Biosensor and Chemical Sensor Technology - American Chemical

Chapter 8

Electroenzymatic Sensing of Fructose Using Fructose Dehydrogenase Immobilized in a Self-Assembled Monolayer on Gold

Downloaded by COLUMBIA UNIV on July 1, 2013 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0613.ch008

Κ. T. Kinnear and H. G. Monbouquette Chemical Engineering Department, University of California, Los Angeles, CA 90095-1592 The hydrophobic enzyme, fructose dehydrogenase (from Gluconobacter sp., EC 1.1.99.11), and coenzyme Q have been co -immobilizedin a self-assembled monolayer (SAM) on gold through a detergent dialysis procedure to create a prototype fructose biosensor. The S A M consists of a mixture of octadecyl mercaptan (OM) and two short chain disulfides, which form -S-CH -CH -CH -COOand -S -CH -CH -NH + on the surface. The short chain, charged modifiers may provide defects, or pockets, in the O M layer where the enzyme may adsorb through electrostatic interactions. At oxidizing potentials, the electrode generates a catalytic current at densities up to about 1 0 μΑ/cm when exposed to fructose solution. The enzyme electrode exhibits a response time well under a minute and the calibration curve is linear at fructose concentrations up to 0.8 m M . The biosensor prototype exhibits low susceptibility to positive interference by ascorbic acid indicating that this construct could be useful for fructose analysis of citrus fruit juice. 6

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A reliable fructose sensor would be of value for the quantitation of this sugar in fruit juice, wine, corn syrup, blood serum, and seminal plasma. Note that a low level of fructose in seminal plasma can be an indicator of male infertility. Several reports have appeared describing the development of prototype amperometric fructose biosensors using Gluconobacter sp. fructose dehydrogenase (EC 1.1.99.11) (7-5), a 140 kDa pyrroloquinoline quinone (PQQ)-containing oxidoreductase. The enzyme has been immobilized on gold, platinum and glassy carbon (3), entrapped in conductive poly pyrrole matrices (4,5), or confined to the surface of a carbon paste electrode behind a dialysis membrane (1,2). Although sensors exhibiting good current density, sensitivity, stability and response time have been described, erroneous signal generation due to electroactive interferents such as ascorbic acid in citrus juice has been a persistent problem. This paper describes an effective approach to the problem of polar, ascorbic acid interference by embedding the enzyme in a mostly hydrophobic, insulating self-assembled monolayer on gold. The hydrophobic nature of membrane-bound redox enzymes, such as this fructose dehydrogenase, provides an obvious route to gentle, stabilizing immobilization in hydrophobic adsorbed layers on electrodes which mimic the cell 0097-6156/95/0613-0082$12.00/0 © 1995 American Chemical Society

In Biosensor and Chemical Sensor Technology; Rogers, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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KINNEAR & M O N B O U Q U E T T E

Electroenzymatic Sensing of Fructose 83

membrane microenvironment. This concept has been demonstrated earlier with the stable immobilization of membrane-bound E. coli fumarate reductase in an alkanethiolate layer on gold (6). In this case, direct electron transfer was achieved between the enzyme and electrode. In contrast, stable immobilization of fructose dehydrogenase required the co-adsorption of short-chain, charged disulfides for electrostatic binding of the enzyme (7); and a mediator, coenzyme was needed for electron transfer. Although others have achieved direct electron transfer between unmodified electrode surfaces and this fructose dehydrogenase (2,3), some of the preparations are very unstable (3). We report a stable and highly electroactive membrane mimetic system where coenzyme Q6 mediates electron flow between the enzyme and electrode.


Experimental Reagents and Materials. D-fructose dehydrogenase (FDH) from Gluconobacter sp., coenzyme Q6, D(-)fructose, and n-octyl β-D-glucopyranoside (n-octyl glucoside) were purchased from Sigma and were used without further purification. Octadecylmercaptan, cystamine dihydrochloride, and 3,3-dithiodipropionic acid were obtained from Aldrich. Other chemicals were reagent grade from Fisher or Sigma and were used as received. A l l electrochemical supplies were purchased from Bioanalytical Systems, Inc. (W. Lafayette, IN). Electrochemical Procedure. Electrochemical data was obtained with a B A S C V 1B instrument and a B A S X - Y recorder. The B A S CV-1B was interfaced to a Macintosh Hex with a National Instruments (Austin, TX) Lab-NB board and software (LabVIEW II). Gold disk electrodes (d = 1.6 mm, A = 0.02 cm ), a Ag/AgCl (3 M NaCl) electrode and a platinum wire were used as the working, reference and counter electrode, respectively. Before each experiment, a gold electrode was polished and electrochemically cleaned as described earlier (6,8). The amperometric determination of fructose was done at 0.5 V vs. Ag/AgCl in stirred deoxygenated 10 m M KH2PO4, pH 4.5 under a blanket of Ar at room temperature unless otherwise stated. 2

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Adsorbed Layer Preparation. A stock solution of F D H (-1.35 μΜ) was prepared with 2.5 mg of FDH and 1 ml of phosphate buffer and 1% octyl glucoside (35 mM) and used within two weeks (stored at 4 °C between experiments). Coenzyme Qo at 170 μΜ was added to the enzyme detergent solution when indicated. Prior to dialysis, freshly polished and electrochemically cleaned electrodes were modified for 2 hours in various thiol/disulfide ethanol solutions. In addition to octadecyl mercaptan (OM), two disulfides were used, cystamine dihydrochloride (CA) and 3,3'dithiodipropionic acid (TP). The disulfides undergo dissociative adsorption at the gold surface and form self-assembled monolayers in much the same way as alkanethiols (2). The total thiol/disulfide concentration was 1 mM with 40% O M , 30% TP, and 30% C A . The F D H stock solution (270-280 μΐ) and the modified electrode were added to a 10,000 M W C O dialysis bag and placed in a 400 ml reservoir of phosphate buffer; dialysis was carried out at 4 °C with stirring. The dialysate was replaced 3 to 4 times over 18 to 48 hours; the last reservoir replacement included approximately 1 ml of the detergent-sorbing resin, Calbiosorb. The electrode was removed from the bag and rinsed with pure water and either used immediately or stored in buffer.


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BIOSENSOR AND CHEMICAL SENSOR T E C H N O L O G Y

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Figure 1. Steady state C V of FDH and Q6 in a mixed thiol monolayer on gold in deoxygenated 10 m M KH2PO4, pH 4.5 buffer (solid curve); and the first C V scan in 28 m M fructose (dashed curve). The scan rate was 50 mV/s.

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0 10 20 30 40 Fructose Concentration (mM) Figure 2. Calibration curve for fructose obtained with an FDH-Q6 modified electrode.


8.

KINNEAR & M O N B O U Q U E T T E

Electroenzymatic Sensing ofFructose


Results and Discussion Coenzyme QG (QO) and fructose dehydrogenase were immobilized simultaneously through detergent dialysis on a gold electrode previously modified using a 40% O M , 30% TP and 30% C A mixture. A strong catalytic current response was observed when this electrode was introduced into fructose solution (Figure 1). Control voltammograms (CVs) of a Q6 mixed monolayer electrode without enzyme do not show catalytic activity in the presence of fructose (data not shown). The enzyme could not be immobilized in an O M layer without TP and C A . As has been demonstrated earlier (7), these charged species can enable stable electrostatic binding of redox proteins to gold. To investigate the potential of this system for sensing applications, various parameters were obtained. Figure 2 depicts calibration curves obtained with a QO-FDH modified electrode. At lower concentrations of fructose, a linear relationship between current and fructose concentration is observed (Figure 2). Saturation values of current are observed at higher fructose concentrations. The current density of this electrode at 10 m M fructose is 11.3 μΑ/cm versus 6.9 μΑ/cm for an F D H carbon paste dialysis membrane electrode reported by Ikeda et al (2). The time response of the Q^-FDH modified electrode was also evaluated as shown in Figure 3. Upon addition of 5 mM fructose to a stirred phosphate buffer solution, the maximum steady-state current response was obtained within about 20 seconds. Another important biosensor feature to evaluate is susceptibility to electroactive interferents. In F D H electrode applications, such as the measurement of fructose in fruit juices or wine, the effects of ascorbic acid should be investigated. Ascorbic acid is oxidized quite readily at bare electrode surfaces. The blocking mercaptan layer of this system may have the advantage of hindering access of ascorbic acid to the electrode surface. Figure 4 illustrates the effect of 100 μΜ ascorbic acid on the measured current of a 2 mM fructose solution. The presence of ascorbic acid at 5% of 2

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Figure 4. Effect of ascorbic acid on current response obtained with an FDH-Q6 modified electrode.


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BIOSENSOR AND CHEMICAL SENSOR T E C H N O L O G Y

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Figure 3. Time dependence of the current response obtained with an FDH-Q6 modified electrode. the fructose concentration results in an error of 9%. Note that ascorbic acid usually is 2-3% of the fructose concentration in citrus fruit juices. The presence of the mixed monolayer does seem to block access of ascorbic acid to some degree. In contrast, the FDH carbon paste electrode of Ikeda et al. gives an error of approximately 80% at 50 μΜ ascorbic acid and 2 mM fructose (2). The membrane mimetic fructose dehydrogenase system for fructose sensing described in this paper is stable for at least several days. Activity loss appears to occur primarily due to desorption of Q6 from the electrode surface since much electrode activity could be restored by exposure of the electrode to a solution containing an alternate lipophilic mediator, decylubiquinone. Efforts are underway to improve mediator retention with the hope that electrode stability can be increased further to a practical level. Acknowledgments This research was supported by National Science Foundation grant BES-9400523. Literature Cited 1. Ikeda, T.; Matsushita, F. and Senda, M. Agric. Biol Chem. 1990, 54, 2919. 2. Ikeda, T.; Matsushita, F. and Senda, M. Biosens. Bioelectron. 1991, 6, 299. 3. Khan, G.F.; Shinohara, H.; Ikariyama, Y. and Aizawa, M. J. Electroanal. Chem. 1991, 315, 263. 4. Khan, G.F.; Kobatake, E.; Shinohara, H.; Ikariyama, Y. and Aizawa, M. Anal. Chem. 1992, 64, 1254. 5. Begum, Α.; Kobatake, E.; Suzawa, T.; Ikariyama, Y. and Aizawa, M. Anal. Chim. Acta 1993, 280, 31. 6. Kinnear, K.T. and Monbouquette, H.G. Langmuir 1993, 9, 2255. 7. Hill, H.A.O. and Lawrance, G.A. J. Electroanal. Chem. 1989, 270, 309. 8. Finklea, H.O.; Avery, S.; Lynch, M. and Furtsch, T. Langmuir 1987, 3, 409. RECEIVED June 5, 1995


Biosensor and Chemical Sensor Technology - American Chemical

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