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Glucose oxidase electrodes via reconstitution of the apo-enzyme: tailoring of novel glucose biosensors
Analytica Chimica Acta 385 (1999) 45±58
Glucose oxidase electrodes via reconstitution of the apo-enzyme: tailoring of novel glucose biosensors Eugenii Katza,*, Azalia Riklina, Vered Heleg-Shabtaia, Itamar Willnera, A.F. BuÈckmannb a
Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Gesellschaft fuÈr Biotechnologische Forschung, Department of Molecular Structure Mascheroder Weg 1, D-38124 Braunschweig, Germany
b
Received 27 May 1998; received in revised form 17 September 1998; accepted 19 September 1998
Abstract Reconstitution of an apo-¯avoenzyme with a relay-FAD-cofactor dyad yields an electrically contacted enzyme, ``electroenzyme''. This is exempli®ed by the reconstitution of apo-glucose oxidase, apo-GOx, with a ferrocene±tethered FAD-cofactor. The resulting semisynthetic protein stimulates the bioelectrocatalyzed oxidation of glucose. Kinetic analysis of the ferrocene±FAD reconstituted enzyme reveals that its electrical communication with an electrode support is superior as compared to a protein randomly functionalized by ferrocene relay units. A pyrroloquinolinoquinone (PQQ)±FAD cofactor monolayer is assembled on a Au-electrode. Apo-GOx is reconstituted on the solid support to yield an aligned enzyme electrode of unprecendently ef®cient electrical contact. The electron-transfer turnover rate with the electrode is estimated to be 900150 sÿ1, a value close to the electron transfer rate between native GOx and molecular oxygen. The effective electrical communication of the integrated enzyme electrode stimulates the ef®cient bioelectrocatalyzed oxidation of glucose, and results in high current densities and high sensitivity for glucose. The effective electrical contact of the enzyme-electrode yields also a speci®c glucose sensing electrode that is not perturbed by oxygen or interferrants. The possible application of the enzyme-electrode as an invasive biosensor is addressed. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Glucose oxidase; Flavin adenine dinucleotide; FAD; Biosensor; Reconstituted enzymes; Enzyme monolayers; Electrical contact of enzymes; Integrated enzyme electrodes; Glucose sensor
1. Introduction The application of redox-enzymes as bioactive matrices for tailoring biosensor [1,2] and bioelectronic [3] devices is of substantial basic and practical importance. Electrical communication between redoxenzymes and electrode surfaces represents the basic process to activate redox-active biocatalysts [4,5]. *Corresponding author. Tel.: +972-2-6585272; fax: +972-26527715; e-mail: [email protected]
Redox sites in proteins, however, lack direct electrical contact with electrodes due to the insulation of the redox-center by the protein [6]. The intrinsic barrier for direct electron transfer between the enzyme redox center and the electrode resulted in the development of various methods to establish electrical communication between redox-enzymes and electrode supports using electron transfer mediators [7]. Diffusional electron transfer mediators, such as hexacyanoferrate(III), ferrocene derivatives, N,N0 -bipyridinium salts, quinones, transition metal complexes, etc. were employed to
0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0003-2670(98)00688-6
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E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
electrically contact biocatalysts and the electrode surfaces in the ®rst generation of biosensors. The architecture of integrated biocatalytic assemblies lacking diffusional components has important advantages in bioelectronic and biosensor technology [4]. Tailoring of bioelectronic systems requires the assembly of biomaterials on solid conductive supports and the design of the appropriate electrical contact between the biological matrices and the support elements. Various chemical means to support biomaterials on conductive surfaces were addressed. These include the immobilization of redox enzymes on electrodes by means of conductive polymers [8,9], redox-relay-tethered polymers [10±12], carbon-paste [13,14] or sol±gel materials [15]. Functionalization of solid surfaces with monolayers of organic compounds and biomaterials has been a subject of extensive research for more than two decades [16±18]. Particularly, thiolated monolayers chemisorbed onto Auelectrodes [19] serve as challenging interfaces for immobilization of redox-enzyme molecules [4,20,21]. Organized mono- and multi-layers of enzymes tethered with redox-relay units and exhibiting electrical communication were assembled onto Au-electrode surfaces primarily functionalized with thiolated monolayers [22±24]. However, random distribution of the relay units at non-optimized positions resulted in inef®cient electron transfer steps and electrical contact. An increase of the transduced currents of enzyme electrodes requires the improvement of the electrical contact between the enzymes and electrodes. A novel means to establish electrical contact between the redox centers of enzymes and the electrode surfaces is based on a reconstitution approach. Reconstitution of enzymes or proteins with semiarti®cial cofactors was applied to generate semi-synthetic proteins of new functionalities [25±27]. Several approaches to reconstitute apo-enzymes on cofactorfunctionalized supports failed to yield electrically contacted enzymes [28±31]. A recent research activity led by our laboratory aims to generate electrically contacted integrated enzyme electrodes for bioelectronic applications [32,33]. Reconstitution of apo¯avoenzymes on relay-FAD-functionalized electrodes yield electrically contacted enzyme electrodes. Crosslinking of af®nity complexes formed between relayNAD monolayers and NAD-dependent enzymes were reported to yield integrated cofactor enzyme
electrodes [34]. Similarly, crosslinking of af®nity complexes between microperoxidase-11 and cytochrome c-dependent enzymes was reported to yield enzyme electrodes exhibiting internal electrical communication [35±37]. The present paper summarizes (preliminary communications [32,33]) a novel method to electrically contact the glucose oxidase via reconstitution of its apo-enzyme with electron-relay-functionalized FAD derivatives. The resulting electrically wired glucose oxidase demonstrates very ef®cient electrical communication with an electrode surface. The reconstitution process on the electron relay-FAD-monolayer-modi®ed electrode results in an integrated enzyme electrode capable to stimulate the bioelectrocatalysed oxidation of glucose with an extremely high ef®ciency. 2. Experimental 2.1. Chemicals N-(2-methylferrocene) caproic acid, (1), [38] and N6-(2-aminoethyl)±¯avin adenine dinucleotide, N6(2-aminoethyl)±FAD, (2), [39] were synthesized and puri®ed as described before. The ferrocene±FAD dyad, Fc±FAD, (3), was prepared by stirring a 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES) buffer solution (0.1 M, pH7.5, 4 ml) that included (2) (10 mg), (1) (18 mg), N-hydroxysuccinimide (13.1 mg) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, EDC, (11.6 mg) for 3 h. The resulting crude product after lyophilization was eluted on a Sephadex G-10 column (118 cm) and further puri®ed by preparative thin layer chromatography (SiO2) (elution with isopropanol:water, 4:7, Rf0.93). N-hydroxysuccinimide ester of thioctic acid, (4), was prepared by coupling thioctic acid and N-hydroxysuccinimide (NHS). To a mixture of dry dichloromethane (5 ml) that included thioctic acid, 1 g, and NHS, 0.7 g, was added a dichloromethane solution (5 ml) that included dicyclohexyl carbodiimide (DCC), 1.7 g, and 120 mg 4-pyrrolidinopyridine. The resulting mixture was stirred overnight and ®ltered. The solution was washed three times with water, three times with 5% acetic acid solution and again three times with water. The organic layer was dried with MgSO4, ®ltered and evaporated to yield 1 g of (4).
E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
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Apo-glucose oxidase, apo-GOx, was prepared by a modi®cation of the reported method [40]. The glucose oxidase, GOx, (from Aspergillus niger, E.C. 1.1.3.4) was dissolved in 0.025 M sodium phosphate buffer, pH6.0, 3 ml, that included glycerol 30% (w/v). The solution was cooled to 08C and acidi®ed to pH1.7 with a 0.025 M sodium phosphate±H2SO4, pH1.1, that included glycerol 30%. The resulting solution was loaded on a Sephadex G-25 column (1.6 cm22 cm). The protein was eluted with 0.1 M sodium phosphate solution, pH1.7, that included glycerol 30%. The eluted fractions were spectrophotometrically analyzed (280 nm) and the samples containing the protein were combined. Dextran-coated charcoal was added to the protein solution. The pH of the mixture was adjusted to pH7.0 with 1 M NaOH and the solution was stirred for 1 h at 48C. The resulting solution was centrifuged (3400 rpm for 5 min) and ®ltered through a 0.45 mm ®lter. The resulting solution was dialyzed against a 0.1 M sodium phosphate buffer solution, pH7.0. Reconstitution of the apo-GOx with (3) was accomplished by mixing (3) (0.8 mg) with the apoGOx (9.6 mg) in 3 ml of 0.1 M phosphate buffer, pH 7.0. The solution was stirred at room temperature (2528C) for 4 h and then for additional 15 h at 48C. The resulting mixture was ®ltered through a 10 000 MW cut-off ®lter and further dialysed against 0.1 sodium phosphate buffer, pH7.0. The solution after dialysis was ®ltered again through a 10 000 MW cut-off ®lter. Pyrroloquinoline quinone, PQQ, (5), and all other chemicals were used as supplied (Sigma, Aldrich). Ultrapure water from Nanopure (Branstead) source was used in all experiments.
i®cation with a base cystamine monolayer and further covalent coupling of the PQQ, (5), was performed according to the published procedure [42]. The covalent coupling of the N6-(2-aminoethyl)±FAD, (2), to the PQQ-monolayer-modi®ed electrodes was performed by soaking the electrodes in the HEPES buffer solution (0.01 M, pH7.2) containing 510ÿ4 M (2) and 110ÿ3 M EDC for 2 h at room temperature. Then the electrodes were thoroughly rinsed with water, examined with cyclic voltammetry and used for reconstitution of the apo-GOx. The reconstituted GOx monolayer electrodes were prepared by treatment of the FAD±PQQ-monolayer-modi®ed electrodes with the apo-GOx, 4 mg mlÿ1, in 0.1 M phosphate buffer, pH7.0, for 4 h at 258C and 12 h at 48C. Then the electrodes were rinsed by shaking in 0.1 M phosphate buffer, pH7.0, at 48C for 1 h to eliminate any non-speci®c adsorbates. In control experiments, the apo-GOx was reconstituted on FAD-monolayer-modi®ed Au-electrodes lacking the PQQ component. A Au-electrode was soaked in 110ÿ2 M (4) in DMSO for 2 h, and then rinsed with pure dimethyl sulfoxide, (DMSO). The resulting electrode was then treated for 1 h in 0.01 M HEPES buffer, pH 7.2, that included (2), 5 mM. The modi®ed electrode was then thoroughly rinsed with water to remove any physically adsorbed components. Reconstitution of the apo-GOx on the (2)-monolayermodi®ed Au-electrode was performed according to the technique described above for the FAD±PQQmonolayer-modi®ed electrode.
2.2. Electrode modification
Electrochemical measurements were performed using a potentiostat (EG&G VersaStat) linked to a personal computer equipped with an electrochemical software (EG&G research electrochemistry software model 270/250). All measurements were carried out in a three-compartment electrochemical cell consisting of the chemically modi®ed Au-electrode as a working electrode, a large area glassy carbon auxiliary electrode isolated by a glass frit, and a saturated calomel electrode (SCE) connected to the working volume with a Luggin capilary. All potentials are reported with respect to the SCE. Argon bubbling was used to remove oxygen from the solution in the electrochemical cell.
Gold wire electrodes (0.5 diameter, ca. 0.4 cm2 geometrical area) were used for electrochemical measurements. Prior to the modi®cation and measurements they were cleaned according to the published technique [41] and, if required, roughened by treatment with mercury followed by dissolution of the amalgam layer in nitric acid [42]. Cleanness of the electrodes and roughness coef®cients of their surface were determined by cyclic voltammetry in 0.1 M H2SO4 [43]. Typical roughness coef®cients of the electrode surfaces were ca. 1.3 and 20 before and after the roughening, respectively. The electrode mod-
2.3. Electrochemical measurements
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E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
3. Results and discussion 3.1. Glucose oxidase reconstitution with the Fc±FAD dyad The synthesis of the Fc±FAD dyad, (3), is outlined in Scheme 1. Fig. 1 (curve a) shows the cyclic voltammogram of the Fc±FAD dyad. It includes two reversible redox waves at E00.35 V for the ferrocene unit and E0ÿ0.50 V for the FAD component (at
pH7.0). Coulometric analysis of the oxidation or reduction waves indicates a 1:2 ratio for the charge associated with the electrochemical processes of the ferrocene and FAD units, respectively. This ratio is consistent with the well-characterized electrochemistry of ferrocenes [44] and FAD [45,46]. Ferrocene derivatives demonstrate a one-electron transfer process, Eq. (1), whereas the redox process of the FAD component is a typical two-electron transfer process followed by protonation, reactions (2) and (3).
Scheme 1. Synthesis of the ferrocene±FAD, (3), semisynthetic cofactor.
E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
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to previous reports [45,46] that observed stepwise single electron redox processes for FAD at pH>9.0, we observed only a single two-electron redox process, indicating that the system lacks the stabilization of the intermediate semi-quinone form (FADH ). It should be noted that the Fc±FAD dyad is strongly adsorbed from aqueous solutions onto the bare Auelectrode. The adsorption is irreversible and Fc±FAD could be removed from the electrode surface only by special cleaning of the electrode. The adsorbed Fc± FAD demonstrates small peak-to-peak separation (E35 and 25 mV for the ferrocene and FAD waves at 0.1 V sÿ1) and linear dependence of the peak currents on the potential scan rate (Ipv) that is typical for surface bound redox species [47,48]. The interfacial electron transfer rates between the electrode and the two redox units of the Fc±FAD dyad were determined using Laviron's method [47]. Fig. 2 shows the peakto-peak separation of the respective two waves as a function of the logarithm of the scan-rate. From the respective curves (taking into account that the electron transfer coef®cients of Fc and FAD are ca. 0.5) the electron transfer rate constants, ket, were estimated to be ca. 250 and 60 sÿ1 for the ferrocene and FAD units, respectively. The difference in the electron transfer rate constants of the ferrocene and FAD units could be attributed to the fact that the redox process of the
Fig. 1. Cyclic voltammograms of: (a) free ferrocene±FAD, (3), dyad adsorbed onto a bare Au-electrode from a 110ÿ5 M stock solution, (b) (3)-reconstituted glucose oxidase in solution (1.75 mg mlÿ1) using a cystamine-modified Au-electrode. Electrolyte solution: 0.1 M phosphate buffer, pH 7.3; scan rate, 1.5 V sÿ1; Ar atmosphere. Inset: Formal redox potential, E0, of the FAD component of the dyad, (3), measured at different pH.
FcÿFAD@Fc ÿFAD eÿ
(1)
FcÿFAD 2eÿ 2H @FcÿFADH2 pH < 6:7
(2)
FcÿFAD 2eÿ 1H @FcÿFADHÿ pH > 6:7
(3)
The redox potential of the FAD wave is pH-dependent (Fig. 1, inset), whereas the redox wave of the ferrocene unit is not affected by the pH of the medium. The redox potentials of the FAD component as a function of pH reveal two linear regions of different slopes: dE0/d(pH) is ca. 60 and 33 mV pHÿ1 below and above pH6.7, respectively. The change of the slope at pH6.7 corresponds to the FADH2/FADHÿ pKa-value. Below pH6.7 the two-electron reduction of FAD is followed by the addition of two protons resulting in the formation of the neutral Fc±FADH2 form. At pH>6.7 the addition of only one proton occurs, resulting in the formation of the negatively charged Fc±FADHÿ. It should be noted that in contrast
Fig. 2. Peak-to-peak separation of the redox processes of: (a) FAD and (b) ferrocene components measured by cyclic voltammetry using different potential scan rates. The measurements were performed at a Au-electrode with the adsorbed dyad immersed in 0.1 M phosphate buffer, pH 7.3.
50
E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
Scheme 2. Electrical contacting of glucose oxidase by its reconstitution with the ferrocene±FAD semisynthetic cofactor.
ferrocene involves an outersphere electron transfer, whereas the FAD component exhibits a quinone-like redox process including a two-electron transfer with an intermediary slow protonation step [49]. Glucose oxidase, GOx, is a ¯avoenzyme that includes two identical subunits, where each subunit contains a strongly non-covalently associated FADcofactor unit [50]. Reconstitution of GOx with the synthetic Fc±FAD, (3), dyad was performed as outlined in Scheme 2. The native FAD-cofactor was excluded from the enzyme, and the Fc±FAD was implanted into the derived apo-enzyme. Fig. 3 shows the absorption spectrum of the (3)-reconstituted GOx. The two bands at max370 and 445 nm are characteristic to the ferrocene and FAD units, respectively. The loading of the (3)-reconstituted GOx with the FAD component was estimated to be ca. 2, using the extinction coef®cient FAD1.41104 Mÿ1 cmÿ1 [50]. Thus, the two subunits of the enzyme are presumably reconstituted with the Fc±FAD. The activity of the (3)-reconstituted GOx was found to be ca. 40% of the native enzyme using the standard assay procedure [51]. It should be noted that the apo-GOx prior to reconstitution did not exhibit any biocatalytic activity, indicating that all of the native cofactor was excluded from the apo-enzyme. The cyclic voltammogram of the (3)-reconstituted GOx is shown in Fig. 1 (curve b). A cystamine
Fig. 3. Absorption spectrum of the (3)-reconstituted glucose oxidase: 210ÿ5 M enzyme in 0.1 M phosphate buffer, pH 7.3.
monolayer-modi®ed Au-electrode was employed to prevent non-speci®c adsorption of the enzyme on the metal surface that leads to denaturation of the biocatalyst. Furthermore, the cystamine monolayer is protonated and positively charged, whereas GOx is negatively charged (isoelectric point pI4 [50]). Thus electrostatic attraction of the modi®ed enzyme to the electrode surface facilitates electrical communication of the redox-relay modi®ed enzyme with the electrode by concentrating the biocatalyst close to the electrode
E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
51
tion of glucose. The main conclusion of these results is that reconstitution of the apo-GOx with Fc±FAD yields an ``electrically wired'' assembly. That is, oxidation of the ferrocene unit located at an exterior position of the protein mediates intraprotein electron transfer that activates the bioelectrocatalytic function of the enzyme and the oxidation of glucose. The bioelectrocatalytic anodic current developed by the (3)-reconstituted GOx depends on the glucose concentration and it is enhanced as the glucose concentration is, elevated (Fig. 5(a)). The anodic current increases up to 410ÿ2 M concentration of glucose and then levels-off to a constant value, indicating that
Fig. 4. Cyclic voltammograms of the system consisting of (3)reconstituted-GOx, 1.75 mg mlÿ1, at different concentrations of glucose: (a) 0 mM, (b) 1 mM, (c) 3 mM, (d) 20.5 mM. All experiments were performed in 0.1 M phosphate buffer, pH 7.3, at 350.58C, using a cystamine-modified Au-electrode; scan rate, 2 mV sÿ1; Ar atmosphere.
interface. It can be seen that the (3)-reconstituted GOx exhibits only a single reversible redox wave corresponding to the ferrocene unit located outside the protein. The FAD component embedded into the protein lacks direct electrical contact with the electrode surface. The fact that the FAD unit lacks electrical response supports the fact that reconstitution indeed occurred and that the FAD is electrically insulated. The redox response of the ferrocene unit suggests that it is positioned at the protein periphery and that mediated electron transfer is feasible. Fig. 4 shows the cyclic voltammograms of the (3)-reconstituted GOx upon addition of glucose at different concentrations. Note that the redox wave corresponding to the ferrocene unit is invisible in the absence of glucose (Fig. 4, curve a) due to the slow scan rate. Upon addition of glucose, an electrocatalytic anodic current is observed at the potential of the ferrocene unit. This result indicates that the ferrocene unit tethered to the FAD cofactor exhibits electron transfer communication with the electrode and mediates the oxidation of the implanted FADH2 moiety that is being reduced enzymatically by glucose. The electrochemical oxidation of the FADH2 and regeneration of the FAD cofactor state stimulate the bioelectrocatalyzed oxida-
Fig. 5. (a) Calibration curve of electrocatalytic anodic current at different glucose concentrations in the presence of (3)-reconstituted-GOx, 1.75 mg mlÿ1. The experiments were performed in 0.1 M phosphate buffer, pH 7.3, at 350.58C; Ar atmosphere. (b) Lineweaver±Burk plot of the electrocatalytic oxidation of glucose by the (3)-reconstituted-GOx.
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E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
the biocatalyst active site is saturated by the substrate. The electrocatalytic anodic current is proportional to the rate of bioelectrocatalyzed oxidation of glucose, Eq. (4). k1
ket
FcÿFADH ÿGOx ! Fc ÿFADH ÿGOx
eÿ to the electrode
(9)
k2
Fc ÿFADH ÿGOx ! Fc ÿ FAD ÿ GOx H
FcÿFADÿGOx Glucose @
(10)
kÿ1
k3
kcat
FcÿFADÿGOx Glucose ! FcÿFADH2 ÿGOx
FcÿFADÿGOx Glucose ! FcÿFADH2 ÿGOx Gluconic acid 1 1 KM 1 I Imax Imax Glucose KM
(4) (5)
kcat kÿ1 k1
(6)
where KMMichaelis±Menten constant. Fig. 5(b) shows the Lineweaver±Burk plot that corresponds to the analysis of the calibration curve shown in Fig. 5(a) according to Eq. (5). A linear relation characteristic to Michaelis±Menten behavior is observed, and the values Imax4 mA and KM2.9 mM (Eq. (6)) characterizing the reconstituted enzyme are derived. Recently, a kinetic scheme for the intramolecular electron transfer rates in randomly modi®ed ferrocene±tethered glucose oxidase was developed [52]. It was assumed that the intramolecular electron transfer from FADH2/FADH to the nearest ferrocenyl cation unit that is generated electrochemically controls the rate of bioelectrocatalyzed oxidation of glucose. The intramolecular rate constants for the randomly modi®ed ferrocene±GOx depend on the loading of the protein by the redox units. The highest observed intramolecular rate constant, kobs0.90 sÿ1, was reported for GOx loaded with 13 ferrocene units. Applying Marcus theory [53] to the measured rate constant, the distance between the nearest ferrocene Ê unit and the FAD cofactor was estimated to be 21.5 A [52]. We adopted a similar kinetic scheme for the characterization of the intramolecular electron transfer in the (3)-reconstituted GOx, Eqs. (7)±(11).
ket
FcÿFADH2 ÿGOx ! Fc ÿFADH2 ÿGOx eÿ to the electrode k1
(7)
Fc ÿFADH2 ÿGOx ! FcÿFADH ÿ GOx H
(8)
Gluconic acid
(11)
where ket is the interfacial electron transfer rate constant corresponding to oxidation of the ferrocene unit by the electrode, k1 and k2 are the rate constants of the ®rst and second steps of the FADH2 oxidation, and k3 is the rate constant of the enzymatic oxidation of glucose by GOx. Applying this kinetic scheme (Eqs. (7)±(11)) and assuming that at a saturation concentration of glucose, the maximum rate of bioelectrocatalaytic oxidation of glucose occurs, and the highest electrocatalytic current, Imax, is observed, then Imax can be expressed by reaction Eq. (12)[52], where F is the Faraday constant, A is the electrode surface area (cm2), DGOx 410ÿ7 cm2 sÿ1 corresponds to the diffusion coef®cient of GOx, and [Fc±FAD±GOx] is the concentration of the (3)-reconstituted-GOx (molecm3). The rate constants kobs and k3 are given by Eqs. (13) and (14), respectively. Imax 2FA DGOx kobs 1=2 FcÿFADÿGOx
(12)
k1 k2 kobs ÿp p2 k1 k2
(13)
k3
kcat
1 KM Glucoseÿ1
(14)
Substitution of the experimentally observed Imax value (4 mA) obtained by the Michaelis±Menten analysis for the (3)-reconstituted-GOx in Eq. (12) yields the kobs40 sÿ1 for the electrical communication rate control in the reconstituted enzyme. It should be noted that the rate constant of the electron transfer between the electrochemically generated ferrocenyl cation and the enzymatically reduced FADH2 in the (3)-reconstituted-GOx is ca. 50-fold higher than the value observed in the randomly-ferrocene-modi®ed GOx exhibiting the highest electrical communication features. The enhancement of electron transfer rates
E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
between the ferrocenyl cation and reduced FAD cofactor is of substantial importance in the design of amperometric glucose biosensors. The turnover rate of the FADH2 cofactor in GOx by the natural electron acceptor, molecular oxygen, (in an aqueous phase equilibrated with air) corresponds to kobs5103 sÿ1 k [52]. This limits the application of GOx-electrodes as amperometric biosensors to deaerated oxygen-free media. The design of fast electrically communicating relay-modi®ed GOx could establish a means to apply the GOx-electrodes under oxygen. Furthermore, knowing the intraprotein electron transfer rate constants in the (3)-reconstituted-GOx and in the randomly-ferrocene-modi®ed GOx (exhibiting highest electrical communication), one could apply Marcus equation, Eq. (15), to estimate the difference in edge-to-edge separation of the redox sites, d, in the two kinds of the proteins, Eq. (16), where k(3)ÿGOx and kFc(n)ÿGOx are the intraprotein electron transfer rate constants in the (3)-reconstituted-GOx and in randomly-ferrocene-modi®ed GOx, respectively: ket / expÿ d ÿ d0 exp ÿG0 2 =4RT ln
k 3-GOx kFc nÿGOx
(15)
ÿ d
(16)
where is the electron transfer reorganization energy Ê ÿ1 is the electron tunneling coeffiand 0.5±1.4 A cient [53]. Using the recently published approach [52] and assuming that the rate constants for oxidation of FADH2 and FADH (k1 and k2 in Eqs. (8) and (10), respectively) are not different, and applying the observed rate constants (kobs in Eq. (13)) to approximate k(3)-GOx and kFc(n)±GOx, we estimated the distance separating the ferrocene unit and FAD cofactor Ê . Thus, the in the (3)-reconstituted-GOx to be ca. 19 A distance separating the redox relay and the redox Ê shorter center in the reconstituted enzyme is ca. 2 A than in the most effective electrically contacted enzyme generated by the random modi®cation of GOx with ferrocene units. Furthermore, the results suggest that by the application of Fc±FAD dyads where the bridging chain separating the ferrocene unit and FAD cofactor is shortened by 3 or 4 methylene groups, and the reconstitution of apo-GOx could yield
53
enzymes of improved intraprotein electrical contact, that eventually could compete with the oxidation of the FADH2 site by oxygen. 3.2. Glucose oxidase reconstitution with the PQQ±FAD monolayer The reconstitution methodology yields a site-speci®c modi®ed bioelectrocatalyst, ``electroenzyme''. To apply such a biocatalyst as active biological material in a sensing device, its integration with an electrode surface is required. Proper alignment of the bioelectrocatalyst on the electrode surface is a crucial element [54]. The electron-relay unit must be positioned at the electrode surface in an optimized con®guration to allow effective electron transport between the biocatalyst active-site and the electrode. With these limiting constraints, we decided to assemble the relay-FAD dyad on the electrode surface and to reconstitute the apo-enzyme on the surface itself. Scheme 3(A) shows the method to assemble the reconstituted GOx on a Au-electrode surface. A primary cystamine monolayer was linked to a roughened Au-electrode and pyrroloquinoline quinone, PQQ, (5), was covalently associated to the base monolayer [42]. N6-aminoethyl±FAD, (2), was coupled to the carboxylic functions of PQQ to yield the relay-FAD dyad on the monolayer. The functionalized monolayer electrode was then reconstituted with apo-GOx to form an active biocatalyst monolayer on the electrode surface. Fig. 6 shows the cyclic voltammogram of the PQQ± FAD dyad monolayer prior to the reconstitution with the apo-GOx (curve a) and after reconstitution with the apo-GOx (curve b). Prior to the reconstitution process, the functionalized monolayer shows two reversible redox waves at E0ÿ0.125 V and E0 ÿ0.50 V (at pH7.0) corresponding to the two-electron redox process of PQQ and FAD, respectively. By integration of the charge associated with the PQQ and FAD redox units, the surface densities of these components were estimated to be ca. 110ÿ11 mol cmÿ2. After reconstitution of the apo-GOx with the functionalized monolayer, the redox wave of the PQQ was almost unaltered, but the redox-wave correspsonding to the FAD unit was substantially decreased in its intensity. This is consistent with the fact that reconstitution of the apo-GOx with the monolayer insulates the FAD-sites towards electrical communication with
54 E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58 Scheme 3. (A) Reconstitution of glucose oxidase onto a PQQ±FAD monolayer Au-electrode and direct oxidation of glucose by the modified electrode. (B) Reconstitution of glucose oxidase onto a FAD-modified Au-electrode and bioelectrocatalyzed oxidation of glucose by the enzyme-electrode in the presence of a diffusional electron mediator (ferrocene carboxylic acid).
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55
Fig. 6. Cyclic voltammograms of (a) the PQQ±FAD monolayer on a Au-electrode before and (b) after reconstitution with apo-glucose oxidase. Ar atmosphere, 0.1 M phosphate buffer, pH 7.0, 250.58C; potential scan rate, 50 mV sÿ1; electrode geometrical area, 0.4 cm2, roughness factor, ca. 20.
the electrode surface. The PQQ redox unit is suf®ciently exposed to the protein periphery and hence reveals non-perturbed electrical contact with the electrode. The residual redox-wave of the FAD units is attributed to the free dyad that was not reconstituted into the apo-GOx. By integration of the depleted charge of FAD units, and assuming that two FADcofactor components participate in the anchoring of the apo-GOx onto the surface in the reconstitution process, the surface density of the protein on the electrode surface was estimated to be ca. 1.710ÿ12 mol cmÿ2. Using the footprint dimension of GOx (58 nm2) [55], the surface coverage of the electrode by the enzyme corresponds to a densely packed enzyme monolayer assembly. Fig. 7(A) shows the cyclic voltammograms of the reconstituted GOx monolayer electrode in the absence of glucose (curve a) and in the presence of glucose (curve b). With glucose, a high-magnitude electrocatalytic anodic current is observed, indicating that the reconstituted GOx on the monolayer yields a bioactive monolayer interface exhibiting direct electrical contact with the electrode. The electrically-contacted enzyme layer stimulates the bioelectrocatalyzed oxidation of glucose. A control system that includes the reconstituted GOx on a single-component FAD monolayer that lacks the PQQ redox-mediating unit was also
Fig. 7. (A) Cyclic voltammograms of: (a) The glucose oxidase reconstituted on the PQQ±FAD monolayer enzyme-electrode. (b) After adding glucose, 80 mM. Ar atmosphere, 0.1 phosphate buffer, pH 7.0; 350.58C; scan rate, 5 mV sÿ1; electrode geometrical area, 0.4 cm2; roughness factor, ca. 20. (B) Amperometric responses of the PQQ±FAD±reconstituted-GOx monolayer electrode at different glucose concentrations. Current determined by chronoamperometry at final potential 0.2 V.
assembled on a Au-electrode (Scheme 3(B)). In this system, no direct electrical communication between the embedded FAD-cofactor and the electrode exists, but the reconstituted GOx exhibits biocatalytic activity, and bioelectrocatalyzed oxidation of glucose proceeds in the presence of a diffusional electron mediator, ferrocene carboxylic acid. These results clearly demonstrate that the reconstitution of the apo-GOx on the FAD-monolayers represents a novel means to link and align ¯avoenzymes onto surfaces. Reconstitution of the apo-GOx onto the PQQ±FAD
56
E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
dyad monolayer yields an electroactive biocatalyst interface, where direct electrical contact between the enzyme-active site and the electrode is achieved. The intermediate PQQ component that acts as a redoxrelay unit, transports the electrons from the active site of the enzyme to the electrode. PQQ, (5), was selected for the role of the intermediate relay because it represents a two-electron transfer mediator that could simplify the mediation process and improve its kinetics. The following reaction scheme (Eqs. (17)± (19)) could be applied to the PQQ-mediated bioelectrocatalyzed glucose oxidation. ElectrodeÿPQQÿFADH2 ÿGOx
kFADÿPQQ
!
ElectrodeÿPQQH2 ÿFADÿGOx
(17) kPQQ
ElectrodeÿPQQH2 ÿFADÿGOx !
ElectrodeÿPQQÿFADÿGOx 2H 2eÿ to the electrode
(18) kcat
ElectrodeÿPQQÿFADÿGOx Glucose ! ElectrodeÿPQQÿFADH2 ÿGOx Gluconic acid (19) In this scheme kFAD±PQQ, kPQQ and kcat represent the rate constants for the electron transfer from the reduced FAD to PQQ, from the reduced PQQ to the electrode and the biocatalyzed reduction of FAD by glucose, respectively. The assumed sequence of reactions does not include one-electron transfer steps for the oxidation of FADH2 (Cf. for comparison FADH2/ FADH oxidation via one-electron transfer steps using ferrocene relay as the mediator, Eqs. (8) and (10)). Fig. 7(B) shows the transduced currents by the PQQ±FAD±reconstituted GOx monolayer electrode at different glucose concentrations. The current responses are almost linear in a concentration range of 1±80 mM of glucose. The upper limit of the turnover-rate of glucose oxidase at 258C is 600100 sÿ1 [56] and the activation energy is 7.2 kcal molÿ1 [57]. At the temperature employed in our measurements (358C), this translates to a limiting turnover rate of 900150 sÿ1. Realizing that the reconstituted GOx surface coverage is 1.710ÿ12 mol cmÿ2, the maximum current density that can be observed for the theoretical turnover of the enzyme is 29060 mA cmÿ2.
Fig. 7(B) shows that at a glucose concentration of 80 mM, the observed current is ca. 1.9 mA and the current density is ca. 300100 mA cmÿ2, taking into account the electrode geometrical area, 0.4 cm2, and its roughness factor, ca. 20. Thus, the experimental current density is within the range of the limiting turnover rate of the native enzyme. This suggests that at the potential employed in the chronoamperometric experiments to analyze glucose (E0.2 V) all FAD sites are electrically connected with the electrode and exist in the oxidized form. The resulting current is then controlled by the diffusion of glucose to the active site. The reconstituted apo-GOx on the PQQ±FADmonolayer electrode exhibits unique electrical communication features, and the theoretical turnover rate of the active center with molecular oxygen is achieved for the electron exchange between the active site and the electrode. Also, the bioelectrocatalyzed oxidation of glucose proceeds at relatively low positive potentials, E>ÿ0.1 V, characteristic to the PQQ electron mediator component. These two features have important implications for the use of the monolayer-modi®ed electrode as a glucose-sensing interface. The high electrical turnover rate of the reconstituted biocatalyst suggests that the enzyme-electrode would not be in¯uenced by oxygen. The presently available amperometric glucose electrodes based on glucose oxidase are sensitive to the oxygen content in the environment, and most analyses are performed in an oxygen-free medium. Fig. 8 shows the amperometric responses of the reconstituted enzyme electrode in the presence of glucose in an oxygen-free environment (curve b) and in the presence of air (curve c). The transduced amperometric signal is only slightly (less than 2%) decreased by oxygen. Similarly, the effective electrical contact between the biocatalyst and the electrode upon application of a low potential suggests that the non-speci®c oxidation of interfering components will be screened by the highly ef®cient bioelectrocatalyzed oxidation of glucose. Ascorbic acid is a common interferant in glucose analysis. Fig. 8 shows the currents transduced by the reconstituted enzyme electrode in the presence of glucose without added ascorbic acid (curve b) and in the presence of ascorbic acid taken at a physiological concentration level (curve d). The transduced current in the presence of ascorbic acid is only slightly increased (less than 5%) implying that the observed current originates essen-
E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
Fig. 8. Amperometric responses produced by the GOx reconstituted with the PQQ±FAD monolayer: (a) in the absence of glucose, (b) in the presence of 50 mM glucose and absence of oxygen, (c) in the presence of 50 mM glucose in a solution saturated with air, and (d) in the presence of 50 mM glucose and 0.1 mM ascorbic acid in the absence of oxygen. Currents determined by chronoamperometry at final potential 0.0 V. Electrolyte composed of 0.1 phosphate buffer, pH 7.0, 350.58C; electrode geometrical area, 0.4 cm2; roughness factor, ca. 20.
tially from the biocatalyzed oxidation of glucose. Thus, the ef®cient electrical contact of the reconstituted enzyme electrode, and the resulting high current densities, as well as the speci®city of the bioelectrocatalytic electrode, enable the future application of the enzyme electrode as a miniaturized invasive glucose sensor. Acknowledgements The research was supported by the BMBF, Germany, and by the Israel Ministry of Science, and in part by the Szold Foundation, The Hebrew University of Jerusalem. References [1] P.N. Bartlett, in: R.P. Buck, W.E. Hatfield, M. Umana, E.F. Bowden (Eds.), Biosensor Technology. Fundamentals and Applications, Chapter 7, Marcel Dekker, New York, 1990, pp. 95±115. [2] H.A.O. Hill, G.S. Sanghera, in: A.E.G. Cass (Ed.), Biosensors. A Practical Approach, Chapter 2, Oxford University Press, Oxford, 1990, pp. 19±46.
57
[3] W. GoÈpel, P. Heiduschka, Biosens. Bioelectron. 9 (1994) 3. [4] I. Willner, E. Katz, B. Willner, Electroanalysis 9 (1997) 965. [5] E. Katz, V. Heleg-Shabtai, B. Willner, I. Willner, A.F. BuÈckmann, Bioelectrochem. Bioeng. 42 (1997) 95. [6] A. Heller, Acc. Chem. Res. 23 (1990) 128. [7] P.N. Bartlett, P. Tebbutt, R.G. Whitaker, Prog. React. Kinet. 16 (1991) 55. [8] S.A. Emr, A.M. Yacynych, Electroanalysis 7 (1995) 913. [9] S.B. Adeloju, G.G. Wallace, Analyst 121 (1996) 699. [10] B.A. Gregg, A. Heller, J. Phys. Chem. 95 (1991) 5970. [11] I. Willner, E. Katz, N. Lapidot, P. BaÈuerle, Bioelectrochem. Bioeng. 29 (1992) 29. [12] E.J. Calvo, R. Etchenigue, C. Danilowicz, L. Diaz, Anal. Chem. 68 (1996) 4186. [13] S.A. Wring, J.P. Hart, Analyst 117 (1992) 1215. [14] L. Gorton, H.I. Karan, P.D. Hall, T. Inagaki, Y. Okamoto, T.A. Skotheim, Anal. Chim. Acta 228 (1990) 23. [15] V. Glezer, O. Lev, J. Am. Chem. Soc. 115 (1993) 2533. [16] R.W. Murray, Acc. Chem. Res. 13 (1980) 135. [17] R.W. Murray, in: A.J. Bard (Ed.), Electroanalytical Chemistry, vol. 13, Marcel Dekker, New York, 1984, pp. 191±368. [18] M.S. Wrighton, Science 231 (1986) 32. [19] H.A. Finklea, in: A.J. Bard, I. Rubinstein (Eds.), Electroanalytical Chemistry, vol. 19, Marcel Dekker, New York, 1996, pp. 109±335. [20] D. Mandler, I. Turyan, Electroanalysis 8 (1996) 207. [21] I. Willner, E. Katz, B. Willner, R. Blonder, V. Heleg-Shabtai, A.F. BuÈckmann, Biosens. Bioelectron. 12 (1997) 337. [22] I. Willner, A. Riklin, B. Shoham, D. Rivenzon, E. Katz, Adv. Mater. 5 (1993) 912. [23] I. Willner, N. Lapidot, A. Riklin, R. Kasher, E. Zahavy, E. Katz, J. Am. Chem. Soc. 116 (1994) 1428. [24] E. Katz, A. Riklin, I. Willner, J. Electroanal. Chem. 354 (1993) 129. [25] I. Hamachi, T. Matsugi, S. Tanaka, S. Shinkai, Bull. Chem. Soc. Jpn. 69 (1996) 1657. [26] I. Hamachi, Y. Tajiri, T. Nagase, S. Shinkai, Chem. Eur. J. 3 (1997) 1025. [27] I. Hamachi, S. Tanaka, S. Tsukiji, S. Shinkai, M. Shimizu, T. Nagamune, Chem. Commun. (1997) 1735. [28] O. Miyawaki, L.B. Wingard Jr., , Biochim. Biophys. Acta 838 (1985) 60. [29] E. Katz, A.Y. Shkuropatov, O.I. Vagabova, V.A. Shuvalov, Biochim. Biophys. Acta 976 (1989) 121. [30] E. Katz, D.D. Schlereth, H.-L. Schmidt, A.J.J. Olsthoorn, J. Electroanal. Chem. 368 (1994) 165. [31] L.-H. Guo, G. McLendon, H. Razafitrimo, Y. Gao, J. Mater. Chem. 6 (1996) 369. [32] A. Riklin, E. Katz, I. Willner, A. Stocker, A.F. BuÈckmann, Nature 376 (1995) 672. [33] I. Willner, V. Heleg-Shabtai, R. Blonder, E. Katz, G. Tao, A.F. BuÈckmann, A. Heller, J. Am. Chem. Soc. 118 (1996) 10321. [34] A. Bardea, E. Katz, A.F. BuÈckmann, I. Willner, J. Am. Chem. Soc. 119 (1997) 9114. [35] V. Heleg-Shabtai, E. Katz, I. Willner, J. Am. Chem. Soc. 119 (1997) 8121.
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[36] V. Heleg-Shabtai, E. Katz, S. Levi, I. Willner, J. Chem. Soc., Perkin Trans. 2 (1997) 2645. [37] F. Patolsky, E. Katz, V. Heleg-Shabtai, I. Willner, Chem. Eur. J. 4 (1998) 1068. [38] I. Willner, A. Doron, E. Katz, S. Levi, A.J. Frank, Langmuir 12 (1996) 946. [39] A.F. BuÈckmann, V. Wray, A. Stocker, in: D.B. McCormick (Ed.), Methods in Enzymology: Vitamins and Coenzymes, vol. 280, Part 1, Academic Press, Orlando, 1997, p. 360. [40] D.L. Morris, R.T. Buckler, in: J.J. Langone, H. Van Vunakis (Eds.), Methods in Enzymology, vol. 92, Part E, Academic Press, Orlando, 1983, pp. 413±417. [41] E. Katz, A.A. Solov'ev, J. Electroanal. Chem. 291 (1990) 171. [42] E. Katz, D.D. Schlereth, H.-L. Schmidt, J. Electroanal. Chem. 367 (1994) 59. [43] R. Woods, in: A.J. Bard (Ed.), Electroanalytical Chemistry, vol. 9, Marcel Dekker, New York, 1978, pp. 1±162. [44] A.E.G. Cass, G. Davis, M.J. Green, H.A.O. Hill, J. Electroanal. Chem. 190 (1985) 117.
[45] Y. Wang, G. Zhu, E. Wang, Anal. Chim. Acta 338 (1997) 97. [46] L. Gorton, G. Johansson, J. Electroanal. Chem. 113 (1980) 151. [47] E. Laviron, J. Electroanal. Chem. 101 (1979) 19. [48] E. Laviron, L. Roullier, J. Electroanal. Chem. 115 (1980) 65. [49] C. Degrand, Ann. Chim. 75 (1985) 1. [50] R. Wilson, A.P.F. Turner, Biosens. Bioelectron. 7 (1992) 165. [51] M. Dixon, E.C. Webb, Enzymes, Longmans, London, 1964. [52] A. Badia, R. Carlini, A. Fernandez, F. Battaglini, S.R. Mikkelsen, A.M. English, J. Am. Chem. Soc. 115 (1993) 7053. [53] R.A. Marcus, N. Sutin, Biochim. Biophys. Acta 811 (1985) 265. [54] E. Katz, J. Electroanal. Chem. 365 (1994) 157. [55] H.J. Hecht, H.M. Kalisz, J. Hendle, R.D. Schmid, D. Schomburg, J. Mol. Biol. 229 (1993) 153. [56] C. Bourdillon, C. Demaile, J. Gueris, J. Moiroux, J.M. SaveÂant, J. Am. Chem. Soc. 115 (1993) 12264. [57] H.G. Eisenwiener, G.V. Schultz, Naturwissenschaften 56 (1969) 563.
Glucose oxidase electrodes via reconstitution of the apo-enzyme: tailoring of novel glucose biosensors Eugenii Katza,*, Azalia Riklina, Vered Heleg-Shabtaia, Itamar Willnera, A.F. BuÈckmannb a
Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Gesellschaft fuÈr Biotechnologische Forschung, Department of Molecular Structure Mascheroder Weg 1, D-38124 Braunschweig, Germany
b
Received 27 May 1998; received in revised form 17 September 1998; accepted 19 September 1998
Abstract Reconstitution of an apo-¯avoenzyme with a relay-FAD-cofactor dyad yields an electrically contacted enzyme, ``electroenzyme''. This is exempli®ed by the reconstitution of apo-glucose oxidase, apo-GOx, with a ferrocene±tethered FAD-cofactor. The resulting semisynthetic protein stimulates the bioelectrocatalyzed oxidation of glucose. Kinetic analysis of the ferrocene±FAD reconstituted enzyme reveals that its electrical communication with an electrode support is superior as compared to a protein randomly functionalized by ferrocene relay units. A pyrroloquinolinoquinone (PQQ)±FAD cofactor monolayer is assembled on a Au-electrode. Apo-GOx is reconstituted on the solid support to yield an aligned enzyme electrode of unprecendently ef®cient electrical contact. The electron-transfer turnover rate with the electrode is estimated to be 900150 sÿ1, a value close to the electron transfer rate between native GOx and molecular oxygen. The effective electrical communication of the integrated enzyme electrode stimulates the ef®cient bioelectrocatalyzed oxidation of glucose, and results in high current densities and high sensitivity for glucose. The effective electrical contact of the enzyme-electrode yields also a speci®c glucose sensing electrode that is not perturbed by oxygen or interferrants. The possible application of the enzyme-electrode as an invasive biosensor is addressed. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Glucose oxidase; Flavin adenine dinucleotide; FAD; Biosensor; Reconstituted enzymes; Enzyme monolayers; Electrical contact of enzymes; Integrated enzyme electrodes; Glucose sensor
1. Introduction The application of redox-enzymes as bioactive matrices for tailoring biosensor [1,2] and bioelectronic [3] devices is of substantial basic and practical importance. Electrical communication between redoxenzymes and electrode surfaces represents the basic process to activate redox-active biocatalysts [4,5]. *Corresponding author. Tel.: +972-2-6585272; fax: +972-26527715; e-mail: [email protected]
Redox sites in proteins, however, lack direct electrical contact with electrodes due to the insulation of the redox-center by the protein [6]. The intrinsic barrier for direct electron transfer between the enzyme redox center and the electrode resulted in the development of various methods to establish electrical communication between redox-enzymes and electrode supports using electron transfer mediators [7]. Diffusional electron transfer mediators, such as hexacyanoferrate(III), ferrocene derivatives, N,N0 -bipyridinium salts, quinones, transition metal complexes, etc. were employed to
0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0003-2670(98)00688-6
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E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
electrically contact biocatalysts and the electrode surfaces in the ®rst generation of biosensors. The architecture of integrated biocatalytic assemblies lacking diffusional components has important advantages in bioelectronic and biosensor technology [4]. Tailoring of bioelectronic systems requires the assembly of biomaterials on solid conductive supports and the design of the appropriate electrical contact between the biological matrices and the support elements. Various chemical means to support biomaterials on conductive surfaces were addressed. These include the immobilization of redox enzymes on electrodes by means of conductive polymers [8,9], redox-relay-tethered polymers [10±12], carbon-paste [13,14] or sol±gel materials [15]. Functionalization of solid surfaces with monolayers of organic compounds and biomaterials has been a subject of extensive research for more than two decades [16±18]. Particularly, thiolated monolayers chemisorbed onto Auelectrodes [19] serve as challenging interfaces for immobilization of redox-enzyme molecules [4,20,21]. Organized mono- and multi-layers of enzymes tethered with redox-relay units and exhibiting electrical communication were assembled onto Au-electrode surfaces primarily functionalized with thiolated monolayers [22±24]. However, random distribution of the relay units at non-optimized positions resulted in inef®cient electron transfer steps and electrical contact. An increase of the transduced currents of enzyme electrodes requires the improvement of the electrical contact between the enzymes and electrodes. A novel means to establish electrical contact between the redox centers of enzymes and the electrode surfaces is based on a reconstitution approach. Reconstitution of enzymes or proteins with semiarti®cial cofactors was applied to generate semi-synthetic proteins of new functionalities [25±27]. Several approaches to reconstitute apo-enzymes on cofactorfunctionalized supports failed to yield electrically contacted enzymes [28±31]. A recent research activity led by our laboratory aims to generate electrically contacted integrated enzyme electrodes for bioelectronic applications [32,33]. Reconstitution of apo¯avoenzymes on relay-FAD-functionalized electrodes yield electrically contacted enzyme electrodes. Crosslinking of af®nity complexes formed between relayNAD monolayers and NAD-dependent enzymes were reported to yield integrated cofactor enzyme
electrodes [34]. Similarly, crosslinking of af®nity complexes between microperoxidase-11 and cytochrome c-dependent enzymes was reported to yield enzyme electrodes exhibiting internal electrical communication [35±37]. The present paper summarizes (preliminary communications [32,33]) a novel method to electrically contact the glucose oxidase via reconstitution of its apo-enzyme with electron-relay-functionalized FAD derivatives. The resulting electrically wired glucose oxidase demonstrates very ef®cient electrical communication with an electrode surface. The reconstitution process on the electron relay-FAD-monolayer-modi®ed electrode results in an integrated enzyme electrode capable to stimulate the bioelectrocatalysed oxidation of glucose with an extremely high ef®ciency. 2. Experimental 2.1. Chemicals N-(2-methylferrocene) caproic acid, (1), [38] and N6-(2-aminoethyl)±¯avin adenine dinucleotide, N6(2-aminoethyl)±FAD, (2), [39] were synthesized and puri®ed as described before. The ferrocene±FAD dyad, Fc±FAD, (3), was prepared by stirring a 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES) buffer solution (0.1 M, pH7.5, 4 ml) that included (2) (10 mg), (1) (18 mg), N-hydroxysuccinimide (13.1 mg) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, EDC, (11.6 mg) for 3 h. The resulting crude product after lyophilization was eluted on a Sephadex G-10 column (118 cm) and further puri®ed by preparative thin layer chromatography (SiO2) (elution with isopropanol:water, 4:7, Rf0.93). N-hydroxysuccinimide ester of thioctic acid, (4), was prepared by coupling thioctic acid and N-hydroxysuccinimide (NHS). To a mixture of dry dichloromethane (5 ml) that included thioctic acid, 1 g, and NHS, 0.7 g, was added a dichloromethane solution (5 ml) that included dicyclohexyl carbodiimide (DCC), 1.7 g, and 120 mg 4-pyrrolidinopyridine. The resulting mixture was stirred overnight and ®ltered. The solution was washed three times with water, three times with 5% acetic acid solution and again three times with water. The organic layer was dried with MgSO4, ®ltered and evaporated to yield 1 g of (4).
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47
Apo-glucose oxidase, apo-GOx, was prepared by a modi®cation of the reported method [40]. The glucose oxidase, GOx, (from Aspergillus niger, E.C. 1.1.3.4) was dissolved in 0.025 M sodium phosphate buffer, pH6.0, 3 ml, that included glycerol 30% (w/v). The solution was cooled to 08C and acidi®ed to pH1.7 with a 0.025 M sodium phosphate±H2SO4, pH1.1, that included glycerol 30%. The resulting solution was loaded on a Sephadex G-25 column (1.6 cm22 cm). The protein was eluted with 0.1 M sodium phosphate solution, pH1.7, that included glycerol 30%. The eluted fractions were spectrophotometrically analyzed (280 nm) and the samples containing the protein were combined. Dextran-coated charcoal was added to the protein solution. The pH of the mixture was adjusted to pH7.0 with 1 M NaOH and the solution was stirred for 1 h at 48C. The resulting solution was centrifuged (3400 rpm for 5 min) and ®ltered through a 0.45 mm ®lter. The resulting solution was dialyzed against a 0.1 M sodium phosphate buffer solution, pH7.0. Reconstitution of the apo-GOx with (3) was accomplished by mixing (3) (0.8 mg) with the apoGOx (9.6 mg) in 3 ml of 0.1 M phosphate buffer, pH 7.0. The solution was stirred at room temperature (2528C) for 4 h and then for additional 15 h at 48C. The resulting mixture was ®ltered through a 10 000 MW cut-off ®lter and further dialysed against 0.1 sodium phosphate buffer, pH7.0. The solution after dialysis was ®ltered again through a 10 000 MW cut-off ®lter. Pyrroloquinoline quinone, PQQ, (5), and all other chemicals were used as supplied (Sigma, Aldrich). Ultrapure water from Nanopure (Branstead) source was used in all experiments.
i®cation with a base cystamine monolayer and further covalent coupling of the PQQ, (5), was performed according to the published procedure [42]. The covalent coupling of the N6-(2-aminoethyl)±FAD, (2), to the PQQ-monolayer-modi®ed electrodes was performed by soaking the electrodes in the HEPES buffer solution (0.01 M, pH7.2) containing 510ÿ4 M (2) and 110ÿ3 M EDC for 2 h at room temperature. Then the electrodes were thoroughly rinsed with water, examined with cyclic voltammetry and used for reconstitution of the apo-GOx. The reconstituted GOx monolayer electrodes were prepared by treatment of the FAD±PQQ-monolayer-modi®ed electrodes with the apo-GOx, 4 mg mlÿ1, in 0.1 M phosphate buffer, pH7.0, for 4 h at 258C and 12 h at 48C. Then the electrodes were rinsed by shaking in 0.1 M phosphate buffer, pH7.0, at 48C for 1 h to eliminate any non-speci®c adsorbates. In control experiments, the apo-GOx was reconstituted on FAD-monolayer-modi®ed Au-electrodes lacking the PQQ component. A Au-electrode was soaked in 110ÿ2 M (4) in DMSO for 2 h, and then rinsed with pure dimethyl sulfoxide, (DMSO). The resulting electrode was then treated for 1 h in 0.01 M HEPES buffer, pH 7.2, that included (2), 5 mM. The modi®ed electrode was then thoroughly rinsed with water to remove any physically adsorbed components. Reconstitution of the apo-GOx on the (2)-monolayermodi®ed Au-electrode was performed according to the technique described above for the FAD±PQQmonolayer-modi®ed electrode.
2.2. Electrode modification
Electrochemical measurements were performed using a potentiostat (EG&G VersaStat) linked to a personal computer equipped with an electrochemical software (EG&G research electrochemistry software model 270/250). All measurements were carried out in a three-compartment electrochemical cell consisting of the chemically modi®ed Au-electrode as a working electrode, a large area glassy carbon auxiliary electrode isolated by a glass frit, and a saturated calomel electrode (SCE) connected to the working volume with a Luggin capilary. All potentials are reported with respect to the SCE. Argon bubbling was used to remove oxygen from the solution in the electrochemical cell.
Gold wire electrodes (0.5 diameter, ca. 0.4 cm2 geometrical area) were used for electrochemical measurements. Prior to the modi®cation and measurements they were cleaned according to the published technique [41] and, if required, roughened by treatment with mercury followed by dissolution of the amalgam layer in nitric acid [42]. Cleanness of the electrodes and roughness coef®cients of their surface were determined by cyclic voltammetry in 0.1 M H2SO4 [43]. Typical roughness coef®cients of the electrode surfaces were ca. 1.3 and 20 before and after the roughening, respectively. The electrode mod-
2.3. Electrochemical measurements
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3. Results and discussion 3.1. Glucose oxidase reconstitution with the Fc±FAD dyad The synthesis of the Fc±FAD dyad, (3), is outlined in Scheme 1. Fig. 1 (curve a) shows the cyclic voltammogram of the Fc±FAD dyad. It includes two reversible redox waves at E00.35 V for the ferrocene unit and E0ÿ0.50 V for the FAD component (at
pH7.0). Coulometric analysis of the oxidation or reduction waves indicates a 1:2 ratio for the charge associated with the electrochemical processes of the ferrocene and FAD units, respectively. This ratio is consistent with the well-characterized electrochemistry of ferrocenes [44] and FAD [45,46]. Ferrocene derivatives demonstrate a one-electron transfer process, Eq. (1), whereas the redox process of the FAD component is a typical two-electron transfer process followed by protonation, reactions (2) and (3).
Scheme 1. Synthesis of the ferrocene±FAD, (3), semisynthetic cofactor.
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49
to previous reports [45,46] that observed stepwise single electron redox processes for FAD at pH>9.0, we observed only a single two-electron redox process, indicating that the system lacks the stabilization of the intermediate semi-quinone form (FADH ). It should be noted that the Fc±FAD dyad is strongly adsorbed from aqueous solutions onto the bare Auelectrode. The adsorption is irreversible and Fc±FAD could be removed from the electrode surface only by special cleaning of the electrode. The adsorbed Fc± FAD demonstrates small peak-to-peak separation (E35 and 25 mV for the ferrocene and FAD waves at 0.1 V sÿ1) and linear dependence of the peak currents on the potential scan rate (Ipv) that is typical for surface bound redox species [47,48]. The interfacial electron transfer rates between the electrode and the two redox units of the Fc±FAD dyad were determined using Laviron's method [47]. Fig. 2 shows the peakto-peak separation of the respective two waves as a function of the logarithm of the scan-rate. From the respective curves (taking into account that the electron transfer coef®cients of Fc and FAD are ca. 0.5) the electron transfer rate constants, ket, were estimated to be ca. 250 and 60 sÿ1 for the ferrocene and FAD units, respectively. The difference in the electron transfer rate constants of the ferrocene and FAD units could be attributed to the fact that the redox process of the
Fig. 1. Cyclic voltammograms of: (a) free ferrocene±FAD, (3), dyad adsorbed onto a bare Au-electrode from a 110ÿ5 M stock solution, (b) (3)-reconstituted glucose oxidase in solution (1.75 mg mlÿ1) using a cystamine-modified Au-electrode. Electrolyte solution: 0.1 M phosphate buffer, pH 7.3; scan rate, 1.5 V sÿ1; Ar atmosphere. Inset: Formal redox potential, E0, of the FAD component of the dyad, (3), measured at different pH.
FcÿFAD@Fc ÿFAD eÿ
(1)
FcÿFAD 2eÿ 2H @FcÿFADH2 pH < 6:7
(2)
FcÿFAD 2eÿ 1H @FcÿFADHÿ pH > 6:7
(3)
The redox potential of the FAD wave is pH-dependent (Fig. 1, inset), whereas the redox wave of the ferrocene unit is not affected by the pH of the medium. The redox potentials of the FAD component as a function of pH reveal two linear regions of different slopes: dE0/d(pH) is ca. 60 and 33 mV pHÿ1 below and above pH6.7, respectively. The change of the slope at pH6.7 corresponds to the FADH2/FADHÿ pKa-value. Below pH6.7 the two-electron reduction of FAD is followed by the addition of two protons resulting in the formation of the neutral Fc±FADH2 form. At pH>6.7 the addition of only one proton occurs, resulting in the formation of the negatively charged Fc±FADHÿ. It should be noted that in contrast
Fig. 2. Peak-to-peak separation of the redox processes of: (a) FAD and (b) ferrocene components measured by cyclic voltammetry using different potential scan rates. The measurements were performed at a Au-electrode with the adsorbed dyad immersed in 0.1 M phosphate buffer, pH 7.3.
50
E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
Scheme 2. Electrical contacting of glucose oxidase by its reconstitution with the ferrocene±FAD semisynthetic cofactor.
ferrocene involves an outersphere electron transfer, whereas the FAD component exhibits a quinone-like redox process including a two-electron transfer with an intermediary slow protonation step [49]. Glucose oxidase, GOx, is a ¯avoenzyme that includes two identical subunits, where each subunit contains a strongly non-covalently associated FADcofactor unit [50]. Reconstitution of GOx with the synthetic Fc±FAD, (3), dyad was performed as outlined in Scheme 2. The native FAD-cofactor was excluded from the enzyme, and the Fc±FAD was implanted into the derived apo-enzyme. Fig. 3 shows the absorption spectrum of the (3)-reconstituted GOx. The two bands at max370 and 445 nm are characteristic to the ferrocene and FAD units, respectively. The loading of the (3)-reconstituted GOx with the FAD component was estimated to be ca. 2, using the extinction coef®cient FAD1.41104 Mÿ1 cmÿ1 [50]. Thus, the two subunits of the enzyme are presumably reconstituted with the Fc±FAD. The activity of the (3)-reconstituted GOx was found to be ca. 40% of the native enzyme using the standard assay procedure [51]. It should be noted that the apo-GOx prior to reconstitution did not exhibit any biocatalytic activity, indicating that all of the native cofactor was excluded from the apo-enzyme. The cyclic voltammogram of the (3)-reconstituted GOx is shown in Fig. 1 (curve b). A cystamine
Fig. 3. Absorption spectrum of the (3)-reconstituted glucose oxidase: 210ÿ5 M enzyme in 0.1 M phosphate buffer, pH 7.3.
monolayer-modi®ed Au-electrode was employed to prevent non-speci®c adsorption of the enzyme on the metal surface that leads to denaturation of the biocatalyst. Furthermore, the cystamine monolayer is protonated and positively charged, whereas GOx is negatively charged (isoelectric point pI4 [50]). Thus electrostatic attraction of the modi®ed enzyme to the electrode surface facilitates electrical communication of the redox-relay modi®ed enzyme with the electrode by concentrating the biocatalyst close to the electrode
E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
51
tion of glucose. The main conclusion of these results is that reconstitution of the apo-GOx with Fc±FAD yields an ``electrically wired'' assembly. That is, oxidation of the ferrocene unit located at an exterior position of the protein mediates intraprotein electron transfer that activates the bioelectrocatalytic function of the enzyme and the oxidation of glucose. The bioelectrocatalytic anodic current developed by the (3)-reconstituted GOx depends on the glucose concentration and it is enhanced as the glucose concentration is, elevated (Fig. 5(a)). The anodic current increases up to 410ÿ2 M concentration of glucose and then levels-off to a constant value, indicating that
Fig. 4. Cyclic voltammograms of the system consisting of (3)reconstituted-GOx, 1.75 mg mlÿ1, at different concentrations of glucose: (a) 0 mM, (b) 1 mM, (c) 3 mM, (d) 20.5 mM. All experiments were performed in 0.1 M phosphate buffer, pH 7.3, at 350.58C, using a cystamine-modified Au-electrode; scan rate, 2 mV sÿ1; Ar atmosphere.
interface. It can be seen that the (3)-reconstituted GOx exhibits only a single reversible redox wave corresponding to the ferrocene unit located outside the protein. The FAD component embedded into the protein lacks direct electrical contact with the electrode surface. The fact that the FAD unit lacks electrical response supports the fact that reconstitution indeed occurred and that the FAD is electrically insulated. The redox response of the ferrocene unit suggests that it is positioned at the protein periphery and that mediated electron transfer is feasible. Fig. 4 shows the cyclic voltammograms of the (3)-reconstituted GOx upon addition of glucose at different concentrations. Note that the redox wave corresponding to the ferrocene unit is invisible in the absence of glucose (Fig. 4, curve a) due to the slow scan rate. Upon addition of glucose, an electrocatalytic anodic current is observed at the potential of the ferrocene unit. This result indicates that the ferrocene unit tethered to the FAD cofactor exhibits electron transfer communication with the electrode and mediates the oxidation of the implanted FADH2 moiety that is being reduced enzymatically by glucose. The electrochemical oxidation of the FADH2 and regeneration of the FAD cofactor state stimulate the bioelectrocatalyzed oxida-
Fig. 5. (a) Calibration curve of electrocatalytic anodic current at different glucose concentrations in the presence of (3)-reconstituted-GOx, 1.75 mg mlÿ1. The experiments were performed in 0.1 M phosphate buffer, pH 7.3, at 350.58C; Ar atmosphere. (b) Lineweaver±Burk plot of the electrocatalytic oxidation of glucose by the (3)-reconstituted-GOx.
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E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
the biocatalyst active site is saturated by the substrate. The electrocatalytic anodic current is proportional to the rate of bioelectrocatalyzed oxidation of glucose, Eq. (4). k1
ket
FcÿFADH ÿGOx ! Fc ÿFADH ÿGOx
eÿ to the electrode
(9)
k2
Fc ÿFADH ÿGOx ! Fc ÿ FAD ÿ GOx H
FcÿFADÿGOx Glucose @
(10)
kÿ1
k3
kcat
FcÿFADÿGOx Glucose ! FcÿFADH2 ÿGOx
FcÿFADÿGOx Glucose ! FcÿFADH2 ÿGOx Gluconic acid 1 1 KM 1 I Imax Imax Glucose KM
(4) (5)
kcat kÿ1 k1
(6)
where KMMichaelis±Menten constant. Fig. 5(b) shows the Lineweaver±Burk plot that corresponds to the analysis of the calibration curve shown in Fig. 5(a) according to Eq. (5). A linear relation characteristic to Michaelis±Menten behavior is observed, and the values Imax4 mA and KM2.9 mM (Eq. (6)) characterizing the reconstituted enzyme are derived. Recently, a kinetic scheme for the intramolecular electron transfer rates in randomly modi®ed ferrocene±tethered glucose oxidase was developed [52]. It was assumed that the intramolecular electron transfer from FADH2/FADH to the nearest ferrocenyl cation unit that is generated electrochemically controls the rate of bioelectrocatalyzed oxidation of glucose. The intramolecular rate constants for the randomly modi®ed ferrocene±GOx depend on the loading of the protein by the redox units. The highest observed intramolecular rate constant, kobs0.90 sÿ1, was reported for GOx loaded with 13 ferrocene units. Applying Marcus theory [53] to the measured rate constant, the distance between the nearest ferrocene Ê unit and the FAD cofactor was estimated to be 21.5 A [52]. We adopted a similar kinetic scheme for the characterization of the intramolecular electron transfer in the (3)-reconstituted GOx, Eqs. (7)±(11).
ket
FcÿFADH2 ÿGOx ! Fc ÿFADH2 ÿGOx eÿ to the electrode k1
(7)
Fc ÿFADH2 ÿGOx ! FcÿFADH ÿ GOx H
(8)
Gluconic acid
(11)
where ket is the interfacial electron transfer rate constant corresponding to oxidation of the ferrocene unit by the electrode, k1 and k2 are the rate constants of the ®rst and second steps of the FADH2 oxidation, and k3 is the rate constant of the enzymatic oxidation of glucose by GOx. Applying this kinetic scheme (Eqs. (7)±(11)) and assuming that at a saturation concentration of glucose, the maximum rate of bioelectrocatalaytic oxidation of glucose occurs, and the highest electrocatalytic current, Imax, is observed, then Imax can be expressed by reaction Eq. (12)[52], where F is the Faraday constant, A is the electrode surface area (cm2), DGOx 410ÿ7 cm2 sÿ1 corresponds to the diffusion coef®cient of GOx, and [Fc±FAD±GOx] is the concentration of the (3)-reconstituted-GOx (molecm3). The rate constants kobs and k3 are given by Eqs. (13) and (14), respectively. Imax 2FA DGOx kobs 1=2 FcÿFADÿGOx
(12)
k1 k2 kobs ÿp p2 k1 k2
(13)
k3
kcat
1 KM Glucoseÿ1
(14)
Substitution of the experimentally observed Imax value (4 mA) obtained by the Michaelis±Menten analysis for the (3)-reconstituted-GOx in Eq. (12) yields the kobs40 sÿ1 for the electrical communication rate control in the reconstituted enzyme. It should be noted that the rate constant of the electron transfer between the electrochemically generated ferrocenyl cation and the enzymatically reduced FADH2 in the (3)-reconstituted-GOx is ca. 50-fold higher than the value observed in the randomly-ferrocene-modi®ed GOx exhibiting the highest electrical communication features. The enhancement of electron transfer rates
E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
between the ferrocenyl cation and reduced FAD cofactor is of substantial importance in the design of amperometric glucose biosensors. The turnover rate of the FADH2 cofactor in GOx by the natural electron acceptor, molecular oxygen, (in an aqueous phase equilibrated with air) corresponds to kobs5103 sÿ1 k [52]. This limits the application of GOx-electrodes as amperometric biosensors to deaerated oxygen-free media. The design of fast electrically communicating relay-modi®ed GOx could establish a means to apply the GOx-electrodes under oxygen. Furthermore, knowing the intraprotein electron transfer rate constants in the (3)-reconstituted-GOx and in the randomly-ferrocene-modi®ed GOx (exhibiting highest electrical communication), one could apply Marcus equation, Eq. (15), to estimate the difference in edge-to-edge separation of the redox sites, d, in the two kinds of the proteins, Eq. (16), where k(3)ÿGOx and kFc(n)ÿGOx are the intraprotein electron transfer rate constants in the (3)-reconstituted-GOx and in randomly-ferrocene-modi®ed GOx, respectively: ket / expÿ d ÿ d0 exp ÿG0 2 =4RT ln
k 3-GOx kFc nÿGOx
(15)
ÿ d
(16)
where is the electron transfer reorganization energy Ê ÿ1 is the electron tunneling coeffiand 0.5±1.4 A cient [53]. Using the recently published approach [52] and assuming that the rate constants for oxidation of FADH2 and FADH (k1 and k2 in Eqs. (8) and (10), respectively) are not different, and applying the observed rate constants (kobs in Eq. (13)) to approximate k(3)-GOx and kFc(n)±GOx, we estimated the distance separating the ferrocene unit and FAD cofactor Ê . Thus, the in the (3)-reconstituted-GOx to be ca. 19 A distance separating the redox relay and the redox Ê shorter center in the reconstituted enzyme is ca. 2 A than in the most effective electrically contacted enzyme generated by the random modi®cation of GOx with ferrocene units. Furthermore, the results suggest that by the application of Fc±FAD dyads where the bridging chain separating the ferrocene unit and FAD cofactor is shortened by 3 or 4 methylene groups, and the reconstitution of apo-GOx could yield
53
enzymes of improved intraprotein electrical contact, that eventually could compete with the oxidation of the FADH2 site by oxygen. 3.2. Glucose oxidase reconstitution with the PQQ±FAD monolayer The reconstitution methodology yields a site-speci®c modi®ed bioelectrocatalyst, ``electroenzyme''. To apply such a biocatalyst as active biological material in a sensing device, its integration with an electrode surface is required. Proper alignment of the bioelectrocatalyst on the electrode surface is a crucial element [54]. The electron-relay unit must be positioned at the electrode surface in an optimized con®guration to allow effective electron transport between the biocatalyst active-site and the electrode. With these limiting constraints, we decided to assemble the relay-FAD dyad on the electrode surface and to reconstitute the apo-enzyme on the surface itself. Scheme 3(A) shows the method to assemble the reconstituted GOx on a Au-electrode surface. A primary cystamine monolayer was linked to a roughened Au-electrode and pyrroloquinoline quinone, PQQ, (5), was covalently associated to the base monolayer [42]. N6-aminoethyl±FAD, (2), was coupled to the carboxylic functions of PQQ to yield the relay-FAD dyad on the monolayer. The functionalized monolayer electrode was then reconstituted with apo-GOx to form an active biocatalyst monolayer on the electrode surface. Fig. 6 shows the cyclic voltammogram of the PQQ± FAD dyad monolayer prior to the reconstitution with the apo-GOx (curve a) and after reconstitution with the apo-GOx (curve b). Prior to the reconstitution process, the functionalized monolayer shows two reversible redox waves at E0ÿ0.125 V and E0 ÿ0.50 V (at pH7.0) corresponding to the two-electron redox process of PQQ and FAD, respectively. By integration of the charge associated with the PQQ and FAD redox units, the surface densities of these components were estimated to be ca. 110ÿ11 mol cmÿ2. After reconstitution of the apo-GOx with the functionalized monolayer, the redox wave of the PQQ was almost unaltered, but the redox-wave correspsonding to the FAD unit was substantially decreased in its intensity. This is consistent with the fact that reconstitution of the apo-GOx with the monolayer insulates the FAD-sites towards electrical communication with
54 E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58 Scheme 3. (A) Reconstitution of glucose oxidase onto a PQQ±FAD monolayer Au-electrode and direct oxidation of glucose by the modified electrode. (B) Reconstitution of glucose oxidase onto a FAD-modified Au-electrode and bioelectrocatalyzed oxidation of glucose by the enzyme-electrode in the presence of a diffusional electron mediator (ferrocene carboxylic acid).
E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
55
Fig. 6. Cyclic voltammograms of (a) the PQQ±FAD monolayer on a Au-electrode before and (b) after reconstitution with apo-glucose oxidase. Ar atmosphere, 0.1 M phosphate buffer, pH 7.0, 250.58C; potential scan rate, 50 mV sÿ1; electrode geometrical area, 0.4 cm2, roughness factor, ca. 20.
the electrode surface. The PQQ redox unit is suf®ciently exposed to the protein periphery and hence reveals non-perturbed electrical contact with the electrode. The residual redox-wave of the FAD units is attributed to the free dyad that was not reconstituted into the apo-GOx. By integration of the depleted charge of FAD units, and assuming that two FADcofactor components participate in the anchoring of the apo-GOx onto the surface in the reconstitution process, the surface density of the protein on the electrode surface was estimated to be ca. 1.710ÿ12 mol cmÿ2. Using the footprint dimension of GOx (58 nm2) [55], the surface coverage of the electrode by the enzyme corresponds to a densely packed enzyme monolayer assembly. Fig. 7(A) shows the cyclic voltammograms of the reconstituted GOx monolayer electrode in the absence of glucose (curve a) and in the presence of glucose (curve b). With glucose, a high-magnitude electrocatalytic anodic current is observed, indicating that the reconstituted GOx on the monolayer yields a bioactive monolayer interface exhibiting direct electrical contact with the electrode. The electrically-contacted enzyme layer stimulates the bioelectrocatalyzed oxidation of glucose. A control system that includes the reconstituted GOx on a single-component FAD monolayer that lacks the PQQ redox-mediating unit was also
Fig. 7. (A) Cyclic voltammograms of: (a) The glucose oxidase reconstituted on the PQQ±FAD monolayer enzyme-electrode. (b) After adding glucose, 80 mM. Ar atmosphere, 0.1 phosphate buffer, pH 7.0; 350.58C; scan rate, 5 mV sÿ1; electrode geometrical area, 0.4 cm2; roughness factor, ca. 20. (B) Amperometric responses of the PQQ±FAD±reconstituted-GOx monolayer electrode at different glucose concentrations. Current determined by chronoamperometry at final potential 0.2 V.
assembled on a Au-electrode (Scheme 3(B)). In this system, no direct electrical communication between the embedded FAD-cofactor and the electrode exists, but the reconstituted GOx exhibits biocatalytic activity, and bioelectrocatalyzed oxidation of glucose proceeds in the presence of a diffusional electron mediator, ferrocene carboxylic acid. These results clearly demonstrate that the reconstitution of the apo-GOx on the FAD-monolayers represents a novel means to link and align ¯avoenzymes onto surfaces. Reconstitution of the apo-GOx onto the PQQ±FAD
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E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
dyad monolayer yields an electroactive biocatalyst interface, where direct electrical contact between the enzyme-active site and the electrode is achieved. The intermediate PQQ component that acts as a redoxrelay unit, transports the electrons from the active site of the enzyme to the electrode. PQQ, (5), was selected for the role of the intermediate relay because it represents a two-electron transfer mediator that could simplify the mediation process and improve its kinetics. The following reaction scheme (Eqs. (17)± (19)) could be applied to the PQQ-mediated bioelectrocatalyzed glucose oxidation. ElectrodeÿPQQÿFADH2 ÿGOx
kFADÿPQQ
!
ElectrodeÿPQQH2 ÿFADÿGOx
(17) kPQQ
ElectrodeÿPQQH2 ÿFADÿGOx !
ElectrodeÿPQQÿFADÿGOx 2H 2eÿ to the electrode
(18) kcat
ElectrodeÿPQQÿFADÿGOx Glucose ! ElectrodeÿPQQÿFADH2 ÿGOx Gluconic acid (19) In this scheme kFAD±PQQ, kPQQ and kcat represent the rate constants for the electron transfer from the reduced FAD to PQQ, from the reduced PQQ to the electrode and the biocatalyzed reduction of FAD by glucose, respectively. The assumed sequence of reactions does not include one-electron transfer steps for the oxidation of FADH2 (Cf. for comparison FADH2/ FADH oxidation via one-electron transfer steps using ferrocene relay as the mediator, Eqs. (8) and (10)). Fig. 7(B) shows the transduced currents by the PQQ±FAD±reconstituted GOx monolayer electrode at different glucose concentrations. The current responses are almost linear in a concentration range of 1±80 mM of glucose. The upper limit of the turnover-rate of glucose oxidase at 258C is 600100 sÿ1 [56] and the activation energy is 7.2 kcal molÿ1 [57]. At the temperature employed in our measurements (358C), this translates to a limiting turnover rate of 900150 sÿ1. Realizing that the reconstituted GOx surface coverage is 1.710ÿ12 mol cmÿ2, the maximum current density that can be observed for the theoretical turnover of the enzyme is 29060 mA cmÿ2.
Fig. 7(B) shows that at a glucose concentration of 80 mM, the observed current is ca. 1.9 mA and the current density is ca. 300100 mA cmÿ2, taking into account the electrode geometrical area, 0.4 cm2, and its roughness factor, ca. 20. Thus, the experimental current density is within the range of the limiting turnover rate of the native enzyme. This suggests that at the potential employed in the chronoamperometric experiments to analyze glucose (E0.2 V) all FAD sites are electrically connected with the electrode and exist in the oxidized form. The resulting current is then controlled by the diffusion of glucose to the active site. The reconstituted apo-GOx on the PQQ±FADmonolayer electrode exhibits unique electrical communication features, and the theoretical turnover rate of the active center with molecular oxygen is achieved for the electron exchange between the active site and the electrode. Also, the bioelectrocatalyzed oxidation of glucose proceeds at relatively low positive potentials, E>ÿ0.1 V, characteristic to the PQQ electron mediator component. These two features have important implications for the use of the monolayer-modi®ed electrode as a glucose-sensing interface. The high electrical turnover rate of the reconstituted biocatalyst suggests that the enzyme-electrode would not be in¯uenced by oxygen. The presently available amperometric glucose electrodes based on glucose oxidase are sensitive to the oxygen content in the environment, and most analyses are performed in an oxygen-free medium. Fig. 8 shows the amperometric responses of the reconstituted enzyme electrode in the presence of glucose in an oxygen-free environment (curve b) and in the presence of air (curve c). The transduced amperometric signal is only slightly (less than 2%) decreased by oxygen. Similarly, the effective electrical contact between the biocatalyst and the electrode upon application of a low potential suggests that the non-speci®c oxidation of interfering components will be screened by the highly ef®cient bioelectrocatalyzed oxidation of glucose. Ascorbic acid is a common interferant in glucose analysis. Fig. 8 shows the currents transduced by the reconstituted enzyme electrode in the presence of glucose without added ascorbic acid (curve b) and in the presence of ascorbic acid taken at a physiological concentration level (curve d). The transduced current in the presence of ascorbic acid is only slightly increased (less than 5%) implying that the observed current originates essen-
E. Katz et al. / Analytica Chimica Acta 385 (1999) 45±58
Fig. 8. Amperometric responses produced by the GOx reconstituted with the PQQ±FAD monolayer: (a) in the absence of glucose, (b) in the presence of 50 mM glucose and absence of oxygen, (c) in the presence of 50 mM glucose in a solution saturated with air, and (d) in the presence of 50 mM glucose and 0.1 mM ascorbic acid in the absence of oxygen. Currents determined by chronoamperometry at final potential 0.0 V. Electrolyte composed of 0.1 phosphate buffer, pH 7.0, 350.58C; electrode geometrical area, 0.4 cm2; roughness factor, ca. 20.
tially from the biocatalyzed oxidation of glucose. Thus, the ef®cient electrical contact of the reconstituted enzyme electrode, and the resulting high current densities, as well as the speci®city of the bioelectrocatalytic electrode, enable the future application of the enzyme electrode as a miniaturized invasive glucose sensor. Acknowledgements The research was supported by the BMBF, Germany, and by the Israel Ministry of Science, and in part by the Szold Foundation, The Hebrew University of Jerusalem. References [1] P.N. Bartlett, in: R.P. Buck, W.E. Hatfield, M. Umana, E.F. Bowden (Eds.), Biosensor Technology. Fundamentals and Applications, Chapter 7, Marcel Dekker, New York, 1990, pp. 95±115. [2] H.A.O. Hill, G.S. Sanghera, in: A.E.G. Cass (Ed.), Biosensors. A Practical Approach, Chapter 2, Oxford University Press, Oxford, 1990, pp. 19±46.
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