Electrical Communication between Components of Self-Assembled

We have succeeded in developing communication between the enzyme glucose oxidase10 and a gold electrode, without direct modification of the enzyme wit...
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Langmuir 1996, 12, 363-370

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Electrical Communication between Components of Self-Assembled Mixed Monolayers Shai Rubin,† Jimmy T. Chow,‡ John P. Ferraris,‡ and Thomas A. Zawodzinski, Jr.*,† Electronics and Electrochemical Materials and Devices Group, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, and Department of Chemistry, University of Texas/Dallas, Richardson, Texas 75083-0688 Received January 31, 1995. In Final Form: August 21, 1995X The use of an alkanethiol-based self-assembled mixed monolayer as an electronic relay system effecting mediated electron transfer between immobilized glucose oxidase (GOx) and a gold electrode is reported. We compare the behavior of mixed monolayers of various compositions of 16-ferrocenylhexadecanethiol (16FAT) and aminoethanethiol, to which GOx is attached, as biosensors for glucose. The amperometric response of such electrodes in the presence of glucose in solution depends on the mole ratio between the 16FAT molecules and the attached protein molecules. The most sensitive system is a mixed monolayer that contains 7% 16FAT. For higher 16FAT concentrations, both a catalytic response and a wave corresponding to reversible 16FAT voltammetry are observed in the presence of glucose. This suggests that there are separate domains of 16FAT and of aminoethanethiol in such a mixed monolayer. When the mixed monolayer contains more than 7% 16FAT, a portion of the 16FAT molecules cannot “feel” the GOx and does not function as relays. The existence of these domains was also characterized by studying the solution voltammetry of Ru(NH3)63+ at electrodes with various proportions of 16FAT and aminoethanethiol.

Introduction Self-assembled monolayers (SAMs) of alkanethiols spontaneously adsorb on gold surfaces to form wellorganized structures.1 Functionalized SAMs can be used to develop image devices,2 photopatterning systems,3 biosensors,4 and affinity biosensors.5 A difficulty in creating biosensors based on some classes of redox enzymes immobilized in such monolayers is the lack of direct electrochemical communication (i.e., electron transfer) between the redox center of the enzyme and the electrode due to the large distance between the two.6 Communication between a redox enzyme and an electrode is the basic feature necessary for the development of biosensors. In the case of enzymes with their redox site close to the periphery of the protein, direct electron transfer between the redox site and the electrode is possible.7 On the other †

Los Alamos National Laboratory. University of Texas/Dallas. X Abstract published in Advance ACS Abstracts, November 15, 1995. ‡

(1) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481. Troughton, E. B.; Bain, C. D.; Whitesides, G. M.; Nuzzo, R. G.; Allara, D. L.; Porter, M. D. Langmuir 1988, 4, 365. Ulman, A. Introduction to Ultrathin Organic Films; Academic Press: New York, 1991. (2) Frisbie, C. D.; Rozsnyai, L. F.; Noy, A.; Wrighton, M. S.; Lieber, C. M. Science 1994, 265, 2071. Wollman, E. W.; Kang, D.; Frisbie, C. D.; Lorkovic, I. M.; Wrighton, M. S. J. Am. Chem. Soc. 1994, 116, 4395. (3) Huang, J.; Dahlgran, D. A.; Hemminger, J. C. Langmuir 1994, 10, 626. (4) Willner, I.; Riklin, A.; Shoham, B.; Revenizon, D.; Katz, E. Adv. Mater. 1993, 13, 912. (5) Willner, I.; Blonder, R.; Dagan, A. J. Am. Chem. Soc. 1994, 116, 9365. Willner, I.; Rubin, S.; Cohen, Y. J. Am. Chem. Soc. 1993, 115, 4937. Willner, I.; Rubin, S.; Cohen, Y. Unpublished results. Willner, I.; Dagan, A.; Rubin, S.; Blonder, R.; Riklin, A.; Cohen, Y. Israel Patent Application No. 108726, Filed on Feb 22, 1994. Willner, I.; Rubin, S. Angew. Chem., in press. (6) Marcus, R. A. Int. J. Chem. Kinet. 1981, 13, 865. Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1985, 811, 265. (7) Willner, I.; Katz, E.; Lapidot, N.; Ba¨uerle, P. Bioelectrochem. Bioenerg. 1992, 29, 29. Kinnear, T. K.; Monbouquette, H. G. Langmuir 1993, 9, 2255. Kinnear, T. K.; Monbouquette, H. G. Biotechnol. Bioeng. 1993, 42, 140. Sucheta, A.; Ackrell, B. A. C.; Cochran, B.; Armstrong, F. A. Nature 1992, 356, 361. Sucheta, A.; Ackrell, B. A. C.; Cammack, R.; Weiner, J.; Armstrong, F. A. Biochemistry 1993, 32, 5455. Guo, L.-H.; Hill, H. A. O.; Lawrance, G. A.; Sanghera, G. S.; Hopper, D. J. J. Electroanal. Chem. 1989, 266, 379.

0743-7463/96/2412-0363$12.00/0

hand, in the case where the redox site of the enzyme is embedded deep within the protein, the direct mode of communication between the enzyme and the electrode is impossible. One demonstrated way of bringing about communication between the active site of redox enzymes of the latter class and the electrode is to create a molecular “wire” from electron relays on the protein backbone.8 An “all-in-one” biosensor must contain three components: the electrode, the enzyme, and the mediator. Two previous papers described such biosensors systems prepared using self-assembly methods. Willner et al.4 described a multilayer system of glucose oxidase (GOx) on a gold electrode that has been modified with N-(2methylferrocene)caproic acid attached directly to the protein backbone. The ferrocene-modified GOx multilayer system has the ability to communicate with the gold electrode. Kajiya et al.9 described a system of a 4-aminothiophenol monolayer absorbed to a gold electrode that was modified simultaneously (using glutaraldehyde) with GOx and 2-(aminoethyl)ferrocene to create a mixed monolayer of ferrocene-modified GOx and a ferrocene derivative of the monolayer component. In this system, shuttling of the electron between the electrode and the flavin adenine dinucleotide (FAD) group of the enzyme is mediated by the surface-attached ferrocene and by the ferrocene molecules that are anchored on the protein backbone, with the latter probably dominating the observed response. In this publication we describe a new solution for the electronic communication problem between the electrode and the enzyme. We have constructed a self-assembled mixed monolayer which contains, as independent components, relay molecules, which mediate the electron transfer between the redox site of the enzyme and the electrode, and functional sites used as anchoring sites for the immobilization of the enzyme. We have succeeded in (8) Heller, A. J. Phys. Chem. 1992, 96, 3579. Degani, Y.; Heller, A. J. Phys. Chem. 1987, 91, 1285. Schumann, W.; Ohara, T. J.; Schmidt, H.-L.; Heller, A. J. Am. Chem. Soc. 1991, 113, 1394. Degani, Y.; Heller, A. J. Am. Chem. Soc. 1988, 110, 2615. Willner, I.; Lapidot, N. J. Am. Chem. Soc. 1991, 113, 3625. (9) Kajiya, Y.; Okamoto, T.; Yoneyama, H. Chem. Lett. 1993, 2107.

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developing communication between the enzyme glucose oxidase10 and a gold electrode, without direct modification of the enzyme with electron relays. We describe in this paper the electrochemical properties of a mixed monolayer of 16-ferrocenylhexadecanethiol (16FAT) and aminoethanethiol and demonstrate the use of this mixed monolayer for the immobilization of GOx and as an electronic relay for its redox center. This represents, to our knowledge, the first case of the use of a synthetically prepared self-assembled mixed monolayer to effect a multistep catalytic reaction based on successive electron transfers. Experimental Section Materials. The gold and the titanium purity is 99.999%. Microscope slides were obtained from Corning. Glutaraldehyde (25% in water) was obtained from Fluka, glucose oxidase (GOx) (E.C 1.1.3.4) (115 000 units/g solid) from Sigma, and 1-hexadecanethiol (16AT) and aminoethanethiol were from Aldrich. 16Ferrocenylhexadecanethiol (16FAT) was synthesized according to the literature.11 (16-mercaptohexadecanoyl)ferrocene was prepared by Friedel-Crafts acylation of ferrocene with 16bromohexadecanoyl chloride and reaction of the bromide with potassium thioacetate (Fluka 99%) (followed by reaction with sodium hydroxide to yield the thiol. 16-Ferrocenylhexadecanethiol was prepared from (16-mercaptohexadecanoyl)ferrocene by borohydride/BF3 etherate reduction of the carbonyl. 16-bromohexadecanoyl chloride was prepared from 16-hydroxyhexadecanoic acid (Aldrich, 98%) by reaction with HBr to form the ω-bromo acid followed by reaction with thionyl chloride (Aldrich, 99%). Electrode Fabrication. Microscope slides were cleaned with piranha solution, (H2SO4:H2O2 4:1) rinsed with distilled water, and dried with argon. The slides were sonicated for 300 s in each of the following solutions: soapy distilled water, distilled water, trichloroethane, acetone, and isopropyl alcohol. The clean glass slides were dried with argon. Gold films (∼2000 Å thick) were prepared by sputter deposition of gold onto the glass slides that had been precoated with a film of titanium (∼50 Å thick). The average roughness factor of the glass/Ti/Au electrodes is 1.7 and was measured by integrating an oxide stripping peak as described by Woods.12 Formation of Monolayers. The electrodes were soaked for 10 min in concentrated nitric acid, rinsed with distilled water and ethanol, and dried with argon. (a) 16FAT:Aminoethanethiol Mixed Monolayer. The electrodes were soaked in an ethanolic solution of 16FAT (0.090.12 mM) and amimoethanethiol (1.9 mM) for 1-6 h and then transferred to an ethanolic solution of aminoethanethiol (2 mM). (b) 16FAT Monolayer. The electrodes were soaked in an ethanolic solution of 16FAT (0.9 mM) 184 h. (c) 16AT:Aminoethanethiol Mixed Monolayer. The electrodes were soaked in an ethanolic solution of 16AT (0.09 mM) and aminoethanethiol (1.9 mM) for 2 h and then transferred to an ethanolic solution of aminoethanethiol (2 mM). Immobilization of the Enzyme. The modified electrodes were soaked for 2 h in an aqueous solution of 1 mg/mL GOx and 7% glutaraldehyde at 30 °C. The imide bonds were subsequently reduced by adding 0.4 mL NaBH4 (4 mg/mL) for 30 min at room temperature. The GOx modified electrodes were soaked for 30 min in sodium bicarbonate buffer (0.1 M, pH ) 7.3, adjusted with HClO4). Electrochemical Studies of the Mixed Monolayers. Electrochemical experiments were conducted with a Princeton Applied Research 273 potentiostat interfaced to a Macintosh II, controlled by the LabVIEW II software program, in a threeelectrode cell that contained a saturated calomel electrode (SCE, Hg/HgCl2/saturated KCl) reference electrode and a platinum mesh as counter electrode. The electrolyte solution was sodium bicarbonate buffer (0.1 M, pH ) 7.3, adjusted with HClO4). For (10) Hecht, H.; Schomburg, D.; Kalisz, H.; Schmid, R. D. Biosens. Bioelectron. 1993, 8, 197. (11) Collard, M. D.; Fox, M. A. Langmuir 1991, 7, 1192. (12) Woods, R. Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker, Inc.: New York and Basel, 1976; Vol 9, p 119.

Figure 1. Illustration of the great size difference between mixed monolayer components 16FAT and aminoethanethiol. probes of the electrode accessibility to solution redox couples, 1 mM Ru(NH3)6Cl3 was added to the buffer solution. Cyclic voltammetric studies of the catalytic amperometric response toward glucose of the mixed monolayer electrodes modified by GOx were carried out at a scan rate of 2 mV/s. All electrochemical measurements were done under argon.

Results and Discussion Preparation of the Mixed Monolayers. To prepare a mixed monolayer that is composed of alkanethiols terminating in two different functional groups with different methylene chain lengths and which has a controlled composition, we had first to consider the different deposition rates of the components.13 The most important parameter in determining this rate is the length of the methylene chain: the longer the methylene chain, the faster chemisorption occurs.13 Figure 1 shows models of 16FAT and aminoethanethiol and emphasizes the huge difference (∼20 Å) in the length of the chains in this case. Since we prepare mixed monolayers of these two compounds, this difference in deposition rate is of particular relevance to our control over the composition of our monolayers. Another parameter that can influence the final ratio of the two components in the mixed monolayer is the interaction between the functional groups. The amine functional group can form hydrogen bonds14 that could stabilize the forming monolayer, while the ferrocene functional groups can participate in a hydrophobic interaction. We empirically determined appropriate solution proportions which yielded specific monolayer compositions. To prepare monolayers that contain 2-36% 16FAT loading, we used for the soaking solution an initial ratio of 1:∼20 16FAT:aminoethanethiol for 1-6 h and then an exchange solution, containing only aminoethanethiol, to achieve the desired final ratio between the mixed monolayer components. Electrochemical Characterization of the Mixed Monolayers. Figure 2 shows the cyclic voltammograms (CVs) of the mixed monolayers with different ratios between the two components (Figure 2a, 36% 16FAT; Figure 2b, 16% 16FAT; Figure 2c, 7% 16FAT; Figure 2d, 2% 16FAT (percentages are relative to a charge of 450 pmol/cm2 for full monolayer coverage of ferrocenylalkanethiol, ref 15)). Several interesting features of the (13) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321. (14) Spril, M.; Delamarche, E.; Michel, B.; Rothlisberger, U.; Klein, M. L.; Wolf, H.; Ringsdorf, H. Langmuir 1994, 10, 4116. Hautman, J.; Klein, M. L. Phys. Rev. Lett. 1991, 68, 2345. Hautman, J.; Bareman, J. P.; Mar, W.; Klein, M. L. J. Chem. Soc., Faraday Trans. 1991, 87, 2031. Klein, M. L. Mol. Phys. 1992, 75, 379. (15) Popenoe, D. D.; Deihammer, R. S.; Porter, M. D. Langmuir 1992, 8, 2521.

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Figure 2. Cyclic voltammograms of different mixed monolayers (a) 16FAT loading is 36%, (b) 16FAT loading is 16%, (c) 16FAT loading is 7%, and (d) 16FAT loading is 2%. For each loading, five different scan rates are presented: curve a, 200 mV/s; curve b, 100 mV/s; curve c, 50 mV/s; curve d, 25 mV/s; and curve e, 10 mV/s. Table 1. Electron Transfer Rate of Each of the Mixed Monolayer Systems Calculated According to the Laviron Method16 mixed monolayer composition (before the modification with GOx)

ket (s-1)

mixed monolayer with 36% 16FAT mixed monolayer with 16% 16FAT mixed monolayer with 7% 16FAT mixed monolayer with 2% 16FAT

3.95 3.52 4.28 2.2

mixed monolayer systems of different compositions can be observed in Figure 2: (i) as the loading of 16FAT is decreased, the charging current increases and (ii) as the proportion of aminoethanethiol in the monolayer increases, the observed current associated with oxidation of the gold surface (∼0.65 V vs SCE in these solutions) increases. This suggests that some bare gold is exposed in the domains under the aminoethanethiol components resulting from disorder in the monolayer prepared from such a short-chain alkanethiol. In Table 1, we summarize the apparent rates of electron transfer, measured by the Laviron method,16 between attached ferrocene and the gold electrode for mixed monolayers of various loadings. In the range of 16FAT loading 7-36%, no significant difference in the electron transfer rate is observed; a somewhat lower electron transfer rate is calculated for the lowest 16FAT loading, though given the method, this may not represent a significant difference from the case with higher loading. This suggests that the 16FAT in these monolayers is exposed to an unchanging environment. Linear Sweep Cyclic Voltammograms of Dissolved Hexaammineruthenium(III) Chloride,17 for Probing the Different Domain Sizes in Different (16) Laviron, E. J. Electroanal. Chem. 1979, 101, 19. Laviron, E. Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker, Inc.: New York and Basel, 1982; Vol 12, p 53. (17) Chailapakul, O.; Crooks, R. C. Langmuir 1993, 9, 884. Sun, S.; Crooks, R. C. Langmuir 1993, 9, 1951. Chidsey, C. E. D.; Loiacono, D. N. Langmuir 1990, 6, 682. Bilewicz, R.; Majda, M. Langmuir 1991, 7, 1951. Ross, C. B.; Sun, S.; Crooks, R. C. Langmuir 1993, 9, 632. Zhang, L.; Lu, T.; Gokel, G. W.; Kaifer, A. E. Langmuir 1993, 9, 786.

Mixed Monolayer Compositions. To further investigate the tendency of the components to segregate, we employed Ru(NH3)6Cl3 as a redox probe of the structure of mixed monolayers with different compositions. Figure 3 shows the CVs of electrodes loaded with several different compositions of 16FAT/aminoethanethiol mixed monolayers exposed to solutions of the redox probe. The ability of the redox probe to penetrate through the mixed monolayer to the electrode changes according to the ratio between the two components of the mixed monolayer. This approach follows that of Crooks and co-workers17 in their probe of the effects of increasing the loading of a shortchain component into a monolayer initially composed of a long-chain alkanethiol. Figure 3a shows the case of a monolayer consisting of only 16FAT. The monolayer completely blocks the penetration of the redox probe. From the shape of the curve, we conclude that electron transfer through the monolayer takes place exclusively by tunneling.17 Figure 3b shows that, for a high proportion of 16FAT in the mixed monolayer (53%), the aminoethanethiol molecules act as a microelectrode domain, allowing the redox probe to penetrate through to the electrode. In Figure 3c, with 49% 16FAT in the monolayer, the shape of the curve is still sigmoidal as in the case of radial diffusion. As shown in parts d and e of Figure 3 (43% 16FAT) and (36% 16FAT), respectively, as the concentration of aminoethanethiol increases, the CV curve shapes suggest an intermediate stage between purely semi-infinite linear and radial diffusion. Figure 3f (8% 16FAT) shows a CV characteristic of semi-infinite linear diffusion. These results suggest that the redox probe can penetrate the mixed monolayer to the electrode through the domains of aminoethanethiol. Since access of Ru(NH3)63+ to the electrode is provided through the aminoethanethiol-rich regions of the monolayer, the transition from voltammograms indicative of radial diffusion to those indicative of semi-infinite linear diffusion suggests that such regions are growing with increasing aminoethanethiol loading in the mixed monolayer. This change of the apparent diffusional mode of

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Figure 3. The electrochemical response of solutions of Ru(NH3)63+ at electrodes coated with 16FAT:aminoethanethiol mixed monolayers of various compositions. (a) Monolayer composed only from 16FAT, (b) 53% loading of 16FAT, (c) 49% loading of 16FAT, (d) 43% loading of 16FAT, (e) 36% loading of 16FAT, and (f) 8% loading of 16FAT.

the redox probe with changing monolayer composition reflects the growing average aminoethanethiol domain size. Comparison between the Electrochemical Response of the 16FAT in the Mixed Monolayer before and after the GOx Modification. Modification of the amine sites in the mixed monolayer by attachment of the enzyme causes changes in electrochemical response of the 16FAT sites. Figure 4 shows the difference between the cyclic voltammograms of the mixed monolayers, recorded at 200 mV/s, before and after the GOx modification. In each case, the area of the redox peaks decreases after modification of the electrode with the enzyme. From Figure 4a, we calculated a reduction of 7% of the area of the redox peak of the 16FAT (initially 36% loading, after the modification with GOx 29%). From Figure 4b we calculate a reduction of 3% of the area of the redox peak of the 16FAT (initially 16% loading). From Figure 4c we calculate a reduction of 4% of the area of the redox peak of the 16FAT (initially 7% loading). From Figure 4d we calculate a reduction of 0.5% of the area of the redox peak of the 16FAT (initially 2% loading). We can imagine two possible explanations for this phenomenon: (1) in the process of modification of the monolayer with the enzyme, some of the 16FAT molecules desorb from the gold surface. However, it is well-known from the literature that to remove this kind of monolayer from the gold surface more drastic conditions are needed.18 (2) Some of the 16FAT molecules interact with the enzyme

layer, and these molecules cannot communication with the electrode because the enzyme layers that block access of compensating ions from the solution to some of the 16FAT monolayer.19 If ferrocenes buried under the GOx are not easily accessible to counterions in solution, the redox process of this population will be hindered. Even for much slower scan rates, the difference ∆QFc (QFc is the charge of the absorbed ferrocene) between electrodes with and without enzyme is still significant. This suggests that the rate of electron transfer between the electrode and the 16FAT population covered by enzyme layers is much slower than that of the uncovered 16FAT population. The effect of this electron transfer rate difference will be discussed below. From Table 2, which compares the peak to peak separation before and after the enzyme modification, it is clear that the electron transfer rate of the ferrocene population that is not covered by enzyme is also slowed down after the enzyme modification. We used Ru(NH3)6Cl3 as a probe to determine if the modification of the mixed monolayer by the enzyme causes changes in the mixed monolayer permeability. Figure 5 shows the difference in the ability of the probe molecules to penetrate through the mixed monolayer (the loading of 16FAT is 8%) before (curve a) and after (curve b) the modification with GOx. The shape of curve a, in the region of the redox procss of the Ru(NH3)6Cl3, suggests almost (18) Zhong, C.-J.; Porter, M. D. J. Am. Chem. Soc. 1994, 116, 11616. (19) Gou, L.-H.; Facci, J. H.; Mclendon, G. J. Phys. Chem. 1995, 99, 4106.

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Figure 4. Comparison of the cyclic voltammograms of the different mixed monolayers before and after attachment of GOx: (a) 36% loading of 16FAT before modification (curve a) and 29% after modification (curve b), (b) 16% loading of 16FAT before modification (curve a) and 13% after modification (curve b), (c) 7% loading of 16FAT before modification (curve a) and 3% after modification (curve b), and (d) 2% loading of 16FAT before modification (curve a) and 1.5% after modification (curve b). All loadings given are based on the charge associated with the ferrocene oxidation wave. Table 2. ∆E of Each of the Mixed Monolayer Systems before and after the Modification with GOx ∆E (mV) scan rate ) 200 mV/s mixed monolayer composition (before the modification with GOx)

only the mixed monolayer

GOx-modified mixed monolayer

mixed monolayer with 36% 16FAT mixed monolayer with 16% 16FAT mixed monolayer with 7% 16FAT mixed monolayer with 2% 16FAT

87 108 90 77

104 145 107 95

pure linear diffusion of the probe toward the electrode. After the enzyme attachment the cyclic voltammogram shape (curve b) suggests an intermediate stage between purely linear and radial diffusion (parts d and e of Figure 3). It can be seen from this figure that the enzyme does not completely block the diffusion of the probe to the electrode, but there is a significant change in the number of redox probe molecules that penetrate through the mixed monolayer. This is evidence that the enzyme sits primarily in the amine domains, as might be expected, because the electroactive probe penetrates to the electrode only through the amine domain of the monolayer (based on the results summarized in Figure 3). Measurement of Glucose Concentration by the Amperometric Response of the 16FAT:Aminoethanethiol GOx Arrays. To create a glucose biosensor based on the enzyme glucose oxidase that can generate a detectable response for glucose, we require (1) enough enzyme to oxidize glucose, (2) enough ferricenium ions sufficiently close to the FADH2 groups for regeneration of the redox active center. Figure 6 shows the voltammetric responses of the 16FAT:aminoethanethiol GOx arrays with different compositions of the absorbed layers toward several concentrations of glucose. Figure 6a, the cyclic voltammetric response of a monolayer containing 36% 16FAT, shows a small catalytic amperometric response (Ep/2(cat) ) 0.04 V, Ep/2(cat) is the half-wave potential of the catalytic oxidation wave, and all potentials are vs SCE)

Figure 5. Cyclic voltammograms obtained for solutions of Ru(NH3)6Cl3 probe (E°′ ) -0.2 V vs SCE) at a gold electrde modified with 16FAT (8% loading) and aminoethanethiol (for 16FAT, E°′ ) 0.39 V vs SCE): curve a, before the modification with GOx; and curve b, after the modification with GOx.

for glucose (45 mM, curve ii) and a large reversible redox response (Ep/2 ) 0.38 V, oxidation wave) due to 16FAT that does not interact with the redox center of the enzyme. From the results shown in Figure 6a, we conclude that most of the 16FAT molecules do not participate in the oxidation of the FADH2 groups. When the basic requirements outlined above are considered, it is clear that, for this composition of mixed monolayer, there is not enough enzyme to interact with all of the electron relays in the monolayer. Figure 6b shows the much larger amperometric response (Ep/2(cat) ) -0.03 V) toward glucose (45 mM, curve iii) of a monolayer containing 16% 16FAT, but the ratio between the monolayer components is still not ideal, and once again, most of the 16FAT molecules are not used as mediators. The non-interacting 16FAT molecules take the place of aminoethanethiol molecules that could be used as anchoring sites for additional enzyme. Figure 6c, the response of an electrode covered with a monolayer with 7% 16FAT, shows the optimal amperometric response (Ep/2(cat) ) 0.02 V) that can be achieved in such a system. In this case most of the 16FAT molecules

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Figure 7. Amperometric response to glucose of (a) 16FAT (36% loading)/GOx-aminoethanethiol array, (b) 16FAT (16% loading)/GOx-aminoethanethiol array, and (c) 16FAT (7% loading)/ GOx-aminoethanethiol array. The inset compares the cyclic voltammetric response of the three systems when 105 mM glucose is present in the solution (scan rate of 2 mV/s).

Figure 6. (a) Cyclic voltammograms of 16FAT:aminoethanethiol (36% 16FAT loading) GOx arrays on a gold electrode in the presence of (i) 0 mM glucose and (ii) 45 mM glucose. (b) Cyclic voltammograms of 16FAT:aminoethanethiol (16% 16FAT loading) GOx arrays on a gold electrode in the presence of (i) 0 mM glucose, (ii) 5 mM glucose, (iii) 45 mM glucose, and (iv) 105 mM glucose. (c) Cyclic voltammograms of 16FAT:aminoethanethiol (7% 16FAT loading) GOx arrays on a gold electrode in the presence of (i) 0 mM glucose, (ii) 15 mM glucose, (iii) 45 mM glucose, and (iv) 105 mM glucose. The scan rate was 2 mV/s.

are used for mediation. This array functions as the most sensitive glucose sensor compared to the other systems presented here (to compare, see the amperometric response toward 45 mM glucose, curve iii). For a case when not enough mediator molecules are included (2% loading of 16FAT), essentially no amperometric response to glucose is observed. Figure 7 illustrates the difference in the sensitivity between the three mixed monolayer systems. It can be seen from this figure that the mixed monolayer containing only 7% 16FAT is the most sensitive system. In the low range of glucose concentrations (from 0 to 30 mM glucose), the mixed monolayer that contains 16% 16FAT and the mixed monolayer that contains 7% 16FAT are similar in their sensitivity toward glucose. However, at higher glucose concentrations, the biosensor array based on the

mixed monolayer 16FAT 16% reached a saturation point. On the basis of the known kinetics of the glucose oxidase catalyzed oxidation of glucose, we conclude that this occurs because the observed Km for the electrode with 16% 16FAT loading is smaller because of the smaller amount of enzyme near the mediator in this case relative to the 7% case. (Km ) Vmax/2 and Vmax ) k2[Et] so if [Et] (considering only the enzyme interacting with the mediator) in the case of 16% loading is smaller than in the 7% case Km of the 16% case should be smaller than the Km of the 7% case.) In the presence of glucose, a substantial shift in the onset potential of the catalytic wave relative to that of the ferrocene oxidation is observed. Thus, the means by which glucose is oxidized in these systems must be clarified. There are three possible explanations for the observation of the shifted redox potential and the presence of both the ferrocene/ferrocenium wave and the catalytic wave: (1) there is direct oxidation of the glucose on the gold electrode20 independent of the presence of the ferrocene; (2) there is direct communication between the redox center of the enzyme and the electrode, due to reorganization of the three-dimensional configuration of the protein in the monolayer environment, again independent of the presence of the ferrocene; and (3) there are two kinds of 16FAT in this system. The first type of 16FAT does not interact with the protein, while the second population of 16FAT does interact with the enzyme. These two types of 16FAT have different apparent redox potentials. We reject the direct oxidation of glucose on the gold electrode on the basis of the experiment, mentioned above, with 2% 16FAT loading in the mixed monolayer. Compared to the other three compositions (see below), this is the most poorly insulated system of the bare gold toward diffusing species in solution (glucose). Nevertheless, there is not any amperometric catalytic response of this system toward glucose. Furthermore, the catalytic wave occurs at a potential inconsistent with that for the direct oxidation of glucose at gold.20 To explore the possibility of direct communication between the redox center of the enzyme and the electrode, we performed a control experiment using a mixed monolayer of 1-hexadecanethiol (16AT) (∼10% loading of 16AT) and aminoethanethiol, to determine if this system is (20) Vassilyev, Yu. B.; Khazova, O. A.; Nikolaeva, N. N. J. Electroanal. Chem. 1985, 196, 105. De Mele, M. L. F.; Videla, H. A.; Arivia, A. J. Bioelectrochem. Bioenerg. 1986, 16, 213.

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sensitive to different glucose concentrations. This system did not show any catalytic amperometric response toward glucose. The explanation of the catalytic response of the mixed monolayers based on the assumption of two 16FAT populations appears to be the most reasonable. There are, however, two additional questions raised from this explanation: (1) in the absence of glucose, why is there no reversible response of the 16FAT population which interacts with the enzyme? and (2) what is the source of the large difference in the redox potential (almost -400 mV in Ep/2 values or -150 to -320 mV in Ep values) between the two populations and among the three different loadings that are sensitive toward glucose? To our knowledge, there are no reports on precisely similar cases or appropriate theoretical predictions that can help to answer these questions. The following discussion summarizes some possible explanations for the potential shift, but it is unclear whether any of these factors can explain by itself such a large shift. The reaction scheme for the catalytic process occurring in the presence of glucose is summarized in the following equations:

with the enzyme so the reduction of the ferrocenium is very rapid. Figure 6 shows that there is no electrochemical response of the 16FAT mediator population in the absence of glucose. The oxidation current appears only in the presence of glucose due to the amplication of the oxidation process caused by the rapid reduction of the ferrocenium by the reduced form of the enzyme, the catalytic process. This explanation may contribute part of the negative potential shift of the catalytic wave but, on the basis of the following discussion, apparently cannot resolve this problem completely. Andrieux and Saveant22 presented a theoretical treatment for the catalytic electrochemical response of a redox catalyst adsorbed on an electrode. Though it treats a diffusion-controlled single step system, the results of this study provide insight into the potential shifts which can be expected in our case. The peak shift for a one-step catalytic process is given by the following equation:

Fc ) Fc+ + e-

(1)

Fc+ + GOxred ) Fc + GOxox

(2)

where Dsub is the glucose diffusion coefficient, Γ is the amount of surface-confined ferrocene that takes part in the catalytic process, ν is the scan rate, and kobs is the rate of the catalytic reaction between the enzyme and the relay molecules. (This equation predicts the value of Ep and not the value of Ep/2. Figure 1 in ref 22 suggests that the shift of Ep/2 is substantially larger than that of Ep.) In our case, the rate of the oxidation of glucose by GOx23 is 1.2 × 107 M-1 s-1, so even if the electron transfer rate from the enzyme to the relay molecule is very rapid, the maximum shift in Ep that can be explained by this model is ∼100 mV. Environmental Explanations for the Potential Shift. There are additional possible explanations for a shift in the ferrocene/ferrocenium redox potential, based not on the ratio between the oxidized and the reduced components but on the other term in the Nernst equation, E°′. Creager and co-workers24 demonstrated this possibility when they showed that the FAT redox potential varies in different environments. In their case, the redox potential of the ferrocene in the monolayer shifts to more positive potentials when the ferrocene is exposed to a hydrophobic environment. In the hydrophobic environment, the ferrocene form is favored relative to the ferricenium form, thus making oxidation less favorable thermodynamically. In our case the potential shift is in the opposite direction. If we claim that the source of the shift is a change of E°′, the mediator population exposed to the enzyme must be in a more hydrophilic environment. It can be argued that, in the uncovered, well-ordered 16FAT domain, the ferrocene is interacting with the solution primarily at the water/monolayer interface. By contrast, the relay 16FAT population is in a domain in which substantial structural disruption of the monolayer is probable, and therefore these Fc’s are more completely surrounded by solution. In our opinion, it is hard to believe that this is the underlying reason in our case since (1) the enzyme aggregate environment is more hydrophobic than

glucose + GOxox ) GOxred + gluconolactone (3) (Fc ) ferrocene, Fc+ ) ferrocenium, GOxred ) the reduced form of GOx (GOx-FADH2), GOxox ) the oxidized form of GOx (GOx-FAD)]. The Kinetic Explanation for the Potential Shift. In reaction 2 of the above scheme, we refer only to the subpopulation of Fc/Fc+ which actively participates in the mediation reaction, while all Fc’s are considered in reaction 1. The Fc population which does not interact with the enzyme is unaffected by reactions 2 and 3. On the basis of the Nernst equation, to observe such a huge negative shift for the catalytic wave, the concentration of ferrocene in the system must be much higher than that of the ferrocenium concentration ([Fc] . [Fc+]), probably by orders of magnitude. Such a high concentration ratio is possible if the formation rate of the ferrocenium is very slow and the elimination rate of the ferrocenium is very fast. This resembles a classic steady-state approximation condition in kinetics. The 16FAT population that acts as a mediator is covered by enzyme layers. From Figure 4, it is clear that, after the enzyme modification process, part of the 16FAT monolayer (i.e., that buried under the enzyme) has no redox response. According to Guo et al.19 when a FAT monolayer is insulated from the solution (by a Langmuir-Blodgett film in the work of Guo et al.), there is no electrochemical response because of the blocking effect of the insulating layer. We also proved, by the Ru(NH3)6Cl3 experiment, that the enzyme layer has a blocking effect (Figure 5) on the penetration of small molecules to the electrode. This blocking effect is in fact a kinetic inhibition process for the formation of the ferrocenium since charge compensation of the oxidation of ferrocene is likely to be inhibited. In the present case, it could be claimed that the enzyme layer is not truly an insulating layer because it is not hydrophobic and therefore will allow solvent and ions to leach into the electrode/SAM interfacial region. However, it is well-known that high molecular weight enzyme aggregates formed by reaction of the enzyme with glutaraldehyde are water insoluble (hydrophobic).21 On the other hand, in this system, the mediator molecules are already placed near or in intimate contact

Ep ) E0 - 0.78(RT/F) + (RT/F) ln[kobsΓ/(DsubFν/RT)1/2]

(21) Broun, G. G. Methods in Enzymology; Mosbach, K., Ed.; Academic Press: New York, San Francisco, London, 1976; Vol 44, p 263. (22) Andrieux, C. P.; Saveant, J.-M. J. Electroanal. Chem. 1978, 93, 163. (23) Bourdillon, C.; Demaille, C.; Moiroux, J.; Saveant, J.-M. J. Am. Chem. Soc. 1994, 116, 10328. (24) Rowe, G. K.; Creager, S. E. J. Phys. Chem. 1994, 98, 5500. Creager, S. E.; Rowe, G. K. Anal. Chim. Acta 1991, 246, 233. Creager, S. E.; Rowe, G. K. Langmuir 1993, 9, 2330. Rowe, G. K.; Creager, S. E. Langmuir 1991, 7, 2307.

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the buffer solution and (2) on the basis of the literature only a dramatic change of the environment can cause such a big shift in the formal potential.24 A second, related environmental explanation for this potential shift can be proposed on the basis of literature reports. If the ferrocene molecules that act as mediators are placed in the enzyme molecule as prosthetic groups, it is possible that interaction with the protein can cause electronic and conformational changes in the ferrocene relay population, which affects the redox features.25 It is important to emphasize that a mediator with redox potential around E1/2 ) 0 V can still can be effective for the oxidation of the GOxred. It was shown by Jiang et al.26 that direct redox reaction of glucose oxidase by a gold electrode occurs at E1/2 ) -238 mV (vs SCE). The environmental effect does not directly explain why, in the absence of glucose, we do not observe a redox peak from the relay population. However, according to Kinnear and Monbouquette,7 it is possible that in the absence of glucose the redox peak of this population is not observed because of lack of sensitivity, assuming that only a small part of the relay population is electroactive. The addition of glucose causes an enhancement in the oxidation peak due to the catalytic process, thus making it detectable. So what is the reason for this potential shift? Such large shifts of the redox potential are not typically reported for enzyme/mediator systems with one or both components soluble23,27 or in systems with relay molecules covalently bonded to the protein.4,8 We report here on a uniquely structured electron transfer assembly which was not demonstrated before. In our case, the relay molecules and the enzyme are both immobilized on the electrode surface and, as shown in Figure 8, the mediator molecules are forced into position between the enzyme and the electrode. This location can cause different environmental effects that can change the native features of the ferrocene. At this stage it is difficult to indicate which of the explanations discussed above is the cause for this negative shift. Additional work on this point is underway in our laboratory. Conclusions We have developed a unique biosensor array based on a mixed monolayer of ferrocenylhexadecanethiol (16FAT) and aminoethanethiol. The mixed layer composition which is optimal as a biosensor included only 7% of the relay component. The huge difference in the size of the enzyme compared to the relay molecule size, as shown in Figure 8, accounts for the small amount of relay molecules needed relative to the anchoring sites of the enzyme. An assumption consistent with our data is that 16FAT and (25) Manners, I. Advances in Organometallic Chemistry; Stone, F. G. A., West, R., Eds.; Academic Press, Inc.: San Diego, 1995; Vol 37, p 131. Manners, I. Adv. Mater. 1994, 6, 68. Foucher, D. A.; Honeyman, C. H.; Nelson, J. M.; Tang, B. Z.; Manners, I. Angew. Chem., Int. Ed. Engl. 1993, 32, 1709. Rulkens, R.; Lough, A. J.; Manners, I. J. Am. Chem. Soc. 1994, 116, 797. Foucher, D.; Ziembinski, R.; Petersen, R.; Pudelski, J.; Edwards, M.; Ni, Y.; Massey, C.; Jeager, C. R.; Vancso, G. J.; Manners, I. Macromolecules 1994, 27, 3992. Brandt, P. F.; Rauchfuss, T. B. J. Am. Chem. Soc. 1992, 114, 1926. (26) Jiang, L.; McNeil, C. J.; Cooper, J. M. J. Chem. Soc., Chem. Commun. 1995, 1293. (27) Hill, H. A. O.; Sanghera, G. S. Biosensors A Practical Approach; Cass, A. E. G., Ed.; Oxford University Press: New York, 1990; p 19.

Rubin et al.

Figure 8. A schematic representation of the GOx-modified mixed monolayer. The sizes of the three components were kept in proportion.

aminoethanethiol segregate into domains on the electrode. Upon modification of the 16FAT/aminoethanethiol mixed monolayer with enzyme, only enzyme molecules that reside near the 16FAT domain can interact with the relay molecules. Ferrocenes deep inside the 16FAT domains cannot interact with the enzyme redox site. Thus, the cyclic voltammetric response is essentially that typical of the adsorbed 16FAT instead of a catalytic amperometric response. Additional characterization, beyond the purely electrochemical study presented here, is necessary to more directly demonstrate the existence of different domains. The primary purpose of this paper was to demonstrate that the electron transfer sequence could be made to work in a mixed monolayer system with no other mediator present. Our immobilization of two different functional units is to be contrasted with the Kajiya9 method, in which a single reactive unit is attached to the surface and subsequently reacted with moieties to be attached. The latter produces an electrode which shows mediated response to glucose, but the reactions used lead to a relatively uncontrolled surface structure in which the mediation undoubtedly occurs via “directly wired” enzymes. The advantage of preparing the mixed monolayer system in the manner presented here is the control one can, in principle, have over placement of functional groups. Eventually, our method could serve as a basis for the development of device structures in which electrons flow through controlled, codeposited structures on surfaces. Acknowledgment. This work was funded by the LANL Chemistry LDRD program. S.R. would like to thank the Director’s funded post-doctoral program at LANL for support. LA950071P