Chronoamperometry-Based Redox Cycling for Application to

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Chronoamperometry-based redox cycling for application to immunoassays Ga-Yeon Lee, Junhee Park, Young Wook Chang, Sungbo Cho, Min-Jung Kang, and Jae-Chul Pyun ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00681 • Publication Date (Web): 25 Dec 2017 Downloaded from http://pubs.acs.org on December 26, 2017

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Ga-Yeon Lee†‡, Jun-Hee Park†‡, Young Wook Chang†, Sungbo Choϕ, Min-Jung Kang§ , Jae-Chul Pyun†* †

Department of Materials Science and Engineering, Yonsei University, Seoul 03722, Korea

ϕ Department §

of Biomedical Engineering, Gachon University, Incheon 21936, Korea

Korea Institute of Science and Technology (KIST), Seoul 02792, Korea

KEYWORDS: redox cycling, chronoamperometry, ELISA kit, 3,3’,5,5’-tetramethylbenzidine, hepatitis B Virus ABSTRACT: In this work, the chronoamperometry-based redox cycling of 3,3’,5,5’-tetramethylbenzidine (TMB) was performed by using interdigitated electrode (IDE). The signal was obtained from two sequential chronoamperometric profiles: 1) with the generator at the oxidative potential of TMB and the collector at the reductive potential of TMB, and 2) with the generator at the reductive potential of TMB and the collector at the oxidative potential of TMB. The chronoamperometrybased redox cycling (dual mode) showed a sensitivity of 1.49 μA/OD, and the redox cycling efficiency was estimated to be 94% (n=10). The sensitivities of conventional redox cycling with the same interdigitated electrode and chronoamperometry using a single working electrode (single mode) were estimated to be 0.67 μA/OD and 0.18 μA/OD, respectively. These results showed that the chronoamperometry-based redox cycling (dual mode) could be more effectively used to quantify the oxidized TMB than other amperometric methods. The chronoamperometry-based redox cycling (dual mode) was applied to immunoassays using a commercial ELISA kit for medical diagnosis of the human hepatitis B virus surface antigen (hHBsAg). Finally, the chronoamperometry-based redox cycling (dual mode) provided more than a 10-fold higher sensitivity than conventional chronoamperometry using a single working electrode (single mode) when applied to a commercial ELISA kit for medical diagnosis of hHBsAg.

Immunoassays have been widely used for the detection of target analytes by using the highly specific interaction between antigens (analytes) and antibodies. Antibodies have usually been immobilized on solid supports, such as particles and plates. When samples were treated on solid supports, the analytes were bound to the immobilized antibodies. Then, the bound analytes were quantified using a treatment of secondary antibodies labeled with enzymes.13 For commercial immunoassays, horseradish peroxidase (HRP) was used as a chromogenic enzyme and 3,3’,5,5’-tetramethylbenzidine (TMB) was used as a chromogenic probe of HRP. TMB (transparent) has two oxidation products: ox1-TMB (blue, λmax = 650 nm) and ox2-TMB (yellow, λmax = 450 nm) (this is a two-step oxidation: TMB → ox1TMB → ox2-TMB). For medical diagnosis, the determination of disease can be carried out using an optical density (OD) cutoff value. Therefore, the deviation at the cutoff value should be as small as possible to increase the sensitivity of the measurement. As shown in Figure 1(a), two oxidation states are observed in the cyclic voltammograms (CVs).4-6

analyte molecule can be obtained from the redox cycling process.7-10 For such redox cycling, one working electrode, called the generator, reduces (or oxidizes) the analyte molecule; then, the reduced (or oxidized) product is oxidized (or reduced) by the other working electrode, called the collector. For redox cycling, the potential of two working electrodes should be controlled to be suitable for the redox cycling process. When the potential of the generator is set to reduce (or oxidize) the analyte molecule, the potential of the collector should be controlled to oxidize (or reduce) the reduced (or oxidized) product from the generator.11 In previous work, redox cycling of TMB from two different CVs with respect to the potential at the collector electrode (i.e., reductive potential of -200 mV and oxidative potential of +600 mV against a Ag/AgCl reference electrode) could achieve a 10-fold higher sensitivity than the conventional amperometries.7-11 From such results, the deviation at the cutoff value could be substantially improved with increased measurement sensitivity when the redox cycling was applied to medical diagnosis. In this work, the redox cycling of TMB was carried out without cyclic voltammetry at the generator using chronoamperometry.12-15 The chronoamperometric signal was obtained at the same potential

Redox cycling usually reduces (or oxidizes) the oxidized (or reduced) product iteratively between two kinds of working electrodes, and the amplified signal for the single ACS Paragon Plus Environment

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conditions of cyclic voltammetry. The chronoamperometric signal was obtained sequentially under two potential conditions: (1) with the generator at the oxidative potential of TMB and the collector at the reductive potential of TMB, and (2) with the generator at the reductive potential of TMB and the collector at the oxidative potential of TMB as shown in Figure 1(b). From such a chronoamperometric method the redox cycling was possible without cyclic voltammetry; therefore, the instrumentation could be simplified and the signal could be substantially amplified with respect to conventional voltammetry. For the redox cycling amperometric methods, interdigitated electrodes (IDE) have been widely used for redox cycling, as shown in Figure 1(c).16-20 One set of finger electrodes is used as the generator and the other set of finger electrode of redox cycling. The distance between the finger electrodes influences the efficiency of the redox cycling because the mass transport of redox products between the generator and collector occurs through diffusion. Therefore, the potential of the generator and collector, and the distance between the finger electrodes of the IDE should be controlled to optimize the efficiency of the redox cycling. In this work, the redox cycling based on chronoamperometry and IDE with improved sensitivity was applied the commercial immunoassay for a human hepatitis B antigen (hHBsAg) ELISA test.21-24

TMB was purchased from Sigma-Aldrich Korea (Seoul, Korea). HBsAg ELISA kits were purchased from Perfemed Group, Inc. (San Francisco, CA, USA). The photoresist (AZ-GXR601) was purchased from Merck Co. (Darmstadt, Germany). Polystyrene microplates were purchased from SPL Co. (Seoul, Korea). Ag/AgCl reference electrodes were purchased from Warner Instruments LLC (Hamden, CT, USA). Pieces of 2-mm-diameter Pt wire were used as the counter electrodes. The IDEs were fabricated on a glass substrate using an ultraviolet (UV) photolithographic process.17,25,26 A 3-µm-thick layer of a positive photoresist (AZGXR-601) was spin-coated onto a 4-inch glass wafer.27,28 After soft-baking the photoresist layer at 105 °C for 1 min, UVphotolithography was performed using a photomask of the IDEs. The IDEs were designed to have 100 pairs of finger electrodes, each with a width of 5 µm (de) and a 5-µm (dgap) space between each. After the IDE pattern was developed, a 5-nm-thick Ti layer was deposited as an adhesive layer; then, a 50-nm-thick Au layer was deposited using sputter deposition. The IDE pattern was obtained after a lift-off process. The amperometric measurements were carried out using a commercial potentiostat from Ivium Technologies (Eindhoven, Netherlands). Cyclic voltammetry was performed at potentials between -200 and +600 mV versus Ag/AgCl at a scan rate of 50 mV/s. Conventional cyclic voltammetry for the oxidation of TMB molecules was carried out, and the CV shown in Figure 1(a) was analyzed.

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The signal for the TMB solution (reduction current) was calculated as the difference in the reduction current for the TMB stock solution (OD = 0 without any oxidized molecules).6–10 To analyze TMB using redox cycling, two distinct potentials were sequentially applied to the collector: an oxidative potential (+600 mV versus Ag/AgCl) and a reductive potential (-200 mV versus Ag/AgCl). For each TMB sample, two CVs were obtained at the generator electrode, one for the oxidative potential and one for the reductive potential of the collector. The signal for the TMB solution during redox cycling was calculated as the difference between the redox cycling signal of the TMB stock solution (OD = 0, without oxidized molecules) and that for the TMB sample at the same reduction potential (-200 mV versus Ag/AgCl). That is, the signal of the TMB solution during redox cycling = TMB0 current − TMB current.11 In this work, the chronoamperometric assay was carried out by application of two kinds of electric pulses to the generator at the fixed potential of the collector. For the first chronoamperometry measurement, the potential of the generator was changed from a reductive potential (-200 mV against a Ag/AgCl reference electrode) for 10 s to an oxidative potential (+600 mV against Ag/AgCl reference electrode) with the same reductive potential at the collector (-200 mV against a Ag/AgCl reference electrode). For the second chronoamperometry measurement, the potential of the generator was changed from reductive (-200 mV against Ag/AgCl reference electrode) for 10 s to oxidative (+600 mV against Ag/AgCl reference electrode) with the same oxidative potential at the collector (+60000 mV against Ag/AgCl reference electrode). The currents at the generator and collector were separately measured, and the signal was determined from the difference between the current at the end of the first amperometric profile and that at the beginning of the second amperometric profile, as shown in Figure 1(b). Commercial ELISA tests were carried out according to the manufacturer’s instructions. The HBsAg (HBV) ELISA test was performed using a 96-well microplate, which was coated with anti-HBV antibodies. The cutoff value was estimated using the positive and negative samples included in the commercial ELISA kit. The standard carcinoembryonic antigen (CEA) samples were prepared by diluting the positive CEA sample to 1–120 ng/mL. Each CEA sample was incubated for 1 h at room temperature. After repeating the washing steps three times, HRPconjugated secondary antibodies were incubated for 1 h at room temperature. The amount of CEA bound to the 96 wells was quantified by a reaction with TMB and a hydrogen peroxide solution for 20 min. After incubation, the OD was measured at 650 nm using an ELISA reader (VersaMax) from Molecular Devices (Sunnyvale, CA, USA). Redox cycling was performed as described in redox cycling section by dipping an IDE into each well of the microplate before the sulfuric acid quenching process.17,27

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As previously mentioned, TMB undergoes a two-step oxidation. From the CV analysis, the oxidation of TMB begins at +100 mV (versus an Ag/AgCl reference electrode) from the oxidative wave and the reduction of TMB begins at +400 mV (versus an Ag/AgCl reference electrode) from the reductive wave. For the redox cycling of TMB, the second working electrode (collector) is used at a fixed potential to reduce (or oxidize) the analyte from the first working electrode (generator). As previously reported, the redox cycling of TMB could be carried out using the collector electrode at the reductive potential of -200 mV and at the oxidative potential of +600 mV against an Ag/AgCl reference electrode.4–6,11 In this work, chronoamperometry was performed by applying two kinds of electric pulses to the generator at the fixed potential of the collector. As shown in Figure 1(b), the redox reaction of TMB could be controlled according to the potentials of the generator and collector. The first chronoamperometry is carried out at the potential range in Figure 1(b)-(1)where the potential of the generator is set to the reductive potential of TMB and that of the collector is also set to the reductive potential of TMB. In this case, the reduced product of TMB at the generator could not be recycled at the collector because the potential of the collector is also set to the reductive potential. At the potential range in Figure 1(b)-(2), the potential of the generator is set to the oxidative potential of TMB and that of the collector is set to the reductive potential of TMB, as shown in Figure 2(a)-chronoamperometry 1. In this case, the oxidized product of TMB at the generator could be recycled at the collector because the potential of the collector is set to the reductive potential. The second chronoamperometry is carried out at the potential range in Figure 1(b)-(3) where the potential of the generator is set to the reductive potential of TMB and that of the collector is set to the oxidative potential of TMB, as shown in Figure 2(a)-chronoamperometry 2. In this case, the reduced product of TMB at the generator could be recycled at the collector because the potential of the collector is set to the oxidative potential. The currents at the generator and collector were separately measured, and the signal was determined from the current at the end of the first profile and that at the beginning of the second amperometric profile. Therefore, redox cycling to reduce (or oxidize) the oxidized (or reduced) TMB molecule at the generator could be carried out iteratively using the collector, and the amplified signal from the redox cycling process is as much as 10-fold higher than that from conventional amperometry with a single working electrode. In this work, the difference in the current from the generator is taken as the redox cycling signal. From the comparison between the signals at the generator and collector, the redox cycling efficiency is estimated to be 94% (n=10). At the potential range in Figure 1(b)-(4), the potential of the generator is set to the oxidative potential of TMB and that of the collector is also set to the oxidative potential of TMB. In this case, the oxidized product of TMB at the generator could not be recycled at the collector because the potential of the collector is set to the oxidative potential.

Such redox cycling processes between the generator and collector, according to the oxidative and reductive potentials, were simulated using a commercial analysis software called COMSOL Multiphysics® (COMSOL).11,17,29-31 For the simulation, two pairs of generator and collector at the same size and electrode distance as IDE in this work. When the potential of the generator is set to the reductive potential of TMB and that of the collector is also set to the reductive potential of TMB, as shown in Figure 2(b)-(1), the simulated reduced product of TMB at the generator is not recycled at the collector, as in the case of Figure 1(b)-(1). The same results are obtained when the potential of the generator is set to the oxidative potential of TMB and that of the collector is also set to the oxidative potential of TMB, as shown in Figure 1(b)-(4). When the potential of the generator is set to the oxidative potential of TMB and that of the collector is set to the reductive potential of TMB, as shown in Figure 1(b)-(2), the simulated oxidized product of TMB at the generator is recycled at the collector. When the potential of the generator is set to the reductive potential of TMB and that of the collector is set to the oxidative potential of TMB, as shown in Figure 1(b)-(3), the simulated reduced product of TMB at the generator is recycled at the collector. These results show that the redox cycling to reduce (or oxidize) the oxidized (or reduced) TMB molecule at the generator could be performed iteratively using the collector. In this work, the redox cycling of TMB was carried out without cyclic voltammetry at the generator using chronoamperometry. As shown in Figure 2(a), the potential of the collector changes from a reductive potential (-200 mV against Ag/AgCl reference electrode) during the first chronoamperometry measurement (Figure 1(b)-chronoamperometry 1) to an oxidative potential during the second chronoamperometry measurement (+600 mV against Ag/AgCl reference electrode) (Figure 1(b)-chronoamperometry 1). As shown in Figure 3, the initial current of TMB analysis increases as the potential of the collector changes from reductive to oxidative because of the oxidation of TMB on the surface of the collector. In addition, the chronoamperometric signal was optimized, which was determined from the difference between the current at the end of the first amperometric profile (Figure 3-(2)) and that at the beginning of the second amperometric profile (Figure 3-(3)). Because the second oxidation step of TMB occurred at a potential of +400 mV against a Ag/AgCl reference electrode, as shown in Figure 1(a), the initial oxidation current of TMB for chronoamperometry is similar above the oxidation potential of the collector (+600 mV against a Ag/AgCl reference electrode). From these results, the optimal potentials of the collector for the first and the second chronoamperometry of TMB were determined to be a reductive potential (-200 mV against an Ag/AgCl reference electrode) and an oxidative potential (+600 mV against an Ag/AgCl reference electrode), respectively. The immunoassay OD value of the TMB solution was measured to quantify the oxidized TMB molecule. The standard TMB solutions at


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different OD values were prepared, and chronoamperometry was performed to quantify oxidized TMB molecules in the standard solutions. As a first step, the current signal from the chronoamperometry-based redox cycling method was compared to the conventional chronoamperometry using a single working electrode for TMB solutions with different OD values. As shown in Figure 4(a), conventional chronoamperometry using a single working electrode was performed at +600 mV against an Ag/AgCl reference electrode. For the chronoamperometry-based redox cycling (dual mode), the potential conditions of Figure 4(a) were applied, and the signal was measured for the same standard TMB solutions. The sensitivities of several kinds of amperometric methods were compared to quantify the oxidized TMB. As shown in Figure 4(b), the chronoamperometry-based redox cycling (dual mode) shows a sensitivity of 1.49 μA/OD for the signal at the generator. The signal at the collector is estimated to be 1.40 μA/OD. As previously mentioned, such a signal difference for the generator and collector could be explained from the redox cycling efficiency which was estimated to be 94%. The sensitivity of the conventional redox cycling with the same IDE is estimated to be 0.67 μA/OD, and the chronoamperometry using a single working electrode is estimated to be 0.18 μA/OD. These results show that the chronoamperometry-based redox cycling could be more effectively used to quantify oxidized TMB than other kinds of amperometric methods. The chronoamperometry-based redox cycling was applied to the immunoassay using a commercial ELISA kit for the medical diagnosis of hHBsAg.17,27 The immunoassay was performed by treating a known concentration of hHBsAg in serum on the microplate coated with the antihHBsAg antibodies as shown in Figure 5(a). To quantify the amount of bound hHBsAg on the microplate, the antihHBsAg antibodies labeled with HRP were treated and then reacted with the TMB solution. The cut-off value for the positive and negative determination of each sample (OD=0.105) was calculated according to the manufacturer’s instructions. Usually, the test sample with higher OD than the cut-off value of ELISA test was determined to be positive. For the hHBsAg ELISA test the cut-off value is known to be quite low in comparison with other infectious diseases because the acute patients of hHBsAg have a wide concentration range of hHBsAg in serum.32,33 From such reasons, the high sensitivity of immunoassay was required for the hHBsAg test. In this work, the oxidized TMB was quantified by inserting an IDE into each well of the microplate for the chronoamperometry-based redox cycling. As shown in Figure 5(b), the chronoamperometry-based redox cycling provided a sensitivity of 5.51 μA/OD for the signal at the generator. The sensitivity is estimated to be 0.30 μA/OD using conventional chronoamperometry with a single working electrode. These results show that the chronoamperometry-based redox cycling could have more than a 10fold higher sensitivity than conventional chronoamperometry using a single working electrode when applied to a commercial ELISA kit for the medical diagnosis of human hepatitis B virus surface antigen (hHBsAg).

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Redox cycling usually reduces (or oxidizes) the oxidized (or reduced) product iteratively between two kinds of working electrodes, and an amplified signal for the single analyte molecule can be obtained from the redox cycling process. In this work, the redox cycling of TMB was performed without cyclic voltammetry at the generator using chronoamperometry. The signal from chronoamperometry was obtained at the same potential conditions of cyclic voltammetry. The signal was obtained from two sequentially performed chronoamperometric profiles: (1) with the generator at the oxidative potential of TMB and the collector at the reductive potential of TMB, and (2) with the generator at the reductive potential of TMB and the collector at the oxidation potential of TMB. The chronoamperometry-based redox cycling showed a signal sensitivity of 1.49 μA/OD at the generator. The signal at the collector was estimated to be 1.40 μA/OD. From the comparison between the signals at the generator and collector, the redox cycling efficiency was estimated as 94% (n=10). In this work, the difference in the currents from the generator was used to determine the redox-cycling signal. The sensitivity of the conventional redox cycling with the same IDE was estimated to be 0.67 μA/OD, and the chronoamperometry using a single working electrode was estimated to be 0.18 μA/OD. These results showed that the chronoamperometry-based redox cycling could be more effectively used to quantify oxidized TMB than other kinds of amperometric methods. The chronoamperometry-based redox cycling was applied to an immunoassay by using a commercial ELISA kit for the medical diagnosis of hHBsAg. Finally, the chronoamperometry-based redox cycling showed a sensitivity of 5.51 μA/OD for the signal at the generator. The sensitivity of conventional chronoamperometry using a single working electrode was estimated to be 0.30 μA/OD. These results showed that chronoamperometry-based redox cycling could have more than a 10-fold higher sensitivity than conventional chronoamperometry using a single working electrode when applied to a commercial ELISA kit for the medical diagnosis of hHBsAg.

* E-mail: [email protected] (J. C. Pyun) Ga-Yeon Lee: 0000-0001-5273-6085 Jun-Hee Park: 0000-0002-2871-0093 Jae-Chul Pyun: 0000-0001-9954-6860 ‡These authors contributed equally.

This work was supported by the Nano-Convergence Foundation (R201602210) funded by the Ministry of Science, ICT and


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Future Planning (MSIP, Korea) and the Ministry of Trade, Industry and Energy (MOTIE, Korea), the Industry Technology Development Program (10063335) funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea), and the National Research Foundation of Korea (NRF-2017R1A2B4004077, NRF2017R1A2B2004398, NRF-2017R1A6A3A11034081), and Yonsei University Research Fund (Yonsei Frontier Lab. Young Researcher Supporting Program) of 2017.

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“for TOC only"


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Figure 1 Redox cycling of TMB. (a) Conventional cyclic voltammogram (CV) obtained using one set of IDEs as a working electrode.


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Figure 1 (b) Two chronoamperomogram from redox cycling with IDEs at oxidative and reductive potential


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Figure 1 (c) Structure of IDE used for redox cycling.


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Figure 2. Chronoamperometry based redox cycling of TMB. (a) Redox reactions of TMB according to the potential of generator and collector. The potential conditions could result in the iterative redox reaction (redox cycling) for a single TMB molecule. (redox cycling at reduction and oxidation with collector)


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Figure 2. (b) Simulation of redox cycling at different potentials of generator and collector


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Figure 3. Optimization of chronoamperometry based redox cycling of TMB. Influence of potential at collector and pulse time


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Figure 4. Comparison of chronoamperometry based redox cycling with the conventional chronoamperometry with a single electrode for the analysis of TMB. (a) Current profiles for standard TMB solutions at OD=0 and OD=2.0.


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Figure 4. (b) Comparison of sensitivities for TMB analysis.


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Figure 5. Application to immunoassay. (a) Procedure of ELISA for the medical diagnosis of human hepatitis B (hHBsAg). (b) Comparison of sensitivities between chronoamperometry based redox cycling and the conventional chronoamperometry with a single electrode for the medical diagnosis of human hepatitis B (hHBsAg).


Chronoamperometry-Based Redox Cycling for Application to

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