A 96-Well Electrochemical Method for the Screening of Enzymatic

Article pubs.acs.org/ac

A 96-Well Electrochemical Method for the Screening of Enzymatic Activities Sofiène Abdellaoui,† Alexandre Noiriel,‡ Robert Henkens,§ Celia Bonaventura,§ Loïc J. Blum,† and Bastien Doumèche*,† †

GEMBAS (Génie Enzymatique, Membranes Biomimétiques et Assemblages Supramoléculaires) and ‡ODMB (Organisation et Dynamique des Membranes Biologiques) ICBMS UMR 5246, Université Lyon 1, CNRS, INSA Lyon, CPE Lyon, 43 bd du 11 Novembre 1918, 69622 Villeurbanne, Cedex, France § Alderon Biosciences Inc., 120 Turner Street, Beaufort, NC 28516, United States S Supporting Information *

ABSTRACT: The rapid electrochemical screening of enzyme activities in bioelectronics is still a challenging issue. In order to solve this problem, we propose to use a 96-well electrochemical assay. This system is composed of 96 screen-printed electrodes on a printed circuit board adapted from a commercial system (carbon is used as the working electrode and silver chloride as the counter/reference electrode). The associated device allows for the measurements on the 96 electrodes to be performed within a few seconds. In this work, we demonstrate the validity of the screening method with the commercial laccase from the fungus Trametes versicolor. The signal-to-noise ratio (S/N) is found to be the best way to analyze the electrochemical signals. The S/N follows a saturation-like mechanism with a dynamic linear range of two decades ranging from 0.5 to 75 ng of laccase (corresponding to enzymatic activities from 62 × 10−6 to 9.37 × 10−3 μmol min−1) and a sensitivity of 3027 μg−1 at +100 mV versus Ag/AgCl. Laccase inhibitors (azide and fluoride anions), pH optima, and interfering molecules could also be identified within a few minutes.

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from the enzyme cofactor) to the electrode. Much effort was done in developing redox polymers (e.g., polyphenazines, Oscomplex containing polymers)4−9 or controlled assemblies of redox species (pyrroloquinoline quinone- or phenazinemodified surfaces) to obtain efficient anodes.10−13 The engineering of the cathode surface is still rarely investigated despite it being often considered as the limiting part of the biofuel cell. In order to operate properly, and to obtain the highest potential in the biofuel cell, a direct electron transfer between the enzyme and the cathode is desirable. The maximal distance between the active site of the enzyme and the electrode surface, providing the highest electron transfer rate, is below 15 Å, according to the Marcus’ equation.14,15 In the primary biofuel cells, the enzyme used at the cathode was generally a peroxidase (horseradish peroxidase or MP-11 peroxidase) but is now replaced by blue-copper oxidases (such as laccase and bilirubin oxidase).16−25 While peroxidases require hydrogen peroxide as the substrate, the blue-copper oxidases use molecular oxygen as an inexpensive and an inexhaustible electron acceptor. Moreover, Fe-porphyrin heme is not synthesized when peroxidases are overexpressed without the coexpression of the enzymes required for their synthesis

espite their relative universality, electrochemical measurements are not much developed compared to optical methods. One of the main reasons is the time required to analyze a large amount of samples with commercial electrochemical apparatus when using single electrodes. Electrochemical screening assays are still necessary to identify and optimize (bio)electrocatalysts and reaction conditions for analytical or (bio)electronic applications. The development of bioelectronic devices such as biofuel cells and biosensors requires optimizing the electron transfer between the biomolecules and the electrode surface. In particular, enzymebased biofuel cells are one of the most promising systems to generate electric current in miniaturized and implantable devices.1−3 Basically, one enzyme (or set of enzyme) is immobilized at the anode and oxidizes an organic fuel, while another enzyme immobilized at the cathode reduces molecular oxygen, using the electrons provided by the electrical circuit. One of the main advantages of biocatalysts over metallic electrodes is their selectivity, thereby preventing the formation of side products (e.g., peroxides). Carbon-based electrodes are preferable to metallic ones in order to avoid nonselective reactions which lead to the poisoning of the electrode, to limit the passivation of the electrode itself, and to decrease substantially the price of the biofuel cell. However, the electrode surface should be carefully functionalized to allow the efficient electron transfer from the enzyme active site (or © 2013 American Chemical Society

Received: December 27, 2012 Accepted: March 6, 2013 Published: March 6, 2013 3690

dx.doi.org/10.1021/ac303777r | Anal. Chem. 2013, 85, 3690−3697

Analytical Chemistry

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Figure 1. Exploded scheme of a single two-electrode system (A) showing the printed circuit board (PCB) overlaid by the screen-printed carbon working electrode (W) and Ag/AgCl counter/reference electrode (ref), by the PF and by the electrolyte drop (E). The red line corresponds to the profile of the topography analysis shown in (B). A photograph of the 96-well PCB is presented in (C), and a comparison between the typical electrochemical response obtained by chronoamperometry (red curve) or by IPA (black dots) of a redox mediator [1 mM Toluidine blue O in 100 mM phosphate buffer (pH 7.0), 0.1 M KCl, applied potential +100 mV vs Ag/AgCl] is given in (D).

containing enzymes.30,31 Directed evolution is probably the most efficient method to optimize and to create new biocatalysts for a specific application, without requiring an extensive comprehension of the enzyme itself. The screening of the library for the desired property is the critical step of the method. Frances Arnold emphasized in her pioneer work that “you get what you screen for”.32 Despite the increasing interest for laccase in bioelectronics, the directed evolution studies only deal with optical methods using colorimetric substrates.33−36 Such assays will only lead to improved mutants for this substrate and not for mutants with improved electron transfer at the electrode surface. Schwaneberg and co-workers intensively studied mutants of Aspergillus niger glucose oxidase (GOx) for biofuel cells. Mutants are screened for soluble redox or absorbing reaction products, but this does not help when looking for an enzyme with an improved direct electron transfer rate.37−41 A library of mutants could easily contain hundreds to thousands of clones. Therefore, a screening performed on a classical carbon electrode is unrealistic. A cheap and rapid method to screen this high number of mutants on carbon surfaces will be of great help in the improvement of biofuel cell properties. In the present work, we present for the first time an electrochemical screening system for the direct electron transfer between a laccase and a carbon surface in a 96-well format. The method is based on a commercial electrochemical reader, and the validity of the assay is shown using the commercial laccase from T. versicolor laccase (TvLac).

and assembly with the apoenzyme. Therefore, functional recombinant blue-copper oxidases are easier to obtain. Structurally, laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2) belong to the multicopper oxidase family characterized by the presence of one mononuclear (T1) and one trinuclear copper site (T2/T3). These oxidases were described for the first time by Yoshida in 1883 and were one of the first enzymes ever described.25 They catalyze the oxidation of a broad range of phenolic compounds at the T1 center coupled to the reduction of oxygen to water at the T2/T3 center (the structure of the Trametes versicolor laccase used in this work is given in Figure S1 of the Supporting Information). An efficient direct electron transfer between cathode and laccase depends on the enzyme orientation onto the electrode surface: the distance between the T1 copper center and the conductive carbon surface should be less than 15 Å, while the T2/T3 center should remain accessible to dissolved molecular oxygen. Blanford et al. (2007),26 first, then Sosna et al. (2010) 27 had modified a carbon electrode surface by anthracene moieties in order to favor the orientation, to immobilize, and to reduce the distance between the electrode surface and the T1 copper center of the laccases of Pycnoporus cinnabarinus, T. versicolor, and Trametes hirsuta.26,27 More recently, the CotA laccase from Bacillus subtilis was efficiently immobilized onto glassy carbon electrodes modified by phenylene diamine diazonium followed by the coupling with a bis-epoxide coupling reagent.28 The electron transfer was achieved with the help of the soluble mediator 2,2′-azinobis(3ethylbenzothiazoline-6-sulfonic acid) (2,2′-ABTS). The same group also entrapped the CotA laccase in Os-complex modified polymers.29 Another approach to enhance the catalytic activity of an enzyme is to introduce beneficial mutations in its primary sequence. To be successful, site-directed mutagenesis requires a deep understanding of the enzyme structure and of its mechanism, which is especially difficult considering metal-

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MATERIAL AND METHODS Chemicals. T. versiclor laccase preparation (TvLac, E.C. 1.10.3.2, 21.7 units mg−1, lot no. 1310430) is from Fluka (Büchs, Switzerland). 2,2′-Azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS) is from Interchim SA (Montluçon, France). Sodium azide is from Prolabo (France). All other 3691



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TvLac presents the same specific activity, meaning the anion exchange chromatography eliminates nonproteic material. Laccase Activity Assay in Solution. The laccase activity is measured by following the oxidation of ABTS at 420 nm (εM420 nm = 36000 M−1 cm−1) with a Tecan Infinite M200 (Salzburg, Austria) microtiter plate reader (200 μL assay). A single well is composed of 2 μL enzyme solution and 188 μL of 20 mM acetate buffer (pH 5.5). Reaction is initiated by the addition of 10 μL of a 20 mM ABTS solution. Optical pathways are determined by measuring the absorbance of water at 975 nm in each individual well (εM975 nm = 3.05 × 10−3 M−1cm−1). Specific activities are expressed in μmol min−1 mg−1. The kinetic parameters of TvLac were determined with a concentration of ABTS, ranging from 1 μM to 1 mM. Kinetic parameters are obtained by fitting the Michaelis−Menten equation. The protein concentration was measured by the Bradford reagent (BioRad protein assay) with BSA as a standard.

chemicals are from Sigma-Aldrich (St-Quentin-Fallavier, France). Electrochemical Screening System. The electrochemical assays were performed with an AndCare 9600 sensor (Alderon Biosciences Inc., Beaufort, NC). The screen-printing of the 96 two-electrode systems is directly performed onto tailored Printed Circuit Board (PCB) (CirLy SA, Brignais, France), displaying 192 copper connectors (see Figure S2 of the Supporting Information). The PCB is used as an electrical connector between the Andcare 9600 sensor and the screenprinted electrodes, as well as a support for the electrodes. Screen-printing is performed with a DEK 248 device and appropriate templates. The inks are from Acheson (Carbon paste is Electrodag PF-407 C and Ag/AgCl paste is Electrodag 6038 SS, Acheson, Scheemda, The Netherlands). The working electrode is made of carbon, while the counter/reference electrode is Ag/AgCl. After printing, electrodes are cured one hour at 100 °C. The wells (6 mm diameter) are obtained by sticking a perforated adhesive plastic foil (PF) of 0.25 mm thickness (3M, Cergy-Pontoise, France) prepared with a craft ROBO (Silhouette America, Inc., Orem, UT) (PF in Figure 1A and Figures S2 and S6 of the Supporting Information). The electrode surface is covered with a drop of 40 μL of electrolyte solution (Figure 1A). Measurements were performed by intermittent pulse amperometry (IPA): a series of millisecond pulses of potentials are applied to the working electrode, separated by longer periods when the electrode is disconnected from the potentiostat. The applied potential is varied from −600 to 600 mV versus Ag/AgCl, keeping the pulse parameters constant ( f = 1 Hz, pulse width 41 ms, measurement time 1 min, 60 data points). The 10 last measurements were averaged and used as representative current intensity on the electrode. Intensity is usually stable during the 30 last seconds. Unless otherwise stated, IPA measurements are performed in a 150 mM phosphate-citrate buffer pH 4.4 containing 100 mM NaClO4 as an additional electrolyte. The enzyme-modified electrode is obtained by the adsorption of 3 μL of the enzyme solution (known protein concentration and specific activity, in 20 mM acetate buffer, pH 5.5, containing 60 mM ammonium sulfate) and is left to dry overnight at 4 °C. Due to the screenprinted method, the electrode surfaces slightly vary from one well to the next. In order to take into account these variations, the electrode signal is measured before (I0) and after (I) the enzyme adsorption on each well. The signal-to-noise ratio (S/ N) is then calculated according to eq 1: S /N =

I − I0 I0

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RESULTS AND DISCUSSION Electrochemical Assay. An appropriate PCB board is used as a substrate for screen printing 96 electrodes pairs: Carbon paste is used as a working electrode, and Ag/AgCl paste is used for counter and reference electrodes (Figure 1A and Figure S7 of the Supporting Information). An electrode area of 7.98 ± 0.17 mm2 (2%) and an average height of 4.56 ± 0.85 μm (18%) is determined by topography analysis (Figure 1B and Figure S6 of the Supporting Information). The high variability between electrode heights is inherent to the screen-printing method. Therefore, a normalization of the electrochemical signal will be applied in order to get comparable data. In the standard commercial plate, the well boundary was obtained with a bottomless 96-well plate of 400 μL. Here, the wells demarcation is obtained with perforated adhesive film (250 μm height), which highly reduces the costs of the assay. This plate configuration allows us to reuse the PCB board after adhesive removal and careful washing with acetone to remove the carbon and Ag/AgCl pastes. With this setup, the volume of electrolyte solution in each well comprises between 35 and 50 μL and forms a drop at the electrode surface (Figure 1A). Furthermore, redox species in solution or adsorbed within the 96 wells could be measured within 1 s with this device, which is the strongest part of the system compared to other classical electrochemical methods. The intensities measured by IPA are also 2.2 times higher than the ones obtained by classical amperometric methods (Figure 1D). Assay Optimization. At first, the feasibility of the assay is studied with the purified commercial laccase from TvLac. Figure 2A shows the current recorded over the time on 96 independent electrodes before (black curves) and after (red curves) adsorption of 1 μg of TvLac, using an applied potential of 100 mV versus Ag/AgCl, with a pulse of 41 ms at a frequency of 1 Hz. The TvLac is prepared in a 20 mM acetate buffer (pH 5.5) containing 60 mM ammonium sulfate before adsorption overnight at 4 °C. Any attempt to adsorb this enzyme in the presence of phosphate buffer or in the presence of sodium chloride was unsuccessful. As could be seen in Figure 2A, nearly no current could be measured onto the electrodes before the laccase adsorption. Once the TvLac is adsorbed, the measured current increases up to ∼280 nA and is attributed to the bioelectrochemical reduction of dissolved oxygen by TvLac. The average intensity is found to be 169 ± 40 nA (CV = 23%). Then, the potential

(1)

The S/N values below 3 are not considered as relevant. T. versicolor Laccase Purification. Commercial TvLac was purified according to the protocol described by Blanford, in order to remove insoluble material.26 Briefly, 40 mg of the commercial laccase preparation was dissolved in 1 mL of 20 mM acetate buffer (pH 5.5) and centrifuged at 14000g at 4 °C for 1 h. The supernatant was purified by anion exchange chromatography (DEAE Sepharose, 3 mL, Pharmacia, Uppsala, Sweden). The TvLac (isoform mixture) was eluted with 60 mM ammonium sulfate in 20 mM acetate buffer (pH 5.5). Fractions showing activity are pooled and concentrated using a 30 kDa cutoff membrane (Amicon Ultra 30 kD, Millipore, Ireland). The SDS−PAGE analysis shows two main bands at 62 and 75 kDa and a minor band around 40 kDa. Crude and purified 3692



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μg of TvLac (7.7 × 10−15 to 2 × 10−10 mol using an average molecular weight of 65 kDa) are adsorbed onto the electrodes before the IPA measurement (Figure 3). The S/N increases

Figure 3. S/N dependence on the amount of adsorbed TvLac. The curve-fitting of the data using a hyperbola as a saturation model is shown as a red line. Enlargement of the linear part of this curve (0−75 ng) is shown in the inset. IPA are recorded at 100 mV vs Ag/AgCl in the 150 mM phosphate-citrate buffer (pH 4.4) supplemented with 100 mM NaClO4. All data points are the average of at least three measurements.

with the TvLac amount, reaching a value of 250 at 0.1 μg. Between 0.1 and 1 μg of TvLac, the S/N remains constant (∼250) but sharply decreases when higher amounts of TvLac are adsorbed (>10 μg), suggesting the formation of catalytically inactive aggregates (see Figure S3 of the Supporting Information). Between 0 and 1 μg, the S/N follows a saturation-like mechanism with a dynamic linear range of two decades, ranging from 0.5 to 75 ng of TvLac. In consideration of the linear part of the graph (Figure 3, inset), the slope allows one to estimate a sensitivity of 3027 μg−1 at 100 mV with a limit of detection of 0.5 ng. pH dependency, Interferences, and Inhibitors. The electrochemical assay also offers the opportunity to quickly explore reaction conditions, such as operational pH and identification of inhibitors. In solution, the TvLac shows an optimal activity at pH values around 3 with ABTS as a substrate, as it is usually described in literature (Figure 4).43 Once adsorbed onto the electrodes, the electrochemical activity of the TvLac is assayed in a phosphate-citrate buffer, with a pH

Figure 2. (A) Typical IPA response at 100 mV vs Ag/AgCl, obtained before (black lines) and after (red lines) laccase adsorption onto the screen-printed electrodes. (B) Average intensities of the last 10 s of the IPA measurement before (black dots) and after (white dots) adsorption of 1 μg of TvLac and the corresponding signal calculated according to eq 1 (red dots). IPA are recorded in 150 mM phosphatecitrate buffer (pH 4.4) supplemented with 100 mM NaClO4.

varies from −600 mV to 600 mV (vs Ag/AgCl) using 1 μg of adsorbed TvLac, in order to find the optima potential for the assays (Figure 2B). Before adsorption of the TvLac, intensities higher than 50 nA are obtained at a potential above 300 mV and below −200 mV (vs Ag/AgCl). After the adsorption, higher intensities are measured for the potential below 300 mV. In consideration of the fact that the T1 copper center of this enzyme has a redox potential of ∼790 mV versus the normal hydrogen electrode (NHE) (e.g., ∼590 mV vs Ag/AgCl),42 as well as the low electronic conductivity of the carbon paste, the increase of the current intensity below 300 mV is considered to be related to the TvLac bioelectrochemical activity. Contrary to classical voltammetry, the IPA measurement does not allow one to observe a clear peak or wave, which could undoubtedly be attributed to the TvLac. Nevertheless, a clear electrochemical activity is observed. Moreover, at potentials below −300 mV, the intensities recorded without laccase are not negligible compared to intensities obtained between −100 and 300 mV. Therefore, the S/N was used instead of the signal (S), using as the noise value (N) the intensity of each electrode before the laccase adsorption. The highest S/N is obtained for a potential of 100 mV versus Ag/AgCl, meaning that the signal of the laccase is specific at this potential. Moreover, an excellent S/N (>20) could also be obtained for potentials ranging from −100 to 300 mV versus Ag/AgCl. The IPA measurements are performed simultaneously in the 96 electrodes, meaning that the whole plate could be read in about one minute with our experimental setup showing a remarkable rapidity of this method. Quantitative Analysis. The S/N should reflect the laccase amount deposited onto the electrode. Therefore 0.5 ng to 13

Figure 4. pH dependency of the TvLac activity assayed in solution with ABTS as a substrate (v in μM min−1, black dots) and absorbed onto the electrodes (S/N, red dots). The x axis is the pH of the 150 mM phosphate citrate buffer (pH 2.2−8) in the bulk. IPA at 100 mV vs Ag/AgCl are performed in the 150 mM phosphate-citrate buffer (pH 2.2−8) containing 100 mM NaClO4. 3693



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Figure 5. Michaelis−Menten plots showing the inhibition of the TvLac by (A) fluoride or (B) azide ions determined by a colorimetric assay [ABTS oxidation followed at 420 nm, 20 mM acetate buffer containing 60 mM ammonium sulfate (pH 5.5)]. (C) S/N measured in the presence of 0−5 mM fluoride or (D) in the presence of 0−100 μM azide in 150 mM phosphate-citrate buffer (pH 4.4) containing 100 mM NaClO4 at 100 mV vs Ag/AgCl.

observed with an apparent Vmax value from 229 ± 4 μM min−1 to 102 ± 4 μM min−1 as the azide concentration goes from 0 to 10 μM and an apparent KMABTS value which is slightly affected (ranging from 38 ± 1 μM to 48 ± 1 μM). The Dixon plot leads to a KIN3− of 10.9 μM. All together, these results are consistent with the generally observed inhibition of laccases by fluoride and azide. Once adsorbed onto screen-printed electrodes, the TvLac electrochemical activity is assayed in the presence of 0−5 mM fluoride anion or in the presence of 0−100 μM azide in the electrolyte (Figure 5, panels C and D). The S/N determined at 100 mV versus Ag/AgCl decreases from nearly 300 to 11 as the fluoride concentration increases from 0 to 5 mM and from 300 to 65 when the azide concentration reaches 100 μM. In the case of azide, the electrochemical signal of TvLac is not totally abolished, probably due to its electro-oxidation into nitrogen gas.48 The hyperbolic behavior of these inhibition curves fit well with a saturation mechanism of the enzyme by these anions. These experiments clearly state that the electrochemical signal measured in this work can be attributed to the electrochemical reduction of molecular oxygen by the TvLac. In these electrochemical assays, the oxygen concentration is not controlled and the determination of the true KI value difficulties are not possible: one should also consider that the electrode is polarized and thus should favor the diffusion of anions to its interface. The real anion concentration in the enzyme microenvironment has been up to now difficult to estimate. Moreover, the real KM of immobilized laccase for molecular oxygen is nearly impossible to determine in an open-cell system. Nevertheless, inhibitors could be compared by determining an approximate IC50, defined as the inhibitor concentration at which half the electrochemical signal is lost. The IC50F− and IC50N3− are found to be 280 ± 14 and 1.2 ± 0.4 μM, respectively. This shows that azide is a better inhibitor than fluoride when the TvLac is assayed in solution or adsorbed

ranging from 2.2 to 8.2 (with 100 mM NaClO4 as electrolyte) at 100 mV versus Ag/AgCl (Figure 4). An apparent optimal pH of 5.2 is observed. With the electrode being polarized at +100 mV versus Ag/AgCl and the enzyme being adsorbed onto the carbon paste, the microenvironment of the TvLac is different than in solution. Adsorption could also modify substrate binding, enzyme secondary structure, or catalytic steps. Altogether, this explains the different pH profile observed. The electrochemical assay could also be useful to screen laccase tolerance toward inhibitors. Most of the laccases are sensitive to halide ions (especially fluoride, F−) or azide (N3−), which bound in the T2/T3 center preventing the reduction of molecular oxygen.44,45 Laccases usually follow a ping-pong mechanism oxidizing and releasing first the oxidized organic substrate before the oxygen is reduced to water.24,46 During the direct electron transfer between the electrode surface and molecular oxygen, only inhibitors that compete with molecular oxygen should lead to a diminution of the electrochemical response. Moreover, most of the other molecules which inhibit laccase activity described in the literature are copper-chelating, copper-reacting (sulphydryl) compounds or molecules which unfold the protein structure (sodium dodecyl sulfate). Therefore, they are not true inhibitors and are not considered in this work. First, the inhibition of TvLac by fluoride and azide anions is verified in solution with ABTS as the substrate (Figure 5, panels A and B). The analysis of kinetic parameters using the classical Michaelis−Menten model shows a decrease of the apparent Vmax value from 229 ± 13 μM min−1 to 87 ± 4 μM min−1 as the fluoride concentration increases from 0 to 1 mM. The apparent KMABTS is not significantly affected, varying from 50 ± 10 μM to 43 ± 8 μM. This supports the idea that fluoride is a competitive inhibitor for oxygen binding on the T2/T3 center with a KIF− of 0.43 mM, as it is described for other laccases.46,47 In the presence of azide, a similar behavior is 3694



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CuCl2) and nickel (Ni2+ from NiCl2) ions are coadsorbed at concentrations of 3, 30, and 170 mM. For both cations, the S/ N value is below 3 when the concentration of cation is below 30 mM. At 170 mM, the S/N for Ni2+ reaches about 5 and the S/N for Cu2+ is about 20. Therefore, the reduction of these ions is not observed on the screen-printed electrodes at 100 mV at low concentration. When TvLac is coadsorbed with the low cation concentration (3 mM), the S/N is not affected but it is strongly decreased at 30 mM of cations (S/N ∼40). There is no signal at a cation concentration of 170 mM. This suggests that laccase is inactivated when adsorbed in the presence of these cations or more probably in the presence of the counterions as observed previously (chloride). Finally, cysteine and DTT are added into the electrolyte solution because they are used as reducing agents during purification, but also because they strongly interfere with colorimetric assays of laccases:52 oxidized ABTS is spontaneously reduced by both DTT and cysteine, while 2,6dimethoxy phenol (2,6-DMP) is only oxidized by DTT. Cysteine and DTT at 10 μM give a S/N of 5 and 1.8 and reach 23 and 15 at 50 μM, respectively. This means they could be oxidized at 100 mV onto a screen-printed carbon electrode, which is consistent with the E°′ of the cystine/cysteine redox couple (estimated between 0.21 and 0.23 V vs NHE).53 Once the TvLac is adsorbed, the S/N obtained in the presence of cysteine is negligible (170) is obtained for the BSA concentration below 1 μg. At higher concentration, S/N strongly decreases and reaches only 15, when 3 μg of BSA is coadsorbed with the laccase. This suggests that protein aggregates are formed at the electrode surface and prevent the direct contact between the laccase and the electrode or prevent oxygen diffusion through this aggregate. Nevertheless, this also means that a minimal purity for the laccase of ∼50% is necessary to obtain a reliable signal. This could be difficult to achieve, depending of the quality of the enzyme overexpression system. The next interfering species studied in this work are those which can be coadsorbed with a his-tagged protein. The type of labeling is now commonly used during protein overexpression. When 3 μL of 100 mM or 250 mM imidazole solutions are deposited onto the electrodes, low S/N values (4−6) are obtained. The TvLac adsorbed in the presence of imidazole at these concentrations presents significant S/N values (139 and 274), meaning that his-tagged laccase samples eluted from a nickel affinity chromatography could be directly analyzed with this electrochemical assay. The higher signal obtained at a high imidazole concentration (250 mM) could be due to the imidazole in the protonated form (∼96% of the imidazole molecules at pH 5.5). This form could be electrochemically reduced and could slightly enhance the electrochemical signal.49 The possible partial unfolding of the laccase during adsorption could also be prevented by the presence of the imidazolium cation.50 During the expression of laccases in bacterial systems, culture media is highly supplemented by copper ions51 and nickel could leak from an affinity chromatography column. Therefore, copper (Cu2+ from

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CONCLUSION We describe for the first time an electrochemical method for the quantitative measurement of the direct electron transfer between an enzyme and an electrode in a 96-well format. It offers the unique opportunity to quickly screen electrochemical media, inhibitors, or reaction conditions for an adsorbed redox enzyme. It is a powerful tool for the screening of enzyme 3695



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libraries because the measured signal is of an electrochemical nature on the contrary to other colorimetric methods. The analysis of interfering species reveals that the composition of the enzyme media during adsorption is crucial, but the rapidity of this method offers a unique opportunity to optimize the adsorption conditions for a defined enzyme. Moreover, this method is reagentless, greatly decreasing the costs of the assay, particularly if one considers that the printed circuit board could easily be reused. In further studies, this method will be applied to screen laccase libraries in order to find mutants with improved direct electron transfer rates.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel:+33 (0)4 72 43 14 84. Fax: +33 (0)4 72 44 79 70. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The French ministry of Science and Education is gratefully acknowledged for the funding of S.A. Dr. B. Leca-Bouvier is acknowledged for fruitful discussions. Dr. C. Marquette is gratefully acknowledged for the SEM imaging.

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REFERENCES

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A 96-Well Electrochemical Method for the Screening of Enzymatic

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