ARTICLE pubs.acs.org/ac
Efficient Direct Electron Transfer of PQQ-glucose Dehydrogenase on Carbon Cryogel Electrodes at Neutral pH Victoria Flexer,† Fabien Durand,† Seiya Tsujimura,†,‡ and Nicolas Mano*,† † ‡
Universite de Bordeaux, Centre de Recherche Paul Pascal, CRPP-UPR 8641-CNRS, 115 Avenue Albert Schweitzer, 33600 Pessac, France Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
bS Supporting Information ABSTRACT: We present a comprehensive study of the direct electron transfer reaction of soluble PQQ-GDH from Acinetobacter calcoaceticus. Wild-type PQQ-sGDH nonspecifically adsorbed on carbon cryogel electrodes retained its enzymatic activity for glucose and maltose oxidation at pH 7.2 and 37 °C. The cyclic voltammograms in the absence of enzymatic substrate showed 2 redox peaks that suggest a two-step, one-electron oxidation/reduction of PQQ. Calibration curves showed a linear amperometric response for a wide glucose concentration range, including the values normally found in blood. At saturation, the catalytic current reached 0.93 mA cm2. Altogether the experimental results suggest that the amperometric output of the electrodes and the shape of the calibration curves represent a combination of the intrinsic enzyme kinetics, the maximum rate of heterogeneous electron transfer and the substrate accessibility to the enzyme’s active center caused by the confinement of the enzyme into the mesoporous structure. A new mutant enzyme, N428C, developed in our group that shows almost twice the maximum catalytic activity in homogeneous experiments in solution, also showed a DET signal on carbon cryogel electrodes for glucose electro-oxidation. The higher activity for the mutant enzyme was also verified on the electrode surface.
’ INTRODUCTION Because of its high activity and insensitivity to O2, soluble PQQ-glucose dehydrogenase (PQQ-sGDH) is widely used in biosensors systems for self-monitoring of blood glucose for diabetic people,1 outrunning glucose oxidase. The structure of soluble PQQ-GDH from the bacteria Acinetobacter calcoaceticus has been determined in apo form (PDB 1QBI) and in complex with its cofactor (PDB 1CQ1), pyrroloquinoline quinone (PQQ).25 The enzyme is homodimeric with a molecular weight of 100.465 kDa. Each of the monomers is organized as a β-propeller fold with six fourstranded antiparallel β-sheets aligned around a pseudo-6-fold symmetry axis. The enzyme binds three Ca ions per monomer: two are located at the interface and are needed for functional dimerization of the protein, and the third is required to bind the PQQ cofactor in the active site.6 The enzyme is rather unspecific, oxidizing β-D glucose, 2-desoxy-glucose, galactose, maltose, cellobiose, and lactose.7 Based on its three-dimensional (3D) structure, site-directed mutagenesis has been successfully carried out to improve its substrate specificity,8,9 thermal stability,10,11 and, most recently, its activity.12 Enzyme electrodes are extensively studied in the fields of biosensors1 and biofuel cells,1315 and commercial PQQ-sGDH electrodes are one of the classical examples. Generally speaking, enzyme electrodes can be classified into a first group that employs mediated electron transfer (MET), where electrons are shuttled from the enzyme to the electrode via a third chemical species, called a redox mediator, such as Os and Ru complexes, ferrocenes, etc.;13 and a second group based on mediator-free direct electron transfer r 2011 American Chemical Society
(DET), where enzymes are able to communicate directly with the electrode.13 Several reports can be found in the literature for mediated electrodes employing GDH.1,1618 DET is desirable over MET because of cost, simpler construction, and, most importantly, in the case of biofuel cells,13 voltage gain and, hence, higher power density. Extensive research on DET between enzymes and electrodes has been carried out in recent years.1924 DET is observed rather easily for molecules with low molecular weight or rather-exposed active centers, such as cytochrome c or multicopper oxidases. This last group of enzymes reduces O2 to water and, consequently, they have been proposed as interesting candidates for biofuel cells cathodes. Several dozens of papers can be found in the recent literature describing high nonmediated catalytic signals for several multicopper oxidases.2531 However, there are only a few reports20,24,3236 on DET for enzymes oxidizing different sugars, usually showing much lower catalytic currents than multicopper oxidases in their respective optimum conditions. DET can be tailored by modifying either the protein (usually the surface or the region around the redox center) or the electrode structure,13,37 to promote the adsorption of the enzyme in a particular configuration or its partial unfolding to increase the accessibility of the redox center of the enzyme.33 Mesoporous materials with pore size close to the enzyme dimensions offer high Received: April 15, 2011 Accepted: June 12, 2011 Published: June 12, 2011 5721
dx.doi.org/10.1021/ac200981r | Anal. Chem. 2011, 83, 5721–5727
Analytical Chemistry specific surface area and are expected to promote enzyme adsorption in a stable configuration.25 Numerous reports suggest that nanostructures materials tend to favor direct electron transfer.24,25,38,39 The first report on DET for PQQ-glucose dehydrogenase was a genetically engineered enzyme obtained by fusing a cytochrome c domain to the C-terminal of PQQ-sGDH.40 Later on, DET for the membrane bound PQQ-GDH from Erwinia sp. was reported.23,41 To the best of our knowledge, only two research groups have succeeded in showing direct electron transfer for the wild-type variety of PQQ-sGDH from Acinetobacter calcoaceticus. First, Razumiene et al.23 showed a bioelectrocatalytic signal for the enzyme adsorbed on carbon paste electrodes. Shortly after, Ivnitski et al.21 reported a much higher direct electron transfer signal for the enzyme covalently attached to modified carbon nanotubes. Cooney et al. showed the PQQ redox peaks of the enzyme in the absence of enzymatic substrate, although no catalytic current was obtained in the presence of glucose.42 Willner et al. also shown that the apo GDH could be reconstituted onto PQQ cofactor sites that were attached to Au-nanoparticles, these nanoparticles acting as a charge-transfer mediator. The bioelectrocatalytic oxidation of glucose occurred with a high turnover number, although at rather high potentials.43 Here, we present a comprehensive study of the direct electron transfer reaction of PQQ-sGDH from Acinetobacter calcoaceticus. Wild-type (WT) PQQ-sGDH nonspecifically immobilized on carbon cryogel electrodes retained its enzymatic activity for glucose and maltose oxidation at pH 7.2 and 37 °C. A new mutant of PQQ-sGDH developed in our group, where an asparagine in position 428 was substituted by a cysteine (N428C)12 that doubles the catalytic activity of the WT enzyme in homogeneous experiments in solution, also showed a DET signal on carbon cryogel electrodes. The higher activity for the mutant enzyme was also verified on the electrode surface.
’ EXPERIMENTAL SECTION Enzyme production and purification are described in detail in the Supporting Information. The preparation of carbon cryogel electrodes has been fully described and characterized elsewhere.24,44 Based on the known dimensions of the enzyme (85 Å 60 Å 61 Å), we chose to work with carbon cryogel with a pore size of 20 nm. The protocol is briefly reviewed in the Supporting Information. Carbon cryogel electrodes were made hydrophilic by exposure to 1 Torr O2 plasma for 10 min. Five microliters (5 μL) of buffer were deposited on the electrode and, after air bubbles came out from the cryogel, the desired amount of enzyme solution was deposited on top of the buffer droplet and allowed to adsorb for 30 min at room temperature. Electrodes were rinsed in buffer for 5 min and tested immediately. To prove that most of the enzyme remained adsorbed on the electrode surface, the amount of enzyme present in the rinsing solution was assessed. The amount of active enzyme was measured by the enzymatic activity, while the total amount of protein (both active and denaturated) was measured by the Bradford method. Data are normalized by the geometrical surface area of electrodes (μA cm2). Electrochemical measurements were performed using a bipotentiostat (Model CHI760C, CH Instruments, Austin, TX) in a thermostatized cell with a platinum gauze as a counterelectrode and a Ag/AgCl reference electrode. All experiments were performed in 20 mM PIPES buffer containing 3 mM CaCl2, pH 7.2. Temperature was thermostatized at 20 °C for the experiments in the absence of sugar and at 37 °C for all other experiments.
ARTICLE
Figure 1. Cyclic voltammogram for WT enzyme adsorbed on carbon cryogel electrode (1 μL loading). Enzyme loading = 7.2 nmol cm2. Conditions: 20 °C, under an argon atmosphere. Scan rate = 1 mV s1. The inner signal corresponds to the redox peaks after the capacitive background signal has been removed. Inner trace has been multiplied by 4, for the sake of clarity.
Results shown in Figures 3 and 5 correspond to data averaged for three independent experiments/calibration curves. Experiments were performed under air, except those in Figure 1.
’ RESULTS AND DISCUSSION We first investigated the possibility of direct electron transfer between the active site of the immobilized enzyme and the carbon cryogel electrode in the absence of enzymatic substrate. Figure 1a shows the cyclic voltammogram for PQQ-sGDH nonspecifically adsorbed on a carbon cryogel electrode at pH 7.2. We observe two redox peaks in the anodic scan: at E = 159 mV and E = +145 mV. The two anodic peaks do not show the same charge: the more negative peak is the largest. In the backward (reductive) trace of the cyclic voltammogram, only one redox peak can be distinguished from the background current. The redox potential of this peak is coincident with the oxidation process occurring at the more-negative potential in the forward scan. The inner trace in Figure 1 shows the background-subtracted current, and it has been multiplied by an arbitrary factor of 4 (for the sake of clarity). None of the peaks observed in Figure 1 were present in the blank experiment performed on the same electrode before enzyme adsorption (see Figure S-1 in the Supporting Information). These peaks are the result of the redox reaction of the PQQ cofactor buried inside the enzyme structure. To the best of our knowledge, Ivnitski et al.21 are the only researchers who have reported direct electron transfer signal for PQQ-sGDH in the absence of enzymatic substrate at pH 6. The shape of our cyclic voltammogram is in excellent agreement with their data; our peak potentials are lower than their report, because our experiments were performed at higher pH. In addition, the shape of our cyclic voltammogram is in also in very good agreement with the shape of the cyclic voltammograms of free PQQ immobilized on SiZr-carbon paste electrodes,45 showing also a redox couple plus an oxidation peak of lower intensity at a more positive potential. The cyclic voltammogram for free PQQ adsorbed on carbon cryogel electrode is shown in Figure S-2 in the Supporting Information. We cannot discard the hypothesis that the redox peaks observed in 5722
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Analytical Chemistry
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Figure 2. Cyclic voltammogram for WT enzyme adsorbed on carbon cryogel electrode of 1 μL loading ( ) in the absence and (—) in the presence of 150 mM glucose. Enzyme loading = 7.2 nmol cm2. Scan rate = 1 mV s1. Conditions: 37 °C, under an argon atmosphere. Inset: Carbon cryogel electrode with a loading of 5 μL. All other conditions are the same as those given for Figure 1.
Figure 1 could arise from PQQ released from some denaturated enzymes, as it has already been shown for other adsorbed enzymes.46 However, the high catalytic current observed in the presence of glucose suggests that most PQQ remains in the active site. In addition, free PQQ does not oxidize glucose. The redox reaction of PQQ is known to be a two-step, oneelectron, and two-proton process.47 The presence of the two peaks would indicate two consecutive one-electron transfer steps,48,49 as depicted in eqs 1 and 2: PQQ OX + e + H+ T PQQ semiquinone
ð1Þ
PQQ semiquinone + e + H+ T PQQ RED
ð2Þ
where the intermediate species would be a rather stable semiquinone free radical.47 This mechanism was suggested by Sato et al.,47 after spectroelectrochemistry studies of the free enzyme in solution. This mechanism is in good agreement with our experimental data, but the redox potentials reported by Sato et al.47 are lower than ours and the two E° values are closer than in our case. This difference could be due to a slower and therefore more irreversible electron transfer on the electrode surface than a mediated electron transfer in solution. We wish to emphasize that the electrochemistry of PQQ is not yet fully understood. It is clear that the electrochemical response of PQQ is strongly dependent on the electrode material, and, in many cases, the electrochemistry is highly irreversible.50 Some authors have shown only one redox wave, which would suggest a one-step, two-electron electron transfer.5052 After proving that the active center of the enzyme can be electrically connected to the electrode surface in the absence of substrate, we investigated the enzyme catalytic activity. Figure 2 shows the cyclic voltammograms at 37 °C and at 1000 rpm of a PQQ-sGDH modified carbon cryogel electrode in the absence (dashed line) and in the presence (solid line) of 150 mM glucose. Upon the addition of glucose, a clear catalytic signal can be observed starting at 0 V and corresponding to the more oxidative peak observed in Figure 1. The maximum catalytic signal is reached at ∼0.35 V where a narrow plateau region appears. Electrodes can be prepared with different loadings of carbon cryogel slurry. Figures 1 and 2 were obtained with electrodes of low loading (1 μL of slurry). These electrodes show a lower background
Figure 3. Dependence of the catalytic current density on the enzyme loading at glucose saturation (300 mM/5400 mg dL1). Chronoamperometry experiments at 0.2 V, 37 °C, 1000 rpm. (Legend: (9) WT enzyme and (4) N428C mutant.)
current, which allowed us to clearly identify the redox peaks of the active center of the enzyme in the absence of a substrate. However, since we wish to maximize the catalytic current of the electrodes, we further prepared electrodes of higher carbon cryogel loading (5 μL of slurry), which allow for an optimized enzyme deposit. The inset in Figure 2 shows data for the same molar amount of enzyme deposited on an electrode with a high loading of carbon cryogel. The capacitive current of this electrode is larger than that in Figure 2. However, the catalytic current is also much higher. For both high and low loading of carbon cryogel, the catalytic wave starts at ∼0 V. The plateau region is not clearly identified, since the background current is already important at 400 mV but can be recovered if the background is subtracted. Further results shown in this paper will all correspond to carbon cryogel electrodes with a loading of 5 μL. Our results are in good agreement with the previously mentioned work on DET for PQQ-sGDH,21,23 which showed the electrooxidation of glucose, starting at ∼0 V and reaching a maximum at ∼0.5 V. If we compare the current normalized with the geometrical surface area, our data are of the same order of magnitude than those obtained by Ivnitski et al.21 and are at least three orders of magnitude higher than those reported by Razumiene et al.23 Furthermore, we immobilized the new mutant enzyme developed in our group12 on similar carbon cryogel electrodes. Electrocatalytic activity was also shown for this enzyme, with the shape of the cyclic voltammogram being very similar to that of the WT enzyme (see Figure S-3 in the Supporting Information). Figure 3 shows the dependence of the catalytic current measured at 0.2 V on the enzyme loading for both the WT (full squares) and the N428C mutant (empty triangles), at glucose saturation and under forced convection. For the WT enzyme, the current increases linearly up to a loading of 14.3 nmol cm2 and does not increase any further for higher enzyme loading. In the case of the N428C mutant, the catalytic current also increased with the enzyme loading and was twice as active as the WT enzyme for loadings of
