Protein-Resistant Electrode for Biosensing

Protein-resistant Electrode for Biosensing

Cheng Jiang

A thesis submitted in fulfilment of the requirements

for the degree of

Doctor of Philosophy

School of Chemistry, Faculty of Science

2016

PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: Jiang

First name: Cheng Other name/s: N/A

Abbreviation for degree as given in the University calendar: PhD

School: School of Chemistry Faculty: Science

Title: Protein-resistant electrode for biosensing

Abstract 350 words maximum: (PLEASE TYPE)

Electrochemical biosensors are of enormous interests in a variety fields including clinical diagnosis and food analysis. However, in biological media the nonspecific adsorption of proteins, referred to as biofouling, interferes with the performance of such devices in terms of reducing sensitivity and selectivity. Hence, antifouling coatings are needed for electrochemical sensors to deliver on their potential when it comes to biologically derived samples. The common effective solutions to the critical issue of biofouling involves using poly(ethyleneglycol) (PEG) or oligo(ethyleneglycol) (OEG)-alkanethiol layers. This is because a highly compressed hydration layer can be formed with these monolayers which is believed to be the reason this chemistry can effectively preventing nonspecific adsorption of protein. However, the use of such long chain self-assembled monolayers (SAMs) or polymeric layers on electrodes is not desirable because such polymers form a high impedance layer on the electrode, effectively passivating the electrode. Surface modifying layers that do not passivate the electrodes typically are also not effective at providing protection against biofouling. In this concern, we have developed an aryldiazonium salt based mixed layer platform. Phenyl phosphorylcholine (PPC) and phenyl butyric acid (PBA) are used for antifouling and bio- recognition component linkage, respectively. Such surface chemistry was demonstrated to be a versatile platform with good antifouling, low impedance and controllable surface composition properties, and was successfully applied to the development of an immunosensor for detecting tumor necrosis factor α in whole blood.

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Acknowledgements

I would like to express my sincere gratitude to my supervisor Scientia professor Justin Gooding for his support and guidance throughout my PhD life. PhD research experience is like a cruise adventure in ocean of knowledge with so many uncertainties. He is like a light tower, leading my right direction and showing me brightness in heart. His profound knowledge and rigorous scholarship impressed me a lot, always giving me power and confidence to overcome any rainstorms and hurdles. His patient guidance on teaching me how to find a gap between current research work and what we need to achieve so as to design PhD project, how to do scientific writing, how to give good presentation in a conference have taught and encouraged me to continue studious and rigorous work in future career.

I would like to acknowledge professor Brynn Hibbert, professor Xuzhi Zhang, professor Guozhen Liu, our postdoctors Muhammad Alam, Nadim Darwish, Xiaoyu Cheng, Stephen Parker, Alexander Soeriyadi, Roya Tavallaie, Abbas Barfidokht and also Xin Chen, Saimon Moraes Silva, Fei Han, Manchen Zhao, Sanjun Fan, Yanfang Wu, Parisa S. Khiabani for their helpful discussions and advices throughout my PhD project.

Many thanks to Safura Taufik, Yong Lu, Ying Yang, Maryam Parviz, we accompanied with each other during the past 4-year PhD period and they helped a lot in research work and daily life. Appreciate to Dr. Yuanhui Zheng, who is always with positive energy and willing to share and help me since he came to our group. His optimistic attitude and sharp insights towards research would influence me a lot in my future career. All other Gooding group members are greatly appreciated for their kind help and helpful talk, makes my PhD period meaningful and colorful.

My beloved parents, relatives and friends always give selfless mental encouragement. My beloved wife Manchen Deng, who made a great sacrifice to wait until my PhD graduation, she is always encouraging me to be fearless to face any situations and

problems when studying overseas. Although we are separated thousands of miles from each other but our hearts always keep close without distance, our love passed the 4-year test!

Special thanks to Tuition Fee Scholarship (TFS) and China Scholarship Council (CSC) for financial support of my PhD study.

Abstract

Electrochemical biosensors are of enormous interests in a variety fields including clinical diagnosis and food analysis. However, in biological media the nonspecific adsorption of proteins, referred to as biofouling, interferes with the performance of such devices in terms of reducing sensitivity and selectivity. Hence, antifouling coatings are needed for electrochemical sensors to deliver on their potential when it comes to biologically derived samples. The common effective solutions to the critical issue of biofouling involves using poly(ethyleneglycol) (PEG) or oligo(ethyleneglycol) (OEG)- alkanethiol layers. This is because a highly compressed hydration layer can be formed with these monolayers which is believed to be the reason this chemistry can effectively preventing nonspecific adsorption of protein. However, the use of such long chain self- assembled monolayers (SAMs) or polymeric layers on electrodes is not desirable because such polymers form a high impedance layer on the electrode, effectively passivating the electrode. Surface modifying layers that do not passivate the electrodes typically are also not effective at providing protection against biofouling. In this concern, an aryldiazonium salt based mixed layer platform have been developed. Phenyl phosphorylcholine (PPC) and phenyl butyric acid (PBA) are used for resistance of nonspecific protein adsorption and linkage of bio-recognition component, respectively. Such surface chemistry was demonstrated to be a versatile platform with good antifouling, low impedance and controllable surface composition properties, and was successfully applied to the development of an immunosensor for sensitively and specifically detecting tumor necrosis factor α in whole blood.

List of Publication and Presentation

Publications

1. Jiang, C.; Alam, M. T.; Parker, S. G.; Darwish, N.; Gooding, J. J.* Strategies to Achieve Control over the Surface Ratio of Two Different Components on Modified Electrodes Using Aryldiazonium Salts. Langmuir 2016, 32 (10), 2509-2517. 2. Taufik, S.; Barfidokht, A.; Alam, M. T.; Jiang, C.; Parker, S. G.; Gooding, J. J.* An antifouling electrode based on electrode–organic layer–nanoparticle constructs: Electrodeposited organic layers versus self-assembled monolayers. J. Electroanal. Chem. 2016. 3. Zheng, Y. H.;* Jiang, C.; Ng, S. H.; Lu, Y.; Han, F.; Bach, U.; Gooding, J. J.*

Unclonable Plasmonic Security Labels Achieved by Shadow‐Mask‐Lithography‐

Assisted Self‐Assembly. Adv. Mater. 2016. 28 (12), 2330-2336

4. Zhang, X. Z.;* Li, Q.; Jin, X.; Jiang, C.; Lu, Y.; Tavallaie, R.; Gooding, J. J. Quantitative determination of target gene with electrical sensor. Sci. Rep 2015, 5. 5. Jiang, C.; Alam, M. T.; Parker, S. G.; Gooding, J. J.* Zwitterionic Phenyl

Phosphorylcholine on Indium Tin Oxide: a Low‐Impedance Protein‐Resistant

Platform for Biosensing. Electroanal 2015, 27 (4), 884-889.

Papers in Preparation

Jiang, C.; Alam, M. T.; Silva, S. M.; Taufik, S, Fan, S. J.; Gooding, J. J.* A unique sensing interface that allows the development of an electrochemical immunosensor for the detection of Tumor Necrosis Factor α in whole blood. ACS Sensors, submitted

Jiang, C.; Silva, S. M.; Fan, S. J.; Wu Y. F.; Alam, M. T.; Liu, G. Z.; Gooding, J. J.*

Aryldiazonium Electrochemistry Derived Mixed Organic Layers: from Surface

Chemistry to Its Applications, J. Electroanal. Chem. submitted vii

Conference Presentations

(1) Poster presentation, 7th International Nanomedcine Conference, Sydney. 2016

(2) Oral presentation, XXIII International Conference on Bioelectrochemistry and Bioenergetics, Sweden. 2015

(3) Poster presentation, ARC Centre for Bio-Nano Science Annual Symposium, Melbourne. 2015

(4) Poster presentation, Nanomaterials and Electrochemistry Symposium, Sydney. 2014

(5) Poster presentation, 19th Australia/New Zealand Electrochemistry Symposium, Melbourne. 2013

viii

Table of Contents

Thesis/Dissertation Sheet ...... i Copyright Statement ...... ii Originality Statement ...... iii Acknowledgements ...... iv Abstract ...... vi List of Publication and Presentation ...... vii Chapter 1 ...... 1 Introduction ...... 1 1.1 General Introduction of Aryldiazonium Salts Based Mixed Layers ...... 2 1.2 Surface Chemistry of Mixed Layers ...... 4 1.2.1 Formation of Mixed Layers Using Aryldiazonium Salts ...... 4 1.2.2 Control over Surface Composition ...... 6 1.2.3 Control over Spatial Distribution ...... 7 1.2.4 Control over Structure of Mixed Layers ...... 9 1.2.5 Characterization of Mixed Layers ...... 15 1.2.6 Other Potential Strategies for Formation of Mixed Monolayers ...... 16 1.3 Applications of Mixed Layers Modified Surfaces ...... 20 1.3.1 Antifouling Coating ...... 20 1.3.2 Analytical Sensing ...... 21

1.3.2.1 Protein Electrochemistry ...... 22 1.3.2.2 Chemical Sensing...... 23 1.3.2.3 Biosensing Applications...... 24 1.3.2.3.1 DNA Sensors ...... 25 1.3.2.3.2 Immunosensor ...... 26 1.3.2.4 Live Cell Electrochemistry ...... 29 1.3.3 Initiator for Surface-Initiated Photopolymerization ...... 31 1.4 Summary and Scope of This Thesis ...... 32 1.5 References ...... 34

Chapter 2 ...... 44 Methods and Materials ...... 44 2.1 Materials ...... 44 2.1.1 Chemicals ...... 44 2.1.2 ITO Coated Glass Slides ...... 47 2.2 Organic Synthesis ...... 47 2.3 ITO Cleaning ...... 47 2.4 X-ray Photoelectron Spectroscopy (XPS) Characterization ...... 48 2.5 Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) Instrumentation ...... 49 2.6 Electrochemical Evaluation of Protein-resistance Behavior ...... 50 2.7 References ...... 50

Chapter 3 ...... 51 Zwitterionic Phenyl Phosphorylcholine on Indium Tin Oxide: a Low-impedance Protein-resistant Platform for Biosensing ...... 51 3.1 Introduction ...... 52 3.2 Experimental Methods ...... 53 3.2.1 Reagents and Materials ...... 53 3.2.2 Cyclic Voltammetry and Electrochemical Impedance Spectroscopy Instrumentation ...... 53 3.2.3 Modification of ITO Electrode Surface ...... 53

3.2.4 Electrochemical Evaluation of Protein-Resistance Behavior ...... 53 3.2.5 X-Ray Photoelectron Spectroscopy Characterization ...... 53 3.3 Results and Discussion ...... 54 3.3.1 Electrografting of PPC on ITO Surface ...... 54 3.3.2 Electrochemical Impedance Spectroscopy and XPS Characterization of the PPC Modified ITO ...... 54 3.3.3 Comparison of Antifouling Behavior of PPC Modified ITO Surfaces ...... 56 3.4 Conclusions ...... 57 3.5 References ...... 57

Chapter 4 ...... 63

Strategies to Achieve Control over the Surface Ratio of Two Different Components on Modified Electrodes Using Aryl-Diazonium Salts ...... 63 4.1 Introduction ...... 64 4.2 Experimental Section ...... 66 4.2.1 Reagents and Materials ...... 66 4.2.2 Modiﬁcation of ITO Electrode Surfaces ...... 66 4.2.3 Electrochemical Apparatus ...... 66 4.2.4 Electrochemical Evaluation of Protein-Resistance Behavior ...... 66 4.2.5.X-ray Photoelectron Spectroscopy Characterization ...... 66 4.3 Results and Discussion ...... 66 4.3.1 PPC−CP/ITO from Simultaneous Electrografting Strategy ...... 66 4.3.2 PPC/CP/ITO from Consecutive Electrografting Strategy ...... 68 4.3.3 Evaluation of Protein-resistant Behavior ...... 68 4.3.4 Functionalization of a Mixed Layer Surface with Ferrocene ...... 69 4.4 Conclusions ...... 70

4.5 References ...... 71

Chapter 5 ...... 77 A Unique Sensing Interface that Allows the Development of an Electrochemical Immunosensor for the Detection of Tumor Necrosis Factor α in Whole Blood ...... 77 5.1 Introduction ...... 78 5.2 Experimental Section ...... 80 5.1.1 Reagents and Materials ...... 80 5.2.2 Fabrication of PPC-PBA Mixed Layers on ITO...... 80 5.2.3 X-ray Photoelectron Spectroscopy Characterization ...... 81 5.2.4 Electrochemical Apparatus ...... 81 5.2.5 Immunoassay Development ...... 81

5.2.6 Electrochemical Evaluation of In-Situ Protein-Resistance Behavior ...... 82 5.3 Results and Discussion ...... 82 5.3.1 Formation of PPC-PBA Mixed Layers on ITO ...... 82 5.3.2 In-Situ Antifouling Evaluation of Sensing Interface ...... 85 5.3.3 Electrochemical Response to Hydrogen Peroxide at the Immunosensor ...... 86

5.3.4 Analytical Performance of the Immunosensor for the Electrochemical Detection of TNF-α ...... 88 5.3.5 Analysis in Blood ...... 90 5.4 Conclusions ...... 91 5.5 References ...... 92

Chapter 6 ...... 97 Conclusions and Future Work ...... 97 6.1 Conclusions ...... 97 6.2 Future Work ...... 99 6.2.1 Multiple Enzyme Strategy for Signal Amplification ...... 99 6.2.2 Application of External Electric Field to Enhance Protein-resistance Behavior .. 100 6.2.3 Link RGD Peptide for Single-cell Study...... 102 6.3 References ...... 103

xii

Chapter 1 Introduction

Publication I

Jiang, C.; Silva, S. M.; Fan, S. J.; Wu Y. F.; Alam, M. T.; Liu, G. Z.; Gooding, J. J.*

Aryldiazonium Electrochemistry Derived Mixed Organic Layers: from Surface Chemistry to Its Applications, J. Electroanal. Chem [submitted]

Declaration

I certify that this publication was a direct result of my research towards this PhD, and that reproduction in this thesis does not breach copyright regulations.

1 Chapter 1 Introduction

1.1 General Introduction of Aryldiazonium Salts Based Mixed Layers

Electrochemical reduction of aryldiazonium salts have attracted considerable attention for surface functionalization with covalent bonding at the substrate–aryl interface since

Pinson and co-workers described the reaction mechanism for the modification of carbon electrodes by aryldiazonium salts in 1992 (Scheme 1.1).1, 2 It has been demonstrated to be an efficient way to introduce of many types of functional groups to decorate, not only all sorts of carbon surfaces (glassy carbon,3 graphite,4, 5 screen printed carbon electrodes,6 carbon nanotubes,7 and diamond8) but also metals,9 silicon,10-12 and indium tin oxide13, 14 with a reaction time scale of seconds to minutes. A surface can be functionalized with either one type or multiple types of aryldiazonium salts, which are defined as single-component or mixed-layers respectively. The modification and application of single-component surface have been reviewed comprehensively by different research groups over the past two decades.15-21

Scheme 1.1 Proposed reaction mechanism for the electroreductive grafting of substituted aryl organic film to electrode surface.

Despite the enormous potential of single-component of aryldiazonium salt modified surfaces, there are applications where the interface is required to perform multiple functions and hence this will often mean multiple chemical species need to be incorporated into the layer (Scheme 1.2). For example, in sensing application, a mixture 2

Chapter 1 Introduction

of antifouling and bio-recognition components are required to permit the biosensor to operate in complex biological fluids. This requires an interface where the recognition component is a minor component within an antifouling ﬁlm. Such an interface could then facilitate interactions with the target analytes that are not sterically hindered and where nonspeciﬁc adsorption of other proteins in the sample are minimized 13, 22-24. In another case, mixed hydrophobic and hydrophilic layers can be modified with controllable surface ratio to adjust surface wetting property.25 These mixed organic layers bearing different substituents can be obtained by electrografting simultaneously

26, 27 or consecutively22, 28, 29 and have been used in diverse applications such as antifouling coating,30 analytical sensing,31-34 and as initiators for surface-initiated photopolymerization.35

Chapter 1 Introduction

Scheme 1.2 Mixed layers produced from electrografting of aryldiazonium cations, where R1 and R2 represent two different substituents.

Thirteen years have passed since the first report on formation of mixed layers using aryldiazonium salts,36 however, formation of mixed layers derived from aryldiazonium salts is still very much in its infancy in which a number of challenges still remain, often related to the fact that the modification reaction is a radical reaction. The first challenge is that the unselective reactivity of two aryldiazonium cations makes it difficult to control the surface composition. Another challenge is that the highly reactive radicals can not only grow on bare surface but also on as-deposited layer resulting in multilayer; thus making it difficult to precisely control the structure of mixed layers to the level possible with alkanethiol based mixed self-assembled monolayers.25

Considerable efforts have been devoted to overcoming these challenges and in the last few years good progress have been made in controlling the surface chemistry and in applying mixed layers derived from aryldiazonium salts. In this chapter, electrografting of aryldiazonium salts, control over surface composition, the morphology of mixed layers, surface characterization and applications of mixed layer are discussed. The first two parts are extremely important since the design of surface chemistry and its properties will fundamentally influence following applications, which will be highlighted.

1.2 Surface Chemistry of Mixed Layers

1.2.1 Formation of Mixed Layers Using Aryldiazonium Salts

The first example on formation of mixed layers via electrografting aryldiazonium salts was reported by Dequaire and co-workers in 2003. Mixed layers were formed by

Chapter 1 Introduction

electrodepositing the aryldiazonium salts mixture of p-nitroaniline and p- aminohippuric acid at several molar ratios.36 This seminal paper was quickly followed by Gooding et al. where the grafting of 4-carboxyphenyl and phenyl onto a glassy carbon surface in one step was achieved by the electrochemical reduction of a mixture of the corresponding aryldiazonium salts (1:1 molar ratio) in acetonitrile. The importance of this study is that it was the first time to then further modify the layer via the covalent attachment of electroactive ferrocene derivatives to the 4-carboxyphenyl moieties (Figure 1.1). This is the first paper to use the mixed layer as the basis upon which more sophisticated molecular assemblies were fabricated.37

The above-mentioned studies focus mainly on formation of mixed layers using aryldiazonium salts mixture with different molar ratios, while the surface ratio of two components was not characterized and the relationship between molar ratio and surface ratio is still unknown. More importantly, questions pertaining to which factors influenced the surface ratio, and how to control surface ratio, were not addressed. Such questions have only begun to be answered in recent years.26, 27, 37

Figure 1.1 Schematic of ferrocenemethylamine immobilized covalently on mixed layers of 4-carboxyphenyl and phenyl moieties. (Adapted with permission from37.

Chapter 1 Introduction

1.2.2 Control over Surface Composition

Some of the important factors in providing control over surface composition were first investigated by the Bélanger group for the electrodeposition of mixed aryldiazonium salts. They have investigated the formation of mixed layers from mixtures of presynthesized aryldiazonium salts (4-nitrophenyl diazonium and 4-bromophenyl diazonium) onto a glassy carbon surface. It was found that the more easily reduced diazonium salt (4-nitrophenyl diazonium at 0.2 V vs. Ag/AgCl) dominated on the surface over the less easily reduced species (4-bromophenyl diazonium at −0.2 V vs.

Ag/AgCl) regardless of the ratio of the two species in solution.27 In a related study,

Gooding and co-workers examined the electrodeposition of eight sets of binary in-situ generated mixed aryldiazonium salts (at a 1:1 molar ratio in the deposition solution) bearing the para-substituents −Br, −COOH, −SH, −NH2, and −NO2 on both carbon and gold.26 Their findings agreed with those work of Bélanger that the surface ratio was dominated by the species with the more positive reduction potential. This observation is attributed to the component with the more positive reduction potential being grafted onto the electrode, thus covering it, before the second component even begun to be reduced, leading to the first-grafted component inhibits reduction of another component.27

Thus, it seems that an arbitrary conclusion can be made that it is difficult to precisely control surface composition to allow poor-reactivity component take domination when using binary species of aryldiazonium salts with different reduction potentials. On the other hand, it is relatively easy to control surface ratio for species with similar reactivity.38 However, the Gooding group has recently reported one exception to the above statements that the surface ratio of two phenyl derivatives was consistently 1:1 regardless of the molar ratios (3:7, 7:3 and 1:1) and similar reduction potentials because of a supramolecular interaction between the two aryldiazonium species bearing 6

Chapter 1 Introduction

− + 39 opposite charges (−SO3 and −N (CH3)3) in solution.

Subsequent to these studies, efforts have shifted to using a consecutive electrografting approach. In one case, the motivation was to being able to obtain layers where the surface is dominated by a component that is more difficult to reduce with a second minor component for sensing applications. What was observed was that from a mixture of the aryldiazonium salts of phenyl phosphorylcholine and carboxyphenyl, even with an extreme molar ratio of 10000:1 in a single step electrodeposition strategy, a surface ratio of only 6:1 was obtained because of the different reduction potentials.22 However, a surface ratio of a ratio of 200:1 was achieved by selectively depositing phenyl phosphorylcholine diazonium salts first, followed by electrodepositing carboxyphenyl diazonium salts on phenyl phosphorylcholine modified surface. This study highlighted that the consecutive electrografting as a good way to achieve better control over surface composition, especially in the case of forming poor-reactivity component dominates surface. For tight control of surface composition, reactivity and charge status of individual component need to be taken into consideration comprehensively.

1.2.3 Control over Spatial Distribution

Besides control over the surface ratio of the components in mixed layers, spatial control over the different components of mixed layers, as distinct from a random distribution, can also be required. Aiming to obtain hybrid surfaces of binary aryl layers with control over the spatial distribution, Downard et al. reported the patterning of carbon surfaces at the nanoscale with organic functionalities.40 Thin films were covalently grafted to the surface via the electrochemical reduction of aryldiazonium salts (first modifier), then areas of the film are partially removed with an atomic force microscopy tip to allow a second modifier electrografting to the exposed surface (Figure 1.2a). In

Chapter 1 Introduction

different but related strategies to generate unmodified surface for a second modifier to be attached to the surface, both poly(dimethylsiloxane)41 (Figure 1.2b) and polystyrene beads42 (Figure 1.2c) have been used as pattern masks that occupy electrode surface in a controlled manner. The first modifier can either be grown on exposed surface in the microchannel or free spaces between polystyrene beads. The second modifier can then electrochemically “fill in” the ungrafted areas after subsequent removal of the molds of poly(dimethylsiloxane) or polystyrene beads. The authors suggested that this simple surface nanopatterning method could be used to create hybrid surfaces with fragile molecules of biological interest such as using the combination of metal particles and biomolecules43 or metallic nanoparticles and yeast cells,44 which will be discussed in detail in the application section.

Figure 1.2 Preparation of patterned mixed layers with aid of (a) atomic force microscopy tip “scratching”, (b) poly(dimethylsiloxane) and (c) polystyrene beads.

(Adapted with permission from references40-42. Copyright (2005), (2006) and (2010) 8

Chapter 1 Introduction

American Chemical Society)

1.2.4 Control over Structure of Mixed Layers

As mentioned above, building mixed layers via electrografting is a radical based surface modification method. The high reactivity of the aryl radicals in solution produces organic films in a very efficient way. A challenge for the electrografting of aryldiazonium salts lies in the difficulty of controlling the vertical extension of the produced layer principally since the highly reactive aryl radicals can react on both the bare electrode surface and on already grafted organic species which leads to multilayer formation with irregular morphology and low homogeneity.45-47 Such disordered multilayers, and often low-conductivity layers, may pose a signiﬁcant barrier to electron transfer between redox centers in solution or attached to the distal end of the layer and the underlying substrate.29

The strategies for prevention of multilayer growth to allow the formation of mixed monolayers can be generally divided into two categories. The first strategy focuses on controlling the experimental conditions such as viscosity of solvent (e.g. use of ionic liquids instead of aqueous or organic solvents), aryldiazonium salt concentration, applied potential or electrolysis time to empirically give mixed near-monolayer structure.10, 37, 48, 49 For example, Ricci et al. have investigated that on gold substrate, by scanning the potential close to the voltammetric peak and recording the infrared spectrum, the authors concluded to the formation of a near-monolayer.50 Liu et al. have shown that mixed thin layers of 4-carboxyphenyl and phenyl moieties can be formed on glassy carbon electrode by reducing the electrodeposition time to just two cycling scans (100 mV/S).37 However, an ultrathin and homogeneous (well-defined) coating is difficult to obtain with this type of strategy since these measures can reduce the chance

Chapter 1 Introduction

of the electrode derivatization with multilayers of aryl groups to some extent but cannot eliminate multilayers.51, 52

Therefore, a more controlled strategy was proposed by selecting suitable molecular systems with an intrinsically or externally bulky steric protection group to avoid attack of radicals on the already grafted species. Mattiuzzi et al. have reported formation of mixed monolayers in one-step electrografting by introducing a calix[4]arene diazonium salt from the corresponding tetra-anilines53, 54 (Figure 1.3). The thickness was examined by atomic force microscopy with an intermittent contact mode55 to scratch a rectangular area (1.3 ± 0.1 nm) and elliposometric measurements (1.09 ± 0.20 nm), indicating the formation of monolayers based on the theoretical height of the calix[4]arenes 2 (c.a. 1.1 nm). The formation of monolayer could be attributed to the already-occupation of 3-/5- substituents of benzene rings in the calix[4]arene structure, which does not allowed for subsequent attack by aryl radicals. Moreover, a strong blocking effect toward redox probe responses was observed which implies the surface construct was composed of a compact-packed layers. Through adequate decoration of the small rim of the calixarenes, up to four different functional molecules (e.g. redox probes or biomolecules) could then be introduced on the immobilized calixarene subunits to give a multifunctional molecular platform. The study provides an efficient approach to build a platform of mixed monolayers through single-step electrografting, while the multiple synthesis steps and fixed surface ratio (because of tetramer structure) of individual component also need to be considered.

Chapter 1 Introduction

Figure 1.3 Representation of the four calixarenes compounds undergoing in-situ transformation to diazonium salts for electrografting. (Adapted with permission from reference53. Copyright (2012) Nature Publishing Group)

Santos et al. have described the formation of a mixed organic layers using bulky β- cyclodextrin as external steric-hindrance protector.23 First, bithiophenephenyl diazonium salt was reduced using host/guest complexation in a water/cyclodextrin solution. The attached cyclodextrin was removed by electrochemical cycling in acetonitrile containing 0.1 mol/L LiClO4 to create surface pinholes with nanometer scale such that nitrophenyl diazoniumcations can be reductively grafted as second component (Figure 1.4). The big advantage is that this method allows electrografting of water-insoluble diazonium salt in aqueous media through the host−guest complexation.

On restriction of this approach is the length of aryldiazonium salts should be greater than the height of the β-cyclodextrin (0.87 nm), otherwise diazonium salts with shorter length may be embedded in the cavity of β-cyclodextrin without exposure of diazonium ions and dramatically reduce the electrografting efficiency.

Chapter 1 Introduction

Figure 1.4 Scheme showing the two-step grafting procedure with the β-cyclodextrin protection-deprotection-“fill in” strategy (Adapted with permission from23. Copyright

(2012) American Chemical Society)

An alternative strategy for using an external protector to resist radicals was recently

reported by Hapiot and co-workers.45, 56 This study took advantage of molecular

system with intrinsic protection group, a bulky and cleavable

((triisopropylsilyl)ethynyl) group, to make robust binary film onto carbon surface. The

first modification consists of the grafting of a protected 4-((triisopropylsilyl)ethynyl)

benzene layer onto the carbon surface. As this layer does not closely pack because of

the bulky protecting group, a second species can be electrodeposited into the free

spaces. After deprotection (removal of the triisopropylsilyl) by soaking the modiﬁed

electrodes in trifluoroacetic acid for 20 min, mixed layers presenting ethynylbenzene

groups (available for further functionalization with ferrocene moieties via “click”

chemistry) and second functional groups (nitrobenzene in this case) were obtained 12

Chapter 1 Introduction

(Figure 1.5). The study highlighted the importance of retaining the chemical protecting

group on the ﬁrst modiﬁer during the second grafting step to avoid degradation of the

functionality of the ﬁrst layer by the second layer. Using mild conditions for the

deposition of the second layer maintains the concentration of active ethynyl groups

similar to that obtained for a one-component monolayer (Γ =2.9 ×10−10 mol/cm2).56

This approach is promising for the preparation of mixed layers on carbon surfaces in

well-controlled conditions without altering the reactivity of either functional group.

Figure 1.5 Principles of stepwise formation of mixed organic layers by the “protection- insertion-deprotection-attachment of ferrocene” strategy. (Adapted with permission from29. Copyright (2013) American Chemical Society)

Very recently, Lee et al. took advantage of the oxidative electrografting of arylhydrazines (similar structure to aniline) to give monolayers on Au and glassy carbon as reported by Daasbjerg group.57, 58 They have proposed a “combined” approach by the sequential reductive electrografting of aryldiazonium salts followed by the oxidative electrografting of arylhydrazine to achieve mixed monolayers29 (Figure 1.6).

The ﬁrst step, which is similar to that of Hapiot and co-workers,56 employs a 4-

((triisopropylsilyl)ethynyl)phenyl ﬁlm electrografted onto the electrode surface,

Chapter 1 Introduction

followed by removal of the triisopropylsilyl group to give a monolayer of phenylethynylene groups with free spaces. Then two strategies were applied to “fill in” the sparse monolayer with a second modifier (Figure 1.6). In the first route, nitrophenyl groups are grafted to the phenylethynylene-modified surface by the oxidation of 4- nitrophenylhydrazine. Ferrocene could be coupled to the terminal alkyne groups on the surface via a click reaction with azidomethyl ferrocene; an electrochemical measurement of the amount of immobilized ferrocene demonstrates that the phenylethynylene layer retains close to full reactivity after the second grafting step. In the alternative strategy, ferrocene was coupled to the phenylethynylene layer first, followed by oxidative grafting of 4-nitrophenylhydrazine. It was reported that multilayers do not form by optimization of the grafting conditions for the second step and arylhydrazines are particularly well suited to this application since they have a low tendency to form multilayers.

Figure 1.6 Two routes (a and b) for the preparation of mixed monolayer films from a deprotected 4-((triisopropylsilyl)ethynyl)phenyl layer. (Adapted with permission from reference29. Copyright (2013) American Chemical Society)

In a later paper, the same group reported another “combined” strategy for formation of mixed monolayers by using different molecular systems, that is, combination of

Chapter 1 Introduction

electrografting of protected aryldiazonium cations and back filling of amine derivative via spontaneous grafting.59 Three protection groups, triisopropylsilyl, fluorenyl methyloxycarbonyl, fluorenyl methyl were examined. Beginning with protected aryldiazonium salts, sparse monolayers of ethynyl-, amino-, and carboxy-terminated tethers were anchored to the surface. The layers were then backﬁlled with a second modiﬁer via the nucleophilic addition (Michael-like addition) of a primary amine derivative to the surface, which has been demonstrated to react directly with carbon substrates by Pinson and co-workers.60

The strategies mentioned above open up a novel horizon for fabricating a surface with multi-functionalities with good control by coupling reductive electrografting with either oxidative grafting or spontaneous grafting methods. The backfilling in such

"combined" strategies can significantly increase the total surface concentration of grafted groups giving concentrations that are up to double those obtained by grafting from a mixture of protected aryldiazonium salts and the backfilling reaction that are unaffected by a near-complete surface coverage by the first modifier.59

Moreover, molecular systems with bulky cleavable groups have been extensively explored in recent years. For instance, besides the tri(isopropyl)silyl29, 45, 61, 62 protection groups, the Hapiot group has further extended this type of molecular system to trimethylsilyl,28 triethylsilyl.29 Similarly, fluorenyl methyloxycarbonyl,63 fluorenyl methyl59, 64 and tert-butyloxycarbonyl65, 66 protecting groups to allow monolayer formation have also been reported.

1.2.5 Characterization of Mixed Layers

Most of the surface properties of mixed layers can be characterized using the same techniques that have been used for single-component surface (e.g. surface roughness 15

Chapter 1 Introduction

using atomic force microscopy,56 electrode passivation using cyclic voltammetry with redox probes,3 and thickness measurement using ellipsometry67), and are beyond the scope of this chapter. The choice of the best technique to characterize a mixed-layer surface depends on different factors including the amount/ratio of compounds presented and the type of information that is required to understand the system and which information will be useful for the given application. X-ray photoelectron spectroscopy

(XPS) is a powerful technique and one of the most commonly used to characterize surfaces. To illustrate that, XPS was used to analyze the deposited 4-nitrophenyl diazonium and 4-bromophenyl diazonium mixed layers, the characteristic signal of the para-substituents in each of these two grafted groups allows the surface concentration of each component to be calculated.27 Electrochemical techniques are also quite useful to obtain and support the information acquired using other techniques, taking the same example mentioned above where the both components are electroactive species with different electrochemistry processes, by using cyclic voltammetry the surface concentration of each component can also be estimated.27

1.2.6 Other Potential Strategies for Formation of Mixed Monolayers

In this section, existing approaches which have already been applied for single- component surface with monolayer (or submonolayer) structure will be covered due to their potential to form mixed layers. Specifically, molecular systems like 3,5-bis-tert- butylbenzene diazonium salt, diaryl disulfide or arylhydrazine can be combined with another type of aryldiazonium salt to produce mixed layers. Radical scavenger can be used for reducing multilayer growth in cases of forming mixed layers.

Introduction of sterically encumbered substituents on the aryl rings has been successfully used to prevent the uncontrolled polymerization process, thus allowing the

Chapter 1 Introduction

formation of a near monolayer. Pinson and co-workers demonstrated that a near- monolayer can easily be obtained using 3,5-bis-tert-butylbenzene diazonium ion

(Figure 1.7).47, 68 They found that the occupation of 3- and 5-positions (next to para- position) by isopropyl groups can prevent subsequent aryl radicals attack at the aryl ring. With the benefits from such molecular system, Feyter et al. have successfully employed such approach by electrografting of 3,5-bis-tert-butyl-benzene diazonium salts to form monolayers on graphene, which is useful for studying nanostructuring surfaces.69

Figure 1.7 Electrografting of aryldiazonium salts and formation of multilayer without steric effect, and monolayer with steric effect. (Adapted with permission from reference68. Copyright (2009) American Chemical Society)

When bulky substituents are used, the polymerization is limited but the surface coverage is lowered and another undesirable counter back is the chemical inertness of these molecules, which precludes further functionalization.53 Therefore, to permit the further functionalizable of this molecular system, it is necessary to redesign and

Chapter 1 Introduction

synthesize 3,5-diisopropylaniline with functional groups like -COOH/-NH2 at papa- position or combine with another type of aryldiazonium cations bearing functionality to produce mixed layers.

Breton et al. have proposed an approach to limit ﬁlm growth to a monolayer during

(electro)grafting of the aryldiazonium ions using a radical scavenger of 2,2-diphenyl-

1- pycrilhidrazyl (Figure 1.8).70, 71 It was shown that 2,2-diphenyl-1- pycrilhidrazyl was remarkably efficient at limiting film growth. This led to a good control of the number of layers in the organic film structure can be achieved, giving rise to organic layers of less than 1 nm thickness with no inhibiting its electrical conductivity.72 The Creus group has successfully shown that a monolayer of nitrophenyl can be obtained on highly oriented pyrolytic graphite by electrografting in the presence of 2 mM 2,2-diphenyl-1- pycrilhidrazyl. Gold nanoparticles can be electrodeposited onto the surface after the nitro moiety is electrochemically converted to amine group.73 A very recent study by

Breton et al. have revealed the control of the layer growth via the use of radical scavenger is dependent on the substituent on the diazonium ions. A monolayer functionalization being only possible when the substituent is an electron withdrawing group.46

Chapter 1 Introduction

Figure 1.8 Grafting of aryl layers on carbon surface with and without radical scavenger.

(Adapted with permission from reference70. Copyright (2013) American Chemical

Society)

Unlike the above-mentioned strategies that use bulky protection groups to prevent multilayer growth, Daasbjerg et al. have explored the possibility of preparing thin layers through a degradation of multilayers formed via reduction of aryldiazonium salts containing cleavable groups like disulfides or hydrazine (Figure 1.9).74, 75 This

“formation-degradation” approach is unique in the sense that it allows the generation of a thin well-defined molecular layer in spite of the involvement of highly reactive radicals in a multilayer formation steps. The resulting thin layer of thiophenol groups can be employed as chemical linkers for anchoring gold nanoparticles through Au-S in a site-selective manner.74, 76, 77 A thin and well-defined film of covalently attached benzaldehyde with an estimated coverage of 4 × 10-10 mol/cm2 was formed and such films can also give well-defined and reproducible electrochemical responses, independent of grafting medium (water or dimethyl sulfoxide).

Chapter 1 Introduction

Figure 1.9 (a) Preparation of thin layers on carbon surfaces through a degradation of multilayers formed via reduction of diazonium salts of diaryl disulfides. (Adapted with permission from reference74. Copyright (2007) American Chemical Society). (b)

Electrografting in aqueous solution of benzaldehyde Girard’s reagent T hydrazonediazonium salt followed by an acidic hydrolysis to form a thin layer of surface-attached benzaldehyde (Adapted with permission from reference75. Copyright

(2009) American Chemical Society)

1.3 Applications of Mixed Layers Modified Surfaces

The majority of applications of mixed layers formed from aryldiazonium salts can be divided into four categories: antifouling coatings, sensing, for protein electrochemistry, and as surface-initiator for photopolymerization. The representative examples of each application will be highlighted in this section.

1.3.1 Antifouling Coating

The development of antifouling coatings on surfaces is vital for application of man- made surfaces in biological matrices such as in biomaterials, tissue engineering and sensing. With this application in mind, the development of antifouling coatings on electrode surfaces using aryldiazonium salt derived layers has received much attention.

For example, Jiang et al. have demonstrated the applications of zwitterionic polymers of phosphorylcholine, carboxybetaine or sulfobetaine as layers with an excellent ability to resist the nonspecific adsorption of protein.78, 79 To render this idea to be compatible with sensing application, zwitterionic component needs to decorated on a substrate for testing their antifouling behavior.13, 80 Gooding et al. drew on the quality of mixed aryl

Chapter 1 Introduction

layers bearing oppositely charged moieties to resist nonspecific protein adsorption to make antifouling layers for electrochemical biosensing.30 Mixed layers of 4- sulfophenyl and 4-(trimethylammonio)-phenyl bearing opposite charges on glassy carbon (Figure 1.10) were prepared using a one-step electrografting approach and results showed that such mixed layer were as effective as oligo(ethylene oxide) self- assembled monolayers formed from alkanethiols (a gold standard) for resisting the nonspecific adsorption of bovine serum albumin and cytochrome.30, 81 The key innovation in the study was to form very thin zwitterionic layers on the electrode surface such that faradaic electrochemical processes were still possible at the underlying electrode.

Figure 1.10 Schematic representation of glassy carbon surface coated with 1:1 mixed layers of 4-sulfophenyl and 4-(trimethylammonio)-phenyl. (Adapted with permission from reference30. Copyright (2013) American Chemical Society)

1.3.2 Analytical Sensing

Chapter 1 Introduction

For analytical sensing, mixed layers will have one component served as anchoring point for linking biomolecules like redox protein, epitope or antibody, another component can act as inactive diluent to reduce steric hindrance or as antifouling layer to resist nonspecific biofoulings.

1.3.2.1 Protein Electrochemistry

Gooding and co-workers have investigated mixed-layers system for protein electrochemistry.82 Mixed oligo(ethyleneglycol) and oligo(phenylethynylene)

(molecular wire) layers were prepared by reduction of a mixture of oligo(ethyleneglycol)- and oligo(phenylethynylene)-based aryldiazonium salts onto a glassy carbon electrode (Figure 1.11). The rigid molecular wires with 20 Å length can interact directly with the protein (horseradish peroxidase) to facilitate efficient electron transfer behavior. The diluent oligo(ethyleneglycol) molecules with three ethylene oxide units was able to resist nonspecific adsorption of proteins in blood serum.82 This strategy was also applied to the modiﬁcation of a carbon electrode by a mixed layer of

4-carboxyphenyl and a oligo(phenylethynylene) molecular wire to interface the electrode with glucose oxidase.83 Liu et al. and Ferri et al. have independently utilized similar methodology by using SWNTs84, 85 or AuNPs86 covalent anchoring to mixed layers of oligo(ethyleneglycol) and benzene to facilate direct electron transfer between

HRP or methyl parathion hydrolase and electrode. The presented sensing interfaces are versatile and could be applied to a large number of enzymes to ensure speciﬁc protein electrochemistry.87, 88

Chapter 1 Introduction

Figure 1.11 Schematic of forming mixed layers of molecular wires and oligo(ethylene glycol) on a glassy carbon electrode interface for protein electrochemistry. (Adapted with permission from reference82. Copyright (2006) American Chemical Society)

1.3.2.2 Chemical Sensing

Bélanger and co-workers reported the electrografting of mixed layers containing 4- sulfophenyl and 4-aminophenyl onto carbon surface31 (Figure 1.12). The composition of the mixed layers was controlled either by molar ratios of the two components in solution and applied deposition potential. The binary organic ﬁlms bearing opposite

2+ 2− charges were used as templates to load bimetallic species of Cu and PtCl6 based on electrostatic interactions, followed by in-situ reduction using NaBH4 as reducing agent.

XPS analysis was performed to conﬁrm the presence of the bimetallic layers on the electrode surface. Stripping voltammetry and adsorption/desorption measurement of hydrogen in sulfuric acid solution were used for determination of loaded amount of Cu and Pt on the surface, respectively. They showed that for the preparation of a surface containing both metallic Pt and Cu, Pt should be loaded ﬁrst to avoid the displacement reaction of Cu by Pt. 23

Chapter 1 Introduction

Figure 1.12 Mixed-layer surface consists of 4-aminophenyl and 4-sulfophenyl groups from mixed solutions of their corresponding diazonium cations and subsequent loading of the metallic Cu and Pt species. (Adapted with permission from reference31.

Another study where nanoparticles were immobilized onto mixed layers derived from aryldiazonium salts was reported by Liu and co-workers for the electrochemical detection of cadmium ions.89 AuNPs modiﬁed with 4-nitrophenyl and 4-carboxyl phenyl were ﬁrst prepared, which were then immobilized in-situ on a gold electrode.

Glutathione was then attached to the nanoparticles used to selectively detect Cd2+ over the concentration range from 0.1 to 100 nM. A detection limit of 0.1 nM was reported.

This electrochemical sensor demonstrated high stability and sensitivity to Cd2+ with the technology having potential for the development of portable devices for the onsite monitoring of trace amounts of heavy metals in environmental samples.

1.3.2.3 Biosensing Applications

Chapter 1 Introduction

Generally, for affinity based biosensors, one component of a mixed layer is further modified with bio-recognition species (e.g. antibodies, nucleic acid) while the other component is used to space the biorecognition species apart, attach redox species or provide resistance to nonspecific adsorption of proteins. Within this general type of interface many variants have been developed for affinity biosensing.

1.3.2.3.1 DNA Sensors

In a recent work, Kuo et al. reported simultaneous reductive deposition of the two aryldiazonium cations to fabricate a bifunctional surface on a gold electrode, in which a maleimide moiety enables regulated number of sites accessible for facile binding to the thiolated DNA probe and a zwitterionic sulfobetaine moiety permits efficient antifouling capability against nonspecific DNA and enzyme.90 Obviously, such dual functional interface is advantageous, compared with a single-component interface, for detect target analyte in complex biological samples in terms of reducing background signals.43 The proposed mixed layer platform was applied to detect three genetic fragments of the new delhi metallo-β-lactamase-coding gene (Figure 1.13). The greatest sensing signal was obtained when the molar ratio of the two anilines was 1:1. The limit of detection was 54 pM with linear correlations over 0−1 nM and in a higher concentration range over 1−100 nM.

Chapter 1 Introduction

Figure 1.13 Schematic diagram of the interface preparation to detect Delhi Metallo-β-

Lactamasegenetic fragments. (Adapted with permission from reference90. Copyright

(2015) American Chemical Society)

Hai et al. have reported their DNA sensor work by simultaneous electrografting mixed layers comprising electroactive 5-hydroxy-1,4-naphthoquinone and 4-aminobenzoic acid and subsequent attaching DNA probes through EDC/NHS coupling reaction. The detection of target complementary DNA was achieved using square wave voltammetry with a detection limit of 10 pM, which demonstrates the potentialities of the aryldiazonium approach for achieving direct electrochemical transducers.32 In another study performed by Hayat and co-workers,91 a highly sensitive label-free ochratoxin A impedimetric aptasensor was developed based on the immobilization of azido-aptamer onto electrografted binary ﬁlms of 4-nitrophenyl and 4-ethynylphenyl via click chemistry.

1.3.2.3.2 Immunosensor

Inspired by protein electrochemistry system using molecular wires,24, 82, 83 single-wall carbon nanotubes85, 92 or gold nanoparticles86, 93 to facilitate electron transfer to allow electric communication between redox protein and underlying electrode, the Gooding

Chapter 1 Introduction

group has reported development of immunosensing interfaces, in which immune recognition components (e.g. epitope, antigen, and antibody) were used to functionalize mixed aryl layer surface for detecting specific biomarkers. As shown in Figure 1.14,

Gooding group has reported an immunosensing interface comprising a mixed layer of an oligo(ethyleneglycol) component, and an oligo(phenylethynylene) molecular. The oligo(ethyleneglycol) controls the interaction of proteins and electroactive interferences with the surface and the molecular wire allows electrochemical communication to the underlying glassy carbon electrode.94 To the distal end of the molecular wire, a redox probe 1,1'-di(aminomethyl)ferrocene is attached, followed by the surface bound epitope to which an antibody would bind. Association or disassociation of the antibody with the sensing interface causes a modulation of the ferrocene electrochemistry. In this work, five sets of molecular wire/oligo(ethyleneglycol) (molar ratios, 1:0, 1:20, 1:50, 1:75 and 1:100) were tested on sensor sensitivity detection for a model analyte (biotin) free in solution, via a displacement assay.95 The ratio of 1:50 was found to give the highest sensitivity and reproducibility.

Figure 1.14 Schematic of a label-free electrochemical immuno-biosensor. Sensor 1

Chapter 1 Introduction

(left) shows the electrode surface before detection of the target analyte with the corresponding electrochemical signal. Sensor 2 (right) shows the detection after the antibody is released with the expected increase in electrochemical signal. (Adapted with permission from reference94. Copyright (2011) Elsevier)

In a recent work, a novel electrochemical immunosensor was developed by Narayanan et al. for the detection of botulinum neurotoxin-E96 (Figure 1.15). In this method, mixed layers of phenyl and aminophenyl were electrografted onto glassy carbon surface. The aminophenyl moiety was diluted in chemical inert phenyl groups to ease following attachment of graphene nanosheet, which can facilitate electron transfer.33 A multiple- enzyme strategy by decorating gold nanoparticles with rabbit anti-mouse IgG-alkaline phosphatase was used to amplify electrochemical signal.97 Silver nanoparticles can be deposited on gold nanoparticles through enzymatic reduction in the presence of 3- indoxylphosphate as substrate. The deposited silver nanoparticles on electrode surface which were correlated with the amount of analyte bind on surface, were determined by stripping signal using linear sweep voltammetry. The developed electrochemical immunosensor could detect the analyte with linear range from 10 pg/mL to 10 ng/mL with the minimum detection limit of 5.0 pg/mL. The immunosensor was successfully evaluated against food samples like orange juice and milk.

Chapter 1 Introduction

Figure 1.15 Steps involved in glassy carbon electrode modification and sandwich immunoassay based detection procedure for analysis of deposited silver nanoparticles generated by rabbit anti-mouse IgG-alkaline phosphatase /gold nanoparticles catalysts and 3-indoxylphosphate. (Adapted with permission from reference96. Copyright (2015)

Elsevier)

1.3.2.4 Live Cell Electrochemistry

In 2009, Brozik and co-workers reported formation of multifunctional thin ﬁlms with dual binding functionalities on gold electrode surface via consecutive electroreduction of aryldiazonium salts with two distinct substituent groups of nitrophenyl and phenyl boronic acid pinacol ester (Figure 1.16).44 The prepared mixed layer gold surface was applied for binding platinum nanoparticles and reversible immobilization of yeast cells after undergoing an electrochemical conversion of nitro to amino group and deprotection of phenyl boronic acid. Electrocatalytic currents from peroxide reduction

Chapter 1 Introduction

at immobilized platinum nanoparticles show that the thin ﬁlm remains conductive, facilitating subsequent electrochemical measurements. Finally, they claimed such multifunctional ﬁlms that maintain conductivity for subsequent electrochemical measurements hold promise for the development of electrochemical or optical platforms for fundamental cell studies, genomic and proteomic analysis, and biosensing.98 In another study, Guo et al. have reported the effects of surface charge on anodic biofilm formation, community composition, and current generation in

+ bioelectrochemical systems using electrografted aryl layers bearing −N (CH3)3 or

– 99 −SO3 . They showed that positively charged and hydrophilic surfaces were more selective to electroactive microbes (e.g. Geobacter) and more conducive for electroactive biofilm formation, suggesting the importance of surface chemistry on bioﬁlm formation, diversity, and would be beneficial to study microbes catalysts driven oxidation and/or reduction in bioelectrochemical systems.99

Figure 1.16. Preparation of the stacked multifunctional thin ﬁlm occurred via consecutive electrodeposition for immobilization of citrate capped platinum

Chapter 1 Introduction

nanoparticles and yeast cells. (Adapted with permission from reference44. Copyright

(2009) American Chemical Society)

1.3.3 Initiator for Surface-Initiated Photopolymerization

Chehimi and co-workers have reported that electrografted 4-benzoyl-phenyl groups can be efficiently served for surface-initiated photopolymerization of α-tert-butoxy-ω- vinylbenzyl-polyglycidol and hydroxyethyl methacrylate35, 100 (Figure. 1.17). The antifouling behavior of the grafted mixed polymer films of α-tert-butoxy-ω- vinylbenzyl-polyglycidol and hydroxyethyl methacrylate was evaluated by surface plasmon resonance using anti-bovine serum albumin. The mixed films exhibit enhanced resistance to anti-bovine serum albumin adsorption compared to the only hydrophilic hydroxyethyl methacrylate grafted surface, and the super hydrophilic α- tert-butoxy-ω-vinylbenzyl-polyglycidol was further modified with bovine serum albumin through the carbonyldiimidazole activation of the ‒OH. The results showed that this new protocol combining aryldiazonium salt based photoinitiators and photopolymerization is a fast, elegant way to prepare ultrathin mixed polymer grafts with dual functionalities of specific protein grafting together with anti-biofouling, presenting great potential for sensing and biomedical applications. This work definitely broadens the enormous possibilities offered by aryldiazonium salts as novel coupling agents for design of polymer based organic coatings.

Chapter 1 Introduction

Figure 1.17 Electrografting derived mixed polymer layers with dual antifouling and specific protein recognition properties. (Adapted with permission from reference35.

1.4 Summary and Scope of This Thesis

This chapter gives an overview of mixed organic layers based multifunctional molecular platform using aryldiazonium salt electrochemistry, in which the significance of formation of mixed layers, strategies to make mixed thin layers, and their existing applications, especially biosensing, are covered. The target of this research work is to utilization of the surface chemistry of mixed layers to develop an antifouling and low- impedance biosensing platform which can allow specifically and sensitively detect target analyte in biological media. In order to achieve the research aims, a number of steps must be investigated and achieved: (1) selection of appropriate antifouling molecule and its evaluation; (2) building a mixed layers molecular system containing antifouling layer and bio-recognition layer, and control over surface chemistry; (3)

Chapter 1 Introduction

developing a final biosensor based on the mixed layers system with a detailed sensing performance examination.

In the first step, indium tin oxide (ITO) was modified by electrografting phenylphosphorylcholine (PPC) and evaluated its protein-resistance performance using electrochemical impedance spectroscopy. During electrografting, significant amount of

PPC gets physically adsorbed on the surface, which cannot be removed by simply rinsing with water or buffer for a short time, consequently the electrochemical response of such electrode is quite unstable. A simple protocol has been developed to remove the physically adsorbed PPC such that a stable interface is produced. It was found that such a stable surface, with appropriate amount of PPC, can highly suppress nonspecific adsorption of protein, giving a platform of performing both electrochemical and spectroscopic studies in biological fluids.

In the second step, aryldiazonium salts of carboxyphenyl (CP) and phenylphosphorylcholine (PPC), generated in-situ from their corresponding anilines, are electrografted to form functionalizable molecular platform. These two components are chosen because CP provides a convenient functionality for further coupling of biorecognition species while PPC offers resistance to nonspecific adsorption of proteins to the surface. Mixed layers of CP and PPC were prepared by grafting them either simultaneously or consecutively. The latter strategy allows an interface to be developed in a controlled way where one component is at levels of less than 1% of the total layer.

In the third step, the effective antibiofouling chemistry that employs short chain zwitterionic species, derived from aryl diazonium salts, that give low impedance layers compatible with amperometry was applied. The application of this surface chemistry to mixed layers of phenyl phosphorylcholine (PPC) and phenyl butyric acid (PBA), to develop immunosensors that can be used in whole blood. The capability of these new modification layers is demonstrated with an immunosensor for detecting tumor necrosis

Chapter 1 Introduction

factor α in whole blood. The immunosensor is shown to specifically and precisely detect

TNF-α in whole blood samples with a minimum detection limit of 10 pg/mL with a wide linear range of 0.01 ng/mL to 500 ng/mL. The results are comparable with those from commercial ELISA kit, indicating the developed immunosensor has great potential for future clinic use.

1.5 References

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2. Bourdillon, C.; Delamar, M.; Demaille, C.; Hitmi, R.; Moiroux, J.; Pinson, J. Immobilization of glucose oxidase on a carbon surface derivatized by electrochemical reduction of diazonium salts. J. Electroanal. Chem. 1992, 336 (1), 113-123.

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Chapter 1 Introduction

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12. Flavel, B. S.; Gross, A. J.; Garrett, D. J.; Nock, V.; Downard, A. J. A simple approach to patterned protein immobilization on silicon via electrografting from diazonium salt solutions. Acs. Appl. Mater. Inter 2010, 2 (4), 1184-1190.

13. Jiang, C.; Tanzirul Alam, M.; Parker, S. G.; Gooding, J. J. Zwitterionic Phenyl Phosphorylcholine on Indium Tin Oxide: a Low-Impedance Protein-Resistant Platform for Biosensing. Electroanal 2015, 27 (4), 884-889.

14. Maldonado, S.; Smith, T. J.; Williams, R. D.; Morin, S.; Barton, E.; Stevenson, K. J. Surface modification of indium tin oxide via electrochemical reduction of aryldiazonium cations. Langmuir 2006, 22 (6), 2884-2891.

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19. Mahouche-Chergui, S.; Gam-Derouich, S.; Mangeney, C.; Chehimi, M. M. Aryl diazonium salts: a new class of coupling agents for bonding polymers, biomacromolecules and nanoparticles to surfaces. Chem. Soc. Rev. 2011, 40 (7), 4143- 35

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24. Michael, N.; áJustin Gooding, J. Protein modulation of electrochemical signals: application to immunobiosensing. Chem. Commun. 2008, (33), 3870-3872.

25. Folkers, J. P.; Laibinis, P. E.; Whitesides, G. M. Self-assembled monolayers of alkanethiols on gold: comparisons of monolayers containing mixtures of short-and long-chain constituents with methyl and hydroxymethyl terminal groups. Langmuir 1992, 8 (5), 1330-1341.

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27. Louault, C.; D'Amours, M.; Belanger, D. The electrochemical grafting of a mixture of substituted phenyl groups at a glassy carbon electrode surface. ChemPhysChem 2008, 9 (8), 1164-1170.

28. Leroux, Y. R.; Hapiot, P. Nanostructured monolayers on carbon substrates prepared by electrografting of protected aryldiazonium salts. Chem. Mater. 2013, 25 (3), 489- 495.

29. Lee, L.; Brooksby, P. A.; Leroux, Y. R.; Hapiot, P.; Downard, A. J. Mixed monolayer organic films via sequential electrografting from aryldiazonium ion and arylhydrazine solutions. Langmuir 2013, 29 (9), 3133-3139.

30. Gui, A. L.; Luais, E.; Peterson, J. R.; Gooding, J. J. Zwitterionic phenyl layers: finally, stable, anti-biofouling coatings that do not passivate electrodes. Acs. Appl. 36

Chapter 1 Introduction

Mater. Inter 2013, 5 (11), 4827-4835.

31. Vilà, N.; Bélanger, D. Mixtures of functionalized aromatic groups generated from diazonium chemistry as templates towards bimetallic species supported on carbon electrode surfaces. Electrochim. Acta 2012, 85, 538-547.

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34. Khor, S. M.; Liu, G.; Peterson, J. R.; Iyengar, S. G.; Gooding, J. J. An electrochemical immunobiosensor for direct detection of veterinary drug residues in undiluted complex matrices. Electroanal 2011, 23 (8), 1797-1804.

35. Gam-Derouich, S.; Gosecka, M.; Lepinay, S.; Turmine, M.; Carbonnier, B.; Basinska, T.; Slomkowski, S.; Millot, M.-C.; Othmane, A.; Ben Hassen-Chehimi, D. Highly hydrophilic surfaces from polyglycidol grafts with dual antifouling and specific protein recognition properties. Langmuir 2011, 27 (15), 9285-9294.

36. Ruffien, A.; Dequaire, M.; Brossier, P. Covalent immobilization of oligonucleotides on p-aminophenyl-modified carbon screen-printed electrodes for viral DNA sensing. Chem. Commun. 2003, (7), 912-913.

37. Liu, G.; Liu, J.; Böcking, 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. Chem. Phys. 2005, 319 (1), 136-146.

38. Esnault, C.; Delorme, N.; Louarn, G.; Pilard, J. F. One‐Pot in Situ Mixed Film Formation by Azo Coupling and Diazonium Salt Electrografting. ChemPhysChem 2013, 14 (9), 1793-1796.

39. Gui, A. L.; Yau, H. M.; Thomas, D. S.; Chockalingam, M.; Harper, J. B.; Gooding, J. J. Using supramolecular binding motifs to provide precise control over the ratio and distribution of species in multiple component films grafted on surfaces: demonstration using electrochemical assembly from aryl diazonium salts. Langmuir 2013, 29 (15), 4772-4781.

Chapter 1 Introduction

40. Brooksby, P. A.; Downard, A. J. Nanoscale patterning of flat carbon surfaces by scanning probe lithography and electrochemistry. Langmuir 2005, 21 (5), 1672-1675.

41. Downard, A. J.; Garrett, D. J.; Tan, E. S. Microscale patterning of organic films on carbon surfaces using electrochemistry and soft lithography. Langmuir 2006, 22 (25), 10739-10746.

42. Corgier, B. P.; Bélanger, D. Electrochemical surface nanopatterning using microspheres and aryldiazonium. Langmuir 2010, 26 (8), 5991-5997.

43. Corgier, B. P.; Li, F.; Blum, L. J.; Marquette, C. A. On-Chip Chemiluminescent Signal Enhancement Using Nanostructured Gold-Modified Carbon Microarrays. Langmuir 2007, 23 (16), 8619-8623.

44. Harper, J. C.; Polsky, R.; Wheeler, D. R.; Lopez, D. M.; Arango, D. C.; Brozik, S. M. A Multifunctional Thin Film Au Electrode Surface Formed by Consecutive Electrochemical Reduction of Aryl Diazonium Salts. Langmuir 2009, 25 (5), 3282- 3288.

45. Leroux, Y. R.; Hui, F.; Noël, J.-M.; Roux, C.; Downard, A. J.; Hapiot, P. Design of robust binary film onto carbon surface using diazonium electrochemistry. Langmuir 2011, 27 (17), 11222-11228.

46. Menanteau, T.; Dias, M.; Levillain, E.; Downard, A. J.; Breton, T. Electrografting via Diazonium Chemistry: The Key Role of the Aryl Substituent in the Layer Growth Mechanism. J. Phys. Chem. C 2016, 120 (8), 4423-4429.

47. Combellas, C.; Kanoufi, F.; Pinson, J.; Podvorica, F. I. Sterically hindered diazonium salts for the grafting of a monolayer on metals. J. Am. Chem. Soc. 2008, 130 (27), 8576-8577.

48. Brooksby, P. A.; Downard, A. J. Multilayer Nitroazobenzene Films Covalently Attached to Carbon. An AFM and Electrochemical Study. J. Phys. Chem. B 2005, 109 (18), 8791-8798.

49. Fontaine, O.; Ghilane, J.; Martin, P.; Lacroix, J.-C.; Randriamahazaka, H. Ionic liquid viscosity effects on the functionalization of electrode material through the electroreduction of diazonium. Langmuir 2010, 26 (23), 18542-18549.

50. Ricci, A.; Bonazzola, C.; Calvo, E. J. An FT-IRRAS study of nitrophenyl mono- and multilayers electro-deposited on gold by reduction of the diazonium salt. Phys. Chem. Chem. Phys. 2006, 8 (37), 4297-4299.

51. Vase, K. H.; Holm, A. H.; Norrman, K.; Pedersen, S. U.; Daasbjerg, K. 38

Chapter 1 Introduction

Electrochemical Surface Derivatization of Glassy Carbon by the Reduction of Triaryl- and Alkyldiphenylsulfonium Salts. Langmuir 2008, 24 (1), 182-188.

52. Kariuki, J. K.; McDermott, M. T. Formation of Multilayers on Glassy Carbon Electrodes via the Reduction of Diazonium Salts. Langmuir 2001, 17 (19), 5947-5951.

53. Mattiuzzi, A.; Jabin, I.; Mangeney, C.; Roux, C.; Reinaud, O.; Santos, L.; Bergamini, J.-F.; Hapiot, P.; Lagrost, C. Electrografting of calix [4] arenediazonium salts to form versatile robust platforms for spatially controlled surface functionalization. Nat. Commun. 2012, 3, 1130.

54. Santos, L.; Mattiuzzi, A.; Jabin, I.; Vandencasteele, N.; Reniers, F.; Reinaud, O.; Hapiot, P.; Lhenry, S.; Leroux, Y.; Lagrost, C. One-Pot Electrografting of Mixed Monolayers with Controlled Composition. J. Phys. Chem. C 2014, 118 (29), 15919- 15928.

55. Anariba, F.; DuVall, S. H.; McCreery, R. L. Mono-and multilayer formation by diazonium reduction on carbon surfaces monitored with atomic force microscopy “scratching”. Anal. Chem. 2003, 75 (15), 3837-3844.

56. Leroux, Y. R.; Fei, H.; Noël, J.-M.; Roux, C.; Hapiot, P. Efficient Covalent Modification of a Carbon Surface: Use of a Silyl Protecting Group To Form an Active Monolayer. J. Am. Chem. Soc. 2010, 132 (40), 14039-14041.

57. Malmos, K.; Iruthayaraj, J.; Ogaki, R.; Kingshott, P.; Besenbacher, F.; Pedersen, S. U.; Daasbjerg, K. Grafting of Thin Organic Films by Electrooxidation of Arylhydrazines. J. Phys. Chem. C 2011, 115 (27), 13343-13352.

58. Malmos, K.; Iruthayaraj, J.; Pedersen, S. U.; Daasbjerg, K. General approach for monolayer formation of covalently attached aryl groups through electrografting of arylhydrazines. J. Am. Chem. Soc. 2009, 131 (39), 13926-13927.

59. Lee, L.; Gunby, N. R.; Crittenden, D. L.; Downard, A. J. Multifunctional and Stable Monolayers on Carbon: A Simple and Reliable Method for Backfilling Sparse Layers Grafted from Protected Aryldiazonium Ions. Langmuir 2016, 32 (11), 2626-2637.

60. Gallardo, I.; Pinson, J.; Vila, N. Spontaneous attachment of amines to carbon and metallic surfaces. J. Phys. Chem. B 2006, 110 (39), 19521-19529.

61. Liu, W.; Tilley, T. D. Sterically Controlled Functionalization of Carbon Surfaces with− C6H4CH2X (X= OSO2Me or N3) Groups for Surface Attachment of Redox- Active Molecules. Langmuir 2015, 31 (3), 1189-1195.

62. Leroux, Y. R.; Fei, H.; Noël, J.-M.; Roux, C.; Hapiot, P. Efficient covalent 39

Chapter 1 Introduction

modification of a carbon surface: use of a silyl protecting group to form an active monolayer. Journal of the American Chemical Society 2010, 132 (40), 14039-14041.

63. Lee, L.; Leroux, Y. R.; Hapiot, P.; Downard, A. J. Amine-Terminated Monolayers on Carbon: Preparation, Characterization, and Coupling Reactions. Langmuir 2015, 31 (18), 5071-5077.

64. Lee, L.; Ma, H.; Brooksby, P. A.; Brown, S. A.; Leroux, Y. R.; Hapiot, P.; Downard, A. J. Covalently anchored carboxyphenyl monolayer via aryldiazonium ion grafting: A well-defined reactive tether layer for on-surface chemistry. Langmuir 2014, 30 (24), 7104-7111.

65. Kocak, I.; Ghanem, M. A.; Al-Mayouf, A.; Alhoshan, M.; Bartlett, P. N. A study of the modification of glassy carbon and edge and basal plane highly oriented pyrolytic graphite electrodes modified with anthraquinone using diazonium coupling and solid phase synthesis and their use for oxygen reduction. J. Electroanal. Chem. 2013, 706, 25-32.

66. Celiktas, A.; Ghanem, M. A.; Bartlett, P. N. Modification of nanostructured gold surfaces with organic functional groups using electrochemical and solid-phase synthesis methodologies. J. Electroanal. Chem. 2012, 670, 42-49.

67. Hetemi, D.; Hazimeh, H.; Decorse, P.; Galtayries, A.; Combellas, C.; Kanoufi, F.; Pinson, J.; Podvorica, F. I. One-Step Formation of Bifunctionnal Aryl/Alkyl Grafted Films on Conducting Surfaces by the Reduction of Diazonium Salts in the Presence of Alkyl Iodides. Langmuir 2015, 31 (19), 5406-5415.

68. Combellas, C.; Jiang, D.-e.; Kanoufi, F.; Pinson, J.; Podvorica, F. I. Steric Effects in the Reaction of Aryl Radicals on Surfaces. Langmuir 2009, 25 (1), 286-293.

69. Greenwood, J.; Phan, T. H.; Fujita, Y.; Li, Z.; Ivasenko, O.; Vanderlinden, W.; Van Gorp, H.; Frederickx, W.; Lu, G.; Tahara, K.; Tobe, Y.; Uji-i, H.; Mertens, S. F. L.; De Feyter, S. Covalent Modification of Graphene and Graphite Using Diazonium Chemistry: Tunable Grafting and Nanomanipulation. ACS Nano 2015, 9 (5), 5520-5535.

70. Menanteau, T.; Levillain, E.; Breton, T. Electrografting via Diazonium Chemistry: From Multilayer to Monolayer Using Radical Scavenger. Chem. Mater. 2013, 25 (14), 2905-2909.

71. Menanteau, T.; Levillain, E.; Breton, T. Spontaneous grafting of nitrophenyl groups on carbon: effect of radical scavenger on organic layer formation. Langmuir 2014, 30 (26), 7913-7918.

Chapter 1 Introduction

72. Menanteau, T.; Levillain, E.; Downard, A.; Breton, T. Evidence of monolayer formation via diazonium grafting with a radical scavenger: electrochemical, AFM and XPS monitoring. Phys. Chem. Chem. Phys. 2015, 17 (19), 13137-13142.

73. González, M.; Orive, A.; Salvarezza, R.; Creus, A. Electrodeposition of gold nanoparticles on aryl diazonium monolayer functionalized HOPG surfaces. Phys. Chem. Chem. Phys. 2016, 18 (3), 1953-1960.

74. Nielsen, L. T.; Vase, K. H.; Dong, M.; Besenbacher, F.; Pedersen, S. U.; Daasbjerg, K. Electrochemical approach for constructing a monolayer of thiophenolates from grafted multilayers of diaryl disulfides. J. Am. Chem. Soc. 2007, 129 (7), 1888-1889.

75. Malmos, K.; Dong, M.; Pillai, S.; Kingshott, P.; Besenbacher, F.; Pedersen, S. U.; Daasbjerg, K. Using a hydrazone-protected benzenediazonium salt to introduce a near- monolayer of benzaldehyde on glassy carbon surfaces. J. Am. Chem. Soc. 2009, 131 (13), 4928-4936.

76. Peng, Z.; Holm, A. H.; Nielsen, L. T.; Pedersen, S. U.; Daasbjerg, K. Covalent Sidewall Functionalization of Carbon Nanotubes by a “Formation− Degradation” Approach. Chem. Mater. 2008, 20 (19), 6068-6075.

77. Antonello, S.; Daasbjerg, K.; Jensen, H.; Taddei, F.; Maran, F. Formation and Cleavage of Aromatic Disulfide Radical Anions. J. Am. Chem. Soc. 2003, 125 (48), 14905-14916.

78. Shao, Q.; Jiang, S. Influence of charged groups on the properties of zwitterionic moieties: a molecular simulation study. J. Phys. Chem. B 2014, 118 (27), 7630-7637.

79. Chen, S.; Zheng, J.; Li, L.; Jiang, S. Strong resistance of phosphorylcholine self- assembled monolayers to protein adsorption: insights into nonfouling properties of zwitterionic materials. J. Am. Chem. Soc. 2005, 127 (41), 14473-14478.

80. Parviz, M.; Darwish, N.; Alam, M. T.; Parker, S. G.; Ciampi, S.; Gooding, J. J. Investigation of the Antifouling Properties of Phenyl Phosphorylcholine ‐ Based Modified Gold Surfaces. Electroanal 2014, 26 (7), 1471-1480.

81. Barfidokht, A.; Gooding, J. J. Approaches toward allowing electroanalytical devices to be used in biological fluids. Electroanal 2014, 26 (6), 1182-1196.

82. Liu, G.; Gooding, J. J. An interface comprising molecular wires and poly (ethylene glycol) spacer units self-assembled on carbon electrodes for studies of protein electrochemistry. Langmuir 2006, 22 (17), 7421-7430.

83. Liu, G.; Paddon-Row, M. N.; Gooding, J. J. A molecular wire modified glassy 41

Chapter 1 Introduction

carbon electrode for achieving direct electron transfer to native glucose oxidase. Electrochem. Commun. 2007, 9 (9), 2218-2223.

84. Arias de Fuentes, O.; Ferri, T.; Frasconi, M.; Paolini, V.; Santucci, R. Highly‐ Ordered Covalent Anchoring of Carbon Nanotubes on Electrode Surfaces by Diazonium Salt Reactions. Angew. Chem. Int. Ed. 2011, 123 (15), 3519-3523.

85. Guo, W.; Jiang, F.; Chu, J.; Song, D.; Liu, G. A stable interface based on aryl diazonium salts/SWNTs modified gold electrodes for sensitive detection of hydrogen peroxide. J. Electroanal. Chem. 2013, 703, 63-69.

86. Liu, G.; Guo, W.; Yin, Z. Covalent fabrication of methyl parathion hydrolase on gold nanoparticles modified carbon substrates for designing a methyl parathion biosensor. Biosens. Bioelectron. 2014, 53, 440-446.

87. Liu, G.; Wang, S.; Liu, J.; Song, D. An electrochemical immunosensor based on chemical assembly of vertically aligned carbon nanotubes on carbon substrates for direct detection of the pesticide endosulfan in environmental water. Anal. Chem. 2012, 84 (9), 3921-3928.

88. Liu, G.; Song, D.; Chen, F. Towards the fabrication of a label-free amperometric immunosensor using SWNTs for direct detection of paraoxon. Talanta 2013, 104, 103- 108.

89. Liu, G.; Zhang, Y.; Qi, M.; Chen, F. Covalent anchoring of multifunctionized gold nanoparticles on electrodes towards an electrochemical sensor for the detection of cadmium ions. Anal. Method 2015, 7 (13), 5619-5626.

90. Kuo, T.-M.; Shen, M.-Y.; Huang, S.-Y.; Li, Y.-K.; Chuang, M.-C. Facile Fabrication of a Sensor with a Bifunctional Interface for Logic Analysis of the New Delhi Metallo-β-Lactamase (NDM)-Coding Gene. ACS Sensors 2016, 1 (2), 124-130.

91. Hayat, A.; Sassolas, A.; Marty, J.-L.; Radi, A.-E. Highly sensitive ochratoxin A impedimetric aptasensor based on the immobilization of azido-aptamer onto electrografted binary film via click chemistry. Talanta 2013, 103, 14-19.

92. Gooding, J. J.; Wibowo, R.; Liu, J.; Yang, W.; Losic, D.; Orbons, S.; Mearns, F. J.; Shapter, J. G.; Hibbert, D. B. Protein electrochemistry using aligned carbon nanotube arrays. J. Am. Chem. Soc. 2003, 125 (30), 9006-9007.

93. Khoo, M. M.; Rahim, Z.; Darwish, N. T.; Alias, Y.; Khor, S. M. Non-invasive control of protein-surface interactions for repeated electrochemical immunosensor use. Sens. Actuator B: Chem. 2016, 224, 683-691.

Chapter 1 Introduction

94. Khor, S. M.; Liu, G.; Fairman, C.; Iyengar, S. G.; Gooding, J. J. The importance of interfacial design for the sensitivity of a label-free electrochemical immuno-biosensor for small organic molecules. Biosens. Bioelectron. 2011, 26 (5), 2038-2044.

95. Gooding, J.; Liu, G. Electrochemical competition sensor. Google Patents, 2014.

96. Narayanan, J.; Sharma, M. K.; Ponmariappan, S.; Shaik, M.; Upadhyay, S. Electrochemical immunosensor for botulinum neurotoxin type-E using covalently ordered graphene nanosheets modified electrodes and gold nanoparticles-enzyme conjugate. Biosens. Bioelectron. 2015, 69, 249-256.

97. Lei, J.; Ju, H. Signal amplification using functional nanomaterials for biosensing. Chem. Soc. Rev. 2012, 41 (6), 2122-2134.

98. Polsky, R.; Harper, J. C.; Wheeler, D. R.; Arango, D. C.; Brozik, S. M. Electrically addressable cell immobilization using phenylboronic acid diazonium salts. Angew. Chem. Int. Ed. 2008, 47 (14), 2631-2634.

99. Guo, K.; Freguia, S.; Dennis, P. G.; Chen, X.; Donose, B. C.; Keller, J.; Gooding, J. J.; Rabaey, K. Effects of Surface Charge and Hydrophobicity on Anodic Biofilm Formation, Community Composition, and Current Generation in Bioelectrochemical Systems. Environ. Sci. Technol. 2013, 47 (13), 7563-7570.

100. Gam-Derouich, S.; Carbonnier, B.; Turmine, M.; Lang, P.; Jouini, M.; Ben Hassen-Chehimi, D.; M. Chehimi, M. Electrografted aryl diazonium initiators for surface-confined photopolymerization: a new approach to designing functional polymer coatings. Langmuir 2010, 26 (14), 11830-11840.

Chapter 2 Methods and Materials

In this chapter, general materials, methods, and instruments used throughout this dissertation are presented, further experimental details are given in the relevant experimental section of each chapter.

2.1 Materials

2.1.1 Chemicals

All chemicals are used as received, unless otherwise specified. Milli-Q water with a resistivity of 18 MΩ cm is obtained from Millipore™ water purification system for aqueous solution preparation. The general chemicals and solutions used in this thesis are summarized in Table 2.1 below.

Table 2.1 General chemicals, and solutions

Name Specification Company dichloromethane (DCM) Analytical grade Ajax Finechem,

Australia

Chapter 2 Methods and Materials

methanol Analytical grade Ajax Finechem,

Australia

potassium carbonate (K2CO3) Analytical grade Ajax Finechem,

Australia

sodium nitrite (NaNO2) Analytical grade Ajax Finechem,

Australia potassium chloride (KCl) Analytical grade Ajax Finechem,

Australia sodium chloride (NaCl) Analytical grade Ajax Finechem,

Australia hydrochloric acid 32 % Ajax Finechem,

Australia

hydrogen peroxide (H2O2) 30 % Ajax Finechem,

Australia potassium dihydrogen phosphate ≥ 99% Sigma, Australia

(KH2PO4)

sodium hydrogen phosphate (Na2HPO4) ≥ 99% Sigma, Australia

N,N-dimethylformamide (DMF) Analytical grade Ajax Finechem,

Australia

4-amino phenyl phosphorylcholine (PPC) ≥ 97% Toronto Research

Chemicals,

Chapter 2 Methods and Materials

Canada

4-(4-aminophenyl) butyric acid (PBA) ≥ 98% Sigma, Australia human serum albumin (HSA) ≥ 97% Sigma, Australia bovine serum albumin (BSA) ≥ 97% Sigma, Australia

1-ethyl-3-(3-dimethylaminopropyl) > 98% Sigma, Australia carbodiimide hydrochloride (EDC)

N-hydroxysuccinimide (NHS) 98% Sigma, Australia ferrocenemethanol 97 % Sigma, Australia hemoglobin (Hb) lyophilized powder Sigma, Australia

TNF-α full length protein (ab9642) > 98 %, lyophilized Abcam, Australia

powder anti-TNF α antibody (Ab1, ab8348) monoclonal, Abcam, Australia

protein A purified horseradish peroxidase conjugated anti- monoclonal, Abcam, Australia

TNF α antibody (HRP-Ab2, ab24473) protein A purified whole blood samples (anticoagulated Innovative

with K2 EDTA) Research, USA

Phosphate-buffered saline (PBS) containing 137 mM NaCl, 2.7 mM KCl, 2 mM

KH2PO4 and 10 mM Na2HPO4 with pH 7.4 was used to dissolve biomolecules (protein, antibodies, etc)

Chapter 2 Methods and Materials

2.1.2 ITO Coated Glass Slides

ITO coated glass slides (15-30 ohm/sq, 18×18 mm, 06480-AB) were obtained from SPI

(West Chester, USA). ITO coated glass slides (8-12 ohm/sq) were obtained from Delta- technologies (Loveland, USA)

2.2 Organic Synthesis

1-Propylamino-1,2,3-triazole-4-ferrocene was synthesized using a “click” reaction with ethynylferrocene and 3-azido-1-propylamine as described previously by Gooding group.1

Figure 2.1 Synthesis of 1-propylamino-1,2,3-triazole-4-ferrocene via “click” reaction of ethynylferrocene and 3-azido-1-propylamine

2.3 ITO Cleaning

Prior to surface modification, ITO electrodes were cleaned by ultrasonication successively with dichloromethane (DCM, 10 min), methanol (10 min), 0.5 M K2CO3 in methanol: H2O (2:1 volume ratio, 30 min), followed by rinsing with copious amount

Chapter 2 Methods and Materials

of Milli-Q water and were dried under a stream of nitrogen.2

2.4 X-ray Photoelectron Spectroscopy (XPS) Characterization

XPS spectra were obtained using an EscaLab220-IXL spectrometer equipped with a monochromated Al Ka source (1486.6 eV), hemispherical analyzer and multichannel detector. The spectra were accumulated at a take-off angle of 90 ° with a 0.79 mm2 spot size at a pressure of less than 1×10−8 mbar. Binding energies of elements were corrected with reference to graphite C1s (284.6 eV). The XPS spectrum was analyzed with the

Avantage software, involving background subtraction using the Shirley routine and a subsequent nonlinear least-squares fitting to mixed Gaussian-Lorentzian functions. The atomic concentration (atom. %) of individual element was determined from the relative peak area of the spectrum and the corresponding sensitivity factors according to equation 1:

퐴 ⁄푠 atom % = 푖 푖 equation 1 ∑퐴푖⁄푠푖

where Ai is the area of the element i, and si is the sensitivity factor for this element. The sensitivity factors for P 2p, O 1s, C 1s and N 1s are 1.19, 2.93, 1.00, and 1.80, respectively. The thickness of the deposited layer on ITO is estimated from the relative attenuation of In 3d signal, using:

In (I/I0) = (-d/λ) × sin (θ) equation 2

Chapter 2 Methods and Materials

where d is the layer thickness, λ is the photoelectron escape depth of In 3d which was

3 estimated to be 3.5 nm, θ is the takeoff angle (90 ° used in the experiment), and I/I0 is the ratio of the In 3d peak intensities (I (modified surface)/I (bare surface)).

2.5 Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) Instrumentation

Cyclic voltammetry measurements were performed with Autolab potentiostat

(Metrohm Autolab B.V., Netherlands) and EIS experiments were conducted using

Solartron SI 1287 electrochemical interface coupled with an SI 1260 frequency response analyser (Solartron Analytical, Hampshire, England). All electrochemical experiments were carried out in a 2.0 mL one-compartment custom made electrochemical cell compromising of a conventional three-electrode system, in which an O-ring was placed on the substrate to prevent solution leakage and to limit the contact area to a circle with surface area of 0.24 cm2. Electrochemical experiments were performed using ITO coated glass as working electrode, an Ag/AgCl (3.0 KCl) as reference electrode, and a platinum wire as counter electrode.

The total surface coverage (Γ) of PPC was derived from the integration of the area under the voltammetric peaks according to the following equation.4

Γ = Q/nFA equation 3

Here, n, F, Q and A represent the number of electrons, Faraday constant, charge, and surface area of the ITO electrode, respectively. Background current was estimated by extrapolation of the baseline capacitive current under the Faradaic peak current.

Chapter 2 Methods and Materials

2.6 Electrochemical Evaluation of Protein-resistance Behavior

EIS to monitor the behavior of PPC-ITO in resisting nonspecific adsorption of protein was performed in a solution of 0.1 M KCl containing 1 mM K3[Fe(CN)6]and 1 mM

K4[Fe(CN)6]. After PBS (pH 7.4) or HSA incubation (1 mg/mL HSA in PBS), the electrodes were rinsed thoroughly with PBS and Milli-Q water, followed by EIS measurement in a fresh solution of 0.1 M KCl containing 1 mM K3[Fe(CN)6] and 1 mM

5 −1 K4[Fe(CN)6]. EIS spectra were recorded in the frequency range of 10 to 10 Hz. An

AC potential with 0.01 V peak to peak separation was superimposed on a DC potential of 0.212 V. Impedance data were recorded and analysed using ZPlot and ZView 3.1 softwares (Scribner Associates, Inc.), respectively.

2.7 References

1. Ciampi, S.; James, M.; Michaels, P.; Gooding, J. J. Tandem “click” reactions at acetylene-terminated Si (100) monolayers. Langmuir 2011, 27 (11), 6940-6949.

2. Chockalingam, M.; Darwish, N.; Le Saux, G.; Gooding, J. J. Importance of the indium tin oxide substrate on the quality of self-assembled monolayers formed from organophosphonic acids. Langmuir 2011, 27 (6), 2545-2552.

3. Seah, M.; Dench, W. Quantitative electron spectroscopy of surfaces: a standard data base for electron inelastic mean free paths in solids. Surf. Interface Anal. 1979, 1 (1), 2-11.

4. Louault, C.; D'Amours, M.; Belanger, D. The electrochemical grafting of a mixture of substituted phenyl groups at a glassy carbon electrode surface. ChemPhysChem 2008, 9 (8), 1164-1170.

Chapter 3 Antifouling Behavior of PPC on ITO

Chapter 3 Zwitterionic Phenyl Phosphorylcholine on Indium Tin Oxide: a Low-impedance Protein-resistant Platform for Biosensing

Publication II

Jiang, C.; Alam, M. T.; Parker, S. G.; Gooding, J. J.* Zwitterionic Phenyl Phosphorylcholine on Indium Tin Oxide: a Low-impedance Protein-resistant Platform for Biosensing. Electroanal 2015, 27 (4), 884-889.

Reproduced with permission. Copyright [2015] ELSEVIER

Declaration

I certify that this publication was a direct result of my research towards this PhD, and that reproduction in this thesis does not breach copyright regulations.

51 Special Full Paper Issue DOI:10.1002/elan.201400557

Zwitterionic Phenyl Phosphorylcholine on Indium Tin R. Oxide:aLow-Impedance Protein-Resistant Platform for G. Biosensing Comptons

Cheng Jiang,[a] Muhammad Tanzirul Alam,[a] Stephen G. Parker,[a] and J. Justin Gooding*[a]

Dedicated to Professor R. G. Compton on the Occasion of his 60th Birthday 60 th Abstract:Inthis study we have modified indium tin oxide unstable.Wehave developed asimple protocol to remove (ITO) by electrografting phenylphosphorylcholine (PPC) the physically adsorbed PPC such that astable interface and evaluated its protein-resistance performance using is produced. It was found that such astable surface,with electrochemical impedance spectroscopy.During electro- appropriate amount of PPC,can dramatically reduce non- grafting, significant amounts of PPC gets physically ad- specific adsorption of protein, giving us aplatform of per- sorbed on the surface,which cannot be removed by forming both electrochemical and spectroscopic studies in simply rinsing with water or buffer, and as aconsequence biological fluids. the electrochemical response of such electrode is quite Keywords: Aryl diazonium salts · Antifouling zwitterionic coating · Impedance spectroscopy · Indium tin oxide · Phenylphosphorylcholine

1Introduction containing phosphorylcholine moiety [13,14].Recently, our group has studied the modification of glassy carbon Electrochemical biosensors are of enormous interest in (GC) electrodes via the electrografting of the aryl diazo- avariety fields including clinical diagnosis and food anal- nium salts,phenylphophorylcholine (PPC) diazonium, to ysis [1–3].However,inbiological media, the nonspecific give an anti-biofoulinglayer that was of sufficiently low adsorption of proteins,referred to as biofouling,inter- impedance that Faradaic electrochemistry could still feres with the performance of such devices in terms of re- occur at the underlying electrode [15].Similar layers on ducing sensitivity and selectivity [4, 5].Hence,protein-re- gold electrodes were shown to be poor at resisting bio- sistant coatings are badly needed for electrochemical sen- fouling [16] which was attributed to alower density of sors to deliver on their potential when it comes to biolog- PPC molecules on the gold surface and the loss of charge ically derived samples [6].The most common solutions to neutrality of the resultant layer. Charge neutrality is the critical issue of biofouling include using poly(ethy- anecessary prerequisite for zwitterionic layers to resist leneglycol) (PEG) or oligo(ethyleneglycol) (OEG)-alka- biofouling [17,18].Similarly,variations in behavior of nethiol layers.Itisbelieved that ahighly compressed hy- agiven aryl diazonium salt derived layer formed on dration layer that forms when these surfaces are exposed metal, semiconductor,and GC electrodes have also been to biological media contributes to their capability in pre- reported in previous studies [19–22].For example,on venting nonspecific adsorption of protein [7,8].However, indium tin oxide (ITO), the electrode material of interest the use of such long chain self-assembled monolayers in this article,electrografting of formylbenzene diazonium (SAMs) or polymeric layers on electrodes is not desirable proceeds with significant physical adsorption of diazoni- because such polymers form ahigh impedance layer on um cations onto ITO surface,with resultant instability in the electrode,which prevents Faradaic electrochemistry the layer [23]. from occurring at the underlying electrode.Toallow elec- Since it is not known how PPC layers on ITOwill trodes to be modified with antibiofouling layers and yet behave as an anti-biofouling layer in electrochemical bio- allow Faradaic electrochemistry to proceed, our group has incorporated molecular wires [9–11] or gold nanoparticles [12] into the modifying layer as conducting channels [a] C. Jiang,M.Tanzirul Alam, S. G. Parker, J. J. Gooding through the OEG layers.However, synthesis of molecular School of Chemistry,Australian Centre for NanoMedicine and ARC Centre of Excellence in Convergent Bio-Nano wires and gold nanoparticles adds complexity to the fabri- Science and Technology,The University of New South Wales cation of the biosensor, and antifouling coatings that do Sydney,NSW 2052, Australia not passivate the electrode are desired. *e-mail:[email protected] An alternative to OEG and PEG that has recently Supporting Information for this article is available on the begun to be actively explored is zwitterionic molecules WWW under http://dx.doi.org/10.1002/elan.201400557 www.electroanalysis.wiley-vch.de 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Electroanalysis 2015,27, 884–889 884 Special Full Paper Issue sensors,the purpose of this paper is to evaluate the per- K2CO3 in 2:1(volume ratio) of methanol:H2O(30 min), formance of PPC layers on ITO electrodes.Inthis regard, followed by rinsing with copious amount of Milli-Q water PPC was electrografted on ITOfrom the in situ genera- and were dried under astream of nitrogen. Theelectro- tion of aryl diazonium cations.Electrochemical impe- des were then modified by cycling potential between 0.1 R. À dance spectroscopy (EIS) was applied to evaluate non- and 0.65 Vat0.1 V/s in a0.1 MHCl solution containing G. specific protein adsorption based on the impedance 1mMPÀ PC and equivalent amount of NaNO .

2 Comptons changes on these surfaces. 2.4 Electrochemical Evaluation of Protein-Resistance 2Experimental Methods Behavior

2.1 Reagents and Materials Theuse of EIS to monitor the behavior of PPC-ITOin

resisting nonspecific adsorption of protein was performed 60

Dichloromethane,methanol, K2CO3,NaNO2,KCl, NaCl, in an solution of 0.1 MKCl containing 1mMK3[Fe(CN)6] th HCl (32 %), KH2PO4 and Na2HPO4 were purchased from and 1mMK4[Fe(CN)6]. After PBS (pH 7.4) or HSA in- Ajax Finecham (Sydney,Australia). 4-amino phenyl phos- cubation (1 mg/mL HSA in PBS), the electrodes were phorylcholine was obtained from Toronto Research rinsed thoroughly with PBS and Milli-Q water, followed Chemicals (Toronto,Canada). Human serum albumin by EIS measurements in afresh solution of 0.1 MKCl

(HSA) was acquired from Sigma (Sydney,Australia). containing 1mMK3[Fe(CN)6]and 1mMK4[Fe(CN)6]. ITO-coated glass slides (15–30 W,6480-AB) were ob- EIS spectra were recorded in the frequency range of 105 1 tained from SPI (West Chester, USA). All chemicals to 10À Hz. An AC potential with 0.01 Vpeak to peak were used as received unless otherwise stated, and aque- separation was superimposed on aDCpotential of ous solutions were prepared using Milli-Q water 0.212 V. Impedance data were recorded and analyzed (18 MWcm, Millipore,Sydney,Australia). using ZPlot and ZView 3.1 softwares (Scribner Associ- ates,Inc.), respectively. 2.2 Cyclic Voltammetry and Electrochemical Impedance Spectroscopy Instrumentation 2.5 X-Ray Photoelectron Spectroscopy Characterization Cyclic voltammetry (CV) measurements were performed XPS spectra were obtained using an EscaLab220-IXL with Autolab PGSTAT 12 potentiostat (Metrohm Auto- spectrometer equipped with amonochromated Al Ka lab B. V. ,Netherlands) and EIS experiments were con- source (1486.6 eV), hemispherical analyzer and multi- ducted using Solartron SI 1287 electrochemical interface channel detector.The spectra were acquired at atake-off coupled with an SI 1260 frequencyresponse analyzer (So- angle of 908 with a0.79 mm2 spot size at apressure of 8 lartron Analytical, Hampshire,England). All electro- less than 110À mbar.Binding energies of elements was chemical experiments were carried out in a2.0 mL one- corrected with reference to graphite carbon C1s (284.6 compartment custom made electrochemical cell compris- eV). TheXPS spectrum was analyzed with the Avantage ing of aconventional three-electrode system, in which an software,involving background subtraction using Shirley O-ring was placed on the substrate to prevent solution routine and asubsequent nonlinear least-squares fitting leakage and to limit the contact area to acircle with sur- to mixed Gaussian Lorentzian functions.The atomic con- face area of 0.24 cm2.Electrochemical experiments were centration (at. %)Àof individual element was determined performed using ITO-coated glass as the working elec- from the relative peak area of the spectrum and the cor- trode,anAg/AgCl (3.0 KCl) as the reference electrode, responding sensitivity factors according to Equation 2: and aplatinum wire as the counter electrode. Thetotal surface coverage (G)ofPPC was derived at: % A=s = A=s 2 from the integration of the area under the voltammetric ¼ð i iÞ ð i iÞðÞ peaks according to the following equation. X

where Ai is the area of theelement i,and si is the sensitivity G Q=nFA 1 ¼ ð Þ factor for this element. Thesensitivity factors forP2p,O 1s,C1s and N1sare 1.19, 2.93,1.00, and1.80,respectively. Here, n, F, Q and A represent the number of electrons, Thethickness of the deposited layer on ITOwas estimat- Faraday constant, charge,and surface area of the ITO ed from therelative attenuation of In 3d signal,using [24]: electrode,respectively.Background current was estimated by extrapolation of the baseline capacitive current under In I=I0 d=l sin q 3 the Faradaic peak current. ð Þ¼ðÀ ÞÂ ð ÞðÞ where d is the layer thickness, l is the photoelectron 2.3 Modification of ITOElectrode Surface escape depth of In 3d and was estimated to be 3.5 nm, q is the takeoff angle (908 was used in our experiment), and ITOelectrodes were sonicated successively with dichloro- I/I0 is the ratio of the In 3d peak intensities (modified sur- methane (DCM, 10 min), methanol (10 min), 0.5 M face/bare surface). www.electroanalysis.wiley-vch.de 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Electroanalysis 2015,27, 884–889 885 Special Full Paper

3Results and Discussion those at gold and glassy carbon prepared from 4-nitro- Issue phenyldiazonium and PPC diazonium cations,respectively [15,25],suggesting aless efficient capture of generated

3.1 Electrografting of PPC on ITOSurface R. radicals and hence grafting reaction.

ITOwas modified with layers formed via the in situ con- G. version of the amino moiety of 4-aminophenyl phosphor- 3.2 Electrochemical Impedance Spectroscopy and XPS Comptons ylcholine to the aryl diazonium species followed by elec- Characterization of the PPC Modified ITO trochemical grafting onto ITO. Cyclic voltammogram (Figure 1) shows awell-defined EIS was used to evaluatethe protein-resistant behavior irreversible peak at 0.55 Vduring the first cycle,indicat- of PPC-ITO surface based on changes in charge transfer À ing the reduction of aryl diazonium cations at the elec- resistance (Rct)induced by interaction between protein

trode surface,followed by gradually diminished currents and electrode surface.After incubating in aprotein solu- 60 in subsequent cycles.Adecay of the reductive adsorption tion, Rct of an excellent protein resistant surface should current is observed with each subsequent scan due to the not change significantly,while for asurface without such th grafting of ablocking layer which severely inhibits further protein resistance, Rct is expected to increase.The expect- electron transfer. It is worth mentioning that the decrease ed increase in Rct is because the access of redox species of current in this case is less pronounced compared to to the electrode surface is hindered due to the adsorption of protein on the surface. In this study,asshown in Figure 2(A),itwas observed 2 that Rct forthe bare ITOelectrode(77 W cm , s=1.6) was low, butafter exposure to proteinsolution(1mg/mLHSA 2 in PBS) for1hour, Rct increasedto686 W cm (s=11), indicating adsorption of proteinonthe unmodified surface, i.e.,without protein-resistance coating. Theimpedance experimental results were consistent with theCVresults (FigureS1SupportingInformation). Figure 2B showsthe

Rct changesfor thePPC modified ITO, prepared using20 CV cycles,beforeand afterincubationinaproteinsolu- tion.Electrodeposited PPCfilmonthe electrodesurface generatedabarriertointerfacial electron transfer conse- 2 quently Rct increasedto703 W cm (s=13.5). Afterincu- bating thesamesurface in HSAsolutionfor 1h,unexpect- 2 edly,the Rct decreasedto523 W cm (s=11)(about25% decrease compared to PPC-ITO surface).Thisdecreasein Fig. 1. Cyclic voltammograms for the in situ electrodeposition Rct is in sharpcontrasttothe responsesobservedatPPC- of PPC diazonium cations on ITO in 0.1 MHCl containing GC wherenosignificant change in Rct wasobservedat

1mMPPC and 1mMNaNO2 at ascan rate of 0.1 V/s. PPC-GCbeforeand afterincubation in protein[15].

Fig. 2. Nyquist plots measured at (A) bare ITO and (B) PPC modified ITO (20 cycles modified) before and after incubation in aprotein solution (1 mg/mL HSA in PBS) for 1h.Experiments were measured in asolution of 0.1 MKCl containing 1mMK3[Fe(CN)6] and 1mMK4[Fe(CN)6]. www.electroanalysis.wiley-vch.de 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Electroanalysis 2015,27, 884–889 886 Special Full Paper

Thedecrease in charge transfer resistance upon expo- Issue sure to HSA implies that the interface is becoming more accessible to redox species in solution rather than less as expected if there was non-specific desorption of protein. R.

We identified three possible reasons for the decrease in G. R after protein incubation. These are (1) the protein ad-

ct Comptons sorption resulted in electrostatic attraction of the redox species to interface,thus giving an accumulation effect, (2) the surface of the ITObeing etched by the ferricyanide used in the EIS experiments thus causing loss of PPC molecules from the electrode surface [26] or (3) de-

sorption of physically adsorbed aryl diazonium salts from 60 the surface. Thefirst possibility can be discounted as,atpH7.4, the th phosphorylcholine moiety of PPC is neutral, while HSA 3 /4 (pI 4.7) and [Fe(CN)6] À À are negatively charged [27], and hence any adsorbed anionic HSA molecules would repel the redox species,consequently Rct should increase. Regarding the second possibility,that ferricyanide etches the ITO and removed part of the modifying layer, this hypothesis was related to the fact that Whitesides and co- workers [26] have reported that ferricyanide can be used to etch gold surfaces.Toascertain whether this hypothesis was viable,experiments were conducted where the PPC- ITOsurfaces were incubated in asolution of 0.1 MKCl containing 1mMK3[Fe(CN)6]and 1mMK4[Fe(CN)6]for 1h,followed by performing the Faradaic EIS measurements.Itwas found that Rct remained stable before and after incubation in ferricyanide solution (data not shown). So the ITO film is not damaged by ferricyanide solution. Concerning the third possibility,ithas been proposed previously that diazonium cations are more inclined to physically adsorb on negatively charged ITOsurfaces during the electro-grafting process compared to other surfaces such as carbon [23,28].Sothe surface could be comprised of both grafted and adsorbed molecules.The decrease in

Rct could therefore be aresult of some adsorbed species (diazonium cations or benzene amines) desorbing from the surface during incubation, giving rise to more pinholes and consequent reduction in charge transfer resistance [28]. To investigate the third hypothesis mentioned above, another control experiment was conducted by incubating the modified surface in PBS instead of HSA solution. As shown in Figure 3(A), Rct decreased even after 1hincu- 2 bation in PBS (421 W cm , s=15), however, Rct became steady after incubating for 2h(after 2h,325 Wcm2, s= 2 21;after 6h,322 W cm , s=4), indicating stable PPC-ITO surfaces can be achieved by incubating in PBS.Asimilar Fig. 3. (A) Nyquist plots measured in asolution of 0.1 MKCl observation was reported by Kim and co-workers in their containing 1mMK3[Fe(CN)6]and 1mMK4[Fe(CN)6]show that study [23],where formylbenzene diazonium (FBD) salt a“stabilized ”PPC-ITO surface can be realized by incubating in was electrografted onto ITO and EIS was used for moni- PBS.XPS core-level spectra of (B) N1sand (C) P2pfor PPC- toring interfacial electron transfer. ITO surfaces (20 cycles modified) with and without incubating in Figure 3B shows XPS characterization of PPC-ITOsur- PBS for 2h. faces with and without PBS incubation. In the N1s narrow scans of PPC-ITO surface,the spectra were fitted ammonium nitrogen (i.e., N (CH ) )ofphosphorylcho- À À 3 3 with two peaks centered at ~403.5 and ~399 eV.The line group,the latter one is often attributed to formation former nitrogen peak is indicative of the presence of the of azo groups (CN=NC) which has been discussed in the www.electroanalysis.wiley-vch.de 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Electroanalysis 2015,27, 884–889 887 Special Full Paper Issue R. G. Comptons 60 th

Fig. 4. (A) Thevariation in charge transfer resistance versus the number of CV cycles used to form PPC modified surfaces before and after HSA incubation and corresponding (B) Nyquist plots recorded at 20 cycles modified PPC-ITOwhere Rct remained unchanged after exposure of a“stabilized” surface (20 cycles modified) to 1mg/mL HSA in PBS for up to 6hours.All the EIS measurements were done at stabilized surfaces (obtained after incubating in PBS for 2h)inasolution of 0.1 MKCl containing 1mM

K3[Fe(CN)6]and 1mMK4[Fe(CN)6]. literatures as aconsequence of deposition of aryl diazoni- HSA, even after prolonged incubation in HSA (before 2 um salts on different electrode surfaces [29,30].Herein, HSA incubation Rct =325 Wcm , s=21;after HSA incu- 2 the XPS intensity of both Pand Nelements (characteris- bation for 1h, Rct =321 W cm , s=1.5;for 6h, Rct = tic elements from the head group of PPC were selected 323 Wcm2, s=2.5, shown in Figure 4B). TheEIS results as XPS markers) decreased after incubating in PBS for were consistent with cyclic voltammograms (see Figure 2h.Atthe same time,the intensity of indium and tin in- S3 Supporting Information), implying that PPC can form creased considerably (Figure S4 Supporting Information). an excellent antifouling layer on ITO surfaces to resist Taken together, the decrease in the levels of Pand Nand protein adsorption whilst maintaining the impedance to the increase in In and Sn indicate that incubation in PBS arelatively low level. removes weakly adsorbed species,giving rise to amore Although the mechanism of how zwitterionic PPC open electrode interface and adecrease in Rct.The film functions as antifouling materials has not been thoroughly thickness of the surfaces was also estimated based on the studied, it is believed that surface hydration (i.e., water In 3d signal attenuation using Equation 3. Before PBS barrier), chain flexibility (i.e., steric repulsion), and pack- treatment, the thickness was estimated to be 3.6 nm ing density can contribute to surface resistance to non- which corresponds to 4–5 layers of PPC on ITO (in this specific protein adsorption [31].Since PPC used in this calculation it is assumed that the length of PPC standing study is asmall molecule containing abenzene ring and normal to the surface is 0.78 nm as determined using the phosphorylcholine group,its structure is not flexible like Chem3D software). Theestimated film thickness de- PEG SAMs or phosphorylcholine based polymer brushes. creased to about 2.5 nm after incubation in PBS for 2h, Turning attention to the density of molecules on the sur- further implying that weakly adsorbed diazonium cations face,Jiang and co-workers have shown that the charge in- of PPC desorb from the surface. teraction of phosphorylcholine groups is the dominant factor for the packing density using both experimental 3.3 Comparison of Antifouling BehaviorofPPC measurements and molecular simulations [18].Inthis Modified ITOSurfaces study,3to 12 cycles of deposited PPC could not offer agood antifouling property,but by 20 cycles of PPC dep- Since a“stabilized” surface can be achieved after incubat- osition, the surfaces showed excellent protein resistance. ing in PBS,such an approach was also applied to all sur- These results suggest that the PPC layer on ITOisalow faces used henceforth for evaluating their ability to resist density layer (will be discussed below) in the first few non-specific of proteins (1 mg/mL HSA in PBS). cycles whereupon it is of sufficient thickness and/or densi- As shown in Figure 4(A), for PPC modified ITOsurfa- ty to give excellentantifouling characteristics to the sur- ces formed using 3, 6, and 12 CV cycles,values of Rct in- face.Regarding the charge of the layer, previous studies creased after protein incubation compared to PBS “stabi- with zwitterionic layers shows that complete charge neu- lized” surfaces,indicating that the PPC layer is only par- trality is vital for these layers to resist non-specific pro- tially effective in resisting protein adsorption. However, tein adsorption. Here the N/P ratio in the XPS data re- once 20 cycles were used to form the “stabilized” surface, mains close to the theoretical value 1:1, suggesting that

Rct remained constant compared to prior to exposure to the PPC layers is charge balanced. Moreover, Jiang and www.electroanalysis.wiley-vch.de 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Electroanalysis 2015,27, 884–889 888 Special Full Paper co-workers showed that the packing density and charge References Issue are interrelated. So we conclude that for the protein resistant with short chain, low impedance zwitterionic [1] E. Bakker, Anal. Chem. 2004, 76,3285–3298.

[2] A. M. J. Haque,H.Park, D. Sung,S.Jon, S. Y. Choi, K. R. layers,the high density and charge neutrality play vital Kim, Anal. Chem. 2012, 84,1871–1878. roles in resisting protein. [3] M. Vestergaard, K. Kerman, E. Tamiya, Sensors 2007, 7, G. As mentioned above,itwas suggested that the grafting 3442–3458. Comptons efficiency of PPC on ITOisnot particularly high, based [4] I. Banerjee,R.C.Pangule,R.S.Kane, Adv.Mater. 2011, 23, on the fact that the CVs recorded during grafting contin- 690–718. ued to show asignificant Faradaic process even after sev- [5] S. Vaddiraju, I. Tomazos,D.J.Burgess,F.C.Jain, F. Papadi- eral cycles whereas on glassy carbon the electrodes were mitrakopoulos, Biosens.Bioelectron. 2010, 25,1553–1565. nd [6] A. Barfidokht, J. J. Gooding, Electroanalysis 2014, 26,1182– passivated by the 2 cycle [15].Exploring this further, 1196. acomparison was made between an estimation of the rel- [7] S. Sharma, R. W. Johnson, T. A. Desai, Biosens.Bioelectron. 60 ative grafting efficiency by XPS and cyclic voltammetry. 2004, 20,227–239. CV gives the total amount of generated radicals as 46.7 [8] E. Ostuni, R. G. Chapman, R. E. Holmlin, S. Takayama, th 10 2 10À mol/cm (estimated by integration of the cyclic vol- G. M. Whitesides, Langmuir 2001, 17,5605–5620. tammetry wave using equation 1), which is obviously an [9] G. Liu, M. N. Paddon-Row,J.J.Gooding, Chem. Commun. 2008,3870–3872. overestimate since not all generated radicals will react [10] G. Liu, J. J. Gooding, Langmuir 2006, 22,7421–7430. with the electrode surface.Using XPS,the coverage was [11] S. M. Khor, G. Liu, C. Fairman, S. G. Iyengar, J. J. Gooding, estimated by assuming that each molecule occupies Biosens.Bioelectron. 2011, 26,2038–2044. 0.18 nm2 based on the size of phosphorylcholine moiety [12] G. Liu, S. G. Iyengar, J. J. Gooding, Electroanalysis 2013, 25, such that the surface coverage of acompact monolayer of 881–887. 10 2 PPC would be 9.210À mol/cm .From XPS,the ratio of [13] T. Goda, M. Tabata, M. Sanjoh, M. Uchimura, Y. Iwasaki, P/In for the “stabilized” PPC-ITO surface was calculated Y. Miyahara, Chem. Commun. 2013, 49,8683–8685. [14] Q. Jin, J. P. Xu, J. Ji, J. C. Shen, Chem. Commun. 2008, to be 0.48, so the actual surface coverage of deposited 3058–3060. 10 2 10 2 PPC is 4.4210À mol/cm (0.489.210À mol/cm ). [15] A. L. Gui, E. Luais,J.R.Peterson, J. J. Gooding, ACSAppl. Considering the XPS and CV numbers,the overall graft- Mater.Interf. 2013, 5,4827–4835. ing efficiency for 20 cycles of PPC modified ITO surface [16] M. Parviz, N. Darwish, M. T. Alam, S. G. Parker, S. Ciampi, is ~9.5%(grafting efficiency=amount of actual deposit- J. J. Gooding, Electroanalysis 2014, 26,1471–1480. ed molecules/amount of total generated radicals=4.42/ [17] S. Chen, F. Yu,Q.Yu, Y. He,S.Jiang, Langmuir 2006, 22, 8186–8191. 46.7100%), further indicating the grafting efficiency is [18] S. Chen, J. Zheng, L. Li, S. Jiang, J. Am. Chem. Soc. 2005, quite low and explaining why more CV cycles are re- 127,14473–14478. quired to deposit enough PPC to achieve good antifouling [19] A. L. Gui, G. Liu, M. Chockalingam, G. Le Saux, E. Luais, properties relative to on glassy carbon electrodes. J. B. Harper, J. J. Gooding, Electroanalysis 2010, 22,1824– 1830. [20] A. L. Gui, G. Liu, M. Chockalingam, G. Le Saux, J. B. 4Conclusions Harper, J. J. Gooding, Electroanalysis 2010, 22,1283–1289. [21] G. Liu, M. Chockalingham, S. M. Khor, A. L. Gui, J. J. We have demonstrated that electrografting of in situgen- Gooding, Electroanalysis 2010, 22,918–926. erated phosphorylcholine based aryl diazonium salts on [22] G. Liu, J. Liu, T. Bçcking, P. K. Eggers,J.J.Gooding, Chem. ITOelectrodes can formexcellentprotein-resistant coat- Phys. 2005, 319,136–146. ings,asevaluatedusing impedance spectroscopy. Unex- [23] A. M. Haque,M.M.Khan, K. Kim, Chem. Commun. 2013, pectedly,adecrease in charge transfer resistance for PPC 49,3802–3804. modified ITOsurface was observed after treatingitwith [24] M. P. Seah, W. A. Dench, Surf.Interf.Anal. 1979, 1,2–11. aprotein solution, whichoriginatesfromphysisorption of [25] A. Laforgue,T.Addou, D. BØlanger, Langmuir 2005, 21, 6855–6865. diazonium speciesduring electrografting process.Astable [26] Y. Xia, X.-M. Zhao,E.Kim, G. M. Whitesides, Chem. surface can be achieved simply by incubating it in PBS for Mater. 1995, 7,2332–2337. 2h.Onstabilized surfaces, the valueofRct remained un- [27] M. C. Rodriguez, A.-N.Kawde,J.Wang, Chem. Commun. changed even afterincubation in aprotein solution was 2005,4267–4269. continuedfor aprolonged time,showing thatPPC on ITO [28] S. Maldonado,T.J.Smith, R. D. Williams,S.Morin, E. can be an appealing protein-resistant platform with low Barton, K. J. Stevenson, Langmuir 2006, 22,2884–2891. [29] S. Baranton, D. BØlanger, J. Phys.Chem. B 2005, 109, impedance for the development of biosensors. 24401–24410. [30] P. Doppelt, G. Hallais,J.Pinson, F. Podvorica, S. Verneyre, Acknowledgements Chem. Mater. 2007, 19,4570–4575. [31] S. Chen, L. Li, C. Zhao,J.Zheng, Polymer 2010, 51,5283– We thank the Australian Research Council Centre of Ex- 5293. cellence in Convergent Bio-Nano Science and Technology (Project Number CE140100036), and the University of Received:October 6, 2014 New South Wales for funding. Cheng Jiang appreciates Accepted:November 25, 2014 China Scholarship Council (CSC) for scholarship. Published online:February 17, 2015 www.electroanalysis.wiley-vch.de 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Electroanalysis 2015,27, 884–889 889 Supporting information

Zwitterionic Phenyl Phosphorylcholine on Indium Tin Oxide: a Low-Impedance Protein-Resistant Platform for Biosensing Cheng Jiang, Muhammad Tanzirul Alam, Stephen G. Parker, J. Justin Gooding * School of Chemistry and Australian Centre for NanoMedicine, The University of New South Wales, Sydney, NSW 2052, Australia *Corresponding author. Tel: +61-2-93855384; fax: +61-2-93856141 Email: [email protected]

Fig. S1 Cyclic voltammograms recorded in 0.1 M KCl containing 1 mM K3[Fe(CN) 6] and 1 mM K4[Fe(CN) 6] at the scan rate of 0.1 V/s for bare ITO before (black line) and after (red line) incubation in HSA solution (1 mg/mL HSA in PBS) for 1 h.

Fig. S2 Cyclic voltammograms recorded in 0.1 M KCl containing 1 mM K3[Fe(CN) 6] and 1 mM K4[Fe(CN) 6] at the scan rate of 0.1 V/s for a bare ITO surface (black line) that have experienced the following treatments, 20 cycles of PPC modification ((red line)), incubation in PBS for 1 h (blue line)), 2 h (dark cyan line) and 6 h (magenta line).

-3/-4 Fig. S3 Cyclic voltammograms recorded in 1 mM [Fe(CN) 6] redox couple at the scan rate of 0.1 V/s for PPC-ITO surface (20 cycles modified) after successive treatment with (cyan line) PBS incubation for 2 h, HSA solution for (blue line) 1 h and (red wine line) 6 h.

Fig. S4 XPS survey scan for bare ITO, PPC-ITO surfaces (20 cycles modified) without PBS incubation and with PBS incubation for 2 h.

Chapter 4 Mixed Layers on ITO

Chapter 4 Strategies to Achieve Control over the Surface Ratio of Two Different Components on Modified Electrodes Using Aryl-Diazonium Salts

Publication III

Jiang, C.; Alam, M. T.; Parker, S. G.; Darwish, N. A.; Gooding, J. J.* Strategies to Achieve Control over the Surface Ratio of Two Different Components on Modified Electrodes Using Aryl-Diazonium Salts. Langmuir 2016. 32 (10), 2509–2517

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Strategies To Achieve Control over the Surface Ratio of Two Diﬀerent Components on Modiﬁed Electrodes Using Aryldiazonium Salts Cheng Jiang, Muhammad Tanzirul Alam, Stephen G. Parker, Nadim Darwish, and J. Justin Gooding* School of Chemistry, Australian Centre for NanoMedicine and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, The University of New South Wales, Sydney, NSW 2052, Australia

*S Supporting Information

ABSTRACT: Controlling the composition of an interface is very important in tuning the chemical and physical properties of a surface in many applications including biosensors, biomaterials, and chemical catalysis. Frequently, this requires one molecular component to a minor component in a mixed layer. Such subtle control of composition has been difficult to achieve using aryldiazonium salts. Herein, aryldiazonium salts of carboxyphenyl (CP) and phenylphosphorylcholine (PPC), generated in situ from their corresponding anilines, are electrografted to form molecular platform that are available for further functionalization. These two components are chosen because CP provides a convenient functionality for further coupling of biorecognition species while PPC offers resistance to nonspecific adsorption of proteins to the surface. Mixed layers of CP and PPC were prepared by grafting them either simultaneously or consecutively. The latter strategy allows an interface to be developed in a controlled way where one component is at levels of less than 1% of the total layer.

1. INTRODUCTION of aryldiazonium salts is a promising alternative that has 16−18 The benefits of introducing multiple functionalities through attracted wide attention in recent times. In this regard, a mixed component from aryldiazonium salts with similar or decorating a surface with organic layers have been well ff demonstrated in a large variety of applications, including di erent electrochemical reactivity can be formed on the 1 2,3 4 electrode by depositing them to give multiple functional- surface wetting, cell biology, chemical sensing, biosens- 4,19−22 5,6 7 ities. Reductive attachment of mixtures of aryldiazonium ing, and photosynthesis. For example, alkanethiols have fi been used successfully in tailoring electrode surfaces with salts leads to a modi ed surface with mixed organic layers, but the final structural composition is hard to control due to the multiple functionalities in a controllable way in order to tune 14 the chemical and physical properties of the surface. This has high and unselective reactivity of the aryl radicals generated. mainly been performed by exposing a gold substrate to a Furthermore, it was found that the ratio of the two components solution containing two alkanethiols with different functional on the surface is dominated by the reduction potentials of the − groups.8 10 The attractiveness of gold−thiol chemistry is the two aryldiazonium salts as distinct from their ratio in solution; ease of forming well-organized monolayers with a controllable the more easily reduced (more positive reduction potential) 11,12 diazonium salt gives greater surface coverage than expected surface composition. Nonetheless, studies of alkanethiols 6,23,24 on gold surfaces have revealed a few limitations with regards to from its concentration in solution. One exception to the the stability of the gold−thiolate bond and hence the stability of above statement about poor control over layer composition due ff the resultant self-assembled monolayers (SAMs). It is reported to di erent redox potentials of reductive adsorption has been that alkanethiols gradually desorb at temperatures over 100 °C, demonstrated by the Gooding group where supramolecular leading to a mobile surface with the monolayer moving across interactions between the two aryldiazonium species bearing − − − + the electrode surface.13,14 Furthermore, the surface-bound SO3 or N (CH3)3 in solution ensured that the ratio on the surface of the two components was always 1:1 even if different alkanethiol is prone to oxidation with resultant loss of the 22 SAM from the surface.15 Such limitations compromises the molar ratios were used. Of course, such a strategy does not viability of using gold−thiol-based SAMs for some applications allow low amounts of one component in a background of where long-term use or high temperatures are required. another to be formed. Gam-Derouich and co-workers have Thus, it would therefore be desirable to develop an approach recently reported that formation of a mixed layer surface with that yields a stronger bond between an electrode surface and an organic layer while maintaining a controllable surface Received: December 14, 2015 composition. Covalent modification of conducting or semi- Revised: February 17, 2016 conducting surfaces via electrochemically reductive adsorption Published: February 22, 2016

Scheme 1. Electrografting Mixed Layer of PPC and CP on ITO Using (a) Simultaneous Electrografting Strategy and (b) a Consecutive Electrografting Strategy

aIn the former strategy, PPC and CP diazonium salts were deposited from mixtures of PPC and CP aryldiazonium salts at different molar ratios. In the latter strategy, PPC diazonium salt was selectively deposited first, followed by depositing CP diazonium salts on PPC modified surface. PPC−CP and PPC/CP were used to distinguish and represent mixed layer deposited by simultaneous and consecutive strategies, respectively. both antifouling and biorecognition properties through electro- ratio of molecular wire to oligo(ethylene glycol) in solution grafting of 4-benzoylphenyl layer (BP) and surface-initiate resulted in only an approximate 1:3 ratio on the surface. This photopolymerization of polyglycidol on BP. In that case the study illustrates that in a simultaneous electrodeposition polyglycidol layer is a high-impedance layer, which blocks strategy there is poor control over the surface composition electron transfer, and the surface composition was not when the two components do not have the same redox controlled in their study, and hence the work is less suited to potentials. electrochemical sensors.25 Hence, a precise control over the surface composition is A sophisticated example of forming mixed layers using required to realize that one component is highly diluted while aryldiazonium salts is where a surface was modified with a another component dominates the surface. This is especially mixture containing a molecular wire and a diluent that provided crucial for making cell chips or electrochemical biosensors resistance to nonspecific adsorption of proteins to give where the recognition component is diluted in an antifouling electrodes for efficient electron transfer to redox proteins26 film so that interactions with target analytes are not sterically − and for immunosensors.6,27 These electrodes were shown to be hindered and nonspecific protein adsorption is minimized.28 30 effective in a complex media. In the immunosensor study, an In this work, we are aiming to achieve control over the oligophenylethynylene molecular wire and an oligo(ethylene surface composition of aryldiazonium salt-derived mixed layers glycol)-terminated antifouling component were attached on a where one component is in excess of the other. Our interest is glassy carbon surface.6 The molecular wire enables electro- in developing an interface for biosensing applications, and very chemical communication between the electrode and a redox recently we have reported that aryldiazonium salts produced species attached to its distal end, and the oligo(ethylene glycol) from zwitterionic 4-aminophenylphosphorylcholine can be layers resists nonspecific adsorption of proteins to the surface. electrodeposited on glassy carbon,31 gold,32 and indium tin For immunosensor applications, the ideal interface was oxide (ITO).33 The resulting modified electrodes are resistant composed of a small amount of molecular wire in a background to nonspecific protein adsorption, but the coating is of of dominating oligo(ethylene glycol). However, because the sufficiently low impedance that the electrode was still capable molecular wire was reductively adsorbed at a more positive of supporting appreciable electron transfer across the layer. redox potential than the oligo(ethylene glycol), even a 1:100 Hence, to render this idea compatible with biosensing

DOI: 10.1021/acs.langmuir.5b04550 Langmuir 2016, 32, 2509−2517 Langmuir Article interfaces requires an additional minor component in the hemispherical analyzer, and multichannel detector. The spectra were interface for attaching biomolecules used as the recognition accumulated at a takeoff angle of 90° with a 0.79 mm2 spot size at a × −8 element in a biosensor. How to fabricate such interfaces is the pressure of less than 1 10 mbar. Binding energies of elements were purpose of this study. Specifically, a mixed layer surface corrected with reference to graphite C 1s (284.6 eV). The XPS spectrum was analyzed with the Avantage software, involving containing a phenylphosphorylcholine (PPC) group and a background subtraction using the Shirley routine and a subsequent carboxyl phenyl (CP) group formed on an ITO surface was nonlinear least-squares fitting to mixed Gaussian−Lorentzian investigated. A comparison was made between the control over functions. The atomic concentration (atom. %) of individual elements the layer composition achieved using simultaneous and was determined from the relative peak area of the spectrum and the consecutive electrodeposition strategies (Scheme. 1). corresponding sensitivity factors according to eq 1: As/ 2. EXPERIMENTAL SECTION atom % = ii 2.1. Reagents and Materials. Dichloromethane, methanol, ∑ Asii/ (1) K CO , NaNO , KCl, NaCl, HCl (32%), KH PO , and Na HPO 2 3 2 2 4 2 4 where A is the area of the element i and s is the sensitivity factor for were purchased from Ajax Finecham (Sydney, Australia). 4-Amino- i i this element. The sensitivity factors for P 2p, O 1s, C 1s, and N 1s are phenylphosphorylcholine was obtained from Toronto Research 1.19, 2.93, 1.00, and 1.80, respectively. The thickness of the deposited Chemicals (Toronto, Canada). Human serum albumin (HSA), 1- layer on ITO is estimated from the relative attenuation of the In 3d ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride signal, using (EDC), and N-hydroxysuccinimide (NHS) were acquired from Sigma (Sydney, Australia). ITO-coated glass slides (15−30 ohm/sq, ln(II /0 )=− ( d /λθ ) sin( ) (2) 6480-AB) were obtained from SPI (West Chester, USA). All chemicals were used as received unless otherwise stated and aqueous solutions where d is the layer thickness, λ is the photoelectron escape depth of − were prepared using Milli-Q water (18 MΩ cm 1, Millipore, Sydney, In 3d which was estimated to be 3.5 nm,35 θ is the takeoff angle (90° Australia). 1-Propylamino-1,2,3-triazole-4-ferrocene was synthesized used in our experiment), and I/I0 is the ratio of the In 3d peak using a “click” reaction with ethynylferrocene and 3-azido-1-propyl- intensities (modified surface/bare surface). amine as described by our group elsewhere.34 Phosphate-buffered saline (PBS) was used to dissolve HSA. 3. RESULTS AND DISCUSSION 2.2. Modification of ITO Electrode Surfaces. ITO electrodes were sonicated successively with dichloromethane (DCM, 10 min), Prior to examining the mixed layers formed by arydiazonium methanol (10 min), and 0.5 M K2CO3 in 2:1 (volume ratio) of salts of CP and PPC, the reductive adsorption behavior of the methanol:H2O (30 min), followed by rinsing with copious amounts of two individual salts and the mixture of the two salts was Milli-Q water and were dried under a stream of nitrogen. The investigated. A typical cyclic voltammogram of a 1 mM CP electrodes were then modified either by cycling the potential between diazonium salt solution on the ITO surface shows a single − 0.2 and 0.65 at 0.1 V/s in a 0.1 M HCl solution containing a irreversible reduction peak at −0.26 V (Figure 1a) during the diazonium salt mixture of PPC and CP (molar ratio 1:1) and first reduction scan whereas PPC diazonium salts shows a equivalent amounts of NaNO2 or by a potentiostatic method through fi − ff reduction peak at −0.54 V (Figure 1b). Importantly, the cyclic xing the potential at 0.6 V for di erent durations. − 2.3. Electrochemical Apparatus. Cyclic voltammetry (CV) voltammogram of a mixed PPC CP diazonium salts solution measurements were performed with an Autolab potentiostat (Figure 1c) exhibits two reduction peaks (appeared at (Metrohm Autolab B.V., Netherlands); ac voltammetry and electro- potentials comparable to those in Figure 1a,b) in the first chemical impedance spectroscopy (EIS) experiments were conducted cycle, while in the following scans only the redox wave using a Solartron SI 1287 electrochemical interface coupled with an SI corresponding to CP diazonium salts was observed. 1260 frequency response analyzer (Solartron Analytical, Hampshire, 3.1. PPC−CP/ITO from Simultaneous Electrografting England). All electrochemical experiments were carried out in a 2.0 Strategy. A mixed layer surface of PPC−CP/ITO was first mL one-compartment custom-made electrochemical cell comprising of obtained by two voltammetric cycles in a solution of PPC−CP a conventional three-electrode system, in which an O-ring was placed on the substrate to prevent solution leakage and to limit the contact aryldiazonium salt mixture with a 1:1 molar ratio. The resultant area to a circle with surface area of 0.24 cm2. Electrochemical surface was characterized by XPS and shown in Figure 2. The experiments were performed using ITO-coated glass as the working characteristic peaks for phosphorus (135 and 132.8 eV) and − + electrode, an Ag/AgCl (3.0 M KCl) as the reference electrode, and a quaternary nitrogen ( N (CH3)3) peak (403 eV) from the P platinum wire as the counter electrode. 2p and N 1s narrow scans, respectively, allow the identification 2.4. Electrochemical Evaluation of Protein-Resistance Be- of PPC on the surface while the 289 eV in the C 1s narrow is havior. EIS to monitor the behavior of PPC-ITO in resisting − fi indicative of the COOH of CP. The surface ratio of P/N nonspeci c adsorption of proteins was performed in a redox probe (using atom % (P)/atom % (−N+(CH ) ) was found to be solution of 0.1 M KCl containing 1 mM K [Fe(CN) ] and 1 mM 3 3 3 6 around 1:1, which is in good agreement with theoretical value K4[Fe(CN)6]. The electrodeposited surfaces (PPC/ITO, CP/ITO, PPC/CP/ITO) were rinsed thoroughly with water before adding them of phosphorylcholine moiety of PPC. Thus, COOH and P were to the solution containing the redox probe, and EIS measurements selected as XPS markers for CP and PPC, respectively. The were then performed. Subsequently, these electrodes were rinsed with surface ratio of PPC:CP was found to be 1:20 by using atom % water and PBS, followed by incubation in an HSA solution (1 mg/mL (P)/atom % (COOH) (eq 1), which was not only significantly HSA in PBS) for 1 h. The electrodes were then rinsed thoroughly with different from the 1:1 molar ratio in the deposition solution but PBS and Milli-Q water, followed by EIS measurement in a fresh redox also a long way from the desired outcome of a low amount of probe solution. EIS spectra were recorded in the frequency range of 5− −1 CP in a layer dominated by PPC. 10 10 Hz. An ac potential with 0.01 V peak-to-peak separation was To further study the relationship between molar ratio in superimposed on a dc potential of 0.212 V. Impedance data was solution and the corresponding surface ratio, a depositing recorded and analyzed using ZPlot and ZView 3.1 software (Scribner − Associates, Inc.), respectively. potential of 0.6 V (at which both PPC and CP diazonium 2.5. X-ray Photoelectron Spectroscopy Characterization. salts can be reduced at same time) was chosen, in which the XPS spectra were obtained using an EscaLab220-IXL spectrometer molar concentration of PPC was fixed at 1 mM while the CP equipped with a monochromated Al Kα source (1486.6 eV), concentration in the deposition solution was varied. For

DOI: 10.1021/acs.langmuir.5b04550 Langmuir 2016, 32, 2509−2517 Langmuir Article

Figure 3. Comparison of molar ratio of PPC−CP (X) in mixed deposition solution to the XPS atomic ratio (Y) of the P 2p narrow scan derived from PPC and the carboxyl peak in the C 1s narrow scan from CP in the PPC−CP/ITO mixed layer surfaces modiﬁed by the simultaneous electrografting approach. The deposition potential and deposition time were ﬁxed at −0.6 V and 5 min, respectively.

example, when the molar ratio of PPC:CP was 1:5, 10:1, and 100:1, the surface ratio was found to be 1:13.8, 1.4:1, and 3.7:1, respectively. Even with 10 000 times greater amounts of PPC over CP, the surface ratio of PPC:CP increased to only 6:1. This can be explained by the more positive reduction potential of the CP diazonium which makes it easier to be reduced on the surface as compared to the PPC diazonium cations. It is Figure 1. Cyclic voltammograms for reductive adsorption of believed that the grafting of the radicals is in competition with aryldiazonium salts on an ITO electrode from (a) 1 mM 4- their reduction,36,37 leading to initially grafted CP species aminobenzoic acid (CP) in 0.1 M HCl, (b) 1 mM 4-aminophenyl- possibly inhibiting the adsorption of PPC to the surface.25 Such phosphorylcholine (PPC) in 0.1 M HCl, and (c) 1 mM mixture of 4- an assertion is also supported by the absence of a peak for PPC aminobenzoic acid (CP) and 4-aminophenylphosphorylcholine (PPC) diazonium salts after the ﬁrst cycle in the cyclic voltammogram with 1:1 molar ratio in 0.1 M HCl, with 1 mM NaNO2 at a scan rate of of the diazonium salt mixture of PPC and CP shown in Figure 0.1 V/s. The black dashed line indicates the reduction peak of the 1c. A similar ﬁnding has been reported by Belangeŕ and co- individual aryldiazonium salt, and the red dashed line indicates the 24 reduction peaks of aryldiazonium salts mixture. workers by depositing a binary mixture of 4-nitrophenyldiazonium cations (reduction potential 0.2 V) and 4-bromo- phenyldiazonium cations (reduction potential −0.2 V) on example, to obtain a mixture solution with 1:5 or 10000:1 glassy carbon. In the cyclic voltammogram recorded in such a molar ratio (PPC:CP), the concentration of CP was adjusted to mixture, only the reduction peak of nitrophenyldiazonium 5 mM or 0.1 μM while keeping the concentration of PPC at 1 cations was observed. They also found that the grafted layer mM. As shown in Figure 3, the increase in surface ratio of was always richer in nitrophenyl groups than in bromophenyl PPC/CP lagged far behind the molar ratios in solution, for groups in comparison to the corresponding concentration of

Figure 2. P 2p, N 1s, and C 1s core level XPS spectra for PPC−CP mixed layer modiﬁed-ITO after two voltammetric cycles (simultaneous electrografting approach). Electrografting is carried out in a solution of 0.1 M HCl containing PPC−CP diazonium salts with molar ratio of 1:1 and − equivalent amount of NaNO2 in the potential range from 0.2 to 0.65 V (vs Ag/AgCl (3 M KCl)) using a scan rate of 0.1 V/s.

DOI: 10.1021/acs.langmuir.5b04550 Langmuir 2016, 32, 2509−2517 Langmuir Article the diazonium cations in the deposition mixture when applying irrespective of what molar fraction it represents in the solution. an over potential of −0.7 V. To achieve better control over the ratio of the two components Interestingly, in the simultaneous electrografted PPC−CP on the surface such that one can enable the poorly reactive mixed layer/ITO surface, the azo group intensity (located at diazonium species to dominate on the ITO surface, a ∼400 eV) seems to be positively correlated with the molar consecutive electrografting strategy was used. In this strategy, fraction of CP in the deposition solution but negatively the PPC diazonium cations were selectively deposited ﬁrst at correlated with that of PPC (Figure 4 and Figure S2). For −0.6 V with higher concentrations and longer deposition times, followed by the electrodeposition of CP diazonium salts with a depositing potential at −0.1 V, lower concentrations, and shorter deposition times. It was found that a condition that deposited 1 mM PPC diazonium salts at −0.6 V for 5 min, followed by depositing CP from highly diluted (1 nM) solution at −0.1 V for 5 s, gave surfaces with a ratio of 200:1 PPC/CP (shown in Table. 1).

Table 1. XPS Characterization of PPC/CP/ITO Surface Composition Derived from the Consecutive Electrografting Approach

PPC/CP surface electrodeposition conditions ratio 1 mM PPC −0.6 V, 30 s, then 1 mM CP −0.6 V, 5 s 2:1 1 mM PPC −0.6 V, 5 min, then 1 μMCP−0.1 V, 5 s 81:1 1 mM PPC −0.6 V, 5 min, then 1 nM CP −0.1 V, 5 s 200:1

The CP layer was believed to grow mainly on the PPC layer due to the steric hindrance of the PC moiety and the multilayer structure of PPC (2.5 nm thickness, assumed to be about three layers considering length of PPC is around 0.8 nm). Evidence from cyclic voltammograms showed that the reduction peak of CP on bare ITO is −0.26 V, while for CP on PPC/ITO (consecutive electrografting) the peak moved more positively to −0.2 V, indicating that CP was mostly growing on the PPC layer. 3.3. Evaluation of Protein-Resistant Behavior. Even Figure 4. N 1s core level spectra of PPC−CP/ITO surfaces modified though this method of forming mixed layers from aryldiazo- by the simultaneous electrografting approach from mixed deposition nium salts should be reasonably general, the purpose of forming solution with different molar ratios (PPC takes more molar fraction mixed layers specifically from PPC and CP was to develop than that of CP): (a) 2:1, (b) 5:1, (c) 10:1, (d) 50:1, (e) 100:1, and biosensing interfaces. In such interfaces, PPC would provide (f) 10000:1. Deposition potential and deposition time were fixed at resistance to nonspecific adsorption of proteins while the CP −0.6 V and 5 min, respectively. The ratio in red represents the relative would allow biorecognition molecules to be attached. In our molar ratios in the deposition solution, and the percentage in green previous study, 20 CV scans on ITO were required to form −  − indicates the proportion of azo group ( N N ) and quaternary PPC layers with good antifouling ability.33 Considering there is − + nitrogen ( N (CH3)3) of the total nitrogen percentage on each a risk that the deposition of CP on PPC may shield the PC surface. moiety and affect the surface antifouling behavior, the deposited amount of CP in the consecutive electrografting example, when the molar ratio of PPC−CP is 1:5, the azo approach was adjusted to retain the antifouling behavior and group dominates the total N, being 78% as shown in Figure accessibility of the carboxylic acid group of the mixed layer/ S2c, while when the molar ratio of PPC−CP is 10000:1 (Figure ITO surface. This was examined by incubating the modified − + 4f), the quaternary nitrogen ( N (CH3)3) peak dominates the surfaces in a HSA solution and further decoration of ferrocene, total N at 88%. These results indicate that CP diazonium respectively. cations can not only graft directly onto the ITO surface but also The antifouling behavior of the resultant mixed layer/ITO graft onto already deposited molecules to form multilayers.38,39 surfaces was studied using electrochemical impedance spec- The results from the deposition of the individual aryl troscopy, in which bare ITO, PPC/ITO, and CP/ITO surfaces diazonium salts also showed that CP diazonium salt tends were used as controls. For functionalized surfaces of PPC/ITO more likely to form the azo group in the layer than PPC (before HSA incubation, 493 ± 4.7 Ω cm2 and after HSA diazonium salt (Figure S1). incubation, 503 ± 10 Ω cm2, Figure 5c) and PPC/CP/ITO 3.2. PPC/CP/ITO from Consecutive Electrografting (before HSA incubation, 542 ± 22.5 Ω cm2, and after HSA, 520 Strategy. As shown above, in the simultaneous electrografting ± 5 Ω cm2, Figure 5d) both showed good antifouling strategy, the relative reactivity of the two aryldiazonium salts properties since the charge transfer resistance (Rct) was similar plays a key role in the surface composition. Thus, when a before and after HSA incubation, indicating nonspecific mixture of aryldiazonium species are present in solution, the proteins can be repulsed from the surface due to a hydrated one with higher reactivity dominates the surface composition layer formed by the interaction between the zwitterionic PPC

DOI: 10.1021/acs.langmuir.5b04550 Langmuir 2016, 32, 2509−2517 Langmuir Article fi signi cantly (for bare ITO, Rct increased by almost 8 times after HSA incubation from 77 ± 1.6 Ω cm2 to 686 ± 11 Ω cm2, ± Ω 2 and for CP/ITO, Rct increased by 50% from 414 3.5 cm to 605 ± 13.7 Ω cm2), indicating adsorption of HSA on such surfaces. It is worth noting that CP itself does not function as an antifouling layer; however, when it was grafted as a small percentage of the layer with PPC, it did not deleteriously affect the antifouling property of the surface as demonstrated by the almost nonvariance of charge transfer resistance in both the cases (Figure 5d). One final concern regarding this method of forming mixed layers was whether the subsequent grafting would increase the charge transfer resistance across these layers by redox species in solution. This is important as the entire purpose was to develop PPC as a low-impedance antifouling layer such that electron transfer could still efficiently proceed.31 Figure S3 showed the blocking effect of such PPC/CP/ITO mixed layer surface from consecutive electrografting strategy with two clear redox peaks still clearly existing, implying that the mixed layer surface does not completely block electron transfer. Figure 5. Nyquist plots comparing Faradaic impedance at surfaces of 3.4. Functionalization of a Mixed Layer Surface with (a) bare ITO, (b) CP/ITO, (c) PPC/ITO, and (d) PPC/CP/ITO fi Ferrocene. To demonstrate that the carboxyl group in CP is (with surface ratio 200:1 modi ed by consecutive electrografting fi approach) in a solution of 0.1 M KCl containing 1 mM K3[Fe(CN)6] free for further modi cation, ferrocene was attached to the and 1 mM K4[Fe(CN)6] before and after 1 h incubation in a protein surfaces of PPC/CP/ITO (200:1 surface ratio from consecutive solution (1 mg/mL HSA in PBS, pH 7.4). electrografting), PPC−CP/ITO (surface ratio 6:1 from the simultaneous electrografting approach), and CP/ITO (control) moieties and water molecules.40,41 While for bare ITO (Figure with the aid of the EDC/NHS coupling reaction (Scheme 2). ff 5a) and CP/ITO (Figure 5b), the Rct value increased The electron transfer kinetics of the di erent surfaces was then

Scheme 2. Chemical Functionalization of (a) PPC−CP/ITO (with Surface Ratio 6:1 Modiﬁed by Simultaneous Electrografting approach), (b) PPC/CP/ITO (with Surface Ratio 200:1 Modiﬁed by Consecutive Electrografting Electrografting Approach), and (c) CP/ITO with 1-Propylamino-1,2,3-triazole-4-ferrocene

DOI: 10.1021/acs.langmuir.5b04550 Langmuir 2016, 32, 2509−2517 Langmuir Article

Figure 6. Plots of I(peak)/I(background) vs log(frequency) prepared using ac voltammetry for (a) Fc/PPC−CP/ITO, (b) Fc/PPC/CP/ITO, and fi × −6 × 5 Ω × −6 (c) Fc/CP/ITO. Solid line shows tted plot using equivalent circuit parameters Cdl = 1.7 10 F, Rct = 1.1 10 , Cad = 8.3 10 F(ket = 2.7 −1 × −6 Ω × −6 −1 × −6 × 5 Ω × −6 s ); Cdl = 5.72 10 F, Rct = 8326 , Cad = 4.75 10 F(ket = 12.6 s ); and Cdl = 1.3 10 F, Rct = 2.5 10 , and Cad =8 10 F(ket = 15.4 s−1) for three surfaces (a−c), respectively. examined using cyclic voltammetry and ac voltammetry simultaneous electrografting strategy, more reactive CP cations (Creager method).42 From the CVs, the coverage of the always showed priority on the surface compared to PPC ferrocene can be estimated to be 3.21 × 10−11, 2.17 × 10−11, cations. A consecutive electrografting strategy was then applied and 0.92 × 10−11 mol cm−2 for the Fc/CP/ITO, Fc/PPC/CP/ by depositing PPC diazonium cations first with higher ITO, and Fc/PPC−CP/ITO, respectively. concentration and favorable potential followed by depositing Ideally, the peak separation in CVs should be negligible for a CP diazonium cations. A 200:1 surface composition of PPC/ well-defined redox system with the ferrocene and ferrocenium CP was achieved using a consecutive electrografting strategy. couple. In this case, the significant peak separation was The resultant mixed layer ITO surface maintains its antifouling noticeable for all surfaces, suggesting the electron transfer properties and has accessible carboxylic acid moieties available kinetics are slow on the time scale of the 0.1 V s−1 voltammetric for ferrocene decoration. In the future, such versatile mixed scan. Smaller peak separation was found in Fc-CP/ITO layer/ITO platforms will be employed by decorating biological compared to Fc/PPC−CP/ITO and Fc/PPC/CP/ITO, species like redox proteins, antibodies, or DNA to develop real implying electron transfer kinetics on both mixed layer/ITO biodevices as has just been demonstrated for the detection of surfaces is much slower than that of CP/ITO surface (Figure DNA sequences.45 S4). The calculated ket values using ac voltammetry are in line ± ■ ASSOCIATED CONTENT with expectation. The value of ket was determined to be 15.4 −1 2.2, 12.6 ± 1.7, and 2.7 ± 0.2 s for Fc/CP/ITO, Fc/PPC/ *S Supporting Information CP/ITO, and Fc/PPC−CP/ITO, respectively (Figure 6). The The Supporting Information is available free of charge on the ket values calculated from the Laviron method were consistent ACS Publications website at DOI: 10.1021/acs.lang- with that from the Creager method (16.9 ± 0.3, 17.2 ± 2.5, and muir.5b04550. ± −1 1.1 0.14 s for three corresponding surfaces mentioned P 2p, N 1s, and C 1s core level XPS spectra for CP/ITO above) (Figure S4). It is expected that ket is independent from and PPC/ITO surfaces; N 1s core level spectra of PPC− the surface coverage of ferrocene but is dependent on the fi 43,44 CP mixed layer/ITO surfaces modi ed by simultaneous distance between the ferrocene and the electrode surfaces. electrografting; cyclic voltammograms recorded on PPC/ For Fc/CP/ITO and Fc/PPC/CP/ITO surfaces the ket value is CP/ITO surface before and after HSA incubation; cyclic comparable to and bigger than that of Fc/PPC−CP/ITO, voltammograms measured in 1.0 M NaClO4 at ITO indicating the distance of ferrocene to the electrode surface is in fi − surfaces modi ed with aryldiazonium cations and the order of Fc/CP/ITO < Fc/PPC/CP/ITO < Fc/PPC CP/ postfunctionalized with ferrocene; procedures for ITO. ff functionalize mixed layer/ITO surface with ferrocene; It is clear that the electron transfer behavior is di erent equation for calculation of total surface coverage (Γ)of between the mixed surface fabricated from simultaneous electroactive ferrocene (PDF) electrografting (Fc/PPC−CP/ITO) and consecutive electrografting (Fc/PPC/CP/ITO) procedures. It is exciting that CP AUTHOR INFORMATION groups on PPC can still allow sufficient electron transfer in the ■ Fc/PPC/CP/ITO surface, while for Fc/PPC−CP/ITO, Corresponding Author * electron transfer is quite slow. Through the rough estimation Tel +61 (02) 9385 5384; e-mail [email protected] of film thickness of the three surfaces using eq 2, it is in the (J.J.G.). order of PPC−CP/ITO (2.9 nm) < CP/ITO (3.1 nm) < PPC/ Present Address ́ ́ CP/ITO (3.5 nm). We believe that in the case of Fc/CP/ITO, N.D.: Departament de Quimica Fisica, Universitat de the lower thickness of the CP layer leads to the shorter distance Barcelona, Diagonal 645, Barcelona 08028, Spain. between the FC and the ITO electrode, resulting in a higher ket Notes compared to the other two surfaces. The authors declare no competing financial interest. 4. CONCLUSIONS ■ ACKNOWLEDGMENTS In this study, PPC and CP aryldiazonium salts with different We thank the Australian Research Council Centre of Excellence reductive potentials were deposited simultaneously or consec- in Convergent Bio-Nano Science and Technology (project utively on ITO to form mixed layer surfaces. Using a number CE140100036) and the University of New South

DOI: 10.1021/acs.langmuir.5b04550 Langmuir 2016, 32, 2509−2517 Langmuir Article

Wales for funding. Cheng Jiang appreciates China Scholarship (19) Santos, L.; Mattiuzzi, A.; Jabin, I.; Vandencasteele, N.; Reniers, Council (CSC) for scholarship. F.; Reinaud, O.; Hapiot, P.; Lhenry, S.; Leroux, Y.; Lagrost, C. One- Pot Electrografting of Mixed Monolayers with Controlled Composi- ■ REFERENCES tion. J. Phys. Chem. C 2014, 118 (29), 15919−15928. (1) Whitesides, G. M.; Laibinis, P. E. Wet chemical approaches to the (20) Mattiuzzi, A.; Jabin, I.; Mangeney, C.; Roux, C.; Reinaud, O.; characterization of organic surfaces: self-assembled monolayers, Santos, L.; Bergamini, J.-F.; Hapiot, P.; Lagrost, C. Electrografting of wetting, and the physical-organic chemistry of the solid-liquid calix [4] arenediazonium salts to form versatile robust platforms for interface. Langmuir 1990, 6 (1), 87−96. spatially controlled surface functionalization. Nat. Commun. 2012, 3, (2) Le Saux, G.; Magenau, A.; Böcking, T.; Gaus, K.; Gooding, J. J. 1130. ̈ The relative importance of topography and RGD ligand density for (21) Leroux, Y. R.; Hui, F.; Noel, J.-M.; Roux, C.; Downard, A. J.; endothelial cell adhesion. PLoS One 2011, 6 (7), e21869−e21869. Hapiot, P. Design of robust binary film onto carbon surface using (3) Roberts, C.; Chen, C. S.; Mrksich, M.; Martichonok, V.; Ingber, diazonium electrochemistry. Langmuir 2011, 27 (17), 11222−11228. D. E.; Whitesides, G. M. Using mixed self-assembled monolayers (22) Gui, A. L.; Yau, H. M.; Thomas, D. S.; Chockalingam, M.; presenting RGD and (EG) 3OH groups to characterize long-term Harper, J. B.; Gooding, J. J. Using supramolecular binding motifs to attachment of bovine capillary endothelial cells to surfaces. J. Am. provide precise control over the ratio and distribution of species in Chem. Soc. 1998, 120 (26), 6548−6555. multiple component films grafted on surfaces: demonstration using (4) Vila,̀ N.; Belanger,́ D. Mixtures of functionalized aromatic groups electrochemical assembly from aryl diazonium salts. Langmuir 2013, generated from diazonium chemistry as templates towards bimetallic 29 (15), 4772−4781. species supported on carbon electrode surfaces. Electrochim. Acta (23) Liu, G.; Chockalingham, M.; Khor, S. M.; Gui, A. L.; Gooding, J. 2012, 85, 538−547. J. A comparative study of the modification of gold and glassy carbon (5) Harper, J. C.; Polsky, R.; Wheeler, D. R.; Lopez, D. M.; Arango, surfaces with mixed layers of in situ generated aryl diazonium D. C.; Brozik, S. M. A multifunctional thin film au electrode surface compounds. Electroanalysis 2010, 22 (9), 918−926. formed by consecutive electrochemical reduction of aryl diazonium (24) Louault, C.; D’Amours, M.; Belanger, D. The electrochemical − salts. Langmuir 2009, 25 (5), 3282 3288. grafting of a mixture of substituted phenyl groups at a glassy carbon (6) Khor, S. M.; Liu, G.; Fairman, C.; Iyengar, S. G.; Gooding, J. J. electrode surface. ChemPhysChem 2008, 9 (8), 1164−70. The importance of interfacial design for the sensitivity of a label-free (25) Gam-Derouich, S.; Gosecka, M.; Lepinay, S.; Turmine, M.; electrochemical immuno-biosensor for small organic molecules. Carbonnier, B.; Basinska, T.; Slomkowski, S.; Millot, M.-C.; Othmane, Biosens. Bioelectron. 2011, 26 (5), 2038−2044. A.; Ben Hassen-Chehimi, D. Highly hydrophilic surfaces from (7) Imahori, H.; Mori, Y.; Matano, Y. Nanostructured artificial − polyglycidol grafts with dual antifouling and specific protein photosynthesis. J. Photochem. Photobiol., C 2003, 4 (1), 51 83. − (8) Banet, P.; Marcotte, N.; Lerner, D. A.; Brunel, D. Single-step recognition properties. Langmuir 2011, 27 (15), 9285 9294. dispersion of functionalities on a silica surface. Langmuir 2008, 24 (26) Liu, G.; Gooding, J. J. An interface comprising molecular wires (16), 9030−9037. and poly (ethylene glycol) spacer units self-assembled on carbon (9) Collman, J. P.; Devaraj, N. K.; Eberspacher, T. P.; Chidsey, C. E. electrodes for studies of protein electrochemistry. Langmuir 2006, 22 − Mixed azide-terminated monolayers: a platform for modifying (17), 7421 7430. electrode surfaces. Langmuir 2006, 22 (6), 2457−2464. (27) Liu, G.; Wang, S.; Liu, J.; Song, D. An electrochemical (10) Lee, L. Y. S.; Sutherland, T. C.; Rucareanu, S.; Lennox, R. B. immunosensor based on chemical assembly of vertically aligned Ferrocenylalkylthiolates as a probe of heterogeneity in binary self- carbon nanotubes on carbon substrates for direct detection of the assembled monolayers on gold. Langmuir 2006, 22 (9), 4438−4444. pesticide endosulfan in environmental water. Anal. Chem. 2012, 84 (11) Bain, C. D.; Whitesides, G. M. Formation of two-component (9), 3921−3928. surfaces by the spontaneous assembly of monolayers on gold from (28) Santos, L.; Ghilane, J.; Lacroix, J. C. Formation of mixed organic solutions containing mixtures of organic thiols. J. Am. Chem. Soc. 1988, layers by stepwise electrochemical reduction of diazonium compounds. 110 (19), 6560−6561. J. Am. Chem. Soc. 2012, 134 (12), 5476−5479. (12) Chapman, R. G.; Ostuni, E.; Yan, L.; Whitesides, G. M. (29) Lee, L.; Brooksby, P. A.; Leroux, Y. R.; Hapiot, P.; Downard, A. Preparation of mixed self-assembled monolayers (SAMs) that resist J. Mixed monolayer organic films via sequential electrografting from adsorption of proteins using the reaction of amines with a SAM that aryldiazonium ion and arylhydrazine solutions. Langmuir 2013, 29 (9), presents interchain carboxylic anhydride groups. Langmuir 2000, 16 3133−3139. − (17), 6927 6936. (30) Liu, G.; Paddon-Row, M.; Gooding, J. Protein modulation of (13) Horn, A. B.; et al. Ageing of alkanethiol self-assembled electrochemical signals: application to immunobiosensing. Chem. − monolayers. J. Chem. Soc., Faraday Trans. 1996, 92 (23), 4759 4762. Commun. (Cambridge, U. K.) 2008, 33, 3870−3872. (14) Delamarche, E. a. A.; Michel, B.; Kang, H.; Gerber, C. Thermal − (31) Gui, A. L.; Luais, E.; Peterson, J. R.; Gooding, J. J. Zwitterionic stability of self-assembled monolayers. Langmuir 1994, 10 (11), 4103 phenyl layers: finally, stable, anti-biofouling coatings that do not 4108. passivate electrodes. ACS Appl. Mater. Interfaces 2013, 5 (11), 4827− (15) Garrell, R. L.; Chadwick, J. E.; Severance, D. L.; McDonald, N. 35. A.; Myles, D. C. Adsorption of sulfur containing molecules on gold: (32) Parviz, M.; Darwish, N.; Alam, M. T.; Parker, S. G.; Ciampi, S.; the effect of oxidation on monolayer formation and stability Gooding, J. J. Investigation of the Antifouling Properties of Phenyl characterized by experiments and theory. J. Am. Chem. Soc. 1995, − Phosphorylcholine-Based Modified Gold Surfaces. Electroanalysis 117 (46), 11563 11571. − (16) Leroux, Y. R.; Hapiot, P. Nanostructured monolayers on carbon 2014, 26 (7), 1471 1480. substrates prepared by electrografting of protected aryldiazonium salts. (33) Jiang, C.; Tanzirul Alam, M.; Parker, S. G.; Gooding, J. J. Chem. Mater. 2013, 25 (3), 489−495. Zwitterionic Phenyl Phosphorylcholine on Indium Tin Oxide: a Low- (17) Mahouche-Chergui, S.; Gam-Derouich, S.; Mangeney, C.; Impedance Protein-Resistant Platform for Biosensing. Electroanalysis Chehimi, M. M. Aryl diazonium salts: a new class of coupling agents 2015, 27 (4), 884−889. for bonding polymers, biomacromolecules and nanoparticles to (34) Ciampi, S.; James, M.; Michaels, P.; Gooding, J. J. Tandem surfaces. Chem. Soc. Rev. 2011, 40 (7), 4143−4166. “click” reactions at acetylene-terminated Si (100) monolayers. (18) Polsky, R.; Harper, J. C.; Dirk, S. M.; Arango, D. C.; Wheeler, D. Langmuir 2011, 27 (11), 6940−6949. R.; Brozik, S. M. Diazonium-functionalized horseradish peroxidase (35) Seah, M.; Dench, W. Quantitative electron spectroscopy of immobilized via addressable electrodeposition: direct electron transfer surfaces: a standard data base for electron inelastic mean free paths in and electrochemical detection. Langmuir 2007, 23 (2), 364−366. solids. Surf. Interface Anal. 1979, 1 (1), 2−11.

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(36) Wildgoose, G. G.; Banks, C. E.; Compton, R. G. Metal nanoparticles and related materials supported on carbon nanotubes: methods and applications. Small 2006, 2 (2), 182−193. (37) Banks, C. E.; Davies, T. J.; Wildgoose, G. G.; Compton, R. G. Electrocatalysis at graphite and carbon nanotube modified electrodes: edge-plane sites and tube ends are the reactive sites. Chem. Commun. 2005, 7, 829−841. (38) Daniel Belanger,́ J. P. Electrografting: a powerful method for surface modification. Chem. Soc. Rev. 2011, 40, 3995−4048. (39) Combellas, C.; Kanoufi, F.; Pinson, J.; Podvorica, F. I. Sterically hindered diazonium salts for the grafting of a monolayer on metals. J. Am. Chem. Soc. 2008, 130 (27), 8576−8577. (40) Chen, S.; Zheng, J.; Li, L.; Jiang, S. Strong resistance of phosphorylcholine self-assembled monolayers to protein adsorption: insights into nonfouling properties of zwitterionic materials. J. Am. Chem. Soc. 2005, 127 (41), 14473−14478. (41) Chen, S.; Liu, L.; Jiang, S. Strong resistance of oligo (phosphorylcholine) self-assembled monolayers to protein adsorption. Langmuir 2006, 22 (6), 2418−2421. (42) Creager, S. E.; Wooster, T. T. A new way of using ac voltammetry to study redox kinetics in electroactive monolayers. Anal. Chem. 1998, 70 (20), 4257−4263. (43) Smalley, J. F.; Feldberg, S. W.; Chidsey, C. E.; Linford, M. R.; Newton, M. D.; Liu, Y.-P. The kinetics of electron transfer through ferrocene-terminated alkanethiol monolayers on gold. J. Phys. Chem. 1995, 99 (35), 13141−13149. (44) Guo, L.-H.; Facci, J. S.; McLendon, G. Distance dependence of electron transfer rates in bilayers of a ferrocene Langmuir-Blodgett monolayer and a self-assembled monolayer on gold. J. Phys. Chem. 1995, 99 (21), 8458−8461. (45) Kuo, T.-M.; Shen, M.-Y.; Huang, S.-Y.; Li, Y.-K.; Chuang, M.-C. Facile Fabrication of a Sensor with a Bifunctional Interface for Logic Analysis of the New Delhi Metallo-β-Lactamase (NDM)-Coding Gene. ACS Sensors 2016, 1, 124−130.

DOI: 10.1021/acs.langmuir.5b04550 Langmuir 2016, 32, 2509−2517 1 Supporting Information

2 Strategies to Achieve Control over the Surface

3 Ratio of Two Different Components on

4 Modified Electrodes Using ArylDiazonium

5 Salts

6 Cheng Jiang, Muhammad Tanzirul Alam, Stephen G. Parker, Nadim Darwish,† J.

7 Justin Gooding*

8 School of Chemistry, Australian Centre for NanoMedicine and ARC Centre of

9 Excellence in Convergent BioNano Science and Technology, The University of New

10 South Wales, Sydney, NSW 2052, Australia

11 †Present address: Departament de Química Física, Universitat de Barcelona, Diagonal

12 645, Barcelona 08028, Spain

2 Figure S1. P 2p, N 1s and C 1s core level XPS spectra for CP/ITO (a1c1) and

3 PPC/ITO (a2c2). Modification condition: 1 mM CP or PPC diazonium salt in a 0.1

4 M HCl containing equivalent amount of NaNO2 was deposited by potential static

5 method with deposition potential of 0.6 V and deposition time of 5 min

6 7 Figure S2. N 1s core level spectra of PPCCP mixed layer/ITO surfaces modified by

8 simultaneous electrografting approach from different ratios in deposition solution: (a)

9 1:1, (b) 1:2, and (c) 1:5. Deposition potential and deposition time were fixed at 0.6 V

10 and 5 min, respectively.

1 2 Figure S3. Cyclic voltammograms recorded in 0.1 M KCl containing 1 mM

3 K3[Fe(CN)6] and 1 mM K4[Fe(CN)6] at the scan rate of 0.1 V/s for a bare ITO surface

4 (black line), and PPC/CP/ITO (surface ratio 200:1 deposited by consecutive

5 electrografting approach) surface before (red line) and after (blue line) 1 h incubation

6 in 1 mg/mL HSA solution.

8 Figure S4. Cyclic voltammograms measured in 1.0 M NaClO4 at ITO surfaces

9 modified with arydiazonium cations and postfunctionalized with ferrocene. The

10 modiﬁed surfaces were (a) PPCCP/ITO (surface ratio of PPC:CP is 6:1 from

11 simultaneous electrografting approach), (b) PPC/CP/ITO (surface ratio of PPC:CP is

1 200:1 from by consecutive electrografting approach, and (c) CP/ITO (1 mM CP

2 diazonium salts deposited at ‒0.6 V for 5 min). Scan rates varied from 0.01 to 7 V/s.

3 Attachment of 1-propylamino-1,2,3-triazole-4-ferrocene to PPC-CP/ITO,

4 PPC/CP/ITO and CP/ITO surfaces

5 The PPCCP/ITO, PPC/CP/ITO and CP/ITO surfaces were incubated in an aqueous

6 solution containing 40 mM EDC and 10 mM NHS for 3 h. The activated surface were

7 then rinsed with copious amount of MilliQ water, followed by incubating in DMF

8 containing 1 mM 1propylamino1,2,3triazole4ferrocene for 12 h. The resultant

9 surfaces were thoroughly rinsed with preheated DMF and copious MilliQ water to

10 remove any nonspecifically adsorbed ferrocene and used for AC voltammetry which

11 was carried on Solartron SI 1287 instrument in 1 M NaClO4 solution.

12 The total surface coverage (Γ) of electroactive ferrocene was derived from the

13 integration of the area under the voltammetric peaks according to the following

14 equation.

16 Γ = Q/nFA S1

18 Here, n, F, Q and A represent the number of electrons, Faraday constant, charge,

19 and surface area of the ITO electrode, respectively. Background current was estimated

20 by extrapolation of the baseline capacitive current under the Faradaic peak current.

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

Chapter 5 A Unique Sensing Interface that Allows the Development of an Electrochemical Immunosensor for the Detection of Tumor Necrosis Factor α in Whole Blood

Publication IV

Jiang, C.; Alam, T. M.; Tauﬁk, S.; Silva, S. M.; Fan, S. J.; Gooding, J. J.* A Unique Sensing Interface that Allows the Development of an Electrochemical Immunosensor for the Detection of Tumor Necrosis Factor α in Whole Blood. ACS Sensors [submitted]

Declaration

I certify that this publication was a direct result of my research towards this PhD, and that reproduction in this thesis does not breach copyright regulations.

77 Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

5.1 Introduction

Developing electrochemical biosensors with interfacial design that achieves high sensitivity and selectivity continues to be a challenge for detecting proteins in complex biological samples despite the incredible promise for the early detection of diseases.1-3 The challenge arises from the abundance of proteins that exist in bodily fluids which nonspecifically adsorb on the sensing interface. The result of this biofouling is changes in sensitivity, specificity, irreproducibility of response and even complete failure of the sensing devices.4-8 Effective solutions are needed that resist this nonspecific protein adsorption. The inhibition of nonspecific protein adsorption is most commonly achieved by modifying the surface with highly hydrophilic poly(ethylene glycol) (PEG) polymer brushes9-11 or oligo (ethylene glycol) (OEG) based self-assembled monolayers (SAMs)12,13 and aryl layers14-16. These layers have excellent antifouling properties due to formation of a hydrated layer by hydrogen bond between the ethylene glycol units and water molecules.17

As good as these molecules, their long-chain organic layers are not desirable for many electrochemical methods because they form a high-impedance layer, which prevents faradaic electrochemistry from occurring with the underlying electrode.18 The most common alternative strategy employed is “backfilling” of non-sensing regions in the biointerface with bovine serum albumin (BSA) to provide resistance of nonspecific protein adsorption to the sensing interfaces.19,20 This strategy also gives high impedance layers and there is concern about the dynamic nature of these protein layers, which can influence the reproducibility and reliability of a sensor.7, 21, 22

A solution to forming antibiofouling layers that are not high impedance comes from recent active exploration of zwitterionic species containing phosphorylcholine (PC) moiety which confers to surfaces high resistance to protein adsorption.18, 23-26 Related to this solution is the development of the application of reductive electrografting of aryldiazonium salts derived layers onto conducting or semi-conducting surfaces which 78

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

can give robust, thick films on electrodes that do not completely passivate the electrode from performing faradaic electrochemistry.27-30 Gooding group has investigated the combination of these ideas by modifying of glassy carbon, gold and indium tin oxide (ITO) electrodes by electrografting the aryldiazonium salts of phenyl phophorylcholine (PPC).18, 24, 25 The resultant PPC layer endows exceptional anti-biofouling performance with sufficiently low impedance that the modified layers exhibit appreciable electron transfer across the layer.24 To make this surface chemistry applicable to biosensing, methods for the electrodeposition of mixed layers containing PPC and a minor amount of carboxyphenyl species for attaching biomolecules have been developed.23 Such mixed layers surfaces have the advantages of 1) low-impedance, 2) anti-biofouling capability and 3) controllable surface composition where the bio-recognition component is diluted in an anti-biofouling layer.5, 23, 31-33

In this chapter, this surface chemistry is developed further to allow affinity biosensing applications in complex samples. Herein, mixed layers of zwitterionic phenyl phophorylcholine (PPC) and phenyl butyric acid (PBA) were developed, in which PPC take responsibility to repel nonspecific protein adsorption while PBA allows the bioconjugation of antibodies to the electrode as the bio-recognition elements. To demonstrate the sensing capability of this electrode interface, the detection of tumor necrosis factor α (TNF-α) in undiluted whole blood was performed using a “sandwich” assay format (Scheme 5.1). TNF-α is a 17.5 kDa pro-inflammatory cytokine that can mediate a variety of biological effects such as immune regulation, antitumor activity, viral replication, and infection resistance.34-36 It is at the low level (pg/mL range) in healthy human blood, whereas a several-fold increase is observed in septic patients.37 Overproduction of TNF-α occurs in numerous pathological conditions, including cancer, heart disease, diabetes and autoimmune diseases. This allows TNF-α to be a typical early-stage indicator of an inﬂammatory reaction, in response to infection or cancer.38,39 Therefore, the development of sensitive and specific assay methods for the detection of the trace biomarkers is very important for the understanding of tumor

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

biological process, inherent mechanism, discovering drugs, and it has a therapeutic potential for the treatment of diseases.40,41

Scheme 5.1 Schematic of the PPC-PBA mixed layers/ITO based immunosensor for the detection of TNF-α.

5.2 Experimental Section

5.1.1 Reagents and Materials

All chemicals and solvent used were obtained and prepared as described in Chapter 2 unless otherwise stated.

5.2.2 Fabrication of PPC-PBA Mixed Layers on ITO.

The cleaning procedure of ITO electrodes is described in Chapter 2.3. The cleaned

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

electrodes were then modified with PPC-PBA mixed layers by holding depositing potential at −0.65 V vs. Ag/AgCl for 2 min in the presence of a 0.1 M HCl solution containing 1 mM PPC-PBA aryldiazonium salts mixture solution (900 μM:100 μM) and 1 mM NaNO2.

5.2.3 X-ray Photoelectron Spectroscopy Characterization

XPS was performed to study the surface chemical composition as described in the Chapter 2.4

5.2.4 Electrochemical Apparatus

All electrochemical apparatus used in this chapter are described in the Chapter 2.5

5.2.5 Immunoassay Development

The capture antibody (Ab1) was immobilized onto the PPC-PBA/ITO surface via the classical EDC/NHS conjugation reactions between‒COOH groups on the mixed-layer surface and residual amino groups of the Ab1. Specifically, the PPC-PBA/ITO surfaces were activated by immersion in 0.01 M PBS (pH 7.4) containing 25 mg/mL EDC and 30 mg/mL NHS for 1 h at room temperature. Subsequently, 40 μL of 10 μg/mL Ab1 was spread onto the resulting electrode surface to allow incubation at 4 °C in an ice bath for 1 h. Next 40 μL of TNF-α with a series of concentrations was dropped onto the Ab1 modified surfaces and incubated at room temperature for 1 h. After binding between Ab1 and TNF-α, the electrodes were finally incubated in 40 μL of 5 μg/mL HRP conjugated detection antibody (HRP-Ab2) to obtain the final sensing interface of HRP-Ab2/TNF-α/Ab1/PPC-PBA/ITO. The electrodes were rinsed thoroughly with PBS after each step. To test the specificity of developed immunosensor, hemoglobin (5

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

μg/mL) and human serum albumin (HSA, 5 μg/mL) were used as interfering proteins in the place of TNF-α. In whole blood assay, 40 μL of blood instead of standard TNF- α was added into Ab1/PPC-PBA/ITO interface, other steps were the same as above- mentioned protocol.

Electrochemical measurements were carried out in 1.0 mL of 0.01 M PBS (pH 7.4) containing 2.0 mM H2O2 and 0. 1 mM ferrocenemethanol as redox mediators with an applied potential of -0.05 V vs Ag/AgCl.

5.2.6 Electrochemical Evaluation of In-Situ Protein-Resistance Behavior

EIS was performed to examine the behaviour of final immunosensor in resisting nonspecific adsorption of proteins using a redox probe solution of 0.1 M KCl containing

1 mM K3[Fe(CN)6] and 1 mM K4[Fe(CN)6]. The final sensing interfaces (HRP- Ab2/TNF-α/Ab1/PPC-PBA/ITO) were rinsed thoroughly with water before incubating in the fresh solution containing the redox probe and EIS measurements were then performed. Subsequently, these electrodes were rinsed with water, followed by EIS measurements in the redox probe solution containing 1 mg/mL HSA. After incubation for 1 h, EIS were performed again. EIS spectra were recorded in the frequency range of 105 to 10−1 Hz. An AC potential with 0.01 V peak to peak separation was superimposed on a DC potential of 0.212 V. Impedance data were recorded and analysed using ZPlot and ZView 3.1 softwares (Scribner Associates, Inc.), respectively.

5.3 Results and Discussion

5.3.1 Formation of PPC-PBA Mixed Layers on ITO

The reductive adsorption behavior of the two individual aryldiazonium salts and the

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

mixture of the two aryldiazonium salts was first investigated. It is important to note the choice of PBA, as distinct from the carboxyphenyl derivative used previously,23 was because the similarity in reductive adsorption potential of PPC and PBA made forming mixed layers of controllable surface composition potentially simpler. A typical cyclic voltammogram of 1 mM PPC diazonium salts solution on the ITO surface shows a single irreversible reduction peak at ‒0.55 V (Figure. 5.1a) during the first reduction scan and PBA diazonium salts shows a similar reduction potential at ‒0.58 V (Figure. 5.1b). Importantly, the cyclic voltammogram of a mixed PPC-PBA diazonium salts solution (Figure. 5.1c) exhibits one broader reduction peak at -0.56 V in the first cycle, which is comparable to those in Figure 5.1a and b.

Figure 5.1 Cyclic voltammograms for reductive adsorption of aryldiazonium salts on 83

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

ITO electrode from (a) 1 mM 4-aminophenyl phosphorylcholine (PPC) in 0.1 M HCl, (b) 1 mM 4-(4-aminophenyl)butyric acid (PBA) in 0.1 M HCl and (c) 1 mM mixture of PBA and PPC (1:1 molar ratio) in 0.1 M HCl, with 1 mM NaNO2 at a scan rate of 0.1 V/s.

Mixed layers of PPC-PBA on ITO were prepared using a fixed potential of ‒0.65 V for 2 min and XPS was used to examine the surface chemical composition (Figure 5.2). The characteristic peaks of phosphorous (~133.8 eV), quaternary nitrogen (~403 eV) from phosphorylcholine moiety of PPC and carboxylic acid group (~289.0 eV) from PBA were observed, indicating successful electrografting of mixed layers of PPC and PBA onto ITO electrode. The azo group located at around 399.7 eV is quite common in the case of aryldiazonium salts modified surfaces, which can be attributed to direct attachment of aryldiazonium cations to the surface or to the as-grafted layers without losing nitrogen.42 The surface ratio of PPC:PBA was calculated to be 8.1:1 by using atomic concentration of P 2p and ‒COOH (Chapter 2.4, equation 1), which is close to their molar ratio of 9:1 in deposition solution. This can be attributed to their similar electrochemical reactivity during the electrografting process. It is reported that the reduction potential plays a key role in determining reactivity of aryldiazonium salt, the more positive reduction potential, the higher reactivity.43 Therefore, it is difficult to adjust surface ratios of two aryldiazonium salts with different reduction potentials, particularly in achieving poor-reactivity layer dominates on surface when simultaneously electrodepositing from a solution of mixed aryldiazonium salts. In latest study, surface ratio of 6:1 was obtained even though the molar ratio of the aryldiazonium salts PPC (reduction potential -0.55 V vs. Ag/AgCl) and carboxyphenyl (reduction potential -0.25 V vs. Ag/AgCl) was 10000:1.23

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

Figure 5.2 P 2p, N 1s and C 1s core level XPS spectra for PPC-PBA mixed layers modified ITO. Electrografting is carried out in a solution of 0.1 M HCl containing PPC- PBA diazonium salts with molar ratio of 9:1 (total concentration is 1 mM) and deposition potential and deposition time were fixed at ‒0.6 V vs. Ag/AgCl and 2 min, respectively.

5.3.2 In-Situ Antifouling Evaluation of Sensing Interface

The antifouling behavior of the final sensing interface (HRP-Ab2/TNF-α/Ab1/PPC- PBA/ITO) was investigated using cyclic voltammetry and impedance spectroscopy based on blocking effect and charge transfer resistance (Rct), respectively. To make the sensor evaluation more compatible with analysis of actual human fluids, solutions containing HSA were employed as biofouling. In the in-situ antibiofouling test, the Rct of final sensing interface (77.9 ± 9.8 Ω cm2) was measured first in redox probe solution

3-/4- of [Fe3(CN)6] , followed by examining Rct instantly after replacement with the redox probe solution containing HSA (1 mg/mL). As shown in Figure. 5.3b, the Rct value increased slightly to 82.4 ± 12.5 Ω cm2, however, the peak separation and peak current kept almost constant. Even after the electrodes were held stationary in the HSA solution

2 for 1 h, the Rct value was found to increase slightly to 104.6 ± 9.8 Ω cm , while the peak separation and peak current in cyclic voltammogram (Figure 5.3a) remained almost unchanged. The above results indicated that the sensing interface represents a high

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

tolerance towards high‒concentration nonspecific protein adsorption (only trace amount of protein can be adsorbed) in a long time scale.

Figure 5.3 Examination of antifouling property using (a) cyclic voltammetry and (b) electrochemical impedance spectroscopy. Black line and red line represent bare ITO

3-/4- and final immunosensor with redox probe solution of [Fe3(CN)6] , respectively. The blue line and dark cyan line represent the final immunosensor after addition of 1 mg/mL HSA, and incubation in 1 mg/mL HSA for 1 h.

5.3.3 Electrochemical Response to Hydrogen Peroxide at the Immunosensor

The electrocatalytical reactivity of the final immunosensor towards H2O2 was investigated by cyclic voltammetry. Figure. 5.4 (a) depicts the current response in the absence and presence of H2O2. A pair of redox peaks were observed in the blank substrate solution, which contributes to the redox reaction of ferrocene. It was observed an increase of both the oxidation current and reduction current upon the addition of

H2O2, which is in accordance with literature report of Fc mediated HRP catalytic

39-41 reaction toward H2O2. This result implies that an enzyme-dependent catalytic

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

current response of H2O2 which originates from the HRP reaction and the soluble redox mediator of ferrocenemethanol could effectively shuttle electrons from the base electrode.

This typical enzyme-dependent catalytic process (shown in Scheme. 5.1) can be expressed as follows:

H2O2 + HRP (red) H2O+ HRP (ox)

HRP (ox) +Fc HRP (red) + Fc+

Fc+ + e- Fc

First, H2O2 in the solution is reduced by the immobilized HRP. Then the reduced HRP is regenerated with the aid of the mediator (Fc), while Fc itself is oxidized in the enzymatic reaction. Finally, the oxidized Fc is electrochemically reduced on the electrode, leading to an increase of the reduction current. Since concentration of TNF- α influences the immobilized amount of HRP, which is further related to reductive current produced, thus a methodology depends on the relationship between concentration of TNF-α and reductive current signal can be developed to monitor TNF- α.

Inﬂuence of H2O2 on the response of immunosensor is of great importance, the amperometric response of the mixed layers based immunosensor for H2O2 reduction was thus examined at an applied potential of 0.05 V upon successive addition of H2O2 in a gentle stirring PBS containing 0.1 mM ferrocenemethanol. As illustrated in Figure.

5.4 (b) the immunosensor displays increasing amperometric responses to H2O2 with the linear ranges from 0.01 mM to 1.07 mM and the maximal cathodic peak current change can be obtained at ~2.0 mM H2O2 and then it started to reach a plateau. In order to avoid the irreversible transition of the HRP to its higher oxidized and inactive form at higher

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

42 H2O2 concentration, 2.0 mM of H2O2 was chosen for following detection.

Figure 5.4 (a) Cyclic voltammograms of the final immunosensor at 0.1 mM Fc, pH 7.4

PBS in the absence (black line) and presence of 50 µM H2O2 (red line) with a scan rate of

20 mV/s. (b) Dependence of the chronoamperometric reduction current on the concentration of H2O2 for the PPC-PBA mixed layers-based immunosensor in pH 7.4 PBS containing 0.1 mM Fc at an applied potential of 0.05 V vs. Ag/AgCl. Error bars represent the standard deviation, n=3.

5.3.4 Analytical Performance of the Immunosensor for the Electrochemical Detection of TNF-α

The performance of the immunosensor for TNF-α was investigated relative to the analytical criteria described in the introduction. As shown in Figure 5.5, the amperometric response of the immunosensor increased with increasing concentration of TNF-α. The calibration curve presented a good linear relationship between the current response and the logarithm of TNF-α concentrations within the range of 0.01- 500 ng/mL. The linear regression equation I = 11.18 lg [TNF-α] + 120.9 was established

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

with a correlation coefﬁcient of 0.9903. The lowest detected concentration was 10 pg/mL. This is similar to a previously reported value using a alkaline phosphatase functionalized gold nanoparticles/poly(styrene-co-acrylic acid) composite based electrochemical immunoassay.36

Figure 5.5 (a) Calibration plot of the PPC-PBA mixed layer based immunosensor toward TNF-α with diﬀerent concentrations in pH 7.4 PBS containing 0.1 mM Fc and

2 mM H2O2 at an applied potential of 0.05 V vs Ag/AgCl. Error bars represent the standard deviation, n=3. (b) Chronoamperometric current response of the immunosensor towards concentrations of TNF-α ranging from 0.01 ng/mL to 500 ng/mL (0.01, 0.05, 0.5, 5, 50, 500, ng/mL).

With the stated goal of developing an electrochemical immunosensor that can operate in complex biological fluids, the selectivity of the chronoamperometric response was studied with regards to proteins such as hemoglobin (Hb), human serum albumin (HSA) that might nonspecifically adsorb onto the sensing interface. The study was implemented by assaying the current response towards the low-concentration (0.5 ng/mL) target TNF-α in the presence of physiologically relevant concentrations (5 ng/mL) of potential proteins that could nonspecifically adsorb onto the sensing 89

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

interface. As shown in Figure 5.6, the current signal from interference proteins (HSA or Hb) is similar to that of negative control (PBS as blank sample) but much lower than the signal from target TNF-α. More importantly, the current signal from mixture of TNF-α and interfering proteins showed comparable results to that of TNF-α alone, indicating that the immunosensor exhibits high specificity towards TNF-α with limited influence from other nonspecifically adsorbed proteins.

Figure 5.6 Selectivity of the proposed mixed layers based immunosensor to 0.5 ng/mL TNF-α by comparing it to the interfering proteins at 5 ng/mL level: HSA and hemoglobin (Hb). Error bars represent the standard deviation, n=3.

5.3.5 Analysis in Blood

With the excellent performance of the electrochemical immunosensor in the presence of interfering proteins the next step was to challenge the device in blood samples. The results obtained from the electrochemical immunosensor were correlated with the

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

analyses performed using a commercially available ELISA kit as shown Table 5.1. The relative deviation between the electrochemical immunosensor and the ELISA was between 2.6 % and 11.7 %. These deviations are quite small and reveal that the experimental data from the developed immunosensor correlated well with those from the commercial ELISA kit. Therefore, the proposed immunosensor could be promising for the clinical determination of TNF-α.

Table 5.1 The comparison of TNF-α levels in whole blood detected by electrochemical assay and commercial ELISA method.

blood samples 1 2 3

Immunosensor (pg/mL)a 22.0 18.8 14.9

ELISA kit (pg/mL)a 19.7 17.9 15.3

Relative deviation (%)b 11.7 5.0 2.6 a The average value of three determinations. b Relative deviation =([TNF-α]immunosensor ‒ [TNF-α]ELISA kit)/ [TNF-α]ELISA kit

5.4 Conclusions

This chapter described an electrochemical immunosensor with integration of PPC antifouling layer and PBA layer for anchoring “sandwich” component of capture antibody-TNF-α-HRP conjugated detection antibody. This mixed layers based immunosensor allows sensitively and specifically detecting vital antigen of TNF-α with an extraordinarily wide dynamic range (0.01‒500 ng/mL) and minimum detection limit of 10 pg/mL. The antifouling behavior benefited from zwitterionic PPC is highlighted

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

as its tolerance of extreme high concentration of nonspecific protein (1 mg/mL of HSA). The developed immunosensor was applied to analyze non-pretreated whole blood samples, the results were comparable to that obtained from commercial ELISA kit, implying its great potential for future diagnosis of clinic biological samples. The mixed layers based sensing platform is versatile that can be extended to detect other biomarkers of interests.

5.5 References

1. Wang, J. Electrochemical biosensors: towards point-of-care cancer diagnostics. Biosens. Bioelectron 2006, 21 (10), 1887-1892.

2. Liu, G.; Qi, M.; Hutchinson, M. R.; Yang, G.; Goldys, E. M. Recent advances in cytokine detection by immunosensing. Biosens. Bioelectron 2016, 79, 810-821.

3. Chen, C.; Xie, Q.; Yang, D.; Xiao, H.; Fu, Y.; Tan, Y.; Yao, S. Recent advances in electrochemical glucose biosensors: a review. RSC Adv. 2013, 3 (14), 4473-4491.

4. Song, L.; Zhao, J.; Luan, S.; Ma, J.; Liu, J.; Xu, X.; Yin, J. Fabrication of a detection platform with boronic-acid-containing zwitterionic polymer brush. ACS Appl Mater Inter 2013, 5 (24), 13207-13215.

5. Huang, C. J.; Brault, N. D.; Li, Y.; Yu, Q.; Jiang, S. Controlled Hierarchical Architecture in Surface‐initiated Zwitterionic Polymer Brushes with Structurally Regulated Functionalities. Adv. Mater. 2012, 24 (14), 1834-1837.

6. Gerritsen, M.; Kros, A.; Sprakel, V.; Lutterman, J.; Nolte, R.; Jansen, J. Biocompatibility evaluation of sol–gel coatings for subcutaneously implantable glucose sensors. Biomaterials 2000, 21 (1), 71-78.

7. Downard, A. J.; Roddick, A. D. Protein adsorption at glassy carbon electrodes: the effect of covalently bound surface groups. Electroanal 1995, 7 (4), 376-378.

8. Barfidokht, A.; Gooding, J. J. Approaches toward allowing electroanalytical devices to be used in biological fluids. Electroanal 2014, 26 (6), 1182-1196.

9. Pelaz, B.; del Pino, P.; Maffre, P.; Hartmann, R.; Gallego, M.; Rivera-Fernandez, S.; de la Fuente, J. M.; Nienhaus, G. U.; Parak, W. J. Surface functionalization of nanoparticles with polyethylene glycol: effects on protein adsorption and cellular uptake. ACS nano 2015, 9 (7), 6996-7008. 92

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10. Riedel, T. s.; Riedelová-Reicheltová, Z.; Májek, P.; Rodriguez-Emmenegger, C.; Houska, M.; Dyr, J. E.; Brynda, E. Complete identification of proteins responsible for human blood plasma fouling on poly (ethylene glycol)-based surfaces. Langmuir 2013, 29 (10), 3388-3397.

11. Haque, A.-M. J.; Park, H.; Sung, D.; Jon, S.; Choi, S.-Y.; Kim, K. An electrochemically reduced graphene oxide-based electrochemical immunosensing platform for ultrasensitive antigen detection. Anal. Chem. 2012, 84 (4), 1871-1878.

12. Li, L.; Chen, S.; Zheng, J.; Ratner, B. D.; Jiang, S. Protein adsorption on oligo (ethylene glycol)-terminated alkanethiolate self-assembled monolayers: the molecular basis for nonfouling behavior. J. Phys. Chem. C. B 2005, 109 (7), 2934-2941.

13. Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G.; Laibinis, P. Molecular conformation in oligo (ethylene glycol)-terminated self-assembled monolayers on gold and silver surfaces determines their ability to resist protein adsorption. J. Phys. Chem. C. B 1998, 102 (2), 426-436.

14. Liu, G.; Gooding, J. J. An interface comprising molecular wires and poly (ethylene glycol) spacer units self-assembled on carbon electrodes for studies of protein electrochemistry. Langmuir 2006, 22 (17), 7421-7430.

15. Liu, G.; Zhang, Y.; Guo, W. Covalent functionalization of gold nanoparticles as electronic bridges and signal amplifiers towards an electrochemical immunosensor for botulinum neurotoxin type A. Biosens. Bioelectron. 2014, 61, 547-553.

16. Khor, S. M.; Liu, G.; Fairman, C.; Iyengar, S. G.; Gooding, J. J. The importance of interfacial design for the sensitivity of a label-free electrochemical immuno-biosensor for small organic molecules. Biosens. Bioelectron 2011, 26 (5), 2038-2044.

17. Xu, F.; Liu, L.; Yang, W.; Kang, E.; Neoh, K. Active protein-functionalized poly (poly (ethylene glycol) monomethacrylate)-Si (100) hybrids from surface-initiated atom transfer radical polymerization for potential biological applications. Biomacromolecules 2009, 10 (6), 1665-1674.

18. Gui, A. L.; Luais, E.; Peterson, J. R.; Gooding, J. J. Zwitterionic phenyl layers: finally, stable, anti-biofouling coatings that do not passivate electrodes. ACS Appl Mater Inter 2013, 5 (11), 4827-35.

19. Zhao, W.-W.; Ma, Z.-Y.; Yu, P.-P.; Dong, X.-Y.; Xu, J.-J.; Chen, H.-Y. Highly sensitive photoelectrochemical immunoassay with enhanced amplification using horseradish peroxidase induced biocatalytic precipitation on a CdS quantum dots multilayer electrode. Anal. Chem. 2011, 84 (2), 917-923.

20. Wang, G.-L.; Shu, J.-X.; Dong, Y.-M.; Wu, X.-M.; Li, Z.-J. An ultrasensitive and 93

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universal photoelectrochemical immunoassay based on enzyme mimetics enhanced signal amplification. Biosens. Bioelectron 2015, 66, 283-289.

21. Kongsuphol, P.; Ng, H. H.; Pursey, J. P.; Arya, S. K.; Wong, C. C.; Stulz, E.; Park, M. K. EIS-based biosensor for ultra-sensitive detection of TNF-α from non-diluted human serum. Biosens. Bioelectron 2014, 61, 274-279.

22. Li, X.; Cao, Y.; Kang, G.; Yu, H.; Jie, X.; Yuan, Q. Surface modification of polyamide nanofiltration membrane by grafting zwitterionic polymers to improve the antifouling property. J. Appl. Polym. Sci. 2014, 131 (23).

23. Jiang, C.; Alam, M. T.; Parker, S. G.; Darwish, N.; Gooding, J. J. Strategies to Achieve Control over the Surface Ratio of Two Different Components on Modified Electrodes Using Aryldiazonium Salts. Langmuir 2016, 32 (10), 2509-2517.

24. Jiang, C.; Alam, M. T.; Parker, S. G.; Gooding, J. J. Zwitterionic Phenyl Phosphorylcholine on Indium Tin Oxide: a Low-Impedance Protein-Resistant Platform for Biosensing. Electroanal 2015, 27 (4), 884-889.

25. Parviz, M.; Darwish, N.; Alam, M. T.; Parker, S. G.; Ciampi, S.; Gooding, J. J. Investigation of the Antifouling Properties of Phenyl Phosphorylcholine-Based Modified Gold Surfaces. Electroanal 2014, 26 (7), 1471-1480.

26. Shao, Q.; Jiang, S. Molecular understanding and design of zwitterionic materials. Adv. Mater. 2015, 27 (1), 15-26.

27. Mahouche-Chergui, S.; Gam-Derouich, S.; Mangeney, C.; Chehimi, M. M. Aryl diazonium salts: a new class of coupling agents for bonding polymers, biomacromolecules and nanoparticles to surfaces. Chem. Soc. Rev. 2011, 40 (7), 4143- 4166.

28. Liu, G.; Guo, W.; Yin, Z. Covalent fabrication of methyl parathion hydrolase on gold nanoparticles modified carbon substrates for designing a methyl parathion biosensor. Biosens. Bioelectron. 2014, 53, 440-446.

29. Taufik, S.; Barfidokht, A.; Alam, M. T.; Jiang, C.; Parker, S. G.; Gooding, J. J. An antifouling electrode based on electrode–organic layer–nanoparticle constructs: Electrodeposited organic layers versus self-assembled monolayers. J. Electroanal. Chem. 2016.

30. Gooding, J. J. Advances in interfacial design for electrochemical biosensors and sensors: aryl diazonium salts for modifying carbon and metal electrodes. Electroanal 2008, 20 (6), 573-582.

31. Santos, L.; Ghilane, J.; Lacroix, J. C. Formation of mixed organic layers by

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

stepwise electrochemical reduction of diazonium compounds. J. Am. Chem. Soc. 2012, 134 (12), 5476-5479.

32. Liu, G.; Paddon-Row, M. N.; Gooding, J. J. Protein modulation of electrochemical signals: application to immunobiosensing. Chem. Commun. 2008, (33), 3870-3872.

33. Keefe, A. J.; Jiang, S. Poly (zwitterionic) protein conjugates offer increased stability without sacrificing binding affinity or bioactivity. Nat. Chem. 2012, 4 (1), 59- 63.

34. Huang, T.; Nallathamby, P. D.; Xu, X.-H. N. Photostable single-molecule nanoparticle optical biosensors for real-time sensing of single cytokine molecules and their binding reactions. J. Am. Chem. Soc. 2008, 130 (50), 17095-17105.

35. Tagawa, M. Cytokine therapy for cancer. Curr. Pharm. Des. 2000, 6 (6), 681-699.

36. Yin, Z.; Liu, Y.; Jiang, L.-P.; Zhu, J.-J. Electrochemical immunosensor of tumor necrosis factor α based on alkaline phosphatase functionalized nanospheres. Biosens. Bioelectron 2011, 26 (5), 1890-1894.

37. Lee, S. J.; Li, Z.; Sherman, B.; Foster, C. S. Serum levels of tumor necrosis factor- alpha and interleukin-6 in ocular cicatricial pemphigoid. Invest. Ophthalmol. Vis. Sci. 1993, 34 (13), 3522-3525.

38. Camussi, G.; Albano, E.; Tetta, C.; Bussolino, F. The molecular action of tumor necrosis factor‐α. Eur. J. Biochem. 1991, 202 (1), 3-14.

39. Chen, X.; Chang, J.; Deng, Q.; Xu, J.; Nguyen, T. A.; Martens, L. H.; Cenik, B.; Taylor, G.; Hudson, K. F.; Chung, J. Progranulin does not bind tumor necrosis factor (TNF) receptors and is not a direct regulator of TNF-dependent signaling or bioactivity in immune or neuronal cells. J. Neurosci. 2013, 33 (21), 9202-9213.

40. Sun, Z.; Deng, L.; Gan, H.; Shen, R.; Yang, M.; Zhang, Y. Sensitive immunosensor for tumor necrosis factor α based on dual signal amplification of ferrocene modified self-assembled peptide nanowire and glucose oxidase functionalized gold nanorod. Biosens. Bioelectron 2013, 39 (1), 215-219.

41. Mainz, E. R.; Serafin, D. S.; Nguyen, T. T.; Tarrant, T. K.; Sims, C. E.; Allbritton, N. L. Single Cell Chemical Cytometry of Akt Activity in Rheumatoid Arthritis and Normal Fibroblast-like Synoviocytes in Response to Tumor Necrosis Factor α. Anal. Chem. 2016. 88 (15), 7786–7792.

42. Mévellec, V.; Roussel, S.; Tessier, L.; Chancolon, J.; Mayne-LʼHermite, M.; Deniau, G.; Viel, P.; Palacin, S. Grafting polymers on surfaces: A new powerful and versatile diazonium salt-based one-step process in aqueous media. Chem. Mater. 2007,

Chapter 5 PPC-PBA Mixed Layers Based Immunosensor

19 (25), 6323-6330.

43. Louault, C.; D'Amours, M.; Bélanger, D. The electrochemical grafting of a mixture of substituted phenyl groups at a glassy carbon electrode surface. ChemPhysChem 2008, 9 (8), 1164-1170.

Chapter 6 Conclusions and Future Work

6.1 Conclusions

This thesis has presented systematic research work to develop a mixed aryl layers based protein-resistant and low-impedance interface for electrochemical biosensing application. It started from surface chemistry design with molecular system derived from electrografting of aryldiazonium salts, then surface characterization using electrochemical and spectroscopic tools, and finally sensing performance evaluation with real sample challenged.

In Chapter 3, zwitterionic layer of phenyl phosphorylcholine (PPC) was electrografted onto ITO surface and its protein-resistance performance was evaluated using electrochemical impedance spectroscopy. For an interface with good antifouling property, the charge transfer resistance (Rct) would keep constant due to rare amount of protein adsorption before and after protein incubation. While in this case, what I have learnt is that during electrografting, significant amounts of PPC gets physically adsorbed on the surface, which cannot be removed by simply rinsing with water or buffer, and as a consequence the electrochemical response of such electrode is quite unstable (the arydiazonium salts tend to have strong physisorption on ITO surface, thus after protein incubation, the Rct value was decreased). A simple protocol has been developed by incubating the modified surface with PBS for 2 h to remove the physically adsorbed PPC such that a stable interface is produced. It was found that such a stable surface, with appropriate amount of PPC, can dramatically reduce nonspecific adsorption of protein without hinder electron transfer.1

Chapter 6 Conclusions and Future Work

In Chapter 4, mixed layers platform was built by introducing another layer of carboxylphenyl (CP) into PPC-ITO. PPC and CP are responsible for antifouling and bio-recognition. To control surface composition of the moieties, the relationship between their molars in deposition solution and surface ratios were studied using XPS. It was found that in simultaneous electrografting, CP layer is always take domination in spite of its proportion in deposition solution due to its more positive reduction potential and thus higher reactivity. In consecutive electrgrafting, PPC was selectively deposit first with higher concentration and longer deposition time, followed by depositing CP with lower concentration and shorter deposition time. More PPC than CP can be achieved using this approach (PPC:CP=200:1). Moreover, good antifouling and accessible -COOH can be obtained by careful experimental condition settings and were examined with impedance spectroscopy and AC voltammetry, respectively.2 The lesson can be learnt is that electrochemical reactivity (dominated by reduction potential) plays an important role in final surface composition in simultaneous electrografting of mixtures of aryldiazonium salt. The component with more positive reduction potential shows greater priority on the surface than the component with less positive reduction potential.

To show the capability of the developed mixed layers platform for sensing application, PPC and phenyl butyric acid (PBA) with similar reduction potential were used. "Sandwich" immune unit of capture antibody-cancer biomarker-enzyme conjugated detection antibody (HRP-detection Ab2-TNF-α-capture Ab1) was attached and was shown in Chapter 5, The catalytic current response towards gradual addition of H2O2 was identical to Michaelis-Menten kinetics, the antifouling perform was very good with in-situ HSA, the dose-response towards different concentrations of TNF-α was found to be a wide dynamic range. The developed immunosensor was challenged with

Chapter 6 Conclusions and Future Work

real blood samples without any pretreatments like centrifugation or dilution. The obtained results were comparable with that from commercial ELISA kit, indicating the developed immunosensor is promising for clinic use and can be extended to detect different types of biomarkers.

In summary, considerable progress has been achieved in the development of electrochemical biosensor using aryldiazonium electrochemistry based mixed layers. The molecular-level control to obtain a multifunctional platform with advantages of easy fabrication, good antifouling, low impedance, robustness and validity for sensing application was achieved. The future work will mainly focus on how to further improve the sensing performance in terms of sensitivity and selectivity, and how to extend applications of mixed-layers platform will be discussed in the following sections.

6.2 Future Work

6.2.1 Multiple Enzyme Strategy for Signal Amplification

To further improve the sensitivity, a multiple-enzyme strategy can be applied.3 Instead of conjugation of enzyme (e.g. HRP) to detection antibody (Ab2), carbon nanomaterials like graphene oxide,4carbon nanotubes,5 gold nanoparticles6-7 can be used as carrier to load detection antibody (Ab2) and enzymes to make a HRP-Ab2@nanomaterials biocomposite. In this biocomposite, Ab2 is served as scaffold to link target biomarker, multiple enzymes are used to amplify catalysis current signal, and nanomaterials with their high conductivity, intrinsic catalytic property can further improve the sensitivity of developed sensors. In this strategy, normally, mixture of enzyme and Ab2 with excess amount of enzymes than Ab2 (tens of to hundreds of times more) are used, and sensitivity can be significantly enhanced with a magnitude of 10-100 times compared to non-multiple enzyme approach as reported in the literatures.

Chapter 6 Conclusions and Future Work

Figure 6.1 Illustration of a multiple enzyme strategy through enzyme-detection antibody@AuNPs biocomposite to enhance sensitivity.

6.2.2 Application of External Electric Field to Enhance Protein-resistance Behavior

PPC based zwitterionic antifouling layers have been demonstrated to effectively resist nonspecific protein adsorption using electrochemical techniques. Very trace amount of protein can be adsorbed onto electrode surface which has minor influence on electrochemical signal. It is clear that all surface chemistry (no matter PPC or PEG based) cannot provide 100% antifouling, so it is necessary to find out other ways to improve the antifouling behavior. Considering that different types of nonspecific proteins have different charges (e.g. BSA is negatively charged while cytochrome C is positively at physiological pH), moreover, the binding events between specific binding (e.g. antibody attaches to antigen) and nonspecific binding (e.g. nonspecific proteins bind to sensing interface) have significant differences. Specific bindings can happen within quicker time scale than nonspecific bindings due to the high bio-affinity while 100

Chapter 6 Conclusions and Future Work

nonspecific protein follow the process of approaching- connect with electrode- unfolding-final adsorption. Thus, it is possible to repel nonspecific protein ways from sensing interface by using a pulsing potential. Specifically, the charge of final sensing interface has a point of zero charge (PZC), when applied potential is higher than PZC, the whole system is positively charged and same charged protein can be repelled and vice versa. Therefore, if a pulsing potential which can sweeping between more positive and more negative potentials than that of PZC, it is possible to resist negatively and positively charged proteins. And for neutrally charged protein at physiological pH, PPC is responsive for defending sensing interface since it is balanced charged at such pH and a hydrated layer is formed. External electric field have been applied to mitigate electrostatics for surface DNA hybridization,8 fouling alleviation in electro-membrane bioreactor,9 modulation of protein behaviors.10 However, there has been no research reported using electric field to enhance surface chemistry based antifouling property in a real biosensing system. So it is necessary and promising to investigate such potential to further enhanced antifouling and thus specificity of a biosensor in complex biological fluids. Another point may need to be considered is realization of electrochemical detection and adjusting electric field using one electrochemical workstation.

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Chapter 6 Conclusions and Future Work

Figure 6.2 Electric filed further enhances antifouling behavior in biosensing.

6.2.3 Link RGD Peptide for Single-cell Study

The mixed layers/ITO can be explored to be ideal platform for single cell study. The ITO electrode is electrically conductive and optically transparent, electrochemical and optical detection can be combined to allow acquisition of multi-dimensional information. Specifically, RGD peptide of Arg-Gly-Asp can be linked to ‒COOH in PBA through EDC/NHS coupling reaction, then cell of interest can be spread onto the interface. If cell is pre-stained with fluorescent dyes and highly diluted, fluorescence imaging combined with an electrochemical technique (e.g. impedance spectroscopy measurement)11-13 can permit single cell real time detection. Different stimuli such as different potentials or drugs can be applied and cell response can be monitored and collected, which would be informative for drug discovery and development of personalized nanomedicine therapy.

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Figure 6.3 Single cell study based on the mixed layers system with a combination of electrochemical workstation and optical imaging.

6.3 References

1. Jiang, C.; Alam, M. T.; Parker, S. G.; Gooding, J. J. Zwitterionic Phenyl Phosphorylcholine on Indium Tin Oxide: a Low-Impedance Protein-Resistant Platform for Biosensing. Electroanal 2015, 27 (4), 884-889.

2. Jiang, C.; Alam, M. T.; Parker, S. G.; Darwish, N.; Gooding, J. J. Strategies to Achieve Control over the Surface Ratio of Two Different Components on Modified Electrodes Using Aryldiazonium Salts. Langmuir 2016, 32 (10), 2509-2517.

3. Lei, J.; Ju, H. Signal amplification using functional nanomaterials for biosensing. Chem. Soc. Rev. 2012, 41 (6), 2122-2134.

4. Song, Y.; Qu, K.; Zhao, C.; Ren, J.; Qu, X. Graphene oxide: intrinsic peroxidase catalytic activity and its application to glucose detection. Adv. Mater. 2010, 22 (19), 2206-2210.

5. Yu, X.; Munge, B.; Patel, V.; Jensen, G.; Bhirde, A.; Gong, J. D.; Kim, S. N.; Gillespie, J.; Gutkind, J. S.; Papadimitrakopoulos, F. Carbon nanotube amplification strategies for highly sensitive immunodetection of cancer biomarkers. J. Am. Chem. Soc. 2006, 128 (34), 11199-11205.

6. Cao, X.; Ye, Y.; Liu, S. Gold nanoparticle-based signal amplification for biosensing. Anal. Biochem. 2011, 417 (1), 1-16.

7. Wang, J.; Meng, W.; Zheng, X.; Liu, S.; Li, G. Combination of aptamer with gold nanoparticles for electrochemical signal amplification: application to sensitive detection of platelet-derived growth factor. Biosens. Bioelectron. 2009, 24 (6), 1598- 1602.

8. Tymoczko, J.; Schuhmann, W.; Gebala, M. Electrical Potential-Assisted DNA Hybridization. How to Mitigate Electrostatics for Surface DNA Hybridization. Acs. Appl. Mater. Inter 2014, 6 (24), 21851-21858.

9. Zhang, J.; Satti, A.; Chen, X.; Xiao, K.; Sun, J.; Yan, X.; Liang, P.; Zhang, X.; Huang, X. Low-voltage electric field applied into MBR for fouling suppression: Performance and mechanisms. Chem. Eng. J. 2015, 273, 223-230.

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10. Xie, Y.; Pan, Y.; Zhang, R.; Liang, Y.; Li, Z. Modulating protein behaviors on responsive surface by external electric fields: A molecular dynamics study. Appl. Surf. Sci. 2015, 326, 55-65.

11. Meunier, A.; Jouannot, O.; Fulcrand, R.; Fanget, I.; Bretou, M.; Karatekin, E.; Arbault, S.; Guille, M.; Darchen, F.; Lemaître, F. Coupling amperometry and total internal reflection fluorescence microscopy at ITO surfaces for monitoring exocytosis of single vesicles. Angew. Chem. Int. Ed. 2011, 50 (22), 5081-5084.

12. Amatore, C.; Arbault, S.; Chen, Y.; Crozatier, C.; Lemaître, F.; Verchier, Y. Coupling of electrochemistry and fluorescence microscopy at indium tin oxide microelectrodes for the analysis of single exocytotic events. Angew. Chem. Int. Ed. 2006, 45 (24), 4000-4003.

13. Miomandre, F.; Meallet-Renault, R.; Vachon, J.-J.; Pansu, R. B.; Audebert, P. Fluorescence microscopy coupled to electrochemistry: a powerful tool for the controlled electrochemical switch of fluorescent molecules. Chem. Commun. 2008, (16), 1913-1915.

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