Biochemical Engineering Journal 156 (2020) 107513

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

A homogeneous magnetic bead-based impedance immunosensor for highly T

sensitive detection of Escherichia coli O157:H7
Shujun Wanga,1, Chongyun Sunb,1, Qiushi Hua, Shuang Lia, Chengbin Wangc,*, Peng Wangd,*,
Lei Zhoua,*
a
National Key Laboratory of Biochemical Engineering, PLA Key Laboratory of Biopharmaceutical Production & Formulation Engineering, Institute of Process Engineering,
Chinese Academy of Sciences, Beijing 100190, China
b
Blood Grouping Laboratory, Beijing Red Cross Blood Center, Beijing 100088, China
c
Department of Clinical Laboratory, Chinese People’s Liberation Army General Hospital, Beijing 100853, China
d
State Key Laboratory of Precision Measurements, Department of Precision Instrument, Tsinghua University, Beijing 100084, China

H I GH L IG H T S

• Electrode modification and samples adsorption restrict the application of electrochemical biosensing.
• Magnetic bead-based impedance biosensor avoids matrix interference and improves electrode reproducibility.
• Interdigital electrode and enzymatic reaction system enhance detection efficiency.
• Escherichia coli O157:H7 could be analyzed with highly sensitivity.

A R T I C LE I N FO A B S T R A C T

Keywords: As foodborne pathogen contamination has become a threat to human health worldwide, early detection of
Impedance immunosensor pathogen is significant. Traditional electrochemical immunosensors for pathogen detection involve electrode
Magnetic bead modification or samples adsorption on the electrode which affect the detection sensitivity and electrode re-
Escherichia coli O157:H7 producibility, thereby limiting their further application in POCT (point of care testing). We established a
homogeneous magnetic bead-based electrochemical impedance system using interdigitated gold electrodes as
sensing elements for high sensitivity detection of Escherichia coli (E.coli) O157:H7. The system avoided electrode
modification and sample matrix adsorption, and greatly improved detection sensitivity. The detection limit of
this method for E.coli O157:H7 was 102 colony-forming units per milliliter (CFU mL−1), coefficient of variation
(CV) < 10 % (n = 3), which was three orders of magnitude lower than the traditional ELISA method, and the
linearity detection range was 102 CFU mL−1 to 106 CFU mL−1. This magnetic bead-based impedance method is
expected to be used for a wider range of pathogen targets analysis.

1. Introduction
Pathogenic microbial infection can cause numerous diseases and has
become one of the principal causes of morbidity and mortality world-
wide [1]. Detection of pathogenic microorganisms is a major challenge,
as early determination of pathogens could avoid most treatments and
unwanted deaths [2–4]. Conventional cultivation and separation
methods cost several days to get results, which are time consuming and
labor intensive. Enzyme-linked immunosorbent assay (ELISA) and
polymerase chain reactions (PCR) nucleic acid detection methods could
also be used for pathogens determination, but tedious procedures and
DNA contamination hinder their application for POCT (point-of-care
testing) [5–11].
As is well known, blood glucose meter based on electrochemical
analysis is one of the most commonly used sensors for glucose POCT
monitoring. Electrochemical sensors exhibit their advantages such as
high sensitivity and low-cost instruments, and have been applied for
pathogens detection [12–18]. Depending on the types of output signals,
electrochemical analysis methods consist of potentiometry, ampero-
metry, voltammetry, impedance, conductimetry, and so on. Among

⁎
Corresponding authors.
E-mail addresses: wangcb301@163.com (C. Wang), peng@mail.tsinghua.edu.cn (P. Wang), zhoulei17@ipe.ac.cn (L. Zhou).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.bej.2020.107513
Received 8 November 2019; Received in revised form 8 January 2020; Accepted 21 January 2020
Available online 26 January 2020
1369-703X/ © 2020 Elsevier B.V. All rights reserved.
S. Wang, et al.
them, impedance-derived immunosensors attract increasing attention
for their lower detection limit and ease of miniaturization. In recent
years, impedance immunosensors have been applied for detection of
biomolecules, proteins, cells, pathogens and viruses [19–26]. However,
many of the developed electrochemical impedance biosensors are de-
pendent of immobilization of antibodies on the electrode surface, which
is time-consuming and does not guarantee reproducibility. Further-
more, the adsorption of sample matrix on the surface of electrode re-
sults in the decrease of detection accuracy, sensitivity and stability
[27]. Therefore, an immunosensor that does not rely on electrode im-
mobilization and sample matrix adsorption should be developed to
improve its overall performance.
Magnetic beads (MBs) have become a powerful tool for biological
targets analysis [28–30]. It has been reported that functional magnetic
beads used for biosensing could increase reactivity area and improve
the reaction kinetics, thereby promoting the development of reaction
performance [31,32]. Herein, we developed a magnetic bead-based
electrochemical impedance immunoassay method for qualitative and
quantitative detection of a representative foodborne pathogen, Escher-
ichia coli (E.coli) O157:H7, with an interdigitated gold electrode. The
combination of immunomagnetic beads and electrochemical detection
provided an efficient strategy for rapid and sensitive sensing of patho-
gens, because magnetic beads were used as homogeneous carriers for
targets capturing and the electrodes were used as sensing elements. The
use of magnetic beads avoided electrode modification and sample
matrix adsorption occurred in traditional electrochemical im-
munoassays [33–38]. This magnetic bead-based electrochemical im-
pedance detection system achieved the goal of low concentrations of
E.coli O157:H7 detection, the enzymatic reaction system enhanced the
detection signal intensity and the detection sensitivity. This detection
system lays a foundation for POCT monitoring of pathogens (Scheme
1).
2. Materials and methods
2.1. Reagents and instruments
MES (C6H13NO4S) ≥99 %, EDC (C8H17N3·HCl), NHS (C4H5NO3), p-
nitrophenyl phosphate disodium (PNPP) and 3, 3′, 5, 5′-tetra-
methylbenzidine (TMB) were obtained from Sigma Aldrich. Magnetic
Scheme 1. Illustration of the magnetic bead-based impedance method for de-
tection of Escherichia coli O157:H7.
2
Biochemical Engineering Journal 156 (2020) 107513
beads with sizes of 1.90 μm (M1-200/20, COOH = 82 μeq g−1) was
purchased from Merck Millipore. E.coli O157:H7 was cultured in Luria-
Bertani (L-B) broth at 37 °C for overnight, afterwards, bacteria cells
were harvested and determined the colony-forming units per milliliter
(CFU mL-1) by plate-counting. E.coli O157:H7 monoclonal antibodies,
alkaline phosphatase (ALP)-labeled goat anti-mouse antibodies, horse
radish peroxidase (HRP)-labeled goat anti-mouse antibodies and horse
radish peroxidase (HRP)-labeled E.coli O157:H7 monoclonal antibodies
were prepared in laboratory. All water used was redistilled water.
Insulated electric thermostat incubator GSP-600 was obtained from
Huangshi medical pharmaceutical equipment Co., Ltd (Hubei Province,
China). Electrochemical work station (Princeton, P4000) was pur-
chased from ParStat4000 (Ametek, USA). Vortex mixers QL-901 was
obtained from Qilinbeier Co., Ltd (Haimen City, China). Microplate
reader (Model 680) was obtained from Bio-Rad, America. Magnetic
separation rack (LSKMAGS08|PureProteome™, 8 holes) was obtained
from Merck Millipore, German.
2.2. Evaluation of two enzymatic reaction systems
Two enzymatic reaction systems were evaluated to determine a
system more suitable for electrochemical impedance sensing. 100 μL
E.coli O157:H7 monoclonal antibody with different concentrations
(0 μg mL−1, 0.5 μg mL−1, 1.0 μg mL−1, 2.5 μg mL−1, 5.0 μg mL−1 and
25.0 μg mL−1), which were multiply diluted from original monoclonal
antibody solutions (10 mg mL−1), were coated on each well of 96-well
microtiter (the protocol was described as “Preparation of standard so-
lutions for impedance detection” in supplementary material). Then
100 μL of alkaline phosphatase (ALP)-labeled goat anti-mouse antibody
working solution (diluted to 1:800) and horse radish peroxidase (HRP)-
labeled goat anti-mouse antibody working solution (diluted to 1:1500)
were respectively added in, and incubated for 20 min at 37 °C.
Afterwards, 100 μL of the corresponding substrates for two enzymes
was respectively added to the microtiter plate (p-nitrophenyl phosphate
disodium (PNPP) for ALP and 3,3′,5,5′-tetramethylbenzidine (TMB) for
HRP), the substrate solution was prepared by mixing PNPP or TMB with
coloring agent A and B in equal amounts, the mixture was subjected to
enzymatic reaction for 10 min away from light. The reaction was ter-
minated by enzyme separation, then collected the product solutions
respectively, following impedance detection and the signals were re-
corded. The procedures were repeated three times.
2.3. Electrode design and evaluation
A planar electrode and four interdigitated electrodes W5D25,
W15D15, W10D20, W5D5 with different sizes (where W is the electrode
width and D is the gap distance between two adjacent electrodes. The
values of W and D were measured in mils (1 mil means 0.0254 mm))
were designed and fabricated. Before electrochemical testing, the
electrodes were immersed in ethanol and distilled water, and then so-
nicated in water. The products of the enzymatic reaction with different
concentrations of E.coli O157:H7 monoclonal antibodies (12.500 μg
mL−1, 3.125 μg mL−1 and 0 μg mL−1) were extracted from the mi-
crotiter plate well and added into a 1.5 mL centrifuge tube, then the
solution was respectively detected by the electrodes, and the impedance
signals were recorded. The procedure was repeated three times and the
impedance values were calculated. The measurement parameters were
set as excitation 50 mV and the frequency range of 1–104 Hz.
2.4. DC Bias optimization
For the investigation of influence of direct current (DC) bias on
impedance detection results, different bias voltages were set including
0 mV, 50 mV, 100 mV, 150 mV, 200 mV, and 250 mV. The impedance
values of enzymatic reaction products at five electrodes were recorded,
and measurements at each electrode was respectively repeated three
S. Wang, et al.
times.
2.5. The establishment of impedance system for E.coli O157:H7
quantitative detection
A series of different concentrations of E.coli O157:H7 bacteria cells
were coated on each well of 96-well microtiter, then the enzyme-la-
beled monoclonal antibody was added, and finally the substrate for
enzyme was added. The enzymatic reaction was terminated by enzyme
separation, and the products were divided into two parts, which were
measured by a microplate reader and an impedance analyzer, respec-
tively. This procedure was repeated three times.
2.6. The establishment of magnetic bead-based E.coli O157:H7 impedance
quantitative analysis system
2.6.1. Magnetic beads labeled antibody
The E.coli O157:H7 monoclonal antibody was linked to magnetic
beads by EDC (C8H17N3·HCl)/NHS (C4H5NO3) method. As a negative
control, a goat Immunoglobulin G (IgG) was linked to the magnetic
beads in the same manner. The effect evaluation was performed as
“Effectiveness evaluation of magnetic bead conjugation” in the sup-
plementary material.
2.6.2. E.coli O157:H7 capturing and impedance analysis
1 mg mL−1 magnetic bead-E.coli O157:H7 monoclonal antibody
solution was added to different concentrations of E.coli O157:H7 solu-
tion for co-incubation. After magnetic separation and supernatant dis-
card, the enzyme-labeled anti-E.coli O157:H7 antibodies were added,
obtaining a double antibody sandwich complex (magnetic bead-E.coli
O157:H7 monoclonal antibody)-(E.coli O157:H7)-(enzyme-labeled anti-
E.coli O157:H7 antibody). Finally, corresponding substrates of the en-
zyme were added and reacted for 10 min, the product was measured by
electrochemical impedance analyzer and the impedance value Z (Ω)
was recorded. For the measurement, the parameters of impedance
analyzer P4000 electrochemical work station were set as: excitation
voltage 50 mV and detection range 1–104 Hz. The measurement was
repeated three times.
3. Results and discussion
3.1. Selection of enzymatic reaction system
In order to determine which enzymatic reaction system exhibited
better performance, we compared the ALP-PNPP system with the HRP-
TMB system by measuring the impedance values of the enzymatic re-
action products at planar electrode. As shown in Fig. 1, in the ALP-
PNPP system, as the concentration of E.coli O157:H7 monoclonal an-
tibody increased, the impedance values of enzymatic reaction product
decreased with minor changing degree, even if 0.5 mM PNPP. In con-
trast, in HRP-TMB system, even 0.1 mM TMB at a low concentration,
the impedance values decreased significantly as the antibody con-
centration increased, as shown in Fig. 1B. In Fig. 1C, we extracted and
compared the impedance values of the two systems at 104 Hz. It was
apparent that the HRP-TMB system was more sensitive to changes of
E.coli O157:H7 monoclonal antibody concentration compared with
ALP-PNPP system. Hence, HRP-TMB enzymatic reaction system was
selected for subsequent measurements.
The mechanisms of the two enzymatic reaction systems were pre-
sented in Figure S1. In ALP-PNPP reaction system, both the product
H3PO4 and reactant PNPP were strong electrolytes. Changing the con-
centration of PNPP had little impact on the products solution ionization
intensity, resulting in negligible changes in the impedance values. In
HRP-TMB reaction system, the reactant TMB was a non-electrolyte but
the product included two photons (H+), changing the concentration of
TMB would cause significant changes of the products liquid ionization
3
Biochemical Engineering Journal 156 (2020) 107513
intensity, as well as the impedance values. Therefore, the HRP-TMB
enzymatic reaction system was more sensitive and more suitable for the
following analysis.
3.2. Optimization of electrode structure
Five electrodes including a planar electrode and four kinds of in-
terdigitated electrodes were used, and the structure of the interdigitated
electrodes in detail were shown in Fig. S2. To determine the optimal
electrode structure for impedance measurement, different concentra-
tions of E.coli O157:H7 monoclonal antibody (12.500 μg mL−1,
3.125 μg mL−1 and 0 μg mL−1) were coated on 96-well microtiter and
the impedance value of enzymatic reaction products were measured at
five electrodes respectively. The impedance values at 104 Hz were
compared, and the results were shown in Fig. 2A. It revealed that the
impedance values at 104 Hz measured by five electrodes all decreased
with the increase of antibody concentration. We calculated the reduc-
tion of impedance values at antibody concentrations of 3.125 μg mL−1
and 12.500 μg mL−1 respectively relative to 0 μg mL−1, and compared
the percentage reductions at different electrodes. As Fig. 2B shown,
with the antibody concentration increased, the impedance values at
W5D5 interdigitated electrode had the greatest extent decrease, fol-
lowed by the planar electrode. It indicated that the sensitivity of the
two electrodes was higher than that of other electrodes, which were
more suitable for determination.
3.3. Influence of DC Bias
The influence of different DC bias voltages on electrochemical
analysis was investigated. In order to compare the effects of different
DC bias on the impedance results, we first determined the frequency
with larger changes when changing antibody concentrations at different
electrodes through Fig. S3. Taking bias voltage 250 mV at planar
electrode as an example, the impedance values changed with the
change of E.coli O157:H7 antibody concentration, as shown in Fig. S3A,
and the biggest changes happened at frequency of 102 Hz. Similarly, at
W5D5 interdigitated electrode, when the bias voltage was 0 mV, the
impedance values changed to the utmost extent at 101.5 Hz, as shown in
Fig. S3B. Therefore, the impedance value at 102 Hz on planar electrode
and 101.5 Hz on W5D5 interdigitated electrode were extracted, and the
relationship between impedance values and antibody concentrations at
different bias voltages was shown in Fig. 3. At each concentration, the
DC bias voltage was set as 0 mV, 50 mV, 100 mV, 150 mV, 200 mV and
250 mV, respectively. Although the increase of DC bias voltage caused
an increase of the impedance value at both electrodes, the tendency of
the impedance value decreasing with the increase of antibody con-
centration was constant, indicating that the DC bias has no significant
effect on the impedance measurement. Therefore, no DC bias was re-
quired and the voltage was set as 0 mV.
3.4. E.coli O157:H7 quantitatively detection by impedance method
The performance of the electrochemical impedance system used for
E.coli O157:H7 measurement was compared with conventional ELISA
method. As shown in Fig. 4A, the impedance value changed with fre-
quency when detecting different concentrations of E.coli O157:H7, and
the linearity was good (R2 was 0.99597). It could be inferred that in the
frequency range of 1 to 102 Hz, the impedance value gradually de-
creased with the increase of E.coli O157:H7 concentration, and the
maximum decrease occurred at frequency of 1 Hz. The impedance va-
lues at 1 Hz were then extracted and compared with the results of ELISA
method, where OD450 nm/630 nm were the detection results of E.coli
O157:H7 with different concentrations. Only the results OD450 nm/630
nm≥0.2 were valid data in ELISA method. As shown in Fig. 4B, the
sensitivity of ELISA for E.coli O157:H7 measurement was 105 CFU
mL−1, as the product solution below 105 CFU mL−1 (OD ＜ 0.2) could
S. Wang, et al. Biochemical Engineering Journal 156 (2020) 107513

Fig. 1. Comparison of ALP-PNPP and HRP-TMB enzymatic reaction systems for impedance measurements. A) ALP-0.1 mM PNPP, ALP-0.3 mM PNPP and ALP-0.5 mM
PNPP; B) HRP-0.1 mM TMB; C) Comparison of impedance values of products after different enzymatic reaction systems at 104 Hz.

Fig. 2. A) Impedance values at 104 Hz measured by five electrodes. B) Comparison of impedance values reduction percentage at different electrodes.

not be detected. In contrast, 102 CFU mL−1 of E.coli O157:H7 could be ELISA method, and was more applicable for analysis of rare samples
detected by electrochemical impedance method, and the detection limit with lower concentration.
was three orders of magnitude lower than the ELISA method. The
coefficient of variation (CV) of this impedance method was less than 10
3.5. E.coli O157:H7 detection by magnetic bead-based impedance method
% (n = 3), proving its good stability and accuracy. It suggested that the
sensitivity of the impedance method was much higher than that of the
Based on the above-mentioned preliminary verification of the

Fig. 3. A) Impedance values at 102 Hz with different DC bias

offsets at planar electrode. B) Impedance values at 101.5 Hz
with different DC bias offsets at interdigitated W5D5 elec-
trode. The concentration of E.coli O157:H7 antibody from 1 to
7 was respectively 0.012 μg mL−1, 0.049 μg mL−1, 0.195 μg
mL−1, 0.780 μg mL−1, 3.125 μg mL−1, 12.500 μg mL−1 and
50 μg mL−1.

4
S. Wang, et al. Biochemical Engineering Journal 156 (2020) 107513

Fig. 4. A) Impedance results for the detection of different concentrations of E.coli O157:H7. B) Comparison of the electrochemical impedance method with ELISA
method for different concentrations of E.coli O157:H7 detection. (n = 3, CV < 10 %).

Fig. 5. A) Impedance result for the detection of different concentrations of E.coli O157:H7 by magnetic bead-based impedance analyzer. B) Comparison of the
magnetic bead-based impedance system with ELISA method for different concentrations of E.coli O157:H7 detection. (n = 3, CV < 10 %).

feasibility of the electrochemical impedance method, we further com- CRediT authorship contribution statement
bined magnetic bead with the impedance system to improve the effi-
ciency of E.coli O157:H7 detection. The introduction of magnetic bead Shujun Wang: Writing - original draft, Writing - review & editing,
avoided tedious electrode modification process and matrix adsorption Data curation. Chongyun Sun: Methodology, Investigation, Data
on the electrode surface. The impedance value recorded by the mag- curation, Visualization. Qiushi Hu: Validation. Shuang Li: Resources.
netic bead-based impedance detection system de with the frequency Chengbin Wang: Project administration. Peng Wang:
when detected different concentrations of E.coli O157:H7, as shown in Conceptualization, Methodology, Software. Lei Zhou:
Fig. 5A. In the frequency range of 1 to 102 Hz, the impedance value Conceptualization, Methodology, Project administration.
gradually decreased with the increase of E.coli O157:H7 concentration,
and the maximum decrease occurred at frequency of 1 Hz. Then the Declaration of Competing Interest
impedance value at 1 Hz was extracted and compared with the ELISA
method. As Fig. 5B shown, the sensitivity of the magnetic bead-based The authors declare that they have no known competing financial
impedance system was 102 CFU mL−1, CV < 10 % (n = 3), which was interests or personal relationships that could have appeared to influ-
three orders of magnitude lower than the ELISA method. Therefore, this ence the work reported in this paper.
magnetic bead-based electrochemical impedance system was more
suitable for the analysis of low concentration samples. This system did Acknowledgements
not require electrode modification and sample adsorption, what’s more,
as the gold interdigitated electrode was an inert electrode, after several This work was supported by the Beijing Nova Program [grant
times of measurements, the surface of the electrode remained smooth number Z151100000315086]; Innovation Program of National Defense
and the electrodes could be reused with high sensitivity. Science and Technology [grant number 17-163-12-ZT-005-029-01];
and National Key Research and Development Program [grant number
4. Conclusions 2016YFC1202502].

In conclusion, we developed a magnetic bead-based electrochemical Appendix A. Supplementary data

impedance immunosensor for E.coli O157:H7 detection. We optimized
the HRP-TMB system as the enzymatic reaction system, the W5D5 in-
terdigitated electrode as sensing element, and proved that no DC bias
voltage should be set for E.coli O157:H7 immunoassay. The limit of the
magnetic bead-based impedance system for detection of E.coli O157:H7
was 102 CFU mL−1, which was three orders of magnitude lower than
the traditional ELISA method. The combination of magnetic beads with
electrochemical impedance system solved the problem of electrode
adsorption, improved sensitivity and reproducibility of the detection.
Therefore, this work opens a new road for the development of elec-
trochemical immunoassays for the detection of low-concentration pa-
thogens.
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.bej.2020.107513.
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