Dr Jason Davis (University of Oxford) has pioneered the application of scanning probe and fluorescence imaging technology to the analysis of bioelectrochemical interfaces; an understanding and control of which is clearly highly beneficial to the development of improved biosensing devices. From the early days of such studies, carried out with Professor Allen Hill FRS, his work has been refined to a level where single, active enzymes and proteins on electrode surfaces can be scrutinised under physiological and electrochemically-controlled conditions. Ground-breaking genetic methodologies are being applied to the generation of enzyme, protein or aptamer molecules which can be self-assembled, in an active form, on metallic electrode surfaces. His research group are also actively engaged in the assembly and construction of host-guest coordination complexes on surfaces, electroanalysis, molecular manipulation and molecular electronics. The group have published more than 80 papers in international journals.

Engineering the Bioelectronic Interface

Applications to Analyte Biosensing and Protein Detection

By Jason Davis

The Royal Society of Chemistry

Copyright © 2009 Royal Society of Chemistry
All rights reserved.
ISBN: 978-0-85404-165-7

Contents

Chapter 1 Communication with the Mononuclear Molybdoenzymes: Emerging Opportunities and Applications in Redox Enzyme Biosensors Paul V. Bernhardt, 1,
Chapter 2 Scanning Probe Analyses at the Bioelectronic Interface Jason J. Davis, Ben Peters, Yuki Hanyu and Wang Xi, 25,
Chapter 3 Electrical Interfacing of Redox Enzymes with Electrodes by Surface Reconstitution of Bioelectrocatalytic Nanostructures Itamar Willner, Ran Tel-Vered and Bilha Willner, 56,
Chapter 4 Single-wall Carbon Nanotube Forests in Biosensors James F. Rusling, Xin Yu, Bernard S. Munge, Sang N. Kim and Fotios Papadimitrakopoulos, 94,
Chapter 5 Activating Redox Enzymes through Immobilisation and Wiring H.A. Heering and G.W. Canters, 119,
Chapter 6 Cytochromes P450: Tailoring a Class of Enzymes for Biosensing Vikash R. Dodhia and Gianfranco Gilardi, 153,
Chapter 7 Label-free Field Effect Protein Sensing Jan Tkac and Jason J. Davis, 193,
Chapter 8 Biological and Clinical Applications of Biosensors Paul Ko Ferrigno, 225,
Subject Index, 243,

CHAPTER 1

Communication with the Mononuclear Molybdoenzymes: Emerging Opportunities and Applications in Redox Enzyme Biosensors

PAUL V. BERNHARDT

Centre for Metals in Biology, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane 4072, Australia

1.1 Introduction – the Three Mo Enzyme Families

The mononuclear Mo-enzymes are remarkable in their coherence of active site structure and function yet equally interesting in the diversity of substrates that they are capable of oxidising or reducing. Apart from the well-studied enzyme nitrogenase, where the Mo ion is found within a S-bridged cluster of metals including Fe, all other enzymes containing Mo bear a single metal at the active site. A recent exception to this may be the novel Mo enzyme CO dehydrogenase, where a Cu ion shares a sulfide bridging ligand with the Mo ion at the active site.

All known enzymes from this family bear either one or two bidentate pterindithiolene (molybdopterin, MPT) ligands bound to the Mo ion at the active site (Figure 1.1). Hille proposed a classification of this group of enzymes into three families based on the coordination environment of the metal as shown in Figure 1.1.

At this time, enzymes from the DMSO reductase family (the most diverse of all) have only been found in bacteria and archea whilst enzymes from the other two families are found in all forms of life.

1.2 Mechanism

Although subtle differences exist in the mechanism of the mononuclear Mo enzymes, as a starting point, the reactions catalysed by this enzyme superfamily can be generalised by eqn (1) written in either the forward (reductase) or reverse (oxidase/dehydrogenase) direction. The substrates represented by the generic symbols Z and ZO are apparent from the names of the respective oxidases/ dehydrogenases (Z) or reductases (ZO) shown in Figure 1.1.

ZO + 2e- + 2H+ -> Z + H2O (1)

The use of Mo enzymes in electrochemically driven (amperometric) biosensors relies on connecting a working electrode with the redox active species involved in the catalytic cycle i.e. the enzyme and/or its substrates and products. The electrons required to sustain catalysis are provided or accepted by the electrode rather than the enzyme’s natural cosubstrate. The various ways in which this can be done are summarised in the following section, but the most relevant point is that the Mo ion at the active site always cycles between its MoVI and MoIV oxidation states during catalysis and an O-donor ligand (oxo or hydroxo) is exchanged with the substrate during turnover. The MoVI form is the catalytically active form of the oxidases/dehydrogenases whilst the reductases must be reduced to MoIV before turnover can commence. The MoV form is a thermodynamically stable intermediate in most, but not all, cases, but it is incapable of turning over substrates in either direction. However, it may become an important rate-limiting intermediate in some cases.

Ligands bonded to the Mo ion are activated by coordination to perform some remarkable bond-breaking and formation reactions that otherwise do not occur in the absence of the enzyme. In the well-studied xanthine oxidor-eductases, a hydroxo ligand participates in a base-assisted nucleophilic attack at C-8 of xanthine coupled with a hydride abstraction by the sulfido ligand (Figure 1.2A). This mechanism is significantly di?erent from that seen in enzymes from the sulfite oxidase (Figure 1.2B) and DMSO reductase (Figure 1.2C) families where an oxo ligand is exchanged directly between the Mo ion and substrate during turnover.

1.3 Amperometric Biosensors

The development of enzyme-based biosensors in general has evolved over recent times as methods for addressing the active sites of enzymes have become better understood. Initially, enzyme electrochemistry relied upon the voltammetric detection of either the product or cosubstrate (so-called first-generation biosensors, Figure 1.3). The most common analyte that has been detected in this way is hydrogen peroxide, a typical product of oxidase enzyme turnover where the cosubstrate dioxygen is reduced in a two-electron proton-coupled reaction by the enzyme after substrate turnover. Alternatively the product itself may be electroactive but this is exceptional. A complementary approach is to monitor the depletion of cosubstrate, e.g. dioxygen, during turnover. However, this approach has limitations as variations in dioxygen concentrations may result from changes to the solution during analysis, e.g. temperature, stirring etc., and thus give false readings.

Second generation biosensors removed the natural cosubstrate from the system altogether, replacing it with a small molecule electron transfer mediator e.g. a redox active coordination compound or organic molecule. This approach has its roots in traditional enzyme solution assays where a mediator is oxidised or reduced chemically rather than electrochemically to drive the catalytic reaction. In electrochemistry, the mediator serves the dual purpose of (i) undergoing homogeneous electron transfer with the enzyme to restore it to its active form following turnover and (ii) undergoing heterogeneous electron transfer with the working electrode to provide the current that quantifies the enzyme-substrate reaction. This approach underpins most commercial enzyme biosensors to date including the glucose oxidase biosensor, which utilised a ferrocenium mediator (rather than dioxygen) as its artificial cosubstrate. The use of redox active polymers adsorbed on the electrode also comes under this classification. In all cases it should be emphasised that the currents observed are due to the mediator and they appear at the formal potential of the mediator and not of the enzyme. Ideally the redox potential of the mediator is in the vicinity of that of the active site. This avoids excessively large overpotentials which may lead to non-specific redox reactions with species in the sample other than the substrate.

The final approach (third generation) is to remove all cosubstrates from the system (natural or artificial) and to achieve direct electron exchange between the enzyme and the electrode. Although this has yet to be applied in a commercial biosensor, it offers significant advantages over (ternary) mediated systems, which each require a certain artificial electron relay tailor-made for the enzyme in question. The challenges in achieving direct enzyme electrochemistry are many. These include avoiding enzyme denaturation of the necessarily electrode-adsorbed enzyme whilst ensuring efficient electronic communication between the active site (or other redox cofactors within the enzyme) and the electrode. However, significant progress has been made in the last 15 years or so and a number of robust enzyme electrode systems have been reported which require no mediators. In the absence of mediators, which mask electron transfer events with the enzyme, some interesting mechanistic studies have been possible which provide new insight to electron transfer pathways in complex enzyme systems.

The following section will review the evolution of Mo enzyme based electrochemical biosensors over recent times with examples of all three types of biosensor being covered. The ordering of sections follows that of the three enzyme sub-families.

1.4 Emerging Applications of Mo Enzymes in Sensing

1.4.1 Xanthine Oxidase Family

1.4.1.1 Xanthine Oxidoreductase

Xanthine oxidase from bovine milk is the most studied of all mononuclear Mo enzymes. The volume of literature on this enzyme alone and its employment in enzyme electrode biosensors far outweighs all other Mo enzymes accordingly. Xanthine oxidase primarily catalyses the oxidation of xanthine to uric acid as part of the process of purine metabolism, but it also is capable of oxidising hypoxanthine to xanthine (Figure 1.4).

Regardless of their origin, all xanthine oxidase/dehydrogenase enzymes contain an active site comprising a single bidentate molybdopterin chelate, an equatorial terminal sulfido, an axial oxo and an equatorial hydroxo/aqua ligand depending on pH (Figure 1.1). The enzymes from this family bear three additional redox cofactors comprising two [2Fe-2S] clusters and an FAD cofactor. Crystal structures of various xanthine oxidoreductases have shown that electrons flow along the pathway Mo[right arrow][2Fe-2S] [right arrow][2Fe-2S] [right arrow]FAD. The FAD cofactor is oxidised by either NADP+ or dioxygen depending on whether it is present in its dehydrogenase or oxidase form.

Like many other oxidase enzymes, hydrogen peroxide is a product of substrate turnover in xanthine oxidase when dioxygen is the cosubstrate. The electroactivity of H2O2 enables its voltammetric detection and provides a method for monitoring turnover without requiring direct or mediated electron transfer with the enzyme itself. A wide variety of electrode systems have been described that utilise immobilised xanthine oxidase to produce H2O2 as an electrochemically detectable product or alternatively to monitor the depletion of cosubstrate dioxygen.

Hypoxanthine in particular is an analyte of interest as its presence is an indicator of spoilage in otherwise fresh fish and beef products. Sol-gel methods were used to produce an electrode coated with a silica-graphite matrix in which xanthine oxidase is entrapped. Following substrate (hypoxanthine) turnover, the H2O2 produced is detected electrochemically by poising the electrode at 0.58 V vs. Ag/AgCl. Alternatively the complementary consumption of dioxygen may be determined at low potential voltammetrically (mediated by viologens) or with an oxygen electrode. In H2O2 production mode the sensor maintained a linear current/concentration response up to 500 µM hypoxanthine. Above this substrate concentration the response saturated following Michaelis–Menten kinetics (KM,app 450 µM). A lower detection limit of 1.3 µM hypoxanthine was reported. The high surface area and biocompatibility of the silica matrix was found to be ideal for encapsulating the enzyme/graphite composite. Furthermore, the mild synthetic sol-gel techniques enabled the enzyme to be incorporated during the synthesis of the matrix.

Screen-printed xanthine oxidase electrodes have also been reported using a vast array of mediators including metal oxides (RuO2, Pd-IrO2) within the matrix to lower the overpotential for H2O2 oxidation. Even more elaborate bienzyme systems have been developed comprising both xanthine oxidase and peroxidase (an enzyme that reduces H2O2 to water) where the H2O2 produced by xanthine oxidase turnover is quantified by the current produced through its ferrocyanide-mediated reduction by peroxidase thus enabling the bi-enzyme system to function at a low potential (ca. -100 mV vs. Ag/AgCl) and minimising possible interferences from oxidation of other analytes at higher potentials.

A miniaturised xanthine oxidase electrode has been developed for monitoring the concentrations of hypoxanthine in myocardial cell culture media. Purines are also associated with signalling in the nervous system and multi-enzyme electrodes (including xanthine oxidase) have been developed to monitor the local changes in purine concentrations in vivo. The enzyme purine nucleoside phosphorylase (NP) catalyses the phosphorylation of inosine (by phosphate) to release hypoxanthine and ribose-1-phosphate (Scheme 1.1). The stoichiometry of the overall reaction coupled with xanthine oxidase activity means that one equivalent of H2O2 is produced for every (hydrogen)phosphate anion present.

A number of groups have developed amperometric bi-enzyme systems of this type. Haemmerli et al. reported a bi-enzyme (NP/xanthine oxidase) system which exhibited a linear response up to 250 µM phosphate. Various ratios of the two enzymes were investigated and the most ideal was found to be 10 : 1 NP : xanthine oxidase. This novel approach provides a viable alternative to otherwise tedious wet chemical (colorimetric) methods for phosphate determination.

Purine analysis can also be achieved with this system. For example, the concentration of inosine (the cosubstrate, with phosphate, in eqn (2a)) can be determined. The so-called hypoxanthine ratio ([hypoxanthine]/([hypoxanthine]+[inosine]+[inosine-monophosphate]) is an important parameter in the analysis of fresh fish as hypoxanthine is a product of nucleotide degradation. Like hydrogen peroxide, uric acid (the product of enzymatic xanthine oxidation, Figure 1.4) is electroactive and it can also be detected electrochemically thus providing a method for quantifying xanthine oxidase turnover.

Electron transfer mediators can be used to great effect in providing a link between the electrode and the enzyme cofactors. There are many approaches that may be taken. Conducting polymers such as poly-p-benzoquinone and poly(mercapto-p-benzoquinone) have been used to provide a redox active matrix in which xanthine oxidase can be both immobilised and addressed electrochemically. The well-studied Os-pyridine-based hydrogel polymers developed by the Heller group effectively mediate electron transfer in horseradish peroxidase (HRP). This polymer has been cast on a glassy carbon electrode and coupled with xanthine oxidase which enables the production of H2O2 from (hypo)xanthine turnover to be monitored electrochemically via the Os-mediated reduction of HRP within the polymeric hydrogel. This is illustrated in Scheme 1.2.

The benefit of using Os-mediated reduction of HRP is that the biosensor functions at a low potential (ca. 0 mV vs. Ag/AgCl) relative to that required for the direct oxidation of H2O2 (4 600 mV), where other species may be oxidised non-specifically as well. The sensor responds to hypoxanthine in a continuous flow system (linear response up to 80 µM with a detection limit of 0.2 µM) and also xanthine but is unaffected by potential interference from glutamate, lactate, glucose and glutathione. Ascorbate interference (significant at a working potential of 0 mV) was negligible when the working electrode was poised at -200 mV vs. Ag/AgCl. Otherwise redox inert polymers such as Nafion® can be made electroactive by the inclusion of small cationic mediators such as methyl viologen, which ion-exchanges within the anionic polymer. Xanthine oxidase adsorbed on such a viologen-modified polymer (cast on a glassy carbon electrode) produces a hypoxanthine biosensor that functions in O2-consumption mode. In this case the viologen mediates the reduction of O2 and lowers the overpotential for reduction by about 100 mV relative to direct oxygen reduction at the electrode.

High potential redox mediators, such as ferrocenium derivatives, have been very effective in acting as artificial electron acceptors for a range of oxidase enzymes; glucose oxidase being the most famous example. Similarly, hydro xymethylferrocenium can accept electrons from xanthine oxidase (replacing dioxygen) to produce a mediated amperometric hypoxanthine biosensor. In this case the enzyme was entrapped within a membrane covering the carbon-paste working electrode. A wide linear range (up to 700 µM hypoxanthine with a detection limit to 0.6 µM) was reported.

Ironically, despite its intensive investigation by enzymologists and spectroscopists for almost 50 years, it is only recently that a direct electrochemical study of a xanthine oxidoreductase was reported, namely the bacterial xanthine dehydrogenase from R. capsulatus. Non-turnover signals from all cofactors were seen and EPR potentiometry was also undertaken to resolve the potentials of the cofactors. Pronounced catalytic voltammetry was seen in the presence of xanthine. The bell-shaped pH profile of the catalytic wave mirrored that seen in solution. A high pH deprotonation of xanthine (pKa 7.7) and a low potential protonation of a glutamate residue (pKa 6) essential for base catalysis each switch off catalysis. An unusual feature of this study was that the potential at which catalysis was observed was ca. 600 mV more positive than that of the highest potential cofactor (FAD). Essentially the same potential “delay” in catalysis has been seen for bovine milk xanthine oxidase when immobilised on a pyrolytic graphite electrode.

(Continues…)Excerpted from Engineering the Bioelectronic Interface by Jason Davis. Copyright © 2009 Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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