Dependent Oxidoreductases Using Boron-Doped Diamond

Article pubs.acs.org/ac

Toward High-Throughput Screening of NAD(P)-Dependent Oxidoreductases Using Boron-Doped Diamond Microelectrodes and Microfluidic Devices Ryo Oyobiki,† Taisuke Kato,‡ Michinobu Katayama,§ Ai Sugitani,‡ Takeshi Watanabe,‡ Yasuaki Einaga,‡,|| Yoshinori Matsumoto,§ Kenichi Horisawa,† and Nobuhide Doi*,† †

Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan § Department of Applied Physics and Physico-Informatics, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan || JST CREST, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan ‡

ABSTRACT: Although oxidoreductases are widely used in many applications, such as biosensors and biofuel cells, improvements in the function of existing oxidoreductases or the discovery of novel oxidoreductases with greater activities is desired. To increase the activity of oxidoreductases by directed evolution, a powerful screening technique for oxidoreductases is required. In this study, we demonstrate the utility of boron-doped diamond (BDD) microelectrodes for quantitative and potentially high-throughput measurement of the activity of NAD(P)-dependent oxidoreductases. We first confirmed that BDD microelectrodes can quantify the activity of low concentrations (10−100 pM) of glucose6-phosphate dehydrogenase and alcohol dehydrogenase with a measuring time of 1 ms per sample. In addition, we found that poisoning of BDD microelectrodes can be repressed by optimizing the pH and by adding L-arginine to the enzyme solution as an antiaggregation agent. Finally, we fabricated a microfluidic device containing a BDD electrode for the first time and observed the elevation of the oxidation current of NADH with increasing flow rate. These results imply that the combination of a BDD microelectrode and microfluidics can be used for high-throughput screening of an oxidoreductase library containing a large number (>106) of samples, each with a small (nanoliter) sample volume.

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a smaller amount of samples. Indeed, Abdellaoui et al. recently performed an electrochemical screening of NAD-dependent dehydrogenase using carbon-based electrodes.20,21 In this study, we used a boron-doped diamond (BDD) electrode22−24 for the detection of NAD(P)-dependent oxidoreductase activity. Because BDD electrodes have a wide potential window (approximately 3.5 V) and a low background current, which allows for highly accurate and quantitative measurements, they have been used for various electroanalytical measurements.25−35 However, to the best of our knowledge, only one study has reported the detection of NAD-dependent alcohol dehydrogenase activity using BDD electrodes.36 Here, we examined the possibility of using a BDD microelectrode for the quantitative and potentially high-throughput measurement of the activities of NAD(P)-dependent oxidoreductases. First, we investigated whether BDD microelectrodes are capable of quantifying the activity of low concentrations of enzymes with a short measuring time. Second, we attempted to minimize

AD(P)-dependent oxidoreductases are widely distributed enzymes that catalyze the transfer of electrons from a broad range of substrates to NAD(P) and thus can be used in many applications, such as biosensors,1,2 biofuel cells,1−4 and cofactor regeneration.5 For example, a large variety of biofuel cells have been developed using NAD-dependent dehydrogenases.4 However, the power output and lifetime of enzymebased biofuel cells are limited by the activity and stability of the enzymes. Therefore, improvements in the properties of existing oxidoreductases or the discovery of novel oxidoreductases with increased activity and stability is desired. Directed enzyme evolution6−11 is one of the most attractive approaches for this purpose. Directed evolution involves repeated rounds of randomization/amplification and screening/selection steps. Currently, several NAD-dependent dehydrogenases with improved activity and stability have been screened from 103−104 clones by monitoring the absorbance of NADH at 340 nm12−14 or by detecting colored formazan products produced by the reduction of tetrazolium salts with NADH15−19 in microtiter plates or on membrane filters. In comparison with conventional spectrophotometric/colorimetric methods, electrochemical methods can be used for more high-throughput detection of NADH in © 2014 American Chemical Society

Received: May 21, 2014 Accepted: September 1, 2014 Published: September 11, 2014 9570

dx.doi.org/10.1021/ac501907x | Anal. Chem. 2014, 86, 9570−9575

Analytical Chemistry

Article

Figure 1. Electrochemical measurements of the activity of NAD(P)-dependent oxidoreductases using boron-doped diamond (BDD) microelectrodes. (A) SEM image of the tip of the BDD microelectrode. (B,C) Cyclic voltammograms of (B) NADH produced by yeast alcohol dehydrogenase (ADH) in the presence of various concentrations of ethanol and (C) NADPH produced by yeast glucose-6-phosphate dehydrogenase (G6PDH) in the presence of various concentrations of glucose-6-phosphate (G6P). The scan rate was 100 mV s−1. (Inset) A Lineweaver−Burk plot for determining the kcat and Km values. (D) The schematic representation of the redox reactions for two enzymes. For the BDD measurements, all components are freely diffusing in the solution.

an MM605 projection camera (Nanometric Technology, Tokyo, Japan). A BDD film was deposited onto a ceramic wafer as described above. The wafers were coated with Omni coat (MicroChem, Newton, MA) using a spin coater and were then baked on a hot plate at 200 °C for 5 min. Similarly, the negative photoresist SU-8 3050 (MicroChem) was spin-coated on the wafers, which were baked at 100 °C for 3 h and then exposed to UV irradiation for 25 s through the emulsion mask using an LA310k mask aligner (Nanometric Technology). After postbaking at 100 °C for 30 min, the wafers were immersed in the SU-8 developer PGMEA (MicroChem). BDD etching using the Bosch process was performed with an inductively coupled plasma reactive ion etching (ICP-RIE) system (SPP Technologies, Hyogo, Japan) under the described conditions.37 Finally, the SU-8 3050 was removed by applying a RemoverPG stripper (MicroChem) at 80 °C for 1 h, and Ag/AgCl ink (BAS, Tokyo, Japan) was then pasted on the BDD as a reference electrode, which was then baked at 120 °C for 5 min. Microfluidic Devices. The wafer of the above-described BDD chip electrode was spin-coated again with a 50 μm layer of SU-8 3050, baked at 100 °C for 15 min, and then exposed to UV irradiation for 10 s through an emulsion mask for microfluidic patterning. After postbaking at 100 °C for 3 min, the wafer was immersed in the SU-8 developer PGMEA. The resulting SU-8 3050 flow cell was covered with a 5 mm × 10 mm PDMS plate whose inlet and outlet holes were punched with a 1 mm diameter biopsy punch (Kai Industries, Tokyo, Japan). Electrochemical Measurements. Voltammograms were recorded using a HSV-100 potentiostat (Hokuto Denko, Tokyo, Japan) with a standard three-electrode configuration.

electrode poisoning by optimizing the pH and adding antiaggregation agents to the enzyme solution. Finally, we fabricated a microfluidic device containing a BDD chip electrode for use in the high-throughput detection of oxidoreductases.

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EXPERIMENTAL SECTION

Enzymes and Chemicals. Yeast alcohol dehydrogenase (ADH), glucose-6-phosphate (G6P), ß-nicotinamido adenine dinucleotide (NAD+), and ß-nicotinamido adenine dinucleotide phosphate (NADP+) were obtained from Oriental Yeast (Tokyo, Japan). Yeast glucose-6-phosphate dehydrogenase (G6PDH), L -arginine (Arg), glycylglycine, and polydimethylsiloxane (PDMS) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Ethanol, 10× PBS (phosphate-buffered saline) buffer (pH 7.4), and MgCl2·6H2O were obtained from Nacalai Tesque (Kanagawa, Japan). Preparation of BDD Microelectrodes and BDD Chip Electrodes. The BDD microelectrodes were prepared as previously described.28,30 Briefly, BDD thin films were deposited on tungsten needles (20−40 μm in diameter) using a microwave plasma-assisted chemical vapor deposition (MPCVD) system (Cornes Technologies, Tokyo, Japan). A mixture of acetone and trimethoxyborane was used as the boron and carbon source at a B/C ratio of 1:100. The surface morphology and crystalline structures of the BDD thin films were confirmed using scanning electron microscopy (SEM). The BDD microelectrode was insulated by prepulled glass capillaries and epoxy resin EP001 (Cemedine, Tokyo, Japan). BDD chip electrodes for microfluidic devices were prepared as previously described,37 with the following modifications. An emulsion mask for a three-electrode system was produced with 9571



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Figure 3. Effect of (A) L-arginine (Arg) and (B) pH on the cyclic voltammograms (CVs) of NADPH produced by G6PDH (26 nM). An almost complete inhibition of electrode poisoning for (C) G6PDH and (D) ADH (monitored by CVs of NADH) was achieved in the presence of 10 mM Arg at pH9.0. Before each measurement, the current level of the BDD microelectrode was recovered by cathodic reduction at −2.0 V in 1 M HCl for 1 min, though the current values were somewhat varied after the reduction treatment.

tubing (int. 0.25 mm; ext. 0.75 mm; Chukoh Chemical Industries, Tokyo, Japan) connected to a 1 mL syringe (Terumo, Tokyo, Japan). The flow rate was controlled by a FP-1000 syringe pump (Melquest, Toyama, Japan) and was maintained between 5 and 20 μL/sec. Enzymatic Reactions. All oxidoreductase reactions were performed at room temperature for 2 h. The ADH reaction was initiated by adding ADH to the assay mixture (1× PBS buffer, pH 7.4, 0.01−1 M ethanol, 10 mM NAD+). The G6PDH reaction was initiated by adding G6PDH to the assay mixture (100 mM glycylglycine buffer, pH 8.0, 20 mM MgCl2, 0.01− 1 mM G6P, 10 mM NADP+). To assess the enzymatic activity, NAD(P)H was quantified based on the change in OD340 using a SAFIRE plate reader (Tecan, Männedorf, Switzerland) or based on the voltammograms, as described above. The kinetic parameters kcat and Km were calculated using a Lineweaver−Burk plot. Each reaction was initiated by the addition of 4.5 nM enzyme to a reaction mixture containing the substrate (2, 2.5, 3.5, 5, and 10 mM) at room temperature. In each case, initial rates were estimated from the change in absorbance or from the voltammograms over the first 5 min of reaction, for which excellent linearity was observed. Linear leastsquares fitting of 1/V0 versus 1/[S] provided Km and Vmax values.

Figure 2. (A) Cyclic voltammograms of NADH solutions (0.1, 0.5, and 1 mM). (B) Peak currents of NADH oxidation detected by a BDD microelectrode with a measurement time between 0.001 and 1 s. The applied potential was 0.7 V. The error bars represent the standard deviation of 10 independent experiments. (C) A calibration curve of the peak current versus NADH concentration for a measurement time of 0.001 s.

All measurements were performed at room temperature (23 ± 2 °C). For BDD microelectrode measurements, the Ag/AgCl electrode was used as the reference electrode, and a Pt wire was used as the counter electrode. The three electrodes were dipped into a sample solution in a microplate well (Thermo Fisher Scientific, Waltham, MA) using a three-dimensional manual manipulator (Bex, Tokyo, Japan). For BDD chip measurements, a sample solution was injected into the microfluidic device via polytetrafluoroethylene (PTFE) 9572



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Figure 4. (A) Schematic for the BDD three-electrode system. The width of the fluidic channel was 200 μm (inset; blue lines), the size of the working electrode was 1 mm × 200 μm, the reference electrode was 100 μm × 100 μm, and the counter electrode was 100 μm × 200 μm. The distance between each pair of electrodes was 100 μm. (B) The BDD three-electrode system was produced by reactive ion etching, and Ag/AgCl ink was pasted on the tip of the reference electrode (left). Then, an SU-8 photoresist channel (light blue) was produced on the BDD three-electrode system by photolithography (middle), which was covered by a flat PDMS plate (dark blue) (right). (C) Cross sections of the illustrations shown in panel (B).

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RESULTS AND DISCUSSION Quantitative Measurements of Enzyme Activities by BDD Microelectrodes. First, we attempted to detect the activity of an NAD-dependent oxidoreductase, yeast alcohol dehydrogenase (ADH), by an electrochemical measurement using the BDD microelectrode (Figure 1A). As shown in Figure 1B, elevation of the oxidation current was observed for increasing concentrations of the substrate ethanol. The kinetic parameters calculated from a Lineweaver−Burk plot (Figure 1B, inset) were comparable to those obtained from a conventional absorbance measurement (kcat = 10.5 s−1 and Km = 3.7 mM for ethanol). We also detected the activity of an NADP-dependent oxidoreductase, yeast glucose-6-phosphate dehydrogenase (G6PDH), using the BDD microelectrode (Figure 1C). The kcat and Km values (Figure 1C, inset) were comparable to those obtained from the OD340 measurement (kcat = 136 s−1 and Km = 0.46 mM for G6P). These results suggest that electrochemical measurements using the BDD microelectrode can effectively quantify the activity of NAD(P)-dependent oxidoreductases on a variety of substrates (Figure 1D). Reduction of Measurement Time and Enzyme Concentration. With the aim of developing a method for high-throughput screening of an oxidoreductase library (>106) using the BDD electrode, we further estimated lower limits of the measurement time and enzyme concentration. When we measured the NADH concentration with a measurement time between 0.001 and 1 s using a standard potentiostat, a quantitative measurement could be achieved in at least 0.001 s per sample (Figure 2).

For the shortest measurement time of 0.001 s, we determined the kcat and Km values again for various concentrations of ADH and G6PDH to estimate the minimum enzyme concentration that could be measured. Consequently, the enzyme activity was quantified at ≥100 pM for ADH (kcat = 8.5− 15.4 s−1 and Km = 1.2−4.4 mM for ethanol) and at ≥10 pM for G6PDH (kcat = 142−234 s−1 and Km = 0.36−0.66 mM for G6P). These results suggest that a BDD measurement system has the potential to screen >106 samples per hour, each containing at least 10−100 pM enzyme. Because the three-electrode system in the microfluidic device described in the last section requires a volume of at least ∼10 nL per sample, the total amount of enzyme required for 106 samples is estimated to be 10 μL of 10−100 nM enzyme solution, which can easily be obtained from one reaction tube of a standard cell-free protein synthesis system. Reduction of Electrode Poisoning. The adsorption of proteins to the electrode surface often leads to a decrease in the response current during repetitive electrochemical measurements.38 Although the effect of electrode “poisoning” is known to be small for BDD electrodes in comparison to other electrodes,25 we examined poisoning of the BDD microelectrode in enzyme solutions during continuous detection of oxidoreductase activity. When we performed five continuous cycles of electrochemical measurements of NAD(P)H in the presence of high concentrations (∼30 nM) of the ADH and G6PDH enzymes, the oxidation current gradually decreased, likely due to electrode poisoning (Figure 3A−D, left panel). Consequently, we attempted to reduce poisoning of the BDD microelectrode. 9573



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Microfluidic Devices Containing BDD Chip Electrodes. In a previous study, a BDD chip electrode was fabricated for single-drop (10 μL) analysis.37 In this study, we constructed a microfluidic channel on a BDD chip electrode for continuousflow detection of a small (nanoliter) reaction volume. First, we designed a three-electrode system in which the size of the working electrode within the fluidic channel was >10-fold larger than those of the other two electrodes (Figure 4A) to enhance the detection sensitivity. The BDD patterning was achieved by reactive ion etching (RIE) as previously described,37 and Ag/AgCl ink was pasted on the tip of the reference electrode. Because the surface of the available ceramic wafer was rough and we could not obtain a smooth BDD-ceramic wafer with a grinder (data not shown), we were concerned that liquid leakage might occur if a normal PDMS flow channel was situated directly on top of the wafer. To overcome this problem, we fabricated a SU-8 photoresist channel on the BDD-ceramic wafer by photolithography, which was covered by a flat PDMS plate with PTFE tubing (Figure 4B, C). In the resulting microfluidic device, no leakage was observed at high flow rates (up to 20 μL/s), allowing us to detect, in principle, a 20 to 30 nL volume of sample at a rate of several hundred samples per second. Next, we attempted to detect NADH by electrochemical measurements using the BDD chip electrode in the microfluidic device. We confirmed an elevation of the oxidation current with increasing NADH concentrations in the nonflow condition (Figure 5A), and the intensity observed was comparable to the value obtained from the BDD microelectrode measurements (Figure 2A); however, the potential peak was slightly shifted, most likely due to the difference in the reference electrode. Moreover, in the flow condition, we found that the oxidation current increased as the flow rate increased (Figure 5B). This result may be attributed to a more efficient supply of fresh NADH to the electrode surface as a result of the higher flow rate. When the peak current was monitored over several hours by continuous amperometry in the flow condition, the relative standard deviation was smaller than 5%.

Figure 5. Electrochemical measurements using the microfluidic device. (A) Cyclic voltammograms (CVs) of NADH solutions (10 μM and 100 μM) in nonflow conditions. (B) CVs of NADH solution (10 μM) in different flow conditions (5 and 20 μL per second).

First, we examined the effect of different concentrations of (Arg), which is known to repress the formation of protein aggregates and stabilize proteins in solution,39 and observed a reduction of electrode poisoning; the most effective result was obtained for 10 mM Arg (Figure 3A, middle panel). This result suggests that BDD microelectrode poisoning can be reduced by inhibiting enzyme aggregation. In contrast, we found that detergents (0.1−1% of Triton X-100, NP-40 and Tween-20) had no effect on electrode poisoning (data not shown). Next, we examined the pH dependence of electrode poisoning because proteins are prone to aggregate at a pH near their isoelectric point (pI). As expected, the extent of electrode poisoning at pH 9.0 was less than that at pH 8.0 for G6PDH (pI = 5.92) (Figure 3B, middle panel). However, electrode poisoning again increased at pH 10.0, which is farther from the pI of the enzyme (Figure 3B, right panel). This result most likely arose because G6PDH has an optimum activity near pH 8.0−9.0, and this activity is drastically decreased at pH 10.0,40 where the enzyme would denature and tend to aggregate. Finally, we combined the two conditions (10 mM Arg and pH 9.0) and observed an almost complete inhibition of electrode poisoning for G6PDH (Figure 3C, right panel). Similar results were obtained for ADH (pI = 6.26) (Figure 3D, right panel).

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L-arginine

CONCLUSIONS

In this study, we demonstrated the utility of BDD electrodes for quantitative and potentially high-throughput measurement of the activity of NAD(P)-dependent oxidoreductases. We first confirmed that the BDD microelectrode can quantify the activity at low concentrations (10−100 pM) of G6PDH and ADH with a measuring time of at least 1 ms. Next, we observed that electrode poisoning can be diminished by changing the pH and adding Arg to the enzyme solution. Finally, we fabricated, for the first time, a microfluidic device containing a BDD chip electrode and observed an enhancement of the oxidation current of NADH with increasing flow rate. These results imply that the combination of a BDD electrode and microfluidics permits repetitive measurements of the NAD(P)-dependent oxidoreductase activity of a large number (>106) of samples per hour and should thus be useful to reduce the time and cost of screening for oxidoreductases with greater activities. Now we are trying to combine our microfluidic device with microbead display41,42 for high-throughput screening of NAD(P)-dependent oxidoreductases with high activity and stability from a mutated gene library or a metagenomic DNA library, and the results will be published in the future. 9574



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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +81 45 566 1440. Tel.: +81 45 566 1772. Author Contributions

R.O. performed all experiments. T.K., T.W., and Y.E. contributed to the preparation of the BDD microelectrodes. M.K., A.S., T.W., Y.E., and Y.M. assisted in preparing the microfluidic devices containing the BDD chip electrodes. R.O., T.W., Y.E., Y.M., K.H., and N.D. contributed to the planning and design of the project and manuscript writing. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank members of our laboratory, especially Shunya Shitara and Shota Shimokihara, for experimental advice and useful discussions. This work was supported by a Grant-in-Aid for Scientific Research (24656508) from the JSPS of Japan, a Keio Gijuku Academic Development Fund, and the Asahi Glass Foundation.

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Dependent Oxidoreductases Using Boron-Doped Diamond

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