Platinum-Catalyzed Enzyme Electrodes Immobilized on Gold Using

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Anal. Chem. 1998, 70, 2396-2402

Platinum-Catalyzed Enzyme Electrodes Immobilized on Gold Using Self-Assembled Layers J. J. Gooding,† V. G. Praig, and E. A. H. Hall*

Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, U.K. CB2 1QT

The bonding of enzymes to self-asembled monolayers (SAMs) of alkanethiols onto gold electrode surfaces is exploited to produce an enzyme biosensor. The attachment of glucose oxidase to a SAM of 3-mercaptopropionic acid was achieved using carbodiimide coupling. The resultant biosensor showed good sensitivity to glucose and a large dynamic range when measured amperometrically via the p-benzoquinone mediator. On the other hand, subsequent platinization of the enzyme-SAM electrode allowed hydrogen peroxide produced in the enzyme reaction to be detected directly, thus obviating the need for an artificial redox mediator. The performance of such sensors constructed on bulk gold electrodes was evaluated and finally compared to that of some preliminary thinfilm gold electrodes. Biosensors constructed using the two alternative electrode surfaces have quite different sensitivities, thus reflecting the influence of the anchoring surface on the performance of the biosensor. One of the greatest obstacles to the success of biosensors is the inability to manufacture reproducible devices. Theoretical models of the classical geometry enzyme electrodes, where the enzyme is immobilized in a three-dimensional reaction matrix placed over a planar electrode, show that the response of the sensor is very sensitive to the thickness of the reaction matrix.1-8 Production of a biorecognition matrix of precisely defined thickness, while still achieving efficient immobilization of the enzyme, reproducible matrix transport properties, and, in a material which is otherwise inert to the biochemical reaction, measurement environment or other external parameters are major contributors to sensor success. The commercial success of different glucose biosensors shows that, with careful manufacture targeted toward a specific analytical environment and accuracy, this problem can be partly overcome. Gooding and Hall have shown that, if a highly

accurate measurement is required for enzyme electrodes7-9 and enzyme optodes,10 then, to avoid variation in response between sensors, enzyme layers must be cast onto the transducer with a precision rarely achieved with current deposition methods.9 An alternative geometry enzyme electrode was developed by Gooding and Hall8,11 and has been shown, both theoretically and experimentally, to have a response independent of the thickness of the reaction layer. However, this geometry is not a generic solution, as it is designed with a long response time, which is too slow for some applications. Our conclusion from the evaluation of the classical geometry and this alternative geometry biosensor is that one must compromise either reproducibility or response time if the sensor involves diffusion through a three-dimensional reaction matrix. An obvious caveat of this would be that threedimensional matrixes should be avoided; in this respect, a twodimensional reaction zone, where the recognition event is a surface reaction, would, perhaps, be a preferred solution. However, for any two-dimensional reaction layer method to be successful, it also requires that the layer be constructed in a highly reproducible manner, which suggests that self-assembled layers of alkanethiols could have considerable potential for enzyme electrodes. The bonding of alkanethiols to metal surfaces has been studied extensively12-19 and shown to produce stable monolayers with a high degree of order comparable to Langmuir-Blodgett films.14 Gold electrodes modified with alkanethiols have been used for studying the theory of heterogeneous electron transfer,20,21 for studing wetting,22,23 and as anchors to immobilize biological

* To whom correspondence should be addressed. E-mail: lisa.hall@ biotech.cam.ac.uk. † Present address: Department of Analytical Chemistry, The University of New South Wales, Sydney, NSW, 205 Australia. (1) Staros, J. V.; Wright, R. W.; Swingle, D. M. Anal. Biochem. 1986, 156, 220222. (2) Leypoldt, J. K.; Gough, D. A. Anal. Chem. 1984, 56, 2896-2904. (3) Martens, N.; Hall, E. A. H. Anal. Chem. 1994, 66, 2763-2770. (4) Mell, L. D.; Maloy, J. T. Anal. Chem. 1975, 47, 299-307. (5) Schulmeister, T. Sel. Electrodes Rev. 1990, 12, 203-260. (6) Parker, J. W.; Schwartz, C. S. Biotech. Bioeng. 1987, 30, 724-735. (7) Gooding, J. J.; Hall, E. A. H. Electroanalysis 1996, 8, 407-413. (8) Gooding, J. J.; Ha¨mmerle, M.; Hall, E. A. H. Sens. Actuators 1996, B34, 516-523.

(9) Hall, E. A. H.; Gooding, J. J.; Hall, C. E. Mikrochim. Acta 1995, 121, 119145. (10) Gooding, J. J.; Hall, E. A. H. Unpublished. (11) Gooding, J. J.; Hall, E. A. H. J. Electroanal. Chem. 1996, 417, 25-33. (12) Ulman, A. An Introduction to Ultrathin Organic Films From LangmuirBlodgett to Self-Assembly; Academic Press: London, 1991. (13) Lotzbeyer, T.; Schuhmann, W.; Schmidt, H.-L. Sens. Actuators 1996, 33, 50-54. (14) Pan, W.; Durning, C. J.; Turro, N. J. Langmuir 1996, 12, 4469-4473. (15) Mrksich, M.; Whitesides, G. M. Annu. Rev. Biomol. Struct. 1996, 25, 5578. (16) Horn, A. B.; Russell, D. A.; Shorthouse, L. J.; Simpson, T. R. E. J. Chem. Soc., Faraday Trans. 1996, 92, 4759-4762. (17) Bain, C. D.; Evans, S. D. Chem. Br. 1995, 31, 46-48. (18) Bain, C. D.; Whitesides, G. M. Science 1988, 240, 62-63. (19) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1988, 110, 6560-6561. (20) Finklea, H. O.; Hansheu, D. D. J. Am. Chem. Soc. 1992, 114, 3173. (21) Becka, A. M.; Miller, C. J. J. Phys. Chem. 1992, 96, 2657. (22) Dubois, L. H.; Zegarski, B. R.; Nuzzo, R. G. J. Am. Chem. Soc. 1990, 112, 570. (23) Evens, S. D.; Urankar, E.; Ulman, A.; Ferris, N. J. Am. Chem. Soc. 1990, 112, 4121.

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molecules.15,24-35 The latter ability has seen them used for both immunosensors15,24-26 and enzyme biosensors.27-35 To construct enzyme electrodes using alkanethiols requires functionality at the terminal end of the molecule, to allow a bond to be formed between the enzyme and the monolayer. In many of the enzyme electrodes in the literature, the alkane end terminates with an amine group, and the biological molecule is immobilized by cross-linking with glutaraldehyde.27-31 However, the cross-linking with glutaraldehyde creates multilayers, as shown by Kajiya et al.27 Thus, a three-dimensional matrix is formed, and the advantages of a well-defined monolayer of immobilized enzyme are lost. Other studies have reported enzymes covalently bound directly to the self-assembled monolayer (SAM) without a crosslinker.32-34,36 With this latter approach, a two-dimensional reaction layer of enzyme at the electrode (Au) surface would be expected to be produced. Self-assembled monolayers of alkanethiols are preferably constructed on gold, rather than other metals such as platinum, copper, or silver, because gold does not have a stable oxide.12 However, an oxidase enzyme-linked electrode, which measures a hydrogen peroxide oxidation current, requires platinum or other peroxide-active electrode material to function. On gold, the peroxide oxidation current is very low and not well suited for analytical use. The formation of the self-assembled layer on gold electrodes, therefore, limits the flexibility of the system. For these oxidase enzyme electrodes, the alternatives to measuring the production of hydrogen peroxide are to monitor the consumption of oxygen by the enzyme reaction or the generation of the reduced form of a synthetic redox mediator. The former choice has been used, but, as oxygen is a substrate for the enzyme reaction, the dynamic range of the measurement in oxidase biosensors is often too limited in this mode.7,8 For an enzyme attached at an electrode via a short carbon chain, the depletion of the oxygen in the vicinity of the enzyme, as a result of the concentration profile next to an electrode polarized at the oxygen reduction potential, is likely to exacerbate this limitation. The use of redox mediators also has associated problems; not only is another reagent required, but it is susceptible to interferences from oxygen. Martens and Hall5 have shown that, unless the cycling of the enzyme by the synthetic mediator is significantly more efficient than that of oxygen, the oxygen will still intercept the redox pathway with the enzyme, particularly at low substrate concentration, and hence (24) Willner, I.; Blonder, R.; Dagan, A. J. Am. Chem. Soc. 1994, 115, 49374938. (25) Willner, I.; Rubin, S.; Cohen, Y. J. Am. Chem. Soc. 1993, 115, 4937-4938. (26) Katz, E.; Willner, I. J. Electroanal. Chem. 1996, 418, 67-72. (27) Kajiya, Y.; Okamoto, T.; Yoneyama, H. Chem. Lett. 1993, 2107-2110. (28) Willner, I.; Lion-Dagan, M.; Marx-Tibbon, S.; Katz, E. J. Am. Chem. Soc. 1995, 117, 6581-6592. (29) Imamura, M.; Haruyama, T.; Kobatake, E.; Ikariyama, Y.; Aizawa, M. Sens. Actuators 1995, B24-25, 113-116. (30) Creager, S. E.; Olsen, K. Anal. Chim. Acta 1995, 307, 277-289. (31) Dong, X.-D.; Lu, J.; Cha, C. Bioelectrochem. Bioenerg. 1995, 36, 73-76. (32) Willner, I.; Riklin, A.; Shoham, B.; Rivenzon, D.; Katz, E. Adv. Mater. 1993, 5, 912-915. (33) Willner, I.; Heleg-Shabtai, V.; Blonder, R.; Katz, E.; Tao, G.; Bu ¨ ckmann, A. F.; Heller, A. J. Am. Chem. Soc. 1996, 118, 10321-10322. (34) Wagner, P.; Hegner, M.; Kernen, P.; Zaugg, F.; Semenza, G. Biophys. J. 1996, 70, 2052-2066. (35) Aizawa, M.; Nishiguchi, M.; Imamura, M.; Kobatake, E.; Haruyama, T.; Ikarijama, Y. Sens. Actuators 1995, B24-25, 1-5. (36) McRipley, M. A.; Linsenmeier, R. A. J. Electroanal. Chem. 1996, 414, 235246.

the associated signal error will be significant. Even with the mediator covalently immobilized to the enzyme in a SAM, Willner et al.33 have reported some interference by oxygen. In this study, a combination is sought whereby an electrode material can be used that is suitable for the self-assembly of thiolated carbon chain enzyme linkers, but where hydrogen peroxide can be monitored via its oxidation current. The first materials of choice here would be Pt from the electrochemistry viewpoint and Au in terms of monolayer assembly. This paper reports on the performance of a “platinium catalyzed” enzymecoupled layer assembled through a thiol on gold. EXPERIMENTAL SECTION Materials and Equipment. The enzyme glucose oxidase (GOx) from Aspergillus niger (EC 1.1.3.4, type VII-S) was purchased from Sigma (Poole, UK). The salts potassium chloride (AR grade), potassium dihydrogen orthophosphate (LR grade), dipotassium hydrogen orthophosphate (LR grade), potassium hydroxide (AR grade), 2-[N-morpholino]ethanesulfonic acid (MES), potassium ferricyanide, and p-benzoquinone were all from Sigma. The potassium hexachloroplatinate (IV) was from Aldrich (Dorset, UK). The 3-mercaptopropionic acid (MPA) was from Fluka (Poole, UK), the N-ethyl-N-(3-dimethylaminopropyl)carbodiimide (EDC) and the N-hydroxysulfosuccinimide (NHS) were from Sigma, and the (3-mercaptopropyl)trimethoxysilane (MPS) was from Aldrich. The glucose was supplied by Fisons (Loughborough, UK), and the glass slides used for making electrodes were supplied by BDH (Poole, UK). All electrochemical experiments were conducted with an EG&G 273 potentiostat with a JJ CR600 Y-t recorder to record current/time traces or a Phillips PM8043 xy recorder for cyclic voltammograms. Evaporated gold films were prepared using an Edwards coating system 306A with an Edwards FTM4 quartzcrystal thickness monitor. Procedures. The formation of reproducible self-assembled monolayers requires a reproducible surface. To prepare the bulk gold electrodes for modification, they were polished to 0.05 µm using alumina powder and then cleaned by boiling in concentrated KOH for 1 h, followed by rinsing with distilled water. The electrodes were soaked for 10 min in concentrated nitric acid and rinsed again with water. The results of this method of electrode preparation were contrasted with results from electrodes which were abrased using emery paper and washed with ethanol prior to modification. Glass slides used to make thin-film electrodes were cleaned in warm chromic acid for 1 h and then rinsed with water. To improve the adhesion of the gold films onto the glass substrates, either a 20-nm thin chromium film was deposited between the gold and the glass or the gold film was coupled using an organosilanethiol.37 In the latter procedure, the glass slides were treated with an alkoxysilyl reagent having a thiol headgroup. The slides were placed in a 95:5 (v/v) methanol/water solution with 0.5% (v/v) of the (3-mercaptopropyl)trimethoxysilane (MPS). The silane is hydrolyzed and condenses with surface -OH groups and with itself, forming a silane polymer at the surface of the slide having exposed thiol groups. The slides were stored in the solution for 24 h to allow sufficient time for polymerization of the (37) Gaus, K.; Hall, E. A. H. J. Colloid Interface Sci. 1997, 194, 364-372.

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silane. Subsequently, the slides were sonicated for 2 min in ethanol to remove excess free polymer. They were placed in the oven for 3 h or more at 150 °C and were coated with gold (100 nm) immediately afterward. The silanized slides or unmodified glass slides were coated with metals. On the latter slides, a chromium underlayer (20 nm) was evaporated before both groups were coated with 100 nm of gold using an Edwards coating system 306 (Buhay, Hemel Hempsted, UK) at a pressure between 2 and 3 × 10-6 mbar at a rate of 0.2 nm s-1. The thickness of the film was measured with an Edwards FTM4 quartz crystal thickness monitor. The resulting gold films were used as the electrodes. The electrode area was defined by the delimiting tubular sample cell (diameter 10 mm), which was glued to the slide. The electrochemical areas of the bare electrodes were determined using cyclic voltammetry and the Randles-Sevcik equation38 for a reversible redox couple, which at 25 °C is

Ip ) (2.69 × 105)n3/2AD1/2C∞ν1/2

(1)

where Ip is the peak current (A), n is the number of electrons transferred, A is the electrode area (cm2), D is the diffusion coefficient of the electroactive species (cm2 s-1), C∞ is the bulk concentration of the same species (mol cm-3), and ν is the scan rate (V s-1). If a reversible electroactive species is used which has a known diffusion coefficient, then the area of the electrode can be calculated from the peak current of a cyclic voltammogram. Potassium ferricyanide, 1 mM in an aqueous solution of 0.2 M KCl and 0.1 M KOH, was used for this purpose. Potassium ferricyanide has a diffusion coefficient of 7.6 × 10-6 cm2 s-1 at 25 °C.39 The polished gold bulk electrodes had a surface roughness of 1.1, while the roughness of the sanded electrodes was 3.3. The gold film electrodes had a roughness factor approaching 1.0. For the current densities quoted, the electrochemically measured area was used. Cleaned gold electrodes were modified by placing them into a 75:25 (v/v) ethanol/water solution with 0.01 M MPA. The slides were stored in the MPA solution for 24 h, as it has been shown that alkanethiols take between 10 and 20 h to approach equilibrium.14 The modified electrodes were then washed in the same ethanolic solution and dried in a nitrogen stream. The MPAmodified electrodes were activated in a solution at pH 5.5 with 0.002 M EDC and 0.005 M NHS in either 0.1 M MES buffer or distilled water. The electrodes were washed in MES buffer or water and placed in a 0.1 M MES buffer or water, pH 5.5, containing 150 µg/mL of the enzyme. The electrodes were left in the enzyme solution for at least 1 h before being used or platinized. The electrodes which were not platinized were known as GOx-SAM-modified gold electrodes, and the platinized ones were Pt-GOx-SAM-modified electrodes. Electrodes were platinized by adaption of the procedure of Gunasingham and Tan40 for the codeposition of platinum and glucose oxidase on glassy carbon electrodes. In this case, the (38) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; John Wiley and Sons: Chichester, UK, 1980. (39) Von Stackelberg, M. V.; Pilgram, M. Z. Elektrochem. 1953, 57, 342-350. (40) Gunasingham, H.; Tan, C. B. Electroanalysis 1989, 1, 223-227.

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Figure 1. Variation of the cyclic voltammogram of a GOx-SAM gold electrode with glucose concentration. The mediator was 1 mM p-benzoquinone in 0.1 M phosphate buffer and 0.1 M KCl at pH 7.0. The electrode had an area of 0.1.67 cm2, and the scan rate was 100 mV s-1.

enzyme-modified electrode was platinized electrochemically by sweeping the electrode potential, in an aqueous solution of 5 mM K2PtCl6 in 0.05 M phosphate and 0.1 M KCl buffered to pH 7.0, between -0.7 and +0.2 V versus a saturated calomel reference at a scan rate of 100 mV/s for 30 min. The platinized electrodes were then washed in phosphate buffer and used. For the measurement of analyte calibration curves, the electrodes were allowed to settle to a stable current over a period of at least 1 h in the background solution. Once the electrode background current was stable, additions of glucose were made from a stock solution. With GOx-SAM electrodes, which were not platinized, a mediator was required: in these instances, the background solution was a pH 7.0 phosphate buffer solution with 1 mM p-benzoquinone as the mediator. The electrode potential was held constant at +0.5 V versus an SCE. Hydrogen peroxide was monitored with the Pt-GOx-SAM electrodes at an electrode potential of +0.65 V versus an SCE. In this latter case, the background solution was pH 7.0 phosphate buffer solution. RESULTS AND DISCUSSION Enzyme Response on Gold Electrodes. Prior to platinizing the GOx-modified gold electrode, it was necessary to determine whether the glucose oxidase had been immobilized on the gold surface such that it retained activity and whether the underlying electrode was still available for electon transfer or was insulated by the immobilized layer. To ascertain the integrity of these enzyme-modified SAMs as transducing layers, they were investigated via an electrochemically active glucose oxidase mediator. p-Benzoquinone was chosen, as it has previously been shown to mediate glucose oxidase both in solution and immobilized in polyaniline films41 and had better solubility characteristics than the more popular ferrocene derivatives. The variation in the cyclic voltammograms, measured at a GOx-SAM-modified gold electrode, with glucose concentrations, is shown in Figure 1. As is (41) Cooper, J. C.; Hall, E. A. H. Electroanalysis 1993, 5, 385-397.

Figure 2. Glucose calibration curve of GOx-SAM modified gold electrodes. The mediator was 1 mM p-Benzoquinone. The potential of the GOx-modified electrode was maintained at +0.5 V, where the benzoquinone oxidation current was in the diffusion-limited region. “Polished” refers to an electrode that was rigorously cleaned, while “sanded” refers to an electrode that was prepared by sanding the surface clean and rinsing.

apparent from the figure, increased glucose concentration results in increased current at +0.5 V, consistent with an immobilized enzyme which retains activity and a SAM which allows the transport of the mediator to the electrode. To confirm that these results were not artifactual, they were compared with those other electrodes and immobilized proteins. No direct oxidation current for glucose was seen on a bare gold electrode in constant potential measurements at +0.5 V, nor on a gold electrode modified with thiol and proteins other than glucose oxidase, but a glucose calibration curve, for a gold electrode modified with a GOx-MPA monolayer, is shown in Figure 2. It has a very large dynamic range (see also Table 1). The large dynamic range is surprising considering that the KM of the enzyme in solution is commonly quoted as 80 >90 30 30

25 10

30 20

Thin-Film Electrodes silanethiol adhesion SAM-GOx Pt-SAM-GOx chromium adhesion SAM-GOx Pt-SAM-GOx Pt-SAM-GOx a

p-benzoquinone hydrogen peroxide p-benzoquinone p-benzoquinone hydrogen peroxide

9.2 6.7 3.5

>60 a 40

Unknown.

by the SAM, possibly reducing the “roughness”; a single enzyme molecule is likely to “sit” across several “headgroups” of the SAM, and thus the immobilization of enzyme may not be influenced by a certain degree of change in topography or order of the SAM. Platinized Electrode Responses. To abolish the need for a mediator and return to hydrogen peroxide determination on these gold thiol-modified electrodes, the strategy adopted was to introduce platinum into the layer, thus providing a catalytic surface for the electrochemical oxidation of enzyme-generated peroxide. The method for electrochemical platinum deposition was adopted from Gunasingham and Tan,40 and, on unmodified gold electrodes, the cyclic voltammogram obtained during platinization was compared with that reported by Gunasingham and Tan. Unlike the gold electrode, the platinized electrode was shown to respond to hydrogen peroxide additions when the electrode potential was set to +0.65 V, and no current response was observed if glucose was added to the solution. The GOx-SAM-modified gold electrodes prepared after rigorous polishing and cleaning were platinized by the same procedure. Platinization develops as a nucleation and growth procedure, so the initial growth requires a seeding point access to the gold surface of the electrode; i.e., defects in the self-assembled monolayer are needed to enable the PtCl6- to diffuse to and be directly reduced at the electrode. Visual inspection of the electrodes under the microscope following the platinization protocol showed that Pt deposited preferentially at the edges of the electrode and that other deposits were irregular and difficult to reproduce. Since the target of this study was to achieve platinization of the GOx-SAM-modified electrode, the short-chain propionic acid thiol had been chosen in preference to the longer chain thiols since they are shown to form less uniform monolayers than longer chain thiols,18 yet even so these layers appeared to inhibit the onset of platinization. In contrast to the polished electrodes, the “sanded” GOxSAM electrodes were completely platinized without difficulty. Under the microscope, these electrodes appeared to have a regular Pt deposition across the surface. This seems to suggest that the sanded electrodes contained more defects within the self-assembled monolayer, which is consistent with previous findings,47 although the GOx-SAM Au electrodes used here with mediators did not reveal evidence for such features. The glucose response 2400 Analytical Chemistry, Vol. 70, No. 11, June 1, 1998

Figure 3. Comparison of glucose response between GOx-SAMmodified gold electrode (electrode potential +0.5 V, and p-benzoquinone used as mediator) and a platinized Pt-GOx-SAM-modified electrode (E ) +0.65 V, no mediator). The electrode was platinized for 30 min. The response is quoted as a current density to allow different electrodes to be compared.

curves obtained with the platinized electrodes are shown in Figure 3. With these calibration curves, the enzyme is recycled using the natural cosubstrate oxygen, and thus the extent of reaction is monitored via the oxidation of hydrogen peroxide. The response curve for the platinized electrode shows both reduced sensitivity (in terms of current output) and reduced dynamic range relative to the unplatinised electrode, where 1 mM benzoquinone reoxidation is recorded. (A sensitivity of 0.88 µA cm-2 mM-1 at low concentrations is recorded, tending toward saturation at 7.5 mM). However, oxygen is present at a concentration of 0.2 mM, and, if the Pt-GOx-SAM electrode was used in the presence of 1 mM p-benzoquinone as mediator, rather than the natural mediator oxygen, the sensor showed a slightly lower saturation current output than that obtained prior to platinization for the GOx-SAM electrode (see Table 1); on the other hand, the dynamic range was reduced and comparable with that for peroxide monitoring on the same electrode. Thus, there is no evidence that platinization directly destroys the enzyme activity, but the reduced dynamic range may indicate a disruption of the diffusion barrier created by the SAM by opening of the structure on deposition of Pt. Thin-Film Electrodes. To manufacture Pt-enzyme-SAM electrodes commercially requires a method of mass-producing the electrodes; to this end, the procedures developed above were exported to the preliminary investigation of a thin-film electrode.

The intention was to create a GOx-SAM layer on a freshly evaporated gold surface, since this technology could be extended to produce photolithographic structures at a later date. However, it is clear from the aforegoing data that the surface roughness of the gold electrode may be paramount to the success and performance of the system. With bulk gold, the alkanethiols can bind to both Au(100) and Au(111) sites, while evaporated gold is composed predominantly of Au(111).12 Furthermore, it has been reported that SAMs of alkanethiols on evaporated gold films are less ordered than those on bulk gold due to defects in the SAM occurring at the grain boundaries present in the evaporated gold films.48 Moveover, the surface topography of the evaporated gold and the real and imaginary parts of the refractive index vary with evaporated thickness and underlying surface. Gold films on glass have to be produced with an “adhesion” layer to prevent the gold from lifting off the glass during use in aqueous media. This is typically provided by an intermediate chromium layer (usually between 5 and 20 nm, depending on application) or by use of reagents such as (3-mercaptopropyl)trimethoxysilane, which hydrolyzes and condenses to couple with the surface -OH groups on the glass and to form a silane polymer with exposed thiol groups to “bind” the evaporated gold. We have shown previously that these latter Au layers have a sharper surface plasmon resonance response,49 indicating a less rough surface than the equivalent chromium interlayer Au films and a slightly different refractive index {(0.185 ( 0.015) + (3.55 ( 0.025)i for Au with the silane adhesion layercompared with (0.19 ( 0.01) + (3.40 ( 0.03)i for the Cr adhesion layer}. Figure 4 shows the response of a GOx-SAM silane adheredgold electrode to glucose challenge before (a) and after (b) platinization, as measured using the benzoquinone mediator. The electrode appears to show a somewhat reduced sensitivity compared with that of the bulk electrodes (Table 1), and, although the surface roughness approaches 1, platinization can be achieved more successfully than for the bulk electrode of equivalent roughness. This is consistent with the idea of a less ordered SAM on the evaporated film.48 The response curve for a GOx-SAM thin-film electrode with a chromium adhesion layer, illustrated in Figure 5, showed a much greater reduction in sensitivity relative to the bulk electrodes (Table 1) but a similar dynamic range. Platinization of this electrode produced a mediated low glucose response similar to that seen prior to platinization, but at high glucose concentrations the response became erratic. On the other hand, the equivalent peroxide response had a smaller dynamic range and a saturation current, which reflected the lower concentration of the natural mediator oxygen (0.2 mM instead of 1.0 mM for benzoquinone). It is clear from these results that the preparation of the gold layer influences the resultant response of the GOx-SAM. Unlike the bulk Au electrodes, GOx-SAM biosensors constructed on the thin-film electrodes cannot correlate performance to the same measure of roughness. These films all approach a roughness factor of 1, but they present different sites for thiol binding to the bulk metal and behave more like the less ordered films on rough bulk electrodes. In particular, a film electrode required a few days following thiol assembly and GOx immobilization before the (48) Creager, S. E.; Hockett, L. A.; Rowe, G. K. Langmuir 1992, 8, 854-861. (49) Gaus, K. A Heparin Biosensor using Surface Plasmon Resonance. M.Ph. Thesis University of Cambridge, UK, 1996, pp 31-40.

Figure 4. Response curves for (a) GOx-SAM-modified gold thinfilm electrode, where the mediator was 1 mM p-benzoquinone, and (b) platinized GOx-SAM-modified thin-film electrode. The electrode area in each case was 0.79 cm2.

Figure 5. Glucose response curves for thin-film electrodes constructed with a chromium adhesion layer between the glass and gold electrode. p-Benzoquinone (1 mM) mediation is compared for the GOx-SAM- and Pt-GOx-SAM-modifed electrodes for different concentrations of glucose.

response on the Pt-deposited electrode stabilized to a maximum, a behavior which would be consistent with an ongoing reorganization after the initial assembly. On the bulk electrodes, it was obvious that a certain amount of disorder in the SAM was required to allow platinization. However, since a lesser order is proposed on film electrodes,48 then platinization “failure” is no longer a consideration. The higher current densities achieved on the silane-adhered gold electrodes may indicate that a more ordered thiol layer is formed than on the chromium interlayer gold films, Analytical Chemistry, Vol. 70, No. 11, June 1, 1998

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but this is a preliminary idea which requires separate investigation with independent methodology. CONCLUSIONS This paper illustrates a preliminary investigation of the application of self-assembled monolayers of alkanethiols to the preparation of covalently immobilized enzyme layers. These thiol monolayers allow two-dimensional reaction matrixes to be created, which confines the biochemical reaction to the surface of the biorecogntion matrix. Therefore, problems associated with diffusion through a three-dimensional reaction layer, namely irreproducibility and poor response time, are overcome, provided the SAM-enzyme layer, and other processing steps, can be prepared in a reproducible manner. The large dynamic range of the mediated SAM-immobilized glucose electrodes is particularly advantageous. The biosensor can, therefore, operate across the entire physiological glucose range of 0-50 mM, with an excellent sensitivity of 1.5 µA cm-2 mM-1, at low glucose concentrations. The sensitivity implies that, with this system, the formation of multilayers is not required, especially as the sensitivity of such electrodes has been reported to be enhanced by roughening gold electrodes using amalgamization prior to modifying the electrode with the SAM,47 so that further improvement may be possible. The platinisation of the gold SAM-GOx electrodes allowed the detection of the enzyme reaction to be monitored via the production of hydrogen peroxide, an approach which is not viable on a gold electrode. The platinization of the electrode obviates the requirement of a synthetic redox mediator and, hence, avoids the problems associated with oxygen interference suffered by redox mediators. In effect, the electrochemically deposited platinum particles serve as an electrochemical mediator. The sensitivity and dynamic range of the monolayer electrodes are

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reduced upon platinization but are still adequate for most applications. Comparison of GOx-SAM and Pt-GOx-SAM biosensors constructed using bulk gold electrodes and evaporated thin-film electrode shows that the nature of the surface upon which the SAM is prepared has considerable influence over the properties of the final biosensor. On bulk electrodes, ordered SAM layers on polished surfaces occlude the electrode, preventing Pt deposition; defects can be introduced by increasing the roughness of the electrode. Au film electrodes have different properties from bulk Au, and the SAM is less ordered. On these electrodes, platinization occurs readily, and the Pt-GOx-SAM electrode can be used to monitor glucose via the H2O2 oxidation current. In contrast with the bulk Au electrodes, the aim here appears to be to increase the ordering of the SAM so as to increase the immobilization efficiency, rather than decrease it to increase defects. The application of self-assembled monolayers for the covalent immobilization of enzymes to gold electrodes, and their subsequent platinization, has considerable potential to produce reproducible enzyme biosensors. However, further work is required with regard to understanding the parameters that influence the reproducibility of the immobilization method, how to control the amount of enzyme that is immobilized, the generation of reproducible defects within the SAM, and the influence of the platinization in the enzyme-SAM layer, for example. This further work is the subject of a current study in our laboratories.

Received for review September 18, 1997. Accepted March 7, 1998. AC971035T