Anal. Chem. 1994,66, 1535-1539

Electrical Communication between Electrodes and NAD(P)+-Dependent Enzymes Using Pyrroloquinolinequinone-Enzyme Electrodes in a Self-Assembled Monolayer Configuration: Design of a New Class of Amperometric Biosensors Itamar Wlllner' and Azalla Rlklln Institute of Chemistty, The Hebrew University of Jerusalem, Jerusalem 9 1904, Israel

The development of an amperometric sensor utilizing the NAD(P)+-cofactor-dependent enzyme, malic enzyme, is de- scribed using a quinone-enzyme monolayer-modified electrode. Pyrroloquinolinequinone (PQQ, 1) was covalently linked to a self-assembled monolayer of cysteamine on an Au electrode. The resulting PQQ-monolayer electrode (PQQ surface cov- erage 1.98 X 10-lo mol.cm-2) catalyzes the electrooxidation of NADPH and NADH. The developed anodic currents are controlled by NAD(P)H concentrations and provide an amperometric sensor for the cofactor. Malic enzyme has been covalently linked to the PQQ-monolayer electrode. The resulting PQQ-enzyme electrode (enzyme coverage 4.01 X 10-l2 mol.cm-2) provides an amperometric biosensor for the determination of malic acid in the presence of the cofactor NADP+. In this system, biocatalyzed oxidation of malic acid generates NADPH that is oxidized by the PQQ component.

Development of amperometric biosensors that apply redox enzymes as the bioactive materials has evoked substantial interest in recent Direct electrical communication between redox centers of enzymes and electrode interfaces is eliminated by the protein shell surrounding the active site. That is, the distance separation of the redox active site from the electrode surface introduces a kinetic barrier for electron transfer. Application of diffusional electron-transfer medi- ators4.5 or immobilization of relay modified enzymes onto electrode^^.^ provides an effective means to establish electrical communication between electrodes and redox proteins. Al- ternatively, immobilization of proteins onto electrode surfaces by means of redox tethered polymers facilitates mediated electron transfer between the enzyme redox site and electrode surfaces.8-10 These approaches have been applied successfully

(1) Wilson, G. In Biosensors: Fundamental and Application; Turner, A. P. F., Karube, I., Wilson, G. S., Eds.; Oxford University Press: New York, 1987.

(2) Biqsensors: A Practical Approach; Cass, A. E. G., Eds.; IRL Press: Oxford, UK, 1990.

(3) Heller, A. Acc. Chem. Res. 1990, 23, 128. (4) (a) Yeh, P.; Kuwana, T. J. Electrochem. SOC. 1976,123, 1334. (b) Cass, A.

E. G.; Davis, G.; Green, M. J.; Hill, H. A. 0. J. Electroanal. Chem. Interfacial Electrochem. 1985, 190, 117.

(5) (a) Crumblis, A. L.; Hill, H. A. O.;Page, D. J. J. Electroanal. Chem. Interfacial Electrochem. 1986,206,327. (b) Ikeda, T.; Hiasa, H.; Senda, M. In Redox Chemistry and Interfacial Behavior of Biological Molecules; Dryhurst, G., Niki, K., Eds.; Plenum Publishing Corp.: New York, 1988; p 193. (c) Taniguchi, I.; Miyamoto, S.; Tomimura, S.; Hawkridgc, F. M. J. Electroanal. Chem. Interfacial Electrochem. 1988, 240, 333.

(6) Degani, Y.; Heller, A. J. Phys. Chem. 1987, 91, 1285. (7) Willner, I.; Katz, E.; Riklin, A.; Kasher, R. J. Am. Chem. Soc. 1992, 114,

10965.

for the development of various amperometric biosensors for glucose, nitrate, or alanine.

A majority of redox enzymes, however, do not communicate electrically with synthetic electron mediators and require specific cofactors for their activities. NAD(P)+-dependent dehydrogenases represent a broad class of redox proteins that require the regeneration of their specific cofactor.I1J2 Elec- trochemical regeneration of NAD(P)+ cofactors is ac- companied by irreversible redox processes degrading the cofactor as well as by overpotential constrains.13 Several modified electrodes have proved to be effective in overcoming these kinetic barriers and inducing specific electrochemical regeneration of NAD(P)+."16 Nonetheless, attachment of enzymes to these electrodes is not straightforward. Thus, the design of enzyme electrodes that are capable of electrical communication between the electrode material and the native NAD(P)+ cofactors is essential to develop amperometric biosensors with this class of biocatalysts.

The pyrroloquinolinequinone, 4,5-dihydro-4,5-dioxo- 1H- pyrrolo [ 2,3-f] quinoline-2,7,9-tricarboxylic acid (PQQ, 1), acts as cofactor for mediated electron transfer in various en- zymes.17J8 Electron-transfer communication between an electrode and the enzyme methanol dehydrogenase containing PQQ was accomplished in the presence of a tetrathiafulvalene tetracyanoquinodimethane-modified electrode, where the redox components act as electron mediators.lg Similarly, it has been established20 that PQQ catalyzes the oxidation of

(8) (a) Hale, P. D.; Inagaki, T.; Karan, H. I.; Okamoto, Y.; Skotheim, T. A. J. Am. Chem. Soc. 1989,111,3482. (b) Maidan, R.; Heller, A. AMI. Chem. 1992, 64, 2889.

(9) Domingues,E.; Lan, H. L.;Okamoto,Y.; Halc,P.D.;Skotheim,T. A.;Gorton, L. Biosens. Bioelectron. 1993, 8, 167.

(10) Willner, I.; Katz, E.; Lapidot, N.; Bluerle, P. Bioelectrochem. Bioewrg. 1992, 29, 29.

(1 1) (a) Chenault, H. K.; Whitesides, G. M. Appl. Biochem. Biotechnol. 1987,14, 147. (b) Dugas, H.; Penney, C. In Bioorganic Chemistry; Cantor, C. R., Ed.; Springer Verlag: New York, 1981; p 395.

(12) Willncr, I.; Mandler, D. Enzyme Microb. Technol. 1989, 12, 467. (13) Jansen, M. A.; Elving, P. J. Biochim. Biophys. Acta 1984, 764, 310. (14) (a) Jaegfeldt, H.; Tortensson, A.; Johansson, G . Anal. Chem. 1981,53,1979.

(b) Laval, J.-M.; Bourdillon, C.; Moiroux, J. J . Am. Chem. Soc. 1984, 106, 4701.

(15) (a) Gorton, L. J. Chem. Soc., Faraday Trans. I 1986,82,1245. (b) Pemon, B. J. ElectroaMI. Chem. 1990,286,61.

(16) Gorton, L.; Persson, B.; Hale, P. D.; Bouguslavsky, L. I.; Karan, H. I.; Lee., H. S.; Skotheim, T.; Lan, H. L.; Okamoto, Y. In Biosensors and Chemical Senrors;Edelman,P.G., Wang, J.,Eds.;ACSSymposiumSerics487;Amcrican Chemical Society: Washington, DC, 1992; Chapter 6, p 56.

(17) Duine, J. A. Biofactors 1989, 2, 87. (18) Duinc, J. A.; Jongejan, J. A. Vitam. Horm. (N.Y.) 1989, 45, 223. (19) Zhao, S.; Lennox, R. B. AMI. Chem. 1991, 63, 1174.

Ana&tical Chemistty, Voi. 66, No. 9, May 1, 1994 1535 0003-2700/94/0366-1535$04.50/0 0 1994 American Chemical Society

,COOH

1

NADH and thus regenerates NAD+. In a series of recent studies, we demonstrated that various proteins could be attached to self-assembled monolayers of functionalized thiols organized on Au electrode^.^.^^ We have shown that electrical communication in the resulting monolayer enzyme electrodes can be attained by modification of the enzyme with long- chain tethered redox relays. The multifunctional carboxylic acid residues attached to the PQQ molecule allow, in principle, the organization of an electron mediator/enzyme monolayer array on electrode surfaces. Thus, modification of an aminothiol self-assembled monolayer with PQQ, followed by covalent linkage of an enzyme to the vacant carboxylic functions of PQQ could lead to the desired electrode configuration. Regeneration of the native NAD(P)H cofactor by PQQ could lead to electrical communication between the electrode and the enzyme redox center, provided that the protein is an NAD(P)+-dependent enzyme. This is anticipated to establish a novel method for the development of ampero- metric biosensors for this broad class of NAD(P)+-dependent enzymes.

Here we report on the development of an enzyme electrode consisting of a covalently linked array of PQQ and the malic enzyme in a self-assembled monolayer on an Au electrode. We demonstrate that PQQ mediates the regeneration of NADP+ and reveal that the PQQ-enzyme electrode allows the amperometric analysis of malic acid.

EXPERIMENTAL SECTION All chemicals were of commercial source (Fluka, Sigma,

Aldrich). Electrochemical measurements were performed by a cyclic voltammetry apparatus (BAS CV-1B). The elec- trochemical cell consisted of three electrodes where the chemically modified electrode acted as the working electrode, a glassy carbon was the auxiliary electrode isolated by a frit, and an Ag/AgCl electrode was used as a reference electrode. The cell was thermostated during the measurements (28 "C), and an inert atmosphere of N2 was used.

Measurements of radioactivity were performed by a Beckman LS2800 scintillator. IR spectra were recorded by a FT-IR apparatus (Nicolet-Impact 400).

Preparation of a Self-Assembled Monolayer (SAM) of PQQ on an Electrode. Gold foils were pretreated by boiling in concentrated KOH solution for 1 h. After washing, the foils were immersed in concentrated H$04 for 12 h (room temperature) and treated with concentrated H N 0 3 for 15 min. The cleaned electrodes were thoroughly rinsed with water prior to modification. A clean Au electrode (0.2-cm2 area) was immersed for 2 h at room temperature in a 0.02 M cystamine dihydrochloride aqueous solution. The modified

(20) Itoh,S.;Kinugawa,M.; Mita,N.;Ohshiro,Y.J. Chem.Soc., Chem. Commun. 1989, 694.

(21) (a) Katz, E.; Riklin, A.; Willner, I. J . Electrmnal. Chem. 1993,354, 129. (b) Willner, I.; Riklin, A.; Shoham, B.; Rivenzon, D.; Katz, E. Ado. Mater. 1993, 5, 912. (c) Israel Patent Appl. No. 102930, 1992.

electrode was rinsed with water several times. It was then incubated overnight at room temperature in 1 mL of 0.1 M phosphate buffer solution, pH = 7.3, that included PQQ (0.3 mg), 1 -ethyl-3 [3-(dimethylamino)propyl]carbodiimide (EDC; 0.8 mg), and N-hydroxysulfosuccinimide sodium salt (NSI; 0.28 mg). The PQQ-modified electrode was rinsed with the same buffer several times.

Covalent Attachment of the Malic Enzyme onto the SAM PQQ Electrode. The PQQ SAM electrode was immersed in 1.2 mL of 0.1 M phosphate buffer (pH = 7.3) that contained malic enzyme (EC 1.1.1.40 from chicken liver, Sigma, 20 units/mg of protein)22 (0.6 mg), EDC (4 mg), and NSI (1.5 mg) and incubated at 4 OC overnight. The electrode was washed repeatedly with the same buffer solution and used for electrochemical measurements.

Radioactive Labeling of Malic Enzyme.23 [ 3H] Iodoacetic acid was anchored to cysteine residues of malic enzyme by reacting the enzyme with an excess of the radioactive label in 0.1 M phosphate buffer, pH = 7.3, at room temperature for 1 h. The resulting solution was chromatographed on Sephadex G25, and the labeled protein was separated. The content of protein in the separated fraction was determined by Lowry and the radioactive labeling of the protein corresponded to 440 000 cpm/mg of enzyme. The extent of immobilized enzyme on the electrode was determined by generation of the monolayer electrodes with the radioactive- labeled malic enzyme and readout of the resulting radioactivity of the electrodes.

RESULTS AND DISCUSSION Construction of the self-assembled monolayer of PQQ on

the Au electrode is shown in Figure la. Treatment of the Au electrode with cystamine results in a cysteamine SAM that is coupled to PQQ.25 Figure 1 b shows the cyclic voltammo- grams of PQQ in the SAM and in a homogeneous solution. While in the homogeneous phase PQQ exhibits an irreversible electrochemical response, presumably due to irreversible adsorption of the quinone to the electrode surface; PQQ in the SAM electrode configuration reveals a reversible redox process. By integration of the cathodic or anodic waves associated with the reduction or oxidation of PQQ, and knowing the surface area of the electrode, the density of PQQ in the SAM is estimated to be 1.97 X 10-10mol.cm-2. the electron-transfer rate constant from the electrode to PQQ was determined by Laviron's method.26 The plot of the anodic to cathodic peak separations as a function of scan rates at pH = 7.3 is shown in Figure 2. The derived value of the apparent electron-transfer rate constant to PQQ, at pH = 7.3, corresponds to 45.5 s-l. The thermodynamic redox potentials of quinones are pH dependent.27 The thermodynamic redox potential of the PQQ

(22) Kun, E. Ensymes 1963, 7, 157. (23) Anderson, P. J. Biochem. J . 1979,179,425. (24) (a) Sigma Diagnostic Procedure No. 690. (b) Lowry, 0. H.; Rosebrough,

N . J.; Lewis Farr, A.; Randall, R. J. J . Biol. Chem. 1951, 193, 265. (c) Ohnishi, S.T.; Barr, J. K. Anal. Biochem. 1978,86. 193.

(25) (a) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J . Am. Chem. Soc. 1987,109,3559. (b) Bain, C. D.; Whitesidcs, G. M. J. Am. Chem. Soc. 1989, 111, 7164. (c) Collard, D. M.; Fox, M. A. h g m u i r 1991, 7, 1192. (d) Walczak, M. M.; Chung, C.; Stole, S. M.; Widrig, C. A.; Porter, M. D. J . Am. Chem. Soc. 1991, 113, 2370. (e) Rubinstein, I.; Steinberg, S.; Tor, Y.; Shanzer, A.; Sagiv, J. Nature, 1988, 332, 426. ( f ) Katz, E.; Itzhak, N.; Willner, I . Lnngmuir 1993, 9, 1392.

(26) Laviron, E. J . Electroanal. Chem. 1979, 101, 19. (27) Mandler, D.; Kaminski. A.; Willner, I. Electrochim. Acta 1992, 37, 2765.

1536 Analytcal Chemistry, Vol. 66, No. 9, May 1, 1994

Figure 1. (a) Sequence of reactions for the organization of the SAM PQQ electrode. (b) Cyclic voltammograms of (a) PQQ In a homogeneous aqueous phase pH = 7.5 using a bare Au electrode and (b) SAM of PQQ electrode at pH = 7.5. Scan rate 300 mV.s-l.

0 -0.8 -0.4 0 0.3 0.6 1

log(v, V/cm)

Flgure 2. Anodic to cathodic peak separations of the SAM PQQ electrode as a function of scan rates.

monolayer electrode is pH dependent. The slope of the plot of the redox potential as a function of pH corresponds to 57 mV/pH unit. This value is very close to the Nernstian value (59 mV/pH unit), suggesting that the reduction process of PQQ involves two electrons and two protons, eq 1. Also, the

PQQ + 2e- + 2H' a PQQH, (1)

cathodic (or anodic) peakcurrents of PQQ are linearly related to the scan rates in the cylic voltammetry experiments, as expected for a redox species attached to the electrode surface.

A further aspect that needs consideration relates to the nature of the carboxylic function that is coupled to the cysteamine monolayer. PQQ includes three carboxylic func- tions at positions 2,7, and 9. The IR spectrum of PQQ was recorded and compared to the IR spectra of a series of PQQ subunits: 2-picolinic acid, isonicotinic acid, pyrrole-2-car- boxylic acid, and 9,lO-phenanthrenequinone. From these

a 1 I . ;!3 it i !

b 400.0

300 0 -2 I

200.0

100.0

0.0

[NADH], mM

Flgure 3. (a) Cycllc voltammograms of SAM PQQ electrodes In the presence of different NADH concentratlons: (1) [NADH] = 0 M; (2) [NADH] = 2 X 109 M; (3) [NADH] = 1 X lo-* M. In all experlments electrolyte solutlon Is composed of 0.1 M phosphate buffer, pH = 7.2; scan rate 2 mV.s-'. (b) Anodic currents developed by the SAM PQQ electrode at different NADH concentratlons. Cwents were extracted from cyclic voltammograms shown In (a) at a potential of -0.08 V vs Ag/AgCI.

Analytical Chembtry. Vol. 66, No. 9, M y 1, 1994 1597

@)

Au

e' NAD(P)H

Flgure 4. (a) Oxidation of NADH by the SAM PQQ electrode. (b) Electron-transfer communication of a SAM of PQQ and enzyme by a diffusional NAD(P)+ cofactor.

studies the carbonyl (M) bands of PQQ at 1722, 1708, 1678, and 1652 cm-l were assigned to the carbonyls of the carboxylic groups in positions 7 and the quinone, and the carboxylic group in position 2, respectively. Reaction of PQQ with butylamine (molar ratio 1: l), under conditions similar to those employed in coupling of PQQ to the cysteamine SAM, results in amidation of PQQ. The IR spectrum of the resulting amide reveals that the band at 1722 cm-l disappears, while the other carbonyl bands are unaffected. Thus, the carboxylic function in position 7 is amidated. We assume that the SAM of PQQ involves a similar covalent attachment of thecarboxylic function in position 7 to the cysteamine monolayer.

Addition of NADH or NADPH to the PQQ SAM Au electrode results in anodic currents that depend on the

0.2M I

0 l ,0' j

0' /'

4y1 ,/' 1 ' .' I0

EDC NSI

(c)

d Wi

200

100 ii 0 -7.5

concentration of added NAD(P)H. Figure 3a shows the cyclic voltammograms of the PQQ electrode at different concen- trations of added NADH. No anodic currents could be detected in this voltage region when NAD(P)H interacted with the unmodified Au electrode. Hence, the SAM of PQQ mediates the electrocatalyzed oxidation of NAD(P)H, Figure 4a. Accordingly, we extracted a calibration curve for the amperometric response of the PQQ electrode as a function of NADH concentrations, Figure 3b. We realize that a linear relation is obtained in the region of NADH concentrations corresponding to 3 X lV-1 X 10-* M. Thus, the PQQ SAM electrode provides a sensor for the quantitative analysis of NAD(P)H in aqueous solutions.

The ability of the SAM of PQQ to oxidize NAD(P)H and yield an amperometric response, and the vacant carboxylic functions associated with the PQQ electron mediator, allow, in principle, the design of amperometric enzyme electrodes utilizing NAD(P)+-dependent biocatalysts, Figure 4b. Here, the enzyme is covalently attached to the PQQ SAM. In the presence of the diffusional cofactor, NAD(P)+, oxidation of the enzyme substrate is accompanied by the formation of NAD(P)H, and this is followed amperometrically through its oxidation by the PQQ SAM. The concentration of the substrate (analyte) is reflected in the concentration of generated NAD(P)H and the resulting observed current. Following this approach, the NADPH-dependent malic enzyme was coupled to the PQQ SAM electrode, Figure Sa. Independently, the malic enzyme was radioactively labeled with [3H ]iodoacetic acid. The labeled protein was covalently linked to the PQQ SAM electrode, and from the resulting

U

4s -5.5 4.5 -3.5 log b a h t e , MI

-0.3 -012 -0.1 0 0.1 E (VI

Figure 5. (a) Sequence of reactbm for the organization of the PQQ-mellc enzyme SAM electrode. (b) Cyclic vottammograms of the PQQ-malic enzyme electrode at different malic acid concentrations corresponding to (1) 0, (2) 1.25 X lo-', (3) 12.5 X 1V , and (4) 1.25 X 1o-S M. Each of the scans is recorded after 30 min of incubation of the electrode with the respecthre malic acid solution to ailow the accumulation of NADPH. In ail experiments electrolyte solution consists of 0.1 M phosphate buffer, pH = 7.2, and [NADP+] = 2 X 1o-S M. Scan rate for all experiments 2 mV.s-l. (c) Anodic currents developed by the PQQ-mallc enzyme SAM electrode as a function of malic acid concentrations. Anodic currents were extracted from a series of cyclic vottammograms simllar to those shown in (b) at the potential corresponding to -0.06 V vs Ag/AgCI. Data of anodic currents represent an average value of two dlfferent electrodes, where the surface area of the two electrodes was normalized by the amperometric response of immobilized PQQ with no NADPH or malic acM.

radioactive counts, the surface coverage of the electrode by the enzyme is estimated to be 4.01 X 10-12 mol-cm-*. An independent IR study revealed that reaction of monoamidated PQQ (amidated position 7) with butylamine (molar ratio 1:l) resulted in the disappearance of the band at 1708 cm-I, characteristic of the carbonyl stretching of the carboxylic acid function of position 9. Thus, it is suggested that the lysine residues of the malic enzyme are covalently linked to the carboxyl function at position 9 of the PQQ SAM.

The malic enzyme catalyzes the oxidation of malic acid according to eq 2. The amperometric responses of the PQQ- malic enzyme SAM electrode were examined in the presence

OH I malic enzyme

HOpC-CHp-CH-C&H + NADP' D

0 I1

CH&CO&I + Cop + NADPH (2)

of the native NADP+ diffusional cofactor at different concentrations of malic acid, Figure 5b. An anodic current is developed in the cell, and its magnitude is controlled by the malic acid concentration. Control experiments indicate that no anodic currents are observed upon interacting the PQQ- malic enzyme electrode with malic acid in the absence of NADP+. Similarly, no anodic currents were detected when the PQQ SAM monolayer (without modification by malic enzyme) interacted with malic acid and NADP+. These experiments clearly indicate that malic acid is neither directly oxidized by the PQQ SAM nor by the PQQ-malic enzyme SAM in the absence of NADP+. Hence, the sequence of biocatalyzed and electrocatalyzed processes that lead to the oxidation of malic acid and result in the anodic currents proceeds according to the cycle outlined in Figure 4b. The

malic enzyme mediates the oxidation of malic acid to pyruvic acid in the presence of NADP+. The resulting NADPH is oxidized by the PQQ SAM and yields the anodic currents. Figure 5c shows the calibration plot for the resulting anodic currents of the PQQ-malic enzyme SAM electrode at different concentrations of malic acid. We see that a linear relationship is observed within a very broad region of malic acid concentrations (10-7-10-3 M).

CONCLUSIONS W.e have demonstrated that a self-assembled monolayer of

the pyrroloquinolinequinone, PQQ , on an Au electrode provides a catalytic interface for the electrocatalyzed oxidation of NAD(P)H. The resulting anodic currents allow the quan- titative amperometric analyses of the native NAD(P)H cofactors. By covalent attachment of the malic enzyme to the PQQ SAM electrode, a bioelectroactive self-assembled mono- layer was organized for the amperometric analysis of malic acid in the presence of the diffusional NADP+ cofactor. Immobilization of other NAD(P)+-dependent enzymes to the PQQ SAM could provide the basis for many new amperometric biosensors. Furthermore, one can envisage the development of bioactive monolayer electrodes that exclude the diffusional cofactor by covalent attachment of modified NAD(P)+ to the enzyme componentz8 of the PQQ-enzyme SAM configuration.

ACKNOWLEDGMENT This research is supported by the Bundesministerium far

Forschung und Technologie, Germany, and the Israeli Council for Research and Development, Ministry of Science and Technology, Israel.

Received for review November 3, 1993. Accepted February 8, 1994."

(28) Blckmann, A. F.; Carrea, 0. Adv. Biochrm. Eng./Biotechnol. 1989,39,97. *Abstract published in Advance ACS Abstracts, March 15, 1994.

Analj.llcal Chemistry, Vol. 66, No. 9, May 1, 1994 1539