binds to the surface of carbon (Fig
1.14).170 A monolayer is believed to be formed by the oxidation of primary amines, whilst secondary amines produce low coverage, implying that the stereochemistry of the alkyl amine being attached are important.170 Bein and co-workers investigated alkyl chains of different lengths and it was observed that those with longer chain length
Chapter 1 | 32
(amino hexanol, decylamine and octadecylamine) did not produce the same amount of coverage as those with shorter chain lengths (butylamine).171 It was noted that the reproducibility of the attachment of the amines was affected by the GC electrode history including its electrochemical performance. The structure of the carbon can also affect the attachment and reproducibility, as it has been shown that the oxidation of amines onto the surface does not occur for basal planes.171
R R CH2 CH2 R R H N 2 H N CH CH2 R - 2 + -e -H H2N HN CH2 HN
Figure 1.14 Oxidation of primary amines onto carbon surfaces.171
The reaction of the amines onto the surface was found to form nitrogen-carbon bonds. The formation of the nitrogen-carbon bond was found to be covalently formed as opposed to the amines being only physisorbed onto the surface. Pinson and co- workers 170 have shown that the bond formed is stable after thermal treatment (110° for
1 hr) under vacuum. However it was shown by Deinhammer et al.172 that after 100 cyclic scans (in 0.1 M HClO4 between 0.2 and 0.8 V vs. Ag|AgCl in saturated LiClO4) a butylamine derived carbon surface gave a 10 % reduction of the N:C ratio. The use of heat as well as time, can be used to modify carbon surfaces with alkyl amines. By refluxing the carbon surface in either neat alkyl amines or concentrated solutions of alkyl amines in terahydrofuran under N2 for 15 hrs to 5 days a monolayer forms, though this is a very harsh method.173 By leaving the surface in the alkyl amine solution
Chapter 1 | 33
without heating the reaction was noted to go slower than the electrochemically assisted modification. Similar electrochemical methods can be used for alcohols. It is believed that a surface radical is produced which then reacts with the alcohols forming an ether bond.
The modification of carbon surfaces using arylacetates is carried out according to Kolbe reaction, through the oxidation of carboxylates.93, 174 This method allows the modification of the basal plane as well as the edge plane of graphite and carbon surfaces. The carboxylate is oxidised releasing CO2 and forming a radical which then reacts with the surface (Fig. 1.15).175-176
R CO2 - R -e -CO2 R
Figure 1.15 Oxidation of arylacetates via the Kolbe reaction.177-178
Several studies have shown that there is the possibility of the formation of multilayers.174, 176 As the arylcarboxylates are oxidised onto the surface a peak in cyclic voltammetry is shown. Upon further scanning this peak is observed to decrease and a secondary peak is observed. If however the potential is held just before the oxidation peak, the removal of the arylcarboxylate is observed.174, 176
The use of amines and arylcarboxylates to modify the surface provides a polar bond and is not as stable as a carbon-carbon bond. The arylcarboxylate is unstable for
Chapter 1 | 34
the use of electrochemical sensors, and also can form multilayers. Can a carbon surface be modified with a covalent bond that is non-polar and thus less prone to oxidation?
1.4.4 MODIFICATION OF CARBON SURFACES BY ARYL DIAZONIUM SALTS
The use of aryl diazonium salts to modify carbon surfaces was first performed by Saveant and Pinson.93, 179 Aryl diazonium salts can be used on both gold and glassy carbon surface to form stable, covalently bound layers on an electrode surface.180-181
The electrochemical reduction of aryl diazonium salts produces a radical, which is then strongly bound to the surface (Fig. 1.16).92 This reduction produces covalently bound aryl groups on the surface through a carbon-carbon bond. The layers produced are stable and have a wider potential range than that of a thiol derived gold surface. Aryl diazonium salts can also be reduced on both GC as well as HOPG, edge and basal planes, though edge planes are more reactive.92
R R
R + e- N - N2 N
Figure 1.16 Reduction of aryl diazonium salts to the surface.92-93, 182
As the aryl diazonium salts are reduced through cyclic voltammetry, the peak for the reduction of the aryl diazonium salt onto the surface becomes passivated and shifts negatively as the surface continues to react towards the radicals. This loss of the
Chapter 1 | 35
reduction peak of the aryl diazonium salt is due to the electron transfer rate becoming slower as the modified layer becomes thicker, as thicker surface modification layers hinder the electron transfer through the layer.92 The saturation of GC is quicker than that seen on HOPG. This again is due to the greater reactivity of edge planes than basal planes, as HOPG in this experiment was mainly basal planes and GC, as it is amorphous, is made of both edge and basal planes. The potential window of aryl diazoniums on carbon is far greater than that of thiolates on gold, -2.6 V to 5.6 V in comparison to +0.8 V to -1.4 V when measured in an aqueous envirnoment.50, 59 The formation of the layer however is not as well defined as that of the gold-thiol monolayer and produces multilayers not monolayers.183-184 On carbon it has been seen that at least three layers of aryl diazoniums are formed on the surface.184 Both monolayers and multilayers have been observed through the reduction of aryl diazonium salts onto the carbon surface. Multilayers can occur as the radical produced reacts with the aryl rings in the ortho positions, either in solution or once they are attached to the surface (Fig.
1.17).184
R R R
R R R R R N + e- N
- N2
Figure 1.17 Formation of multilayers through diazonium salt reduction.184-186
It has been noted that the scan rate can control the number of these layers. The use of steric hindrance to control the formation of monolayers or multilayers has been
Chapter 1 | 36
investigated by Pinson and co-workers187-188 by using different aryl diazonium salts where either the para position was blocked, one of the meta positions or both the meta positions or the ortho position or a combination of the three (Fig 1.18). It was seen that those in the ortho position 2,6-dimethylbenzene diazonium (Fig 1.18a) and 2- ethylbenzene diazoniums (Fig 1.18b) showed no surface modification. Blocking of the two meta positions with a bulky butyl group limited the modification layer to a monolayer (Fig 1.18d).187-188 Aryl diazoniums modified at either the two meta positions with methyl groups (Fig 1.18c) or the ortho and para positions also with methyl groups showed multilayers (Fig 1.18e).
ab c
N2 N2 N2
d e
N2 N2
Figure 1.18 Aryl diazonium salts used to investigate steric hindrance and modification of
carbon surfaces.187-188 a) 2,6-dimethylbenzene diazoniums. b) 2-ethylbenzene diazonium. c)
3,5-dimethylbenzene diazonium. d) 3,5-dibutylbenzene diazonium. e) 2,4-dimethylbenzene
diazonium.
The layers derived by the reduction of aryl diazonium salts are highly stable to both air and heat as it was seen that the layers are still present after heating to 700 K.
The electrochemical reduction of aryl diazonium salts can be performed in one of two
Chapter 1 | 37
ways, firstly via “pre-making” the aryl diazonium salt and carrying out electrochemical reduction. Secondly with an in situ method by forming the aryl diazonium salt in solution from the aniline derivative and immediately reducing the compound to the surface.189-190 It was shown by Liu et al. that the surface produced by in situ generation of aryl diazonium salts is the same as that produced by “pre-made” aryl diazonium salts.191 It has also been seen that they can form stable layers through spontaneous formation on the surface.192-194 By simply immersing the surface into the aryl diazonium solution with an open-circuit potential, spontaneous reduction is carried out and a covalent bond is formed on the surface.176 It is yet to be understood how these spontaneously formed layers are attached. However, this spontaneous reaction does allow the patterning of the carbon surface through micro contact printing.192-193
Like the gold-alkanethiol layers it is possible to use mixed layers with aryl diazonium salts. This has been shown in a number of studies.56, 180, 193, 195 Using mixed layers gives a modified surface the ability to have a multi functional aspect, and different aspects of the surface can have differing functions, such as detecting different analytes with the same electrode. Mixed layers also have the ability to combine a biorecognition element with a diluent that not only spaces out the biorecognition element but provides resistance to non-specific protein adsorption.43, 180, 195
Aryl diazonium derived carbon surfaces have been shown to be applicable to many electrochemical sensor techniques. It has been shown that through the use of a phenyl acetate aryl diazonium derived surface the concentration of dopamine in high ascorbic acid concentrations can be measured.196 The measurement of dopamine in the presence of ascorbic acid is difficult as generally the response is small and both
Chapter 1 | 38
compounds oxidise at nearly the same potential, and dopamine can oxidise in the presence of ascorbic acid.197 Once the carbon surface is modified with phenyl acetate aryl diazonium salt dopamine is distinguishable from ascorbic acid.196
The use of aryl diazonium salts allows for the surface of the electrode to be modified with a stable, covalently bound layer. The electrochemical reduction of aryl diazonium salts provide a more stable layer over those formed from the reduction of amines and arylacetates, this is provided by the carbon-carbon bond. The stability of this modification layer and the ability to partially control the number of layers formed through differing functionalities at the ortho and para positions makes this surface modification technique useful in the construct of an affinity biosensor.
1.4.5 HYDROSILYLATION, HYDROGENATION AND THE ATTACHMENT OF
ALKENES
Although we have the ability to produce sensitive biosensors on smooth carbon surfaces, we still have to use aryl diazonium salts to attach the particular sensor parts to the surface.43 The use of aryl diazonium salts has the ability to produce multilayers instead of monolayers on the surface, which has the problem of again placing the biorecognition element and redox mediator in different environments on the sensor.
If there was a way to form controlled monolayers like the gold-thiolate derived layers, with the added stability of the aryl diazonium salts it would greatly increase the capacity of an affinity sensor. Add to these attributes the ability to pattern the surface
Chapter 1 | 39
then the possible uses of the sensor can be greatly expanded to cover multianalyte- sensors.
Silicon has the ability to be hydrogenated via the exposure to hydrofluoric acid, this means that the surface instead of being covered in oxide is covered in hydrogen.
This surface can then be exposed to alkenes and either with heat or UV activation be modified with a stable, covalently bound monolayer as was first shown by Linford et al..90, 198-200 The layer formed has a monolayer surface coverage, which can then be further modified. This can be seen in the work of Böcking et al, where they showed that a formed monolayer of alkenes can then be modified by the attachment of oligo(ethylene glycol) which reduces the amount of protein adsorbed onto the surface.78,
201-202 Hamers and co-workers have also shown that hydrolysed silicon can be used for subsequent modification with DNA strands.90
Through the use of hydrogenated diamond surfaces stable, covalently bound monolayers can be formed through the reactions of alkenes with the surface. Hamers and co-workers have shown that through the exposure of alkenes to a hydrogenated diamond surface with UV excitation at the wavelength 254 nm that the diamond surface can be modified with a monolayer.94 Hamers and co-workers203-204 investigated the process through which alkenes are covalently attached to the surface and suggested that it was not the activation of the alkene but of the surface causing ejection of a photoelectron (Fig. 1.19). The method of alkene addition to diamond surfaces is similar to that of alkene addition to silicon surfaces.205-206 It was also noted that the modification depended on the alkene used and that alkenes that have a high electron affinity are more likely to react with the surface.203 The hydrogenated amorphous
Chapter 1 | 40
carbon can also be further modified with alkenes in the similar manner to that of diamond surfaces.
Figure 1.19 Alkene addition to the surface of hydrogenated diamond surfaces. Hamers and co-
workers suggest that instead of the alkene being activated with UV light, the surfaces is
activated. UV light activates the surface causing ejection of an electron from the surface to the
alkene, this causes rearrangement of the alkene and the loss of an hydrogen of the surface
leaving an activated site for the attachment of the alkene.203-204, 207
The use of alkenes to modify hydrosilylated surfaces and diamonds surfaces would work well for an affinity biosensor as they provide a well defined stable monolayer. This thesis though is focusing on the use of carbon as and electrode surface, which rules out the use of hydrosilylated silicon for an affinity biosensor construct. It was also previously noted that diamond surfaces are not nearly smooth enough if the sensor sensitivity is to be enhanced. To be able to use alkenes on PPF as a monolayer would require the PPF to be hydrogenated or activated in a similar fashion allowing UV attachment of alkenes, this will be investigated further in chapter 5.
Chapter 1 | 41
1.5 WHICH CARBON AND WHICH MODIFICATION METHODS TO
USE?
It is the hope of this project to improve on both the electrode surface and the methods of modifying the surfaces, whilst at the same time improving the sensor construct. The use of thin carbon films or PPF will provide a near atomically smooth surface, by modifying this surface with aryl diazonium oligo(ethylene glycols) will reduce the non-specific protein adsorption. By combining these to factors it will allow the biorecognition elements to be in the same environment without hindrance from unwanted proteins. The use of iodinated PPF will allow the formation of stable monolayers on the electrode surface. These elements combined hope to produce a more sensitive affinity biosensor.
Chapter three investigates the preparation of thin carbon film electrodes and their usability for biosensors. The effect of varying the heating parameters during the pyrolysis procedure and the effect of gas flow are investigated. This chapter hopes to understand the affects of the heating profile on the pyrolysis of spin-coated photoresist to produce almost atomically smooth, electrochemically conducting carbon surfaces.
The application of these surfaces towards biosensors is also shown through the modification with 4-carboxyphenyl diazonium salt, and the attachment of the peptide
Gly-Gly-His to measure the concentration of copper.
Chapter four reports the use of new oligo(ethylene glycol) aryl diazonium salts to further investigate the reduction of protein adsorption on the thin carbon film surface.
Through the investigation of the chain length of oligo(ethylene glycol) aryl diazonium
Chapter 1 | 42
salts and the affect of the distal end on non-specific protein adsorption. By comparison to those formed on gold surfaces provides a comparison to their effectiveness.
In chapter five, iodination of the carbon surface and alkene attachment through
UV activation is used to improve surface modification, whilst retaining the surface attributes of smoothness, and electrical conductivity. By producing stable, covalently bound monolayers on the almost atomically smooth PPF surface, the two surface modification techniques of the monolayer formation from gold-thiol modification and the stability from electrochemical reduction of aryl diazonium salts have been combined. This will give the potential for a more sensitive ad stable affinity sensor than those formed previously with gold-thiol surface modification or through aryl diazonium reduction on carbon. The ability to pattern the surfaces to increase functionality is shown.
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207. Colavita, P. E.; Sun, B.; Tse, K.-Y.; Hamers, R. J., Photochemical Gafting of n-Alkenes
onto Carbon Surfaces: the Role of Photoelectron Ejection. J. Am. Chem. Soc. 2007, 129
(44), 13554-13565.
Chapter 1 | 62
Chapter Two
Experimental Methods
Chapter 2 | 63
The focus of this work is on the preparation of smooth thin carbon films and subsequent modification. This chapter describes the various instrumentation used for this project. The procedures and chemicals used are also described. Further specific experimental are described in the relevant chapters. The instrumentation used in this research is mainly electrochemical and is described in this chapter. Other experimental techniques used such as X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), contact angle and scanning electron microscope (SEM) are also described.
2.1 MATERIALS
2.1.1 CHEMICALS AND REAGENTS
All chemicals used were reagent grade or higher and were used as received unless otherwise stated. All aqueous buffers and solutions were prepared using Milli-Q water (18 MΩ cm; Millipore, North Ryde, Sydney).
Chemical Formula Grade Supplier
Acetone CH3COCH3 - Ajax
Acetonitrile CH3CN - Ajax
Cambridge Isotope Acetonitrile-d CD3CN 99.8% Laboratories Inc.
Ammonia solution NH3 28% Ajax
4-Aminobenzoic acid C7H7NO2 99% Sigma
Chapter 2 | 64
Chemical Formula Grade Supplier
Ascorbic acid C6H8O6 - Unilab
Celite - 501 Fluka
Cambridge Isotope Chloroform-d CDCl3 D 98.8% Laboratories Inc.
Copper (II) sulfate CuSO4•5H2O - Ajax
1-Decanol C10H22O - Sigma
N-(-3- dimethylaminopropyl)-Nʼ- C8H17N3•HCl - Sigma ethylcarbodiimide hydrochloride (EDC) Disodium hydrogen Sigma- Na2HPO4 phosphate Aldrich 98% Dipotassium hydrogen K2HPO4 Sigma- ACS phosphate Aldrich reagent
Dichloromethane CH2Cl2 - Ajax
Ethanol CH3OH - Ajax
Ethylenediaminetetraacetic (HO2C2H2)2NC2H4N(CH2CO2H)2 99% Aldrich acid EDTA
Ethyl acetate CH3CO2C2H5 - Ajax
4-Fluoro-1-nitrobenzene C6H2FNO2 99% Aldrich
Forming gas 5% H2 in 95% N2 - Air liquide
Gold chloride HAuCl4•3H2O 99.9% Aldrich
Hexa(ethylene glycol) C18H38O7 Aldrich
Hexa(ethylene Sigma- glycol)mono-11- C23H48O7S 95% Aldrich mercaptoundecyl ether
Chapter 2 | 65
Chemical Formula Grade Supplier Hexaamine ruthenium(III) Ru(NH3)6Cl3 98% Aldrich chloride N Hydroxysuccinimide C4H5NO3 98% Aldrich (NHS) Hydrochloric acid HCl 32% Ajax
Hydrogen peroxide H2O2 30% Aldrich
Hydrogen gas H2 - Air liquidé
Iodine I2 Sublimed Fluka
Isopropanol (CH3)2CHOH - Ajax
2-(N-morpholino)
ethanesulfonic C6H13NO4S 99.5% Sigma acid (MES)
Magnesium sulphate MgSO4•3H2O - Ajax
Methyl iodide CH3I - Sigma
Methanol CH3OH - Ajax
2-Mercaptoethanol C2H6OS - Sigma
Mowiol 4-88 - - polysciences
Nitric acid HNO3 70% Ajax
Nitrosonium NOBF4 - Aldrich tetrafluoroborate
Nonadiyne C9H12 98% Sigma Ammonium iron(II) (NH4)2SO4·FeSO4·6H2O - Ajax sulphate 10 % Palladium on carbon Pd - Lancaster AZ electronic Positive photoresist AZ - - materials 4620, (Japan). Potassium dihydrogen KH2PO4 >99% Aldrich phosphate
Chapter 2 | 66
Chemical Formula Grade Supplier
Potassium chloride KCl - Sigma
3-inch <100> n-type silicon Micro wafer (500-550 μm thick Materials and - - and 2.0-9.0 Ω cm Research resistivity) Consumables
Silica Grade 9385, 230-400 - Merck
Sodium chloride NaCl - Ajax
Sodium citrate C6H5Na3O7•2H2O ACS Sigma
70% in Sodium hydride NaH Aldrich oil
Sodium hydroxide NaOH - Ajax
Sodium tetrafluoroborate NaBF4 98% Aldrich
Sodium nitrite NaNO2 97% APS
Sodium sulphate Na2SO4 - Ajax
Sulphuric acid H2SO4 - Ajax
Tetrabutylammonium (CH3CH2CH2CH2)4N(BF4) 99% Aldrich tetrafluoroborate Tetrabutyl ammonium (C4H9)4N(BF4) 98% Aldrich hydrogen sulphate Tri(ethylene glycol) C7H16O4 95% Aldrich monomethylether
Tri(ethylene glycol) O(C2H4O)2C2H4OH - Aldrich Triton X - - Sigma Triethylene glycol mono- Sigma- HS(CH2)11(OCH2CH2)3OH 95% 11-mercaptoundecyl ether Aldrich
Table 2.1 A list of chemicals and reagents used.
Chapter 2 | 67
2.1.2 PREPARATION OF BUFFERS AND STANDARD SOLUTIONS
A range of buffer solutions was prepared for this work. The pH of the buffer solutions was measured using a Futura pH electrode connected to a pH meter (both from Beckman Instruments, Fullerton, CA, USA). The pH meter was calibrated using standard pH 4.0 (± 0.05) and pH 7.0 (± 0.05) buffer solutions (Asia Pacific Specialty,
Sydney, Australia). Buffer solutions used were 50 mM phosphate buffer (pH 7.0) with
1.0 M KCl, 100 mM MES (pH 6.8), 50 mM ammonium acetate (pH 7.0) with 50 mM
NaCl and phosphate buffered saline (PBS).
Peptide modification was carried out in 100 mM MES buffer (pH 6.8).
Electrochemical measurements of peptide-modified electrodes were carried out using 50 mM ammonium acetate buffer (pH 7.0) with 50 mM NaCl. The accumulation of copper was carried out in stock copper sulphate solutions made with 50 mM ammonium acetate buffer (pH 7.0).
For the exposure of modified PPF and gold surfaces to BSA-FITC, a solution of
BSA-FITC in PBS was used. PBS was prepared from sodium chloride (8 g, 0.14 mol), potassium chloride (0.2 g, 2.7 mmol), disodium hydrogen phosphate (1.44 g, 0.010 mol) and potassium dihydrogen phosphate (0.24 g, 1.8 mmol) in Milli-Q water (1 L) at pH
7.4.
Electrochemical measurements to investigate the affect of iodination and modification with alkenes and aryl diazonium salts were carried out in phosphate buffer
Chapter 2 | 68
with 1.0 M KCl. Potassium dihydrogen phosphate (1.7 g, 9.8 mmol), dipotassium hydrogen phosphate (2.2 g, 16.2 mmol) and potassium chloride (3.7 g, 50 mmol).
Solutions of the redox species, 10 mM hexamine ruthenium(III) chloride, 10 mM
Potassium ferricyanide, and 10 mM ascorbic acid, were all made with 50 mM phosphate buffer (pH 7.0) and 1.0 M KCl unless otherwise stated.
2.2 INSTRUMENTATION
2.2.1 ANALYSIS
2.2.1.1 CHARACTERISATION OF SYNTHETIC COMPOUNDS
2.2.1.1.1 NMR
1H NMR spectra were obtained on a Bruker DPX300F (300 MHz) spectrometer.
Data is reported as follows: chemical shifts (δ) are measured in parts per million (PPM) down field from TMS; multiplicity: observed coupling constants (J) in Hertz (Hz).
Multiplicities are reported as singlet (s), broad singlet (bs), doublet (d), broad doublet
(bd), triplet (t), broad triplet (bt), quartet (q), doublet of doublets (dd), doublet of doublet of doublets (ddd), doublet of triplets (dt), doublet of doublet of triplets (ddt), pentet (p), heptet (h), multiplet (m), and double multiplet (dm).
13C NMR spectra were obtained on a Bruker DPX300F (75.6 MHz) spectrometer. C13 chemical shifts (δ) were reported in parts per million (PPM) down field from TMS.
Chapter 2 | 69
2.2.1.2 SURFACE ANALYSIS
2.2.1.2.1 ELECTROCHEMISTRY
The main experimental technique used in this body of work has been voltammetric measurements. This is the application of a potential to an electrochemical cell and the monitoring of the output current, as can be observed in cyclic voltammetry.
In voltammetry, a three electrode cell system is used. The cell is then connected to a potentiostat, which controls the input waveform and acts as a current measuring device.
The three electrodes used are a working electrode, counter electrode, and reference electrode. The potential applied to the working electrode is measured relative to the reference electrode. An electrochemical current of a species can be measured upon applying a potential that can cause its oxidation or reduction.
Electrochemical measurements were predominantly performed with an Autolab
PGSTAT 12 potentiostat (Eco Chemie, Netherlands) interfaced to a personal computer.
Data acquisition was performed using GPES and Nova software. Cyclic voltammetry and Osteryoung square wave voltammetry experiments were carried out with a conventional three electrode system, comprising a bare or modified working electrode, a platinum flag counter electrode and a Ag|AgCl 3.0 M NaCl reference electrode (from
Bioanalytical Systems Inc., Lafayette, IN, USA). All potentials are reported versus this reference at 25°C unless otherwise stated.
Chapter 2 | 70
a
b
Figure 2.1 a) Autolab potentiostat used for electrochemical measurements. b)
Electrodes used from right to left PPF, GC, reference and counter.
Redox species that either are in solution or attached to the surface can be detected from the current flowing between the working electrode and counter electrode.
The working electrodes were either Pyrolysed photoresist films (PPF), as prepared per the general procedure discussed in section 2.3.1.1 or as described per chapter, glassy carbon (GC) electrodes (Bioanalytical Systems Inc., Lafayette, USA), and gold foil.
Chapter 2 | 71
2.2.1.2.1.1 Cyclic Voltammetry
Cyclic voltammetry (CV) is a potential sweep technique.1-2 The potential is swept back and forth between two potential limits (E1 and E2) at a set sweep rate, otherwise known as the scan rate (Vs-1), and is the rate of change of potential with time
(Fig. 2.1). The current is measured as the potential is swept continuously between E1 and E2. The use of multiple scans can give an indication to the stability of the redox species.3 The variation of current with scan rate can give an indication as to whether the redox species is surface bound or in solution.1-2
a
b
Figure 2.1 a) Potential-time profile for cyclic voltammetry. b) Representation of a current (I)
vs potential (E) plot for cyclic voltammetry.
Chapter 2 | 72
2.2.1.2.1.2 Osteryoung Square Wave Voltammetry
Osteryoung square wave voltammetry (OSWV) is a sensitive technique, combining large amplitude square wave modulation with a staircase waveform (Fig.
2.2a).4-6 Higher sensitivity is achieved by removing the background currents. The current is sampled at the end of each pulse; the forward current sampled at t1 and the reverse current at t2. The net current is the difference between the two (Fig 2.2b) and removes the response due to the charging current.6 The redox species is continuously reduced and oxidised. The conditions for OSWV were a pulse amplitude of 0.025 V, a step of 0.004 V and frequency of 25 Hz.
a
b
Figure 2.2 a) Diagram of the square wave potential waveform where Esw is the pulse
amplitude, ΔE is the step height and τ is the square wave period. b) Representative ΔI vs E plot
for OSWV.
Chapter 2 | 73
2.2.1.2.2 CONTACT ANGLE
All contact angles were measured on a ramé-hart standard contact angle goniometer and data acquisition collected on a personal computer. The angles were processed with dropimage.
The contact angle can be measured by producing a drop of liquid on a solid surface. For these studies only milli-Q is used. The angle formed between the liquid/solid interface and the solid/air interface and the surface of the interface is the contact angle (Fig 2.3).7 The contact angle is measured using the Young equation
(equation 1.1) and results from the interface/surface tensions between the water and solid surface surrounded by air.
Figure 2.3 Contact angle of a water droplet on a solid surface. γ1 is the solid/water interface
free energy, γ2 is the solid surface free energy and γ12 is the water surface free energy.
γ 2 = γ12 +γ1 cosθ equation 1.1
A water drop with a large contact angle is hydrophobic (Fig 2.4a), and a drop with a small contact angle is hydrophilic (Fig 2.4b). By measuring the advancing and
Chapter 2 | 74
receding angles information about the surface topology, i.e. surface modification heterogeneity, effect of surface treatments and cleanliness, can be gained. The advancing angle can be measured by adding volume to the drop without increasing the surface area of the drop (Fig. 2.4c) and the receding angle is the reverse where volume is removed without changing the surface area (Fig 2.4d). The difference between the two gives the hysteresis.7
a b
c d
Figure 2.4 a) Hydrophobic surface with a large contact angle. b) Hydrophilic surface with
small contact angle. c) Advancing angle, where water is added to the droplet without increasing
the surface area. d) Receding angle, where water is removed form the droplet without
increasing the surface area.
The contact angles of modified surfaces were measured three times, on three different areas of the surface. The averages of all three measurements were used to gain
Chapter 2 | 75
an understanding of the modification of the entire surface. The advancing and receding angles were measured four times on one area of the surface.
2.2.1.2.3 X-RAY PHOTOELECTRON SPECTROSCOPY
X-ray photoelectron spectroscopy (XPS) was used to determine the presence of species on surfaces, such as iodine and fluorine. XP spectra were obtained using a VG
ESCALAB 220-iXL spectrometer (from VG Scientific, West Sussex, UK) with a monochromated Al Kα source (1486.6 eV). The spectra were accumulated at a take-off angle of 90° and an analyser pass energy of 20 eV. The pressure in the analysis chamber was less than 10-8 mbar. Survey spectra (0 - 1100 eV) were obtained followed by high resolution spectra and both were analysed by XPSPEAK41 software.
In XPS x-rays are used as a fixed point of energy to inject electrons into the inner-shell orbitals from the sample.8-9 The kinetic energy of the ejected electrons is then analysed. Each element has its own characteristic binding energy and is dependent on the chemical environment of the element, e.g. the oxidation state or the chemical bonding and what is bound to. The intensity of the peaks in XPS is also related to the concentration.9
Chapter 2 | 76
2.2.1.2.3 SCANNING ELECTRON MICROSCOPY
Scanning electron microscopy (SEM) uses energetic electrons to scan the surface in a raster pattern. This produces several signals including backscattered, secondary and Auger electrons.8 A Hitachi S-900 'in-lens' field emission scanning electron microscope with a cold emission source (12 kV) was used to image the samples.
The samples were prepared as described in chapter 5. The samples were mounted on a brass sample base with conducting carbon tape to ensure conducting and stability of the sample on the brass mounting stage. Samples were chromium coated before being mounted using an Emitech K575x Chromium Sputter Coater.
2.2.1.2.4 ATOMIC FORCE MICROSCOPY
Atomic force microscopy (AFM) images were taken using a Digital Instruments
Dimension 3000 scanning probe microscope. All images were acquired in tapping mode using commercial Si cantilevers/tips (Olympus) used at their fundamental resonance frequencies, which typically varied between 275-320 kHz. AFM images were performed on PPF substrates.
In AFM a laser is reflected off the back of a countilever into a segmented photodiode that is sensitive to the position of the reflected beam.8 For tapping mode AFM the cantilever is oscillated at a set frequency that has been optimised for the cantilever used.
Chapter 2 | 77
The amplitude of the oscillation is continuosly measured and and the cantilever “taps” back and forth over the sample and the change in amplitude is measured thus an image of the surface can be obtained. The tip is of atomic propotions which allows atomic resolution of the surface.
Photodiode, position
sensitive deflector
Laser
Laser beam
Cantilever Tip
Sample
Figure 2.5 Basic side view of an AFM, showing laser and photodiode, cantilever (not to scale)
and sample.
2.2.1.2.5 FLUORESCENCE MICROSCOPY
Fluorescence microscopy was carried out on a Leica DM IL inverted microscope. The DM IL is an inverted epifluorescence microscope, with transmitted light LED back illumination and an EL6000 Fluoro system. The images were taken with a ProgRes CFscan CCD camera attached to the Leica DM IL inverted microscope.
Chapter 2 | 78
The filter set GFP was used for blue excitation and the objective 63x NA1.25 oil was used to investigate the surfaces. The inverted microscope works in a similar fashion to that of a normal microscope, instead of normal UV light the fluorescence microscope shines fluorescent light at the surface of the sample, at differing wavelengths depending on the excitation needed. The fluorescent species on the surface are excited and the light emitted is measured.
2.2.2 SURFACE PREPARATION
2.2.2.1 SPIN COATER
The spin coater used was from Electronic Microsystems LTD and was multi- programmable. A diaphragm pump was used to hold the samples on the chuck. Spin coating was carried out under a yellow light. 3-4 drops of photoresist were applied before spinning so that any bubbles in the photoresist could be removed before coating.
All samples were spin coated at 3000 rpm for 30 s.
2.2.2.2 OVEN
The oven used was from Haraeus Instruments which was able to be set at the required temperatures. Samples were soft baked at 95° for 20 min and left to rest in the oven at 45° overnight.
Chapter 2 | 79
2.2.2.3 TUBE FURNACE
A tube furnace, k897 1300° tube furnace (purchased from ceramic engineering), was used as a pyrolysis program was able to be stored in the furnace. A quartz tube runs through the middle where ceramic boats containing the samples can be placed.
Aluminum caps are connected on either end so as to allow copper tubing to be used to provide the reducing atmosphere of 5% hydrogen in nitrogen (Fig 2.6). A flow rate gauge was connected to the copper piping in between the tube furnace and gas cylinder to adjust the flow rate used.
Figure 2.5 Tube furnace used to for pyrolysis of photoresist films and ceramic boats.
Chapter 2 | 80
2.2.2.4 PLASMA CHAMBER
Plasma is formed when a radio frequency potential is applied to a gas at reduced pressure.10 This forms a partially ionized gas in equal positive and negative charges.
As the gas is ionized a bound electron from the gaseous species is ejected from the atom. A glow is seen from the plasma which is attributed to the electronically excited species producing an optical emission. For each gas the colour of the discharge is different; for water the colour is blue and for iodine it is a purple colour.10
Solid resublimed iodine was placed in the bottom of the chamber. PPF samples are placed in the centre and the chamber is sealed and evacuated down a pressure of 0.7 to 2.8 mbar. The vacuum is applied using a two stage pump (Edwards 5) with a liquid nitrogen trap applied to prevent iodine entering the pump. The chamber is allowed to come to equilibrium with the iodine before the radio frequency generator (AG0201HV-
OS “high voltage” power source from T and C Power Conversion) is turned on, and allowed to warm up. The power is applied till the forward power reaches 10 Watts, the frequency is then optimized so that the reverse power is at it’s lowest reading. The power is then turned to the desired amount and the surface is exposed to the plasma for the desired period of time. The power is then turned down and off before the generator is switched off. The vacuum is gently released, the samples removed and the chamber cleaned with ethanol then dried under vacuum.
Chapter 2 | 81
2.3 PREPARATION OF ELECTRODES AND ANALYSIS
2.3.1 PREPARATION OF ELECTRODES
2.3.1.1 GENERAL PROCEDURE FOR PPF PREPARATION
A 3-inch <100> n-type silicon wafer was cut into 1.4 × 1.4 cm2 pieces using a diamond scribe. The wafer pieces were then cleaned by sonicating in successive baths of acetone, methanol and isopropanol. These samples were then dried with nitrogen.
Under yellow light, positive photoresist AZ 4620 was spin coated (3-4 drops/0.3-0.5 mL) onto the wafer pieces at 3000 rpm for 30 s. The wafer pieces were then heated at
95 ºC for twenty minutes in an oven (Chapter 2.2.2.2), to remove excess solvent but the films were still soft to touch. The samples were then kept overnight at 45 ºC.
The photoresist-coated silicon pieces were then placed in ceramic boats which were then placed in the centre of a tube furnace. The following heating program was run T1- 550 °C, t1 – 20 min; T2 – 680 °C, t2 – 15 min; T3 – 1100 °C, t3 – 60 min., with fifteen minutes between each step to allow for the temperature increase. This heating program was carried out under a reductive atmosphere of 5% hydrogen in 95% nitrogen. The pyrolysed samples were allowed to cool in this atmosphere before being stored in a vacuum desiccator.
Chapter 2 | 82
2.3.1.2 GLASSY CARBON
Glassy carbon electrodes were obtained from Bioanalytical systems Inc. (USA) and were 3 mm diameter rods encased into solvent resistant epoxy resin. The electrodes were hand-polished successively in 1.0, 0.3 and 0.05 mm alumina slurries (5 minutes each) made from dry Buehler alumina (Buelher, Lake Bluff, IL, USA) and Milli-Q water on microcloth pads (Buelher, Lake Bluff, IL, USA). The electrodes were thoroughly rinsed with Milli-Q water and sonicated in Milli-Q water for 5 minutes after the final polish.
2.3.2 ANALYSIS OF SURFACES
2.3.2.1 ELECTROCHEMICAL MEASUREMENTS
Electrochemical measurements to probe the surface modification of PPF were carried out as follows unless stated otherwise. A Ag|AgCl|3.0 M NaCl reference electrode and a platinum wire counter electrode were placed in the top of a custom- made electrochemical cell. The PPF is sandwiched in the base of the electrochemical cell which gives a controlled area of PPF by allowing only a disc with a 3 mm diameter to be exposed as shown in Fig 2.6, this gives a working electrode area of 7.1 mm2.
Chapter 2 | 83
3 mm opening to surface O-ring
10 mL solution space Gold contact to PPF surface Lid
Base with nylon PPF screws to give a tight seal of the PPF to the cell 1 cm
Figure 2.6 Electrochemical teflon cells used to control exposure area for PPF
3+ The redox systems used were 10 mM Ru(NH3)6 , in 50 mM phosphate buffer
3- with 1.0 M KCl from [Ru(III)(NH3)6]Cl3; 10 mM Fe(CN)6 in 1.0 M KCl from
2+ K3Fe(III)(CN)6; 10 mM Fe in 0.2 M HClO4 from (NH4)2Fe(III)(SO4)2·6H2O and 70%
HClO4; and 10 mM ascorbic acid in 50 mM phosphate buffer with 1.0 M KCl. All
solutions were degassed with dry nitrogen for 15 minutes prior to use. For each redox
probe, three cycles at 100 mV s-1 were run employing CV techniques. General scan
ranges are shown in table 2.2.
Redox species Scan range
50 mM Phosphate buffer -1.0 V to +1.0 V
3+ 10 mM Ru(NH3)6 -0.6 V to +0.2 V
3- 10 mM Fe(CN)6 -0.6 V to +1.0 V
10 mM Fe2+ -0.5 V to +1.5 V
10 mM ascorbic acid -0.5 V to +1.2 V
Table 2.2 General scan ranges employed for redox species.
Chapter 2 | 84
2.4 REFERENCES
1. Forster, R. J.; Walsh, D. A., Encyclopedia of Analytical Science. Academic Press:
London, 2005; Vol. 9.
2. Pletcher, D.; Greef, R.; peat, R.; Peter, L. M.; Robinson, J., Instrumental Methods in
Electrochemistry. 1st Ed. ed.; Horwood Publishing Limited: Chichester, 2001.
3. Laviron, E., General expression of the linear potential sweep voltammogram in the case
of diffusionless electrochemical systems. J. Electroanal. Chem. 1979, 101, 19-28.
4. O'Dea, J. J.; Osteryoung, J. G.; Osteryoung, R. A., Analytical Chemistry 1981, 53, 695.
5. Osteryoung, J. G., Journal of Chemical Eductaion 1983, 60, 296.
6. Osteryoung, J. G.; Osteryoung, R. A., Analytical Chemistry 1985, 57, A101.
7. Zisman, W. A., ACS Adc. Chem. Ser. 1964, 43, 1.
8. Skoog, D. A.; Holler, F. J.; Nieman, T., Principles of Instrumental Analysis. 5th ed. ed.;
Brooks/Cole, Thompson Learning: Crawfordsville, 1998.
9. Briggs, D.; Seah, M. P., Practical Surface Analysis. 2nd Ed. ed.; Wiley: New York,
1995.
10. Dendy, R. O., Plasma Dynamics. 2nd Ed. ed.; Oxford University Press: New York,
2002.
Chapter 2 | 85
Chapter Three
The Exploration of Variables in the
Fabrication of Pyrolysed Photoresist
3.1 INTRODUCTION
Pyrolysed photoresist films (PPF) are used as an alternative to glassy carbon
(GC) for both bare electrode electrochemical studies and to provide carbon surfaces for modification.1 Previous studies by McCreery and co-workers2 and the Kinoshita group3 have shown that thin carbon films can be formed by the pyrolysis in a reducing atmosphere of a thin film of photoresist laid down on a substrate to give a conducting carbon film that gives far smoother carbon surfaces than are achievable with conventional methods of fabricating GC (with a rms value less than 0.5 nm compared with 4.5 - 44 nm for GC respectively).3-4 The attractiveness of PPF is that it not only provides a much smoother surface but also has the good electrochemistry of GC and is compatible with bulk manufacture of a range of conducting carbon structures.5 In common with surfaces of GC, films formed from this process are mainly amorphous with graphitic regions.6 This can be seen by the presence of the relative D (disordered) and G (graphitic) bands in the Raman spectra around 1360 cm-1 and 1600 cm-1.3 The use of the ratio between the D and G bands can give an idea as to how much disorder is present in the structure.
In general photoresists are made of light sensitive polymers and are a material used in the microfabrication industry.5, 7 There are two types of photoresist, positive and negative. Positive photoresist is already a long chain polymer, which on exposure to light degrades. There are two types of positive photoresist used for the development of pyrolysed photoresists and they are based on novolak resins, which are phenolic formaldehyde based. Negative photoresist, on the other hand, is an epoxy based resist that is not a long chain polymer, until exposed to light.8 Cross linking of negative
Chapter 3 | 87
photoresist occurs due to light activation, in which many reactions can occur but generally they are ring expansions or insertations.9
Pyrolysed photoresist films have been shown to have low oxygen content (O/C ratios of approximately 0.05 for films pyrolysed at temperatures greater than 700 ºC)3 and are slow to be oxidised in ambient atmospheres.2 The low O/C is important if these materials are to be used as micro electrodes and micro batteries as the O/C ratio influences the electron transfer rate of some redox probes.2-3
The effect of the final pyrolysis temperature on PPF properties has been investigated by McCreery and co-workers,4 Lyons and co-workers,10-11 and Kinoshita and co-workers.3 It has been shown that PPF prepared at pyrolysis temperatures of
1000 ºC to 1100 ºC has lower resistivity and faster electron transfer rates12 than those prepared at lower temperatures. It has also been shown that the peak separation is
3-/4- reduced as the final pyrolysis temperature increases. For Fe(CN)6 , ΔEp at 800 ºC is
-1 277 mV whilst at 1100 ºC ΔEp is 80 mV, both at a scan rate of 200 mV s , and for
2+/3+ Ru(NH3)6 ΔEp at 800 ºC is 88 mV whilst at 1100 ºC ΔEp is 70 mV, both at a scan rate of 200 mV s-1.4 These pyrolysis effects though are only applicable for PPF prepared when the temperature is increased in one step at a rate of 10 °C/min13 to the specified temperature, instead of in a step wise manner.
Furthermore, a positive photoresist is preferred as less film shrinkage is observed when pyrolysed compared with negative photoresist,14-15 and graphite-like areas begin to form at lower temperatures in positive photoresist films (600 ºC) than in negative photoresist films (2700 ºC).14-15 These studies also show the importance of the
Chapter 3 | 88
pyrolysis heating program. Mass loss from the film is observed to begin from temperatures above 150 ºC, and between 250 ºC and 500 ºC there is the greatest loss of mass from the photoresist film as oxygen and nitrogen species are removed and carbonization begins. At temperatures above 600 ºC graphitisation occurs and the films begin to display nanocrystalline structures that have a significant contribution from both sp2 and sp3 type carbon.3, 10 Consistent with these structural changes in PPF, the rate of electron transfer increases as pyrolysis temperatures are increased.12
The atmosphere in which the photoresist is pyrolysed has also been studied by
McCreery and co-workers,4 and Lyons and co-workers,6, 10-11 where it was observed that a low O/C ratio is produced in an atmosphere of “forming gas” (95% N2, 5% H2).
Madou and co-workers12 and Lyons and co-workers6 examined the initial thickness of the photoresist and found the resistivity was independent of film thickness when pyrolysed at 1100 ºC. Pyrolysis of photoresist in inert environments, either under vacuum, N2, H2 or forming gas, leads to low oxide surfaces. In the case of pyrolysis under vacuum, the surfaces produced have the lowest O/C ratio of all. However, these films cannot be heated to temperatures above 1000 ºC4 and the final PPF films have higher resistance than those pyrolysed at 1100 ºC. A way to combat this is to use forming gas as the PPF produced has similarly low O/C ratio but this process allows the films to be heated to higher temperatures with the concomitant lower film resistance.4
A 10% increase in mass loss is seen between PPF samples pyrolysed in H2 rather than
6, 10 N2. Different gas flows have also been used for the fabrication of PPF films: 0.1 L min-1 by McCreery and 6 L min-1 by Downard and co-workers.2, 16
Chapter 3 | 89
There are two heating programs commonly used for pyrolysing photoresist. The first is the continuous heating of the photoresist directly to the required temperature at a constant rate (e.g. 10 ºC/min or 5 ºC/min) as performed by McCreery2 and Kinoshita.17
The second method is in a three step heating program used by Downard and co- workers.18-19 In this heating program the temperature is increased to a certain point and held there for a set amount of time. This process is repeated twice more until the temperature reaches 1050 ºC.18
Increasing electrochemical usage of PPF films has lead to a need to understand and control properties imparted during fabrication.14-15 This chapter explores the effects of different variables used during Downard’s three-step pyrolysis program,20 on the behaviour of PPF, and to understand how the combination of times and temperatures the
PPF is held at during this process can be used to make the production of PPF less of a
“dark art” and more reliable for mass production. Physical parameters of the films such as resistivity, film thickness and roughness were measured as well as electrochemical properties using a range of redox species to provide information on surface oxides, surface contamination and heterogeneous electron transfer kinetics. The ability of the
PPF to be modified and applied was also investigated.
3.2 EXPERIMENTAL METHOD
3.2.1 PPF PREPARATION
A 3-inch <100> n-type silicon wafer was cut into 1.4 × 1.4 cm2 pieces using a diamond scribe. The wafer pieces were then cleaned by sonicating in successive baths of acetone, methanol and isopropanol. These samples were then dried with nitrogen.
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Under yellow light, positive photoresist AZ 4620 was spin coated (3-4 drops/0.3-0.5 mL) onto the wafer pieces at 3000 rpm for 30 s. The wafer pieces were then heated at 95 ºC for twenty minutes to remove excess solvent but the films were still soft to touch. The samples were then kept overnight at 45 ºC.
The photoresist-coated wafer pieces were placed in three ceramic boats, five wafer pieces per boat. The boats were then placed in the furnace as illustrated in Fig.
3.1. The furnace was flushed with forming gas for 30 min before the pyrolysis program was started. Pyrolysis proceeded with forming gas flowing through the tube continuously at a flow speed of either 600 mL min-1 or 0.64 mL min-1. The pyrolysis programs used are shown in Table 3.1. The PPF samples were then left to cool down to room temperature slowly whilst still under forming gas. The PPF samples were removed and placed under vacuum until used.
Figure 3.1 Position of samples in tube furnace. The arrows show the gas flow direction.
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The PPF used for the modification with aryl diazonium salts was pyrolysed under run 6 conditions, T1 550 °C, t1 20 min, T2 680 °C, t2 15 min, T3 1100 °C, and t3 60 min. A gas flow of 0.64 mL min-1 was used.
3.2.2 EXPERIMENTAL DESIGN
For complex systems such as the one investigated here, there are too many factors to investigate by traditional ‘change one factor at a time’ methods. Here we demonstrate the use of a simple screening design, which allows us to study seven factors in only eight experiments. Even with these few experiments the effect of each factor is obtained as the average of four comparisons. Plackett-Burman designs21 are highly fractionated two-level designs that give main (independent) effects only. They require 4n experiments to obtain 4n – 1 main effects. Although the design gives no information about interactions among effects (which could well exist in the present system), and cannot offer optima of responses or a set of optimised parameters, they are valuable as a first step to understanding how changes in influence factors affect the characteristics of the carbon films. The efficiency of the design is particularly important when there is a cost, in time or money, in performing an experiment in a given set of conditions. Here the fabrication of a PPF electrode takes several hours, to which must be added the subsequent characterisation making time a cost to consider.
Two values (coded ‘-’ and ‘+’ in the design shown in Table 3.1) are chosen for each factor. By convention the ‘-’ level of a factor is the usual value applied in experiments and the ‘+’ is a value that is chosen to be sufficiently different to cause a measurable change in the response (if indeed the factor causes a change), but not so
Chapter 3 | 92
great as to take the experiment into nonsensical factor values. The ‘-’ value represents the usual value used, in this case we are using the temperature and time values investigated by Liu in her PhD thesis22 and Downard and co-workers values used for their preparation of PPF.16, 23 The ‘+’ values is one significantly different from the usual ‘-’ value. The main effect for each variable measured has the units of the response variable and is the average change in the variable when that factor goes from its ‘-’ value to its ‘+’ value. When a response (e.g. resistivity) is measured for each experiment, the main effect of a factor is calculated by summing over all experiments the product of the coded level of the factor (+1 or -1) and the response, and then dividing this sum by 4 (half the number of experiments). To negate the effects of uncontrolled variables, the order of performing the experiments is randomised.
A Plackett-Burman seven-factor screening design (Table 3.1) was used to investigate six heating parameters and the position of the sample in the heating tube
(centre, ends). Two flow rates were also investigated (0.64 mL min-1 and 600 mL min-
1) in separate Plackett-Burman designs. Five response variables were measured: three
2+/3+ 3-/4- cyclic voltammetry peak separations (ΔEp Ru(NH3)6 , ΔEp Fe(CN)6 , and ΔEp
Fe2+/3+) and the resistivity and the roughness of the carbon film. Measurement of peak separation provides information on heterogeneous electron transfer kinetics, and
2+/3+ 3-/4- indirectly, on surface oxides (ΔEp Fe ) and surface cleanliness (ΔEp Fe(CN)6 ).
The information obtained by the experiments described here could have been provided in a more efficient manner by a 12 experiment, 11 factor Plackett-Burman design (i.e. the next in the 4n series, n = 3). In this eight factors, namely 3 times, 3 temperatures, position and flow rate could have been investigated, with the remaining
Chapter 3 | 93
three factors being ‘dummy’ variables to give estimates of the variance of factor effects directly. However, at the planning stage, there was sufficient interest in the effect of flow rate that it was decided to obtain more information on this factor as described previously.
A main effect design, as used here, cannot give information about correlations among factors. It is expected that time and temperature will be correlated; to some extent, higher temperatures can be traded off against longer times. Factorial or composite designs can give such information at the expense of a greater number of experiments.
3.2.3 PHYSICAL CHARACTERISATION OF FILMS
Resistance of the PPF was measured using copper conducting wires set in a square arrangement 1 cm by 1 cm with reproducible pressure of the copper wires onto the carbon surface.24 The sheet resistance (R) for each PPF film was calculated as the average of 4 independent measurements. Resistivity (ρ) is
(R − R ) × w × d ρ = Cu equation 3.1 L
where RCu is the internal resistance of the copper bars, w is the distance between copper bars, L is the length of each bar (measured with vernier calipers) and d is the film thickness. Film thickness was obtained using a Sloan Dektak II profilometer.
Three PPF samples, one from each end of the furnace and one from the middle were
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scratched down to the substrate using a needle and the depth of the scratch was measured three times in different places and the average of these was calculated.
Atomic force microscopy (AFM) was performed on a Digital Instruments
Dimension 3000 AFM in tapping mode. Images of 10 μm by 10 μm were recorded and used for all roughness data presented herein.
3.2.4 ELECTROCHEMICAL CHARACTERISATION
Electrochemical measurements were carried out according to the methods in chapter section 2.3.2.1 the solutions were made though without the phosphate buffer.
3+ The redox systems used were 1.0 mM Ru(NH3)6 in 1.0 M KCl from
3- [Ru(III)(NH3)6]Cl3; 1.0 mM Fe(CN)6 in 1.0 M KCl from K3Fe(III)(CN)6; and 1.0 mM
2+ Fe in 0.2 M HClO4 from (NH4)2Fe(III)(SO4)2·6H2O and 70% HClO4. All solutions were made in Milli-Q water (resistivity > 18 MΩ cm). All solutions were degassed with dry nitrogen for 10 minutes prior to use. For each redox probe 5 cycles at 100 mV s-1 were run employing cyclic voltammetry techniques.
3.2.4.1 GLASSY CARBON
Glassy carbon electrodes were prepared as stated in chapter 2 section 2.3.1.2.
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3.2.4.2 ELECTRON TRANSFER RATES
Electron transfer rates were calculated according to the Nicholson method25 where a theoretical working curve (Fig. 3.2) showing the variation was used to provide the equation used to calculate 1/ψ for the ΔE values found for the redox systems.
Figure 3.2 Theoretical working curve from the Nicholson method showing the theoretical
variation of peak separation with a change in the charge transfer parameter ψ for ruthenium
hexaamine.25
From this the results are used in the following equation:
γ α k ψ = s equation 3.2 πaD0
Equation 3.2 can be rearranged to find the electron transfer rate
ψ × πaD k = 0 equation 3.3 s γ α
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nFυ a = equation 3.4 RT
Where for equation 3.3 ks = electron transfer rate, ψ = charge transfer parameter as found using the equation from Fig 3.2, π = pi, D0 = diffusion coefficient, γ = 1
(variable), α = 0.5 the transfer coefficient. The value a is shown in equation 3.4, n = number of electrons, F = Faradays constant, υ = scan rate, R = gas constant (J mol-1
-1 K ), T = temperature (K). The diffusion coefficient (D0) used for each redox systems is
3-/4- -6 2+/3+ -6 as follows: Fe(CN)6 D0 = 7.09 × 10 and Ru(NH3)6 D0 = 6.00 × 10 .
3.2.5 SYNTHESIS OF 4-CARBOXYPHENYL DIAZONIUM TETRAFLUOROBORATE
The synthesis of 4-carboxyphenyl diazonium tetrafluoroborate was carried out according to the method by Saby et.al..26 Para-aminobenzoic acid (1 g, 6.62 mM) was dissolved in hydrochloric acid (2 mL, 20 mM) and water (3 mL) was added slowly.
The mixture was then heated over a steam bath whilst stirring to dissolve the amine.
Once the solid was dissolved the solution was then cooled to –40 ºC in a dry ice/acetonitrile bath. A solution of sodium nitrite (0.52 g, 1.28 mM) in water (1 ml) was added dropwise. The solution was then filtered and the remaining filtrate was kept. To this sodium tetrafluoroborate (0.96 g) was added and dissolved and the solution allowed to reach room temperature. Colourless needle like crystals were obtained.
The mixture was then filtered to obtain the crystals, which were then rinsed with a cold solution of sodium tetrafluoroborate and then cold diethyl ether. They were then re-crystallised in a two solvent step of acetonitrile and diethyl ether. Colourless crystals
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1 where obtained in a 80% yield. H NMR (300 mHz, CDCl3): O δ 8.61 (d, J 12.9 Hz, 2H), δ 8.45 (d, J 12.9 Hz, 2H), δ 3.83 (bs, F4B N2 OH 1H).
3.2.6 MODIFICATION OF ELECTRODES
3.6.2.1 ELECTROCHEMICAL REDUCTION OF 4-CARBOXYPHENYL DIAZONIUM
TETRAFLUOROBORATE
A solution of the 4-carboxyphenyl diazonium tetrafluoroborate (10 mM) was made up in acetonitrile with tetrabutylammonium tetrafluoroborate (16 mM) to act as the electrolyte. The carbon electrodes were exposed to the aryl diazonium solution and a cyclic voltametry scan was carried out scanning from 0.2 V to -1.0 V for 2 cycles against a Ag|AgCl [3 M] reference electrode. Investigation into the passivation of the surface was carried out using a solution of 10 mM ruthenium hexamine in phosphate buffer (pH
7) and a solution of 10 mM ferricyanide in phosphate buffer (pH 7) with the same electrochemical conditions as previously mentioned.
3.2.6.2 ATTACHMENT OF PEPTIDE TO SURFACE
Once the aryl diazonium salt is attached the carboxylic acid is activated with 1-
40 mM ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 20 mM
N-hydroxysuccinimide (NHS) for an hour then exposed to a solution of the peptide 50 mg mL-1 glycyl-glycyl-histidine (Gly-Gly-His) overnight in 0.1 M 2-(N- morpholino)ethanesulfonic acid (MES) buffer solution (pH 6.8).
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3.2.6.3 COPPER ACCUMULATION
The electrodes were exposed to a solution of copper (II) for ten minutes at 37°C.
The electrodes were removed from this solution and placed in a copper free ammonium acetate buffer (pH 7) and NaCl (50 mM) solution. An Osteryoung squarewave voltametry (OSWV) was carried out in this solution to measure the amount of copper.
The copper was removed from the peptide by holding at a set potential of +0.5 V in
HClO4 (0.1 M) for 15 seconds. The electrode can then be reused for the accumulation of copper.
A calibration was carried out using various concentrations of copper sulphate solution in Milli-Q water. These concentrations were 0 nM, 1 nM, 5 nM, 10 nM, 50 nM,
0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 1mM. The order the standards were measured in was randomised. Each electrode was used four times before being remade, and each concentration was measured three times with different electrodes, each with their own order for measuring the standards. After each accumulation an OSWV was measured, the copper was then removed by holding at a potential of +0.5 V in HClO4 (0.1 M) for
15 seconds. Another OSWV was carried out in copper free ammonium acetate buffer
(pH 7) and NaCl (50 mM) solution to measure the base line for the next measurement. If the copper was not fully removed in the first pulse, a second pulse was carried out and the base line re-measured.
The same method was carried out on both GC and PPF electrodes.
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3.3 RESULTS AND DISCUSSION
The general method of preparation, including type of photoresist to use, general heating program, and spin speed to use, has already been investigated previously by
Guozhen Liu.27 However the effect of the different heating stages in the preparation and their effect on the properties of the PPF will be examined here. The change in gas flow effects will also be examined in the following chapter. For this investigation, as there are eight different effects to be investigated an experimental design is required.
For PPF to be used in affinity biosensors we require the PPF to be near atomically smooth, with a rms value of 0.3 to 1 nm, they also need to have a low resistivity < 10 mΩ cm. The PPF also needs to have good electrochemical behaviour and low surface impurities. Liu found that using 2-3 drops of the photoresist AZ4620, and spin coating for 30 s at 6000 rpm gave a decent thickness of photoresist on the silicon wafer. The pyrolysis program 550ºC for 40 min, then 750ºC for 30 min, and
1050ºC for 60 min was used. From using these settings the PPF produced had a final film thickness 0.8 μm with a surface roughness of 0.4 – 0.6 nm.
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3.3.1 CHOICE OF FACTOR VALUES
Run T1 /ºC T2 /ºC T3 /ºC t1 /min t2 /min t2 /min position
1 500(+) 680(+) 1050(−) 40(−) 15(+) 90(+) centre (−)
2 550(−) 750(−) 1050(−) 40(−) 30(−) 60(−) centre (−)
3 550(−) 750(−) 1050(−) 20(+) 15(+) 90(+) end (+)
4 500(+) 750(−) 1100(+) 20(+) 30(−) 90(+) centre (−)
5 500(+) 680(+) 1050(−) 20(+) 30(−) 60(−) end (+)
6 550(−) 680(+) 1100(+) 20(+) 15(+) 60(−) centre (−)
7 550(−) 680(+) 1100(+) 40(−) 30(−) 90(+) end (+)
8 500(+) 750(−) 1100(+) 40(−) 15(+) 60(−) end (+)
Table 3.1 Levels of seven factors in eight preparations of a PPF electrode in a randomised
Plackett-Burman experimental design. The ‘−’ indicates the usual value applied in the
experiments, whilst the ‘+’ indicates a value significantly different to cause a measurable
change in the response (if indeed the factor causes a change), but not so great as to take the
experiment into nonsensical factor values.
The staggered heating program of Downard and co-workers16, 18 is used which gives six factors to be investigated, three times and three temperatures as shown in Fig.
3.3.
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Figure 3.3 Heating profile for pyrolysis of photoresist films. T1, T2 and T3 refer to heating
temperatures and t1, t2 and t3 refer to heating times.
The three temperatures were chosen as they fall into roughly the three areas of the pyrolysis process. Between 250 ºC and 500 ºC the greatest mass loss occurs during pyrolysis.4 Carbonisation starts from 600 ºC3 and it has been shown that the best result in terms of resistance and electron transfer for final pyrolysis temperatures is between
1000 ºC and 1100 ºC. Table 3.1 gives the values corresponding to the Plackett-Burman levels -1 and +1.
In addition, the last factor in the design was the position of the PPF in the furnace. The positions of the PPF and direction of gas flow are schematically illustrated in Fig. 3.1. Two gas flows were chosen in this study; 0.64 mL min-1 and 600 mL min-1 with the full Plackett-Burman design being repeated at each gas flow.
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The number of potential factors in the production of a PPF film that can be studied (several temperatures, times, flow rate and position) requires a carefully designed experimental campaign. The chosen Plackett-Burman design minimises the number of experiments at the expense of knowledge of two-way and higher order interactions.28 As with all experimental designs, the random variation in the response variables arising from uncontrolled factors in the experiment must be minimised, as any effect that is less than the random variation might not be identified as significant. The raw results from the response variables measured in accordance with the position
(middle or edge position in tube furnace) as a variable (voltammetric peak separation
3-/4- 2+/3+ 2+/3+ for Fe(CN)6 , Ru(NH3)6 and Fe , the films resistivity and film roughness) for the two flow rates are shown in Table 3.2 and Table 3.3.
ΔEp ΔE Fe(CN) 3- ΔE Fe2+/3+ Resistivity Roughness p 6 2+/3+ p Run Ru(NH3)6 /4- /mV /mV /(mΩ cm) /nm /mV 1 105 88 579 3.82 0.38 2 413 276 564 4.22 0.44 3 400 107 337 11.00 0.44 4 322 195 432 5.85 1.15 5 215 327 542 9.68 1.29 6 134 110 457 3.72 0.98 7 969 127 610 6.51 3.33 8 383 103 149 5.46 8.54
Table 3.2 Measured values of response variables for preparation of PPF electrodes with a gas
flow rate of 600 mL/min per position variable.
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ΔEp ΔE Fe(CN) 3-/4- ΔE Fe2+/3+ Resistivity Roughness p 6 2+/3+ p Run Ru(NH3)6 /mV /mV /(mΩ cm) /nm /mV 1 918 134 806 2.40 2.75 2 820 115 571 3.13 5.17 3 132 122 357 6.60 0.67 4 632 68 615 1.93 1.92 5 649 115 610 15.10 0.76 6 264 186 782 1.93 1.34 7 737 176 664 5.05 23.59 8 474 110 550 0.43 0.90
Table 3.3 Measured values of response variables for preparation of PPF electrodes with a gas
flow rate of 0.64 mL/min per position variable.
2+/3+ Generally the faster gas flow of 600 mL/min gave lower ΔEP values for Fe
2+/3+ and had an over all lower surface roughness. The low ΔEP values for Fe suggest a smaller number of oxides on the surface of the PPF formed in the faster gas flow. For both fast and slow gas flows the resistivity was low and below a working value of 30
2+/3+ mΩ cm. The ΔEP values for Ru(NH3)6 in both slow and fast gas flows are generally low except for runs 2 and 5, this indicates that both gas flows produce PPF surfaces that
3-/4- are able to be used for electrochemistry. The ΔEP values for Fe(CN)6 show no consistency between runs or gas flows, run one in the fast gas flow shows a value of
105 mV whilst for the slow gas flow has a value of 918 mV which a large difference between the two. This is different from run 6 in which both gas flows give a low value
3-/4- for ΔEP Fe(CN)6 134 mV and 264 mV for the fast and slow gas flow respectively.
The main effects are calculated from this data and shown in Table 3.4 for the faster flow rate and Table 3.5 for the slower flow rate. The signs have been adjusted to
Chapter 3 | 104
show the effect of moving from the smaller value to the greater value. In the case of position the ‘positive’ direction of the change is from middle to edge.
Response variable T1 T2 T3 t1 t2 t2 position 3-/4- ΔEp Fe(CN)6 223 24 169 200 224 163 248 /mV 2+/3+ ΔEp Ru(NH3)6 -23 7 -66 -37 129 -74 -1 /mV 2+/3+ ΔEp Fe 266 -706 -374 134 628 247 -393 /mV Resistivity 0.16 0.70 -1.79 -2.56 0.57 1.02 3.76 /(mΩ cm) Roughness 1.56 -2.22 0.83 -3.14 -1.21 -2.58 -1.62 /nm
Table 3.4 Main effects for preparation of PPF electrodes with a gas flow rate of 600 mL/min,
where the effect is the average change in response variable as the parameter shifts from it’s ‘-’
value to it’s ‘+’ value.
Response variable T1 T2 T3 t1 t2 t2 position 3-/4- ΔEp Fe(CN)6 -180 -128 -103 318 263 53 -160 /mV 2+/3+ ΔEp Ru(NH3)6 43 -49 13 11 -19 -6 5 /mV 2+/3+ ΔEp Fe -52 -192 67 57 -9 -18 -148 /mV Resistivity -0.79 -3.10 -4.47 -3.64 3.46 -1.15 4.45 /(mΩ cm) Roughness 6.11 -4.94 4.60 6.93 6.45 5.19 3.68 /nm
Table 3.5 Main effects for preparation of PPF electrodes with a gas flow rate of 0.64 mL/min,
where the effect is the average change in response variable as the parameter shifts from it’s ‘-’
value to it’s ‘+’ value.
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The main effects calculated show little correlation between the different variables of time, temperature and position. The positive and negative signs indicate a positive or negative effect of the variable on the measured response variables. Both fast and slow gas flows show different effects indicating that as gas flows change, different heating parameters become important and others less important. Some of the more evident effects are discussed here. Position in the furnace has the greatest effect on ΔEp
3-/4- Fe(CN)6 , making the peak separation greater, but reducing the peak separation for
2+/3+ 2+/3+ ΔEp Fe at the faster flow rate. For ΔEp Fe the peak separation decreases, and hence the rate of electron transfer increases, at the higher gas flow. For most variables the effect of the change of factor level is reduced when there is a slower flow. We also found that the reproducibility of the responses was reliable for the slower flow rate.
The peak separation of the ruthenium couple was the most unstable.
3.3.3 UNCERTAINTY IN PREPARATION AND MEASUREMENT
Where possible the repeatability of the measurement of each response has been estimated from replicate measurements on a single prepared electrode. Their values are reflected in the significant figures reported for the results in Table 3.2 and Table 3.3.
For example, for the first run with a slow gas flow the repeatability relative standard deviation (RSD) of the resistivity for all positions in the furnace was 2.7% (n = 4).
Typical confidence intervals calculated from these data are shown in Fig. 3.4. There is an art in preparing reproducible electrodes that depends critically on day to day variations in gas flow, temperature, position in the furnace and so on. The data given here reflects this, but despite the variability it does allow some conclusions to be drawn.
Chapter 3 | 106
3.3.4 PHYSICAL CHARACTERISTICS OF PPF SAMPLES
3.3.4.1 RESISTIVITY
In general, it can be seen that the resistivity of the films is greatly affected by the position of the sample within the furnace (Fig. 3.4). The heating capacity of the furnace is at a maximum in the middle of the tube. Although, according to the supplier of the furnace, the heat should be uniform within the region in which the PPF samples are located (i.e. the heat in the centre of the tube should be the same as within 4 cm of either end of the tube). It can be seen that positioning at the ends of the furnace leads to greater resistivity, whilst those in the middle that have been subjected to a greater heat have a lower resistance. It is however important to note that within this range of resistivity there is no direct correlation with the electron transfer rate as can be seen in
Table 3.2. Furthermore, it is clear from Table 3.2 that the rate of gas flow does not influence the resistivity. Resistivity in slower gas flows was favoured by higher temperature and longer times for t1 and t3. At higher flow rate the position was less clear; what remained the same was the influence of position.
Chapter 3 | 107
Figure 3.4 Resistivity of electrodes prepared from PPF carbon films as a function of position in
the furnace. Positions refer to area in furnace, gas inlet describes the positions 1-5, middle
describes the positions 6-10, and gas outlet describes the positions 11-15. Error bars are 95% confidence intervals of the mean of four separate measurements. Upper line; flow rate 600 = mL
min-1, lower line; flow rate = 0.64 mL min-1.
3.3.4.2 FILM THICKNESS
Film thickness is also not affected by either gas flow or heating parameters.
Within the variables assessed film thickness is only influenced by the initial amount of photoresist spin coated onto the silicon wafer, and the spin speed. The greater the amount of photoresist on the wafer either due to too much applied during spin coating or the spin speed being too slow, flaking of the pyrolysed photoresist occurs as seen in
Fig. 3.5. Conversely, roughness is affected by the gas flow.
Chapter 3 | 108
a 1cm b c
Figure 3.5 Effect of spin speed and amount of photoresist on PPF stability. a) Good spin
speed and amount of photoresist, PPF surface is smooth with no pealing of PPF from silicon
wafer. b) Too much photoresist during spin coat, entire surface of PPF is pealing from the
silicon wafer. c) Slow spin speed, inside area of PPF is smooth and stable, however the outer
edges are pealing from the silicon wafer.
3.3.4.3 ROUGHNESS
Roughness generally increased (the main effect was positive) at the slower flow rate as can be seen by comparing Table 3.2 and Table 3.3, while at the greater flow rate longer pyrolysis times promoted negative main effects. For each flow rate, higher T1 and T3 favoured greater roughness. The ranges of the roughness increased as the gas flow decrease, for the fast gas flow the roughness ranged between 0.38 nm to 8.54 nm
(Fig. 3.6a) whilst for the slow gas flow the roughness varies between 0.76 nm to 23.59 nm (Fig. 3.6b and c respectively). Higher temperatures during the carbonisation step
(T2) gave smaller roughness values (Fig. 3.6d). As a smooth, reproducible surface that has a lower surface roughness or rms value than glassy carbon is required, if the surface is near atomically smooth even better. For these desired roughness properties we require the surface roughness to be less than 2 nm.
Chapter 3 | 109
a b
c d
Figure 3.6 Cross section of AFM image showing roughness a) Fast gas flow run 8. b) Slow
gas flow runs 5. c) Slow gas flow 7. d) Fast gas flow run 6.
3.3.5 ELECTROCHEMICAL CHARACTERISTICS OF PPF SAMPLES
2+/3+ It can be seen the effects on ΔEp Ru(NH3)6 are smaller than the other redox
3-/4- couples such as Fe(CN)6 (Fig. 3.7). As ΔEp is indicative of the kinetics of electron transfer,25 this result is consistent with our knowledge of the electrochemistry as the
2+/3+ Ru(NH3)6 couple is regarded as truly outer sphere and hence the electrochemistry should not be greatly influenced by the nature of the surface of the electrode.29-30
As noted in Tables 3.2 and 3.3 no correlation can be seen between larger ΔEp for
2+/3+ Ru(NH3)6 and resistance. From these results it can be observed that the faster gas
2+/3+ flow of 0.64 mL/min gives a more consistent ΔEp Ru(NH3)6 value. In comparison those PPF surfaces produced with a slower gas flow gave a larger variation in response
(a difference or 239 mV). The consistency of the fast gas flow can be seen in
Chapter 3 | 110
comparing fig. 3.8a and 3.8b were both run 1 and run 6 for the fast gas flow show
2+/3+ smaller ΔEp Ru(NH3)6 values, whilst the inconsistency seen in the slow gas flows
2+/3+ can also bee seen in fig. 3.8a and 3.8b were run 1 shows a small ΔEp Ru(NH3)6
2+/3+ value where as run 6 has a large ΔEp Ru(NH3)6 value. This suggests a difference in carbon structure which was investigated further in this study.
a
b
2+/3+ Figure 3.7 Electrochemistry of 1 mM Ru(NH3)6 in 1 M KCl scanned at 100 mV/s, for the
different gas flow rates, fast gas flow 600 mL min-1 and slow gas flow 0.64 mL min-1)
a) Run 6 fast (blue line) and slow (red line) gas flow rate. b) Run 1 fast (blue line) and
slow (red line) gas flow rate.
3-/4- The Fe(CN)6 couple is classed as inner sphere but insensitive to surface
3-/4- oxides. Fe(CN)6 is however sensitive to surface cleanliness in regard to adventitious
Chapter 3 | 111
impurities.29-32 These results provide an indication that the range of resistivities observed as a function of position in the tube furnace, do not have a significant effect on
3-/4- the electron transfer kinetics. In comparison ΔEp for the Fe(CN)6 couple is, for the majority of runs, smaller at the faster gas flow rate. An example of the electrochemical behaviour is illustrated in Fig. 3.8 for the two different gas flow rates for run 1 and run
3-/4- 6. In general the “pattern” of ΔEp of Fe(CN)6 was repeated throughout both gas flow rates which indicates that the heating parameters have some influence on the response
3-/4- of Fe(CN)6 .
a
b
3-/4- Figure 3.8 Cyclic voltammograms of 1 mM Fe(CN)6 , 1 M KCl scanned at 100 mV/s, for the
different gas flow rates, fast gas flow 600 mL min-1 and slow gas flow 0.64 mL min-1)
a) Run 1 fast (blue line) and slow (red line) gas flow rate. b) Run 6 fast (blue line) and slow
(red line) gas flow rate.
Chapter 3 | 112
In Table 3.6 a comparison with other studies is made for the surface with the
3-/4 -1 fastest rate of electron transfer for Fe(CN)6 , run 1 for the 600 mL min flow rate.
The electron transfer rates are comparable to those obtained for previous studies carried out by the groups of both McCreery2 and Downard16 for their best set of conditions for
PPF fabrication. McCreery and co-workers have also noted that the ΔEp of
2+/3+ -1 Ru(NH3)6 were slightly greater on PPF (ΔEp = 222 mV, scan rate 20 Vs , kº 0.020
-1 -1 -1 2 cm s ) than on GC (ΔEp = 125 mV, scan rate 20 Vs , kº 0.037 cm s ) with a difference of 93 mV which is contrary to observations from the current study.
This study PPF Gas flow PPF Gas flow Glassy PPF2 PPF16 0.64 mL min-1 600 mL min-1 carbon 3-/4- -6 -1 ks Fe(CN)6 / 10 cm s 1300 7000 400 100 1500 2+/3+ -6 -1 ks Ru(NH3)6 / 10 cm s 300 3200 200 200 2600 2+/3+ -6 -1 ks Fe / 10 cm s 15700 18200 34100 210000 -
Table 3.6 Comparisons of electron transfer rate constant for certain redox probes calculated
25 from ΔEp according to the Nicholson method as stated in the experimental method.
To examine the oxide content, the redox probe Fe2+/3+ was used as it has been shown that as oxide content increases on the surface the rate of electron transfer
30, 32 increases. It was seen that, in general, the runs with the higher T2 of 750 ºC had a
2+/3+ 2+/3+ greater ΔEp for Fe . It was observed that the ΔEp for Fe did not correlate with
3-/4- either fast or slow electron transfer kinetics for Fe(CN)6 thus implying there is no correlation between surface cleanliness and oxide content, this has also been shown by
McCreery on glassy carbon.30
Chapter 3 | 113
2+/3+ This trend in ΔEp for Fe was also seen in the pyrolysis runs with a slower gas flow rate; those runs with a lower T2 showed higher ΔEp values. It was also observed that the change in gas flow does not significantly influence the surface oxides (see
Table 3.2 and Table 3.3).
3.3.6 ELECTROCHEMICAL USABILITY AND FUNCTIONALITY
Since the PPF was seen to behave well for the redox active complex of
3-/4- 2+/3+ Fe(CN)6 and Ru(NH3)6 , it was decided to investigate the ability of the PPF to be functionalised and used for biosensor applications. One of the main surface modification techniques used for carbon as seen in Chapter 1, is the electrochemical reduction of aryl diazonium salts.33-36 Liu et.al. has shown that an aryl diazonium salt with a para substitution of the peptide glycine glycine histidine (Gly-Gly-His) can be used for the detection of copper (II) in water.37
The application of the peptide Gly-Gly-His in the detection of copper (II) in water has been noted in previous studies,38-40 where the complexation of the copper occurs via the deprotonation of the amide nitrogens and a 4N sequence planar complexation occurs as shown in Fig 3.11. Electrodes modified with this peptide have been shown to be highly sensitive copper sensors with good selectivity.39-40 It has been shown that sensors constructed using gold-thiol layers which are then modified with
Gly-Gly-His have instability in the gold-thiol bond. The possibility of under-deposition of the copper giving a gold-copper-thiol relationship which can cause interference of the sensor and thus a Cu2+/Cu0 reduction is also seen.41 The use of aryl diazonium salts on
GC instead of the gold-thiol sensors prevents under-deposition of the copper producing
Chapter 3 | 114
a Cu2+/Cu1+ reduction and less interference.22 If PPF’s are an alternative to glassy carbon then sensors fabricated using these films should perform in a similar manner to the same sensor fabricated on glassy carbon electrodes.
This section shows that sensors formed on PPF performs similarly to the same sensor formed on GC. To do this PPF will be modified with a simple aryl diazonium salt and the further modification with the peptide Gly-Gly-His will be carried out. This surface will then be compared to that of an electrode surface of GC that has been modified in the same manner. For PPF to be equivalent to GC as electrode substrate the behaviour and response needs to be studied on both.
The comparison of the sensor based on the peptide Gly-Gly-His was used as it has been intensively studied by Gooding and co-workers.22, 37-41 The sensor surface is modified with 4-carboxyphenol diazonium salt, which is then activated and the attachment of the peptide Gly-Gly-His occurs as seen in Fig. 3.9. Once the peptide is attached to the surface the un-complexed peptide shows no electrochemical signal, but when Cu (II) is complexed with the peptide an electrochemical signal due to the reduction of Cu2+ to Cu1+ is seen and corresponds to the amount of copper in the sample solution.
Chapter 3 | 115
Figure 3.9 Modification of Carbon substrates with 4-carboxyphenol diazonium salt, followed
by the attachment of the peptide Gly-Gly-His. The OSWV show no signal without copper, and
a signal once the electrode has been exposed to copper.
3.3.6.1 COMPLEXATION OF COPPER(II) WITH THE PEPTIDE GLY-GLY-HIS BOUND TO
A CARBON SUBSTRATE.
It was seen from cyclic voltammetry that the electrochemical reduction of the aryl diazonium salt, 4-carboxyphenol diazonium salt, had occurred, due to the loss of the reduction peak. The surface was assumed to be passivated on both the glassy carbon and the pyrolysed photoresist film electrodes as seen in Fig. 3.10, due to the observation of a decrease in size of the reduction peak for the aryl diazonium salt.
Chapter 3 | 116
Figure 3.10 Cyclic voltammogram of the electrochemical reduction of 4-carboxyphenyl
diazonium salt in acetonitrile at a scan rate of 0.1 V/s. both electrodes, GC and PPF, have a
working electrode area of 7.1 mm2. a) PPF (first scan in red, second scan in blue). b) GC (first
scan in red, second scan in blue).
Further investigation into the surface modification of PPF with 4-carboxyphenol diazonium salt, was carried out by observing the passivation with ferricyanide. These investigations showed that the negative charge on the carboxylic acid of the bound
3-/4- diazonium salt to the surface prohibited the Fe(CN)6 from coming close to the
3-/4- surface. As mentioned previously Fe(CN)6 is an inner sphere complex, therefore it needs to approach the surface for it to undergo oxidation and reduction. The thickness
3-/4- of the film produced would also hinder Fe(CN)6 as the thickness of the modified layer, as the reduction of aryl diazonium salts form multilayers, would also hinder access to the electrode surface. The decrease of the ferricyanide peaks indicates the presence of the 4-carboxyphenyl bound to the surface. This can be seen in Fig. 3.11
3-/4- where the Fe(CN)6 produces peaks before surface modification and after modification the surface has been passivated and the reduction and oxidation of
3-/4- Fe(CN)6 is “blocked” or reduced.
Chapter 3 | 117
Figure 3.11 Cyclic voltammogram of 10 mM ferricyanide in phosphate buffer (pH 7) on PPF
before (red trace) and after (blue trace) surface modification with 4-carboxyphenyl diazonium
salt, the scan rate was 0.1 V/s.
3.3.6.2 COMPLEXATION OF COPPER (II) TO GLY-GLY-HIS
Copper accumulation was carried at a maintained pH of 7 using ammonium acetate buffer at a temperature of 37 °C as was previously performed on glassy carbon and gold electrodes.37, 40 It was seen that on both the GC surfaces and the PPF surfaces that the accumulation of copper could be achieved, as determined from the redox peaks in the square wave voltammograms (Fig 3.12). Furthermore, the copper could be removed from the electrode surfaces by holding the electrode at the +0.5 V in HClO4
(Fig. 3.12).
Chapter 3 | 118
Figure 3.12 Osteryoung square wave voltametry of Gly-Gly-His on a PPF electrode in
ammonium acetate buffer (pH 7) after 10 min Cu2+ accumulation in 0.05 M ammonium acetate
buffer (pH 7), blue line; and after the electrode has been held at the se;;t potential +0.5 V in 0.1
M HClO4 solution to release the copper from the peptide, red line.
A calibration curve was carried out on both the glassy carbon and the pyrolysed photoresist film to compare the behaviour of the surfaces as electrodes, and the feasibility of the comparison. The GC behaved as expected and produces a calibration curve (Fig. 3.13) with a detection limit of around 5 nM. This was comparable to previous studies carried out by Liu et al.37
It was seen that in the PPF samples there was some variability between electrodes this can be put down to position in the furnace, but on the whole a calibration curve could be achieved (Fig. 3.13). The detection limit was estimated to be 5 nM which is similar to that found of glassy carbon. It is also noted that PPF surfaces have a lower sensitivity towards the detection of copper as can be seen in Fig. 3.14 where the linear region is higher in comparison to that of the GC linear region. Although for the
PPF electrodes there are not enough points to determine the exact linear region in comparison to the GC linear region they are relatively close.
Chapter 3 | 119
Figure 3.13 Curve of average current density versus concentration of copper(II) for the
detection of copper on GC (blue) and PPF (red).
Figure 3.14 Linear region of average current density versus concentration of copper(II) curve
for Cu detection on PPF and GC.
Chapter 3 | 120
3.4 CONCLUSION
The use of the Plackett and Burman design makes it possible to study the influence of the different factors on the final PPF produced and has served as a guide to the types of experiments to perform. However, from the results presented in Table 3.2 and Table 3.3 it is clear there is a high degree of variability between runs which compromises the depth of information that can be extracted from the design. However, general trends can be observed and the important and unimportant factors identified.
This information allows fabrication guidelines for PPF to be stated for specific end-user requirements (Table 3.7). If the surface roughness and cleanliness are not important but good electron transfer kinetics are, then fast gas flow rates are not needed. Faster gas flow rates will, however, give far cleaner surfaces and better electron transfer kinetics.
Further surface characteristics such as surface roughness and oxide content can be controlled through heating variables as seen in Table 3.7.
It can also be seen from these studies that both GC electrodes and PPF’s are comparable as electrode substrates. The reduction of aryl diazonium salts onto pyrolysed photoresist film surfaces is comparable to that of glassy carbon, and they are both able to be applied to sensing applications.
Chapter 3 | 121
Temp 1 Temp 2 Temp 3 Time 1 Time 2 Time 3 Desired surface Gas Flow General (ºC) (ºC) (ºC) (min) (min) (min) properties mL min-1 statements T1 T2 T3 t1 t2 t3 • Fast electron transfer, High final • clean surface, 600 temperature 550 680 1100 20 15 60 • low surface (fast) for a shorter roughness <0.6 time nm • Fast electron transfer, Lower final clean surface, 600 temperature • 500 680 1050 40 15 90 • higher surface (fast) for a longer roughness > 1.0 time nm • low surface oxide Low T2, and 600 • low surface 500 680 1050 20 30 60 T1 held for a (fast) roughness <0.6 short time nm • low surface oxide Low T2, and 600 • higher surface 550 680 1100 40 30 90 T1 held for a (fast) roughness > 1.0 longer time nm • Higher surface oxide 600 • Low surface 550 750 1050 20 15 90 High T2 (fast) roughness < 0.6 nm Generally the same trends • Fast electron occur for the 0.64 transfer 550 750 1050 40 30 60 slower gas (slow) • Rough surface flow but the surfaces are rougher
Table 3.7 General guidelines for PPF surface properties.
Chapter 3 | 122
3.5 REFERENCES
1. Yu, S. S. C.; Downard, A. J., Dynamic behavior of organic thin films attached to carbon
surfaces. e-J. Surf. Sci. Nanotech. 2005, 3, 294-298.
2. Ranganathan, S.; McCreery, R. L., Electroanalytical performance of carbon films with
near-atomic flatness. Anal. Chem. 2001, 73 (5), 893-900.
3. Kostecki, R.; Schnyder, B.; Alliata, D.; Song, X.; Kinoshita, K.; Kotz, R., Surface
studies of carbon films from pyrolyzed photoresist. Thin Solid Films 2001, 396, 36-43.
4. Ranganathan, S.; McCreery, R. L.; Majji, S. M.; Madou, M., Photoresist-derived carbon
for microelectromechanical systems and electrochemical applications. Journal of the
Electrochemical Society 2000, 147 (1), 277-282.
5. Schueller, O. J. A.; Brittain, S. T.; Marzolin, C.; Whitesides, G. M., Fabrication and
Characterization of Glassy Carbon MEMS. Chemistry of Materials 1997, 9 (6), 1399-
1406.
6. Lyons, A. M.; Wilkins Jnr., C. W.; Robbins, M., Thin pin hole free carbon films. Thin
solid films 1983, 103, 333-341.
7. Schueller, O. J. A.; Brittain, S. T.; Whitesides, G. M., Fabrication of glassy carbon
microstructures by pyrolysis of microfabricated polymeric precursors. Advanced
Materials (Weinheim, Germany) 1997, 9 (6), 477-480.
8. Tao, S. L.; Popat, K. C.; Norman, J. J.; Desai, T. A., Surface modification of SU-8 for
enhances biofunctionality and nonfouling properties. Langmuir 2008, 24 (6), 2631-
2636.
9. Roy, D.; Basu, P. K.; Raguhunathan, P.; Eswaran, S. V., Photo-induced cross linking
maechanism is azide-novolac negatice photoresists: molecular level investigation using
NMR spectroscopy. Magnetic Resonance in Chemistry 2003, 41, 671-678.
10. Lyons, A. M., Photodefinable carbon films: Electrical properties. J. Non-Cryst. Solids
1985, 70, 99-109.
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11. Lum, R.; Wilkins Jnr., C. W.; Robbins, M.; Lyons, A. M.; Jones, R. P., Thermal
Analysis of Graphite and Carbon-Phenolic Composites by Pyrolysis-Mass
Spectrometry. Carbon 1983, 21 (2), 111-116.
12. Kim, J.; Song, X.; Kinoshita, K.; Madou, M.; White, R., Electrochemical studies of
carbon films from pyrolysed photoresist. J. Electrochem. Soc. 1998, 145 (7), 2314-
2319.
13. Ranganathan, S. Preparation, Modification and Charcterisatio of a Novel Carbon
Electrode Material for Applications in Electrochemistry and Molecular Electronics.
Ohio State University, Ohio, 2001.
14. Park, B. Y.; Taherabadi, L.; Wang, C.; Zoval, J.; Madou, M. J., Electrical properties and
shrinkage of carbonized photresist films and the implications of carbon
microelectromechanical sytems devices in conductive media. J. Electrochem. Soc.
2005, 152 (12), J136-J143.
15. Singh, A.; Jayaram, J.; Madou, M. J.; Akbar, S., Pyrolysis of Negavtive photoresists to
fabricate carbon structures for micromechanical systems and electrochemical
applications. J. Electrochem. Soc. 2002, 149 (3), E78-E83.
16. Brooksby, P. A.; Downard, A. J., Electrochemical and Atomic Force Microscopy Study
of Carbon Surface Modification via Diazonium Reduction in Aqueous and Acetonitrile
Solutions. Langmuir 2004, 20 (12), 5038-5045.
17. Kostecki, R.; Song, X.; Kinoshita, K., Electrochemical analysis of carbon interdigitated
microelectrodes. Electrochem. solid-state lett. 1999, 2 (9), 465-467.
18. Brooksby, P. A.; Downard, A. J., Nanoscale Patterning of Flat Carbon Surfaces by
Scanning Probe Lithography and Electrochemistry. Langmuir 2005, 21 (5), 1672-1675.
19. Tan, E. S. Q. Assembly of Organic Layers onto Carbon Surfaces. University of
Canterbury, Canterbury, 2006.
20. Tan, E. S. Q. Assembly of Organic Layers onto Carbon Surfaces. University of
Canterbury, Christchurch, 2006.
Chapter 3 | 124
21. Plackett, R. L.; Burman, J. P., The Design of Optimum Multifactorial Experiments.
Biometrika 1946, 33 (4), 305-325.
22. Liu, G. Creating Stable and Versatile Monolayer Systems on Carbon Substrates for
Sensors and other Applications. PhD., The University of New South Wales, Sydney,
2006.
23. Downard, A. J., Electrochemically assisted covalent modification of carbon electrodes.
Electroanalysis 2000, 12 (14), 1085-1096.
24. Brooksby, P. A.; Downard, A. J.; Yu, S. S. C., Effect of applied potential on arylmethyl
films oxidatively grafted to carbon surfaces. Langmuir 2005, 21 (24), 11304-11311.
25. Nicholson, R. S., Theory and application of cyclic voltammetry for measurement of
electrode reaction kinetics. Anal. Chem. 1965, 37 (11), 1351-1355.
26. Saby, C.; Ortiz, B.; Champagne, G. Y.; Belanger, D., Electrochemical modification of
glassy carbon electrode using aromatic diazonium salys. 1. Blocking effect of 4-
nitrophenyl and 4-carboxyphenyl groups. Langmuir 1997, 13 (25), 6805-6813.
27. Liu, G.; Liu, J.; Boecking, T.; Eggers, P. K.; Gooding, J. J., The modification of glassy
carbon and gold electrodes with aryl diazonium salt: The impact of the electrode
materials on the rate of heterogeneous electron transfer. Chemical Physics 2005, 319 (1-
3), 136-146.
28. Hibbert, D. B., Quality assurance for the analytical chemistry laboratory. 1 ed.; Oxford
university press: 2007; p 306.
29. Kneten Cline, K. R.; McDermott, M. T.; McCreery, R. L., Anomalously slow electron
Transfer at ordered graphite electrodes: Influence of electronic factors and reactive
sites. J. Phys. Chem. 1994, 98 (20), 5314-5319.
30. McCreery, R. L.; Cline, K. K.; McDermott, C. A.; McDermott, M. T., Control of
reactivity at carbon electrode surfaces. Colliods Surf., A 1994, 93, 211-219.
31. McCreery, R. L., Electrochemical properties of carbon surfaces. In Interfacial
electrochemistry, 1 ed.; Wieckowski, A., Ed. Marcel Dekker: New York, 1999.
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32. Chen, P.; McCreery, R. L., Control of electron transfer kinetics at glassy carbon
electrodes by specific surface modification. Anal. Chem. 1996, 68 (22), 3958-3965.
33. Allongue, P.; Delamar, M.; Desbat, B.; Fagebaume, O.; Hitmi, R.; Pinson, J.; Saveant,
J.-M., Covalent Modification of Carbon Surfaces by Aryl Radicals Generated from the
Electrochemical Reduction of Diazonium Salts. Journal of the American Chemical
Society 1997, 119 (1), 201-207.
34. Downard, A. J.; Roddick, A. D.; Bond, A. M., Covalent modification of carbon
electrodes for voltammetric differentiation of dopamine and ascorbic acid. Analytica
Chimica Acta 1995, 317 (1-3), 303-10.
35. Liu, G. Z.; Paddon-Row, M. N.; Gooding, J. J., A molecular Wire Modified Glassy
Carbon Electrode for Achieving Direct Electron Transfer to Native Glucose Oxidase.
Electrochemistry communications 2007, 9, 2218-2223.
36. Pinson, J.; Podvorica, F., Attachment of organic layers to conductive or semiconductive
surfaces by reduction of diazonium salts. Chem. Soc. Rev. 2005, 34, 429-439.
37. Liu, G.; Nguyen, Q. T.; Chow, E.; Bocking, T.; Hibbert, D. B.; Gooding, J. J., Study of
factors affecting the performance of voltammetric copper sensors based on Gly-Gly-His
modified glassy carbon and gold electrodes. Electroanalysis 2006, 18 (12), 1141-1151.
38. Yang, W.; Chow, E.; Willett, G. D.; Hibbert, D. B.; Gooding, J. J., Exploring the use of
the tripeptide Gly-Gly-His as a selective recognition element for the fabrication of
electrochemical copper sensors. Analyst (Cambridge, United Kingdom) 2003, 128 (6),
712-718.
39. Gooding, J. J.; Chow, E.; Finlayson, R., Biosensors for detecting metal ions: new
trends. Australian Journal of Chemistry 2003, 56 (2-3), 159-162.
40. Yang, W. R.; Jaramillo, D.; Gooding, J. J.; Hibbert, D. B.; Zhang, R.; Willett, G. D.;
Fisher, K. J., Sub-ppt detection limits for copper ions with Gly-Gly-His modified
electrodes. Chem. Commun. 2001, (19), 1982-1983.
41. Chow, E. Peptide Modified Electrochemical Sensors for the Detection of Heavy Metal
Ions. PhD., The University of New South Wales, Sydney, 2005.
Chapter 3 | 126
Chapter Four
Protein resistance of Oligo(Ethylene
Glycol) Aryl Diazonium Derivatives
4.1 INTRODUCTION
The previous chapter illustrated that PPF can be used in place of GC for the use of biosensors. It has similar electrochemical properties; it has lower, more reproducible, surface roughness, and it can be modified with aryl diazonium salts.
For a biosensor that targets biological species in the blood system it is desired that the sample to be tested comes directly from the body. This will expose the sensor to a complex media in which there are many species that have the ability to foul the surface1 as blood contains not only the target analyte but also various other species as well, mostly these are proteins. There is then a need, to reduce the amount of fouling of the electrode from these unwanted proteins. Interference can be caused in two ways if unwanted proteins stick to the surface. Firstly, they could inhibit the desired protein from interacting with the surface giving a false reading, and secondly they could block electrons from getting to the redox species preventing it from being reduced and thus producing interference.2 This raises the question, how can we prevent non-specific protein adsorption on the electrode surfaces?
There are several approaches that have been shown to reduce non-specific protein adsorption. Generally these include diluting the detection peptide and redox species with a compound that will provide resistance to unwanted proteins and prevent them from sticking to the surface as discussed in chapter 1 section 1.3.2.1. There are three common types of resistant layers. Firstly zwitterionic layers in the form of self assembled monolayers (SAM’s) on gold surfaces.3 The second is an ethylene glycol polymer layer on the surface and the third uses small chain lengths of 2 to 6 ethylene
Chapter 4 | 128
glycol units, oligo(ethylene glycols), whereby the single strand is attached to the surface. By varying the length and distal end of the oligo(ethylene glycol), the response can be controlled.4-6
4.1.1 ZWITTERIONIC SPECIES
A zwitterionic species contains both a positive and negative charge on the same molecule but has an over all neutral charge. There has been an increase in the use of these species as SAMs to reduce the amount of non-specifically adsorbed proteins.
There are two types of SAMs formed from zwitterionic species. The first of these is referred to as single components zwitterionic species.7-8 Another form of zwitterionic species is to produce zwitterionic-like surfaces. A mixture of positive and negative species in a 1:1 ratio is attached to the surface and they are known as zwitterionic mixed
SAMs.3 It is thought that both surface types have a large hydration layer which causes repulsion of the protein because they are both neutral in overall charge and are highly hydrophilic.9 Whitesides et al. has shown that surfaces of both single component and zwitterionic-like SAMs have the ability to reduce non-specific protein adsorption.7, 10 A surface modified with positive and negative charges needs an overall neutral charge for effective resistance to non-specific protein adsorption. A surface of either only the positive or only the negative molecules shows non-specific protein adsorption.3 The ionic strength of the buffer does not affect the resistant properties of the mixed surface.
Single component zwitterionic SAMs though started to absorb protein at low ionic strengths (0.01 to 0.05 M) however these results did vary as the pH was changed. This variation was due to the compounds not being fully deprotonated at certain pH’s and therefore not fully zwitterionic.3
Chapter 4 | 129
4.1.2 POLYMERS
There are several uses for polymers in protein resistance. One method is using the polymer as a membrane over the recognition layer, therefore the polymer acts as a physical barrier. Zwitterionic polymers can also be used and these behave in much the same way the zwitterionic SAMs that were previously discussed.11-12 Zwitterionic polymers are used in the biomedical field as it has been shown that they are biocompatible.
Polymers of ethylene glycol are otherwise known as poly(ethylene oxides) or poly(ethylene glycols) (PEGs). It has been seen by Jeon et al.13 that a polymer layer of
PEG’s on an electrode surface has a better ability to resist non-specific protein adsorption on the surface in comparison to no anti fouling layer. The long chained ethylene glycol units are attached to the surface in one of two ways, either as a polymer coating or they can be secured covalently to the surface through surface oxides.14 It is thought that through hydrophobic interactions, steric repulsion and van der Waals forces, the PEG polymer gains its protein resistance properties.15-18 This occurs due to a more favourable enthalpic interaction between the polymer and water but at the same time sacrifices entropy. As the protein approaches the surface, it presses down on the surface causing the PEG’s to compress and the concomitant exclusion of water.13, 17, 19
This process undergoes an energy change due to the transfer of water from the PEG to the bulk solution. An entropic penalty occurs and causes steric repulsion of the protein as the PEG holds onto the water and maintains shape causing repulsion forces to form from the entropic sacrifice.4, 20 The polymers can be grafted to the surface and the structure the polymer can take is varied. Generally at lower densities a mushroom
Chapter 4 | 130
formation occurs; this is where an individual chain is grafted and has room to pack down.21 At a higher polymer density, a more brush like formation occurs in which the polymer strands behave in a linear arrangement of helical strands that are overlapping.
A third arrangement, called a star formation, occurs where the strands are at a high density and overlapping of the helical chains occur4, 14 It was shown that the limit of packing density where the PEG change formation from mushroom to brushes is the minimum density required for protein resistance. As the density of PEG’s increases so too does protein resistance.18, 22-23 The same can be said with the molecular weight of the PEG, the higher the molecular weight, the better the non-specific protein absorption resistance.24
For the brush formation, if an appropriate solvent, such as water, is used, the chains become more stretched as they repel each other and the surface, compared to those formed in toluene.19 The other observation to note is that the packing density of the polymers and its effect on the protein resistance does not change when the protein size changes for the “brush” PEGs.14
For in situ sensors, the sensitivity is of great importance. This implies a requirement of only a monolayer of the epitope on the surface. This will require single molecule coverage using oligo(ethylene glycol), in comparison to a polymer coverage of PEG described above, with the epitope.
Chapter 4 | 131
4.1.3 OLIGO ETHYLENE GLYCOLS
Oligo (ethylene glycols) (OEGs) are able to be thiolated, which allows them to be attached to many surface types including gold and silver surfaces. They can also be linked to alkenes allowing them to be attached to silicon and diamond surfaces.5, 25-27
There are many factors that will affect the adsorption of protein; these include oligo unit length, the distal group and the packing conformation. The reasons behind the ability of
OEGs to resist non-specific protein adsorption are similar to those of PEGs, described above, and can be linked to steric repulsion. Steric repulsion is made of two parts, an osmotic part, and an elastic part. The osmotic part includes the solvation of the ethylene chain, which is a dominant part of the osmotic component; the elastic part is the conformational energy of the OEG, as the protein approaches the OEG layer the ethylene units compress.13, 17, 20 Electrostatic forces also play a role, as can be seen by
Grunze et al.28
Grunze et al. has previously investigated OEGs and other oligoethers on both gold and silver surfaces and has concluded three general guidelines as to what is required for SAM’s to have good protein resistance.15 Firstly, the hydrophilicity of the termination is important. The more hydrophilic the distal end is, the better the protein resistance.15, 29-30 Secondly, the hydrophilicity of the internal units is important; the less hydrophilic the internal structure is, the less protein resistant the surface is.15, 31 Thirdly the lateral packing density is also of importance,15, 32-33
Research into the importance of the hydrophilicity of the distal end has shown that hydroxy terminated OEGs resist non-specific proteain adsorption better than
Chapter 4 | 132
methoxy terminated OEGs.34 Decreasing the hydrophilicity of the distal end by changing from a hydroxy to either a methoxy, ethoxy, propoxy and butoxy decreases the protein resistance of the surface.5, 15, 34 As the hydrophobicity increases, it decreases also the ability of the water layer to penetrate into the OEG layer (Fig 4.1).15, 20
O O HS O OH
O O HS O O Resistance to nonspecific protein O O adsorption HS O O Increasing O O HS O O
Figure 4.1 Affect of resistance to non-specific protein adsorption.15
The second guideline states that the hydrophilicity of the internal units can affect the ability of the SAM to resist protein. A certain amount of hydrophobicity is important as changes from tri(ethylene glycol) to tri(methylene glycol) increased the amount of adsorbed protein on a gold surface. However when changing to tri(propylene glycol) the amount of protein absorbed on a gold surface increased from 0% on tri(ethylene glycol) methoxy terminated to 49% on tri(propylene glycol) methoxy terminated surface. This is because the propylene glycol unit is unable to pack as densely due to steric hindrance and is also highly hydrophobic in its nature.1, 15 This increase in the amount of non-specific protein on the modified gold surface can be due to the ability of the SAM to absorb water and the amount of water bound in the glycol chains due to the hydrophilicity of the internal structure. The number of repeating units in the chain length of the OEGs can also affect the amount of protein adsorption; a single ethylene unit does not resist protein adsorption whilst units of two or more are
Chapter 4 | 133
able to resist protein. In general, a greater amount of ethylene glycol units provides better protein resistance as the amount of protein adsorbed decreases as the chain length increases from a tri(ethylene glycol) to a hexa(ethylene glycol).6 It has also been seen that temperature also plays a role in the protein resistance of OEGs.35
a
O O
HS O O
O O O Resistance to HS nonspecific protein
adsorption Increasing O O HS O O
b
O O O OH HS O O O
Resistance to O O nonspecific protein HS O OH adsorption Incresing O OH HS O
Figure 4.2 Affect of hydrophilicity of internal units on resistance to non-specific protein
adsorption.15 a) Change in oligo unit from ethylene to methylene and propylene. b) change in
the number of OEG units, from six to three and then two.
Lateral packing density is the third guideline from Grunze and co-workers, and shows that the lateral packing of the OEGs was important. By investigating the differences in the SAM formation of alkane thiol OEGs on both gold and silver, it was seen that although the SAMs were made of the exact same molecule, the non-specific adsorption behaviour varried.15, 36 Resistance to non-specific protein adsorption on
Chapter 4 | 134
OEG modified gold surfaces was higher than that on similar silver modified surface.
This is due to the OEGs packing differently on the silver surface in comparison to the gold. On silver surfaces, the OEGs pack in a more orderly, crystalline manner and are in a planar trans formation, due to the alkane thiols having a higher packing density as they orientate with a small tilt of 7-14°. This makes helical conformation hard.36
Whilst on the gold surface the OEGs pack less densely and have a tilt angle of 30° which allows more room for the glycols, thus they can take up a helical structure.20, 36-40
It was shown by Whitesides and co-workers that not only pure hydroxy terminated hexa(ethylene glycol) gave protein resistance, but SAM’s diluted with n-alkanethiols with a ratio of up to 65:35 hydroxy terminated hexa(ethylene glycol) to alkane also produced protein resistance and the effectiveness was maintained.36 This observation extends to the packing density of the OEG on silicon, as can be seen by work carried out by Böcking et al.41-42 and Yam et al.25 It was seen that in comparison to gold surfaces, OEGs on silicon substrates did not give as effective protein adsorption. This was thought to be due to the lower packing density of the OEGs on silicon than they are on gold, thus a looser SAM formation is formed and not that of the helical conformation.41-43
The lateral packing of the SAM is also relevant to the amount of water that is able to diffuse into the layer.6, 44-45 This is important as the amount of water that is able to diffuse into the OEG layer plays an large role in the resistance of the OEG to non- specific protein adsorption. From computational studies, it was seen that the helical formation allowed one water molecule to every ethylene unit as the two hydrogen atoms on one water molecule interact with the OEG oxygen atoms in succession.20, 33 The water binds strongly to the OEG in helical formation and the OEG acts as a template for
Chapter 4 | 135
water nucleation, which can be initiated by the last oxygen in the glycol chain nearest the distal end as it generates a dipolar field.20 In comparison to OEGs packed in a trans planar conformation, the solvation is difficult and the OEG-water interaction is far weaker in comparison to the helical formation, thus less water is solvated into the OEG layer.
Some recent work of aryl diazonium OEGs grafted to a carbon surface has been carried out by Lui et al.46-47 and Downard and co-workers.48-49 Lui showed that a tri(ethylene glycol) aryl diazonium with a methoxy on the distal end gave a reduction in protein adsorption in comparison to a bare glassy carbon.47 Whilst Downard and co- workers investigated the difference in protein adsorption of the same OEG aryl diazonium salt as well as various other PEG molecules attached to glassy carbon surfaces through electrochemical reduction. Downard and co-workers also studied a diamine PEG and an OEG with a propyl amine distal end.48-49 From this study it was noticed that the aryl diazonium OEG did not have good protein resistance and showed more protein on the modified surface than that of the bare glassy carbon. This was also thought to be due to the loose packing of the aryl diazonium salt derived layers, and is consistent with previous studies showing that nitroazobenzene films have a greater than
50 % free volume.48
From these previous studies it has been shown that OEG packing, and water solvation are important towards protein resistance. Protein resistance via OEGs has been seen on all surfaces, however only the difference of chain length and distal ends has been thoroughly explored on gold and silicon. This chapter explores the effects of changing the distal end of aryl diazonium OEGs and the chain length to investigate
Chapter 4 | 136
whether there was an improvement of the protein resistance such as was seen for the thiol OEG mentioned previously. The protein resistance of these aryl diazonium salt derived layers on thin carbon films was also compared to aryl diazonium OEGs on gold as well to SAM’s of thiolated OEGs on gold surfaces.
4.2 EXPERIMENTAL METHOD
4.2.1 GENERAL SYNTHESIS OF ARYL DIAZONIUM DERIVATIVES OF
ETHYLENE GLYCOL
All aryl diazonium salts used in this chapter were synthesised by the candidate specifically for this project. The same method for the synthesis of the aryl diazonium salts was carried out for all OEG derivatives. The previous method for the synthesis of
OEG3OMe46-47 has been modified, with the first two steps of tosylation of the OEG and subsequent addition of a nitro benzene, removed and replaced with a single step of addition of the nitrobenzene by reacting with 1-fluoro-4-nitrobenzene, and four new compounds have been made. With the only changes to the method being due to the solubility of the decanol plus the modification of the distal end of the hexa(ethylene glycol) from a hydroxy to a methoxy. Previously, only the OEG3OMe aryl diazonium salt derivative had been synthesised.46-47 All chemicals and reagents used in this chapter are described in Chapter 2, section 2.1.1. Silica gel (grade 9385, 230-400 mesh,
Merck) was used for column chromatography and silica gel on aluminium (60 F254,
Merck) for thin layer chromatography (TLC).
Chapter 4 | 137
4.2.1.1 SYNTHESIS OF 2-(2-(2-(4-NITRO-PHENOXY)-ETHOXY)-ETHOXY)-ETHANOL
Tri(ethylene glycol) (11.2481 g, 74.89 mmol), 4-fluoro-1-nitrobenzene (2.1727 g, 15.40 mmol) and tetrabutylammonium hydrogen sulphate (146.9 mg, 0.43 mmol) were placed in a round bottom flask and allowed to stir. Sodium hydroxide (18 mL, 25
M) was then added slowly, and the solution was heated to 40 ºC and left to stir overnight under argon. The solution was then extracted with ethyl acetate (3×50 mL).
The organic layers were then combined and dried over anhydrous magnesium sulphate, which was then filtered through cotton wool and the solvent removed in vacuo to give a yellow solid.
The resulting solid was then TLC’d using ethyl acetate. The compound was seen via short wave U.V. and had an r.f. value of 0.6. A column was then run using ethyl acetate and the desired compound collected, the fractions containing the compound were combined and the solvent removed in vacuo to give a pale yellow oil,
1 90% yeild. H NMR (300 mHz, CDCl3): δ 8.17
(d, J 9.03 Hz, 2H), δ 6.97 (d, J 9.42 Hz, 2H), δ
4.23 (m, 2H), δ 3.88 (m, 2H), δ 3.70 (m, 6H), δ
3.60 (m, 2H).
46-47 4.2.1.2 TRI(ETHOXY-(ETHOXY(ETHOXY))) ANILINE
2-(2-(2-(4-nitrophenoxy)ethoxy)ethoxy)ethanol (2 g, 7.3 mmol) was dissolved in acidified ethanol (10 % hydrochloric acid in ethanol) in a dry round bottom flask. 10
% palladium on carbon (catalytic amount) was added to the reaction. The vessel was
Chapter 4 | 138
evacuated and purged three times with nitrogen and a forth evacuation and purge is carried out with hydrogen gas. The reaction was left for 5 days at room temperature and pressure. Once complete the reaction was quickly filtered through filter aid. The solution was stirred over sodium bicarbonate for 1 hour before being filtered and the solvent is removed in vacuo to give a brown oil.
The resulting brown oil is column purified using ethyl acetate, 5 % methanol and 1 % ammonia. The fractions were collected and those containing product were combined and the solvent removed in vacuo to give a red/brown oil, yield 80%. 1H
NMR (300 mHz, CDCl3): δ 6.97 (d, J 9.03 Hz, 2H),
O O δ 6.75 (d, J 9.42 Hz, 2H), δ 4.47 (m, 2H), δ 3.88 O OH
H2N (m, 2H), δ 3.70 (m, 6H), δ 3.60 (m, 2H).
4.2.1.3 SYNTHESIS OF 4-(2-(2-(2-HYDROXY-ETHOXY)ETHOXY)ETHOXY)
46-47 BEZENEDIAZONIUM (OEG3OH)
A solution of nitrosonium tetrafluoroborate in dry, degassed acetonitrile (500 mg, 4.28 mmol) was placed in a dry two-necked round bottom flask, and placed under argon. The solution was then cooled to -40 ºC via an ethanol/liquid nitrogen bath whilst being stirred. A solution of degassed, dry acetonitrile (3 mL) and 2-(2-(2-(4-amino- phenoxy)-ethoxy)-ethoxy)-ethanol (300 mg) was made up and added to the first solution slowly over 30 min. The solution was stirred at – 40 ºC for 30 min after all the solution was added, then left to come to room temperature before being washed with diethyl ether and the solvent removed in vacuo to give a red/brown oil. No further purification was carried out due to the sensitivity of the aryl diazonium and the red oil was used as
Chapter 4 | 139
1 obtained. H NMR (300 mHz, CDCl3): δ 8.38
O O (d, J 9.03 Hz, 2H), δ 7.34 (d, J 9.42 Hz, 2H), δ O OH
F4B N2 4.41 (m, 2H), δ 3.88 (m, 2H), δ 3.70 (m, 6H), δ
3.60 (m, 2H).
4.2.1.4 NITROBENZENE DECANOL
Tetrabutylammonium hydrogen sulphate (0.446 g, 1.31 mmol) was added with
1-decanol (2.0282 g, 12.8 mmol) and 1-fluoro-4-nitrobenzene (mg, mmol) to a round bottom flask. A sodium hydroxide solution (2 mL, 25 M) was added slowly to avoid heat build up. The reaction was left to sir under argon for 2 days. Water was then added to the yellow mixture, and the solution is extracted with ethyl acetate (3 × 50 mL). The organic phases were combined and dried over magnesium sulphate and filtered. The solvent was removed in vacuo to give a yellow solid.
A column was run using light petroleum and ethyl acetate to purify the solid obtained, the resulting fractions containing the desired product were combined and the solvent removed to give fine colourless crystals, yield 98%. 1H NMR (300 mHz,
CDCl3): δ 8.21 (d, J 9.3 Hz, 2H), δ 6.95 (d, J 9.3 O Hz, 2H), δ 4.04 (m, 2H), δ 1.82 (m, 2H), δ 1.44
O2N (m, 19H).
Chapter 4 | 140
46-47 4.2.1.5 4-(DECYLOXY)ANILINE
1-(decyloxy)-4-nitrobenzene (600 mg, 2.06 mmol) was dissolved in acidified ethanol (10 % hydrochloric acid in ethanol) in a dry round bottom flask. 10 %
Palladium on carbon (catalytic amount) was added. The vessel was evacuated and purged three times with nitrogen a forth evacuation and purge is carried out with hydrogen gas. The reaction was left for 5 days at room temperature and pressure. Once complete, the reaction was quickly filtered through filter aid to give a brown filtrate which was then stirred over sodium bicarbonate for 1 hour before being filtered and the solvent removed to give a brown mixture.
The resulting brown mixture was columned using ethyl acetate and 2 % methanol and 0.5 % ammonia and the resulting fractions combined and the solvent
1 removed in vacuo to give a red/brown oil. H NMR (300 mHz, CDCl3): δ 7.34 (d, J 9.
3 Hz, 2H), δ 6.94 (d, J 9.3 Hz, 2H), δ 4.42 (m, 2H), O δ 1.82 (m, 2H), δ 1.44 (m, 19H). H2N
4.2.1.6 1-(DECYLOXY)-4-ARYL DIAZONIUM TETRAFLUOROBORATE (DECANE
46-47 DERIVATIVE)
A solution of nitrosonium tetrafluoroborate in dry, degassed acetonitrile (400 mg, 3.42 mmol) was placed in a dry two-necked round bottom flask, and placed under argon. The solution was stirred and cooled to -40 °C using an ethanol/liquid nitrogen bath. A solution of 4-(decyloxy)aniline (350 mg, 1.33 mmol) in acetonitrile (2 mL) was added dropwise with a syringe over half an hour. The solution was allowed to stir for a
Chapter 4 | 141
further half an hour at -40 ºC and then come to room temperature, were the flask was placed carefully under vacuum to remove some solvent. The reaction was then exposed to air and ether was added to crash out the aryl diazonium salt as an oil, the top solvent was removed and more ether is added and the procedure repeated. The remaining red oil was dried on the vacuum line, the product was used with out further purification. 1H
NMR (300 mHz, CDCl3): δ 8.40 (d, J 13.2 Hz, O 2H), δ 7.66 (d, J 13.2 Hz, 2H), δ 4.42 (m, 2H), F4B N2 δ 1.82 (m, 2H), δ 1.44 (m, 19H).
4.2.1.7 MONOMETHYLETHER(ETHOXY-(ETHOXY(ETHOXY))) NITROBENZENE
Tetrabutylammonium hydrogen sulphate (2.0579 g, 12.5 mmol) was added to monomethylether triethylene glycol (0.2816 g, 0.83 mmol) and 1-fluoro-4-nitrobenzene
(1.9959 g, 14.1 mmol) in a round bottom flask with a magnetic stirrer bar. A sodium hydroxide solution (4 mL, 25 M) was added slowly to avoid heat build up. The reaction was left to stir under nitrogen for 2 days. Water was then added, and the solution was extracted with ethyl acetate (4 × 50 mL). The organic phases were combined and dried over magnesium sulphate. The mixture was filtered through cotton wool and the solvent removed on the rotary evaporator to give a yellow oil.
A column was run using 30 % light petroleum and 70 % ethyl acetate to purify the yellow oil. The desired fractions were combined and the solvent removed in vacuo
1 to give a yellow oil, yeild 97%. H NMR (300 mHz, CDCl3): δ 8.17 (d, J 9.25 Hz, 2H),
δ 6.97 (d, J 9.25 Hz, 2H), δ 4.23 (m, 2H), δ 3.88 O O O O
O2N
Chapter 4 | 142
(m, 2H), δ 3.70 (m, 6H), δ 3.60 (m, 2H), δ 3.27 (s, 3H).
46-47 4.2.1.8 MONOMETHYLETHER(ETHOXY-(ETHOXY(ETHOXY))) ANILINE
1-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-4-nitrobenzene (3.0221 g, 11.2 mmol) was dissolved in acidified ethanol (10 % hydrochloric acid in ethanol) in a dry round bottom flask. A catalytic amount of 10 % palladium on carbon was added to the solution. The vessel was evacuated and purged three times with nitrogen a forth evacuation and purge was carried out with hydrogen gas. The reaction is left at room temperature and pressure for 5 days. Once complete, the reaction was quickly filtered through filter aid to give a pink filtrate. The resulting solution was stirred over sodium bicarbonate for 1 hour before being filtered and the solvent is removed. The resulting brown oil was column purified using ethyl acetate and 5 % methanol and 1 % ammonia.
The desired fractions were combined and the solvent removed in vacuo to give a red oil,
1 85% yeild. H NMR (300 mHz, CDCl3): δ 7.45 (d, J 9.3 Hz, 2H), δ 7.23 (d, J 9.3 Hz,
O O 2H), δ 4.40 (m, 2H), δ 3.84 (m, 2H), δ 3.70 (m, O O
6H), δ 3.60 (m, 2H), δ 3.29 (m, 3H) H2N
4.2.1.9 MONO METHYLETHER(ETHOXY-(ETHOXY(ETHOXY))) ARYL DIAZONIUM
46-47 TETRAFLUOROBORATE (OEG3OME)
A solution of nitrosonium tetrafluoroborate (56.1 mg, 0.48 mmol) was made up in degassed, dry acetonitrile under argon. The solution was stirred and cooled to -40 °C using an ethanol/liquid nitrogen bath and the temperature was monitored. A solution of
Chapter 4 | 143
4-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)aniline (122.1 mg, 0.39 mmol) in degassed, dry acetonitrile was added drop wise with a syringe over half an hour. The solution is allowed to stir for another half an hour under argon at -40 ºC before being allowed to come to room temperature, were the flask was placed carefully under vacuum to remove some solvent. The reaction was then exposed to air and a small amount of diethyl ether
(1 mL) was used to crash out the aryl diazonium salt as an oil, the top solvent was removed leaving and red oil and more diethyl ether was added to rinse the oil and the procedure repeated. The remaining oil was dried on the vacuum line, the product was
1 used with out further purification. H NMR (300 mHz, CDCl3): δ 8.40 (d, J 14.4 Hz,
2H), δ 7.36 (d, J 15.9 Hz, 2H), δ 4.23 (m, 2H), δ O O O O
3.84 (m, 2H), δ 3.70 (m, 6H), δ 3.60 (m, 2H), δ F4B N2
3.29 (m, 3H).
4.2.1.10 HEXAETHYLENE GLYCOL NITROBENZENE
Tetrabutylammonium hydrogen sulphate (0.2443 g, 0.72 mmol) was added with hexaethylene glycol (4.5965 g, 16.3 mmol) and 1-fluoro-4-nitrobenzene (2.0100 g, 14.1 mmol) in a round bottom flask with a magnetic stirrer bar. A sodium hydroxide solution (2 mL, 25 M) was added, slowly to avoid heat build up, a bright yellow mixture was formed. The reaction was left to stir under nitrogen for 2 days to give a partial yellow emulsion. Water was then added, and the solution was extracted with ethyl acetate (4 × 60 mL). The organic phases were then combined and dried over magnesium sulphate. The mixture was filtered through cotton wool and the solvent removed on the rotary evaporator to give a yellow oil.
Chapter 4 | 144
A column was run using a graduated column starting at 50 % light petroleum, 50
% ethyl acetate and finishing at 100 % ethyl acetate. The fractions containing the desired compound were combined and the solvent removed in vacuo to give a light
1 yellow oil, 68% yield. H NMR (300 mHz, CDCl3): δ 8.15 (d, J 9. 3 Hz, 2H), δ 6.96
(d, J 9.3 Hz, 2H), δ 4.18 (m, O O O OH O O O 2H), δ 3.84 (m, 2H), δ 3.61 (m, O2N 16H).
46-47 4.2.1.11 HEXAETHYLENE GLYCOL ANILINE
17-(4-nitrophenoxy)-3,6,9,12,15-pentaoxaheptadecan-1-ol (1.0216 g, 3.0 mmol) was dissolved in acidified ethanol (10 % hydrochloric acid in ethanol) in a dry round bottom flask. To this yellow solution 10 % palladium on carbon (catalytic amount) was added to the reaction. The reaction was evacuated and purged three times with nitrogen whilst a forth evacuation and purge was carried out with hydrogen gas. The reaction was left to stir at room temperature and pressure under hydrogen gas for 4 days. Once complete the reaction was quickly filtered through filter aid to give a brown solution.
The solution was stirred over sodium bicarbonate for 2 hours before being filtered and the solvent removed. The resulting brown oil was columned using ethyl acetate and 5
% methanol and 1 % ammonia. The desired fractions were combined and the solvent
1 removed in vacuo to give a red brown oil, yield 78%. H NMR (300 mHz, CDCl3): δ
6.97 (d, J 9.0 Hz, 2H), δ 6.78 (d, J 9.0 Hz, 2H), δ 4.46 (m, 2H), δ 3.88 (m, 2H), δ 3.84
(m, 2H), δ 3.61 (m, 16H). O O O OH O O O
H2N
Chapter 4 | 145
4.2.1.12 HEXAETHYLENE GLYCOL ARYL DIAZONIUM TETRAFLUOROBORATE
46-47 (OEG6OH)
A solution of nitrosonium tetrafluoroborate (56.1 mg, 0.48 mmol) in dry, degassed acetonitrile was made in a dry two necked round bottom flask, under argon.
The resulting red/brown solution was stirred and cooled to -40 °C using an ethanol/liquid nitrogen bath and the temperature monitored. A solution of 17-(4- aminophenoxy)-3,6,9,12,15-pentaoxaheptadecan-1-ol (179.8 mg, 0.53mmol) in dry, degassed acetonitrile (mL) was added dropwise with a syringe over half an hour. The solution was allowed to stir under argon for half an hour at -40 ºC and then allowed to reach room temperature. The round-bottom was placed carefully under vacuum to remove some solvent. The reaction was then exposed to air and ether was added to crash out the aryl diazonium salt as a red oil, the top solvent was removed and more acetonitrile was added (0.5 mL) was added followed by more diethyl ether (2 mL) again crashing out the oil and the top solution was removed. The remaining oil was dried on the vacuum line to give a red oil, the product was used without further purification. 1 H
NMR (300 mHz, CDCl3): δ 8.40 (d, J 14.4 Hz, 2H), δ 7.36 (d, J 15.9 Hz, 2H), δ 4.46
(m, 2H), δ 3.88 (m, 2H), δ O O O OH O O O 3.8 (m, 2H), δ 3.61 (m, 16H). F4B N2
4.2.1.13 MONOMETHYLETHER HEXAETHYLENE GLYCOL NITROBENZENE
17-(4-nitrophenoxy)-3,6,9,12,15-pentaoxaheptadecan-1-ol (1.4786 g, 3.67mmol) was dissolved in THF with sodium hydride (0.9032 g, 37.6 mmol). The resulting yellow mixture was allowed to stir for an hour before a solution of iodomethane (0.35
Chapter 4 | 146
mL, 5.58 mmol) in THF (mL) was added dropwise over an hour. The reaction was allowed to stir under argon for 2 days. The reaction was quenched by adding water.
The resulting yellow mixture was extracted with ethyl acetate (5 × 50 mL). The organic phases were combined and dried over magnesium sulphate. The mixture was filtered through cotton wool and the solvent was removed via rotary evaporator, to give a yellow oil, 87% yeild. An NMR was collected showing pure compound, no further
1 purification was required. H NMR (300 mHz, CDCl3): δ 8.17 (d, J 9.03 Hz, 2H), δ
6.97 (d, J 9.42 Hz, 2H), δ 4.23
O O O O (m, 2H), δ 3.88 (m, 2H), δ 3.70 O O O
O2N (m, 18H), δ 3.29 (m, 3H).
4.2.1.14 MONOMETHYLETHER HEXAETHYLENE GLYCOL ANILINE
19-(4-nitrophenoxy)-2,5,8,11,14,17-hexaoxanonadecane (0.6036 g, 2.6 mmol) was dissolved in acidified ethanol (10 % hydrochloric acid in ethanol) in a dry round bottom flask. 10 % Palladium on carbon was added to the reaction in a catalytic amount. The reaction was evacuated and purged three times with nitrogen, a forth evacuation and purge was carried out with hydrogen gas. The reaction was left for 5 days at room temperature and pressure under hydrogen. Once complete the reaction was quickly filtered through filter aid to give a maroon solution. This solution was then stirred over sodium bicarbonate for 2 hours before being filtered and the solvent removed.
The resulting brown oil was columned using ethyl acetate and 5 % methanol and
1 % ammonia. The desired fractions were combined and the solvent removed in vacuo
Chapter 4 | 147
1 to give a red/brown oil, 40% yeild. H NMR (300 mHz, CDCl3): δ 6.97 (d, J 9.0 Hz,
2H), δ 6.78 (d, J 9.0 Hz, 2H), δ 4.46 (m, 2H), δ 3.88 (m, 2H), δ 3.84 (m, 2H), δ 3.61
O O O O (m, 16H), δ 3.29 (m, 3H). O O O
H2N
4.2.1.15 MONOMETHYLETHER HEXAETHYLENE GLYCOL ARYL DIAZONIUM
46-47 TETRAFLUOROBORATE (OEG6OME)
A solution of nitrosonium tetrafluoroborate (74.8 mg, 0.64 mmol) in dry, degassed acetonitrile was made in a dry two necked round bottom flask, under argon.
The resulting red/brown solution was stirred and cooled to -40 °C using an ethanol/liquid nitrogen bath and the temperature monitored. A solution of 4-
(2,5,8,11,14,17-hexaoxanonadecan-19-yloxy)aniline (177.7 mg, 0.44 mmol) in degassed, dry acetonitrile (2 mL) was added dropwise with a syringe over half an hour under argon. The solution is allowed to stir for half an hour at -40 ºC and then allowed to come to room temperature, were the flask was placed carefully under vacuum to crash out the aryl diazonium salt as a red oil, the top solvent is removed and more dry acetonitrile (0.5 mL) was added followed by more diethyl ether the procedure was repeated. The remaining red oil was dried on the vacuum line. 1 H NMR (300 mHz,
CDCl3): δ 8.40 (d, J 14.4 Hz, 2H), δ 7.36 (d, J 15.9 Hz, 2H), δ 4.46 (m, 2H), δ 3.88 (m,
2H), δ 3.84 (m, 2H), δ 3.61 O O O O O O O
(m, 16H), δ 3.29 (m, 3H). F4B N2
Chapter 4 | 148
4.2.2 MODIFICATION OF SURFACES
4.2.2.1 PREPARATION OF SURFACES
4.2.2.1.1 PYROLYSED PHOTORESIST FILMS
1.4 cm × 1.4 cm square pieces of PPF were used that were prepared as per chapter 2 section 2.3.2.1. For the measurement of proteins on the surface a 1 cm diameter area was exposed to the modification solution, using Teflon cells with a circular exposure area of 1 cm and a 1.5 mL well. The samples were secured in the cell as seen in Fig. 4.3.
Workingng electrode contactct O-ringO
Wellell BottomBBo plate Top platette
Screws to securere top and bottomm PlatinumPPl counter plates electrodeel
Figure 4.3 Teflon cell used to expose a 1 cm diameter circle of PPF to a small volume of
solution.
For samples used for the elution of proteins, 1.5 cm by 0.5 cm PPF samples were used. The top of the surfaces were clamped in alligator clips wrapped in foil and then wrapped in Teflon tape to protect the alligator clip from the modification solution.
Chapter 4 | 149
Once modified, the Teflon tape and alligator clip was removed and the non-modified area was removed.
4.2.2.1.2 GOLD
1 cm by 0.5 cm gold foil pieces were used as the surface substrates. The foil pieces were cleaned by leaving them in a solution of piranha (sulphuric acid and hydrogen peroxide with a 2:1 ratio respectively) over night. The surfaces were then rinsed with copious amounts of Milli-Q water followed by ethanol.
For aryl diazonium salt modification the the top of the foil pieces was attached with an alligator clip that had been wrapped in foil to prevent surface damage and ensure contact. The alligator clip and foil under it was wrapped with Teflon tape to protect the clip from exposure to the modification solution. The rest of the foil (roughly
0.8 cm by 0.5 cm) was left exposed to be modified.
4.2.2.2 THIOL ATTACHMENT TO GOLD SURFACES
Thiol modification was carried out by Joshua Z. Ginges. The gold foil pieces were cleaned as per previously described using piranha solution. They were then soaked for 1 hr in redistilled ethanol to remove oxides. The foil pieces were then exposed to a solution of the thiols in ethanol (1 mM, 3 mL) for 18 hrs at room temperature before being removed and rinsed with ethanol. The thiols used were
Chapter 4 | 150
decanthiol, triethylene glycol mono-11-mercaptoundecyl ether and hexa(ethylene glycol)mono-11-mercaptoundecyl ether.
4.2.2.3 PREMADE ARYL DIAZONIUM ATTACHMENT
Each surface modification solution was made separately for each surface due to the large surface area required. Acetonitrile was degassed before making the solution to reduce volume loss. Tetrabutyl ammonium tetrafluoroborate (17 mg) was used as the electrolyte and 10 - 20 mg of OEG was used (Table 4.1). The two dry ingredients were placed in a small glass sample vial and degassed acetonitrile (1 mL) was added and the solution was added to the Teflon cell. A platinum counter electrode and a Ag|AgCl reference electrode was used and the cell was cycled from 0.2 V to 1.2 V several times depending on the OEG aryl diazonium salt used (Table 4.1).
Cycles used to Cycles used to OEG aryl diazonium salt Mass of OEG / mg modify the PPF modify the gold surface surface
Decane 10.0 15 7
OEG3OMe 12.0 7 7
OEG3OH 10.0 5 5
OEG6OMe 15.0 30 30
OEG6OH 18.0 20 15
Table 4.1 Mass of OEG aryl diazonium salts used and the number of cycles used to modify the
PPF and gold surfaces.
Chapter 4 | 151
4.2.2.4 ACETONITRILE IN SITU FORMATION OF ARYL DIAZONIUM SALTS AND
50 ATTACHMENT
A solution of the OEG3OH aniline derivative (10 mg, 0.029 mmol) was made with terabutylammoinium tetrafluoroborate (17 mg) as the electrolyte in degassed acetonitrile. A solution of sodium nitrite (25 μL of a 69 mg/mL aqueous solution) was added as well as perchloric acid (75 μL).
Each modified surface had an individual solution. The surface was exposed and cyclic voltammetry employed against a Ag|AgCl 3 M NaCl reference electrode and a platinum counter electrode. The solutions were scanned from -0.2 V to -1.4 V for 5 cycles.
4.2.2.5 AQUEOUS IN SITU FORMATION OF ARYL DIAZONIUM SALTS AND
ATTACHMENT
Individual solutions of the OEG3OH aniline derivative (10 mg, mol) was made up with potassium chloride as the electrolyte in a degassed solution of water per each surface to be modified. Hydrochloric acid (75 μL) and sodium nitrite (25 μL of a
69 mg/mL aqueous solution) solutions were added to each solution and cyclic voltammetry was carried out by cycling between 0.2 V and -1.4 V for 5 cycles.
Chapter 4 | 152
4.2.2.6 ELECTROCHEMISTRY OF MODIFIED SURFACES
3-/4- Each surface was tested before and after with ferricyanide (Fe(CN)6 ) and
2+/3+ ruthenium hexamine (Ru(NH3)6 ) in accordance to the method in chapter 2 section
2.3.2.1.
4.2.3 NON-SPECIFIC PROTEIN ADSORPTION MEASUREMENTS
4.2.3.1 ELUTION OF PROTEIN FROM THE SURFACE
The modified surfaces were exposed to a solution of BSA-FITC in PBS (1 mg/mL) for one hour. The surfaces were then rinsed with PBS and soaked for 18 hrs in an elution buffer (300 mM NaCl, 20 mM Na2HPO4, 2 mM EDTA, 1% Trion X and 1% v/v 2-mercaptoethanol). For the last hour the surfaces were heated to 37 ºC to ensure complete desorption of the protein from the surface. The PPF pieces were removed from the elution buffer solutions containing protein removed from the different surfaces. The amount of BSA-FITC in the elution buffer solutions was measured using a fluorescence spectrophotometer with excitation of the BSA-FITC at 494 nm and emission at 525 nm. Concentration standards were also made to quantify the amount of protein desorbed from the surface. The concentrations of the standard solutions were as follows; 0.01 mg/mL, 0.005 mg/mL, 0.001 mg/mL, 0.0005 mg/mL, 0.0001 mg/mL,
0.00005 mg/mL, and 0.00001 mg/mL.
Chapter 4 | 153
4.2.3.2 MEASUREMENT OF PROTEIN ON THE SURFACE
For each OEG aryl diazonium derivative, nine PPF surfaces were made and four gold surfaces. The PPF surfaces were modified in the middle of the wafer leaving an unmodified section for comparison on the outside. Gold foil pieces were completely modified on both sides leaving no unmodified section for comparison. Exposure to protein was carried out in a dimly lit laboratory to prevent the BSA-FITC from fading.
Modified surfaces were exposed to 1.5 mL of BSA-FITC (1 mg/mL) in PBS solution for 1 hour in plastic Eppendorf tubes. The reactions were protected from light by covering with foil. The BSA-FITC solution was filtered through a 0.22 μm sized filter before exposure to the modified surface to remove large clumping of the protein before it is exposed. After an hour of exposure, the solution was then removed. A solution of
PBS was subsequently added to the tubes containing and the surfaces were soaked for 7 min. The PBS solution was removed and replaced with Milli-Q water, twice, to remove any salts from the surface. The surfaces were gently dried with nitrogen and mounted surface side down onto glass microscope slide covers that were cleaned via sonication in ethanol and rinsing with ethanol and dried under nitrogen. The surfaces were fixed to the glass slide coverslip with mowiol, an antifade fixative. The samples were left over night in the dark to set.
The amount of BAS-FITC adsorbed onto the surface was recorded by measuring the intensity of fluorescence with a Leica DM IL inverted microscope as discussed in
Chapter 2 section 2.2.1.2.5, and images were taken with ProgRes CFscan CCD camera.
The surfaces were placed on the microscope stage and images were recorded of the surface. An image of the surface was recorded with a 60 × focus with oil, the images
Chapter 4 | 154
were exposed for 100 ms. For the PPF surfaces six images were captured on the non- modified outer areas of the PPF and six images taken from the modified inner area. By taking images on the same surface of modified and bare surfaces exposed the BSA-
FITC allows an internal standard for the adsorption of protein. Six images were taken any where on the gold surfaces, as the entire surface was modified and the intensity was standardised against a surface modified with the alkane, either the aryl diazonium decane derivative and the dodecane thiol derivative which was measured on the same day.
For the PPF the bare outer surface was used to standardise the samples for the halogen lamp, as not all of the surfaces could be measured at the one time. They were then compared to the decane surface. For the gold surfaces, all types of surface modification (thiol or aryl diazonium salt) were measured at the same time, and were then directly compared to the decane modified surface for comparison with the PPF surface. Bare gold was hard to quantify due to quenching of the BSA-FITC.
Quenching on the modified gold surfaces did not occur as readily due to the surface modification providing a “buffer zone” between the BSA-FITC and the gold.
The amount of fluorescence was then processed using the image processing program, ImageJ. For the analysis of the images the average amount of grey scale was measured for each image of the intensity of fluorescence on the surface and divided by the area measured.
Controls were run of each OEG aryl diazonium derivative on both PPF and gold surfaces. For each OEG aryl diazonium salt three PPF surfaces were modified and three
Chapter 4 | 155
gold surfaces were modified, as per the premade diazonium aryl diazonium salt attachment as discussed above.
The modified surfaces were exposed to a solution of PBS for 1 hour in plastic tubes. The solution was then removed and another solution of PBS was added and the surfaces were soaked for 7 min. The PBS solution was then removed and replaced with
Milli-Q water, this was repeated to remove any salts from the surface. The surfaces were gently dried with nitrogen and mounted surface side down onto glass microscope slide covers with mowiol. The glass microscope slide covers were cleaned via sonication in ethanol and rinsing with ethanol before being used. The samples were then left over night in the dark to set.
The fluorescence of each surface were measured on the fluorescence microscope in the same manner as the modified surfaces exposed to protein.
4.2.3.3 NON-SPECIFIC PROTEIN ADSORPTION WITH RELATIONSHIP TO TIME
hen rinsed twice with Milli-Q water but removing the liquid from the tube and gently adding more solution. The surfaces were then gently dried with nitrogen and mounted on clean microscope cover slips with mowiol.
The amount of BAS-FITC adsorbed onto the surface was recorded by measuring the intensity of fluorescence with a Leica DM IL inverted microscope as discussed in
Chapter 2 section 2.2.1.2.5, and images were taken with ProgRes CFscan CCD camera.
The surfaces were placed on the microscope stage and images were recorded of the
Chapter 4 | 156
surface. An image of the surface was recorded with a 60 × focus with oil, the images were exposed for 100 ms. Six images were captured on the non-modified outer areas of the PPF and six images taken from the modified inner area. By taking images on the same surface of modified and bare surfaces exposed the BSA-FITC allows an internal standard for the adsorption of protein.
For the PPF the bare outer surface was used to standardise the samples for the halogen lamp, as not all of the surfaces could be measured at the one time. They were then compared to the decane surface. For the gold surfaces, all types of surface modification (thiol or aryl diazonium salt) were measured at the same time, and were then directly compared to the decane modified surface for comparison with the PPF surface.
The amount of fluorescence was then processed using the image processing program, ImageJ. For the analysis of the images the average amount of grey scale was measured for each image of the intensity of fluorescence on the surface and divided by the area measured.
4.2.4 CONTACT ANGLE
Each surface contact angle with water was measured as per chapter 2 section
2.2.1.2.2.
Chapter 4 | 157
4.3 RESULTS AND DISCUSSION
The use of OEGs for protein resistance, as previously discussed, is well known on gold and silicon. The use of these molecules on carbon has recently been carried out by Hammers and co-workers5, 26 through the use of alkenes on hydrogenated diamond
48-49 46-47 surfaces. Downard et al. and Lui et al. have shown the use of the OEG3OMe aryl diazonium derivative, although the effectiveness of the protein resistance varied.
Here we look at the protein resistance of aryl diazonium derivatives of OEGs and whether chain length and distal end vairation affect the protein resistance as seen on gold. This was previously discussed by Grunze and co-workers,15 as these factors are important to the protein resistance of OEG thiol derivatives on gold surfaces. Two chain lengths were explored; the tri(ethylene glycol) and the hexa(ethylene glycol). The distal end was also varied from methoxy to hydroxy. An alkane was used for a comparison between OEGs and different surfaces (PPF and gold) as it is not expected to be resistant to non-specific protein adsorption (Fig. 4.4).
O O O O O OH O OH O O O 1 2
F4B N2 F4B N2
O O O O O 3 4
F4B N2 F4B N2
O O O O O O O 5 F4B N2
Figure 4.4 Oligo (ethylene glycol) aryl diazonium salt derivatives used for protein resistance
on gold and PPF surfaces. 1) OEG3OH 2) OEG6OH 3) OEG3OMe 4) Decane 5) OEG6OMe
Chapter 4 | 158
The general method for the synthesis of the aryl diazonium salts was carried out for all OEG derivatives. With the only changes to the method being due to the solubility of the decanol plus the modification of the distal end of the hexa(ethylene glycol) from a hydroxy to a methoxy. Previously, only the OEG3OMe aryl diazonium salt derivative had been synthesised.46, 48 The previous method carried out by Liu et al. was changed from a two step tosylation of the monomethyl ether triethylene glycol followed by reacting with benzyl bromide, to a single step reaction of the oligo(ethylene glycol) with 1-fluoro-4-nitrobenzene, under basic reducing conditions. An amine variation, 1-fluoro-4-aniline, was unable to be used as the amine was to electron donating for the reaction and an electron withdrawing group is required on the benzene for the reaction to continue.
The modification of the method reduced the amount of workup required to purify the compounds and the amount of time. By using this method it was also easier to modify only one end of the tri(ethylene glycol) and the hexa(ethylene glycol). Using a 4:1 ratio of Tri(ethylene glycol) or hexa(ethylene glycol) to 1-fluoro-4-nitrobenzene mono substitution was carried out giving an hydroxy terminated OEG diazonium salt.
As monomethylether hexa(ethylene glycol) was unable to be purchased at the time the nitrobenzene modified hexa(ethylene glycol) was reacted with methyl iodine to give a monomethylether hexa(ethylene glycol) derivative. This reaction though proved difficult due to the small amounts being used. So far from these five compounds only the mnomethyl ether tri(ethylene glycol) aryl diazonium derivative has previously been synthesised and the others are new compounds.
Chapter 4 | 159
Alkane thiol derivatives (Fig. 4.5) for the modification of gold surfaces as a comparison with carbon surfaces were used as purchased.
O O HS 6 HS O OH 7
O O O OH 8 HS O O O
Figure 4.5 Alkane thiol derivatives for the modification of gold surface to be used for protein
resistance comparisons.
The ability of a modified surface to resist non-specific protein adsorption for the current study is defined as the ability of the OEG modified surface to reduce the amount of BSA-FITC adsorbed onto the surface when compared to an alkane modified surface.
This ability to reduce the amount of protein can be referred to as protein resistance.
4.3.1 ELECTROCHEMISTRY OF MODIFIED SURFACES
The PPF and gold surfaces were modified with the OEG aryl diazonium salt derivatives via the electrochemical reduction of the diazonium to a radical which can then react to the surface to form a stable covalent bond, as previously discussed in chapter 1. As shown in chapter 3, the modification of the surface can be probed
3-/4- 2+/3+ electrochemically with the use of Fe(CN)6 and Ru(NH3)6 . The aryl diazonium salt derived surfaces were investigated before and after modification.
Chapter 4 | 160
4.3.1.1 ATTACHMENT OF OLIGO(ETHYLENE) GLYCOLS
The reduction of the OEG aryl diazonium salts was carried out by cycling the potential between 0.2 V to -1.5 V for PPF surfaces and 0 V to -1 V for gold surfaces. It can be noted that as the aryl diazonium salt are reduced to form radicals that bond with the surface a voltammetric peak is observed, this reduction peak is becomes more passivated each cycle due to more radicals reacting with the surface to form a modified layer. The passivation of the surface by the attachment of the aryl diazonium salts to the surface took longer in comparison to the shorter derivatives, 5 to 20 cycles for OEG aryl diazoniums in comparison to the 2 cycles for the 4-carboxyphenyl diazonium salt.
To achieve similarly passivated surfaces the number of cycles was varied for each OEG diazonium derivative.
This increase in the cycles required for passivation could be linked to the conformation of the OEG. At first it is loosely packed with no structure and partially could block the surface from further modification until more molecules are attached to the surface to give a more structured conformation. As observed by Whitesides and co-
36 workers that for loosely packed OEG3OMe on gold surfaces, the OEG tail takes on an amorphous like structure. As the surface becomes more densely packed it takes up a helical structure, and as it becomes more densely packed, a planar trans formation is taken. It was also discussed by Downard et al. that the layers formed by the reduction of aryl diazonium salts are loosely packed.48
Similar passivation of the PPF surfaces was achieved by OEG3OH, OEG3OMe,
OEG6OH and the decane aryl diazonium salt derivatives (Fig 4.6). This allowed for
Chapter 4 | 161
some control in the consistency of the surface. The OEG3 aryl diazonium surfaces required fewer number of scans in comparison to those of OEG6 and the decane aryl diazonium derivatives.
a b
c d
Figure 4.6 Electrochemical reduction of premade OEG aryl diazonium salts onto PPF surfaces in acetonitrile with 10 mM tetrabutyl ammonium tetraflluoroborate. a) OEG6OH b) OEG3OMe
c) OEG3OH d) Decane
The aryl diazonium salts on gold require a smaller scan range due to the potential window of gold, and thus the stability of the aryl diazonium on the surface was assured. The passivation of the surface for both the decane derivative on gold did not show any further passivation after five cycles as observed by the similarity of cycle five
Chapter 4 | 162
and seven in Fig. 4.7d. This is similar to the OEG6OH derivative on gold, no difference is observed between the fifth cycle and the tenth cycle (Fig. 4.7a)
a b
c d
Figure 4.7 Electrochemical reduction of premade OEG aryl diazonium salts onto gold surfaces
in acetonitrile with 10 mM tetrabutyl ammonium tetraflluoroborate against a Ag|AgCl 3M NaCl
reference electrode. a) OEG6OH b) OEG3OMe c) OEG3OH d) Decane
From Fig 4.6 and Fig 4.7 it can be observed that the electrochemical reduction of the aryl diazonium salts gave more well-defined voltammetric peaks on PPF surfaces than those observed on the gold surfaces. It has been observed previously by Gooding and co-workers that aryl diazonium salts behave differently towards gold surfaces than carbon surfaces.51-53 For small aryl diazonium salts the reduction peak tends to be more well defined on gold than carbon surfaces, but for larger aryl diazonium salts the behaviour on gold surfaces in comparison to carbon surfaces can vary. This variation in
Chapter 4 | 163
the behaviour of the electrochemical reduction large aryl diazonium salts towards gold surfaces and carbon surfaces is noted by the broadness in the voltammetric reductions peaks on the gold surfaces as seen in Fig 4.7.
4.3.1.2 ELECTROCHEMICAL PASSIVATION OF MODIFIED SURFACES
To evaluate the modification of surfaces with aryl diazonium salts a comparison of the electrochemistry at the underlying PPF electrode before and after modification
3+/2+ was performed with both 1.0 mM Ru(NH3)6 in the form of ruthenium hexamine
3-/4- trichloride and 1.0 mM Fe(CN)6 in the form of potassium ferricyanide. Both solutions were prepared in phosphate buffer (50 mM, pH 7) with potassium chloride (1
M). It was observed that the aryl diazonium salt derived layers passivated the
3-/4- 3+/2+ electrodes towards Fe(CN)6 (Fig. 4.8b) but not towards Ru(NH3)6 (Fig. 4.8a).
As discussed in chapter 3, the passivation with regards to ferricyanide is to be expected as it is an inner sphere complex and requires contact with the surface.54-55 The ethylene glycol layers would be reasonably thick due to the long chain lengths,
3+/2+ therefore some slowing of the electron transfer from the surface to Ru(NH3)6 maybe expected. Although it is an outer sphere complex and is not required to contact the surface to undergo reduction or oxidation,54-55 some slowing of electron transfer
3+/2+ resulting in an increase in ΔEp due to the distance of Ru(NH3)6 to the surface. It can be seen that the length of the ethylene glycol aryl diazonium has no effect on the ΔEp of
3+/2+ 3-/4- Ru(NH3)6 . The passivation of the surface towards Fe(CN)6 and no change in
3+/2+ ΔEp of Ru(NH3)6 indicates that the surface has been modified with OEG aryl
Chapter 4 | 164
diazonium derivatives. Fig 4.8b also shows that all three OEGs tend to passivate the surface to near the same extent.
a b
Figure 4.8 a) Ruthenium hexamine before and after surface modification on PPF. b)
Ferricyanide before and after surface modification on PPF.
The aryl diazonium salt derived layers on gold are observed to behave in a
2+/3+ similar manner to those on PPF. The redox chemistry of Ru(NH3)6 (Fig. 4.9a) can
3-/4- be seen as well as the passivation of Fe(CN)6 (Fig. 4.9b). It can be noted that in both
Fig 4.8a and Fig 4.9a that there is some to complete passivation of the surface towards
2+/3+ Ru(NH3)6 . This passivation is most likely due to the packing arrangement of the decane aryl diazonium layers on surface, due to the straight alkane chain of the decane it is less likely to hinder as the amorphous as the oligo(ethylene glycol) chains. OEG chains take on an amorphous structure until the packing density is such that helical structures are formed, making reaching the surface for aryl diazoniums harder, in comparison to alkane chains which will more readily take on a straight chain formation allowing easier access to the electrode surface. As such, the decane will not hinder the attachment of the aryl diazonium radicals to the surface as much, producing a better packed layer, with the decane chain in a vertical orientation which will give a greater
Chapter 4 | 165
2+/3+ distance to the surface thus causing hindrance of the electrons from Ru(NH3)6 to the electrode. It is also noted that both the decane and OEG6OH aryl diazonium modified
3-/4- layers give complete passivation towards Fe(CN)6 (Fig. 4.9b) whilst both OEG3OH and OEG3OMe aryl diazonium modified layers give similar passivation results.
a b
Figure 4.9 a) Ruthenium hexamine before and after surface modification on gold. b)
Ferricyanide before and after surface modification on gold.
4.3.2 CONTACT ANGLES
To examine the hydrophobicity and hydrophilicity of the modified surfaces contact angles on the surface with water were measured and compared with literature values (Table 4.2). From literature it was observed that by increasing the number of ethylene glycol units the contact angle decreased, the contact angle also decreased as the distal end changed from a hydroxy to a methoxy.
The aryl diazonium salt derived layers on gold surfaces recorded lower surface contact angles than both bare gold and decane aryl diazonium derived modification layers (Fig 4.10). This reduction in the contact angles of the OEG aryl diazonium
Chapter 4 | 166
derived layers can be attributed to the hydrophilicity of the OEGs. The similarity of the contact angles of OEG aryl diazonium derived modified gold surfaces suggests that a densely packed layer is not forming, thus a similar surface is being presented to the water giving similar contact angles. This is not in accordance to literature values of
OEG thiol derived layers where the longer chain lengths on gold give a comparatively lower contact angles to those with shorter chain lengths.
Monomethylether Monomethylether triethylene hexaethylene Surface Bare Decane hexaethylene triethylene glycol glycol glycol glycol Gold 82.2 ± Aryl 84.8 º 51.3 ± 4.5 º 57.0 ± 3.3º 37.5 ± 20.5 ° 54.8 ± 2.9 º 1.5 º diazonium 105.4 Gold thiol 30.0 ± 4.0 ° 45.8 ± 9.4 ° ± 2.5 ° Gold Reported 63 ± 2° 30-35 ± 2º thiol15, 36 76.6 ± PPF 90.8 ° 51.9 ± 3.1° 68.5 ± 4.5 ° 17.0 ± 4.8 ° 69.4 ± 7.6 ° 6.0 ° Silicon reported 109 ° 45 ° angles41
Table 4.2 Comparison of contact angles of aryl diazonium derived layers on gold and PPF
surfaces, and thiol derived surfaces. Literature values were also noted for comparison. Where
± is the standard deviation of three measurements.
Chapter 4 | 167
a b c
d
Fig 4.10 a) decane aryl diazonium derivative on gold b) triethylene glycol derivative on gold
c) monomethylether triethylene glycol on gold d) hexaethylene glycol derivative on gold
The contact angles of the aryl diazonium derived PPF surface again have lower contact angles for that of the OEG derivatives to those of both the bare PPF and the decane aryl diazonium derived surface. The contact angles of the OEG aryl diaznoum derived layers on the PPF were larger in comparison to those on gold suggesting that again the packing of the OEGs is less dense though the gold surfaces show slightly better packing. This difference in contact angles from PPF to gold and the suggested better packing of the aryl diazonium derived layers on gold than PPF is not reflected in the comparison of protein resistance, as will be discussed later.
4.3.3 EFFECT OF MODIFICATION METHOD ON RESISTANCE TO NON-SPECIFIC
PROTEIN ADSORPTION
Aryl diazonium salts can be formed and reduced to the surface in several ways.
The first requires the aryl diazonium salt to be pre-made before a solution is made in
Chapter 4 | 168
acetonitrile with tetrabutylammonium tetrafluoroborate as an electrolyte. The aryl diazonium is then electrochemically reduced and reacts with the surface. This method is one of the standard methods used but there has been an increase in the use of in situ formation of the aryl diazonium from the aniline and direct electrochemical reduction to the surface. This increase is due to the aniline being more stable than the aryl diazonium salt. The first in situ method uses the aniline instead of the aryl diazonium salt. The solvent is also acetonitrile with tetrabutyl ammonium tetrafluoroborate as an electrolyte, but perchloric acid is added together with sodium nitrite solution to reduce the aniline to the aryl diazonium salt which is then electrochemically reduced to the surface. The second in situ method again requires the aniline, but uses an aqueous environment. Potassium chloride is used as the electrolyte, hydrochloric acid and sodium nitrite are added to the solution to reduce the aniline. Electrochemical reduction is carried out directly afterwards on the aryl diazonium formed, forming the radical and reacting it to the surface to form a modified layer.
It was shown by Liu et al.50 that the method of aryl diazonium formation has little difference in the amount of aryl diazonium on the surface on the surface with a mixed monolayer. Before comparisons of the resistance to non-specific protein adsorption of the different OEG aryl diazoniums could be conducted, the affect of the modification method towards the resistance of non-specific protein adsorption was explored. Using OEG3OMe as a base surface, the protein absorbed on the surface was measured using fluorescent microscopy.
The amount of BSA-FITC was measured on the outside bare PPF surfaces and the inside modified PPF surface by measuring the mean grey scale of each resulting
Chapter 4 | 169
image taken of the surfaces using a fluorescent microscope. As shown in Fig. 4.11, premade diazonium salts of OEG3OH on PPF gave a lower protein resistance and smaller uncertainty in comparison to those that were made in situ. PPF surfaces modified via the in situ formation of aryl diazonium salts in acetonitrile gave a greater uncertainty in comparison to those formed in an aqueous environment. The use of the premade aryl diazonium salt for the modification of the surfaces was continued to be used as the method to modify the surfaces. The premade aryl diazonium salt gave the smallest ratio of the measured mean grey scale of bare PPF to modified PPF, it also had the smallest standard deviation.
Figure 4.11 Level of protein resistance of OEG3OH aryl diazonium salt derived PPF surfaces
attached to the surface using different modification methods in comparison to bare PPF surfaces
from fluorescent microscope measurements. (n= 4 errors bars are the standard deviation)
4.3.4 EXPOSURE TIME AND EFFECT ON NON-SPECIFIC PROTEIN ADSORPTION
Biosensors are intended for use with complex matrix samples including blood and it is assumed that the use of the sensor should require minimum time. It is useful
Chapter 4 | 170
therefore to study the effects of time on a surface exposed to protein. The OEG3OMe aryl diazonium on PPF was employed as it has previously been studied. The intensity of fluorescence on each surface exposed to BSA-FITC was imaged with a fluorescent microscope and the mean grey scale was measured. The modified area on the PPF samples were standardised to the bare PPF, and the ratios of the modified surfaces to the bare surfaces where compared in regards to the exposure time. It was observed that a maximum amount of protein was adsorbed onto the surface within two minutes. The fluorescence ratios of bare to modified PPF decreases after two minutes as seen in Fig.
4.12. It can be assumed then that surfaces ability to resist non-specific protein adsorption is reached within 20 minutes, and that the ability to resist is maintained for an hour. An equilibrium time is required for the maximum resistance to protein adsorption to be obtained.
Figure 4.12. Ratios of the mean grey scale of PPF OEG3OMe aryl diazonium salt derived
compared to bare PPF surfaces exposed to 1 mg/mL BSA-FITC in PBS for different periods of
time (from 2 min to 60 min, n = 4 and error bars are standard deviation). The fluorescence of
the modified area is standardised to the surrounding bare PPF surface.
Chapter 4 | 171
4.3.5 RESISTANCE TO NON-SPECIFIC PROTEIN ADSORPTION ON PPF
SURFACES
Two methods were trialled to investigate the ability of the modified surfaces to resist protein adsorption. The first method was an elution method where the modified surface was exposed to the fluorescently labelled protein, which was then eluted off the surface and the corresponding solution was measured using a fluorescent spectrometer.5,
41, 56 The second method measured the amount of protein on the surface directly with a fluorescent microscope.48
4.3.5.1 ELUTION OF FLUORESCENTLY LABELLED PROTEIN FROM THE SURFACE
By eluting the protein off a modified surface, and measuring the amount of protein obtained, the amount of protein absorbed onto the surface can be quantitatively assessed.5, 56 A calibration curve was made to calculate the amount of protein adsorbed by the PPF as seen in Fig. 4.13. PPF surfaces were modified with the OEG aryl diazonium salts and the non-modified areas of PPF were removed. The entire surface was exposed to the protein solution including both back and front.
Chapter 4 | 172
Figure 4.13 Calibration curve of the fluorescence of BSA-FITC in elution buffer.
It was observed from Fig. 4.14, that the results varied greatly for surfaces prepared under the same conditions, and that little difference was seen between modified and non-modified surfaces. However, a reduction of 50% of absorbed protein on the OEG surfaces can be seen in comparison to both the bare PPF and the decane aryl diazonium derived surfaces. The measured eluted protein from the decane modified PPF surfaces, is 3.8 times greater than that absorbed on an alkene derived porous silicon surface (0.25 μgcm-2 PPF to 0.065 μg cm-2)56 methods. This lack of difference between the OEG modified surfaces and the large difference between alkane modified PPF surfaces and alkane modified silicon surfaces, was thought to be due to the entire PPF piece being exposed to the protein solution. This includes both the modified regions as well as the rough bare silicon underside. It is believed that the rough bare silicon underside is absorbing too much protein and also producing too much variable in the result to see the difference between the different OEG aryl diazonium derived surfaces.
Chapter 4 | 173
Figure 4.14 Measured fluorescence and mass results of BSA-FITC that was eluted from the
OEG aryl diazonium derived PPF surfaces per cm2.
4.3.5.2 FLUORESCENT MICROSCOPY
Downard et al. have previously used a fluorescent microscope to look directly at the modified surface to measure the amount of protein on the surface qualitatively against the bare surface.48-49 It is thought that perhaps this method can be applied to confirm the results seen above. Unlike the elution method, where the fluorescence of the eluted BSA-FITC can be compared to a standardised calibration curve to quantatively measure the amount of the protein on the surface, the use of the fluorescence microscope does not allow the comparison to a known amount of BSA-
FITC. As the elution method was inappropriate for the PPF surfaces, it is believed using the fluorescence microscope to measure the BSA-FITC absorbed on the surface will allow a more reproducible qualitative comparison of the different OEG aryl
Chapter 4 | 174
diazonium derived surfaces by the comparison of the modified areas of PPF to the surrounding bare PPF surface. By using the large area electrochemical cells the PPF surfaces could both have a bare outer edge and a modified inner section as illustrated in
Fig. 4.15. This allowed for internal calibration of the samples.
Bare PPF
Modified PPF
Figure 4.15 Illustration showing a PPF sample exposed to BSA-FITC with a bare PPF outer
edge and a modified inner circle.
Images are taken of both the modified and unmodified areas (Fig. 4.16). The fluorescence of each surface is calculated by measuring the mean grey scale of each image. For each surface the mean grey scale of the modified area is compared to either the bare surface, as carried out for PPF surfaces, or the decane modified surface, as for the gold surfaces; this will be discussed in more detail later.
Chapter 4 | 175
a b c
d e f
g h
i j
Figure 4.16 Images of bare and modified PPF surfaces taken with a fluorescence microscope
with a 60×60 oil lens. a) Bare PPF exposed to BSA-FITC. b) Decane modified PPF exposed to
BSA-FITC. c) OEG3OMe modified PPF exposed to BSA-FITC. d) Bare PPF control, not
exposed to BSA-FITC. e) Decane modified PPF control, not exposed to BSA-FITC. f)
OEG3OMe modified PPF control, not exposed to BSA-FITC. g) OEG3OH modified PPF
exposed to BSA-FITC. h) OEG6OH modified PPF exposed to BSA-FITC. i) OEG3OH
modified PPF control, not exposed to BSA-FITC. j) OEG6OH modified PPF control, not
exposed to BSA-FITC.
The aryl diazonium derived surfaces on PPF are compared to both the OEG aryl diazonium derived gold modified surfaces and the thiol derived gold surfaces. The use
Chapter 4 | 176
of the OEG thiol derived surfaces, as a comparison will provide a well understood OEG modified surface. This will provide a better indication of the performance of the OEG aryl diazonium derivatives as they can be compared.
Due to fluctuation of the power in the mercury lamp on a day-to-day basis the measured fluorescence needed to be standardised to ensure that all measurements were comparable, because the fluorescence of the OEG aryl diazonium derived PPF surfaces were not all measured on the same day. The fluorescence intensity was normalised to overcome any day to day variability by dividing by the intensity of the surrounding bare surface. Once the fluorescence measurements on the OEG aryl diazonium derived PPF surfaces had been compensated for lamp fluctuation they were then compared to the decane surface (Fig 4.17). Since the gold surfaces were measured on the same day, no compensation for lamp fluctuation was required before the OEG aryl diazonium derived gold surfaces were compared to the decane aryl diazonium derived gold surface (Fig
4.17). It was observed that quenching of the fluorescence of the FITC occurred on the bare gold surface, but once modified quenching of the fluorescence was slowed due to the modified surfaces providing a “buffer zone” between the FITC and the gold surface.
Chapter 4 | 177
Figure 4.17 Ratio of mean grey scale of OEG aryl diazonium derived surfaces on gold and PPF
and OEG thiol derived gold surfaces in comparison to decane aryl diazonium derived PPF and
gold surfaces plus dodecane thiol derived gold surfaces. By comparing to decane modified
surfaces and indication as to the resistance of the surface to non-specific protein adsorption is
gained. As were recorded from the measurement of the fluorescence of BSA-FITC absorbed
onto the surface via a fluorescence microscope. Error bars indicate the standard deviation.
It can be seen from the results shown in Fig. 4.17, that alkane thiol terminated ethylene glycols gave the biggest reduction in protein resistance of around 90% in comparison to decane modified surfaces. Aryl diazonium derived ethylene glycol derivatives on PPF showed an increase in protein resistance in comparison to those on gold, but still exhibited good protein adsorption with a reduction of between 50-70%.
Although neither of these exhibit as much resistance to protein as the thiol modified surfaces which gave a reduction of 90% in comparison to the dodecane thiol derived
41 gold surface. It was seen from Böcking et al. that OEG3OMe modified silicon surface
Chapter 4 | 178
gave a 70-80% reduction of protein in comparison to octadecyl Si-C layers. From this it was concluded that OEG aryl diazonium derived PPF surface produce comparable protein resistance to the OEG modified silicon surfaces.
The changes in the distal end and amount of ethylene glycol units had little effect on protein resistance for OEG aryl diazonium derived modification layers on PPF
The effect of the charge of the distal end of OEG-aryl-diazonium-modified-gold surfaces shows a decrease in the non-specific adsorbed protein from methoxy to hydroxy. The variation of protein adsorption between alkane modified surfaces was also investigated. It can be seen that little difference can be observed between the thiol- derived-gold surfaces and the aryl-diazonium-derived-PPF surfaces in the intensity of fluorescence measured. The aryl diazonium derived gold surfaces though, were twice the intensity of measured fluorescence in comparison to both the aryl diazonium derived
PPF surface and the thiol derived gold surfaces as seen in Fig. 4.18. This difference in intensities suggests that the aryl diazonium derived gold surfaces absorb greater amounts of protein in general. However when compared to a non-protein resistant surface, modified the same way, protein resistance is still achieved, although there may be more interference produced.
Chapter 4 | 179
Figure 4.18 Comparison of the measured mean grey scale of fluorescence measured on the surface of the different alkane comparisons for the OEG protein resistance for each modification
method and surface, error bars are the standard deviation.
4.4 CONCLUSION
From this study it can be concluded that the use of OEG-aryl-diazonium-derived layers on both gold and PPF surfaces were able to reduce the amount of protein adsorbed onto the surface when compared to alkane-aryl-diazonium-derived layers. The protein resistance of the OEG aryl diazonium derived PPF surface is consistent with previous studies34, 36, 41, 49, 57 showing that loosely packed surfaces produce lower protein resistance. As seen by the similarity of the contact angles, it is concluded that the packing of the OEG aryl diazonium derived modification layers on PPF are loosely packed.
The length of time that the surface is exposed to the protein solution has an effect on the surfaces ability to resist proteins and it was shown that 10 minutes is required before the amount of protein adsorbed or resisted becomes stable. Using the in
Chapter 4 | 180
situ method of aryl diazonium derived modification also produces greater uncertainty in the protein resistance of the aryl diazonium derived modification layers on PPF.
The use of a fluorescent microscope to measure qualitatively the amount of protein on the surface is useful for surfaces that are only able to be modified on one side. In comparison, measuring the amount of protein eluted from the surface can also show the relative effectiveness of the surface modification, although a greater uncertainty is observed as the underside or non-modified side is a large factor in the amount of protein adsorbed.
4.5 REFERENCES
1. Herwerth, S.; Rosendahl, T.; Feng, C.; Eck, W.; Himmelhaus, M.; Dahint, R.; Grunze,
M., Covalent coupling of antibodies to self-assembled monolayers of carboxy-
functionalised poly(ethylene glycol): Protein resistance and specific binding of
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33. Pertsin, A. J.; Hayashi, T.; Grunze, M., Grand Canonical Monte Carlo Simulations of
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36. Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E., Molecular
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Chapter 4 | 188
Chapter Five
Iodination and Alknene
Chapter 5 | 189
5.1 INTRODUCTION
The use of aryl diazonium salts to modify a surface as discussed in Chapter 1 has the potential to produce multilayers instead of monolayers.1 Most commonly, the formation of monolayers on gold is by the use of organothiols. The gold thiolate bond though is polar, with a bond energy of 167 kJ /mol,2 and is prone to oxidation from light and heat3-4 as well as thermal desorption.5-7 The ability to combine the stability of the diazonium layers on carbon and the formation of monolayers as well-defined as the gold-thiols system would allow for a versatile surface modification method for electrochemical biosensors and many other applications.8
5.1.1 COMBINING STABILITY OF COVALENT BONDS AND MONOLAYERS
5.1.1.1 ALKENES AND ALKYNES ON SILICON
The use of alkenes to modify silicon surfaces has been well documented.9 It has been shown that hydrogenation of the surface produces a hydrogen terminated silicon surface.10 This can be carried out chemically using hydrofluoric acid or ammonium hydrogen fluoride.11 When the hydrogenated silicon is exposed to alkenes or alkynes the surface can be further modified by the use of either light or heat to give a stable carbon-silicon bond.11-14 The modification of silicon though needs to be carried out in inert atmospheres, as exposure to oxygen before or during modification will produce silicon oxides on the surface. This formation of oxides on the surface can cause surface degradation, influence the electrical properties of the device being fabricated15-16 and can make the surface unstable for electrochemical sensors.17-18 On a well-formed
Chapter 5 | 190
alkene derived silicon surface though, further modification can be carried out with several different methods.10 One method is by activating the surface followed by attachment of the next modified layer, as seen with the use of EDC/NHS to activate acid terminated surfaces (Fig 5.1 a). Another method is the use of “click” chemistry where diazo derivatives can be added to alkyne surfaces (Fig 5.1 b).17
OH OH
a O O
OH O O
O O O
OH O O O O
O N O O N O O OH OH O O HN HN O O O O O O O
H2N EDC/NHS Δ
H H H H H H H H H H Silicon Silicon Silicon Silicon
b HO HO
O O OH
O O O
O O O
N N O N N N N
N3
Δ
H H H H H H H H Silicon Silicon Silicon
Figure 5.1 Further modification of silicon surfaces. a) The use of activating the surface. b)
The use of “click” chemistry.
Chapter 5 | 191
5.1.1.2 ALKENES ON DIAMOND
The use of diamond as a carbon surface was previously discussed in Chapter 1.
It was shown that the hydrogenation of boron-doped diamond surfaces produces a conductive surface that can be further modified with alkenes via ultra violet (U.V.) light. The modification of the diamond surface is thought to be from photoelectron ejection from the surface to the alkene forming a radical on the surface that then binds to an alkene.19-20 The activation of the hydrogenated diamond surface via photo ejection is similar to the proposed method by which hydrogenated silicon reacts with alkenes with U.V. light.10, 13-14 By using an alkene with an electron withdrawing group, a more densely packed surface is produced.21-22
The use of alkene modified diamond surfaces for DNA detection has been shown by Hamers and co-workers23 where a diamond surface was modified with trifluoroacetamide-protected-10-aminodec-1-ene (TFAAD) under U.V. light for 12 hours. The TFAAD was deprotected to expose the amine; the amine was then reacted with a thiol terminated single strand DNA (Fig 5.2). The surface was then exposed to fluorescently labelled complementary and non complementary DNA and the binding was investigated via the fluorescence emitted from the surface.
Chapter 5 | 192
S S
S O O O O O
CF3 N O NH O CF3 CF3 O O O O NH NH NH2 NH2 NH NH O O N O
SO2Na HCl
Methanol hv H H H H H 254 nm Diamond Diamond Diamond Diamond
Figure 5.2 Alkene addition to hydrogenated diamond surfaces and subsequent modification
with sDNA.
For the preparation of diamond surfaces for electrochemical sensors, boron doped diamond needs to be deposited on the surface via microwave plasma.24-25 Boron doped diamond forms nano-crystalline surfaces, which then need to be further modified with hydrogen plasma. The use of plasma to hydrogenate the diamond produces a rough surface.26-27
The hydrogen-carbon bond produced is reactive towards alkenes with the addition of U.V. light, forming a stable bond.19-20, 28-31 The hydrogen-carbon bond in hydrogenated-amorphous carbon is very stable, in air these surfaces do not oxidise as quickly in comparison to freshly prepared bare amorphous carbon. The modification layer therefore requires a high energy input to break these bonds. Is it possible to form a halogenated surface which will provide a weaker surface modification and which will react with the alkene easier? It is hypothesized that the versatility of the smoothness of
Chapter 5 | 193
the PPF can be combined with the ability to modify these surfaces with alkenes. This is where iodination of the surface fits in; iodination could provide a surface modification similar to that of hydrogenation whilst providing a less stable bond to react with alkenes.
5.1.2 IODINATED SURFACES
Iodinated carbon and silicon surfaces have previously been prepared via chemical reactions.32 It has been shown by Cai et al. that silicon surfaces can be iodinated through the immersion of hydrogenated silicon into a solution of iodine and benzene.33
Iodination of carbon has also been shown to be achieved with polymers and amorphous films.32 More recently, iodination has been used to modify carbon nanotubes via heating single walled nanotubes in iodine at 250°C for 3 days.34
Amorphous carbon films were iodinated during formation via pyrolysis of maleic anhydride and solid iodine. A vapour is formed by the two reactants which is then pyrolysed to the surface at temperatures of 700 - 980°C.35 Diamond films have also been grown with iodine incorporated instead of boron. The use of a mixture of iodobenzene and benzene to form polymers has also been used to incorporate iodine.36
The possibility of iodinating PPF through radio-frequency (RF) generated plasma, would allow the smoothness of PPF to be combined with the ability to react alkenes to the surface to form covalently bound monolayers.
Chapter 5 | 194
5.2 EXPERIMENTAL METHODS
5.2.1 PREPARATION OF PPF
PPF was prepared as per chapter 2 section 2.3.1.1.
5.2.2 IODINATION OF PPF
Iodination of the PPF surfaces was carried out using a radio-frequency (RF) plasma generator in a reaction chamber (Fig. 5.3). To investigate the effect of iodine plasma on the PPF it was exposed for different time and power settings (Table 5.1).
Pressure Reactor readoutr chamber RFR generator Liquid nitrogen
trap and
vacuum line
Figure 5.3 Plasma reactor set up with RF generator.
Chapter 5 | 195
1 min 2 min 5 min 10 min 30 min 60 min
0 W 30 W 60 W - - - 100 W - - - -
Table 5.1 Power settings and time that PPF was exposed to.
For each power setting and time three repeats were run, except for 10 min at 30
W where 6 were run. The iodination time and power settings are denoted as, X min Y
W, where X is the time exposed to the plasma and Y is the power of the plasma.
Solid iodine was put in the bottom of the chamber in a small beaker. The samples were positioned in the middle of the chamber which was then sealed and placed under vacuum. The chamber was evacuated down to 0.8 mbar and left for 5 min. The
RF generator was turned on and the frequency set to 116.5 kHz. The power was increased to the set wattage and the samples left in the plasma for the required time.
Once finished the power was turned off and the vacuum slowly released, the samples where removed and rinsed with ethanol, they were then dried with nitrogen.
5.2.3 ALKENE AND ALKYNE ADDITION TO AN IODINATED PPF SURFACE
A thin layer of neat liquid alkenes or alkynes was placed on the iodinated surface and placed in a quartz tube, which was then flushed with argon gas (Fig. 5.4).
Two different wavelengths were used 254 nm and 514 nm. Undecylenic acid was used as well as nonadiyne and S-undec-10-enyl-2,2,2-trifluoroethanethioate. The surfaces
Chapter 5 | 196
were then exposed to the required alkene or alkyne for 12-16 hrs before the U.V. light was switched off. The surfaces were washed with dichloromethane and ethanol and dried under nitrogen. In the case of undecylenic acid, the surfaces were also rinsed with water and then hot acetic acid, followed by water and then ethanol before being dried with nitrogen.
Figure 5.4 U.V. reaction chamber.
For the investigation in to the U.V. light wavelength to use for surface modification with alkenes not all exposure times and plasma power setting were used. For surface modified with U.V. light at wave length 254 nm with undecylenic acid, six PPF surfaces were iodinated for 10 min at 60 W, and also six untreated PPF surfaces were used. For surfaces exposed to undecylenic acid with U.V. light at wavelength 514 nm the following exposure times and power settings were used, three repeats of each were also run; untreated PPF, 5 min at 30 W, 10 min at 30 W, 30 min at 30 W, and 30 min at
60 W.
Chapter 5 | 197
5.2.4 ANALYSIS OF THE IODINATED AND UV REACTED SURFACES
5.2.4.1 CONTACT ANGLE
Static contact angles were carried out according to chapter 2 section 2.2.1.2.2, as were advancing and receding angles.
5.2.4.2 ELECTROCHEMICAL CHARACTERISATION
Electrochemical investigation into the passivation of the surface was carried out according to chapter 2 section 2.3.2.1. The surfaces were placed in Teflon cells limiting the exposure area for each sample to a circle of 3 mm diameter. For iodination of the
PPF the electrochemical probes used were ruthenium hexamine, ferricyanide, ascorbic acid and ammonium iron sulphate. These redox complexes were used as changes in their behaviour reflect changes in the surface.
For the investigation of alkene and alkyne modified surfaces only ruthenium hexaamine and ferricyanide were used.
5.2.4.3 X-RAY PHOTOELECTRON SPECTROSCOPY
X-ray photoelectron spectroscopy (XPS) investigations were carried out on the surface by Muthukumar Chockalingam and Erwann Luais. The spectra were calibrated to carbon, and the atomic percentages were made against the bulk carbon as per chapter
2 section 2.2.1.2.3.
Chapter 5 | 198
5.2.4.4 ATOMIC FORCE MICROSCOPY
Tapping mode atomic force microscopy (AFM) was used to measure the roughness of the surface as per chapter 2 section 2.2.1.2.5.
5.2.4.5 “CLICK” CHEMISTRY
PPF surfaces were iodinated at 30 W for 30 min. The surfaces were then modified with nonadiyne with 514 nm U.V. light. The surfaces were then reacted with tetra(ethyleneglycol) azide (40 mg), in an aqueous solution of copper sulphate (1 mol% relative to the azide) with ascorbic acid (25 mol % relative to the azide) in ethanol (1 mL), and N,N,N’,N”-tetramethylethane-1,2-diamine (1 mol % relative to the azide) as the ligand over night. The surfaces were removed and rinsed with water and ethanol and were dried with nitrogen. This experiment was repeated to give a sample set of four.
5.2.4.5 PATTERNING OF IODINATED PPF
5.2.4.5.1 SYNTHESIS OF GOLD NANO PARTICLES
All glassware was cleaned first with aqua regia (1:1 hydrochloric acid and nitric acid) followed by piranha (1:2 hydrogen peroxide to sulphuric acid). After acid cleaning to remove all organic and inorganic contaminants, the glassware was rinsed thoroughly with Milli-Q water.
Chapter 5 | 199
A solution of gold chloride (40 mg) in Milli-Q (150 mL) water was brought to boiling. A solution of sodium citrate (20 mg) was added and the solution turned red.
The solution was allowed to boil until the volume was reduced by half. Subsequently the reaction was cooled to room temperature and transferred to a 100 mL volumetric flask and made to the mark with Milli-Q water.37 This procedure produces gold nano particles with a diameter of 67 ± 11 nm.
5.2.4.5.2 PATTERNING OF THE IODINATED PPF SURFACE WITH S-UNDEC-10-
ENYL-2,2,2-TRIFLUOROETHANETHIOATE
Three PPF surfaces were iodinated at a power setting of 30 W for 30 min. The surfaces were rinsed with ethanol and dried under nitrogen. Neat S-undec-10-enyl-
2,2,2-trifluoroethanethioate (C11-S-TFA) (10 μL), synthesised by Guillaume Le Saux, was placed on the surface and a quartz cover slip was placed over this. On top of the quartz cover slip a TEM grid was placed (Fig. 5.5). The surface was exposed to 514 nm
U.V. light for 12 hours. The surfaces were removed and rinsed with dichloromethane then ethanol and dried with nitrogen.
TEM grid
Quartz cover slip
PPF
Figure 5.5 Patterning set up with iodinated PPF and C11-S-TFA.
Chapter 5 | 200
5.2.4.5.3 DEPROTECTION OF C11-S-TFA MODIFIED SURFACES
To deprotect the amine of C11-S-TFA the surfaces were left in a 10 % ammonium hydroxide solution (10 % ammonia in water) for ten minutes before being removed and rinsed with water then ethanol and dried with nitrogen.
5.2.4.5.4 ATTACHING GOLD NANOPARTICLES TO THE MODIFIED SURFACE
The now amine terminated surfaces were exposed to gold nanoparticle solution, prepared in section 5.2.4.5.1, solution for 2.5 hrs before being removed and washed with water then ethanol and dried with nitrogen.
5.2.4.5.5 SEM IMAGING
SEM images were collected by Guozhen Lui. The surfaces were coated with chromium before the images were taken. SEM was carried out according to chapter 2 section 2.2.1.2.4.
5.3 RESULTS AND DISCUSSION
The ability to iodinate the surface and carry out subsequent alkene/alkyne modification would allow the formation of covalently bound monolayers. The use of
RF plasma to modify surfaces has been shown for the use of diamond like films and
Chapter 5 | 201
polymer formation.36, 38 Can PPF be iodinated using iodine plasma in a similar manner to the way diamond like films are hydrogenated?
Plasma is formed from the ionisation of the gas. Hydrogenation of amorphous carbon via hydrogen plasma has been previously shown by Swain and co-workers,26, 39 and Kuo et al..27 From these experiments it was observed that the plasma roughened the surface.27 As plasma is highly energised ionised gas, the surface is bombarded which causes bond breaking. It has been shown by Hamers and co-workers26 that glassy carbon (GC) exposed to hydrogen plasma becomes rough and the height of the GC is reduced from exposure to the plasma. To find an appropriate power setting for the plasma, and the appropriate exposure time the PPF pieces were exposed to different combinations of times and power settings (Table 5.1). The affect that iodine plasma has on the surface was studied. Electrochemical effects as well as surface effects were investigated. Electrochemical redox probes were used to monitor how the iodine affects the surface behaviour. The affect on surface roughness was measured via AFM. The use of XPS allowed investigation of the binding of the iodine to the surface.
5.3.1 IODINATION OF PYROLYSED PHOTORESIST FILMS
In the work presented herein, it was regarded as desirable that the iodination of
PPF was achieved whilst maintaining the smoothness, and electrochemical properties, of PPF but at the same time allowed the further modification of the surface with alkenes. Different power settings were used, and the PPF surfaces were exposed to each setting for different amounts of time (Table 5.1). The amount of iodination of the
PPF was quantified using XPS, whilst the effect of the iodine on the surface
Chapter 5 | 202
electrochemistry was measured using surface sensitive redox probes. The effect on the iodination on the topography of the surfaces was investigated with AFM, to observe any increase in roughness, and by using a contact angle goniometer. In general the colour of the PPF surfaces became a darker grey, with hints of orange, though they were still reflective. However, surfaces exposed to 100 W for 30 min and 60 min, became blue in colour and passivation of the surface was observed. Occasionally PPF exposed to 60 W plasma for 30 min became blue. The orange colour observed is due to iodine being deposited on the surface. The increase in the change of colour from orange to blue on the PPF surfaces after exposure to iodine plasma of higher powers and times can be attributed to the surface becoming more diamond like, but this it yet to be confirmed.
After exposure to the iodine plasma the samples also contain a little heat ~ 20 ºC, as the exposure time is increased and the power of the iodine plasma is increased the samples become hot. For the lower plasma power of 30 W no heat is felt in the samples, the same is observed for samples exposed to 60 W plasma power for 0 to 30 min, those exposed to 60 W for 60 min, contain some heat ~ 25 ºC. Samples exposed to 100 W for longer than 30 min also contain heat when being removed from the reactor ~ 30 ºC.
This is expected and plasma is a highly energetic process as well as the formation of iodine-carbon bonds.
5.3.1.1 XPS OF IODINATED SURFACE
Upon iodination of PPF a wide scan was carried out and several peaks were observed corresponding to carbon, oxygen and iodine (Fig. 5.6a). In the narrow scan of the I3d region two types of iodine peaks were observed. One at 618.5 eV for the I3d5, and a corresponding peak for the I3d3 at 629 eV, and one at 620 eV for the I3d5 with a
Chapter 5 | 203
corresponding peak at 631 eV for I3d3 (Fig. 5.6b). It was observed that upon rinsing with ethanol the peak at 618.5 eV diminished and is attributed to physisorbed iodine.38
The peak at 620 eV is attributed to iodine bound to carbon.36 Surfaces left under vacuum with iodine in the chamber, but without plasma formation, only presented iodine peaks at 618.5 eV which also supports the hypothesis that the peaks at 620 eV were due to iodine bound to carbon.35, 40
a
b
Figure 5.6 XPS spectrum of iodinated PPF. a) Wide scan of the sample showing two I3d peaks
around 600 eV and 1 C1s around 280 eV. b) A narrow scan of the I3d region showing
physisorbed iodine (618 eV and 629 eV) and iodine bound to carbon (620 eV and 632 eV).
Chapter 5 | 204
As the power of the plasma increased it was observed that the amount of bound iodine increased relative to the intensity of the C1s peak at 280 eV (Fig. 5.7). It was also noted that the longer the PPF was left in the plasma, the greater the amount of surface carbon bound iodine (Fig 5.8).
a
b
c
Figure 5.7 XPS spectrum showing wide scans of PPF iodinated with different power settings
plasma for 10 min. The higher the power the greater the amount of iodine bound to carbon (~
600 eV) is relative to carbon (~280 eV). a) 30 W. b) 60 W. c) 100 W.
Chapter 5 | 205
Figure 5.8 Amount of iodine bound to carbon in relation to time exposure and plasma power,
the errors bars reflect the standard deviation of n = 3 measurements.
For the higher plasma power settings it was noted that the amount of iodine was found to be greater than a monolayer for a given surface area of a PPF sample, which suggests that roughening of the surface due to the plasma. Multilayers of iodine are shown not to form as there is no increase in peak at 618.5 eV for the iodine bound to iodine, but the iodine bound to carbon does increase. For shorter exposure times, 1-5 min, significantly less carbon bound iodine was observed.
5.3.1.4 ROUGHNESS OF PPF AFTER IODINATION
Roughening of the surface was observed as power increased as well as the length of time that the PPF was exposed to the iodine plasma (Fig. 5.9).
Chapter 5 | 206
Figure 5.9 Roughness as measured by the root mean square (rms) value as determined via
AFM, of PPF exposed to iodine plasma at different powers for different exposure times.
PPF exposed to iodine plasma at 100 W was shown to dramatically increase the surface roughness; it was not investigated further as the smoothness of PPF is required to be retained to form well defined molecular constructs on PPF surfaces. PPF exposed to RF plasma at 30 W was not observed to increase in roughness (Fig. 5.10). Nor did the time the PPF was exposed to iodine influence the roughness of these surfaces when
30 W power was used in the plasma reactor. Higher power plasma produced much greater variability in roughness. As with the amount of carbon bound iodine, the greater the plasma power, and longer exposure time, the greater was the increase in surface roughness. For both the 30 W plasma power and the 60 W power gave a general roughness, root mean square (rms), under 1 nm which is still significantly smoother than most glassy carbon surfaces.
Chapter 5 | 207
Figure 5.10 Roughness of iodinated PPF surfaces using 0 W, 30 W and 60 W iodine plasma.
5.3.1.2 ELECTROCHEMICAL BEHAVIOUR OF THE SURFACE
Cyclic voltammetry was carried out on the iodinated PPF surfaces. Ruthenium
3+/2+ hexamine (Ru(NH3)6 ) was used as it is not surface sensitive, ferricyanide
3-/4- (Fe(CN)6 ) was used as it is sensitive to adventitious species adsorbed onto the surface. Ammonium iron sulphate (Fe2+/3+) was used as it is oxide sensitive.41 By using these three redox active species the affect of iodinating the surface on the electrochemical behaviour of PPF can be investigated. Any passivation of the surface due to the addition of iodine can be observed. Phosphate buffer was also used to examine the electrochemical behaviour of iodinated PPF. Tables 5.2, 5.3 and 5.4 and
3+/2+ 3-/4- 2+/3+ figures 5.12, 5.13 and 5.14 show the ΔEp for Ru(NH3)6 , Fe(CN)6 , and Fe and the corresponding peak heights. It was observed that a slight shift in the ΔEp for
3+/2+ Ru(NH3)6 was observed from the iodination of PPF. Passivation of the surface
Chapter 5 | 208
3-/4- 2+/3+ towards both Fe(CN)6 , and Fe was observed from the increase in both the ΔEp’s and from the decrease in peak heights.
Figure 5.11 Cyclic voltammogram of 50 mM phosphate buffer and 1 M KCl on iodinised PPF
at different power and time settings.
Cyclic voltammetry in phosphate buffer of the iodinated surfaces (Fig 5.11) shows a CV lacking in any peaks from reduction or oxidation. The CV of the iodinated
PPF in phosphate buffer revealed that the iodinated surface exhibited no redox activity due to the iodine on the surface. The lack of any redox activity on the surface is good as any unreacted iodine left on the surface will not produce interfering peaks. It was shown that iodination of the PPF does not have a major influence on the behaviour of
3+/2+ Ru(NH3)6 (Fig. 5.12, Table 5.2). The observation of a small change in the ΔEp
3+/2+ potential of Ru(NH3)6 suggests that some change in the electrical behaviour occurs.
This effect does have a small amount of influence, as the peak height is observed to increase after iodine plasma exposure except PPF exposed to 100 W plasma for 60 min.
Chapter 5 | 209
The 100 W iodine plasma seemed to cause passivation of the surface towards all three
3+/2+ 3-/4- 2+/3+ redox active molecules (Ru(NH3)6 , Fe(CN)6 and Fe ). From the AFM data it was observed that as the power and time exposure to iodine plasma increased so did the roughness, this may be due to a layer of iodine forming on the surface, this may form a
3+/2+ barrier thus affecting the electrochemical behaviour of Ru(NH3)6 . The XPS data though, observed that there was mostly iodine bound to carbon with very little iodine bound to iodine, this discounts the barrier theory. Perhaps the iodine plasma is degrading the PPF surface and the electrochemical behaviour as well.
2+/3+ Figure 5.12 Cyclic voltammograms of 10 mM Ru(NH3)6 in 50 mM phosphate buffer (pH
7) and 1 M KCl scanned at 100 mV/s for PPF exposed to iodine plasma for different times and
powers. Where x min Y W refers to time exposed to plasma and power of plasma, and bare PPF
refers to unreacted PPF.
Chapter 5 | 210
3+/2+ Power setting / W Exposure Time / min ΔEp Ru(NH3)6 / mV 10 188.00 ± 27.62 0 30 163.57 ± 6.90 60 268.56 ± 75.68 1 233.56 ± 39.17 2 224.61 ± 31.08 5 224.61 ± 40.35 30 10 297.85 ± 50.74 30 238.44 ± 32.51 60 203.45 ± 26.78 10 362.14 ± 74.75 60 30 207.52 ± 55.25 60 155.04 ± 32.80 10 207.52 ± 34.18 100 30 152.18 ± 14.10 60 209.96 ± 31.07
3+/2+ Table 5.2 Voltametric peak separation for Ru(NH3)6 for PPF exposed to iodine plasma at
different power settings (W) and different periods of time (min), n = 3.
For samples exposed to 30 W and 60 W iodine plasma the electrochemical behaviour of the PPF is retained. The effect of the iodine plasma on the surface the redox behaviour of surface sensitive complexes was then investigated.
3-/4- As mentioned previously Fe(CN)6 is sensitive to adventitious species
3-/4- absorbed onto the surface. The electrochemical behaviour of Fe(CN)6 was affected as iodine was bound to the surface (Fig. 5.13 and Table 5.3). As the iodine is reacted with the surface it provides a barrier on the surface blocking the ferricyanide from reaching the surface. As the iodine plasma power increased as well as the exposure
Chapter 5 | 211
3-/4- time, the surface became more passivated towards Fe(CN)6 than PPF surfaces only exposed to 30 W iodine plasma for 5 min and 10 min. These observations are
3+/2+ concurrent with that observed for Ru(NH3)6 , as plasma power and time increases the
3-/4- electrochemical response of the PPF diminishes, for Fe(CN)6 there is also the affect
3-/4- of the amount of iodine also increasing prohibiting Fe(CN)6 from coming towards the surface. PPF exposed to 30 W plasma power for shorter periods of time, 1 to 2 min, showed a larger uncertainties as seen in Table 5.3.
3-/4- Figure 5.13 Cyclic voltammograms of 10 mM Fe(CN)6 in 50 mM phosphate buffer (pH 7)
and 1 M KCl scanned at 100 mV/s for PPF exposed to iodine plasma for different times and
powers. Where x min Y W refers to time exposed to plasma and power of plasma, and bare
PPF refers to unreacted PPF.
Chapter 5 | 212
3-/4- Current of reduction peak of Power setting / W Exposure Time / min Ep Fe(CN)6 / mV Δ 3-/4- Fe(CN)6 / mA 10 408.94 ± 46.61 161.50 ± 26.88 0 30 352.78 ± 164.00 124.20 ± 43.74 60 363.77 ± 151.91 231.13 ± 87.31 1 446.77 ± 108.66 170.34 ± 72.06 2 665.283 ± 433.31 150.49 ± 8.72 5 272.22 ± 39.71 156.36 ± 49.29 30 10 454.10 ± 133.20 177.90 ± 38.64 30 547.69 ± 366.54 79.11± 79.0 60 612.80 ± 277.89 8.95 ± 5.2 10 570.48 ± 209.51 83.15 ± 64.8 60 30 423.59 ± 298.66 130.68 ± 47.7 60 599.37 ± 309.02 18.68 ± 15.3 10 469.97 ± 284.84 39.52 ± 32.3 100 30 222.98 ± 59.07 8.32 ± 7.4 60 236.82 ± 82.87 6.42 ± 5.6
3-/4 Table 5.3 Voltammetric peak separation for Fe(CN)6 for PPF exposed to iodine plasma at
different power settings (W) and different periods of time (min). Reduction peak height for
3-/4- Fe(CN)6 . n = 3.
Peak separation of Fe2+/3+ was shown to have increased as the amount of oxide decreased (Fig. 5.14 and Table 5.4). The decrease in oxide species occurred as more iodine was bound to the surface. This was also noted in the XPS as only a small amount of oxygen species were present. Passivation of the surface toward Fe2+/3+ was also observed as the iodine plasma power increased, and the length of exposure increased. Complete passivation of the surface towards Fe2+/3+ was noted for PPF surfaces at 60 W and 100 W plasma power, though it is noted from Tables 5.2 and 5.3
3+/2+ that these surfaces are still electrochemically active towards Ru(NH3)6 and
Chapter 5 | 213
3-/4- 2+/3+ Fe(CN)6 . The passivation of the iodinated PPF surfaces towards Fe can be attributed to the presence of the iodine on surface.
2+/3+ Figure 5.14 Cyclic voltammograms of 10 mM Fe in 0.1 M HClO4 and 1 M KCl scanned at
100 mV/s for PPF exposed to iodine plasma for different times and powers. Where x min Y W
refers to time exposed to plasma and power of plasma, and bare PPF refers to unreacted PPF.
Chapter 5 | 214
Exposure Time / 2+/3+ Current of reduction peak Power setting / W ΔEp Fe / mV min of Fe2+/3+ / mV 10 598.15 ± 130.81 159.95 ± 74.9 0 30 605.47 ± 124.82 ± 20.6 60 693.36 ± 93.22 256.86 ± 47.7 1 756.03 ± 15.55 194.29 ± 74.8 2 771.50 ± 27.65 205.53 ± 54.3 5 668.95 ± 44.88 190.54 ± 80.1 30 10 709.63 ± 93.21 161.22 ± 74.0 30 764.16 ± 15.04 ± 13.1 60 993.68 ± 15.94 ± 11.2 10 - 0 60 30 - 0 60 - 0 10 1058.31 ± 364.72 58.65 ± 47.2 100 30 - 0 60 - 0
Table 5.4 Voltametric peak separation for Fe2+/3+ for PPF exposed to iodine plasma at different
power settings (W) and different periods of time (min). Reduction peak height for Fe2+/3+. n = 3.
5.3.1.3 HYDROPHOBICITY OF IODINATED PPF
The hydrophobicity of the PPF surfaces exposed to the iodine plasma was investigated using contact angle measurements. By examining, the angle formed between water and the iodinated surface the tendency of the surface to be hydrophobic or hydrophilic can be determined. From the results it was observed that the surface after iodination became a little more hydrophilic than a surface exposed to but not
Chapter 5 | 215
reacted with the iodine. In general, regardless of the modification procedure explored, the surfaces remained similar to each other with regards to contact angles (Fig. 5.15).
Figure 5.15 Contact angles of iodinated PPF surfaces exposed for different times at different
iodine RF plasma powers. Error bars are the standard deviation, n = 3.
From the results of exposing PPF to iodine plasma at different power settings and exposure times, not all iodinated settings will be continued with. Iodine plasma of
100 W for any length of time will not be used, as the surface roughness is greatly increased in comparison to other plasma power settings. Surfaces exposed to 30 W and
3-/4- 60 W for 60 min are also not to be used further due to the passivation of Fe(CN)6 and Fe2+/3+. Due to the inconsistency of the iodination of surfaces exposed to 30 W iodine plasma for 1 and 2 min, these samples will also not be used. For further modification PPF exposed to 30 W iodine plasma for 5 min, 10 min, and 30 min will be used and also those exposed to 60 W iodine plasma for 10 min and 30 min. The stability of the iodinated surfaces to heat and exposure to air for longer periods than 12 hours requires further investigation.
Chapter 5 | 216
For the current work, as the samples are being characterised and further modified directly after exposure to the iodine plasma, stability of the surfaces over a long time is not required. Further more as it is thought that by iodinating the surfaces, it creates a less stable surface than those created by the hydrogenation of carbon surfaces.
In saying this an XPS characterisation of surfaces iodinated with 60 W for 10 min, 30 min and 60 min was carried out and it was observed that at 60 % of the iodine was retain on the surface, though further repeats are required.
5.3.2 MODIFICATION OF IODINATED PPF BY U.V. ACTIVATION
As discussed in the introduction for this chapter and in chapter one section 1.4.5, the modification of hydrogenated diamond films with alkenes via U.V. light at a wavelength of 254 nm, can be achieved. The modification of iodinised silicon has also been shown to occur, but with U.V. light at a wavelength of 514 nm, as the iodine- silicon bond is weaker than that of the hydrogen-silicon bond and thus the amount of energy required was less.
To investigate the ability of the iodinated PPF surface to be modified with alkenes, undecylenic acid was used. The iodinated PPF surfaces were exposed to neat undecylenic acid and U.V. light (Fig. 5.16) under argon for 12 to 16 hours.
Chapter 5 | 217
HO O HO HO HO O O O
Iodine
Plasma I I I I I hv PPF PPF PPF
Figure 5.16 Reaction of iodinated PPF with undecylenic acid
5.3.2.1 REACTION OF IODINATED PPF WITHUNDECYLENIC ACID AT 254 NM
Each surface was exposed to undecylenic acid for 14 hrs. The surface hydrophobicity/hydrophilicity was investigated. The passiviation of the surface towards an inner sphere redox probes was investigated electrochemically with ferricyanide, whilst the general electrochemical behaviour was investigated with ruthenium hexamine, an outer sphere redox probe. By investigating how much the electron transfer rate is decreased due to the modification of the surface will give some insights as to how well the surface has been modified with undecylenic acid under U.V. light at wavelength 254 nm. Untreated PPF, and PPF exposed to iodine plasma for 10 min at
60 W (referred to as 10 min 60 W) will be modified with undecylenic acid first. The untreated PPF will act as a control and the 10 min 60 W PPF is used as the electrochemical behaviour of the surface is maintained and it has a high iodination level.
Chapter 5 | 218
5.3.2.1.1 ELECTROCHEMISTRY OF IODINATED PPF EXPOSED TO UNDECYLENIC
ACID AT U.V. WAVELENGTH 254 NM
The passivation of the iodinated PPF surfaces exposed to undecylenic acid was
3+/2+ 3-/4- examined using Ru(NH3)6 and Fe(CN)6 . It was observed for both untreated PPF and 10 min 60 W iodinated PPF surfaces exposed to undecylenic acid that passivation
3-/4- 3-/4- towards Fe(CN)6 was observed (Fig 5.17). The passivation of the Fe(CN)6 for surfaces not iodinated suggest the untreated PPF is also able to be reacted with alkenes.
3+/2+ The behaviour of Ru(NH3)6 was observed to not be affected for either surface (Fig.
5.18).
Figure 5.17 Cyclic voltammograms of iodinated PPF surfaces exposed to undecylenic acid and
3-/4- U.V. light at 254 nm wave length. 10 mM Fe(CN)6 in 50 mM phosphate buffer (pH 7) and 1
M KCl scanned at 100 mV/s.
Chapter 5 | 219
Figure 5.18 Cyclic voltammograms of iodinated PPF surfaces exposed to undecylenic acid and
3+/2+ U.V. light at 254 nm wave length. 10 mM Ru(NH3)6 in 50 mM phosphate buffer (pH 7) and
1 M KCl scanned at 100 mV/s.
The suspected modification of the untreated PPF with undecylenic acid with
U.V. is due to the way PPF is prepared. PPF is prepared in a reducing atmosphere of
5% H2 in N2, this allows the surface to be partially hydrogenated. It has previously been shown by Downard and co-workers42 that freshly made PPF could be modified with alkenes and alkynes with U.V. light at wavelength 254 nm, they surmised that the surface was hydrogenated during the pyrolyses process of the photoresist.
5.3.2.1.2 CONTACT ANGLE OF IODINATED PPF EXPOSED TO UNDECYLENIC ACID
AT U.V. WAVELENGTH 254 NM
Iodinated 10 min 60 W PPF exposed to undecylenic acid and UV light of wavelength 254 nm was noted to have slightly reduced contact angle. Untreated PPF surfaces exposed to undecylenic acid with U.V. light at wavelength 254 nm, was also
Chapter 5 | 220
observed to have a smaller contact angle than fresh untreated PPF. For the iodinised
PPF the contact angle was recorded to be 75.6 ± 2.0° whilst the untreated PPF exposed to undecylenic acid gave a recorded contact angle of 79.9 ± 1.6°. The contact angle for modified undecylenic acid derived silicon surfaces is between 30-60°.43 The angles observed on both the 10 min 60 W iodinated PPF and untreated PPF surfaces that have been exposed to undecylenic acid is far greater than the angle on silicon modified with undecylenic acid. This difference in contact angle suggests that the undecylenic acid derived layer on 10 min 60 W iodinated PPF is not as well packed as a well formed, tightly packed monolayer of undecylenic acid gives a small contact angle in comparison to those less densely packed.
From the electrochemical measurements, passivation of the surface towards
3-/4- Fe(CN)6 was observed whilst no change was seen in the electrochemical behaviour
3+/2+ 3-/4- of Ru(NH3)6 . As Fe(CN)6 is sensitive to species on the surface, due to it being an inner sphere complex, passivation on a modified surface is to be expected. As the
3- surface is modified with undecylenic acid this will form a barrier preventing Fe(CN)6
/4- from contacting the surface and slowing down the rate electrons are transferred to the
PPF surface. Thus the surface will become more passivated as the layer of undecylenic acid becomes better packed, as a loosely packed surface will allow holes where
3-/4- 3-/4- Fe(CN)6 can contact the surface allowing electrons to the surface. Fe(CN)6 is also negatively charged and will be repelled by the surface thus increasing the affect on
3+/2+ passivation. No change in the electrochemical behaviour of Ru(NH3)6 is to be expected, however, on surfaces that have been modified with layers that have a
3+/2+ negatively charged distal end, as Ru(NH3)6 is a positively charged molecule, it will be attracted to the surface, and since it is outer sphere there will be no change.
Chapter 5 | 221
Both the 10 min 60 W iodinated PPF surface and the untreated surface exposed to undecylenic acid with U.V. light at wavelength 254 nm showed passivation of the
3-/4- 3+/2+ surface towards Fe(CN)6 and no change in the electrochemistry of Ru(NH3)6 .
Modification of both surfaces with a layer of undecylenic acid is suggested. The contact angles, however, suggest that the modification layer is not well packed. The slight differences though in both the contact angle and the passivation infer that a more densely monolayer of undecylenic acid has been formed on the 10 min 60 W iodinated
PPF than the untreated PPF.
The modification of untreated PPF with undecylenic acid via U.V. light at 254 nm wavelength has been shown by Downard and co-workers, as mentioned earlier.42 It was observed that freshly made PPF was able to be modified with alkenes and alkynes with U.V. light at wavelength 254 nm. Modification with undecylenic acid gave a
3-/4- contact angle of 60° and also passivation of the surface towards Fe(CN)6 was observed. These observations confer the modification of the untreated PP with undecyleneic acid via U.V. light at wavelength 254 nm.
5.3.2.2 REACTION OF IODINATED PPF WITH UNDECYLENIC ACID AT 514 NM
Iodination of silicon has been shown by Hamers and co-workers33 followed by subsequent modification with alkenes. Hamers and co-workers showed that due to the iodine-silicon bond being weaker to that of the hydrogen-silicon bond, the energy needed to react the surface with alkenes was less. Alkenes were reacted with the iodine modified silicon surface using light at wavelength 514 nm to form a monolayer.
Chapter 5 | 222
Because PPF is formed in a reducing atmosphere of nitrogen and hydrogen, PPF has a low oxide content on the surface but a high hydrogen content, thus leading to the untreated surfaces reacting with the alkenes.44-45 By using U.V. light at wavelength 514 nm to modify the iodinated PPF surfaces with alkenes, a difference should be seen between the untreated PPF and the iodinated PPF after exposure to undecylenic acid and
U.V. light. Both iodinated surfaces and untreated PPF surfaces were exposed to U.V. light at wavelength 514 nm for 12 hours under argon. Control surfaces were carried out of iodinated PPF exposed to undecylenic acid without U.V. light. Iodine plasma power and exposure times are indicated by X min Y W, where X is the time, 5, 10, or 30 min, and Y indicates power of the plasma, 30 or 60 W.
5.3.2.2.1 CONTACT ANGLE OF IODINATED PPF EXPOSED TO UNDECYLENIC ACID
AT U.V. WAVELENGTH 514 NM
Iodinated PPF exposed to undecylenic acid with U.V. light at 514 nm showed a lower contact angle than untreated PPF exposed in a similar manner (Table 5.5). The contact angles seen on the iodinated PPF are not as small as that seen on hydrogenated silicon that has been modified with undecylenic acid.43, 46 This suggests that the monolayer formed is not as tightly packed as those formed on silicon. Iodinated surfaces were also exposed to undecylenic acid without U.V. to ensure that the U.V. light had an effect on the attachment and that spontaneous attachment was not occurring. It was shown that the angle of iodinated PPF exposed to undecylenic acid without U.V. light had little change in the contact angle.
Chapter 5 | 223
Power and time settings PPF Exposed to undecylenic acid at Contact angle was exposed to iodine plasma U.V. light at wavelength 514 nm Untreated PPF (0 min 0 W) Yes 81.4 ± 1.8° 5 min 30 W Yes 80.0 ± 6.7° 10 min 30 W Yes 75.8 ± 3.3° 30 min 30 W Yes 74.3 ± 4.2° 30 min 30 W No 81.3 ± 4.5° 30 min 60 W Yes 73.9 ± 4.8°
Table 5.5 Contact angles of iodinated PPF exposed to undecylenic acid and 514 nm U.V. light
where the error is the standard deviation of n = 3 measurements.
5.3.2.2.2 ELECTROCHEMISTRY OF IODINATED PPF EXPOSED TO UNDECYLENIC
ACID AT U.V. WAVELENGTH 514 NM
The results discussed in this section are more a generalisation of the results as due to the continuing variation in PPF produced it was difficult to obtain precise results to make an exact statement. From the results obtained a good overall picture of the reaction of alkenes with iodinated PPF surfaces under U.V. light at wavelength 514 nm can be obtained.
3-/4- Passivation of the surfaces towards Fe(CN)6 was observed on all iodinated
PPF exposed to undecylenic acid with U.V. light wavelength 514 nm (indicated from here on as λ = 514 nm). The extent of passivation was observed to increase with increased iodine plasma exposure settings exposed undecylenic acid with U.V. light (λ
= 514 nm) (Fig. 5.19). 10 min 30 W, 30 min 30 W and 30 min 60 W iodinated PPF
3-/4- samples show the greatest amount of surface passivation towards Fe(CN)6 , resulting
Chapter 5 | 224
3-/4- in the decrease of the electron transfer rate of Fe(CN)6 . Some passivation of the
3+/2+ surface towards Ru(NH3)6 was observed (Fig. 5.20), the layer produced form undecylenic acid becomes better packed on the surface, this produces a thick layer
3+/2+ 3+/2+ slowing down the electron transfer rate of Ru(NH3)6 , Although Ru(NH3)6 is an outer sphere redox complex the larger the modifying layer produces a greater distance from the surface and slows the electron transfer rate. The results show that longer exposure times and higher iodine plasma powers should produce a better formed layer on the PPF surface.
3-/4- Figure 5.19 Cyclic voltammograms of Fe(CN)6 in 50 mM phosphate buffer (pH 7) and 1 M
KCl scanned at 100 mV/s on iodinated surfaces exposed to undecylenic acid and U.V. light (λ =
514 nm). PPF surfaces used were exposed to iodine plasma for 5 min, 10 min and 30 min at a
plasma power of 30 W and for 30 min at a plasma power of 60 W. Untreated PPF was not
exposed to iodine plasma or undecylenic acid and U.V. light.
Chapter 5 | 225
2+/3+ Figure 5.20 Cyclic voltammograms of Ru(NH3)6 in 50 mM phosphate buffer (pH 7) and 1
M KCl scanned at 100 mV/s on iodinated PPF exposed to undecylenic acid and U.V. light (λ =
514 nm). PPF surfaces used were exposed to iodine plasma for 5 min, 10 min and 30 min at a
plasma power of 30 W and for 30 min at a plasma power of 60 W. Untreated PPF was not
exposed to iodine plasma or undecylenic acid and U.V. light.
PPF iodinated for 30 min at 30 W (30 min 30 W) was exposed to undecylenic acid with and without U.V. light (λ = 514 nm). Passivation of the surface towards
3-/4- Fe(CN)6 was observed for the iodinated surface with U.V. light (λ = 514 nm), whilst the iodinated surface not exposed to U.V. light (λ = 514 nm) show no passivation of the
3-/4- surface towards Fe(CN)6 (Fig. 5.21). The iodinated surface not exposed to U.V. light (λ = 514 nm) showed slight passivation but this could be just variation in the PPF samples.
Chapter 5 | 226
3-/4- Figure 5.21 Cyclic voltammograms of Fe(CN)6 in 50 mM phosphate buffer (pH 7) and 1 M
KCl scanned at 100 mV/s on iodinated surfaces exposed to iodine plasma for 30 min at 30 W.
one set of surfaces was exposed to undecylenic acid and U.V. light (λ = 514 nm) (30 min 30 W
undecylenic acid). The other set of surfaces were exposed to undecylenic acid but without the
U.V. light (λ = 514 nm) (30 min 30 W undecylenic acid no U.V.). Both were compared to
untreated PPF.
The surface also produced similar passivation of the surface towards
3+/2+ Ru(NH3)6 (Fig 5.22). The 30 min 30 W iodinated surface exposed to undecylenic acid with U.V. light (λ = 514 nm) showed slowing of the electron transfer rate of
3+/2+ Ru(NH3)6 resulting in passivation of the surface. As discussed previously if the
3+/2+ modification layer is large Ru(NH3)6 , although an outer sphere redox probe, will
3- slow the electron transfer rate. The lack of passivation of the surface towards Fe(CN)6
/4- 3+/2+ and Ru(NH3)6 for the iodinated surface not exposed to U.V. (λ = 514 nm) indicates no alkene has reacted with the surface and strongly suggests that U.V. is required for the iodinated surfaces to be modified with alkenes.
Chapter 5 | 227
2+/3+ Figure 5.22 Cyclic voltammograms of Ru(NH3)6 in 50 mM phosphate buffer (pH 7) and 1
M KCl scanned at 100 mV/s on iodinated PPF exposed to iodine plasma for 30 min at 30 W.
one set of surfaces was exposed to undecylenic acid and U.V. light (λ = 514 nm) (30 min 30 W
undecylenic acid). The other set of surfaces were exposed to undecylenic acid but without the
U.V. light (λ = 514 nm) (30 min 30 W undecylenic acid no U.V.). Both were compared to
untreated PPF.
As discussed in section 5.3.2.1 both untreated PPF surfaces and iodinated PPF surfaces reacted with undecylenic acid under U.V. light (λ = 254 nm). This is thought to be due to the untreated PPF surfaces being slightly hydrogenated during the pyrolysis procedure. Untreated PPF surfaces were exposed to undecylenic acid again but this time U.V. light (λ = 514 nm) was used to activate the surface. Both figures 5.23 and
5.24 show that the surfaces do not react with undecylenic acid either: under U.V. light
(λ = 514 nm) or without UV light. This suggests that the results observed for the passivation of the PPF surfaces exposed to both iodine plasma and undecylenic acid under U.V. light (λ = 514 nm), is due to the modification of surface by alkenes reacting with iodinated carbon.
Chapter 5 | 228
3-/4- Figure 5.23 Cyclic voltammograms of Fe(CN)6 in 50 mM phosphate buffer (pH 7) and 1 M
KCl scanned at 100 mV/s on untreated PPF surfaces. One set of surfaces was exposed to
undecylenic acid and U.V. light (λ = 514 nm) (Untreated undecylenic acid). The other set of
surfaces was exposed to undecylenic acid but without the U.V. light (λ = 514 nm) (untreated
undecylenic acid no U.V.). Both were compared to untreated PPF.
2+/3+ Figure 5.24 Cyclic voltammograms of Ru(NH3)6 in 50 mM phosphate buffer (pH 7) and 1
M KCl scanned at 100 mV/s on untreated PPF surfaces. One set of surfaces was exposed to
undecylenic acid and U.V. light (λ = 514 nm) (Untreated undecylenic acid). The other set of
surfaces was exposed to undecylenic acid but without the U.V. light (λ = 514 nm) (untreated
undecylenic acid no U.V.). Both were compared to untreated PPF.
Chapter 5 | 229
For PPF surfaces iodinated for 30 min at 60 W plasma power it was noticed that
2+/3+ 3-/4- iodinated surfaces passivated both Ru(NH3)6 (Fig. 5.25) and Fe(CN)6 (Fig. 5.26) more before reaction with alkenes and U.V. light (λ = 514 nm). The passivation of the
2+/3+ iodinated surface towards Ru(NH3)6 may be due to the surface being less well packed once it is reacted. A lower packing density is also inferred by the contact angles being greater than those observed for undecylenic acid on silicon surfaces. This lower density packing may produce areas that have lost iodine and are more accessible to
3- redox species. The surface however remains completely passivated towards Fe(CN)6
/4- after exposure to undecylenic acid with U.V. light (λ = 514 nm). The continued
3-/4- 2+/3+ passivation of Fe(CN)6 and the increase of the electron transfer rate of Ru(NH3)6 suggests surface modification of the iodinated PPF with undecylenic acid.
2+/3+ Figure 5.25 Cyclic voltammograms of Ru(NH3)6 in 50 mM phosphate buffer (pH 7) and 1
M KCl scanned at 100 mV/s on iodinated PPF exposed to iodine plasma for 30 min at 60 W (30
min 60 W iodine plasma), and on iodinated PPF exposed to iodine plasma for 30 min at 60 W
followed by exposure to undecylenic acid and U.V. light (λ = 514 nm) (30 min 60 W
undecylenic acid). Both were compared to untreated PPF.
Chapter 5 | 230
2+/3+ Figure 5.26 Cyclic voltammograms of Ru(NH3)6 in 50 mM phosphate buffer (pH 7) and 1
M KCl scanned at 100 mV/s on iodinated PPF exposed to iodine plasma for 30 min at 60 W (30
min 60 W iodine plasma), and on iodinated PPF exposed to iodine plasma for 30 min at 60 W
followed by exposure to undecylenic acid and U.V. light (λ = 514 nm) (30 min 60 W
undecylenic acid). Both were compared to untreated PPF.
3-/4- The passivation of the iodinated PPF surface towards of Fe(CN)6 and less passivation after the surface has been modified with undecylenic acid with U.V. light (λ
= 514 nm) is also apparent in PPF surface exposed to 30 W iodine plasma for 5 min
2+/3+ (Fig. 5.27). The same surfaces though produced no passivation towards Ru(NH3)6
(Fig. 5.28). This is slightly different to the results observed in section 5.3.1.2 where
3-/4- 2+/3+ little passivation of the surface towards both Fe(CN)6 and Ru(NH3)6 was noted.
It is unsure as to whether the iodine is removed from the surface after the alkene has reacted with the iodine surface or if it remains bond to the alkane chain that is formed.
These suggests that further investigation into the surface modification with iodine plasma is required if these surfaces are to be used for reliable affinity biosensors.
Chapter 5 | 231
3-/4- Figure 5.27 Cyclic voltammograms of Fe(CN)6 in 50 mM phosphate buffer (pH 7) and 1 M
KCl scanned at 100 mV/s on iodinated PPF exposed to iodine plasma for 5 min at 30 W (05 min
30 W iodine plasma), and on iodinated PPF exposed to iodine plasma for 5 min at 30 W
followed by exposure to undecylenic acid and U.V. light (λ = 514 nm) (05 min 30 W
undecylenic acid). Both were compared to untreated PPF.
2+/3+ Figure 5.28 Cyclic voltammograms of Ru(NH3)6 in 50 mM phosphate buffer (pH 7) and 1
M KCl scanned at 100 mV/s on iodinated PPF exposed to iodine plasma for 5 min at 30 W (05
min 30 W iodine plasma), and on iodinated PPF exposed to iodine plasma for 5 min at 30 W
followed by exposure to undecylenic acid and U.V. light (λ = 514 nm) (05 min 30 W
undecylenic acid). Both were compared to untreated PPF.
Chapter 5 | 232
On a side note, the iodinated 30 min 30 W PPF did produce a blue coloured surface (Fig. 5.29a). On subsequent modification of the iodinated 30 min 30 W surface with undecylenic acid via U.V. light (λ = 514 nm) this surface became yellow/orange, with a rainbow developing on the edge of the PPF (Fig. 5.29b). The surface though maintained reflectivity (Fig. 5.29c).
c a b
Figure 5.29 a) Iodinated 30 min 30 W PPF. b) Iodinated 30 min 30 W PPF, modified with
undecylenic acid via U.V. light (λ = 514 nm). c) Reflection of UNSW library building 500 m
away from chemistry building on iodinated 30 min 30 W PPF, modified with undecylenic acid
via U.V. light (λ = 514 nm).
3-/4- From the passivation of the surface towards Fe(CN)6 results from those surfaces that have been exposed to 30 W iodine plasma for 30 min will continue to be used. 30 min 30 W iodine plasma PPF surface exposed to undecylenic acid and U.V. light (λ = 514 nm), gave a low contact angle in comparison to the 5 and 10 min exposure times inferring a more densely packed monolayer. 30 min 30 W iodinated
PPF exposed to undecylenic acid without U.V. light showed no passivation towards
3-/4- Fe(CN)6 and gave a large contact angle comparable to untreated PPF not exposed to undecylenic acid.
Chapter 5 | 233
The use of U.V. light at both 254 nm and 514 nm with iodinated PPF surfaces exposed to alkenes, allows the modification of the surfaces. Untreated PPF surface though can also be modified with alkenes via U.V. light at wavelength 254 nm, though no modification was observed with U.V. light at wavelength 514 nm.
The ability of the iodinated PPF surfaces to be modified with alkenes makes them amenable to further modification via the attachment of other chemistry, biochemistry or nanomaterials. Furthermore, using light to modify the surfaces with alkenes makes the surface modification chemistry highly compatible with the formation of patterns using standard photolithographical strategies. Using 30 min 30 W iodinated
PPF further reactions of this surface via U.V. light (λ = 514 nm), will be examined.
5.3.3 MODIFICATION OF IODINATED PPF WITH ALKYNES
5.3.3.1 MODIFICATION WITH NONADIYNE AND USE OF “CLICK” CHEMISTRY
Gooding and co-workers have showed the use of nonadiyne on silicon surfaces.
Once the nonadiyne is attached to the surface via one end of the nonadiyne, the remaining alkyne is available to undergo further reactions.17-18 “Click” reactions occur between azides and alkynes forming a covalent bond. Previous studies by Ciampi et al. have shown that through the use of “click” chemistry a ferrocene azide derivative can be attached to the surface producing fast electrochemistry on the surface.18 The use of
“click” chemistry to attach an azide derivative of tetra(ethylene glycol), produces a surfaces that reduce the amount of non-specifically adsorbed protein.17
Chapter 5 | 234
To investigate the versatility of iodinated PPF and the modification via U.V. light (λ = 514 nm), the 30 min 30 W iodinated PPF surfaces was exposed to nonadiyne.
The ability of the these surfaces to undergo further modification was carried via “click” chemistry of an tetra(ethylene glycol) azide to the surface (Fig. 5.30).
HO HO HO
O O O
O O O HO
O O O O
N N N O N N N N N N
O
N Iodine 3
Plasma I I I I I hv PPF PPF PPF PPF
Figure 5.30 Reaction of nonadiyne with an iodinated surface and subsequent “clicking” of
tetra(ethylene glycol) to the surface.
Iodinated PPF was exposed to nonadiyne and U.V. light (λ = 514 nm). Contact angles were measured on the modified surface (Fig 5.31) due to the nonadiyne derived surface being terminated with alkynes; little change was expected of the surface and an angle of 80.9 ± 2.9° was recorded. This contact angle is comparable to those observed by Ciampi et al.. Silicon surfaces modified with nonadiyne give a contact angle of 87 ±
3°. Upon reaction of the nonadiyne derived surface with tetra(ethylene glyol) azide an angle of 53.4 ± 8.1° was recorded. This decrease of the contact angle after the
“clicking” of tetra(ethylene glycol) azide derivative compares to literature values as
Ciampi et al. have shown that these surfaces provide contact angles of 54 ± 4°. This is expected as the ethylene glycol derived surface is more hydrophylic due to the presence
Chapter 5 | 235
of the ethers from the glycol chain and the hydroxyl termination, in comparison to the carbon chain of the nonadiyne.
a b
Figure 5.31 Contact angle of water on a) a nonadiyne derived iodinated PPF surface, and b) a
nonadiyne derived iodinated PPF surface “clicked” with tetra(ethylene glycol) azide.
3+/2 The passivation of the surface was investigated with Ru(NH3)6 and
3-/4- 3+/2 Fe(CN)6 . Passivation of the surface towards Ru(NH3)6 , and the decrease in the electron transfer rate, was observed (Fig. 5.32). This passivation of the modified iodinated surfaces with U.V. light (λ = 514 nm), could be attributed to the surfaces
3+/2 providing a densely packed layer, with a neutral distal end, stopping the Ru(NH3)6
3-/4- transferring electrons to the surface. Passivation of the surface towards Fe(CN)6 was observed (Fig. 5.33) for both 30 min 30 W iodinated PPF after modification with nonadiyne via U.V. (λ = 514 nm). Further modification with tetra(ethylene glycol)azide derivative, also produces passivation.
Chapter 5 | 236
3+/2+ Figure 5. 32 Cyclic voltammograms of Ru(NH3)6 in 50 mM phosphate buffer (pH 7) and 1
M KCl scanned at 100 mV/s of iodinated PPF reacted with nonadiyne and 514 nm U.V. light,
and subsequent “click” reaction with OEG.
3-/4- Figure 5.33 Cyclic voltammograms of Fe(CN)6 in 50 mM phosphate buffer (pH 7) and 1 M
KCl scanned at 100 mV/s on iodinated PPF reacted with nonadiyne and 514 nm U.V. light, and
subsequent “click” reaction with OEG. 10 mM Fe(CN)6 in phosphate buffer against Ag|AgCl
reference.
Chapter 5 | 237
From these results it is concluded that modification of 30 min 30 W iodinated
PPF with nonadiyne via U.V. light (λ = 514 nm), has been carried out as well. The
3- nonadiyne-derived surface shows the passivation of the surface towards both Fe(CN)6
/4- 3+/2+ and Ru(NH3)6 . It can be inferred that the surface is well densely packed, however, further investigations are needed. Subsequent reaction with the alkyne distal end and a tetra(ethylene glycol) azide, to give an tetra(ethylene glycol) terminated surface proved to be successful. Upon the modification of the nonadiyne surface with tetra(ethylene glycol) azide derivative with a “click” reaction the contact angle was observed to decrease which was comparable to values shown in literature. Passivation of the surface was maintained.
5.3.4 PATTERNING OF IODINATED PPF
The use of S-undec-10-enyl-2,2,2-trifluoroethanethioate (C11-S-TFA) to modify a surface has been shown by Gooding and co-workers.47-48 The deprotection of this modified layer reveals a thiol moiety which can be employed to couple to further functionality such as noble metal and colloids as shown by Gooding and co-workers.
The Patterning of iodinated PPF can be carried out using trifluoroacetyl thioldecene.
The fluorine group serves as a good XPS marker. The distal thiol produced after deprotection can also be used to bind to gold nanoparticles as shown by Le Saux et al. on silicon surfaces modified with C11-S-TFA (Fig. 5.34).
Chapter 5 | 238
F3C O S F3C F3C F3C O O O S S S HS HS HS HS S HS
10% Ammonium hydroxide Gold nanoparticles Iodine
Plasma I I I I I hv PPF PPF PPF PPF PPF
Figure 5.34 Modification of PPF with Thiol.
To investigate the ability to pattern iodinated 30 min 30 W PPF surfaces with alkenes using U.V. light (λ = 514 nm) a way to differentiate the modified surface from the bare surface not exposed to U.V. light is needed. The use of gold nanoparticles allows the patterning of the surface to be investigated with scanning electron microscopy (SEM). By patterning a thiol terminated alkene onto the surface and exposing the patterned surface to a solution of gold nanoparticles the surface can be imaged using SEM. Exposing 30 min 30 W iodinated PPF to neat C11-S-TFA and only exposing the outer area of the surface to U.V. light at (λ = 514 nm) whilst protecting the middle area with a TEM grid, the surface may be able to be patterned with a thiol terminated alkene.
5.3.4.1 MODIFICATION WITH C11-S-TFA
Modification of the surface is confirmed by XPS. A wide scan shows peaks in the S2p region as well as the C1s, F1s and I3d. A narrow scan in the F1s region shows the fluorine bound to carbon at 689 eV. The narrow scan of the S2p region indicates a
Chapter 5 | 239
sulphur bound to carbon at 164 eV (Fig 5.35a). After deprotection of the thiol, the
narrow scan of the S2p region, sulphur bound to carbon at 164 eV is still observed. The
narrow scan of the F1s region however, shows the fluorine bound to carbon at 689 eV is
lost (Fig 5.35b). When the iodinated PPF surfaces are exposed to C11-S-TFA but not
the U.V. light no S2p or F1s is seen but in the peaks corresponding to the I3d region at
620 eV are still present (Fig. 5.35c) indicating there is still iodine bound to carbon.
a F1s I3d S2p
b
F1s I3d S2p
c
F1s I3d S2p
Figure 5.35 XPS of C11-S-TFA derived iodinated PPF surface, fluorine 1s, iodine 3d, and
Sulphur 2p. a) Before deprotection of the thiol. b) After deprotection of the thiol. c) Exposure
to C11-S-TFA without U.V. light.
Chapter 5 | 240
5.3.4.2 PATTERNING WITH TRIFLUOROACETYL THIOLDECENE
Iodinated PPF was exposed to C11-S-TFA with U.V. (λ = 514 nm), except for the centre which was protected form the light with a TEM grid. The surface was deprotected exposing the thiol group. By exposing the modified surface to gold nanoparticles patterning of the surface can be probed.
The gold nanoparticles will attach to the thiol terminated areas but not to those that were not exposed to U.V. light and thus not modified. SEM was used to image the surfaces. Iodinated PPF was exposed to gold nanoparticles, but none attached to the surface (Fig 5.36a), whilst thiol terminated derived iodinated PPF exposed to gold nanoparticles showed that they had attached to the surface (Fig 5.36b). For the patterned surface it was seen that the area that was not exposed did not have nanoparticles whilst the area exposed to U.V. light was modified with gold nanoparticles (Fig. 5.36c).
It can be observed that the nanoparticles are well dispersed on the surface on the modified areas of the PPF surface, as the nanoparticles were formed in a citrate solution they are citrate capped. Citrate capping produces and overall negative charge thus the particles attach to the surface isolated rather than forming aggregates. The dispersal of citrate capped gold nanoparticles on surfaces, rather than the formation of aggregates, has been previously noted by Shein et al.49 and Liu et al.50, where both gold and carbon surfaces have been modified with either amine terminated thiols in the case of gold surfaces or sulphur or amine terminated aryl diazonium salts in the case of carbon surfaces. The gold nanoparticles are firmly bound to the PPF surface through the Au-S
Chapter 5 | 241
bond. No sonication has been carried out on these surfaces though it has been noted by
Liu et al.50 that gold nanoparticles bound to carbon surfaces through a sulphur bond are stable even after sonication of the surface.
a b
500 nm 500 nm
c d Modified
Line indicating boundary between modified 500 nm and non- modified
Non-Modified
Figure 5.36 SEM images of patterned C11-S-TFA derived iodinated PPF surfaces. a) Iodinated
PPF not exposed to U.V. light, C11-S-TFA, and gold nanoparticles . b) Iodinated PPF exposed
to U.V. light, C11-S-TFA, and gold nanoparticles. c) Patterned iodinated PPF with U.V. light,
C11-S-TFA, and gold nanoparticles. d) Boundary between modified and unmodified area, the
line indicates the boundary between the two areas.
Chapter 5 | 242
5.4 CONCLUSION
The iodination of PPF can be carried out using RF generated plasma, producing an iodinated surface. The iodinated surface produced maintains its electrochemical attributes as well as its physical attribute of smoothness, if iodinated with a 60 W or 30
W plasma.
Modification of the iodinated PPF surface with alkenes can be carried out with both shortwave (254 nm) and long wave light (514 nm) U.V. light. The use of long wave U.V. light (514 nm) allows the modification of iodinated PPF with both alkenes and alkynes and not the modification of the untreated PPF surface. The modification of iodinated PPF forms monolayers that are stable. The layers formed are able to be further modified for sensing applications, whilst maintaining integrity. The use of nonadiyne allows further modification via “click” chemistry. Use of C11-S-TFA allows investigation of the layer on the surface due to the fluorine marker. Deprotection of the thiol on C11-S-TFA allows for the attachment of gold nanoparticles, which can be used as a replacement to molecular wires.
The layers formed can then be used for further applications with out the degradation of the electrochemistry of the surface. Due to the low energy of the U.V. wavelength used (514 nm over 254 nm) it also allows patterning of the iodinated PPF surface.
Chapter 5 | 243
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42. Yu, S. S. C.; Downard, A. J., Photochemical grafting and activation or organic layers on
glassy carbon and pyrolysed photoresist films. Langmuir 2007, 23 (8), 4662-4668.
43. Boecking, T.; Kilian, K. A.; Gaus, K.; Gooding, J. J., Modifying porous silicon with
self-assembled monolayers for biomedical applications: the influence of surface
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44. Kostecki, R.; Schnyder, B.; Alliata, D.; Song, X.; Kinoshita, K.; Kotz, R., Surface
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46. Kilian, K., A. Chemical and Biological Modification of Porous Silicon Photonic
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47. Cahen, D.; Salomon, A.; Boecking, T.; Gooding, J. J., Effects of Molecule - Electrode
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Chapter Six
Conclusions and Future Work
6.1 SUMMARY
For electrochemical affinity biosensors to be efficient they are required to be highly sensitive and selective. Recent developments in affinity biosensors require well- defined interfaces fabricated on the molecular level.1-3 This thesis has reported the preparation of smooth thin carbon films that are able to be used as a substrate in electrochemical affinity biosensors. The modification of the electrode surface has also been reported through the use of OEG aryl diazonium derivatives. The ability of these
OEG aryl diazonium-dervied surfaces to resist non-specific protein adsorption has also been reported. A new method of modification for surface stability and sensitivity, has been shown through the use of iodination of the carbon surface and subsequent modification with alkenes.
The surface for an affinity sensor, in theory, should be smooth less than 0.5 nm rms. The smoothness/roughness of the surface can affect the sensitivity as all biorecognition elements should be in the same environment. This allows for maximum binding to the biorecognition element as none are “hidden” due to being in a lower area of the surface. The use of PPF provides a covalently modifiable surface that is far smoother than that provided by GC. The preparation of PPF though provides a variable surface and can be labelled a “black art”. Chapter 3 investigated the factors involved in the preparation of PPF. The investigation into the effects of gas flow rate and the pyrolysis program on the electrochemical behaviour and smoothness of PPF was conducted. A slow gas flow affected the smoothness of the surface, the faster the gas flow during pyrolysis the smoother the surface. The effects of the three step pyrolysis program, three heating steps and holding times, were investigated with a Plackett-
Chapter 6 | 251
Burman design. The effects can be arranged into some general guidelines. Cross correlation between the different temperatures cannot be extracted due to variability within the samples. In general though the faster gas rates give smoother, cleaner surfaces, in regards to oxides and adventicious material, surfaces GC. By varying the final step temperature, and time held the surface roughness is also affected. High temperatures (1100°C in comparison to 1050°C) and shorter times (60 min rather than
90 min) for the final heating step give smoother surfaces in general. The middle step in the pyrolysis program can affect surface oxides, generally lower temperatures here give low surface oxides.
Affinity biosenors are required to be highly selective when being exposed to complex matrixes. The adsorption of unwanted material, in particular proteins, causes interference. The use of OEG’s on gold, silver and silicon surfaces through thiol and alkene modification respectively, has been well studied. Chapter 4 reported the use of new OEG aryl diazonium salt derivatives and their ability to resist proteins from non- specific adsorption onto the electrode surface. From previous studies by Gunze and co- workers4 it was shown that several guidelines were important to the ability of the OEG modified surface to resist non-specific protein adsorption.5 The use of a hydroxyl distal end instead of a methoxy distal end improved the resistance to non-specific adsorption.
The length of the OEG chain was also shown to affect the surface’s ability to resist non- specific adsorption, a chain length of six OEG units improved the resistance of surfaces modified compared to a chain length of three OEG units. These guidelines were investigated on PPF surfaces with OEG aryl diazoniums salts (Fig. 6.6) that varied in length from three OEG units to six OEG units and the distal end was changed from methoxy to hydroxy.
Chapter 6 | 252
O O O O O OH O OH O O O 1 2
F4B N2 F4B N2
O O O O O 3 4
F4B N2 F4B N2
O O O O O O O 5 F4B N2
Figure 6.1 Oligo(ethylene glycol) aryl diazonium salt derivatives.
The ability of the modified surface to resist non-specific protein adsorption was investigated using fluorescently labelled bovine serum albumin (BSA-FITC). Two fluorescence techniques were also used. The ability to measure the amount of BAS-
FITC adsorbed onto the surface was carried out using a fluorescence microscope. The use of a fluorescence microscope allowed for a direct measurement of the BSA-FITC on the modified surface. By measuring the mean grey scale of the images taken the ability of each surface to resist non-specific protein adsorption can be qualitatively measured.
From the results it was observed that the OEG aryl diazonium salt derived PPF surfaces were able to resist non-specific protein adsorption, though in relation to the guidelines that Grunze and co-workers,4, 6 little difference was observed between the
OEG’s in regards to chain length and distal end. The non-specific resistance of the
OEG aryl diazonium salt derived PPF surfaces gave a 50-70% reduction of adsorbed protein in comparison to alkane modified surfaces. OEG thiol-derived gold surfaces gave a 90% decrease of adsorbed protein. For gold surfaces modified with OEG aryl diazonium salts a difference was observed; as chain length increased the amount of non-
Chapter 6 | 253
specific adsorbed protein decreased and as the distal end changed from a methoxy to a hydroxy the amount of non-specific adsorbed protein also decreased. Though in comparison to alkane modified surface, there was a 5% decrease for OEG3OMe whilst the OEG3OH gave a 70 % reduction and OEG6OH gave a 50% reduction.
Chapter 5 investigated the use of iodine plasma to iodinate PPF surfaces and subsequent modification with alkenes and alkynes. This modification allows the formation of a monolayer instead of multilayers as seen in aryl diazonium modification.
The use of a monolayer allows further insurance that the biorecognition elements are all in the same environment.
The use of iodine plasma to iodinate the surface produced two peaks in the I3d spectra that corresponded to the I3/5d and I3/2d binding energies of iodine bound to carbon. Plasma powers of 60 and 30 W were found not to increase the roughness of the
PPF to greater than an rms value of 1 nm. Due the large amount of iodine bound to carbon and no increase in iodine bound to iodine it assumed that some roughening of the surface is still occurring.
Exposure of iodinated PPF to alkenes showed that both short wave (λ = 254 nm) and long wave (λ = 514 nm) U.V. light modified the surfaces. Though the use of short wave U.V. light with alkenes on bare PPF also modified the surface as seen by the
3-/4- passivation of Fe(CN)6 , though not to the same extent as iodinated PPF. The use of long wave U.V. light (λ = 514 nm) allowed the modification of the iodinated PPF but not the bare PPF. Through the use of U.V. activation of the iodinated PPF alkynes can also be used to modified PPF. Nonadiyne was used to modify the iodinated PPF and
Chapter 6 | 254
further modification was carried out through the use of “click” chemistry of tetra(ethylene glycol) azide. After “clicking” the azide tetraethylene glycol to the nonadiyne derived surface it was observed that the contact angle decreased.
The use of U.V. light to modify the iodinated PPF surface can also be applied further to pattern the surface. Using S-undec-10-enyl-2,2,2-trifluoroethanethioate (C11-
S-TFA) to on the surface and the use of a TEM grid as a mask, the areas that were exposed to U.V. light (λ = 514 nm) were modified whilst those under the grid were not.
By deprotecting the thiol group and exposing the surface to gold nanoparticles SEM images showed the patterning of the surface.
6.2 FUTURE WORK
6.2.1 FURTHER INVESTIGATING THE EFFECTS OF PLASMA IODINATION AND
ALKENE/ALKYNE ADDITION
Due to the large amount of iodine bound carbon formed on the PPF in comparison to the roughness of the PPF, more research needs to be carried out on the structural effects on the PPF. As the amount of iodine bound to carbon on iodine plasma modified PPF is more than a monolayer coverage on the surface, there is no large increase in the surface roughness. Using gas sorption Brunauer, Emmett & Teller technique (BET) the microporosity of the surface can be investigated. The stability of the surfaces also needs to be investigated, as the iodinated PPF may be prone to oxidation and adventitious addition. The stability of the surface over time should also be investigated.
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The modification of alkenes and alkynes also require further investigation. As of yet the exposure time to U.V. light at both wavelengths 254 and 514 nm requires optimisation so that the minimum amount of U.V. light is used. The types of alkenes that are able to be used to modify the surface also requires attention as it has been observed by Hamers and co-workers7-10 that alkenes that have an electron withdrawing group are better for modifying the surface. A more comprehensive study into the nature of the alkene modified iodinated PPF needs to be carried out to investigate the packing density of alkenes, how chain length and functionalities can affect the reaction of alkenes with the surface and their packing structure and density.
The ability to form mixed monolayers is important as it enables the surface with several different functionalities. The use of mixed layers has been shown on silicon surfaces, combining both a biorecognition element and a diluent that also resists non- specific protein adsorption.11
Protein resistance of oligo(ethylene glycol) alkene derivatives should also be investigated, as each surface allows for different packing densities that can affect the ability of the surface to resist non-specific protein adsorption. Further modification of nonadiyne modified surfaces can be investigated as well, through the “click” reaction of different azides.
6.2.1.1 PHOTOLITHOGRAPY
The observation that iodinated PPF can be patterned with photolithography requires further investigation. The use of photolithography on the iodinated PPF
Chapter 6 | 256
surfaces allows a defined attachment of molecules on the surface. This can give rise to multi-analyte biosensors as different biorecognition elements can be patterned onto the surface.
Before multi-analyte patterning of the surface can be undertaken, study into the patterning of iodinated PPF needs to be undertaken. The extent of patterning the surface should be investigated. This requires an understanding of the patterning resolution, how small the patterns can be before diffraction of light interferes with the pattern boundaries. The ability of the surface to be patterned multiple times to allow controlled modification of multiple biorecognition elements. This will also link into the stability of the iodinated PPF surface.
The understanding of these aspects of photolithograpy on iodinated PPF and the modification via U.V. light (λ = 514 nm), will allow the production of well defined interfaces for biosensors.
6.2.2 BIOLOGICAL APPLICATIONS
6.2.2.1 AFFINITY SENSORS
The use of PPF for an affinity sensor will allow for the biorecognition elements to be placed in the same environment and should allow for greater sensitivity of the detection of analytes. Work carried out by Liu et al. showed that an affinity sensor can be used for the detection of small analytes in particular biotin.2, 12, 13 By applying PPF as the surface substrate it may be possible to allow for greater sensitivity of the sensor.
Chapter 6 | 257
The use of PPF as an interface for biosensors also allows mass production. PPF surfaces are transferable to mass production, and the formation of monolayers that are covalently bound may also allow the production of simple biosensors for the general public. PPF is cheaper than gold and iodination of PPF allows the formation of well defined monolayers similar to that of gold-thiol monolayer but with greater stability.
By combining the ability to modify PPF surfaces with alkenes, an affinity sensor for analytes in complex matrixes may be investigated. For example, the detection of oxidised low-density lipoproteins (LDL’s) will allow better determination into the threat of heart disease in a patient.
Oxidation of LDL’s causes the inability of receptors in the lining of blood vessels to stop the deposition of cholesterol in the blood system.14 The use of the peptide Glycine-Cystine-Serine-Aspartic acid-Glutamine, has been shown to have a high affinity towards ox-LDL’s.15, 16 Through the combination of a mixed alkyne/alkene modification and subsequent “click” reactions of tetra(ethylene glycol) azide on an iodinated PPF surface, a smooth surface which is resistant to non-specific protein adsorption can be combined with the recognition element in a similar manner to the affinity biotin sensor discussed at the beginning of this thesis.
The use of nanoparticles on surfaces has been shown to act as a replacement for molecular wires.17, 18 Gooding and co-workers have shown that by attaching nanoparticles to a passivating surface, it becomes electrochemically active again. By using C11-S-TFA as part of a mixed monolayer gold nanoparticles can be attached to the surface. The use of nonadiyne as the other part of the mixed alkene/alkyne modifying
Chapter 6 | 258
layer allows “clicking” of tetra(ethylene glycol) azide derivative to the surface, providing resistance to non-specific protein adsorption. This combination on a surface allows the use of the smoothness of PPF, the definition of a monolayer, and the functionality of a mixed layer surface with multi-functions.
OH
O O OH
O O N O N N O N
PPF HO N N O HO O O S O HS N OH S N Fe H O NH H H N S O N O O H H O N N N O O O OH
Figure 6.2 Suggested ox-LDL affinity biosensor.
6.2.2.2.1 DEVELOPMENT OF A MUTLIANALYTE SENSOR
Through the ability to pattern the PPF surface, the development of a sensor that has the ability to detect multianalytes in the same sample can be produced (Fig. 6.3).
The ability to control the modification process will allow greater reliability of the surface. The concentration of each biorecognition element can be defined as each is individually attached to the surface. This ability to understand the modified surface will allow greater sensitivity. By using different biorecognition elements attached to different redox probes individual signals could be seen corresponding to each analyte being detected.
Chapter 6 | 259
Figure 6.3 Idealist construct of a multi array sensor using photolithography of iodinated PPF.
6.3 CONCLUDING REMARKS
In summary smooth reliable thin carbon film electrodes in the form of PPF have been produced and further understanding into factors affecting pyrolysis have been investigated. Reduction of non-specific protein adsorption has been achieved through
OEG aryl diazonium salt derivatives on gold and PPF surfaces. A new method of modification producing monolayers on PPF surfaces using iodination and U.V. light has been shown. The use of iodinated PPF and the modification with alkenes and alkynes allows space for patterning the surface. This ability combined with the smoothness and electrochemical capabilities of PPF will allow for further improvements for affinity biosensors and surface modification.
This work allows greater definition and control of the modification of carbon surfaces. Through the construction of stable monolayers on smooth thin carbon films, further advances for affinity biosensors can be achieved.
Chapter 6 | 260
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