Microfluidic Separation of Redox Reactions for Coulometry Based on

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Microfluidic Separation of Redox Reactions for Coulometry Based on Metallization at the Mixed Potential Kazuhiro Ikemoto, Takafumi Seki, Shohei Kimura, Yui Nakaoka, Shinnosuke Tsuchiya, Fumihiro Sassa, Masatoshi Yokokawa, and Hiroaki Suzuki Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01234 • Publication Date (Web): 31 Aug 2016 Downloaded from http://pubs.acs.org on September 3, 2016

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Analytical Chemistry

Microfluidic Separation of Redox Reactions for Coulometry Based on Metallization at the Mixed Potential Kazuhiro Ikemoto, Takafumi Seki, Shohei Kimura, Yui Nakaoka, Shinnosuke Tsuchiya, Fumihiro Sassa, Masatoshi Yokokawa, Hiroaki Suzuki* Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan ABSTRACT: Coulometric detection of an analyte in a solution at nanoliter scale was conducted by having redox reactions proceed simultaneously on a platinum electrode. The analyte was oxidized on a part of the electrode in one flow channel and silver was deposited on an array of circular microelectrodes formed in another flow channel at a mixed potential. Coulometric determination of the deposited silver showed a steep change in the generated charge as a result of the complete oxidation of silver. The short measurement time after the start of the coulometry suppressed the increase in background charge, resulting in significant lowering of the detection limit. The lower detection limit for H2O2 was 30 nM (3σ). To improve selectivity and minimize the influence of coexisting interferents, the shifting of the mixed potential, application of a permselective membrane, and electrochemical elimination of the interferents were effective modifications.

The reduction of sample and reagent volumes is becoming a critical requirement in chemical analyses, particularly in those that are targeted toward cells. For the quantitative analysis of molecules in small droplets, the utility of electrochemistry has been demonstrated. Even electroanalyses at picoliter scale have been successfully conducted, with a particular emphasis on sin1-4 gle-cell analyses. As evidenced by several previous studies, the use of microfabricated electrodes promises to expand the scope of this technology. Concomitantly, the recent progress in microfluidic technology, particularly that based on the transport of droplets (or 5-8 plugs), widely expands the possibility of realizing highly sophisticated, multiplexed analytical systems, which is the final goal of our present study. For the electroanalysis of molecules in very small volumes, cyclic voltammetry and amperometry are typically used. Although voltammetric techniques are indispensable in revealing the behavior of redox systems, amperometry is more suitable for multiplex analysis and/or for on-site analysis. Nevertheless, as the volume of a solution decreases to the nano- or picoliter order, highly sensitive, reliable amperometric detection becomes difficult because of the nonnegligible consumption of the analyte, which is manifested as a rapidly decaying current after the applica2,4,9 tion of a potential. To avoid this complication and achieve highly sensitive detection, recovery of the analyte by redox cycling using an interdigitated microelec10-12 13-16 trode array or a micro- or nano-cavity can be a solution. However, a problem with this method is that

the analytes are limited to those that undergo reversible redox reactions. In this respect, coulometry is more general and applicable to even irreversible redox compounds with simple electronics for operation and signal 17 acquisition. In coulometry, a generated charge, or current integrated over time, increases gradually, in contrast to the case of amperometry, facilitating the measurement even when the current decay is rapid. Furthermore, unlike other techniques that may be affected by changes in conditions such as temperature and pH, calibration is not necessary if all the analytes 9 are consumed exhaustively during the measurement. This is also beneficial for batch-fabricated disposable single-use analytical devices because incorporating the means for calibration always poses a problem. In conventional coulometry, the generated charge increases monotonically as time elapses and gradually 9,18 levels off as the analyte is depleted (Figure 1A). In contrast to amperometry, however, background charge also increases as time elapses because the background current is integrated as well before obtaining the change originating from the Faradaic current. Because of this, the standard deviation (σ) of the background charge increases, and the detection limit, often defined as the signal that is comparable with 3σ of the variation of the background, increases. Therefore, although the detection sensitivity can be improved by measuring the charge after a sufficient time elapses, it does not necessarily lead to a lower detection limit. In this regard, if the Faradaic current is saturated within a very short time and the background charge can be ACS Paragon Plus Environment


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Figure 1. Typical changes in charge observed by conventional coulometry (A) and using our coulometric method and device (B). The dashed lines indicate the times at which charge measurements are made. minimized, as shown in Figure 1B, both the sensitivity and detection limit can be improved significantly by obtaining data shortly after the start of the measurement. To realize this concept, we developed a system in which redox reactions simultaneously take place in different areas of a single electrode to convert the analyte concentration information in one area into the amount of metal deposited in another. This technique, in the form of silver metallization, has been used in 19-29 various electrical and electrochemical methods. Also, in attempts to improve the sensing performance, the locations for analyte oxidation and metal ion reduction were separated, and the amount of deposited metal was measured by anodic stripping 30,31-33 voltammetry. The technique to separate the oxidation and deposition of the metal would also be advantageous in coulometry for realizing the responses described in Figure 1B. We used two microfabricated flow channels (A and B) to separate the redox reactions and allow spontaneous metallization to proceed (Figure 2). A critical feature of our device is the use of different geometries for the shapes of the electrodes that oxidize the analyte and deposit the metals (Figure 2C), which affords high sensitivity due to the excellent collection efficiency and effectively lowers the detection limit. In this study, we demonstrate that analytes can be collected efficiently and the background charge can be minimized simultaneously with this approach. Using our device, the detection limit was lowered by one order of magnitude compared with conventional coulometry.

Figure 2. Structure of the coulometric device (type I). (A) Top view of the type I device. Dimensions of the device are 17 mm × 30 mm. (B) Cross section of the electrode area in flow channel A along X – X′. (C) Magnified view of the microelectrode array (MEA) in flow channel A (left) and the pinhole structure of the working electrode in flow channel B (right). (D) Top view of the type II device. WE: working electrode. RE: reference electrode. AE: auxiliary electrode.

EXPERIMENTAL SECTION Reagents and Materials. Reagents and materials used for the fabrication and characterization of the devices were obtained from the following commercial sources: Glass wafers (no. 7740, 3 inch, 500 µm thick) from Corning Japan (Tokyo, Japan); a positive photoresist, S1818G, from Dow Chemical (Midland, MI); a thick-film photoresist, SU-8 25, from MicroChem (Newton, MA); poly(dimethylsiloxane) (PDMS), KE-

1300T, from Shin-Etsu Chemical (Tokyo, Japan); a photocurable polymer gel, PVA-SbQ, SPP-H13, from Toyo Gosei (Chiba, Japan); H2O2 (30 %), L-glutamate, Nafion (5%), and silver nitrate from Wako Pure Chemical Industries (Osaka, Japan); bovine serum albumin (BSA) and p-aminophenol (PAP) from Sigma-Aldrich Japan (Tokyo, Japan); and L-glutamate oxidase from Strep-1 tomyces sp. (330 U mL ) from Yamasa (Chiba, Japan).

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Table 1. Structure of the electrodes used in flow channel A Electrode

Total area (mm2)

Number of strips

Width of a strip (µm)

Interstrip distance (µm)*

1

0.24

40

10

130

2

0.24

20

20

260

3

0.24

10

40

520 * Edge to edge

silver pattern, and AgCl was grown from there. Details of formation of the AgCl are described in the Supporting Information. The Ag/AgCl structure realized a du34 rable thin-film reference electrode. Contact pads were formed on one side of the chip to connect the electrodes to an electrochemical workstation (Autolab PGATAT12, Metrohm Autolab, Netherlands). To apply pressure to move the solution, two microsyringe pumps (IM-9B, Narishige, Tokyo, Japan) were connected to flow channel A and the auxiliary flow channel through silicone tubes (inner diameter: 500 µm) connected to the inlets of the flow channels. Silicone tubes were also connected to the outlets of the main flow channel for wastes. To demonstrate the effect of the removal of electrochemical interferents that may exist in the analyte solution, a Nafion layer was additionally formed on the electrode in flow channel A. Details of the formation of this layer are described in the Supporting Information. Another device shown in Figure 2D (type II) was also used to remove electroactive interferents for biosensing. The basic structures used for detection in flow channels A and B were the same as those of the type I device. In this device, however, an additional flow channel (flow channel C) and a platinum electrode that extended between flow channels A and C were added to deposit silver for the oxidative removal of interferents in flow channel A. The exposed areas of the electrode additionally formed in flow channel A 2 and C were 3.4 mm . The flow channels A and C were also connected with each other via a liquid junction, similar to the one between flow channels A and B. The enzyme L-glutamate oxidase (L-GlOx) was immobilized on the electrode of flow channel A. Equal volumes of 10-units/mL L-GlOx, 0.1 wt% bovine serum albumin (BSA), and 0.1 wt% glutaraldehyde solutions were mixed. Then, 4 µL of the mixture was spread on the surface of the array of platinum strips using a micropipette. Subsequently, the chip was immersed in 0.1 M glycine solution for 30 min to treat the unreacted glutaraldehyde termini, and then rinsed well with distilled water. Measurement Procedures and Principle of Detection. To start the coulometric analysis, flow channel B was initially filled with a solution containing metal ions. In most of the experiments, a 100 mM KNO3 solution containing 1.0 M AgNO3 was used. For comparative experiments, a 100 mM KNO3 solution containing 1.0 M CuSO4 or a 100 mM KCl solution containing 1.0 M NiCl2 was alternatively used. Then, a plug of an analyte solution was formed in flow channel A, using an array of rhombuses in the auxiliary flow 18 channel. Details of the volume measurement and the formation of the plug are described in the Supporting Information. In our present experiment, seven rhombuses were used to make a plug of 700 nL. Immediately after the plug was moved onto the electrode in flow channel A and connected to the solution in flow chan-

Other reagents were obtained from Wako Pure Chemical Industries. All the chemicals were reagent grade unless otherwise noted. All solutions were prepared with Milli-Q water. The concentration of H2O2 in the standard solution was checked using a calibrated refractometer (PAL-39S, Atago, Tokyo, Japan). Device fabrication. The flow channel structure was created with PDMS by replica molding using patterns of the thick-film photoresist (SU-8 25) as a template. The structure was closed with a glass substrate with electrodes (vide infra). The flow channels were 600 µm wide and 540 µm high. Only the sensing region of flow channel A was constricted to a height of 70 µm (Figure 2B). The flow channels were connected via a liquid junction formed with a photocurable polymer gel (PVA-SbQ) containing KCl. Details of the formation of the junction are described in the Supporting Information. To form the electrodes, metal thin-films were deposited on the glass substrate by sputtering and were patterned by lift-off using a photoresist protecting layer. First, a layer of platinum electrodes was patterned to form the electrode in flow channel A, the working and auxiliary electrodes in flow channel B, and the base layer for the reference electrode. Then, silver patterns were formed only on the reference electrode areas. The electrode in flow channel A consisted of an array of strips (Figure 2C). Unless otherwise noted, an array of electrodes, 10 µm wide, with 130 µm inter-electrode distances (edge to edge) was used (#1 in Table 1). The electrode geometry and dimensions were the same as those of our previous device, for comparison. Other than this, two different electrode structures (#2 and #3 in Table 1) were used. The electrode in flow channel B was used as the working electrode in the coulometric measurements and had 26 pinholes, 10 µm in diameter, formed in an insulating layer of the positive photoresist (Figure 2C). The pinholes delineated the microelectrodes. To apply a potential to the working electrode and to detect generated current, a Ag/AgCl reference electrode and a platinum auxiliary electrode were also formed in flow channel B. For the reference electrode, three pinholes, 30 µm in diameter, were formed in the positive photoresist insulating layer on a ACS Paragon Plus Environment


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Figure 3. Coulometric detection of hydrogen peroxide. (A) Response profiles observed with H2O2 solutions of different concentrations. The dashed lines are a representative response curve for 10 µM H2O2 and the change in the background (BG) recorded by conventional coulometry using the same electrode and flow channel structures. (B) Relationship between the charge and the concentration of H2O2. The dashed line is a guide for the eyes. Five measurements were made for each data point in the figure, and averages and standard deviations are shown. nel B via the liquid junction, oxidation of the analyte started. At the same time, metal deposition started on the electrode in flow channel B connected to the electrode in flow channel A. Unless otherwise noted, the metal deposition time was 5 min. The solutions in flow channels A and B were flushed by introducing air from the inlets. Then, flow channel B was washed with distilled water, dried with an air flow for 10 s, and filled with a 100 mM KCl solution. The amount of silver or other metals deposited on the platinum electrode was measured by coulometry by applying +0.7 V using the on-chip reference and auxiliary electrodes formed in flow channel B. The charge was measured at 2 s after the application of the potential. Since the response decreased gradually when the same device was used repeatedly (see Figure S-1), different chips were used to obtain data under fixed conditions.

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Figure 4. Dependence of the measured charge on deposition time. (A) Charges observed when deposited metal and the analyte are changed. Materials oxidized and deposited in flow channel A and B are 10 µM H2O2 and silver (▲), 10 µM H2O2 and copper (●), and 10 µM L-ascorbic acid and silver (■). (B) Dependence of the charge on the deposition time measured using electrode No. 1 (▲), No. 2 (■) and No. 3 (◆) with 10 µM H2O2 and silver deposited as in (A). The horizontal broken lines indicate the calculated value from the total amount of H2O2 in the 700 nL solution. Five measurements were made for each data point in the figure, and averages and standard deviations are shown. Measurement of the mixed potential. To measure the mixed potential of the electrodes connected and exposed in the two flow channels, the inlet of flow channel B was made larger in order to insert the liquid junction of a commercial Ag/AgCl reference electrode (2080A-06T, Horiba, Kyoto, Japan). Diffusion of the internal solution of the reference electrode was avoided by forming a PVA-SbQ gel containing 0.1 M KNO3 at the entrance of the flow channel. The potential was


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measured using an electrometer (AutoLab PGSTAT12, Eco Chemie, Utrecht, Netherlands). All the experiments were conducted at room temperature, and measurements were made five times for each point in the graphs.

RESULTS AND DISCUSSION Collection of Analytes and Deposition of Silver. The device shown in Figure 2 (type I) was characterized using H2O2 as a representative analyte. Figure 3A shows the time courses of charge generation observed following the oxidation of the deposited silver with H2O2 standard solutions of different concentrations. For comparison, the time course of the current obtained by directly oxidizing 10 µM H2O2 via ordinary coulometry using our previous device is also shown, along with the background charge (dashed lines in 18 Figure 3A). The geometry and dimensions of the working electrode and the structure of the sensing region of that device were the same as those of flow channel A. The slow increase in charge observed by the conventional method reflects the diffusion of H2O2 to the electrode in flow channel A. Also, the background charge was not negligible throughout the experiment. In contrast, the response profiles of the type I device feature curves with an elbow. The charge increases rapidly within 2 s, and the increase stops abruptly. The background charge is significantly smaller than that of conventional coulometry because of the significantly small electrode area used for measurement (the microelectrode array in flow channel B). It should be noted that the rate of the very gradual charge increase that follows the rapid charge increase in the detection of H2O2 is close to the increase of the background charge, which indicates that silver was completely removed from the working electrode in flow channel B and only the background current contributed to the measured charge in this time period. Therefore, if the charge is measured immediately after the rapid charge increase, the influence of the background charge will be minimized effectively. Although our strategy was to separate the locations of the oxidation and reduction reactions into two different flow channels, the deposition of silver on the electrode is also observed when silver ions are present in the H2O2 solution. This common technique, known as metallization, has been used to enhance sensitivity for various biochemical analyses. Therefore, we considered whether the separation of the solutions was actually necessary. To investigate this point in more detail, a solution containing both 10 µM H2O2 and 1 M AgNO3 was introduced into only flow channel B, and the amount of deposited silver was measured using the three-electrode system. A comparative study was also conducted by introducing the 10 µM H2O2 and 1 M AgNO3 solutions separately into flow channels A and B, respectively. The results were surprising. The charge measured with a single solution was only 0.37% of that

Figure 5. Dependence of the mixed potential on the concentration of H2O2 obtained with AgNO3 (A) and CuSO4 (B) solutions in flow channel B. Concentrations of AgNO3 and CuSO4 were 1 M (■), 100 mM (●), and 10 mM (▲), respectively. Five measurements were made for each data point in the figure, and averages and standard deviations are shown. measured with the separate solutions on average (n = 5). With separated electrodes and solutions, the efficiency for the reduction of silver ions accompanying the oxidation of hydrogen peroxide is high because of the much larger area that the electrode spans in flow channel A, which actually demonstrates the advantage of separate electrodes and reactions to achieve both sensitivity and a lower detection limit. Figure 3B shows the dependence of the charge on the concentration of H2O2. Here, silver was deposited for 5 min. A clear concentration-dependence for the charge was observed. The detection limit was 30 nM (3σ), which was lower than that achieved by conventional coulometry using the same microelectrode array and flow channel structure (410 nM) by one order of 18 magnitude. It should be noted that the sensitivity and lower detection limit depend on the deposition time



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Figure 6. (A) Changes in charge measured with p-aminophenol (PAP, 10 µM) and H2O2 (10 µM) in combination + 2+ 2+ with the deposition of Ag , Cu , and Ni ions on the working electrode of flow channel B. The inset shows the ratio of the response to H2O2 with respect to that to PAP. (B) Effect of a Nafion layer formed on the electrode of flow channel A. Responses to L-ascorbic acid (AA, 10 µM) and H2O2 (10 µM) in the absence and presence of the Nafion layer are shown. (C) Change in charge measured in the presence of 100 µM AA using the type II device. The inset shows charges measured in the presence of 100 µM L-glutamate (L-Glu) and a mixture of 100 µM L-Glu and 100 µM AA. “ON” and “OFF” indicate whether a mechanism for the removal of AA was used or not, respectively. Five measurements were made for each set of conditions, and averages and standard deviations are shown. and total volume of the sample droplet, and can be improved by simply increasing them. On the other hand, the length of time required for deposition could be shortened by improving the collection efficiency. This is in marked contrast to redox cycling, in which reversible redox compounds that exist in close proximity to interdigitated electrodes are involved. The background charge is directly related to the dimensions of the working electrode for the deposition of silver and coulometric analysis. Although circular microelectrodes of 10 µm in diameter were used here, the dimensions of each microelectrode can be reduced to the order of nanometers by electron beam lithography. Therefore, there is still room to further reduce the background charge and lower the detection limit. Dependence of Metal Deposition Time on Metal, Analyte, and Electrode Structure. Figure 4A shows the relationship between the charge and deposition time for silver. In the graph, the calculated charge anticipated from the volume and concentration of the H2O2 solution is added as a horizontal broken line. When 10 µM H2O2 was used and silver was deposited, the measured charge increased with the increase in the deposition time but leveled off after 20 min, indicating that H2O2 was entirely consumed. Figure 4A also shows the change measured with the same H2O2 and 1 M CuSO4 solution instead of the AgNO3 solution. In this case, copper rather than silver was deposited. The change was much slower than that for silver, and the charge did not saturate, even after 30 min. The rapidness of the change also depended on the analyte. When L-ascorbic acid was used and silver was deposit-

ed, the charge was more rapid than for the H2O2 case, becoming saturated within 5 min (Figure 4A). Figure 4B shows the changes when the three electrode structures shown in Table 1 were used for the detection of 10 µM H2O2. As the strips became thinner and closer, the charge increased, demonstrating the higher collection efficiency of the thinner microelectrodes. As mentioned above, the result suggests an effective method to improve the collection efficiency, shorten the deposition time, and enhance detection sensitivity. Control of the Mixed Potential. A problem with the present method is that the potential of the electrode for the oxidation of the analyte cannot be controlled intentionally, as is the case when using a potentiostat. To investigate this influence in more detail, the mixed potential of the electrodes in flow channels A and B that were connected with each other was measured with H2O2 standard solutions of different concentrations. First, AgNO3 was used for flow channel B in concentrations fixed at 10 mM, 100 mM, and 1 M (Figure 5A). Here, the potentials at 5 s after both electrodes were placed in contact with the solutions are plotted. The measured values of the potential clearly show a dependence on the concentration of silver ions, and shift in the negative direction as the silver ion concentration decreases. On the other hand, the dependence on H2O2 concentration was weak, reflecting a significant difference in the exchange current density between the reactions that took place in the two flow channels. The results apparently indicate that the mixed potential can be adjusted within a range of 100 mV by simply changing the concentration of silver ions.


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The mixed potential can also be changed by changing the metal deposited in flow channel B. Figure 5B shows the mixed potentials when the concentrations of H2O2 and CuSO4 are changed, as in the case of Figure 5A. In this case, the concentration dependence of the mixed potential is weak for both H2O2 and CuSO4. Compared with the case of 1 M AgNO3, the potential was more negative by approximately 0. 1 V and was comparable with the case of 10 mM Ag NO3. The results demonstrate that the mixed potential or the overpotential for the oxidation of the analyte molecules can be changed by changing the deposited metal. Eliminating the Influence of Electroactive Interferents. The foregoing experiments and discussions involved cases in which only a single analyte, H2O2, was present in the solution. This is sufficient for situations in which an enzymatic reaction product in an immunoassay or a single electroactive compound such as a DNA intercalator is detected in the final stage of affinity sensing. However, in general, the determination of a single analyte in solutions containing electroactive interferents may be required. Although the problem is common for all electrochemical techniques based on the measurement of the progress of a redox reaction, we investigated three possible compensatory approaches applicable to our method and device. The first approach is to change the mixed potential so that the influence of interferents is negligible. This approach is often used in mediator-type amperometric biosensors. For this purpose, we used copper or nickel ions in addition to silver ions based on the same procedure mentioned earlier. Figure 6A shows the charges measured for p-aminophenol (PAP) or H2O2 along with the deposition of silver, copper, or nickel ions. The data were obtained over the same deposition time (5 min). The absolute values of the charge are smaller in the cases of copper and nickel. This is reasonable, considering the mixed potentials at which the oxidation of H2O2 or PAP proceeds and the difference in the length of time needed to deposit the same amount of metal originating from the difference in the exchange current densities. The inset in Figure 6A shows the ratio of the response to H2O2 with respect to that of PAP calculated from the averaged values. By changing the metal from silver to copper or nickel, the response to H2O2 decreased relatively, suggesting that selectivity can be improved based on this strategy. Although the changes in the redox potentials and exchange current densities for Cu and Ni decreased the response relative to that of Ag, they were still useful. The use of other metals with more negative redox potentials should be more effective for this purpose. Here, a problem is that the measured charge decreases as the redox potential of the deposited metal becomes more negative. Although a simple solution would be to extend the deposition time, the improvement of the collection efficiency of the analyte by the use of a better electrode structure could solve the problem.

As the second approach, we tested the application of a permselective membrane (Nafion) on the electrode. Nafion is negatively charged and excludes anions in addition to its size-selective transport properties. Figure 6B compares the changes observed with and without a Nafion layer on the electrode in flow channel A. Without the Nafion layer, the observed charge for the L-ascorbic acid was larger than that observed with a solution containing only H2O2. On the other hand, with the Nafion layer, the measured charge decreased by 73% for L-ascorbic acid while the decrease was only 30% for H2O2, which indicates that the Nafion layer effectively blocked the diffusion of L-ascorbic acid. Note that the charges are compared at the same concentration of these compounds. Therefore, in situations in which H2O2 produced from an enzymatic reaction is determined in the presence of L-ascorbic acid of lower concentrations, the application of the permselective membrane would be more effective. The third approach is to eliminate electroactive interferents electrochemically prior to the analysis. To examine the effectiveness of this method and demonstrate its application to biosensing, we used the enzymatic reaction of L-glutamate oxidase (L-GlOx) in the type II device. L-GlOx

L-glutamate + O2 + H2O → α-ketoglutarate + NH3 + H2O2 Flow channels C of the device were first filled with a 1.0 M AgNO3 solution and 700 nL of 100-µM L-glutamate solution was brought in contact with the electrode to remove the interferents from flow channel A. As in the coulometric analysis with the type I device, electroactive interferents were oxidized and removed while silver was deposited on the electrode in flow channel C. After a predetermined time, the solution in flow channel C was flushed and the flow channel was dried by flowing air. Then, the deposition of silver and the coulometric analysis were performed by moving the solution to the electrode for detection in flow channel A and injecting a 1.0 M AgNO3 solution in flow channel B. Figure 6C shows the changes in charge when a solution containing only 100 µM L-ascorbic acid was placed on the electrode for removal and the length of time to remove L-ascorbic acid was changed. The charge decreased rapidly and nearly reached the background level within 10 min, suggesting that this mechanism actually worked. The inset to Figure 6C compares the results obtained with a solution containing both 100 µM L-glutamate and 100 µM L-ascorbic acid with those for a solution containing only 100 µM L-glutamate. Charges originating from H2O2 produced by the enzymatic oxidation of L-glutamate were measured with and without the removal of L-ascorbic acid. The length of time for the removal of L-ascorbic acid was 10 min. The obtained result shows that the measured charges



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were virtually the same when the mechanism to remove the interferent was applied, suggesting that the influence of the interferent could be removed effectively. Although this method would not be applicable for the detection of electroactive compounds that are present initially, it would be effective when an enzymatic reaction product is detected after the removal of the interferent. Issues for General Use. Although we have presented a basic sensing strategy and techniques for solution processing, further simplification of the process will be necessary prior to general use. In this device, although we used an array of rhombuses to show the possibility of changing solution volumes, volume measurement using simple hydrophobic valves would be more suitable if the solution volume is fixed. In that case, solution injected from an additional injection port located at a point in flow channel A would penetrate a hydro35 philic bottom area and be stopped by two valves. The solution fraction could be a plug used for the analysis when it moved in the hydrophobic flow channel, which would extend to the sensing region. Although the current device is designed for single use, repetitive measurements using a single device may be required, depending on the style of use. Figure S-1 shows a typical change in the output charge when measurements are repeated using the same device. The output charge decreases gradually after several measurements. The causes of this change are not clear at present, but the fouling of the electrode surfaces and/or decrease in the conductivity of the liquid junction may be possible. We believe that this is a technical problem and will be solved by modification of the device structure and further optimization. One simple solution may be to integrate a number of the unit structures—including the electrodes and flow channels—on a centimeter-scale chip and use a fresh device for each use.

CONCLUSIONS In the conventional coulometric analysis of components in a solution plug, the charge originating from the Faradaic current tends to saturate as the analyte is depleted, whereas the background charge increases monotonically as time elapses. By converting the analyte in the plug to silver deposited on an electrode in another flow channel and conducting coulometry for the deposited silver, the charge generated in the coulometric analysis can be measured within a short time before the background charge increases, which is effective in improving sensitivity and lowering the detection limit significantly. The lowest observed detection limit was 30 nM (3σ) for hydrogen peroxide, which was lower than that achieved using the same electrode and flow channel structures by conventional coulometry by one order of magnitude. Although electroactive interferents can pose a problem, their influence can be minimized by shifting the mixed potential in the negative

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direction, using a permselective membrane, or incorporating another flow channel and electrode to eliminate the interferents prior to the enzymatic reaction used for detection. As demonstrated, there are still opportunities to improve sensitivity and lower the detection limit, including changing the electrode structure and deposition time. The next challenging issue will be to investigate the extent to which the sample volume can be minimized while maintaining good performance.

AUTHOR INFORMATION Corresponding Author [email protected]

Author Contributions The manuscript was written through contributions of all authors.

ACKNOWLEDGMENT This study was supported by a Grant-in-Aid for Scientific Research (No. 26560365) under the Japan Society for the Promotion of Science (JSPS).

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Figure 1. Typical changes in charge observed by conventional coulometry (A) and using our coulo-metric method and device (B). The dashed lines indicate the times at which charge measurements are made. 86x41mm (300 x 300 DPI)


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Figure 2. Structure of the coulometric device (type I). (A) Top view of the type I device. Dimensions of the device are 17 mm × 30 mm. (B) Cross section of the electrode area in flow channel A along X – X′. (C) Magnified view of the microelectrode array (MEA) in flow channel A (left) and the pinhole structure of the working electrode in flow channel B (right). (D) Top view of the type II device. WE: working electrode. RE: reference electrode. AE: auxiliary electrode. 86x175mm (300 x 300 DPI)



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72x117mm (300 x 300 DPI)


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Figure 4. Dependence of the measured charge on deposition time. (A) Charges observed when depos-ited metal and the analyte are changed. Materials oxidized and deposited in flow channel A and B are 10 µM H2O2 and silver (▲), 10 µM H2O2 and copper (●), and 10 µM L-ascorbic acid and silver (■). (B) Dependence of the charge on the deposition time measured using electrode No. 1 (▲), No. 2 (■) and No. 3 (◆) with 10 µM H2O2 and silver deposited as in (A). The horizontal broken lines indicate the cal-culated value from the total amount of H2O2 in the 700 nL solution. Five measurements were made for each data point in the figure, and averages and standard deviations are shown. 143x246mm (600 x 600 DPI)



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Figure 5. Dependence of the mixed potential on the concentration of H2O2 obtained with AgNO3 (A) and CuSO4 (B) solutions in flow channel B. Concen-trations of AgNO3 and CuSO4 were 1 M (■), 100 mM (●), and 10 mM (▲), respectively. Five measurements were made for each data point in the figure, and averages and standard deviations are shown. 131x249mm (600 x 600 DPI)


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179x57mm (160 x 160 DPI)


Microfluidic Separation of Redox Reactions for Coulometry Based on

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