Sensors & Actuators: B.

Chemical 371 (2022) 132532

Contents lists available at ScienceDirect

Sensors and Actuators: B. Chemical

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

Dual-enzyme cascade amplification electrochemical biosensor for human

papillomavirus based on DNA nanoflower structure
Hongzhang He a, 1, Lingjun Cheng a, 1, Yinghao He a, Jiaming Chen a, Liang Song b, c,
Yuanyuan Yang a, Yun Zhang b, c, d, *, Zhenyu Lin e, **, Guolin Hong a, ***
a
Department of Laboratory Medicine, Xiamen Key Laboratory of Genetic Testing, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen
University, Xiamen 361005, China
b
State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002, China
c
Xiamen Key Laboratory of Rare Earth Photoelectric Functional Materials, Xiamen Institute of Rare Earth Materials, Haixi Institute, Chinese Academy of Sciences,
Xiamen 361021, China
d
University of Chinese Academy of Sciences, Beijing 100049, China
e
Ministry of Education Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety,
Department of Chemistry, Fuzhou University, Fuzhou, Fujian 350116, China

A R T I C L E I N F O A B S T R A C T

Keywords: The lack of large-scale human papillomavirus (HPV) DNA screening is a major contributor to the high incidence
DNA hydrogel and mortality of cervical cancer in economically undeveloped areas. The development of sensitive, rapid, and
DNA nanoflowers low-cost screening techniques is urgently needed. Here, DNA nanoflowers encapsulating glucose oxidase and
Electrochemical biosensor
horseradish peroxidase (GHDFs) were synthesized by one-pot rolling circle amplification, and then the GHDFs
Enzyme cascade catalytic amplification
Human papillomavirus
were used as the cargo of DNA hydrogel and applied for HPV DNA detection. When target DNA was present, the
DNA hydrogel cross-linking structure was disrupted, releasing GHDFs, which then catalysed the oxidation of
glucose and tetramethylbenzidine in a cascade, generating a significant electrical signal. Signal intensity had a
linear relationship with the logarithm of target DNA concentration in the range of 10 fM-1 nM with a detection
limit of 3.76 fM. The detection time of the proposed biosensor was 25 min, which was suitable for large-scale
HPV DNA screening in economically underdeveloped areas and provides a blueprint for the detection of other
DNA of interest.

1. Introduction
Cervical cancer is a malignant tumour caused by persistent infection
with human papillomavirus (HPV). Among gynaecological tumours,
cervical cancer ranks second in the world in incidence and mortality,
seriously threatening women′ s health [1]. Cervical cancer can be largely
affected by screening, which reduces the risk of death. Compared with
other methods, the most effective cervical cancer screening method is
HPV nucleic acid detection [2,3]. Currently, the main clinical HPV
nucleic acid screening methods are PCR and Hybrid Capture 2 (HC2).
However, these methods are time-consuming, and many developing and
economically underdeveloped regions still lack the required specialized
equipment and skilled personnel. As a result, these regions have the
highest number of cases in the world, accounting for approximately
90%. Therefore, the development of a sensitive, rapid, and affordable
HPV nucleic acid detection strategy for cervical cancer screening is ur­
gently needed.
Electrochemical biosensors have been widely developed due to their
advantages of fast signal readout speed, affordable sensing element and
simple sensing platform [4], which are expected to address this chal­
lenge for the development of practical HPV screening strategies. The low
concentration of HPV DNA in cervical swab samples poses a challenge to
the accurate diagnosis of early HPV infection [5]. To achieve the
required detection sensitivity, amplification techniques such as

* Correspondence to: No.155 Yangqiao Road West, Fuzhou, Fujian, People’s Republic of China.
** Correspondence to: No.2 Xue Yuan Road, University Town Fuzhou, Fujian, People’s Republic of China.
*** Correspondence to: No.55 Zhenhai Road, Xiamen, Fujian, People’s Republic of China.
E-mail addresses: zhangy@fjirsm.ac.cn (Y. Zhang), zylin@fzu.edu.cn (Z. Lin), xmhgl9899@xmu.edu.cn (G. Hong).
1
These authors contributed equally to this work

https://doi.org/10.1016/j.snb.2022.132532
Received 8 July 2022; Received in revised form 12 August 2022; Accepted 16 August 2022
Available online 18 August 2022
0925-4005/© 2022 Elsevier B.V. All rights reserved.
H. He et al.
nanomaterial amplification [6], enzyme-catalysed oxidation amplifica­
tion [7], target cycle amplification [8,9], DNA self-assembly amplifica­
tion [10] and isothermal amplification technology [11] have been
widely used. The high efficiency of enzyme-catalysed oxidation, high
specificity for catalytic substrates, and mild reaction conditions make an
enzyme-catalysed oxidation signal amplification strategy one of the
most popular methods used in electrochemical signal amplification
technology. This method has been frequently applied to realize sensitive
detection [12,13]. For example, Yuan et al. developed a photochemical
electrochemical (PEC-EC) dual-mode biosensor of miRNA-21 by using
the cascade catalytic amplification of glucose oxidase mimic enzyme
and peroxide mimic enzyme [14]. This strategy achieves good sensi­
tivity. However, the distance between the two enzymes is relatively far,
and there is a shortcoming of ineffective diffusion of the reaction sub­
strate, which fails to give full play to all the advantages of enzyme
cascade catalytic amplification. Therefore, detection sensitivity can be
further improved by bringing the two enzymes into close enough
proximity.
DNA nanoflowers (DFs) have been proven to be a desirable scaffold
for enzyme cascade catalysis due to their porous material structure and
high loading efficiency [7,15]. Apart from that, studies have reported
that the negatively charged DNA phosphate backbone may also enhance
enzyme activity [16]. More importantly, it has been shown to be stable
even in harsh environments, including nuclease treatment, high tem­
perature, and urea treatment [17]. For example, Yan et al. prepared DFs
by rolling circle amplification (RCA) with simultaneous coencapsulation
of glucose oxidase (GOx) and horseradish peroxidase (HRP) [7] and
realized highly sensitive target detection.
DNA hydrogels have attracted extensive attention because of their
excellent biocompatibility, portability, and programmability. Many
stimu-responsive hydrogels have been designed and frequently applied
in biosensors. In this study, a sensitive, rapid and economical electro­
chemical biosensor strategy for HPV DNA screening based on a DF-
encapsulated enzyme cascade catalytic amplification system and DNA
hydrogels was developed. During rolling circle amplification to syn­
thesize DFs, GOx and HRP were also simultaneously encapsulated into
the DFs, obtaining the GHDFs. They were then entrapped as signalling
molecules by the stimuli-responsive hydrogel to construct the HPV
biosensor. The sensor has been applied to HPV DNA detection in cervical
brush samples.
2. Experimental section
Materials and reagents, characterization of GHDFs, etc. have been
described in the Supplementary Material.
2.1. Preparation of ligated DNA template
Slightly modified from the previous protocol [17]. Mix 5 µl of 100 µM
padlock probe and 10 µl of 100 µM primer in 1 × T4 DNA ligase buffer
(containing 50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT and 1 mM ATP,
pH 7.5). Subsequently, the mixture was heated at 95 ◦ C for 5 min and
cooled slowly to 25 ◦ C for 3 h. Later, 0.4 µl of T4 DNA ligase (400 U/µl)
was added to the annealed mixture. The mixture was incubated at 16 ◦ C
for 12 h. The mixture was incubated at 65 ◦ C for 10 min to inactivate the
ligase. Add 6 µl of Exo I (20 U/µl) and a final concentration of 1 × Exo I
buffer (67 mM glycine-KOH, 6.7 mM MgCl2, 10 mM 2-mercaptoethanol;
pH 9.5 @ 25 ◦ C) to digest residual padlock probes and primers and then
incubate at 37 ◦ C for 1.5 h. Exo I final inactivation at 80 ◦ C for 20 min.
2.2. Preparation of GHDFs
The GHDFs were self-assembled by the rolling circle amplification
(RCA) process. Reactions were performed in phi29 DNA polymerase
buffer (33 mM Tris-acetate, 10 mM Mg (CH3COO)2, 66 mM CH3COOK,
1% Tween 20, 1 mM DTT; pH 7.9) containing ligated DNA template (22
2
Sensors and Actuators: B. Chemical 371 (2022) 132532
μM), GOx (50 mg/ml), HRP (25 mg/ml), phi29 DNA polymerase (1000
U/ml) and dNTPs (1 mM) and incubated at 30 ◦ C for 20 h. GHDFs were
then purified by centrifugation at 10,000 g for 10 min and further
washed three times with ultrapure water. Resuspend the purified GHDFs
in ultrapure water and store them at − 20 ◦ C.
2.3. Preparation of hydrogel encapsulated with GHDFs
Strands A or B and 6 wt% acrylamide were mixed at 37 ◦ C and
vacuum dried for 10 min to remove air. Then 0.2% TEMED and 0.2%
APS were added, mixed well, and dried for 15 min to form P-SA (poly­
acrylamide A strand) or P-SB (polyacrylamide B strand). Then, P-SA, P-
SB, GHDFs and 1 ×NEbuffer 2 were mixed well, heated at 60 ◦ C for 30
min, and then slowly cooled to room temperature to form DNA
hydrogels.
2.4. Construction of HPV electrochemical biosensor
The gold electrode (AuE) was polished to a mirror surface with a
mixture of 0.30 and 0.05 µm Al2O3 and water, and then ultrasonically
cleaned with anhydrous ethanol and ultrapure water in turn and blow
dried with N2. Various concentrations of target DNA sample were added
to the encapsulated GHDF hydrogel prepared above. After incubating
the mixture at 25 ◦ C for 15 min, the supernatant of the hydrogel was
pipetted and mixed, 2.5 µl of the supernatant was pipetted onto the
preprepared gold electrode, and the mixture was dried to construct an
electrochemical biosensor interface at 10 min.
2.5. Electrochemical assay procedure
Electrochemical measurements were performed on a computer-
controlled CHI 660 electrochemical system (CH Instruments,
Shanghai). The three-electrode system contained Au as the working
electrode, platinum as the reference electrode, and Ag/AgCl (saturated
KCl) as the reference electrode. During measurement, the prepared
electrodes were immersed in 1 mM PBS containing glucose (4 mM) and
TMB (1.5 mM). Thereafter, the redox signals of glucose and TMB cata­
lysed by oxidative double enzymes were measured with the ampero­
metric curve. The initial voltage was 0 V, the sample interval was 0.1 s,
and the running time was 100 s. All electrochemical experiments were
performed at room temperature.
3. Results and discussion
3.1. Principle of a double enzyme cascade signal amplification
electrochemical biosensor for HPV detection
The highly conserved sequences of HPV16 E6 and E7 genes provide
excellent targets for large-scale screening of cervical cancer [18,19].
Therefore, HPV16 E6 and E7 sequences were chosen as hydrogel target
DNA sequences. A DNA nanoflower structure encapsulating both HRP
and GOx was synthesized by the RCA reaction. The constructed elec­
trochemical biosensor uses DNA nanoflowers as signal-responsive mol­
ecules, and DNA hydrogels specifically respond to target DNA as a
sensing strategy for the rapid and sensitive detection of HPV16. The
principle is given in Scheme 1. Large amounts of GHDFs were synthe­
sized by the RCA reaction, and then the synthesized GHDFs were trap­
ped by the hydrogel to construct the hydrogel sensors. When target DNA
was present, the target DNA can hybridize with the P-SA end and replace
the P-SB through the strand displacement interaction, resulting in the
collapse of the cross-linked structure of P-SA and P-SB, releasing the
GHDFs signal molecules encapsulated in the hydrogel. The collapse of
the hydrogel was proportional to the target DNA concentration. Then,
the supernatant containing GHDFs was aspirated and modified on a gold
electrode to obtain an HPV electrochemical sensor. Through the cascade
catalytic oxidation of glucose and TMB by GHDFs, a significant
H. He et al. Sensors and Actuators: B. Chemical 371 (2022) 132532

Scheme 1. Schematic illustration of DNA detection based on GHDFs for hydrogel biosensors. (ⅰ) The synthesis procedure of GHDFs; (ⅱ) The construction of a
hydrogel biosensor based on GHDFs as signal molecules.

electrochemical signal can be detected. This has some relationship with
the target concentration. By this means, a sensitive electrochemical
biosensor for HPV detection can be designed.
3.2. Comparison of the signal amplification
To evaluate the superiority of the proposed signal amplification
strategy, mixtures of HRP and GOx, mixtures of HRP, GOx and DFs,
GHDF- catalysed glucose and TMB amperometric current signals were
recorded. The results are shown in Fig. S3. Under the same experimental

Fig. 1. Optimization of the experimental conditions. (A) Concentration of SA-SB dsDNA; (B) percentage of acrylamide; (C) response time; (D) pH value. The con­
centration of target DNA was 100 pM. Each data point is the mean of three repeated measurements, and error bars represent the standard deviation.

3
H. He et al. Sensors and Actuators: B. Chemical 371 (2022) 132532

conditions, compared with the free enzyme, the electrochemical signal
generated by GHDF catalytic oxidation was 2.81 times that of the free
enzyme, indicating that the encapsulated double enzyme improved the
catalytic oxidation efficiency due to the cascade amplification [20]
effect.
3.3. Optimization of the experimental conditions
To obtain the best analytical performance of the sensing platform,
four experimental parameters that most influence this sensing strategy
were optimized. As shown in Fig. 1A, when the concentration of SA-SB
dsDNA was between 50 μM and 150 μM, the S/B (signal blank ratio)
value increased with increasing crosslinking concentration. The S/B
value peaked when the concentration of DNA crosslinking was 150 μM.
In the range of 150–250 μM, the S/B value decreased with increasing
SA-SB dsDNA concentration. Therefore, the optimal concentration of
SA-SB dsDNA was 150 μM. The percentage of acrylamide is also a key
factor in the stability of hydrogels. In Fig. 1B, S/B increases with the
increase of percentage, peaked at 6%, then as the percentage increase
gradually, S/B value smaller and smaller. Consequently, the percentage
of acrylamide under optimal sensor performance in this study was 6%.
Fig. 1C reveals that S/B increased with the response time from 5 to
15 min, while decreased with the extension of reaction time from 15 to
25 min. Therefore, the sensor response time was set to 15 min Fig. 1D
demonstrates that the electrochemical signal peaked at a pH of 5.5.
Accordingly, we obtained the maximum GHDF catalytic activity at a pH
of 5.5. Therefore, the final optimization condition of the pH value was
set to 5.5.
3.4. The performance of the hydrogel sensor for DNA analysis
3.76 fM. As shown in Table 1, compared with the previously reported
HPV biosensor, this strategy has a lower detection limit and shorter
detection time (25 min), which is attributed to the amplification effect
of the double-enzyme cascade in the DNA nanoflowers and the contin­
uous catalytic oxidation of the substrates, which greatly amplifies the
signal and shortens the detection time.
To further study the anti-interference performance of the sensor in a
complex environment, the response signals of HPV genotypes 18, 31, 51
and 58 with similar pathogenicity to HPV 16 were experimentally
compared, and the specificity of the HPV16 biosensor was evaluated. In
addition, the concentration of the above interfering DNA sequence was
much higher (100 times) than the target DNA concentration. As shown
in Fig. 2B, the analysis results of the solution containing the interfering
DNA sequence were basically consistent with that of the blank sample.
These results show that the DNA strands that make up the hydrogel
specifically recognize only the HPV 16 E6 E7 gene, and do not recognize
other interfering sequences, even when the concentration of the inter­
fering sequence was 100 times the concentration of the target sequence.
Therefore, the electrochemical biosensor based on hydrogel-
encapsulated GOx-HRP DFs has a good ability to identify target DNA
in actual detection scenarios.
3.5. Clinical sample analysis
To further evaluate the applicability and reliability of the proposed
method, we used this strategy to detect HPV 16 DNA in cervical brush
specimens. As shown in Table S1, the recoveries detected by the sensor
were 91.40%− 112.46%, and the RSD was 3.39%− 8.45%. These results
confirm that electrochemical biosensors can be used in biomedical
research and have great potential for clinical analysis. The sensor has

Under these optimized conditions, the sensor performance was

Table 1
studied by detecting different amounts of HPV E6 and E7 genes. As Analytical performance comparison with other HPV 16 biosensors.
shown in Fig. 2A, the current increases with the concentration of HPV 16
Detection limit (mol L− 1) Time (min) Technique Reference
DNA. The illustration in the linear graph shows a linear relationship
10
between the current magnitude and the logarithm of the target con­ 2.00 × 10− 103.5 PCR + Electrochemistry [22]
12
1.75 × 10− 70 Electrochemistry [23]
centration in the range of 10 fM-1 nM. The linear relationship is 13
1.50 × 10− 120 Electrochemistry [24]
expressed as follows: 4.03 × 10− 14
180 Electrochemistry [25]
10
2 1.50 × 10− 50 Electrochemistry [26]
ΔCurrent= 176.84+65.78lgCHPV16 R = 0.997 3.00 × 10− 11
240 Electrochemiluminescence [27]
9
2.39 × 10− 60 Electrochemistry [28]
Where ΔCurrent is equal to the current generated by the target DNA 2.30 × 10− 9
47 Electrochemistry [29]
concentration minus the blank signal current. R is the correlation coef­ 7.60 × 10− 15
320 Electrochemiluminescence [30]
ficient. By calculating the mean signal plus three times the standard 3.76 × 10− 15
25 Electrochemistry This work
deviation from the blank sample [21], the limit of detection (LOD) was

Fig. 2. (A) Correlation between amperometric current and HPV16 concentration. The insert picture is the fitting curve between ΔCurrent and the logarithm of
HPV16 concentration. (B) Selectivity of the biosensor, 100 ×CHPV16 = CHPV18 = CHPV31= CHPV51 = CHPV58= 10 nM. The error bars show the standard deviation of
three replicate determinations.

4
H. He et al.
been applied to HPV detection in cervical brush samples.
4. Conclusions
In this work, a specific responsive HPV16 E6 and E7 oncogene
hydrogel electrochemical sensor was developed. By aggregating the two
enzyme constraints together, the catalytic synergy formed by DFs en­
sures the high efficiency and convenience of the enzymatic reaction.
Compared with noncascade catalysis, it eliminates the ineffective
diffusion of the reaction substrate, improves the local concentration of
intermediates, and obtains advantages over traditional multistep re­
actions. Taking advantage of the simple preparation and rapid reaction
of hydrogels, as well as the significant signal amplification properties of
GHDFs in substrate catalysed oxidation, the developed biosensor has the
advantages of an affordable and short detection time, and the sensitivity
meets the needs of HPV detection. Therefore, it provides a protocol for
large-scale HPV DNA screening in economically impoverished areas.
Furthermore, the method was versatile and can be used as a powerful
tool for the detection of other target DNA by simply changing the DNA
sequence in the hydrogel. This strategy has great potential in clinical
testing, biomedical research, and POCT.
CRediT authorship contribution statement
Hongzhang He: Conceptualization, Investigation, Data curation,
Writing – original draft. Lingjun Cheng: Conceptualization, Investiga­
tion, Data curation, Writing – original draft. Yinghao He: Conceptual­
ization, Data curation, Writing – original draft. Jiaming Chen: Data
curation, Writing – original draft. Liang Song: Resources. Yuanyuan
Yang: Resources. Yun Zhang: Supervision, Project administration.
Zhenyu Lin: Supervision, Project administration, Writing – review &
editing. Guolin Hong: Supervision, Project administration, Writing –
review & editing, 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.
Data availability
The data that has been used is confidential.
Acknowledgements
The authors are sincerely grateful for financial support by the Na­
tional Natural Science Foundation of China (82171474, 81772287,
81371902) and the Natural Science Foundation of Fujian Province of
China (2020J011241).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.snb.2022.132532.
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Hongzhang He is presently pursuing for his master’s r degree in laboratory medicine in
Xiamen University. His research focuses on electrochemical biosensor for disease fore­
warning & diagnosis.
Lingjun Cheng is presently pursuing for his Ph.D. in laboratory medicine in Xiamen
University. His research focuses on electrochemical biosensor for disease forewarning &
diagnosis.
Yinghao He obtained a M.S. in laboratory medicine from Xiamen University and now
works in the department of laboratory medicine at the First Affiliated Hospital of Xiamen
University. His research focuses on electrochemical biosensor and electro­
chemiluminescence biosensor.
Jiaming Chen obtained a Ph.D. in Analytical Chemistry from Fuzhou University and now
works in the department of laboratory medicine at the First Affiliated Hospital of Xiamen
University. His research focuses on biosensors for food safety analysis and disease fore­
warning & diagnosis.
Sensors and Actuators: B. Chemical 371 (2022) 132532
Liang Song obtained a Ph.D. in Analytical Chemistry from Fuzhou University. He joined
the Fujian Institute of Research Structure of Matter, Chinese Academy of Sciences since
2018. His research focuses on design and synthesis of rare earth biomedical materials,
research and development of in vitro diagnostic methods and kits for major diseases.
Yuanyuan Yang obtained a M.S. in laboratory medicine from Xiamen University and now
works in the department of laboratory medicine at the First Affiliated Hospital of Xiamen
University. Her research focuses on biosensor for disease forewarning & diagnosis.
Yun Zhang is currently a full professor in the University of Chinese Academy of Science.
He obtained his Ph.D. in Technische Universität München. He joined the Fujian Institute of
Research Structure of Matter, Chinese Academy of Sciences since 2013. His research fo­
cuses on bio-nanomaterials, molecular diagnostics and molecular imaging.
Zhenyu Lin is currently a full professor in College of Chemistry at Fuzhou University
(China). He obtained his B.S. degree in polymer material and engineering from Beijing
Institute of Technology (China) and PhD in analytical chemistry from Fuzhou University.
He joined the Ministry of Education Key Laboratory of Analysis for Food Safety & Biology
at Fuzhou University since 2007. Subsequently, he worked as a postdoctoral fellow in
Graduate School of Environmental Studies & School of Engineering, Tohoku University
(Japan). He is a 2013 recipient of the National Science Fund for Outstanding Young
Scholar and a 2014 recipient of Natural Science Found of Fujian Province for Distinguished
Young Scholar. His research focuses on biosensors for food safety analysis and disease
forewarning & diagnosis.
Guolin Hong is a professor at Xiamen University School of Medicine and the head of the
department of laboratory medicine of the First Affiliated Hospital at Xiamen University. He
obtained a Ph.D. from Xiamen University. His research focuses on biosensors for disease
forewarning & diagnosis.