Reversible Associative and Dissociative Interactions of Glucose

Jan 15, 1996 - controlling electrical communication of glucose oxidase, GO, and ... also serve as a model for mimicking basic functions of the vision ...
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Langmuir 1996, 12, 946-954

Reversible Associative and Dissociative Interactions of Glucose Oxidase with Nitrospiropyran Monolayers Assembled onto Gold Electrodes: Amperometric Transduction of Recorded Optical Signals Itamar Willner,* Amihood Doron, Eugenii Katz, and Shlomo Levi Institute of Chemistry and Farkas Center for Light-Induced Processes, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

Arthur J. Frank The National Renewable Energy Laboratory, Golden, Colorado 80401 Received August 21, 1995. In Final Form: October 24, 1995X Photoisomerizable nitrospiropyran monolayers assembled onto Au surfaces provide active electrodes for controlling electrical communication of glucose oxidase, GO, and ferrocene-modified glucose oxidase, FcGO, with the electrode interface. A thiolated nitrospiropyran monolayer was assembled onto Au electrodes by treatment with 1-(4-mercaptobutyl)-3,3-dimethyl-6′-nitrospiro[indolin-2,2′-[1-2H]benzopyran] (3). The monolayer undergoes reversible photoisomerization to nitrospiropyran (SP (3)) monolayer state and nitromerocyanine (MRH+ (4)) monolayer. With ferrocenecarboxylic acid as a diffusional electron mediator, the electrobiocatalyzed oxidation of glucose by GO is inhibited in the presence of the MRH+-monolayer electrode as compared to the system that includes the SP-monolayer electrode. The inhibition phenomenon originates from electrostatic binding of GO to the MRH+-monolayer electrode that perturbs the interfacial redox process with the diffusional electron mediator, ferrocenecarboxylic acid. The interfacial electrontransfer rate between ferrocenecarboxylic acid and the electrode is 10-fold slower in the presence of the MRH+-monolayer electrode and GO as compared to the SP-monolayer electrode and GO. Quartz crystal microbalance analyses and determination of the respective capacity currents reveal electrostatic attraction of GO to the MRH+-monolayer electrode. With ferrocene-modified glucose oxidase, Fc-GO, the electrobiocatalyzed oxidation of glucose is enhanced in the presence of the MRH+-monolayer electrode as compared to the SP-monolayer electrode. The modified enzyme, Fc-GO, exhibits direct electrical communication with the electrode surface. Electrostatic attraction of Fc-GO to the MRH+-monolayer electrode increases the biocatalyst concentration at the electrode surface and facilitates electrochemical oxidation of glucose. Similar results are observed upon organization of the nitrospiropyran monolayer on Au electrodes by a stepwise method that includes covalent linkage of 1-(β-carboxyethyl)-3,3-dimethyl-6′-nitrospiro[indolin2,2′-[1-2H]benzopyran] (1) to a cystamine monolayer-modified Au electrode. The reversible photoisomerizable properties of the nitrospiropyran monolayer electrodes allow the cyclic modulation of the electrocatalytic anodic currents in the systems that include GO and Fc-GO as biocatalysts. The assemblies represent systems for the amplification and amperometric transduction of optical signals recorded by monolayer electrodes. They can also serve as a model for mimicking basic functions of the vision process.

Transduction of recorded optical signals by means of a physical output provides the basis for electronic devices.1 Photoisomerizable molecular2,3 and macromolecular assemblies4 have been used as optical recording media that switch “ON-OFF” chemical or physical functions that are transduced as secondary physical signals. Photochemically controlled ion-binding to photoisomerizable crown ethers5,6 and optically stimulated physical parameters, such as viscosity or wettability, of photoisomerizable * To whom correspondence may be addressed: Fax, 972-26527715; Tel, 972-2-6585272. X Abstract published in Advance ACS Abstracts, January 15, 1996. (1) (a) Lehn, J.-M. Angew. Chem., Int. Ed. Engl. 1988, 27, 90-112. (b) Feringa, B. L.; Jager, W. F.; de Lange, B. Tetrahedron 1993, 49, 8267-8310. (c) Lehn, J.-M. Supramolecular Chemistry; VCH: Weinheim, 1995, Chapter 8, pp 89-138. (2) Kawai, S. H.; Gilat, S. L.; Lehn, J.-M. J. Chem. Soc., Chem. Commun. 1994, 1011-1013. (3) Willner, I.; Marx, S.; Eichen, Y. Angew. Chem., Int. Ed. Engl. 1992, 31, 1243-1244. (4) Irie, M. Adv. Polym. Sci. 1990, 94, 27-67. (5) (a) Irie, M.; Kato, M. J. Am. Chem. Soc. 1985, 107, 1024-1028. (b) Shinkai, S. Pure Appl. Chem. 1987, 59, 425-430. (c) Shinkai, S.; Ishihara, M.; Ueda, K.; Manabe, O. J. Chem. Soc., Perkin Trans. 2 1985, 511-518. (6) (a) Shinkai, S.; Nakamura, S.; Nakashima, M.; Manabe, O.; Iwamoto, M. Bull. Chem. Soc. Jpn. 1985, 58, 2340-2347. (b) Shinkai, S.; Manabe, O. Top. Curr. Chem. 1984, 121, 67-104. (c) Winkler, J.; Deshayes, K. J. Am. Chem. Soc. 1987, 109, 2190-2192.

0743-7463/96/2412-0946$12.00/0

polymers,7 represent molecular and macromolecular systems for the transduction of recorded optical signals. Lightmodulated transport of substrates across photoisomerizable vesicles represents a microheterogeneous assembly for the transduction of optical information.8 Amperometric transduction of recorded optical signals is of specific interest in bioelectronics because it provides the grounds for constructing optoelectronic devices by coupling photosensitive biomaterials with electrode surfaces. This has recently been demonstrated by the application of photoisomerizable biomaterials that switch “ON-OFF” their redox properties upon exposure to light.9 Of special interest is the development of systems in which the transduced output of the recorded optical signal is amplified. For this purpose, the optical signal must activate a catalytic cascade that is subsequently transduced as a physical output. Rebek and co-workers10 have recently demonstrated a photoisomerizable H-bonding (7) Applied Photochromic Polymer Systems; McArdle, C. B., Ed.; Blackie: Glasgow and London, 1992. (8) Sato, T.; Kijima, M.; Shiga, Y.; Yonezawa, Y. Langmuir 1991, 7, 2330-2335. (9) Willner, I.; Willner, B. In Bioorganic Photochemistry - Biological Applications of Photochemical Switches; Morrison, H., Ed.; Wiley: New York, 1993; Vol. 2, pp 1-110. (10) Wu¨rther, F.; Rebek, J. Angew. Chem., Int. Ed. Engl. 1995, 34, 446-448.

© 1996 American Chemical Society

Reversible Interactions on Au Electrodes

receptor that stimulates a self-replicating catalytic process. Similarly, the use of a mixed PQQ/nitrospiropyran monolayer electrode has been used to amplify recorded optical signals by the catalyzed electrooxidation of NAD(P)H.11 Enzymes have recently been applied as biocatalysts for the amplification of recorded optical signals.11a,12 Photostimulated redox enzymes assembled as monolayers on electrode surfaces were applied as biocatalysts for amperometric transduction and amplification of recorded optical signals. In these studies, chemical modification of redox enzymes by photoisomerizable units,11a,12 or the application of photoisomerizable electron mediators,13 led to light-stimulated activation and deactivation of the biocatalysts. The latter photoswitchable biocatalyst states were transduced amperometrically, and the transduced signal reflected the “ON-OFF” electrical communication of the enzyme with the electrode. Recently, a mixed pyridine-nitrospiropyran monolayer electrode was reported as an active interface for the recording of optical signals and their amperometric transduction by means of the cytochrome c/cytochrome c oxidase system.14 Organization of proteins on electrode surfaces by means of electrostatic interactions has been demonstrated by Kunitake and co-workers.15 Monolayers and multilayers were assembled on surfaces using polyelectrolytes as linking units. Here we wish to describe a novel approach for controlling the electrical communication of glucose oxidase, GO, with an electrode by means of a nitrospiropyran monolayer assembled onto the electrode. The effectiveness of electrical communication between GO and the electrode is controlled by electrostatic interactions between the monolayer photoisomer states and the protein. The system provides a means for the amperometric transduction of optical signals recorded by the monolayer. The system also provides an insight into the design of new configurations of amperometric devices and demonstrates the principles of regenerative enzyme electrodes. Experimental Section Cyclic voltammograms were recorded using an electroanalyzer (VersaStat, EG&G) linked to a PC computer (Research Electrochemical Software Model 270/250, EG&G). The electrochemical cell consisted of three electrodes in which a gold wire (0.5 mm diameter Au wire of geometrical area ca. 0.2 cm2, roughness coefficient, ca. 1.2) was used as a working electrode. A glassy carbon electrode separated by a glass frit from the working volume was used as a counter electrode, and a saturated calomel electrode (SCE), connected to the working volume with a Luggin capillary, was used as reference electrode. A thermostated cell was employed. The electrolyte solution consisted of a 0.1 M phosphate buffer solution, pH ) 7.0. Argon bubbling was used to remove oxygen from the solutions in the electrochemical cell. Mass adsorption and desorption onto and from the electrode were monitored by frequency changes of a quartz crystal microbalance analyzer, QCM (Seiko EG&G Model QCA917), connected to a PC computer. For QCM measurements, quartz crystals (AT-cut, EG&G) sandwiched between two Au electrodes (A ) 0.196 cm2, roughness factor ca. 3.5) were used. The fundamental frequency of the crystals was ca. 109 Hz. (11) (a) Lion-Dagan, M.; Katz, E.; Willner, I. J. Am. Chem. Soc. 1994, 116, 7913-7914. (b) Katz, E.; Lion-Dagan, M.; Willner, I. J. Electroanal. Chem. 1995, 382, 25-31. (12) Willner, I.; Lion-Dagan, M.; Marx-Tibbon, S.; Katz, E. J. Am. Chem. Soc. 1995, 117, 6581-6592. (13) Lion-Dagan, M.; Marx-Tibbon, S.; Katz, E.; Willner, I. Angew. Chem., Int. Ed. Engl. 1995, 34, 1604-1606. (14) Lion-Dagan, M.; Katz, E.; Willner, I. J. Chem. Soc., Chem. Commun. 1994, 2741-2742. (15) (a) Lvov, Y.; Ariga, K.; Kunitake, T. Chem. Lett. 1994, 23232325. (b) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117-6123.

Langmuir, Vol. 12, No. 4, 1996 947 Scheme 1

Absorption spectra were recorded on a Uvikon-860 (Kontron) spectrophotometer. Photochemical isomerization of nitrospiropyran derivatives to nitromerocyanine was stimulated by illumination at 320 nm < λ < 350 nm using an 18 W mercury pencil lamp (Oriel-6042). Photoisomerization of nitromerocyanine derivatives to nitrospiropyran states was induced by irradiation with a Xe-arc lamp (150 W Oriel), fitted with a 495 nm cut-off filter. The electrodes modified with photoisomerizable monolayers were protected from room light during the electrochemical measurements. Chemicals (Aldrich) and glucose oxidase (Aspergillus niger, E.C. 1.1.3.4, Sigma) were used without further purification. Ultrapure water from a Nanopure (Barnstead) source was used throughout this work. 1-(β-Carboxyethyl)-3,3-dimethyl-6′-nitrospiro[indolin-2,2′-[12H]benzopyran] (1)16 and 1-(4-iodobutyl)-3,3-dimethyl-6′-nitrospiro[indolin-2,2′-[1-2H]benzopyran] (2)17 were prepared according to the literature and their structures are shown in Scheme 1. 1-(4-Mercaptobutyl)-3,3-dimethyl-6′-nitropsiro[indolin-2,2′[1-2H]benzopyran] (3) was synthesized according to Scheme 2. Thioacetic acid potassium salt (1.2 g, 3.0 mmol) and 2 (421 mg, 7.5 mmol) were stirred in ethanol solution for 4 h under reflux. The resulting dark red solution was filtered, and KOH in ethanol was added (the molar ratio of KOH to 2 was 2:1). The solution was then stirred at room temperature under argon for 2 h, filtered, and evaporated to dryness. The yellow crude product was dissolved in CH2Cl2. The resulting solution was washed with an aqueous HCl solution (pH ) 4.0) and evaporated to dryness. The resulting product was analyzed by 1H-NMR and gave satisfactory elemental analysis. N-(2-Methylferrocene)caproic acid (5) was prepared as described previously.18 Glucose oxidase, GO, was modified with ferrocene units by treating GO (100 mg) with 5 (16 mg, 0.048 mmol) in a HEPES buffer solution (0.1 M, 3 mL, pH ) 7.3) in the presence of 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide methiodide, EDC-methiodide (24 mg) and urea (180 mg) for 10 h (4 °C). The resulting mixture was dialyzed against sodium phosphate buffer (0.01 M, pH ) 7.3) for 24 h (cf. Scheme 5). The extent of loading of GO with ferrocene units was determined by comparing of the fluorescence in the presence of fluorescamine of native GO to that of ferrocene-modified GO. The loading corresponded to 24. Gold electrodes were pretreated by immersing them in boiling 0.2 M aqueous KOH for 3 h followed by rinsing with water. The resulting electrodes were transferred to concentrated H2SO4 and stored in the acid overnight. Prior to modification, the electrodes were washed with water, immersed in concentrated HNO3 for 10 min, and then washed thoroughly with water. The thiolate nitrospiropyran monolayer was assembled onto the Au electrodes by treatment of the gold wire with a solution consisting of 3 in ethanol, 5 × 10-4 M, for 2 h. The resulting electrodes (cf. Scheme 3) were washed with ethanol and then with water. All cyclic voltammograms were recorded using freshly prepared modified electrodes. For QCM measurements, Au-coated quartz crystals were washed with ethanol and water prior to modification. The AuQCM electrodes were then treated with dry dichloromethane that contained 3, 5 × 10-4 M, for 2 h. The resulting electrodes were washed sequentially with dichloromethane, ethanol, and water and then dried with argon. (16) (a) Namba, K.; Suzuki, S. Bull. Chem. Soc. Jpn. 1975, 48, 13231324. (b) Hassner, A.; Alexanian, V. Tetrahedron Lett. 1978, 6, 44754478. (17) Vandewyer, P. H.; Smets, G. J. Polym. Sci., A-1 1970, 8, 23612374. (18) Shoham, B.; Migron, Y.; Riklin, A.; Willner, I.; Tartakovsky, B. Biosensors Bioelectron. 1995, 10, 341-352.

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3

Stepwise chemical assembly of a nitrospiropyran monolayer on Au electrodes was performed by covalent linkage of the photoisomerizable units to a cystamine-monolayer-modified electrode (cf. Scheme 7). Au electrodes were immersed in a 0.01 M cystamine hydrochloride aqueous solution for 2 h followed by rinsing with water. The resulting electrodes were treated with a 0.01 M ethanolic solution of EDC-methiodide and 1, 5 × 10-4 M, for 1 h. The resulting electrodes were rinsed several times with water. Cyclic voltammograms were recorded with freshly prepared electrodes.

Results and Discussion The substrate, 1-(4-mercaptobutyl)-3,3-dimethyl-6′-nitrospiro[indoline-2,2′-[1-2H]benzopyran] (3) SP, exhibits reversible photochromic properties, Figure 1. Its irradiation with 320 nm < λ < 350 nm light yields the protonated nitromerocyanine isomer (4), MRH+, eq 1, exhibiting a maximum absorbance band at λ ) 550 nm. Further

is saturated with 3. The Sauerbrey equation,19 eq 1, relates the measured frequency change of the crystal, ∆f (Hz), to the mass change ∆m (g), where F0 is the fundamental frequency of the QCM, A is the electrode geometrical area (0.196 cm2), Fq is the density of quartz (2.65 g‚cm-3), and µq is the shear modulus (2.95 × 10-11 dyn‚cm-2). Inserting these numerical values into eq 2 gives the simplified expression, eq 3, that relates the observed frequency change with the mass associated on the electrode (ng). Using eq 3, and taking into account the roughness factor of the Au electrode (kr ≈ 3.5), we estimate the mass change on the Au electrode as a result of association of (3) to be ∆m ) 97 ng‚cm-2 which corresponds to a surface density of 2.48 × 10-10 mol‚cm-2 of 3.

∆f )

(1)

irradiation of the MRH+ isomer, λ > 495 nm, regenerates 3. From the absorbance spectra and 1H-NMR spectra of the two isomers, we estimate that each of the states corresponds to >95% of the respective isomer. Treatment of a bare Au electrode with 3 results in the thiolate nitrospiropyran self-assembly of the SP monolayer on the electrode surface, Scheme 3. The formation of the monolayer on the gold surface was characterized by QCM analysis where the quartz crystal is sandwiched between two Au electrodes. Figure 2 shows the decrease in the crystal frequency upon incubation of the crystal in the presence of a dichloromethane solution of 3. A decrease in the crystal frequency is observed, implying the association of 3 to the surface. The frequency change of the crystal reaches a constant value that is ca. 60 Hz below its fundamental frequency, indicating that the Au surface

Figure 1. Absorption spectra of (a) 3, 1 × 10-4 M, and (b) 4, 1 × 10-4 M.

-2F02 ∆m A(ρqµq)1/2

∆f ) (2.26 × 10-15)F02 ∆m/A

(2) (3)

The nitrospiropyran monolayer electrode was used as an active interface to photostimulate the electrical communication of glucose oxidase, GO, with the electrode. This enzyme lacks, as in the case of most of the other redox proteins, direct electrical communication with the electrode. In the presence of diffusional electron mediators such as ferrocene carboxylic acid, electrical communication between the enzyme and electrode is attained.20 Also, chemical modification of GO with a ferrocene electron transfer mediator leads to electrical communication between the redox-active enzyme site and the electrode surface.21 We have examined the effects of the photoisomerizable nitrospiropyran monolayer electrode on electrical communication with GO, in the presence of diffusional electron mediators, and with ferrocene-modified GO, respectively. Figure 3 shows the cyclic voltammograms obtained upon application of the photoisomerizable thiolate nitrospiro-

Figure 2. Frequency changes upon assembly of 3 monolayer in the QCM analysis. A bulk CH2Cl2 solution of 3, 5 × 10-4 M, was applied to assemble the monolayer.

Reversible Interactions on Au Electrodes

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Scheme 3. Organization of a Self-Assembled Nitrospiropyran Monolayer on a Au Electrode

Scheme 4. Inhibited Electrical Communication between GO and the Electrode, in the Presence of a Diffusional Electron Mediator, by Electrostatic Association of GO to the Photoisomerizable Monolayer Electrode

pyran monolayer electrode, as working electrode, in the bioelectrocatalyzed oxidation of glucose by GO using ferrocene carboxylic acid as a diffusional electron mediator. In the presence of the nitrospiropyran monolayer electrode, SP-state (3), an electrocatalytic anodic current density of

Figure 3. Cyclic voltammograms of photoisomerizable electrodes: (a) 3-modified electrode in the presence of ferrocenecarboxylic acid, 4 × 10-4 M and GO, 1.5 mg‚mL-1. (b) and (d) 3-modified electrode in the presence of ferrocenecarboxylic acid, 4 × 10-4 M, GO, 1.5 mg‚mL-1, and glucose, 5 × 10-2 M. (c) 4-modified electrode in the presence of ferrocenecarboxylic acid, 4 × 10-4 M, GO, 1.5 mg‚mL-1, and glucose, 5 × 10-2 M. All experiments were performed in a phosphate buffer, 0.1 M, pH ) 7.0, 35 °C, under argon. Potential scan rate, was 2 mV‚s-1. (Inset) Cyclic bioelectrocatalytic amperometric responses of the photoisomerizable electrode: (2) in the presence of the nitrospiropyran (3)-modified electrode; (b) with the nitromerocyanine (4)-modified electrode.

imax ) 65 µA‚cm-2 is developed (Figure 3, curve b). Photoisomerization, 320 nm < λ < 350 nm, of the monolayer to the nitromerocyanine state, MRH+ state (4), significantly lowers the electrocatalytic anodic current density, imax ) 52 µA‚cm-2 (Figure 3, curve c). Further irradiation of the nitromerocyanine monolayer electrode with λ > 495 nm light restores the SP-monolayer state (3) and raises the electrocatalytic anodic current in the cell to its original value (Figure 3, curve d). By reversible photoisomerization of the monolayer electrode between the SP and MRH+ states, the electrocatalytic anodic current, resulting from the biocatalyzed oxidation of glucose by GO, is switched and cycled between a high and low amperometric output (inset of Figure 3). Thus, optical signals recorded by the monolayer are amperometrically transduced by the bioelectrocatalyzed oxidation of glucose. The photoswitchable transduction of amperometric signals by GO/glucose and the monolayer electrode is attributed to different electrostatic interactions of the enzyme with the distinct isomer states of the monolayer electrode, Scheme 4. Because the nitromerocyanine monolayer state exists at pH ) 7.0 in the protonated form, MRH+ (the pKa for MR is ca. 8.622), the surface is charged positively. Upon photoisomerization of the MRH+ state to the nitrospiropyran monolayer state, SP (3), the monolayer interface becomes neutral. Glucose oxidase is negatively charged at pH ) 7.0 (isoelectric point, pI ) 4).23 As a result, GO is electrostatically attracted to the MRH+-monolayer (19) Sauerbrey, G. Z. Phys. 1959, 155, 206-215. (20) Bartlett, P. N.; Tebbutt, P.; Whitaker, R. G. Prog. Reaction Kinet. 1991, 16, 55-155. (21) Degani, Y.; Heller, A. J. Phys. Chem. 1987, 91, 1285-1289. (22) Kato, S.; Aizawa, M.; Suzuki, S. J. Membr. Sci. 1976, 1, 289300. (23) Wilson, R.; Turner, A. P. F., Biosensors Bioelectron. 1992, 7, 165-185.

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Figure 4. Frequency changes of the QCM crystal modified by the photoisomerizable monolayer in the presence of GO, 1.5 mg‚mL-1, in phosphate buffer solution 0.1 M, pH ) 7.0: (a) with the SP-(3)-modified crystal; (b) with the MRH+-(4)-modified crystal.

electrode. Previous studies indicate that association of proteins to monolayer electrodes can be used to control the interfacial electron transfer between the electrode and a diffusional redox probe solubilized in the electrolyte.24,25 Specifically, adsorption of proteins to monolayer assemblies on electrodes insulate the electrodes from diffusional redox probes resulting in a substantially lower interfacial electron-transfer rate. This phenomenon was applied to follow amperometrically the antibody-antigen binding at a monolayer electrode.25 Thus, in the presence of the SP-monolayer electrode, only partial nonspecific adsorption of GO to the monolayer occurs (vide infra). The partial association of GO to the monolayer permits improved electrical communication between the electrode and the solubilized ferrocene carboxylic acid electron mediator. Oxidation of the redox relay generates the ferrocenylium cation that acts as electron mediator for GO. Thus, in the presence of the SP monolayer electrode, the mediated biocatalyzed oxidation of glucose yields a high electrocatalytic anodic current. In the presence of the MRH+ monolayer electrode, electrostatic association of GO to the monolayer interface occurs. This electrostatic binding of negatively charged GO to the MRH+ monolayer produces a much higher surface loading than the nonspecific adsorption of GO to the SP-monolayer electrode. Association of GO to the MRH+ monolayer blocks the electrical communication between the electrode and the ferrocenecarboxylic acid redox mediator. The inefficient interfacial oxidation of the electron relay (ferrocenecarboxylic acid) retards the mediated oxidation of GO and the biocatalyzed oxidation of glucose. This is reflected by a lower electrocatalytic anodic current. Thus, the electrostatic interaction of the MRH+-monolayer electrode with GO controls the interfacial oxidation of the diffusional electron mediator and the resulting mediated electrobiocatalyzed oxidation of glucose. Further insight into the associative interactions of GO with the SP and MRH+ monolayer electrodes is obtained by QCM analyses. Treatment of the neutral SP monolayer assembled on the Au-QCM electrode with GO results in a frequency decrease of ∆f ) 65 Hz. In turn, interaction of the MRH+ monolayer Au-QCM crystal with GO results in a frequency decrease of ∆f ) 100 Hz. By cyclic photoisomerization of the monolayer electrode between the SP and MRH+ states in the presence of GO, the crystal frequency is reversibly modulated between the respective frequencies, Figure 4. Using the Sauerbrey equation and (24) Katz, E.; Solov’ev, A. A. Anal. Chim. Acta 1992, 266, 97-106. (25) Willner, I.; Blonder, R.; Dagan, A. J. Am. Chem. Soc. 1994, 116, 9365-9366.

Figure 5. Cyclic voltammograms of ferrocenecarboxylic acid, 4 × 10-4 M, in the presence of GO, 1.5 mg‚mL-1, and the MRH+(4)-monolayer electrode (b) and SP-(3)-monolayer electrode (a): (A) potential scan rate, 200 mV‚s-1; (B) 800 mV‚s-1. All experiments were recorded in a 0.1 M phosphate buffer solution, pH ) 7.0.

knowing the surface area and roughness factor (A ) 0.196 cm2, kr ) 3.5) of the electrode, we estimate the surface coverage of the SP and MRH+ monolayer electrodes by GO to be 7.4 × 10-13 and 9.7 × 10-13 mol‚cm-2, respectively. The control of interfacial electron transfer to the ferrocenecarboxylic acid by the photoisomerizable monolayer electrode and associated GO was confirmed by characterizing the electrochemistry of the electron mediator in the absence of glucose. Figure 5 shows the cyclic voltammograms of ferrocenecarboxylic acid in the presence of the SP- and MRH+-monolayer electrodes and GO at a slow scan rate, Figure 5A, and a high scan rate, Figure 5B. At a high scan rate, the peak-to-peak separation of the oxidation and reduction waves of ferrocenecarboxylic acid differ substantially in the presence of the SP- and MRH+-monolayer electrodes. However, the cyclic voltammograms of ferrocenecarboxylic acid in the presence of SP- and MRH+-monolayer electrodes without GO are almost identical at high or low scan rates. These results clearly indicate that the interaction of GO with the photoisomerizable monolayer electrode strongly affects the interfacial electron transfer with the diffusional electron mediator. The redox reactions of ferrocenecarboxylic acid in the presence of GO and the MRH+monolayer electrode is retarded as compared to the SPmonolayer electrode and GO. Figure 6 shows the peakto-peak separation of the redox waves of ferrocenecarboxylic acid, as a function of scan rates, using the SP and MRH+ states of the monolayer electrodes, respectively. The interfacial electron transfer rate constants in the presence of SP- and MRH+-monolayer electrodes were

Reversible Interactions on Au Electrodes

Figure 6. Dependence of peak-to-peak separation as a function of scan rate for the redox process of ferrocenecarboxylic acid, 4 × 10-4 M, in the presence of GO, 1.5 mg‚mL-1 with (a) SP(3)-modified electrode and (b) MRH+-(4)-modified electrode.

calculated from these plots using the Nicholson method,26 assuming a diffusion coefficient of D ≈ 7 × 10-6 cm2‚s-1 for ferrocenecarboxylic acid.27 For the SP-monolayer electrode and GO, the interfacial electron transfer rate constant for the redox processes of ferrocenecarboxylic acid corresponds to ket ) 4.7 × 10-2 cm‚s-1, while that for the MRH+-monolayer electrode is ket ) 5.6 × 10-3 cm‚s-1. Thus, when GO is associated with the MRH+-monolayer electrode, the interfacial electron transfer to ferrocenecarboxylic acid is 10-fold retarded as compared to the GO SP-monolayer electrode. It should be noted that the ferrocenecarboxylic acid acting as electron transfer mediator is dissociated from the ferrocenylcarboxylate anion at the pH conditions employed in our experiments (pH ) 7.0). This might result in electrostatic repulsion of the electron mediator from the MRH+ monolayer with adsorbed GO being negatively charged. This electrostatic repulsion could contribute to the lower interfacial electron transfer rate constant to the ferrocene mediator in the presence of the MRH+-monolayer electrode, in addition to the insulating effect of the protein. Previous studies indicated, however, that protein adsorption to the monolayer retarded the interfacial electron transfer rates even for neutral redox components.24 Hence, we believe that the retarded electron transfer rate to ferrocenecarboxylate in the presence of the MRH+-GO monolayer is primarily due to the insulating effect of the adsorbed enzyme and the additional electrostatic repulsions could provide a secondary influence. Modification of glucose oxidase, GO, with ferrocene electron mediator units yields an “electrically-wired” protein assembly that maintains electrical communication with electrodes.21 GO was modified with N-(2-methylferrocene)caproic acid (5), Scheme 5. The loading of the protein by ferrocene groups corresponds to 24. Electrobiocatalyzed oxidation of glucose by ferrocene-modified GO, Fc-GO, was studied in the presence of the SPmonolayer and MRH+-monolayer electrodes, Scheme 6. (26) Nicholson, R. S. Anal. Chem. 1965, 37, 1351-1355. (27) Bourdillon, C.; Demaille, C.; Moiroux, J.; Save´ant, J.-M. J. Am. Chem. Soc. 1993, 115, 2-10.

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Figure 7. Cyclic voltammograms of ferrocene-modified GO, Fc-GO, 1.5 mg‚mL-1 in the presence of the photoisomerizable monolayer electrode: (a) with the SP-(3)-modified electrode and absence of glucose; (b) and (d) with the SP-(3)-modified electrode and glucose, 5 × 10-2 M; (c) With the MRH+-(4)modified electrode and glucose, 5 × 10-2 M. All experiments were recorded in 0.1 M phosphate buffer, pH ) 7.0, 35 °C, under argon. Potential scan rate was 2 mV‚s-1. (Inset) Cyclic bioelectrocatalytic amperometric responses of the photoisomerizable electrode: (b) In the presence of SP-(3)-monolayer electrode; (2) with the MRH+-(4)-monolayer electrode.

In the presence of the SP-monolayer electrode, an electrocatalytic current density of imax ) 11 µA‚cm-2 is observed (Figure 7, curve b). Photoisomerization of the SP monolayer to the MRH+ state, 320 nm < λ < 350 nm, results in an almost 2-fold increase in the electrocatlytic anodic current (Figure 7, curve c). Further irradiation of the MRH+-monolayer electrode, λ > 495 nm, regenerates the SP-monolayer electrode and results in a decrease of the electrocatalytic anodic current to its original value (Figure 7, curve d). By cyclic photoisomerization of the monolayer electrode between the SP and MRH+ states, the transduced anodic current as a result of glucose oxidation is reversibly switched between a low and a high value, respectively (Figure 7, inset). Note that in contrast to the previous system that employed GO and the diffusional electron mediator, ferrocenecarboxylic acid, where an enhanced electrocatalytic current was observed with the SP-monolayer electrode, the amplitude of the current switch is reversed in the system that includes Fc-GO. Here a low amperometric response is observed in the presence of the SP-monolayer electrode, and the electrocatalytic oxidation of glucose is enhanced in the presence of the MRH+monolayer electrode, as reflected by the higher transduced current. This result is consistent with the effect of the electrostatic attraction of the enzyme to the charged monolayer and the consequent control of electrical communication between the biocatalyst and the electrode. In contrast to GO that lacks direct electrical communication with the electrode, the covalently-linked electron mediator groups in Fc-GO facilitate electron transfer between the redox protein and the electrode. The observed electron flow (current) in the system, as a result of glucose oxidation, correlates with the association of the enzyme to the electrode surface. In the presence of the MRH+monolayer electrode, electrostatic attraction of Fc-GO to the monolayer increases the biocatalyst concentration at the electrode surface, Scheme 6. Upon photoisomerization of the MRH+ state to the SP-monolayer electrode, dissociation of Fc-GO from the monolayer electrode occurs. Thus, localization of Fc-GO at the charged monolayer

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Scheme 5. Modification of GO by Ferrocene Relay Units

Scheme 6. Enhanced Electrical Communication of Ferrocene-Modified GO and the Electrode by Electrostatic Attraction of the Biocatalyst to the Photoisomerizable Monolayer Electrode

Scheme 7. Stepwise Organization of a Nitrospiropyran Monolayer on a Au Electrode

interface facilitates electrooxidation of glucose. This is reflected by the higher transduced electrocatalytic anodic current in the presence of the MRH+-monolayer electrode. Similar results are observed for a nitrospiropyran monolayer deposited onto a Au electrode by a stepwise chemical synthesis, Scheme 7. A cystamine monolayer was first assembled onto a Au electrode. The photoisomerizable unit 1-(β-carboxyethyl)-3,3′-dimethyl-6′-nitrospiro[indolin-2,2′-[1-2H]benzopyran] (1) was coupled to the base monolayer as recently described.11 The surface density of the photoisomerizable units associated with the electrode corresponds to 3 × 10-10 mol‚cm-2. Figure 8 shows the cyclic voltammograms obtained upon the application of the photoisomerizable monolayer electrode for the electrobiocatalyzed oxidation of glucose in the presence of GO, Figure 8A, and Fc-GO, Figure 8B. In the presence of the SP-monolayer electrode and GO, a significantly higher amperometric response is detected. For Fc-GO, the photochemically switched currents are reversed: an almost 2-fold lower amperometric signal is observed in the presence of the SP-monolayer electrode, while the electrocatalytic anodic current is enhanced in the presence of the MRH+-monolayer electrode. The amperometrically transduced signals can be reversibly

switched by photochemical cycling of the monolayer across the SP and MRH+ states (insets of Figure 8). The reversible photostimulated activities of the systems were observed for 10 cycles without noticeable degradation. Upon further cycling, the systems slowly degraded and their amperometric responses decreased in the presence of SP- and MRH+-monolayer states. As the spiropyran monolayer electrodes reveal high photochemical isomerization turnovers,14 we believe that the slow degradation of the present systems originates from the deactivation of the enzyme in the systems. These results further confirm that electrostatic attraction or release of GO or Fc-GO to and from the monolayer electrode control the electrical communication between the biocatalysts and the electrode as reflected by the effectiveness of the electrobiocatalyzed oxidation of glucose. Further insight into the adsorption of GO to the MRH+monolayer electrode and its release from the SP-monolayer electrode is obtained by inspection of the capacity currents of the different electrode states in the absence and presence of GO. Figure 9 shows the capacity currents of the MRH+monolayer and SP-monolayer electrode in the absence of GO (curves a and b, respectively). The capacity current of the MRH+-monolayer electrode is higher than that of

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Figure 9. Cyclic voltammograms of background solution of 0.1 M phosphate buffer, pH ) 7.0: (a) in the presence of MRH+ electrode; (b) in the presence of SP electrode; (c) with the SP electrode and GO, 1.5 mg‚mL-1; (d) with the MRH+ electrode and GO, 1.5 mg‚mL-1. Potential scan rate was 200 mV‚s-1.

Figure 8. Cyclic voltammograms corresponding to the bioelectrocatalyzed oxidation of glucose by the photoisomerizable electrode prepared according to Scheme 7. (A) In the presence of ferrocenecarboxylic acid, 4 × 10-4 M, and GO, 1.5 mg‚nL-1 (a) with the MRH+ electrode without glucose, (b) with MRH+ electrode and glucose, 5 × 10-2 M, and (c) with SP electrode and glucose, 5 × 10-2 M. (Inset) Cyclic amperometric responses of the electrode: (2) MRH+ electrode; (b) SP electrode. (B) In the presence of ferrocene-modified GO, 1.5 mg‚mL-1; (a) with the SP electrode and in the absence of glucose, (b) with the SP electrode and glucose, 5 × 10-2 M, and (c) with the MRH+ electrode and glucose, 5 × 10-2 M. (Inset) Cyclic amperometric responses of electrodes (b) with the SP electrode and (2) with the MRH+ electrode. All experiments were recorded in 0.1 M phosphate buffer, pH ) 7.0; 35 °C, under argon, potential scan rate 2 mV‚s-1.

the SP-monolayer electrode consistent with the fact that the SP-monolayer exhibits less polar character than that of the MRH+ monolayer. Addition of GO results in a decrease in the capacity current for both electrodes, indicating the adsorption of the insulating protein onto the electrode surface. The capacity current of the MRH+monolayer electrode in the presence of GO (curve d) is, however, lower than that of the SP-monolayer electrode in the presence of GO (curve c). By reversible photoisomerization of the monolayer between the SP and MRH+ states in the presence of GO, the capacity currents can be cycled between a high and a low value, respectively. Thus, although the MRH+-monolayer electrode represents a charged interface and exhibits a higher capacity current as compared to the SP-monolayer electrode, the capacity current of the MRH+ monolayer electrode in the presence of GO is lower than that observed with the SP-monolayer electrode. This is attributed to enhanced binding of the

hydrophobic GO protein to the MRH+-monolayer as a result of electrostatic attraction. Our results demonstrate that an electrode modified with a photoisomerizable SP/MRH+ monolayer can photostimulate the electrobiocatalyzed oxidation of glucose by GO or Fc-GO. These systems mimic several functions of the vision process.28 In the vision apparatus, photoisomerization of the retinal chromophore induces a conformational change in the surrounding protein that provides a binding site for protein G. Association of the latter protein to the membrane activates an enzymatic cascade that generates c-GMP that ultimately activates the neural response. Thus, photostimulated binding of a protein to the membrane and enzymatic amplification of the photochemical event represent two basic features of the vision process. To this extent, the photoisomerizable monolayer mimics the functions of the retinal membrane. Photoisomerization of the monolayer to the MRH+ state provides a route for the association of Fc-GO to the membrane-mimetic assembly. Binding of Fc-GO to the monolayer electrode facilitates the enzyme-catalyzed oxidation of glucose. The biocatalytic process amplifies the absorbed optical signal which is transduced as an electrocatalytic current. Thus, our systems provide basic features of an optoelectronic device in which optical signals recorded by the monolayer assemblies are amplified and amperometrically transduced. The results presented here have significant implications in biosensor technology. Enzyme electrodes assembled, via immobilization of biocatalysts onto conductive surfaces, are the basis for numerous electrochemical sensing devices.29 Also, bioactive electrodes have important applications in biotechnological transformations. The stabilities of immobilized enzymes on the electrode surfaces are often a limiting factor in their practical application. We suggest a rapid means to recycle and reassemble enzyme electrodes by the application of photoisomerizable monolayer-modified electrodes. Reversible photoisomerization of the monolayer provides a method to deplete the denaturated biocatalyst from the electrode and reassemble the enzyme electrode. Electrostatic association of Fc-GO to the MRH+ monolayer provides the principle for organizing the enzyme electrode. (28) Stryer, L. Biochemistry, 3rd ed.; W. H. Freeman & Co.: New York, 1988; p 1034. (29) Biosensor Technology. Fundamentals and Applications; Buck, R. P., Hatfield, W. E., Umana, M., Bowden, E. F., Eds.; Dekker: New York, 1990.

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The denaturated enzyme can then be released from the electrode surface by photoisomerization to the SP-monolayer state. Further isomerization of the monolayer to the MRH+ state, followed by treatment with the active enzyme solution, reassembles the active enzyme electrode. Conclusions The present study employed photoisomerizable nitrospiropyran monolayer electrodes as a means to control electrical communication of glucose oxidase with the electrode interface. The reversible photoisomerization of the monolayer between the SP and MRH+ states provides a means to electrostatically attract the protein to the positively-charged MRH+-monolayer electrode. For native GO, that communicates with the electrode by a diffusional electron relay, association of the biocatalyst to the MRH+monolayer retards interfacial electron transfer with the electron mediator and perturbs the electrical communication of GO with the electrode. Desorption of GO from the SP-monolayer electrode facilitates electrical communication between the redox relay and the electrode, and the bioelectrocatalyzed oxidation of glucose is enhanced. The switching amplitude is reversed upon interaction of the photoisomerizable monolayer electrode with the “electri-

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cally-wired” ferrocene-modified GO, Fc-GO. Association of Fc-GO to the MRH+-monolayer electrode increases the enzyme concentration at the electrode interface and, as a result, the bioelectrocatalytic oxidation of glucose is enhanced as compared to the system that employs the SP-monolayer electrode. The monolayer-modified electrodes allow gating of the electrical processes at electrode interfaces by means of external light signals. This allows the enzyme-amplified amperometric transduction of optical signals recorded by the monolayer. To this extent, the systems comprised of photoisomerizable nitrospiropyran monolayer electrodes and Fc-GO mimic the basic function of the vision process and represent a novel approach to construct optoelectronic devices. A further practical implication of the present study includes the introduction of a new methodological concept to regenerate enzyme electrodes. Acknowledgment. This research was supported by the Bundesministerium fu¨r Forschung und Technologie (BMFT), Germany, and the Ministry of Science and Arts, Israel. We are grateful to Dr. P. Weaver (NREL) for his valuable comments. LA9507038