Cancer Cell Detection‐Based on Released Hydrogen Peroxide Using a Non‐Modified Closed Bipolar Electrochemical System

Abstract

In this study, a non‐modified closed bipolar electrochemical system for ultrasensitive cancer cell detection by quantitative measurement of released hydrogen peroxide in the presence of ascorbic acid has been developed. This system includes two separated chambers (reporting and sensing), a piece of a gold archival CD as a bipolar electrode and two platinum driving electrodes. By following ECL intensities of luminol oxidation in the reporting chamber, the released hydrogen peroxide by cancer cells in the presence of ascorbic acid was monitored. Under the optimum conditions, the biosensor delivers the wide liner ranges from 2.5×10 −9 ‐ 1×10 −6 M, and the detection limit as low as 1.8 × 10 −9 M of hydrogen peroxide. Also, this system can detect cancer blood cells (T‐lymphoblastic leukemia CCRF‐CEM) as low as 12 cells in 300 µL of cell suspension. The present ECL‐BPE system demonstrates a simple and low‐cost device with excellent performance without any electrode surface modification for the detection of cancer cells just based on different levels of released hydrogen peroxide by normal and cancer cells. The present biosensor can be applied as a promising alternative method for the detection of H 2 O 2 in the field of pathology, cancer diagnosis, and environmental monitoring.

Full text

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Cancer Cell Detection-Based on Released Hydrogen
Peroxide Using a Non-Modified Closed Bipolar
Electrochemical System
Maryam Shokouhi[b] and Masoud A. Mehrgardi*[a]
In this study, a non-modified closed bipolar electrochemical
system for ultrasensitive cancer cell detection by quantitative
measurement of released hydrogen peroxide in the presence of
ascorbic acid has been developed. This system includes two
separate chambers (reporting and sensing), a piece of a gold
archival CD (compact disc) as a bipolar electrode, and two
platinum driving electrodes. By following the ECL (electro-
chemiluminescence) intensities of luminol oxidation in the
reporting chamber, the hydrogen peroxide released by cancer
cells in the presence of ascorbic acid was monitored. Under the
optimum conditions, the biosensor delivers a wide linear range
from 2.5 × 109to 1 × 106M, and a detection limit for hydrogen
peroxide as low as 1.8× 109M. Also, this system can detect
cancer blood cell concentrations (CCRF-CEM: T-lymphoblastic
leukemia cell line) as low as 12 cells in 300 μL of cell suspension.
The present bipolar electrode-electrochemiluminescence (BPE-
ECL) system demonstrates a simple and low-cost device with
excellent performance without any electrode surface modifica-
tion for the detection of cancer cells based on different levels of
released hydrogen peroxide by normal and cancer cells. The
present biosensor can be applied as a promising alternative
method for the detection of H2O2in the field of pathology,
cancer diagnosis, and environmental monitoring.
Introduction
Hydrogen peroxide (H2O2) is one of the most important forms
of reactive oxygen species (ROS) and it is produced by different
biochemical reactions and oxygen metabolism in cells.[1] Also,
H2O2has a substantial role in cell growth, signal transduction,
and apoptosis.[2] The concentration level of hydrogen peroxide
in living tissues is one of the most important parameters for the
monitoring of physiological balance and pathological diagnosis
in living cells.[3] Accordingly, monitoring and maintaining the
physiological balance of the H2O2concentration level in living
cells are too vital.[1] On the other hand, pathological events can
be detected in the living cells by the quantitative measuring of
H2O2concentration levels. Therefore, the detection and quanti-
fication of H2O2are one of the main studies in biological,
environmental, clinical, and pharmaceutical fields.[2a,4] Investiga-
tion of diagnostic methods for accurate scrutiny of hydrogen
peroxide levels attracted high attention in recent years. While
the previous researches demonstrated the released hydrogen
peroxide levels in cancerous and normal tissues are significantly
different from,[5] to the best of our knowledge, there are no
reports for discrimination between cancer and normal cells
based on quantitative measurement of released hydrogen
peroxide levels by living tissues.[5c,6] Just a few articles focused
on cancer detection by capturing cancer cells on antibody or
aptamer modified electrodes and subsequently monitoring of
released hydrogen peroxide as an analytical signal. Various
analytical methods have been applied for the determination of
hydrogen peroxide in cellular tissues, including
spectrophotometry,[7] electrochemical sensors,[8] automatic po-
tential titration,[9] photoelectrochemical sensors,[10] fluorescence
assay.[11] Among electrochemical methods, fewer researches
focused on the development of BPE-ECL for the monitoring of
hydrogen peroxide in living tissues.[12]
BPE-ECL sensing platform offers intrinsic advantages such as
low cost, without any direct contact between external potential
instruments and sensing electrodes, simplicity, portability and
the capability of multiplex analysis. Thus, closed-BPE-ECL is an
appropriate method for biochemical analysis BPE platforms
include a conductor such as a gold electrode which is in the
uniform electric field.[13]
In a closed-BPE, cathode and anode are in separate
chambers as sensing and reporting with different fluids, and
current only passed through BPE.[4a,14] The reporting chamber
reflects reaction occurred in the sensing chamber.[12a,15] Various
optical methods such as fluorescence, Electrochemilumines-
cence (ECL) and anodic dissolution applied as a readout in the
reporting chamber. Electrochemiluminescence (ECL) reporting
offers some advantages such as near zero-background signal,
better control for position or time of light emission, compati-
bility with solution-phase and high selectivity and sensitivity.[13]
[a] Prof. M. A. Mehrgardi
Department of chemistry
University of Isfahan
Isfahan 81746-73441, Iran
Fax: (
+
9)8 313 6689732
E-mail: m.mehrgardi@chem.ui.ac.ir
m.mehrgardi@gmail.com
[b] Dr. M. Shokouhi
Department of chemistry
University of Isfahan
Isfahan 81746-73441, Iran
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/celc.202000535
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In this study, a biosensor based on coupling BPE and ECL
methods for quantitative measurement of hydrogen peroxide
and discrimination between cancer and normal blood cells on
“an unmodified bipolar electrode” has been introduced. The
principal superiority of the present biosensor is the ultra-
sensitive detection of cancer cells on a disposable bare
electrode without any further complicated surface modifica-
tions like aptameric or antibody as a trap for capturing cancer
cells. As a result, this biosensor not only offers a simple and
low-cost device but also shows a good detection limit and a
wide linear range. Moreover, the gold bipolar electrode surface
has not been modified for cell capture. The present biosensor
can be used for the indirect measurement of the other
biomolecules through the detection of hydrogen peroxide like
glucose since H2O2is a product of many oxidative biological
reactions.[16]
Result and Discussion
In the present manuscript, a closed bipolar electrochemical
system integrated with ECL for selective discrimination between
cancer and normal cells based on the released hydrogen
peroxide has been designed and investigated.
As shown in Figure 1, in the reporting chamber, the
luminol-hydrogen peroxide system in alkaline medium was
utilized and a photomultiplier tube (PMT) was applied to detect
emitted photons in the reporting side. As a consequence of
H2O2electrochemical oxidation on the electrode surface, some
reactive oxygen species (ROSs) are produced. The reaction
between ROSs like super-oxide anion (O2
*) and hydroxyl radical
(OH*) and luminol cause to generate 3-aminophthalate in the
excited state. Coming back to the ground state leads to light
emission.[14b,17] In the sensing side, hydrogen peroxide or
released hydrogen peroxide by living cells were detected.
ð1Þ
ð2Þ
Ascorbic acid, as hydrogen peroxide inducer, was intro-
duced to the sensing chamber. There is a direct relationship
between the oxidation of luminol in the presence of hydrogen
peroxide (as co-reactant) on the reporting side [Eq. (1)] and the
hydrogen peroxide reduction reaction in the sensing side
[Eq. (2)].
Due to cathode and anode connection in the BPE system,
ECL reaction was accompanied to the electron transfer proc-
esses at the closed BPE.[14b] Therefore, the ECL signals are
quantitatively associated with electrochemical oxidation is
occurring at the anode reservoir. By following emitted photons
using PMT, the concentration of hydrogen peroxide or released
hydrogen peroxide by the cells can be measured.[13,15]
Optimization of Effective Parameters in the Closed BPE-ECL
System
All effective parameters on ECL intensities for the detection of
hydrogen peroxide including, the applied voltage to the driving
electrodes, pH and concentration of luminol solution and
concentration of H2O2in the reporting chamber have been
optimized.
The applied voltage on driving electrodes has an impressive
effect on the electrochemical oxidation rate of luminol to 3-
aminophthalate and accordingly ECL signals.[17a,b,18] Figure 2
illustrates the ECL responses versus different driving voltages in
the presence and absence of hydrogen peroxide in the sensing
Figure 1. Schematic illustration of the closed- BPE-ECL system for the
detection of leukemia cancer cells. (AA: ascorbic acid).
Figure 2. The effect of applied voltages on ECL intensities in the absence (A) and presence (B) of hydrogen peroxide in the sensing chamber and the
differences of ECL signals (C). Luminol concentration: 5.5 mM, pH 10.
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chamber. In the presence of hydrogen peroxide (Figure 2B), a
weak ECL signal is observed even when the applied voltage is
0 V. This can be attributed to the emitted photons from
background reactions like reduction reaction of environmental
oxygen in the sensing side also obtrusive photons detected by
PMT. While, by increasing applied voltage up to 0.9 V, the ECL
intensities have been increased, the voltages higher than 0.9 V
lead to the diminishing of ECL signals. It can be attributed to
the bubble formation on the bipolar electrode surface due to
the water oxidation that both chemically and physically
interferes with ECL emission.[18] Moreover, this is worthy to note
that the applied voltage with the highest ECL intensity is not
optimum voltage necessarily. In optimum voltage, the ECL
signal differences in the presence and absence of hydrogen
peroxide in the sensing chamber should be maximum. In 0.7 V,
the maximum differences were achieved and therefore, this
potential was chosen as an optimum power supply voltage for
further investigations.
Another effective parameter on the ECL signal is the pH of
the luminol-hydrogen peroxide solution on the reporting side.
The electrochemical oxidation of luminol and electro-gener-
ation of ROS from H2O2leads to ECL signals in the reporting
chamber. These reactions are more efficient in the alkaline
medium.[19] Therefore, the pH of the luminol solution in the
reporting chamber has been optimized (Figure S1). Because of
the faster luminol oxidation reaction, ECL signals are enhanced
by increasing pH up to 10.[4a,17d,20] In higher pHs, the
fluorescence quantum yield of phthalate ion decreases and the
ECL signal is reduced.[12b,20–21] By changing pH, the backgrounds
were also changed. The maximum difference between blank
and sample was achieved at pH 10 and this was selected as
optimum pH for luminol and H2O2mixture on the reporting
side.
The concentration of luminol is another effective parameter
on the alteration of ECL intensity in BPE-ECL platforms. As
illustrated in (Figure S2), the effect of luminol concentrations on
the ECL signals has been investigated as well. Increasing
luminol concentration causes an increase of aminophthalate
ions and subsequently enhancement of ECL signals for both
blank and sample solutions in the sensing side. In higher
concentrations (more than 7.5 mM) of luminol, the intensity of
the ECL signal decreases. It could be attributed to the self-
quenching effect in the high concentration of luminol.[12b,19,22]
Also, the maximum difference between blank and sample
solutions has been achieved at 7.5 mM luminol and it was
selected as the optimal concentration for subsequent experi-
ments.
In the reporting chamber of the closed-BPE-ECL platform,
the decomposition of H2O2leads to generate ROSs (such as
OH**and O2
-**) and they react with luminol to produce exciting
luminol; therefore, the concentration of hydrogen peroxide in
the reporting chamber is another efficient parameter on
luminol ECL reactions.[21a,23] As shown in (Figure S3), ECL signals
are enhanced by increasing H2O2concentration up to 3.0 mM,
but in higher concentrations, it decreases. It may be due to H+
formation, which produced from the electrochemical oxidation
of H2O2, and the production of H+leads to a decline in pH and
suppression of ECL reactions and signals.[24] Also, the maximum
signal difference between blank and sample solution was
observed at 3 mM and this was chosen as the optimal
concentration of H2O2in the reporting side.
Analytical Performance of the Closed BPE-ECL System for the
Quantitative Measurement of Hydrogen Peroxide
The efficiency of this BPE-ECL sensor for the detection of
hydrogen peroxide at very low concentrations and its reprodu-
cibility was demonstrated. The responses of this sensor to
various concentrations of H2O2have been followed by record-
ing ECL signals of the bipolar system Figure 3. By increasing
H2O2concentration, the ECL intensities are increased linearly
over 2.5 × 109–1 × 106M with a limit of detection (LOD) as
low as 1.8 × 109M (Based on 3SDblank). The linear dynamic
ranges and the detection limit of this biosensor are better than
Figure 3. A) ECL responses to various concentrations of H2O2. B) Calibration curve of the ECL response to the different H2O2concentration in optimum
parameters: driving voltage: 0.7, luminol concentration: 7.5 mM, pH 10; concentration of H2O2as a co-reactant in reporting side: 3 mM. The error bar is the
standard error from 3 independent experiment
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previously reported BPE-ECL sensors for the detection of
hydrogen peroxide (Table S1).
Detection of Released Hydrogen Peroxide by Living Cells
As mentioned earlier, hydrogen peroxide is one of the common
ROS molecules that is present in different biological tissues.[25]
H2O2concentration is rather low under normal physiological
conditions[26] and a high concentration level of H2O2could be a
sign of pathological conditions like cardiopathy, tumor, neuro-
degeneration, etc.[5a,27] On the other hand, previous studies have
demonstrated that high doses of ascorbate, as pro-oxidant and
in presence of free transition metals, induce oxidative stress by
the generation of H2O2.[28] The main enzymes for the decom-
position of extracellular H2O2are catalase, glutathione
peroxidase (GPx) and peroxiredoxins (Prx).[1,29] Biomedical
studies have demonstrated that antioxidant enzyme levels in
cancer cells are lower than normal cells. Thus, elimination of
extracellular H2O2and its rate constant in normal cells
approximately 2-fold higher than cancer cells[5a,b, 30] and it can
be concluded that H2O2is an implicit biomarker of cancer. In
This Closed-BPE-ECL biosensor, when ascorbic acid stimulates
living cells to release H2O2, respiratory burst occurred with H2O2
as the final product. Afterward, the ECL signals are monitored
as the quantitative signals for the detection of cancer cells.
Under the optimum conditions for the detection of hydrogen
peroxide, the amount of released extracellular H2O2by different
numbers of T-lymphoblastic leukemia CCRF-CEM cancer blood
cells were determined using the present closed-BPE-ECL
biosensor. These samples were prepared by Sequential dilution
from the initial sample contain 106cells. mL1.
As shown in Figure 4, a linear calibration range (327–8 × 104
cells in 300 μL) was achieved from the logarithmic diagram of
cell concentration. Moreover, the LOD of 12 cells in 300 μL was
obtained (based on 3SDblank). The mean released H2O2by a
single cancer cell was found equal to 4.22 × 1015 mol based on
a linear relationship between the ECL intensities and the
concentration of H2O2Figure 3B. This value is consistent with
the previously reported values.[5c,10a,31].
Based on the evidence in previous studies, extracellular
H2O2levels in normal cells are lower than cancer cells, and
therefore by increasing numbers of normal blood cells changes
have been observed in ECL intensities and they are as big as
the background signal Figure 5. A comparison of the present
method with previous reports on cancer cell detection demon-
strates that the figure of merits for present biosensor is
comparable or better than previous studies (Table S2).
Conclusions
In summary, we have developed an unmodified closed BPE-ECL
sensing system for the quantitative detection of the as-released
Figure 4. A) and B) ECL signals for the various T-lymphoblastic leukemia CCRF-CEM cells number. C) Calibration curve for detection of T-lymphoblastic
leukemia CCRF-CEM with optimum parameters: driving voltage: 0.7, luminol concentration: 7.5 mM, pH 10; concentration of H2O2as a co-reactant in reporting
side: 3 mM, 10 μL of ascorbic acid (1 mM) was added to cells (as an inducer). The error bar is the standard error from 3 independent experiments
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hydrogen peroxide through cancer cells in the presence of
ascorbic acid, by following ECL intensity of luminol oxidation.
The main advantage of the present biosensor is the ultra-
sensitive detection of cancer cells on a disposable bare
electrode without any further complicated surface modifica-
tions like aptameric or antibody as a trap for capturing cancer
cells. In comparison with prior biosensors that modified the
surface of electrodes using various complex biomolecules, this
biosensor is a very simple, inexpensive, biocompatible, and a
label-free diagnostic tool for the detection of the T-lympho-
blastic leukemia CCRF-CEM cells and offers a comparable or
even better sensitivity, dynamic range, and detection limit.
Looking to the future, this closed-BPE-ECL system (based on
ability in H2O2detection) will probably find broad applications,
such as in the clinical diagnosis, environmental monitoring, and
bimolecular and high-throughput biochemical analysis.
Experimental Section
Materials
Sodium chloride, potassium chloride, sodium hydrogen phosphate,
potassium dihydrogen phosphate, sodium tetraborate, sodium
hydroxide, luminol, and hydrogen peroxide 30 % were procured
from Merck or Sigma-Aldrich. All chemicals were of analytical grade.
All solutions were prepared using deionized water.
Instrumentation
A DC power supply (DAZHENG, China) was used to apply the
potential on the platinum driving electrodes (Metrohm, Switzer-
land). ECL signals were recorded using a photomultiplier tube
(Hamamatsu, USA) connected to an oscilloscope (Tektronix, USA).
Fabrication of closed BPL-ECL device
The BPE cell was fabricated by a 3D printer (Monkati, China) with
FDM technology using Polylactic Acid (PLA) polymer. PLA, with
bioresorbability and biocompatible properties, is one of the most
common polymers used in biomedical applications.[32]
A slice of the archival CD (Memorex, USA) with the dimensions of
10 mm × 4 mm was used as a gold bipolar electrode.
Two platinum electrodes, as the driving electrodes, were placed in
the same distances (1 cm) from BPE in each sensing and reporting
chambers. Subsequently, 300 μL luminol solution (7.5 mM) in the
borate buffer (0.025 M, pH =10) (Luminol should be dissolved in
alkaline pH)[33] and 50 μL H2O2(3 mM), as the oxidation catalyst of
luminol was added to reporting chambers. On the other side, the
sensing chamber was filled by 300 μL containing various concen-
trations of H2O2in the 1X PBS buffer.
Cell culture and detection
T-lymphoblastic leukemia CCRF-CEM cells were cultured in RPMI
1640 (Sigma, USA) containing 10 % fetal bovine serum (FBS) (Sigma,
USA) and 1 % antibiotics mixture containing penicillin (Sigma-
Aldrich, Germany) and streptomycin (Sigma-Aldrich, Germany). The
cells were incubated in a humidified incubator at 37 °C in a 5 % CO2
atmosphere. Then, the cells were detached using trypsin (Sigma,
USA) and counted. Finally, ~ 1 × 106cells were dispersed in 1 mL
PBS 1X (pH =7.4) and the suspensions with various numbers of
cells were prepared using serial dilution. Afterward, 290 μL aliquot
of cell suspension was added to the sensing chamber, then 10 μL
of ascorbic acid (1 mM) was added for inducing the release of
hydrogen peroxide by the cells. The final concentration of AA in the
sensing chamber was 33 μM and it was in the proper range for
inducing realization of hydrogen peroxide from living cells.[29c,33]
Also, blood mononuclear cells (MNC) were used as control blood
cells. Firstly, a healthy human blood sample was prepared. After-
ward, blood MNC were extracted from whole blood by Ficoll[34] and
were counted using a cell counter.
Figure 5. A) ECL response to the different blood mononuclear cells (MNC) number, B) calibration curve with optimum parameters: driving Voltage: 0.7, luminol
concentration: 7.5 mM, pH 10; concentration of H2O2as a co-reactant in reporting side: 3 mM, 10 μL of ascorbic acid (1 mM) was added to cells (as an inducer).
The error bar is the standard error from 3 independent experiments.
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Conflict of Interest
The authors report no conflict of interest.
Acknowledgments
The authors are grateful for the financial support of this project by
Iran National Science Foundation (Grant # 94026958 ) and the
research council of the University of Isfahan. Also, the authors
would like to express their sincere thanks to Miss Hajar Termebaf
for the creative designing of the cover feature.
Conflict of Interest
The authors declare no conflict of interest.
Keywords: bipolar electrochemistry ·hydrogen peroxide ·
leukemia ·CCRF-CEM ·cancer
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Manuscript received: April 12, 2020
Revised manuscript received: June 26, 2020
Accepted manuscript online: July 1, 2020
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