Article

Cite This: ACS Sens. 2018, 3, 106−112 pubs.acs.org/acssensors

Chronoamperometry-Based Redox Cycling for Application to

Immunoassays
Ga-Yeon Lee,†,# Jun-Hee Park,†,# Young Wook Chang,† Sungbo Cho,‡ Min-Jung Kang,§
and Jae-Chul Pyun*,†
†
Department of Materials Science and Engineering, Yonsei University, Seoul 03722, Korea
‡
Department of Biomedical Engineering, Gachon University, Incheon 21936, Korea
§
Korea Institute of Science and Technology (KIST), Seoul 02792, Korea
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ABSTRACT: In this work, the chronoamperometry-based

redox cycling of 3,3′,5,5′-tetramethylbenzidine (TMB) was
Downloaded via UNIV FED DO AMAZONAS on March 30, 2021 at 13:52:03 (UTC).

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 chronoamperometry-based
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.
KEYWORDS: redox cycling, chronoamperometry, ELISA kit, 3,3′,5,5′-tetramethylbenzidine, hepatitis B virus

I mmunoassays 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.1−3 For commercial immuno-
assays, 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 → ox1-TMB → 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
1a, two oxidation states are observed in the cyclic voltammo-
grams (CVs).4−6
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 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 5-
fold higher sensitivity than the conventional amperome-
tries.7−11 From such results, the deviation at the cutoff value
could be substantially improved with increased measurement
Received: September 13, 2017
Accepted: December 25, 2017
Published: December 25, 2017

© 2017 American Chemical Society 106 DOI: 10.1021/acssensors.7b00681

ACS Sens. 2018, 3, 106−112
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Figure 1. Redox cycling of TMB. (a) Conventional cyclic voltammo-
gram (CV) obtained using one set of IDEs as a working electrode. (b)
Two chronoamperomograms from redox cycling with IDEs at
oxidative and reductive potential. (c) Structure of IDE used for
redox cycling.
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 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 1b. From such a
chronoamperometric method the redox cycling was possible
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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 1c.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 the 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 to the commercial immunoassay for a human hepatitis
B antigen (hHBsAg) ELISA test.21−24
■ EXPERIMENTAL SECTION
Materials. 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.
IDE Fabrication. 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 (AZ-GXR-601) was spin-coated
onto a 4 in. glass wafer.27,28 After soft-baking the photoresist layer at
105 °C for 1 min, UV-photolithography 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.
Redox Cycling. The amperometric measurements were carried out
using a commercial potentiostat from Ivium Technologies (Eind-
hoven, 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 1a was
analyzed. 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 measure-
ment, 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
DOI: 10.1021/acssensors.7b00681
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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). (b) Simulation of redox cycling at different potentials of generator and collector.

oxidative (+600 mV against Ag/AgCl reference electrode) with the (Sunnyvale, CA, USA). Redox cycling was performed as described in
same oxidative potential at the collector (+600 mV against Ag/AgCl redox cycling section by dipping an IDE into each well of the
reference electrode). The currents at the generator and collector were microplate before the sulfuric acid quenching process.17,27

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 1b.
ELISA. 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, HRP-conjugated 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
108
■
RESULTS AND DISCUSSION
Redox Cycling and Modified Chronoamperometry. 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
DOI: 10.1021/acssensors.7b00681
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Figure 3. Optimization of chronoamperometry based redox cycling of TMB. Influence of potential at collector and pulse time.
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 1b, the redox
reaction of TMB could be controlled according to the
potentials of the generator and collector. The first chro-
noamperometry is carried out at the potential range in Figure
1b-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 1b-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 2a-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 1b-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 2a-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 1b-4, the potential of the generator is
set to the oxidative potential of TMB and that of the collector is
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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 the
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 simu-
lation, 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 2b-1, the simulated reduced product of TMB
at the generator is not recycled at the collector, as in the case of
Figure 1b-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 1b-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 1b-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 1b-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 chronoam-
perometry. As shown in Figure 2a, the potential of the collector
changes from a reductive potential (−200 mV against Ag/AgCl
reference electrode) during the first chronoamperometry
measurement (Figure 1b-chronoamperometry 1) to an
oxidative potential during the second chronoamperometry
measurement (+600 mV against Ag/AgCl reference electrode)
(Figure 1b-chronoamperometry 1).
DOI: 10.1021/acssensors.7b00681
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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. (b) Comparison of sensitivities for TMB analysis.

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).

110 DOI: 10.1021/acssensors.7b00681

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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 chronoampero-
metric 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
1a, the initial oxidation current of TMB for chronoamperom-
etry 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.
Application to Immunoassay. The immunoassay OD
value of the TMB solution was measured to quantify the
oxidized TMB molecule. The standard TMB solutions at
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 chronoamperom-
etry-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 4a, 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 4a 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 4b, 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 chronoamper-
ometry 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 anti-hHBsAg antibodies as shown in
Figure 5a. To quantify the amount of bound hHBsAg on the
microplate, the anti-hHBsAg antibodies labeled with HRP were
treated and then reacted with the TMB solution. The cutoff
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 cutoff value of ELISA test was determined
to be positive. For the hHBsAg ELISA test the cutoff value is
known to be quite low in comparison with other infectious
diseases because the acute patients of hHBsAg have a wide
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concentration range of hHBsAg in serum.32,33 From such
reasons, the high sensitivity of the 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 5b, 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 10-fold 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).
■ CONCLUSIONS
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 chronoamperomet-
ric 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 chronoamper-
ometry 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.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: jcpyun@yonsei.ac.kr.
ORCID
Ga-Yeon Lee: 0000-0001-5273-6085
Jun-Hee Park: 0000-0002-2871-0093
Jae-Chul Pyun: 0000-0001-9954-6860
DOI: 10.1021/acssensors.7b00681
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Author Contributions
# microelectrodes for electrochemical sensing. Sens. Actuators, B 2000,
Ga-Yeon Lee and Jun-Hee Park contributed equally.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by the Nano-Convergence
Foundation (R201602210) funded by the Ministry of Science,
ICT and 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, NRF-2017R1A2B2004398, NRF-
2017R1A6A3A11034081, NRF-2017R1D1A1A09000712), and
Yonsei University Research Fund (Yonsei Frontier Lab. Young
Researcher Supporting Program) of 2017.
■
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DOI: 10.1021/acssensors.7b00681
ACS Sens. 2018, 3, 106−112