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Anal. Chem. 1986, 58, 2984-2987
Enhanced Direct Electron Transport with Glucose Oxidase Immobilized on (Aminopheny1)boronic Acid Modified Glassy Carbon Electrode Krishna Narasimhan and Lemuel B. Wingard, Jr.*
Department of Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
The immobilization of enzymes In a manner that promotes direct electron transfer between the enzyme cofactor and an electrode surface is of considerable Interest for the develop ment of biosensors having high substrate specificity. Direct electron transfer between immobilized glucose oxidase (GO) and (aminopheny1)boronic acid modified glassy carbon (GC) electrodes was observed in the present study. Surface carboxylic acid groups were generated on gtassy carbon rods by chemical oxidation. (3-Aminophenyi)boronlc acid (APBA) was coupled to the carboxylic acid functions after actlvatlon with water-soluble carbodiknkle to give GC-APBA electrodes. Interaction with GO furnished GC-APBA-GO electrodes with enzyme immobillred presumably by complex formation of the sugar portlon of the enzyme with the boronic acid groupings on the GC surface. This is the first report of the lmmoblllzation of an enzyme to an electrode surface through boronate groups. Direct electron transfer between the enyme cofactor and the electrode was demonstrated by cyck and differential pulse voltammetry. No significant electron transfer was observed with GO in solution either alone or with added APBA. Possible explanatlons are examlned for the direct electron transfer.
The direct electron transfer (without the use of mediators) between the cofactor portion of a flavoenzyme and an electrode surface is of interest for development of novel enzyme biosensors or enzyme electrochemical reactors. The possibilities of achieving direct electron transfer in principle should be enhanced by bringing the flavoenzyme close to the electrode surface, such as by immobilization. Although many studies have been reported wherein the flavoenzyme glucose oxidase or L-amino acid oxidase have been immobilized on electrode surfaces, very few of these studies have included an investigation of the possibilities for direct electron transfer in the absence of mediators. With glucose oxidase immobilized by adsorption on graphite electrodes, no evidence for direct electron transfer has been obtained by using cyclic voltammetry ( I , 2). However, a reduction peak has been observed with glucose oxidase adsorbed on graphite using the more sensitive differential pulse voltammetry ( 2 , 3 ) . Direct electron transfer has been claimed for glucose oxidase covalently attached via a cyanuric chloride bridge to graphik and measured using differential pulse voltammetry (3). The voltammograms that showed direct electron transfer were attributed to oxidation-reduction of the flavin cofactor while still complexed with the apoenzyme and not to flavin molecules that had become dissociated from the apoenzyme and adsorbed onto the graphite surface. We have been interested for over 8 years in the immobilization and characterization of glucose oxidase on electrode surfaces ( 4 - 7 ) , especially for the development of an in vivo glucose sensor. Much of our recent work has dealt with methodology for the attachment of the enzyme to the electrode
surface in such a way as to achieve rapid and direct electron transfer. The present paper reports the attachment of glucose oxidase to (aminopheny1)boronic acid modified glassy carbon electrodes and the resulting enhanced direct electron transfer. Glassy carbon was selected because of the relative ease of removal of adsorbed materials. Phenylboronic acid forms complexes with compounds containing cis-1,2-diols, and the use of supports containing immobilized boronate groups is increasingly popular for affinity separations (8). The formation and stability of the phenylboronate-diol complexes varies with the specific diol, the support matrix, and the reaction conditions; but for some systems the complex is highly stable (8). Boronate supports have proven quite valuable for the separation of small molecular weight biomolecules; recently they have been used for the separation of large molecular weight glycoproteins and glycosylated proteins through complex formation of boronate with sugar residues (9,lO). Since glucose oxidase is a glycoprotein, containing 16% carbohydrate (Il), the possibility exists for the immobilization of this enzyme through the formation of a boronate-diol complex. So far, no reports of boronate modified enzyme electrodes have appeared. EXPERIMENTAL SECTION Materials. Type GC-A Tokai glassy carbon (GC) rods, 0.545 cm diameter by 1.5 cm long and 1-2% porosity, were obtained from International Minerals and Chemicals Corp. The rods were polished sequentially with No. 600 silicon carbide paper and 1-wm diamond powder, cleaned ultrasonically in methanol and methanol-water, extracted for 18 h with methanol, and vacuum dried prior to use. The extraction and drying steps were repeated after each chemical treatment of the rods. High-purity flavin adenine dinucleotide (FAD) (monosodium salt) from Boehringer Mannheim; glucose oxidase (EC 1.1.3.4) (type VII, from Aspergillus niger, ca. 125 U/mg solid), horseradish peroxidase (EC 1.11.1.7) (Type 11, ca. 150 U/mg solid), and o-dianisidine from Sigma; (n-aminopheny1)boronicacid hemisulfate and HEPES (N-(2hydroxyethyl-l-piperizine-N’-2-ethanesulfonic acid) sodium salt, carbodifrom Aldrich; and l-ethyl-3-[3-(dimethylamino)propyl] imide hydrochloride from Pierce were used as received. The GO did not have any free FAD, as discussed in a later section. Solutions were prepared with Millipore “Milli &” grade water and were purged for 10 min with nitrogen for deoxygenation prior to use in making the electrochemical measurements. Preparation of (Aminopheny1)boronic Acid Modified Glassy Carbon Electrodes (GC-APBA). Chemical oxidation of the glassy carbon electrode surface to create carboxylic acid groups was carried out as described previously (12). The hemisulfate salt of (m-aminopheny1)boronicacid was converted into the free base (APBA) (13)prior to use. The carboxylic acid groups on the glassy carbon surface were activated by shaking each electrode at room temperature with 3 mL of 5 mg/mL aqueous carbodiimide solution. The electrodes were washed with water and then treated with an aqueous solution of 10 mg/mL APBA. This was followed by washing with water, extracting with methanol, and drying to give the GC-APBA electrodes. Attachment of Glucose Oxidase (GO) to GC-APBA. Each GC-APBA electrode was shaken with 3 mL of 2 mg/mL glucose oxidase in 50 mM HEPES buffer containing 0.1 M magnesium chloride hexahydrate, pH 8.45, for 10 h at 4 “C. The electrode
0003-2700/86/0358-2984$01.50/06 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
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Figure 2. Effect of scan rate on peak current for cyclic voltammograms of GC-APBA-GO in buffer A at 25 "C. Data were obtained by using results recorded with expanded vertical scale, as shown in Figure 1 insert.
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Figure 1. Cyclic voltammograms obtained under anaerobic conditions at 25 "C in buffer A at 50 mV/s sweep rate (potentials are with reference to Ag/AgCI (1 M KCI)): GC-APBA-GO (-); untreated GC in 1 mg/mL GO (---), untreated GC in 0.83 mg/mL FAD and 0.29 mglmL APBA (X). Insert is for GC-APBA-GO electrode at scan rate of 20 mV/s and vertical scale as shown.
was washed with the same buffer until no protein could be detected in the washings, when tested with the Bio-Rad Bradford reagent. This washing would also have removed any loose FAD or any FAD that had been adsorbed on the GC (2). The electrode (GC-APBA-GO) was characterized electrochemically. Electrochemical Measurements. A jacketed electrochemical cell was fitted with a 2-cm2platinum gauze auxiliary electrode, a Ag/AgCl (1 M KCl) reference electrode, and the working electrode. Cyclic and differential puke voltammetry was carried out with a Princeton Applied Research Model 174A polarographic analyzer and a Houston Instrument X-Y recorder. Although the entire surface of the GC electrodes was derivatized, only one polished, flat end was used for the electrochemicalmeasurements. HEPES buffer (50 mM) containing 0.1 M MgCl2-6H20,pH 8.45, called buffer A, served as the electrolyte for the electrochemical measurements. R E S U L T S AND DISCUSSION Evidence for Direct Electron Transfer. The presence of cyclic or differential pulse voltammetric peaks attributable to oxidation or reduction of the flavin cofactor portion of glucose oxidase is taken as positive evidence for direct electron transfer between the immobilized holoenzyme and the electrode surface. This definition is appropriate because the flavin cofactor of glucose oxidase is essentially irreversibly bound to the apoenzyme in the absence of glucose (14). Therefore, the flavin cofactor molecules are not able to dissociate from the apoenzyme and diffuse to the electrode surface but must undergo oxidation or reduction while still complexed within the apoenzyme matrix. The cyclic voltammetry data showed well-defined reduction and oxidation peaks at an E"' of -505 mV (defined as midway between the oxidation and reduction peaks) for the GCAPBA-GO electrodes in buffer A (Figure l),thus indicating that electron transfer occurred. The results shown in Figure 1were obtained at a scan rate of 50 mV/s and a vertical scale of 10 bA/cm. Much sharper peaks were obtained at slower rates and with vertical scale expansion (Figure 1 insert); but the capacitive effect made the plots excessively large to include in the paper. Thus, the 50 mV/s and insert at 20 mV/s results are shown. In control runs, no peaks were observed for a plain,
untreated glassy carbon electrode immersed in a 1 mg/mL solution of glucose oxidase in buffer A (Figure 1). Thus, the electron transfer was not observed for GO in solution. From other control results discussed later, it is concluded that the redox peaks were not caused by FAD extracted from GO to form APBA-FAD or free FAD in solution. These results suggest that the immobilized boronic acid residues and the enzyme interacted in a manner that resulted in enhanced electron transfer. An additional control run (Figure l),in which an untreated GC electrode was immersed in a solution of 0.83 mg/mL FAD and 0.29 mg/mL APBA, showed strong FAD reduction and oxidation peaks at an E"' of -475 mV. In a related experiment, FAD was immobilized on a GC-APBA electrode by substituting FAD for GO in the electrode preparation procedure. The resulting GC-APBA-FAD electrode was examined by cyclic voltammetry. The E"' of -495 mV compared favorable with the value of -505 mV for the GCAPBA-GO electrode and -475 mV for FAD in solution. The work with the GC-APBA-FAD electrode and its interaction with apoglucose oxidase will be dealt with in a subsequent publication. The high capacitance of the modified electrode was attributed to the increase in surface area brought about by treatment of the GC with hot acid to generate surface carboxylic acid groups. Integration of the reduction peak area for the GC-APBAGO cyclic voltammogram (Figure 1)gave an estimated surface concentration of redox species of 4.6 X mol/cm*, based on the geometrical surface area of the electrode. This is approximately equivalent to monolayer coverage (15). Additional evidence was obtained to support the conclusion that the redox species was immobilized on the electrode surface and not free in solution. For redox groups attached to the electrode surface, the cyclic voltammetry peak current should vary with the sweep rate to a power of 1.0 (16). This compares to a power of 0.5 for redox materials in solution (16). The results are shown in Figure 2. The peak currents were obtained by using the cyclic voltammetry data obtained with expanded vertical scales in order to obtain greater accuracy. The excellent linearity (r = 0.999) and slope of 0.994 leave no doubt that the redox species was attached to the electrode surface. The results from the cyclic voltammetry studies were corroborated very strongly by the more sensitive differential pulse voltammetry (DPV) runs. The GC-APBA-GO electrode in buffer A exhibited an intense reduction peak centered at -510 mV, with a small shoulder at -280 mV and a very small peak a t -870 mV (Figure 3). In comparison, no peaks or shoulders were observed by DPV for an untreated glassy carbon electrode in 1 mg/mL GO in solution in buffer A. The Figure 3 data show that enhanced electron transfer definitely
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
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Flgure 3. Differential pulse voltammograms obtained under anerobic cordtiins at 25 "C in buffer A at 5 mV/s, 25 mV pulse amplitude, and 0.5 s between pulses (reference AgIAgCI, 1 M KCI): GC-APBA-GO (-); untreated Gc in 1 mg/mL GO (---); untreated GC in 0.83 mg/mL FAD and 0.29 mg/mL APBA (X).
occurred with the APBA immobilized GO. The minor DPV peaks, also seem at low cyclic voltammetry scan rates, were not investigated. Redox Species Accounting for GC-APBA-GO Electron Transfer. FAD is the cofactor that is responsible for the enzymatic redox properties of GO. Therefore, FAD is the most likely source of the direct electron transfer observed with the GC-APBA-GO electrodes. The cyclic and differential pulse voltammograms for FAD in buffer A, containing 2 mol of APBA/mol of FAD, at pH 8.45 are shown in Figures 1 and 3, respectively. Eo'for GC-APBA-GO occurred at -505 mV. This compares well with an Eo' of -495 mV for GC-APBAFAD. The E"' of -475 mV (Figure 1)agreed favorably with a previously reported value of -520 mV for FAD in 0.1 M Tris buffer, pH 8.0, as measured with a plain glassy carbon electrode (2). The more sensitive differential pulse voltammetry measurements for the reduction peak gave E, values of -485 mV for the FAD/APBA mixture in solution in buffer A at pH 8.45 (Figure 3), -520 mV for FAD in solution in 0.1 M Tris buffer a t pH 8.0 (2), and -530 mV for GO adsorbed on a spectroscopic graphite electrode and measured in 0.1 M Tris buffer at pH 8.0 ( 2 ) . These differential pulse measurements agree closely with the E , of -530 mV observed for the GCAPBA-GO electrode (Figure 3). These results are highly consistent with attributing the direct electron transfer to the reduction or oxidation of the FAD associated with the immobilized GO in the GC-APBA-GO electrodes. Additional control experiments were carried out to show that the APBA was not the source of the redox peaks. When a GC-APBA electrode was subject to cyclic voltammetry at 50 mV/s in buffer A a t pH 8.45, no peaks were observed between +200 mV and -800 mV. Similarly, no evidence of oxidation or reduction peaks was seen in the cyclic voltammogram of APBA dissolved in buffer A, as determined with a polished, unmodified glassy carbon electrode scanned at 50 mV/s over the same range of potentials. Mechanisms of APBA-GO Binding and Enhanced Electron Transfer. Preliminary ESCA studies indicated that the level of boron was elevated on the GC-APBA electrodes as compared to an unmodified glassy carbon electrode. The B 1s peak occurred at 190.0for plain glassy carbon and at 191.4 for GC-APBA. The corresponding B ls/C 1s ratio was 0.0026 for the plain electrode and 0.0041 for the GC-APBA unit, representing a 57% increase in the boron level. As discussed
in the previous section, the redox peaks associated with the GC-APBA-GO electrodes did not originate with the APBA. Moreover, the cyclic voltammetry data (Figure 2) show that the redox peaks came from a species attached to the electrode surface. Therefore, it appears that APBA bound the GO, along with its tightly attached FAD cofactor, to the electrode surface. The possibility that APBA extracted FAD from the added GO to give APBA-FAD or free FAD was rejected based on differential pulse as well as spectroscopic measurements. GO in the buffer solution used in this study did not show any differential pulse voltammetric response (Figure 3), even when increasing amounts of APBA were added to the solution. If APBA was able to extract FAD from GO, then the characteristic differential pulse peak of FAD or APBA-FAD should have been seen at the higher levels of added APBA. The absence of such peaks clearly indicates that APBA is not capable of extracting FAD from GO. A similar conclusion based on absorbance spectrophotometry is discussed below. The most likely sites for complexation between APBA and GO are through the carbohydrate portion of GO or through the ribityl sugars of FAD. Both electrochemical and spectroscopic techniques were used to assess whether or not FAD-APBA complexation occurred. Cyclic and differential pulse voltammetry were carried out with FAD solution in buffer A. No difference was seen in the peak oxidation or reduction potentials when APBA was added to the solution to give the voltr.mmograms shown in Figures 1 and 3. Thus, if complexation occurred between FAD and APBA in solution, the results did not influence the E, values for FAD. In aqueous solution, FAD forms intramolecular complexes in which the adenine and isoalloxazine ring systems are stacked (171,although it is not known if such stacking exists for FAD in GO. It has also been reported recently that there is substantial charge transfer interaction between the aromatic rings in FAD (18). If APBA forms a complex with the ribityl portions of FAD, this may interfere with the charge transfer interactions, and such perturbations may show up as changes in the UV spectrum. This was observed experimentally in the present study. A solution of FAD (0.83 mg/mL) in buffer A containing 0.29 mg/mL APBA showed a 19% decrease in absorbance for the peak at 374 nm and a 20% decrease in the peak at 446 nm when compared with the values for FAD alone (0.83 mg/mL) in buffer A. No change either in absorbance or in the peak position at 380 nm and 450 nm was observed for 23 pM GO in buffer A without and with 0.33 mg/mL APBA. These data indicate that complex formation between APBA and the ribityl portion of FAD may not be a factor in the binding of GO to the GC-APBA electrodes. These data also corroborate the above conclusion that APBA is not capable of extracting FAD from GO since no change in absorbance of the charge transfer bands occurred on adding APBA to GO. Complex formation between APBA and diol groupings of the carbohydrate portion of GO appears to be the most likely mechanism for attachment of GO to the GC-APBA electrodes. The tetrahedral boronate anion is an important element for the formation of stable cyclic complexes (19). The reaction scheme is shown in Figure 4. We postulate that complexation of GO by APBA causes a change in the conformation of the apoenzyme. This conformational change leads to greater solvent exposure of the isoalloxazine ring system of FAD, presumably enhancing the accessability of the ring system to electron transfer. Differential pulse voltammograms of GO in solution showed no evidence of oxidation or reduction peaks in the range -450 mV to -550 mV for 1.0 mg/mL GO mixed with buffer A containing 0 or 1.0 pg/mL APBA (1 mol of APBA/mol of FAD in GO).
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
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Flgure 4. Reaction scheme for complex formation between APBA and diol grouping on carbohydrate of GO. A, B, D, and E represent H, OH, or other groups of the carbohydrate portions of GO.
It was necessary to carry out the complexation studies at p H values that were much more alkaline than 5.5-6.0, where soluble glucose oxidase activity is a maximum. The need for the alkaline pH was generated by the stability-pH relationship for boronate complexes. Phenylboronic acid has its pK, a t 8.86 (20). Thus the medium must be buffered a t pH values in excess of about 8.0 for the formation of stable complexes. The addition of salts, such as NaCl or MgC12, was included for buffer A since this aids in complex stabilization (21). The GC-APBA-GO complex was stable for at least several days in buffer A at room temperature, as determined by periodic differential pulse voltammetry measurements (longer stability tests were not made). Tris buffer at pH 8.0 also could be used but the voltammetry peak heights were less than when measured at pH 8.45. Determinations at pH values less than 8.0 resulted in still smaller peak heights. When citratephosphate buffer of pH 4.0 was used with a GC-APBA-GO electrode, the redox peaks disappeared. At pH 4.0, the complexation was nearly totally reversed, as evidenced by microassay of the solution, using the Bradford reagent, after removal of the electrode. The released protein amounted to 66% of the GO attached to the electrode. This determination mol of was based on (1)the measured loading of 4.6 X FAD/cm2, (2) the geometrical area of the electrode, (3) the assumption that the redox peaks were due to the FAD in the GO, and (4) a ratio of 1 mol of FAD for each mole of GO (22). Several attempts were made to observe a catalytic current by adding glucose to the HEPES buffered solution in which the GC-APBA-GO electrode was immersed. The efforts were frustrated by the appearance of a greatly increased background current that masked any catalytic wave upon addition of glucose. This increase in background current also occurred upon glucose addition with a plain unmodified GC electrode. Considerable additional work will be needed to understand these results and to demonstrate a catalytic current. For this study it appears valid to conclude that the redox peaks associated with the GC-APBA-GO electrode came from FAD-apoenzyme and not from FAD that had dissociated from the apoenzyme and then been adsorbed on the electrode surface. The following results are used to support this conclusion: (1) protein could be extracted from the electrode surface by a major pH change, (2) a better defined differential pulse voltammetry peak with increasing peak current should
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have appeared as more APBA was added to GO in solution if APBA influenced the extraction of FAD from the enzyme, and (3) the UV extinction coefficient for GO did not change with the addition of APBA whereas a definite change in the extinction coefficient exists between FAD free in solution and FAD complexed with the apoenzyme. In summary, the results indicate that GO can be immobilized on GC-APBA, presumably by complex formation with the carbohydrate portion of the enzyme. The complexation apparently improves the accessability of the FAD cofactor to direct electron transfer interaction with the electrode surface. The simplicity of this method of immobilization makes this an attractive procedure for the immobilization of other glycoproteins and for the electrochemical exploitation of the resulting enzyme electrodes. It may even be possible to modify the pK, values of the boronate complexes by the use of appropriate substituents on the phenyl ring of the phenylboronic acid (23). Examination of the effects of GC-APBA-immobilization on GO enzymatic activity will be dealt with in a separate paper. ACKNOWLEDGMENT A. Proctor of the University of Pittsburgh Department of Chemistry Surface Science Center very kindly carried out the ESCA measurements. Regietry No. GO, 9001-37-0;FAD, 146-14-5;D-glucose,50-99-7. LITERATURE CITED Ikeda, T.; Katasho, I.; Kamer, M.; Senda, M. Agric. B i d . Chem. 1984,4 8 , 1969-1976. Miyawaki, 0.;Wingard, L. B., Jr. Biotechnol. Bioeng. 1984, 26,
1364-1371. Ianniello, R. M.; Lindsay, T. J.; Yacynych, A. M. Anal. Chem. 1982, 5 4 , 1098-1101. Wingard, L. B., Jr.; Schiller, J. G.; Wolfson, S. K., Jr.; Liu, C. C.; Drash, A. L.;Yao, S.J. J . Biomed. Mater. Res. 1979, 73,921-935. Wingard, L. B., Jr.; Narasimhan, K.; Miyawaki, 0.In Flavins and Falvoproteins ; Bray, R. C., Engel, P. C., Mayhew, S. G., Eds.; Walter de Gruyter: Berlin, 1984;pp 893-896. Narasimhan, K.; Wlngard, L. B., Jr. Enzyme Microb. Techno/. 1985,7 ,
283-286. Castner, J. F.; Wingard, L. B., Jr. Biochemistry 1984,23, 2203-2210. Bergold, A.; Scouten, W. H. I n SolidPhase Biochemistry; Scouten, W. H., Ed.; Wiiey: New York, 1983; pp 149-187. Williams, G. T.; Johnstone, A. P.; Bouriotis, V.; Dean, P. D. G. Biochem. Soc. Trans. 1981,9 , 137-139. Middle, F. A.; Bannister, A.; Bellingham, A. J.; Dean, P. D. G. Biochem. J . 1983,209, 771-779. Zaborsky, 0.R.: Ogletree, J. Biochem . Biophys . Res. Commun. 1974,61, 210-216. Wingard, L. B., Jr.: Guercka, J. L., Jr. J . Mol. Catal. 1980, 9 ,
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749-756. Lorand, J. P.; Edwards, J. 0. J . Org. Chem. 1959, 2 4 , 769-774. Branch, G. E. K.; Yabroff, D. L.; Bettman, B. J . Am. Chem. SOC. 1934,5 6 , 937-941. Bouriotis, V.; Galpin. I. J.; Dean, P. D. G. J . Chromatography 1981,
210, 267-278. Stankovich, M. T.; Schopfer, L. M.; Massey, V. J . B i d . Chem. 1978, -2.-5-3 , 4971-4979 . - . . . - . -. Torssell, K.; Mc Clendon, J. H.; Somers, G. F. Acta Chem. Scand. 1958, 12, 1373-1385.
RECEIVED for review October 16,1985. Resubmitted July 17, 1986. Accepted July 28, 1986. This work was supported by Contract DAAG2982K0064 from the Army Research Office.
