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1. Sensors & Actuators: B. Chemical 347 (2021) 130630
Available online 20 August 2021
0925-4005/© 2021 Elsevier B.V. All rights reserved.
Ultrasensitive detection of pathogenic bacteria by CRISPR/Cas12a coupling
with a primer exchange reaction
Shengjun Bu , Xiu Liu , Ze Wang , Hongguo Wei , Songling Yu , Zhongyi Li , Zhuo Hao ,
Wensen Liu *
, Jiayu Wan *
Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun 130122, China
A R T I C L E I N F O
Keywords:
CRISPR/Cas12a
Primer exchange reaction
Signal amplification
Bacteria detection
ECL signal
A B S T R A C T
The detection of pathogenic bacteria is extremely important to maintain food safety. In the present study, we
constructed an electrochemical biosensor for point-of-care testing (POCT) of pathogenic bacteria through the
cascade signal amplification of CRISPR/Cas12a and primer exchange reaction (PER) without nucleic acid
extraction. Functional DNA aptamers locked the hairpin of PER, thereby preventing primer extension in the
absence of target pathogenic bacteria. The presence of target pathogenic bacteria triggered the unlocking of the
hairpin of PER and extended the primer into a long single-stranded DNA (ssDNA), which then activated the
cleavage of ssDNA modified on an Au electrode by Cas12a, resulting in the decrease of the electrochemical signal
detectable by an electrochemical workstation. The sensor could quantify Escherichia coli O157:H7 concentration
from 10 to 106
CFU mL− 1
, with a detection limit of 19 CFU mL− 1
. Target bacteria in milk samples can be suc­
cessfully detected by this method. On the basis of these characteristics, the proposed electrochemical biosensor
based on CRISPR/Cas12a and PER could serve as a prospective tool for new point-of-care (POC) applications in
food safety.
1. Introduction
Foodborne diseases caused by pathogenic bacteria are a serious
threat to public health and state economy. Escherichia coli O157:H7
(E. coli O157:H7), a food contaminant, is one of the most common
foodborne pathogenic bacteria [1]. In USA, over 63,000 cases of E. coli
O157:H7 foodborne infection are estimated to occur each year, with
approximately 2100 hospitalizations and up to 20 deaths. The early
detection of pathogenic bacteria is important to ensure food quality and
safety [2]. The traditional culture-based approach for detecting E. coli
O157:H7 has low sensitivity and is labor-intensive and time-consuming,
and it usually requires a long culture time (2–3 days). Some other
methods such as real-time quantitative PCR (qPCR) and enzyme-linked
immunosorbent assay (ELISA) have reduced the assay time from days to
several hours, but they are relatively expensive and need specialized
equipment and trained personnel, which limit their widespread appli­
cation [3]. Therefore, it is critical to develop innovative methods that
are low cost, sensitive, and simple to detect pathogenic bacteria.
Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR)-Cas system is a complex immune system in archaea and
bacteria that confers resistance to foreign nucleic acids [4]. (CRISPR)-­
Cas systems mainly use Cas effector proteins under the guidance of a
guide-RNA (gRNA) to recognize and destroy the target nucleic acid [5].
Recently, Cas12a (previously named as Cpf1) was shown to exhibit a
collateral cleavage activity on surrounding nonspecific, nontarget
single-stranded DNA (ssDNA) as reporters providing signal readout after
gRNA recognizes the target DNA. This unique property shows that
Cas12a has great potential for developing novel biosensors for pathogen
detection [6–8]. To improve the sensitivity of Cas12a-based detection
method, it is usually combined with some isothermal amplification
techniques [9]. For instance, by coupling with recombinase polymerase
isothermal amplification technique (RPA), Cas12a was used to construct
DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter)
methods for rapid and sensitive visual analysis of human papillomavirus
nucleic acid [10]. Cas12a combined with RPA has also been used for
electrochemical detection of Listeria monocytogenes nucleic acid [11].
Some Cas12a-based biosensors, which incorporate other isothermal or
non-isothermal amplification techniques (e.g., loop-mediated
isothermal amplification (LAMP) [12], PCR [13], and multiple cross
displacement amplification [14]) with Cas12a/gRNA complexes to
* Corresponding authors.
E-mail addresses: Liuws85952@163.com (W. Liu), wanjiayu@hotmail.com (J. Wan).
Contents lists available at ScienceDirect
Sensors and Actuators: B. Chemical
journal homepage: www.elsevier.com/locate/snb
https://doi.org/10.1016/j.snb.2021.130630
Received 29 June 2021; Received in revised form 27 July 2021; Accepted 17 August 2021

2. Sensors and Actuators: B. Chemical 347 (2021) 130630
2
allow rapid detection of nucleic acids of pathogens such as SARS-CoV-2,
have recently been successfully established [15]. However, all these
Cas12a biosensing methods depend on nucleic acid extraction and
amplification for sensitive detection of the target pathogen. Hence, they
require intricate reagent/sample treatment steps and sophisticated sys­
tems. This can render the system to become less robust and practically
cumbersome [16].
A robust signal isothermal amplification strategy, primer exchange
reaction (PER), has recently attracted wide attention in the field of
biosensors and biological imaging [17]. In PER, short DNA primers are
programmed to autonomously synthesize ssDNA in vitro. A single PER
hairpin serves as a catalytic template, and a nascent single-stranded
sequence is attached to the short DNA primer through DNA polymer­
ase. The autonomously synthesized ssDNA can spontaneously detach
from the PER hairpin, which becomes free and reacts with another
primer to initiate the next cycle of ssDNA synthesis. A series of
high-performance biosensors based on PER have been developed for the
amplification and imaging of proteins, RNA, and DNA in cells and tissues
[18,19].
Herein, we introduced CRISPR/Cas12a into an electrochemical
biosensor for detecting pathogenic bacteria, combined with a structure-
switching aptamer and PER, to establish a cost-effective, specific, and
ultrasensitive method (Scheme 1). Functional DNA aptamer molecules
can lock hairpin of PER, thus preventing primer extension in the absence
of target pathogenic bacteria. The presence of target pathogenic bacteria
can lead to binding to aptamers and expose the complementary domain
of the primer. This will trigger the PER reaction and extend the primer to
grow nascent strands of ssDNA (PER product). This PER product can
generate the formation of a Cas12a-crRNA-target DNA ternary complex,
which can then activate the ssDNase activity of Cas12a. This will cause
the loop region of the MB probe to divide into short fragments, resulting
in the breakage of the stem part of MB. Finally, the MB will be released
from the Au electrode and weaken the peak current, resulting in a
remarkable decrease in the electrochemical signal. Even without nucleic
acid extraction from the target pathogen, target-responsive functional
DNAs (structure-switching aptamer) and PER reaction can switch the
target pathogen recognition event into the collateral cleavage activity of
Cas effectors, which is usually activated through the specific integration
of the crRNA with its target DNA (as “activator”). Based on PER reaction
and the Cas12a system, the electrochemical biosensor achieved a change
in the electrochemical signaling probe (ssDNA of the MB probe), thus
causing an obvious change in the electron transfer of the electro­
chemical tag depending on the presence or absence of the target.
2. Experimental section
2.1. Reagents and materials
All DNA oligonucleotides used in this study were synthesized and
modified from Sangon Biotech Co., Ltd (Shanghai, China). The corre­
sponding sequences are shown in Table S1 of Supporting Information.
HiScribe™ T7 Quick High Yield RNA Synthesis Kit (NEB #E2050),
Monarch® RNA Cleanup Kit, Engen Lba Cas12a, and 10 × NEBuffer 2.1
(0.5 M NaCl, 0.1 M Tris-HCl, 0.1 M MgCl2, and 0.01 M BSA, pH 7.9)
were purchased from New England Biolabs (Ipswich, MA, USA). The
storage buffer used for DNA oligonucleotides and crRNAs was HEPES
buffer (20 mM HEPES, 8 mM MgCl2, 20 mM KCl, 200 mM NaCl, pH 7.4)
and RNase-free water, respectively. EnGen®Lba Cas12a (Cpf1) and
1 × NEBuffer™ 2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2,
100 µg mL− 1
BSA, pH 7.9) were purchased from New England Biolabs
Ltd. (Beijing). Streptavidin-coated Dynabeads (DBs) were purchased
from Themo Fisher Scientific Co., Ltd. (Norway). Bst DNA Polymerase
(Bst), dNTP mixture, Tris(2-carboxyethyl) phosphine hydrochloride
(TCEP), and Water-DEPC Treated Water were obtained from Sangon
Biotech Co., Ltd (Shanghai, China). K3[Fe(CN)6] and K4[Fe(CN)6] were
obtained from Sigma-Aldrich (St. Louis, MO, USA). An RNase inhibitor
and RNase-free water were obtained from Tiangen Biotech Co., Ltd.
(Beijing, China). E. coli O157:H7 (CICC 10907), Escherichia coli (CICC
Scheme 1. Illustration of the PER-assisted CRISPR/Cas12a system for E. coli O157:H7 detection. Abbreviations: Hp: Hairpin, Ap: Aptamer, MB: MB probe,
DBs: Dynabeads.
S. Bu et al.

3. Sensors and Actuators: B. Chemical 347 (2021) 130630
3
10389), Salmonella typhimurium (CICC 21484), Vibrio parahaemolyticus
(CICC 21617), and Staphylococcus aureus (CICC 21600) were provided
by the China Center of Industrial Culture Collection (Beijing, China). All
buffer solutions were prepared with Water-DEPC Treated Water and
sterilized. All centrifuge tubes and tips used were RNase-free. All other
chemicals were purchased from Chemical Works Co., Ltd. (Beijing,
China) and used without further purification.
2.2. Apparatus
Differential pulse voltammetry (DPV), electrochemical impedance
spectroscopy (EIS), and cyclic voltammetry (CV) experiments were
performed on a Shanghai Chenhua CHI660E electrochemical worksta­
tion (Shanghai, China). All electrochemical experiments were conducted
with a conventional three electrode system including an Au electrode
(working electrode, Φ = 2 mm), an Ag/AgCl electrode (reference elec­
trode), and a platinum wire (counter electrode) and were performed at
room temperature. CV measurement was performed stepwise in 0.1 M
KCl solution containing a 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] mixture at
potentials ranging from − 0.2 to 0.6 V, and the scan rate was 50 mV/s.
EIS was also performed in 0.1 M KCl solution containing a 5 mM K3[Fe
(CN)6]/K4[Fe(CN)6] mixture at the frequency range of 0.1 Hz to
100 kHz with a given open circuit potential of 5 mV. DPV measurement
was performed in 5 mL of 20 mM PBS containing 50 mM NaCl and
50 mM KCl (pH 7.4) as the supporting electrolyte, and the experiment
parameters were as follows: voltage within the range of − 0.5 to − 0.1 V;
pulse amplitude of 50 mV.
2.3. Preparation of probes
First, the functionalization probe of MB with the thiol group at one
end and the methylene blue group at the other end was incubated at
95 ◦
C for 5 min and cooled to room temperature for 2 h to form a stem-
loop structure. Then, the MB probe was dissolved in 100 μL of 20 mM
PBS buffer (containing 100 mM TCEP, pH 7.4) to the final concentration
of 300 nM, and the mixture was incubated in dark at room temperature
for 1 h to cleave the disulfide bond. Finally, the prepared MB was stored
at 4 ◦
C for further studies.
Next, 0.2 μM hairpin probe was mixed with 5 μM AP probe, incu­
bated at 95 ◦
C for 5 min, and cooled to room temperature to lock the
hairpin Hp of PER. The prepared AP-Hp (the probe of Hp was modified
with biotin at the 3′
-end) was then incubated with washed streptavidin-
coated DBs (pure water for three times to remove the preservative) at
37 ◦
C for 60 min. Through the specific recognition of biotin and strep­
tavidin, the DBs and AP-Hp were tightly linked together. Subsequently,
these nonfunctional DBs-AP-Hp were washed three times with HEPES
buffer to remove the uncombined AP-Hp and resuspended in the same
buffer, followed by storage at 4 ◦
C for further use.
2.4. Electrode cleaning and modification
Before the modification of the functional probe, the working elec­
trode of Au was carefully polished and cleaned according to a previously
reported method [20]. The Au electrode was then immersed in the
prepared MB probe to self-assemble on the Au surface through an Au-S
covalent bond and kept overnight at room temperature to create the
MB/Au electrode. Finally, 10 μL MCH (1 mM) was added to the MB/Au
electrode and incubated at 37 ◦
C for 1 h to passivate the Au electrode
surface and to obtain well-aligned DNA monolayers. After rinsing and
drying, the treated electrode was labeled as the MCH/MB/Au. To
characterize the construction of the electrode interface stepwise, CV and
EIS were performed in a 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] mixture.
2.5. CRISPR/Cas12a platform setup
The HiScribe Quick T7 High Yield RNA Synthesis Kit was used to
composite crRNAs according to a specific protocol. In brief, the DNA
templates containing the T7 promoter were incubated at 95 ◦
C for 5 min
and then left at room temperature for 2 h. A typical reaction that
included DNA templates with the other transcription components was
performed at 37 ◦
C for 4 h. The Monarch RNA Cleanup Kit was used to
purify the as-obtained crRNA. Finally, the prepared crRNA was quanti­
fied by NanoDrop 1000 (Thermo Fisher Scientific) and stored at − 80 ◦
C
before use.
2.6. Bacterial strains
Strains of E. coli O157:H7, E. coli, S. typhimurium,
V. parahaemolyticus, and S. aureus were cultured in sterile LB broth
(180 rpm) at 37 ºC for 12 h. The resulting bacterial culture was quan­
tified by measuring the optical density at 600 nm.
2.7. Protocol for E. coli O157: H7 detection
Briefly, different concentrations of E. coli O157: H7 (50 μL) were
added to the pretreated DBs-AP-Hp (3 μL) and incubated at 37 ◦
C for
30 min. The mixture was then rinsed by repeated magnetic separation
thrice using 30 μL HEPES solution (pH 7.4) by using a magnetic rack and
resuspended in HEPES. The prepared DBs-AP-Hp were released based on
the specific interaction between aptamer and target bacteria [21]. As
shown in Fig. 1A, the unlocked Hp could expose the complementary
domain 1 * of the primer (the step of primer binding). Then, with the
assistance of Bst, the primer was subsequently bounded with domain
2 * and extended to a nascent ssDNA (2 domain) through to the strand
displacing elongation response (the step of strand displacing elonga­
tion). The neonatal 2 domain was then allowed to connect with the 2
domain on the Hp through the random walk procedure of three-way
branch migration [17] (the step of branch migration). The new strand
then autonomously segregated from the PER hairpin of Hp once the
copied domain 2 has been replaced (the step of transcript dissociation).
Next, the other primer was reacted with the PER hairpin of Hp in another
cycle of PER reaction. The PER reaction cycle was performed by adding
20 μL mixed liquor (4 μL 2 × HEPES buffer, 5 μL 5× Bst reaction buffer,
5 μL dNTP mixture (0.2 mM), 4 μL primer (1 µm), 2 μL Bst DNA Poly­
merase (8000 U)) and incubated with the cleaned DBs-AP-H at 37 ◦
C for
60 min. The Bst was then deactivated by incubation at 80 ◦
C for 5 min.
Subsequently, the supernatant (PER product, 4 μL) was separated from
the mixture as an ssDNA activator to activate the Cas12a-mediated
cleavage reaction by adding 10 μL of LbaCas12a-crRNA complexes
(including 4 μL crRNA (500 nm), 4 μL LbaCas12a (300 nM), 2 μL
RNAsin Inhibitor (2 U)). Subsequently, the above mixed liquor was
dropped onto the prepared MCH/MB/Au electrode surface at 37 ◦
C for
1 h. The interfacial cleavage activity of Cas12a was activated, which
caused the MB reporter to separate from the Au electrode surface,
thereby reducing the MB signal transduction. After rinsing and drying,
the ECL measurement of DPV was performed in 5 mL PBS (20 mM, pH
7.4) containing 50 mM NaCl and 50 mM KCl.
2.8. Gel electrophoresis
Initially, 10 μL of each sample was mixed with 6× loading buffer
(2 μL). Next, different mixtures (10 μL) were instilled into the native
polyacrylamide gel electrophoresis (PAGE) system. The PAGE was per­
formed on a 15% gel in 1× TBE (89 mM Tris, 89 mM boric acid, and
2.0 mM EDTA, pH 8.2) at 100 V constant voltage for 50 min using the
above prepared samples (10 μL). The gels were stained with SYBR for
20 min and then visualized on a gel imaging system (Beijing Sage Cre­
ation Science Co., Ltd., Beijing, China).
2.9. Real sample preparation
To simulate real food samples, pure milk was purchased from a local
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4. Sensors and Actuators: B. Chemical 347 (2021) 130630
4
supermarket. The detection of pathogens in food processing was based
on the national standards protocol (GB 4789) of China [22]. Briefly,
25 mL of pure milk sample was homogeneously mixed with 225 mL of
PBS buffer. Next, different amounts of E. coli O157:H7 cells were added
to the mixture to prepare artificially contaminated samples. Thus, spiked
samples containing different concentrations (103
to 107
CFU mL− 1
) of
E. coli O157:H7 were acquired. Finally, the procedure for target detec­
tion was the same as that described above. The recovery rate was
calculated by the ratio of the measured E. coli O157:H7 signal and the
amount of E. coli O157:H7 added to the system.
3. Results and discussion
3.1. Feasibility of the PER-Cas12a biosensor
To verify the validity of the PER-CRISPR/Cas12a approach to detect
pathogenic bacteria, PAGE and DPV were performed to examine the
feasibility of this system. The PER product used in this study activated
the CRISPR/Cas12a system. The loop-stem structure of the MB probe
was designed to serve as the substrate of Cas12a/crRNA trans-cleavage.
Hence, it was necessary to verify whether the PER product efficiently
activated the CRISPR/Cas12a system to digest the MB. The PER primer
led the DNA replication machinery to produce the PER product
continuously in the presence of hairpin H through the mechanism shown
in Fig. 1A and B. After assembling the PER product, the CRISPR/Cas12a
system was directly activated, and the MB could be digested. The re­
action products were investigated by PAGE. As shown in Fig. 1C, lanes a
and b represent the crRNA and MB, respectively. After the addition of
the PER system, the MB band was dissociated by the activated CRISPR/
Cas12a system with a lower molecular weight. A negligible MB band was
observed in the absence of the PER product (line 3), which indicated that
the loop-stem structure of the MB probe was cut. In the absence of the
PER system, the band of MB reporter did not show any obvious changes
in the control group (line 4). In general, these results verified that the
PER approach successfully led to the ssDNase activity of Cas12a and
triggered the degradation of the MB probe. The electrochemical bio­
sensing platform was developed based on the Cas12a system, and DPV
was applied to evaluate the MB signal on the Au electrode surface. In
short, different DPV responses were compared to determine the capacity
of Cas12a to perform cleavage of the assembled MB reporter on the Au
electrode surface. As shown in Fig. 1D, the peak current signal was
dramatically decreased only in the presence of the target E. coli O157:
H7, Cas12a, and crRNA. If any element was missing, the Cas12a trans-
cleavage activity was silenced, thus retaining the ssDNA of MB on the
Au electrode surface (Fig. 1D).
3.2. Electrochemical characterization of the modification of the electrode
EIS and CV were performed to verify whether the electrochemical
biosensor was successfully fabricated. The Nyquist plots of impedance
Fig. 1. Feasibility analysis of PER/CRISPR for E. coli O157:H7 detection. (A) Schematic representation of the mechanism of the PER reaction. (B) PAGE to char­
acterize the PER reaction. (C) PAGE on a 15% gel to characterize oligonucleotides used for signal amplification: lane a, crRNA; lane b, ssDNA of MB; lane c, positive
PER reacted with MB; lane d, negative PER reacted with MB. (D) DPV characterization of the electrochemical signal with and without CRISPR/Cas12a.
S. Bu et al.

5. Sensors and Actuators: B. Chemical 347 (2021) 130630
5
were analyzed to represent the stepwise assembly process to detect the
target bacteria on the MB-modified Au electrode. The transfer kinetics of
[Fe(CN)6]3− /4−
was represented by the charge transfer resistance (Rct),
which is equal to the change in the semicircle diameter of each EIS
curve. As shown in Fig. 2B and Table S2, the bare Au electrode showed a
very small Rct value, a nearly straight line (Rct = 156 Ω, curve a), thus
showing excellent electrochemical conductivity and indicating good
preparation of the clean electrode. When the MB reporter was fixed onto
the surface of bare Au electrode, the Rct increased distinctly (Rct =
23,060 Ω, curve c) because the negatively charged phosphate backbone
of oligonucleotides of MB promoted electrostatic repulsion between
K3[Fe(CN)6]/K4[Fe(CN)6]. These results proved that the MB reporter
was successfully fixed onto the surface of the Au electrode by the Au-S
bond. The Rct value was further increased when the MCH on the elec­
trode was blocked; this was because the MCH compact layer could
preclude electron permeation to the electrode surface (Rct = 28,640 Ω,
curve d). In contrast, following the addition of the target bacteria to
initiate the PER/CRISPR approach, the Rct significantly increased (Rct =
8250 Ω, curve b), thus demonstrating the successful cleavage of ECL
signal tags by the target E. coli O157:H7. After incubation with the target
E. coli O157:H7, the Ap were carried in the prepared hairpin structure of
H-Ap, revealing the toehold parts of the PER primer. The DNA ma­
chinery was triggered to produce the PER product. The PER product as
the target sequence activated the Cas12a system and cut the loop region
of the MB reporter into short fragments on the electrode. The MB was
then discharged from the electrode, indicating successful working of the
entire detection mechanism. CV measurements were conducted to
further characterize the fabrication of the biosensor step-by-step and
performed in a potential range of − 0.2 to 0.6 V (Fig. 2A). A pair of well-
defined redox peaks were obtained for the bare Au electrode. After the
modification, the electrode surface showed a similar tendency to that
obtained for the EIS measurements, in which the peak currents varied
upon the processes of binding and assembly. The results of CV and EIS
confirmed the successful fabrication of MB on the Au electrode surface.
3.3. Optimization of the experimental conditions
To achieve better performance of the biosensor, some relevant
experimental conditions were optimized. Here, (I - I0)/I0 was defined as
the current response signal, where I and I0 represented the current in­
tensity of presence and absence of target bacteria, respectively. First, the
trans-cleavage activity was crucial for signal transduction, as it deter­
mined the sensitivity of the entire experiment. Therefore, we first
investigated different reaction conditions such as concentration of
LbaCas12a, incubation time of LbaCas12a, incubation temperature, and
amount of crRNA. As shown in Fig. S2A, the current signal (I - I0)/I0
increased rapidly as the concentration of LbaCas12a increased from 0.01
to 0.3 µm and then became stable. Hence, an optimized LbaCas12a
concentration of 0.3 µm was chosen to prepare the LbaCas12a-crRNA
duplex. Next, the effect of incubation time on the trans-cleavage effi­
ciency was monitored by DPV. The (I-I0)/I0 continuously increased with
the increase in incubation time, thus demonstrating that the trans-
cleavage mechanism is an ongoing response (Fig. S2B). With regard to
the timeliness of this assay, the appropriate response time was within
60 min. When the detection temperature changed from 15 ◦
C to 37 ◦
C
(Fig. S2C), the current signal increased. Thus, the signal had obviously
increased as the incubation temperature increased. This may be because
high temperature affects the activity of Cas12a. Thus, the optimal
temperature of 37 ºC was used in this study. Furthermore, to test the
influence of the amount of crRNA on the performance of CRISPR for
E. coli O157:H7 detection, different concentrations of crRNA from 0.05
to 5 μM were added to the CRISPR system. As shown in Fig. S2D, the
current signal reached the maximum value when crRNA concentration
was 0.5 μM; with further increases in crRNA concentration, the current
signal remained steady. Thus, 0.5 μM was selected as the appropriate
concentration.
Some other critical experimental conditions were chosen for inves­
tigation to achieve an excellent analytical capability. For instance, the
incubation time of target bacteria and PER reaction time highly affected
the sensitivity and accuracy of this assay. When the incubation time of
target bacteria increased from 5 min to 30 min, the ECL signal also
significantly increased and then showed a tendency to plateau
(Fig. S3A). Thus, the optimal incubation time of target bacteria was
30 min. In the PER reaction system, the reaction time of the Bst was
another relevant parameter that affects the performance of the
biosensor. The current signal increased as the reaction time of the Bst
progressed from 15 min to 60 min (Fig. S3B). However, when the re­
action time of PER increased to 60 min, the current intensity did not
increase. Thus, the optimal reaction time of PER of 60 min was used in
this study. The amount of DBs used to grab the PER hairpin of Hp. As
shown in Fig. S3C, with the increase in the concentration of DBs, the
currents in DPV curves reached the maximum and did not increase
further or reduced, thus revealing that a high amount of DBs could
inhibit the effectiveness of PER due to the effects of steric hindrance.
Therefore, the optimal amount of DBs was 3 μL. Finally, the pH of re­
action buffer played a crucial part in this assay. As illustrated in Fig. S3D,
the current intensity reached the maximum and plateaued when the pH
value reached 6.0.
3.4. Performance of the E. coli O157:H7 detection system
Under the optimal experimental conditions, the current signal for a
Fig. 2. The characterization of the fabricated electrochemical biosensor based on CV graphs (A) and EIS measurements (B): a, bare Au electrode; b, MB modified on
Au electrode; c, MB modified on Au electrode after blocking with MCH; d, the proposed electrochemical biosensor after PER/CRISPR reaction.
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6. Sensors and Actuators: B. Chemical 347 (2021) 130630
6
series of diluted E. coli O157: H7 culture was recorded to evaluate the
analytical capability of this assay through DPV measurements. As shown
in Fig. 3, the MB oxidation peak intensity exhibited a gradual decrease
with the increase in different amounts (10–108
CFU mL− 1
) of the target
E. coli O157:H7 concentration. A strong linear relationship was observed
between the peak current in DPV signal and the logarithmic value of
E. coli O157:H7 concentration in the serially concentration range from
103
to 108
CFU mL− 1
, with a correlation equation of Y= 0.1304 *X-
0.1072 (R2
= 0.959), where X represent the logarithmic value of E. coli
O157:H7 and Y is the peak current (μA). The lowest limit of detection
(LOD) of 19 CFU mL− 1
was obtained based on the signal-to-noise ratio of
3. Different concentrations of target bacteria (105
-107
CFU mL− 1
) were
then used to evaluate the reproducibility performance of this biosensor.
As shown in Fig. S4A, the relative standard deviations (RSDs) were
2.72% for 105
CFU mL− 1
, 2.59% for 106
CFU mL− 1
, and 3.67% for 107
CFU mL− 1
in five independent measurements. The detection was per­
formed using the same batch electrode through intermittent DPV
monitor for 10 ten times after incubation with 108
CFU mL− 1
E. coli
O157:H7, and the response of DPV was steady (Fig. S4B), thus con­
firming good repeatability and stability. Comparison of this strategy
with some reported methods is shown in Table 1. The results indicated
that the sensitivity of this proposed strategy for bacterial detection was
better than most recently reported methods. Among the recent methods,
V. Mazzaracchio et al. reported an electrochemical aptasensor for
detecting B. cereus spores [23]. The electrochemical aptasensor
demonstrated the capability to detect B. cereus spores, with a detection
limit of 3 × 103
CFU mL− 1
. Compared to this aptasensor, our method
combines the cascade signal amplification of CRISPR/Cas12a with PER
for the first time to detect pathogenic bacteria, and the sensitivity was
almost increased by 150 times. The immunological assays developed by
R. F. Ye et al. [24] were based on the interaction between the immo­
bilized antibody and pathogenic bacteria and achieved an LOD of
10 CFU mL− 1
. The main disadvantages of this immunoassay are that the
antibodies used in the assay need to be produced in animals and the shelf
life of antibodies under working and storage conditions was often very
short. Aptamers for biosensing applications overcome these drawbacks
because of their distinct performance of specificity and stability for the
target analyte. On the basis of structure-switching mechanisms, the
aptamer of the biosensor specifically bound to the target bacteria and
released the closed hairpin structure, further initiating the amplification
of the cascade signal coupling PER and CRISPR/Cas12a. The biosensor
innovatively combines aptamer with CRISPR/Cas12a and PER to ach­
ieve portable and sensitive detection of pathogenic bacteria.
To evaluate the selectivity of this proposed biosensor, L. mono­
cytogenes, S. typhimurium, E. coli, V. parahaemolyticus, and a negative
control (PBS) were introduced as nontarget bacteria at the same con­
centration of 107
CFU mL− 1
(Fig. 4). As expected, only E. coli O157:H7
triggered a conspicuous change in the current signal as compared to the
other tested strains of pathogenic bacteria.
To investigate the potential application of the designed system in real
world, the detection of target bacteria in pure milk samples was per­
formed. Different concentrations of E. coli O157:H7 cells were spiked
into 10 × diluted milk samples, and the samples were tested by the
proposed method. The recovery (%) was defined as the ratio of the mean
tested concentration by this assay and the spiked concentration of E. coli
O157:H7. The results of five independent samples are given in Table S3.
The obtained average recovery (%) for milk samples ranged from 91.3%
to 128.0%, with an RSD of 1.2–14.1%. These findings confirmed that
this assay had good reproducibility to detect E. coli O157:H7 in actual
samples. To summarize, the obtained results showed that the micro­
electrode immunosensor could provide an effective electrochemical
platform to detect E. coli O157:H7.
4. Conclusion
In summary, an electrochemical biosensor for highly sensitive
detection of pathogenic bacteria E. coli O157:H7 by combining CRISPR/
Cas12a with PER was successfully established for the first time without
nucleic acid extraction of pathogenic bacteria. This biosensor showed
the LOD of 19 CFU mL− 1
for E. coli O157:H7. The developed strategy
also showed high selectivity to E. coli O157:H7 and can be applied to real
food samples with good recovery and accuracy. The successful combi­
nation and application of CRISPR/Cas12a with PER provides a new
insight to improve the use of CRISPR/Cas12a and PER for extensive
applications. In general, the proposed biosensor under isothermal con­
ditions is nucleic acid extraction-free and has low dependence on
analytical instruments, which meets the requirements of rapid bacterial
detection for food safety. Presently, we are focusing on minimizing the
requirements of operation procedure in order to make the
manufacturing process more convenient. Thus, the biosensor meets the
need for rapid diagnosis of E. coli O157:H7 in a variety of settings,
especially in resource-limited environments.
CRediT authorship contribution statement
Shengjun Bu: Methodology, Investigation, Writing – original draft.
Xiu Liu: Validation, Methodology. Ze Wang: Methodology. Hongguo
Wei: Investigation. Songling Yu: Investigation. Zhongyi Li: Investiga­
tion. Zhuo Hao: Investigation. Wensen Liu: Supervision, Writing – re­
view & editing. Jaiyu Wan: Conceptualization, Methodology, Writing –
Fig. 3. Performance of PER/CRISPR for target E. coli O157:H7 detection. (A) The DPV curve of MB in the presence of different concentrations (10–108
CFU mL− 1
) of
E. coli O157:H7 and HEPES as the control signal. (B) Linear analysis results for different concentrations of E. coli O157:H7 (103
, 104
, 105
, 106
, 107
, and 108
CFU mL− 1
). All plots are expressed as mean ± SD for n = 3 replicates.
S. Bu et al.

7. Sensors and Actuators: B. Chemical 347 (2021) 130630
7
review & editing, Supervision, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was financially supported by the Science and Technology
Development Plans of Jilin Province (20150101105JC).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.snb.2021.130630.
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Comparison of different electrochemical techniques to detect pathogenic bacteria.
Assay Target Linear range (CFU mL− 1
) LODa
(CFU mL− 1
) Recognition elements Reference
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Shengjun Bu is now studying for Ph.D. degree in Academy of Military Medical Sciences.
Her major is Preventive Veterinary Medicine. Her current research interests are biosensors
and pathogen diagnosis.
Xiu Liu is currently studying at Changchun University of Science and Technology as a
master’s student. Her major is Bioengineering. Her current research interests are bio­
sensors and pathogen diagnosis.
Ze Wang is now a graduate student at Jilin Agricultural University. Her major is Micro­
biology. Her current research interests are biosensors and pathogen diagnosis.
Hongguo Wei is studying for a master’s degree in Changchun University of Science and
Technology. Her major is Biochemistry and Molecular Biology. Her current research in­
terests are biosensors and pathogen diagnosis.
Songling Yu is now a graduate student at Jilin Agricultural University. Her major is
Microbiology. Her current research interests are biosensors and pathogen diagnosis.
Zhongyi Li obtained his M.S. degree in biochemistry and molecular biology from Jilin
University. He is currently an associate professor at Academy of Military Medical Sciences.
His current research interests are biosensors and pathogen diagnosis.
Zhou Hao obtained her M.S. degree in biochemistry and molecular biology from Jilin
University. She is currently an associate professor at Academy of Military Medical Sci­
ences. Her current research interests are biosensors and pathogen diagnosis.
Wensen Liu obtained his Ph.D. degree in veterinary medicine from Academy of Military
Medical Sciences. He is currently a professor at Academy of Military Medical Sciences. His
current research interests are biosensors and pathogen diagnosis.
Jiayu Wan obtained his Ph.D. degree in biochemistry and molecular biology from Jilin
University. He is currently an associate professor at Academy of Military Medical Sciences.
His current research interests are biosensors and pathogen diagnosis.
S. Bu et al.