Self-assembled monolayers and enzyme electrodes: Progress, problems and prospects

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CHIMICA ACTA ELSEVIER

Analytica

Chimica Acta 307 (1995) 277-289

Self-assembled monolayers and enzyme electrodes: progress, problems and prospects Stephen E. Creager *, Kimberly G. Olsen Department of Chemistry, Indiana University, Bloomington, IN 47405, USA Received 8 August 1994; revised 20 September

1994; accepted

12 October 1994

Abstract Initial results on the combined use of self-assembled monolayers and redox enzymes on electrodes to prepare electrochemical sensors are presented. Specifically, electrodes coated with self-assembled monolayers of 6-mercaptohexanol and 11-mercaptoundecanol are shown to exhibit dramatically reduced background currents relative to uncoated electrodes, and addition of a glucose oxidase layer on top of the self-assembled monolayer yielded electrodes which responded to glucose (in the presence of a soluble redox mediator) while still retaining the diminished background currents. It is shown that oxidation of ascorbate, urate, 4-acetamidophenol and hydrogen peroxide, and reduction of oxygen, are strongly suppressed at monolayer-coated gold electrodes relative to uncoated gold electrodes. This suppression is the source of the reduced background currents at the monolayer-coated electrodes, however, it also dictates that sensor strategies based on detection of hydrogen peroxide produced by enzyme-catalyzed reactions will not work with these electrodes. It is furthermore shown that oxidation of selected redox mediators, e.g. hydroxymethylferrocene, can proceed at monolayer-coated gold electrodes at which other redox reactions are suppressed. This suggests that an enzyme-based sensor could operate at a monolayer-coated gold electrode provided that an appropriate redox mediator was used to shuttle charge between the enzyme and the electrode. Data on the response of 6-mercaptohexanol-glucose oxidase-modified electrodes to changes in glucose concentration, and data which address the stability of the self-assembled monolayers on continuous contact with a bioactive medium (a yeast fermentation), the effect of homogeneous redox reactions between oxidized mediators and ascorbate, interference by molecular oxygen, and the effect of local hydrodynamics, are presented. Strategies for preparing improved sensors that overcome some of the problems with the present configuration are discussed. Keywords:

Biosensors;

Electrode coatings;

Enzymatic

methods; Glucose oxidase; 6-Mercaptohexanol;

1. Introduction Self-assembly techniques provide a powerful means for controlling the chemical nature of electrode-solution interfaces [l]. Monolayers of func-

* Corresponding

author.

0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIOOO3-2670(94)00506-O

11-Mercaptoundecanol;

Voltammetry

tionalized alkanethiolates on gold electrodes show particular promise and have been widely studied [2,3]. In particular, there have been several reports on the use of self-assembled alkanethiolate monolayers to improve selectivity and/or sensitivity of gold electrodes in certain electroanalytical applications. Recent examples include the selective reduction of copper in the presence of iron at electrodes coated

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with a copper-binding chelate [4], selective oxidation of dopamine in the presence of ascorbate at wmercaptoalkanoic acid-coated electrodes [5], selective oxidation of hexammineruthenium(I1) in the presence of hexacyanoferrate(I1) at a thiooctic acidcoated electrode 161, and selective oxidation of the hydrophobic drugs chlorpromazine and promethazine at alkanethiolate-coated electrodes at which hydrogen peroxide and hexacyanoferrate(I1) oxidation were inhibited [7]. Strategies involving molecular “gate sites” at which specific electron-transfer reactions could take place have also been proposed and tested [S,o]. The two primary analytical advantages associated with coating an electrode with a self-assembled monolayer are as follows: (i) non-Faradaic background currents are dramatically reduced, largely because the monolayer prevents close approach of solvent and ions to the electrode surface and therefore decreases the double-layer capacitance [lo]; and (ii) Faradaic background currents can be reduced because inter-facial electron-transfer reactions are forced to occur either by long-range electron transfer across the monolayer [ll] or at microscopic defect sites in the monolayer [12]. Suppression of undesired Faradaic background current comes with a price, however, that price being that the current for desired redox reactions, i.e. those of the analyte, may also be suppressed. It therefore becomes the goal of the analyst to design an interface at which redox reactions of analytes are allowed but those of interferers are inhibited. The examples cited above all used variations of self-assembly chemistry involving (for example) electrostatic forces [5,6], dispersion forces [7], and chemical binding affinities of species in solution with species in the monolayer [4] to achieve a selective response. Designed electrochemical interfaces such as those described above can sometimes be made exquisitely selective by incorporating very specific catalysts, typically biocatalysts such as redox enzymes, into the interphase region. This strategy has become quite well developed for electrodes without self-assembled monolayers, resulting in electrochemical biosensors for a wide variety of substances [13-151. It would be desirable to incorporate enzyme catalysts into selfassembled monolayers on electrodes; one might then hope to gain the advantages of diminished back-

Chimica Acta 307 (1995) 277-289

ground currents and suppression of undesired redox reactions while also benefiting from the selectivity of the enzyme catalyst. There have in fact been a few preliminary reports on electrochemical devices incorporating both organic monolayers and enzyme layers on electrodes [16-201; even so, much remains to be learned about the behavior of such electrodes, particularly regarding the ways in which performance could be optimized to yield analytically useful sensors. Another advantage of using self-assembled monolayers with enzyme catalysts is that one has considerably more flexibility (relative to a bare metal or carbon electrode) regarding the choice of immobilization chemistry for the enzymes [21]; this can result in more stable and active immobilized enzyme layers. Of course, one must also ensure that the redox-enzyme-catalyzed reaction is strongly coupled to the electrode, such that an analytically useful current is obtained for oxidation/reduction of the analyte. This paper will present the results of our initial studies of gold electrodes modified with self-assembled monolayers and redox enzymes. Specifically, we have begun to explore the possibility of co-immobilizing glucose oxidase with ferrocene-based redox mediators atop self-assembled alkanethiolate monolayers on gold electrodes so as to generate reagentless electrochemical glucose sensors with improved performance characteristics. Self-assembled monolayers of 6-mercaptohexanol and 1 lmercaptoundecanol were found to inhibit the redox reactions of oxygen, hydrogen peroxide, and some common interfering substances in glucose analysis. Alcohol-terminated alkanethiols were chosen for reasons of biocompatability and for their anticipated resistance to fouling in bioactive media. The inhibition of oxygen and hydrogen peroxide electrochemistry at monolayer-coated electrodes dictates that a redox mediator must be used to shuttle charge back and forth between the electrode and the redox enzyme. Preliminary data are presented and discussed for electrodes in which relatively thick layers of glucose oxidase are immobilized on gold electrode that had been previously coated with monolayers of 6-mercaptohexanol. Soluble redox mediators were used to shuttle charge between the electrode and the enzyme catalysts. Because this paper is to some extent a description of work in progress, we present

S.E. Creager, KG. Olsen /Analytica

surface of an inverted electrode and allowing the water to evaporate. The resulting films covered the gold and some of the surrounding epoxy encasement, and were clearly visible to the naked eye. The films appeared visibly to be homogeneous, albeit with a slight build-up of material at the edges. Films were cross-linked by holding the coated electrodes over several droplets of glutaric dialdehyde (Aldrich, 25 wt.% in water) in a test tube for approximately one hour.

data which highlight some of the potential problems associated with the combined use of enzyme catalysts and self-assembled monolayers on electrodes, and discuss some possible solutions to those problems.

2. Experimental Electrode preparation and coating

Gold electrodes were prepared by sealing polycrystalline gold wires (nominally 1 mm diameter, area = 0.0079 cm2) in epoxy [22]. Electrodes were polished with alumina, sonicated in soapy water followed by distilled water, and then etched for l-2 min in a 1:3:4 (v/v/v) solution of concentrated HNO,-concentrated HCl-water prior to coating [23]. Self-assembled monolayers were formed by immersing the freshly etched electrodes into a millimolar solution of alkanethiol in ethanol for not less than 12 h. Glucose oxidase coatings were prepared by applying a 3.0-~1 droplet of an aqueous solution of glucose oxidase (5 or 15 mg ml-‘) onto the exposed

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Materials

Glucose (Baker, analyzed), glucose oxidase (Sigma, grade VII from aspergillus niger), sodium chloride (Baker, analyzed), sodium dihydrogenphosphate (Mallinckrodt), disodium hydrogenphosphate (Fisher, ACS certified), hydroxymethylferrocene (HMFc, Strem), ferrocene carboxylic acid (FCA, Aldrich), ethanol (Midwest Grain Products, absolute), L-( + )-ascorbic acid (Baker, analyzed), uric acid (MCB), and 4-acetamidophenol (Aldrich, 98%) were used as received. House distilled water was purified before use with a Bamstead Nanopure water purification system. 6-Mercaptohexanol and 1 l-

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Fig. 1. Cyclic voltammetry (100 mV s-‘) in phosphate-buffered saline (PBS, 0.09 M NaCl and 0.01 M NaH,PO,-Na2HP0,, pH 7) containing (left) 1.25 mM uric acid, (center) 1.22 mM 4-acetamidophenol and (right) 1.1 mM ascorbic acid. Top: uncoated gold electrodes; bottom: electrodes coated with 6-mercaptohexanol monolayers.

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mercaptoundecanol were prepared from the corresponding o-bromoalcohols (Aldrich) by reaction with thiourea [24]. Yeast fermentations were run unstirred in Erlenmeyer flasks at ambient temperature using a commercial growth medium (Sigma, YPD broth) seeded with conventional baker’s yeast (Fleischmann’s RapidRise). Electrochemical

cells and instrumentation

Electrochemical experiments were performed in single-compartment three-electrode cells housed in a Faraday cage. An Ag/AgCl/saturated KC1 reference electrode was isolated from the main cell compartment via a double Vycor glass junction. A platinum coil served as auxiliary electrode. Potentials were applied using a single channel of a Pine Model RDE-4 bipotentiostat, and current-voltage and current-time curves were recorded using a Yokagawa Model 3025 recorder. Solutions were sparged with water-saturated nitrogen to remove dissolved oxygen, except when indicated otherwise. In some instances, a small magnetic stir bar was placed in the cell to ensure rapid and reproducible mixing as glucose and/or HMFc were added to the cell, usually via microliter syringe. Glucose was added from 0.5 M or 0.05 M stock solutions in aqueous 0.09 M sodium chloride-O.01 M phosphate buffer, pH 7.0, and HMFc was added from a 0.02 M stock solution in ethanol to avoid problems with slow dissolution of solid HMFc.

Chimica Acta 307 (1995) 277-289

acquired in the same solutions using electrodes coated with a monolayer of 6-mercaptohexanol. In each case the voltammetric background current at the monolayer-coated electrode is reduced by at least an order of magnitude relative to that at an uncoated electrode. There are two reasons for this background current reduction, both of which have to do with the barrier properties of the monolayer. First, the non-Faradaic double-layer charging current is reduced at the coated electrode because its interfacial capacitance is much smaller than that of an uncoated electrode. This is a consequence of the monolayer preventing close ap-

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3. Results and discussion

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electrodes

Probably the most obvious and analytically important consequence of coating an electrode with a self-assembled organic monolayer is the dramatic suppression of the background current relative to that at an uncoated electrode. Fig. 1 illustrates this for solutions containing three electroactive compounds (uric acid, 4-acetamidophenol, and ascorbic acid) that often interfere with electrochemical glucose analyses in blood. The top voltammograms were acquired at freshly polished and uncoated gold electrodes, whereas the bottom voltammograms were

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Fig. 2. Cyclic voltammetry (100 mV s-l) of (A) oxygen-saturated PBS and (B) PBS containing 0.5 mM hydrogen peroxide. In both (A) and (B) the top voltammogram corresponds to an uncoated gold electrode, and the bottom voltammogram to an electrode coated with a monolayer of 6-mercaptohexanol.

SE. Creager, K.G. Olsen/Analytics

preach of solvent molecules and electrolyte ions to the metal electrode surface. Second, the Faradaic current associated with oxidation of redox-active solutes is reduced at the coated electrode because the monolayer impedes access of redox-active solutes to the electrode surface. This forces oxidation (or reduction) to occur either by long-range electron transfer across the monolayer (an inherently slow process [ll]) or by electron transfer at a few microscopic defect sites in the monolayer [12]. In either case the net effect is that the apparent rate constant for electron transfer is reduced by many orders of magnitude relative to that at a bare electrode, and Faradaic currents are diminished. Two other redox-active molecules for which selfassembled organic monolayers inhibit electrochemical reactivity are oxygen and hydrogen peroxide. Fig. 2 illustrates this for oxygen reduction and hydrogen peroxide oxidation at a 6-mercaptohexanolcoated gold electrode. The voltammograms in Fig. 2A show that irreversible reduction of oxygen proceeds cleanly at a freshly polished gold electrode (top, E,, = -0.2 V), however, the cathodic current is almost completely eliminated at the coated electrode at potentials less negative than -0.40 V (bottom). (Note the change in current scales between the uncoated and coated electrodes.) The voltammograms in Fig. 2B illustrate the same point for the irreversible oxidation of hydrogen peroxide; the anodic current corresponding to peroxide oxidation at bare gold (top) is again strongly suppressed at the coated electrode (bottom). The slight rise in current at the coated electrodes at the positive and negative potential limits may represent the foot of a wave for the redox processes that readily occur at the bare electrodes; alternatively, they may simply reflect a potential-dependent breakdown of the barrier properties of the monolayer 1251. This suppression of oxygen reduction and hydrogen peroxide oxidation may also be caused by the monolayer preventing close approach of these molecules to the electrode surface. However, given the relatively small sizes of the oxygen and hydrogen peroxide molecules, it seems likely that they should be able to at least partially penetrate the monolayer to get closer to the electrode than could the somewhat larger molecules considered in Fig. 1. Another possible contributing factor might be the fact that

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most of the potentially active sites on the electrode surface are blocked by thiolates. The electrochemistry of oxygen and hydrogen peroxide at solid electrodes is dominated by the chemistry of surfacebound intermediates, for example surface oxides, hydrides, or hydroperoxides [26]. These intermediates cannot form at an alkanethiolate-coated electrode unless thiolates are displaced to open up active sites. This is unlikely to occur on gold (at least at these modest applied potentials) since the goldthiolate bond is quite strong [27]. It is worthwhile to consider the consequences of these effects on the utility of monolayer-coated electrodes in electroanalysis. Reducing background currents is usually desirable in electroanalytical chemistry because it results in improved analytical sensitivity. Reducing non-Faradaic currents is most important in voltammetry, where the potential applied to an electrode is constantly changing and the electrical double-layer is continuously being charged or discharged. It is less important in conventional amperometric sensors, for example enzyme-based electrochemical biosensors, since non-Faradaic processes should contribute little to the steady-state current. Reducing “ undesired” Faradaic currents is important in any electroanalytical device since such currents always interfere with the “desired” Faradaic currents associated with oxidation/reduction of the analyte. Unfortunately, it can be difficult to bring about such selective inhibition of undesired redox reactions since electrode treatments that suppress redox reactions of interferers will often also suppress

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Fig. 3. Cyclic voltammetry (100 mV s- ’ ) at 6-mercaptohexanolcoated gold electrodes in PBS solutions containing (top) 100 /.LM hydroxymethylferrocene (HMFc) and (bottom) LOOPM ferrocene carboxylic acid (FCA).

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S.E. Creager, K.G. Olsen/Analytics Chimica Acta 307 (1995) 277-289

the redox reactions of analytes. Of particular importance in this regard is the fact that oxygen and hydrogen peroxide electrochemistry is inhibited at monolayer-coated electrodes; this means (among other things) that conventional enzyme-based electrochemical sensor schemes that rely on amperometric detection of the oxygen consumed and/or hydrogen peroxide generated by an enzyme-catalyzed reaction with analyte will not work at monolayer-coated electrodes. Fortunately, not all interfacial redox reactions are as thoroughly suppressed by organic monolayers on electrodes as are the processes considered above. Fig. 3 shows voltammograms for oxidation/reduction of two water-soluble ferrocene derivatives, hydroxymethylferrocene (HMFc, top) and ferrocene carboxylic acid (FCA, bottom), at a 6-mercaptohexanol-coated gold electrode. (Voltammograms acquired independently at uncoated electrodes were reversible for both of these molecules, with E”‘(HMFc) = +0.24 V and E”‘(FCA) = +0.34 V vs. Ag/AgCl.) The peak splitting of 110 mV in the top voltammogram indicates that HMFc oxidation is quasi-reversible at this electrode. This is in contrast to the bottom voltammogram, which shows no peak on either the forward or reverse scan but which clearly indicates that FCA oxidation is more strongly suppressed than HMFc oxidation at the same electrode. The reasons for the different behavior for HMFc and FCA are not entirely clear, though it is possible that the negative charge on FCA (FCA will be deprotonated at the pH of the buffered electrolyte used in this experiment) may play a role. In any case, both of these redox reactions are much less perturbed by the presence of the 6-mercaptohexanol monolayer than were the reactions considered in Figs. 1 and 2. The observation that a 6-mercaptohexanol monolayer can strongly suppress the redox reaction of one electroactive species (e.g. ascorbate, an interferer) while the reaction of another species (e.g. HMFc, an analyte), is much less affected, suggests that it is possible to design electrode surfaces with a selective response. In particular, the use of HMFc (or another redox mediator with fast redox kinetics at a coated electrode) to shuttle charge back and forth between an immobilized redox enzyme and an electrode may offer a means of exploiting the specificity of an

enzyme catalyst while still preserving the advantages of background suppression by the monolayer. 3.2. Self-assembled

monolayer-glucose dox mediator electrodes

oxidase-re-

As mentioned in the Introduction, we have targeted the glucose oxidase-redox mediator system for study with monolayer-coated electrodes. As a first pass at preparing a combined self-assembled monolayer and glucose oxidase-coated electrode, we have prepared electrodes with a relatively thick coating of glutaraldehyde-cross-linked glucose oxidase atop a 6-mercaptohexanol monolayer that had been previously assembled onto a gold electrode. Electrical communication between the electrode and the glucose oxidase was achieved via freely diffusing HMFc in solution. Ultimately, we hope to prepare electrodes in which a thinner layer of enzyme is co-immobilized along with a redox mediator, perhaps be direct covalent binding to the monolayer surface, to create a self-contained sensor. For the present, however, we have studied the simpler but more easily prepared electrodes with thick glucose oxidase layers and soluble mediators to begin learning about the behavior of electrodes coated with an enzyme layer atop a thin barrier layer.

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Fig. 4. Cyclicvoltammetry(100mV s- ’ ) at 6-mercaptohexanolcoated gold electrodescoated with glutaraldehyde-cross-linked glucoseoxidaseas describedin the text. The solutionis PBS containing20 PM HMPc;the solutionfor topvoltammogram also contains0.5 M glucose.

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thereby indicating that the glucose oxidase layer does not present a significant barrier to the diffusional transport of HMFc (or HMFc+). The top voltammogram was acquired in the presence of 0.5 M glucose; the increase in oxidative current and the corresponding disappearance of the reductive return peak both strongly indicate that electrogenerated HMFc+ is being consumed in a following chemical

Fig. 4 shows a pair of voltammograms acquired at a 6-mercaptohexanol-glucose oxidase-coated electrode prepared as described in the experimental section and studied in the presence of 20 PM HMFc. The bottom voltammogram, which was acquired in the absence of glucose, is not greatly different than the voltammogram that would be observed at an electrode without the glucose oxidase coating,

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Fig. 5. Response to glucose of gold electrode coated with 6-mercaptohexauol and glucose oxidase. Top: amperometry (Eqp, = +0.45 V) for serial addition of glucose to a stirred PBS solution containing 100 PM HMPc; bottom: calibration curves for glucose in solutions containing (solid circles) 20 PM and (open squares) 100 PM HMFc.

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reaction that regenerates HMFC. Undoubtedly that reaction is the enzyme-catalyzed oxidation of glucose by HMFc+. In support of this postulate, no response to glucose was observed for electrodes without the glucose oxidase layer, and no significant oxidative current was observed for solutions without HMFc. The limiting current in the presence of glucose and HMFc was approximately independent of the scan rate for voltammograms between 10 and 1000 mV s-l, although it must be noted that some variation in the non-Faradaic background current over this range of scan rates prevented a careful quantitative comparison. Interestingly, tripling the amount of glucose oxidase on the electrode had a negligible effect on the excess current, suggesting that glucose oxidation is occurring entirely within a relatively thin reaction layer inside the glucose oxidase film. There have been several reports of glucose oxidation using soluble mediators at electrodes coated with glucose oxidase [28-301, however, none has involved an electrode that was also coated with a self-assembled monolayer. It is of interest to know how these electrodes respond to changes in the concentration of glucose. Fig. 5 presents data on the steady-state current at a 6-mercaptohexanol-glucose oxidase-coated electrode in solutions containing different concentrations of glucose. The data in Fig. 5 were acquired at a static potential (+0.45 V vs. Ag/AgCl) in a stirred solution to which was added a series of aliquots of a concentrated stock solution of glucose. The top trace of current vs. time is for a solution containing 100 PM HMFc, and is included to illustrate both the speed of response and the noise level of the steadystate current. (The non-zero steady-state current present before addition of glucose is due to oxidation of HMFc mediator that is present in bulk solution and is transported to the electrode by stirring. This current would of course be absent in a device in which the mediator was co-immobilized with the enzyme.) The response to a change in glucose concentration is quite rapid (within the time of mixing), however, the noise level is much higher than the noise in the voltammograms in Fig. 4. Much of this noise can be traced to the stirring of the solution (vide supra), and would not be a problem for a sensor operating in a quiescent solution. The bottom curves in Fig. 5 were obtained for

Chimica Acta 307 (1995) 277-289

two solutions, one containing 20 PM HMFc and one containing 100 PM HMFc. In each case the steadystate current in response to added glucose increases approximately linearly with glucose at low glucose concentrations but approaches a limiting value at high glucose concentrations. This is as expected for a system following Michaelis-Menten kinetics for an enzyme-catalyzed reaction between two substrates, in this case glucose and HMFc+ [31]:

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According to Eq. 1, for a given concentration of HMFc mediator, a calibration curve of Z vs. Cgl,, should be linear with a slope of Zmax/Kglu at very low glucose concentration [when K&C,,, 3 1 + HMFc)], it should exhibit a limiting current (KHMFC/~ of Z,,,/[l + (KHMFc/CHMFc)l at high glucose concentrations [when Kglu/Cg,,, e 1 + >] and it should exhibit a current of (KHMFc/CHMFc 7 half the limiting current when Kglu/Cglu = 1 + >. This is precisely the behavior ex( KHMFc/CHMFc hibited in Fig. 5 for two independent experiments with different values of CHMFc.The fact that the limiting current depends on the mediator concentration suggests that the reaction between mediator and the reduced form of the enzyme is at least partially rate-limiting; however, this fact (and the overall adherence of the system to Eq. 1) should not be given too much significance, since the apparent Michaelis constants for such a system are a function not only of the enzyme kinetics but also of the transport dynamics within the surface layers [32,33]. It is for this reason that we have not attempted a more quantitative analysis of the data in Fig. 5. Still, the fact that the overall system behaves in such a simple and predictable way with respect to changes in glucose and HMFc concentration suggests that the presence of the glucose oxidase overlayer has not dramatically affected the interfacial redox reaction of the redox mediator, and that the presence of the self-assembled monolayer has not changed in any major way the mechanism of the enzyme-catalyzed reaction.

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3.3. Potential problems with the monolayer-glucose oxidase-mediator electrodes The results presented above hold out the promise that improved enzyme electrodes could be prepared by using self-assembled monolayers as underlayers between an electrode and an enzyme layer. This promise is not without its potential problems, however, and the electrodes described above do have problems that would probably make them unsuitable for use in a real sensor. This section of the paper will review several of these problems, and will discuss possible strategies that could be (and are being) used to overcome them. One factor affecting the utility of self-assembledmonolayer-coated electrodes in electroanalysis is the stability of the monolayer, particularly on continuous exposure to potentially aggressive bioactive media. This is particularly important for enzyme electrodes since many such electrodes are ultimately intended for use in such media. There is in fact some evidence that alkanethiolate monolayers on gold can be slowly degraded by continued exposure to light and/or oxygen [34,35]. There is also evidence that a dynamic on-off exchange process occurs at coated electrodes that can result in material being lost from an electrode surface when it is in contact with a medium in which the material comprising a monolayer is soluble [22,36]. These problems can obviously limit the useful lifetime of an electrochemical sensor. Fig. 6 presents the results of our initial stability tests of 6-mercaptohexanol and ll-mercaptoundecanol monolayers on gold on continuous contact with a representative bioactive medium, an active yeast fermentation, over a timescale of several days. This medium was chosen partly because it is particularly “dirty” and aggressive, and partly because it was convenient. Voltammetric background scans taken after four days’ exposure of the monolayercoated electrodes to the fermentation (Fig. 6, left) showed considerable degradation in the capacity of the 6-mercaptohexanol layer to suppress the background current. We attribute this to a partial loss of material from the electrode surface, as discussed above. With this in mind, a parallel experiment was conducted with an electrode coated with a monolayer of ll-mercaptoundecanol, our postulate being that a

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Fig. 6. Cyclic voltammetry in an active yeast fermentation; see Experimental section for details. Left: after four days: (top) bare gold, (middle) 6-mercaptohexanol-coated gold, and (bottom) llmercaptoundecanol-coated gold. Right: after seven days, at llmercaptohexanol-coated gold, (top) after and (bottom) before spiking with 10 PM HMFc.

monolayer comprised of longer-chain molecules would be less likely to desorb and would therefore be more stable. The voltammogram in Fig. 6 strongly supports this postulate; the background current at the 1 l-mercaptoundecanol-coated electrode after four days exposure to the fermentation (Fig. 6, bottom left; note the expanded current scale relative to the voltammogram at bare gold) is much smaller than that at either the uncoated electrode or the 6mercaptohexanol-coated electrode. In fact, the background current at this electrode was virtually unchanged even after seven days continuous exposure to the active fermentation (Fig. 6, bottom right). After seven days, the fermentation broth was spiked with 10 PM of HMFc and the voltammogram at the top right of Fig. 6 was recorded; the Faradaic current for HMFc oxidation is easily distinguished from background in this voltammogram, indicating that the 11-mercaptoundecanol monolayer provides the

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ers on gold in contact with bioactive media can be improved by increasing the alkanethiol chain length. A potential pitfall in the use of self-assembled monolayers to selectively inhibit undesired redox reactions at coated electrodes comes about when a homogeneous redox reaction involving the interferer and the analyte can occur. Fig. 7 illustrates the problem for the particular combination of HMFc oxidation in the presence of ascorbate. Oxidation under such conditions at a 6-mercaptohexanol-coated gold electrode produces a sigmoidal current-voltage curve (Fig. 7, top) that is indicative of electrogenerated HMFc+ being consumed in a catalytic regeneration reaction with ascorbate (a known reaction [37]) thereby oxidizing ascorbate indirectly. This happens despite the fact that independent voltammetric studies showed that HMFc oxidation is allowed but ascorbate oxidation is inhibited at the coated electrode. Similar tests with uric acid and 4-acetamidophenol did not show this effect. The degree to which such a homogeneous redox reaction would contribute to the current in an enzyme-based sensor would depend on many factors, including the relative

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Fig. 7. Cyclic voltammetry (100 mV s-l) at 6-mercaptohexanoicoated gold electrode in PBS containing (bottom) 20 /.LM HMFc, (middle) 1.1 mM ascorbic acid, and (top) both 20 PM HMFc and 1.1 mM ascorbic acid.

same sort of selective inhibition of background currents that was obtained with 6-mercaptohexanolcoated electrodes in more controlled media. Hence, it appears that the stability of alkanethiolate monolay-

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concentrations of enzyme, substrate, mediator, and interferer in the reaction layer, and the relative rate constants for reaction of mediator with interferer and substrate (or with the appropriate form of the enzyme). The effect would obviously be most important in applications where low concentrations of the enzyme substrate must be detected. Modifications of the self-assembled layer are unlikely to have much effect on this process, since it occurs away from the electrode surface. The magnitude of the effect for the specific case of an ascorbate interference in glucose sensing could be reduced by applying a passive layer of a cation-exchange polymer, for example Nafion, to electrostatically exclude ascorbate from the reaction layer where the oxidized form of the mediator is generated. Another factor that must be considered for most sensors based on redox enzymes is the possibility of interference by oxygen. Mediator-based sensors do not require oxygen to operate, and oxygen electroactivity is suppressed at a monolayer-coated electrode; still, oxygen can diminish sensor response by consuming substrate via a non-current-producing pathway, namely the enzyme-catalyzed reaction between oxygen and substrate [38]. Fig. 8 illustrates this for an electrode similar to that used in Figs. 4 and 5. The current at low glucose concentration in the presence of oxygen is strongly suppressed relative to that in the absence of oxygen. At higher glucose concentrations the effect is less pronounced, and the current in both the presence and absence of oxygen appears to approach the same limiting value at very high glucose concentrations. This behavior is explained qualitatively by noting that the mediated reaction occurs in a relatively thin reaction layer near the electrodeglucose oxidase interface, and that glucose oxidation occurring via an oxygen-dependent pathway in the outer portions of the film will diminish the flux of glucose reaching the inner reaction layer, thereby diminishing the response. This phenomenon will be a problem for any mediator-based sensor, not just sensors that incorporate self-assembled monolayers. Still, it must be considered in the design of improved sensors, and is therefore relevant to the present discussion. One possible solution is to decrease the thickness of the glucose oxidase layer such that the reaction layer is thicker than the glucose oxidase layer and the oxi-

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0.6 -

3 E

Fi

3

0.4 -

0.2 -

OOLJ 0

I

20

40

60 Time (set)

Fig. 9. Effect of stirring rate on the current for mediated glucose oxidation at a gold electrode coated with 6-mercaptohexanol and glucose oxidase. The solution is PBS containing 0.5 M glucose and 20 PM HMFc. The initial rise in current just after t = 0 corresponds to application of a potential step from 0.0 V to + 0.45 V vs. Ag/AgCl. The solution is initially unstirred; stirring was initiated at around t = 25 s and was stopped just before t = 40 s.

dized mediator has access to all of the glucose oxidase. (The present enzyme layers are thought to be between 5 and 15 pm thick, based on the approximate amount of material deposited per unit area, a projected area per molecule of between 60 and 100 nm’, and a thickness per enzyme layer of between 6 and 10 nm.) Then, oxygen-dependent substrate oxidation would have to compete directly with mediator-dependent substrate oxidation to have any effect on the current. The oxygen interference will also be minimized when the rate constant for mediator-dependent glucose oxidation is greater than that of oxygen-dependent glucose oxidation, and/or when the mediator is present in a large excess over oxygen. Changes in local hydrodynamic conditions (i.e., stirring) at the electrode surface also affected the response, as illustrated in Fig. 9. In unstirred solution, the current increased rapidly after stepping to a potential where HMFc would be oxidized, then slowly decreased on a timescale of some tens of seconds. When the solution was stirred, the current

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rose slightly to achieve a steady-state value, however, when stirring was stopped, the current again began to slowly decrease. The falling current in unstirred solution can be attributed to depletion of glucose at the electrode surface; glucose is replenished by diffusion from bulk solution, however, the flux in the absence of stirring is insufficient to replace the glucose as fast as it is consumed. The glucose flux also decreases with time as the depletion layer extends further out into the bulk of solution; hence, the current falls. When the solution is stirred, the local hydrodynamics are such that the flux of glucose to the surface is higher and no longer varies with time; hence, a steady-state current is obtained. An exact description of this phenomenon should account for diffusion of both glucose and HMFc in the film and the exact hydrodynamic profile at the electrode surface, and has not been attempted. Still, the observation that current depends on stir rate highlights a potential problem for a sensor, namely that the current will be sensitive to local hydrodynamic conditions near the sensor. Those conditions must therefore be carefully controlled if the sensor is to be reliable. A possible solution to this problem is to make a sensor from a microelectrode for which the diffusion profile of substrate is radial rather than linear [39,40]. Then, the diffusional flux of substrate to the electrode surface would be higher and would occur in a steady-state fashion, thereby eliminating the slow decrease in response as glucose is depleted. Also, the diffusion layer would then be smaller that the boundary layer associated with hydrodynamic flow near the electrode surface; this should made the sensor response less sensitive to local hydrodynamic conditions, and therefore more reliable.

4. Summary Gold electrodes coated with self-assembled monolayers of 6-mercaptohexanol and ll-mercaptoundecanol exhibit dramatically reduced background currents relative to uncoated electrodes, due in part to a reduction in the double-layer capacitance and in part to a suppression of Faradaic current associated with oxidation/reduction of electroactive species in solution. Suppression of hydrogen peroxide oxida-

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tion at these electrodes makes it impossible to employ sensor strategies with them involving electrochemical detection of hydrogen peroxide. However, oxidation of selected redox mediators, e.g. hydroxymethylferrocene, can proceed at coated electrodes at which other redox reactions are suppressed; this enables the use of enzyme catalysts with monolayer-coated electrodes provided that an appropriate redox mediator was used to shuttle charge between the enzyme and the electrode. Gold electrodes coated with both glucose oxidase and a monolayer were prepared and shown to respond to glucose when used in this fashion. The stability of self-assembled monolayers on continuous contact with a bioactive medium (a yeast fermentation), the effect of homogeneous redox reactions between oxidized mediators and ascorbate, interference by molecular oxygen, and the effect of local hydrodynamics, were all investigated, and strategies suggested for improving the performance of subsequent generations of devices.

Acknowledgements This work has been supported in part by a grant from the Eli Lilly Company, by the United States Department of Education for a National Needs Fellowship for Kimberly Olsen, and by the Eli Lilly Company, also for a fellowship for Kimberly Olsen.

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