Sensors & Actuators: B.

Chemical 331 (2021) 129422

Contents lists available at ScienceDirect

Sensors and Actuators: B. Chemical

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

DNAzyme biosensors for the detection of pathogenic bacteria

Xiaoyi Ma a, b, Wen Ding a, b, Cang Wang a, b, Hangjie Wu a, b, Xiaopeng Tian a, b,
Mingsheng Lyu a, b, c, *, Shujun Wang a, b, c, *
a
Jiangsu Key Laboratory of Marine Bioresources and Environment /Jiangsu Key Laboratory of Marine Biotechnology, Jiangsu Ocean University, Lianyungang, 222005,
PR China
b
Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang, 222005, PR China
c
Collaborative Innovation Center of Modern Biological Manufacturing, Anhui University, Hefei, 230039, PR China

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

Keywords: Pathogenic bacteria contamination can cause many problems, such as infections in humans and animals and
Pathogenic bacteria environmental pollution. Early prevention and detection of pathogenic bacteria outbreaks is crucial to managing
DNAzyme these problems. Some traditional detection methods such as cell culturing and polymerase chain reaction (PCR)
Biosensors
are time-consuming, labor-intensive, insensitive, and inconvenient for detection. Therefore, simple and efficient
Detection methods
pathogenic bacteria detection methods involving DNAzymes signaling mechanisms have been reported,
including fluorescence and color detection. DNAzymes are divided into cases as a molecular recognition element
(RNA-cleaving DNAzyme) and as a reporter element (peroxidase mimicking DNAzyme), which have proven
compatible with isothermal amplification technology for pathogenic bacteria detection. In this review, we
summarize the current methods of detecting pathogenic bacteria based on various DNAzyme sensors. We include
the design principles, working mechanisms, and detection applications of these sensors and discuss the experi­
mental and theoretical bases for their application in the diagnosis and prevention of diseases caused by path­
ogenic bacteria.

1. Introduction
The growth of pathogenic bacteria and other microbes not only has a
great impact on food but also seriously pollutes the environment,
increasing the probability of diseases in animals both on land and in the
ocean [1,2]. Foodborne, waterborne, and airborne microbes enter the
human body through various routes and lead to diseases, resulting in a
great threat to human health [3–6]. For example, Helicobacter pylori
infection is related to many gastrointestinal diseases, such as acute
gastritis, gastric cancer, and duodenal ulcers [7–9]. Vibrio anguillarum is
a common pathogenic marine bacterium that exists in water and aquatic
animals. It can infect marine animals such as eels, perch, and crucian
carp [10]. Salmonella is a zoonotic pathogen, and eating food contami­
nated with it can cause poisoning, leading to diseases such as typhoid
fever, enteric fever, sepsis, and gastroenteritis [11–13].
To avoid pathogenic bacteria outbreaks, it is important to detect
viable pathogenic bacteria cells with high sensitivity and specificity in a
short time on site [14]. While traditional detection methods have made
great contributions to the prevention and detection of pathogenic
bacteria, they have many disadvantages [15]. For example, the bacterial
culturing method is time-consuming and labor-intensive, and serolog­
ical tests are extremely susceptible to environmental impact [16].
Enzyme-linked immunosorbent assay (ELISA) based on specific
antigen-antibody interactions can be used to detect a wide variety of
pathogens, but its sensitivity needs to be improved, and antigen
cross-reactions are likely to occur [17,18]. PCR technology requires the
extraction of bacterial DNA as well as the use of a thermocycler, making
it inconvenient for on-site applications [19,20]. Finally, cell
activity-based tests are difficult to judge and are not suitable for field
applications. Point-of-care PCR technology is being developed into a
new type of diagnostic tool that can ensure sensitivity and specificity,
and plays a great role in bacterial detection [21]. Compared to these
technologies, DNAzymes are preferred for pathogenic bacteria detec­
tion. DNAzymes have been used broadly in the fields of micro-sensors
because of their special inexpensive, stable, tolerable performance and
advantages in technology [22,23]. The ability to identify targets quickly
and accurately is critical for environmental monitoring and food safety
[24–27].

* Corresponding authors at: Jiangsu Key Laboratory of Marine Bioresources and Environment, Jiangsu Ocean University, Lianyungang, 222005, PR China.
E-mail addresses: mingshenglu@hotmail.com (M. Lyu), shujunwang86@163.com (S. Wang).

https://doi.org/10.1016/j.snb.2020.129422
Received 8 September 2020; Received in revised form 13 December 2020; Accepted 26 December 2020
Available online 4 January 2021
0925-4005/© 2021 Elsevier B.V. All rights reserved.
X. Ma et al.
With the development of Systematic Evolution of Ligands by expo­
nential enrichment (SELEX) and in vitro selection technology [28], DNA
sequences with catalytic functions DNAzymes have been obtained [29,
30]. Although no natural DNAzymes have been discovered, the DNA­
zymes screened in vitro have been widely used in the detection of metal
ions and biomolecules due to their excellent catalytic activity [31–33].
DNAzymes can be directly bound to various fluorescent labels [34],
functional molecules, and solid surfaces, and can also be used as signal
transduction elements to amplify detection signals in combination with
rolling circle amplification (RCA), loop-mediated isothermal amplifi­
cation technique (LAMP), and other technologies [35–37]. DNAzymes’
strong chemical and thermal stabilities are utilized in biosensors and
suggest they have great potential for the detection of pathogenic
bacteria.
RNA-cleaving DNAzymes (aptazymes) were used for target recog­
nition to achieve a specific cleavage reaction [38]. Another type of
DNAzyme named peroxidase mimicking DNAzyme (PMD) was also used
for pathogenic bacteria detection for a signal generation [39]. A
G-quadruplex (G4) is a structure formed spontaneously by a group of
guanine-rich DNA sequences and exhibit peroxidase-like activity after
binding to hemin, which can catalyze the H2O2-mediated oxidation of 2,
2’-azino-bis diammonium salts (ABTS2− ) to a green-colored radical
cation (ABTS.+) for generating chemiluminescence and colorimetric
signals (Fig. 2B) [40,41]. DNAzyme is easy to modify and has strong
compatibility with various isothermal amplification technology, and can
be used for signal amplification in designing biosensors [42–44]. It is
very convenient to perform site modification [45–47], which is an
advantage for different detection needs.
DNAzyme has attracted great attention in the field of biosensing
because of its high activity and selectivity [48,49], as shown in Fig. 1.
This review summarizes the latest developments in the design of
DNAzyme-based sensors for pathogenic bacteria detection, hoping to
provide a reference for the detection, supervision, prevention, and
control of pathogens in food and the environment. Besides, the discus­
sion further extends to the possible detection range of these DNAzymes,
providing a basis for future research.
2. In vitro selection of bacterial sensing aptazymes
Since the discovery of aptazymes, many studies have been conducted
to isolate aptazymes in vitro [50–52]. In vitro selection is the process of
enriching a large number of random sequences in a DNA pool into one or
several sequences with high activity under a certain reaction condition
[53]. A selection process is shown in Fig. 2A. It starts with a library
containing around 1014 different single-stranded DNA sequences.
Fig. 1. DNAzyme-based bacteria detection timeline. This scheme shows major the development of DNAzymes in bacterial detection.
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Sensors and Actuators: B. Chemical 331 (2021) 129422
Appropriate addition of negative selections in the selection process can
increase target selectivity and can remove catalytically active DNA se­
quences, not due to the presence of target molecules. Negative selections
are typically carried out by incubating the library with closely related
samples, and the active DNA sequences are discarded. The inactive ones
are collected for the next round of positive selection using the intended
target. Through the PCR technology, the active sequences in the DNA
library can be effectively amplified and a large number of active se­
quences can be enriched. After multiple rounds of selection, the activity
of the library will eventually saturate, and the library can then be
sequenced. The mixed DNA sequences (substrate sequences and apta­
zymes sequences) obtained by the screening has the ability to cleave the
fluorogenic substrate and produces a fluorescent signal [54].
In Li’s study, aptazymes could be isolated from random-sequence
DNA pools. A fluorophore and a quencher were designed on both sides
of the adenine ribonucleotide (rA) embedded in the substrate chain for
selection [54]. The PAGE-based experiments were used to separate
cleaved and uncleared bands before amplification. Gu’s screening
principle was to use micro-magnetic beads for DNA ligation, reaction,
and separation [55]. This work used an unmodified DNA library to
obtain a aptazyme that could be activated by bacteria. Using
PAGE-based methods to select aptazymes required expensive library
modifications at an early stage and required gel recovery. The selection
process was relatively complicated, but the experimental process was
clear and the experimental conditions could be monitored at every step.
Using the magnetic bead methods, the whole experiment was carried out
in an EP tube before amplification, which was more convenient to
operate, but for reverse screening, poorly treated bacteria on EP tube
walls would produce nonspecific results.
Since bacterial cells are quite large, it is unlikely that the entire cell
can be used to activate the aptazymes. Li’s lab proposed the use of crude
extracellular mixture (CEM) and crude intracellular mixture (CIM) of
bacteria as a target for selection. CEM refers to the cell culture after
removing the cells, while CIM refers to the crushed cells with the in­
ternal contents released [56]. This experiment does not need to set a
specific target for the selected aptazymes. Any molecule in CEM/CIM
may become a target to activate matching aptazymes. Another advan­
tage is that the aptazymes-based sensor can directly identify the target
from the complex mixture to achieve the detection purpose. Studies
have shown that both CEM and CIM can be used as targets for obtaining
aptazymes. However, the cleaving efficiency of the aptazymes in the
presence of the CIM was higher than CEM [56,57]. CEM is the super­
natant collected from the cell culture by centrifugation as a sample
containing the target, and the bacterial cells must be destroyed to obtain
CIM, such as by applying bacterial lysozyme or ultrasonic destruction, to
X. Ma et al.
Fig. 2. (A) In vitro screening process and the mechanism of aptazymes. (B) The mechanism of peroxidase mimicking DNAzyme for pathogenic bacteria detection.
obtain intracellular lysate. This may be the reason why, compared with
CEM, CIM can obtain more targets from broken cells to activate the
aptazymes reaction. However, if the potential targets could exist in the
sample matrix (for example lake water or milk) in a high enough con­
centration, it can be directly sampled and detected without additional
bacterial processing, such as broken cells. This is a major advantage of
using CEM as a screening target.
The RFD-EC1 (RNA-cleaving fluorescent DNAzyme) was selected by
Li’s lab, and experiments verified that the fluorescent aptazyme acti­
vated by CEM produced from Escherichia coli could be isolated from the
DNA library [56]. Further work confirmed that the target of RFD-EC1
was a protein. Clostridium difficile, which was confirmed to be the
BI/027 strain, CEM was prepared to form BI/027-H for positive selec­
tion. After an in-depth study, the target was identified to be a tran­
scription factor (TcdC) that could activate aptazyme [58]. The DHp3
aptazyme was obtained for H. pylori detection with the use of the CEM of
H. pylori (CEM-HP) as the positive selection target [59]. They used the
cell lysate of Klebsiella pneumoniae as a complex target (thus a mixture of
CEM and CIM) and obtained an aptazyme (RFD-KP6) that recognized
K. pneumoniae in 2018 [60]. Later, VAE2 was selected for V. anguillarum
detection using its CEM [55]. Such an approach was also used for the
selection of aptamers to bind cell surface markers since the binding
aptamers and non-binding sequences can be readily separated via
centrifugation, but aptazymes are more appropriate to use CEM/CIM.
3. Molecular recognition element DNAzymes
3.1. Fluorescent sensing bacterial cells
After obtaining bacteria-sensing aptazymes, the presence of a spe­
cific target is converted into a signal in the next step. Fluorescent la­
beling is simple for DNA, and it is also flexible in design, highly sensitive,
and fast in analysis speed. Many fluorescent catalytic beacon sensors
have been designed using selected DNAzymes [34,53]. The most com­
mon method is to position a fluorophore and a quencher close to each
other in the initial state, resulting in the initial fluorescence being
quenched. After the cleavage reaction is induced by the target, the flu­
orophore and quencher are separated to increase the fluorescence in­
tensity [61]. Aptazymes combined with magnetic beads have been
studied as conductive elements to induce RCA and hybrid chain re­
actions to achieve the final detection purpose of bacteria [57]. The
signaling aptazymes selected by Li’s group were already fluorescent
sensors since a fluorophore and a quencher was labeled right next to the
cleavage RNA junction. When no target was present, it showed a low
background fluorescence signal. In the presence of the target, the
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Sensors and Actuators: B. Chemical 331 (2021) 129422
aptazymes were activated, and a fluorescent signal was released. The
RFD-EC1 aptazyme can detect 103 CFU/mL E. coli in a solution using
fluorescent labeling. In another example, the VAE2 screened by Gu’s
group could detect 4 × 103 CFU/mL of V. anguillarum in optimal con­
ditions [55]. Li’s group used fluorescent labels and quenching groups to
label the DNA throughout the experiment, whereas Gu’s group only
modified the aptazyme after successfully obtaining the aptazyme
(Fig. 3A). Finally, the detection results are determined according to the
fluorescence signal. Different strains were selected for specific detection
of aptazymes, and it was found that aptazymes were still able to main­
tain their specificity for E. coli and V. anguillarum respectively. The
successful selection of aptazymes provides the basis for the DNAzyme
research of other pathogenic bacteria.
To transform this solution-based assay into an easy-to-use sensor
device, several simple and stable aptazymes-based biosensors have been
reported for pathogenic bacteria detection. Two examples are shown in
Fig. 3B, Brennan et al. researched a simple all-in-one paper sensor for the
detection of E. coli [62]. They chose nitrocellulose paper (NCP, with a
thin plastic layer) with low background fluorescence, and made micro­
pores on the paper, then printed wax on the paper and heat at high
temperature to melt the wax into the micropores and provide a hydro­
phobic barrier around the microdomains. A mixed solution of pullulan,
trehalose, and aptazyme was added to the micro-zone and air-dried
overnight in the dark to permanently fix the aptazyme. A strong fluo­
rescent signal was displayed in the micro-zone in the presence of
CIM-EC. The sensor generated a significant fluorescent signal within 5
min, and the detection limit was as low as 100 CFU/mL. The fluorescent
signal was captured using a fluorescent scanner. Based on the same
principle, as shown in Fig. 3C, Zhao et al. developed the same sensor for
detecting K. pneumoniae [60]. This sensor can directly detect 105
CFU/mL bacteria. These two examples showed the feasibility of
aptazymes-based sensors. Aptazymes could be designed as a simple and
inexpensive device, and it can be stored for a long time (six months) at
ambient temperature and can be used for monitoring the pathogenic
bacteria of water, milk, etc.
Liu’s group also studied an aptazyme/graphene hybrid material for
the optical detection of pathogenic bacteria [63]. As shown in Fig. 3D,
the sensor mainly included two parts: a fluorescently labeled E. coli
specific aptazyme and graphene. In brief, because the sp2-conjugated
domains of graphene materials allow for the direct interfacing of
nucleic acid moieties, the aptazyme was non-covalently adsorbed onto
graphene via π-π stacking interactions, forming aptazymes-graphene
element. Graphene could quench the fluorescent signal of aptazyme
such that the entire element showed a low fluorescent signal. In the
presence of E. coli, the target competed with graphene to bind aptazyme.
X. Ma et al.
Fig. 3. (A) Principle of fluorescence sensing directly in the solution of V. anguillarum. Reprinted with permission from Ref [55]. Copyright (2019) American Chemical
Society. (B) Schematic showing for E. coli detection. (C) Aptazyme-based sensor for K. pneumoniae detection. Reprinted with permission from Ref [60]. Copyright
(2018) WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. (D) Schematic diagram of the graphene-aptazyme complex for detecting E. coli. Reprinted with permission
from Ref [63]. Copyright (2018) Materials Research Society.
When aptazyme was activated by the target, it released fluorescent DNA
fragments. Short fluorescent DNA fragments could not be adsorbed onto
graphene again. However, compared to the reaction of aptazymes
modified with fluorescent groups and quenching groups, the E. coli
target must be at a sufficient concentration to first overcome the binding
of aptazyme to the graphene surface and then bind to aptazyme to
activate the cleavage reaction, so for pathogenic bacteria, the sensitivity
of the obtained concentration detection was reduced. At least 105
CFU/mL E. coli was required to activate the aptazyme connected to the
graphene to cause the cleavage reaction. This novel method provided
more thought for the design of aptazymes-based sensors for the detec­
tion of pathogenic bacteria. Graphene can interact with the bases in
nucleic acids so that DNA can be attached to the surface of graphene.
The combination of DNA and graphene can be used as a biosensor as a
sensitive tool for bacterial detection after obtaining the DNA probes.
3.2. Aptazymes combined with magnetic beads and enzymes
The Colorimetric sensors represent an attractive strategy, which uses
chemical reactions to produce a visible change of color for the detection
of pathogenic bacteria. At one end of aptazyme, biotin was connected to
the magnetic beads coated with streptomycin, while at the other end was
modified with urease. Aptazyme was activated by the target for cleavage
reaction. The cleavage fragment brings urease into the contained urea
and litmus (phenol red dye) solution causes the color to change gradu­
ally, thus achieving the detection process (Fig. 4A). 500 CFU/mL of
E. coli can be detected after two hours using this method [64]. This
method is 2 folds more sensitive than PCR and ELISA, which are two
popular methods for pathogen detection, and the detection time is
shorter [65,66].
The same principle was used for the detection of H. pylori. The dif­
ference is that aptazyme was designed as a portable sensor for H. pylori
detection [59]. As shown in Fig. 4B, the sensor consisted of three
spherical areas interconnected by flow channels. Area 1 carried acetate
buffer solution. The aptazyme connected to magnetic beads and urease
was fixed in area 2. Area 3 contained urea and phenol red for a colori­
metric reaction. In the presence of H. pylori, the acetic acid buffer in zone
1 could bring the reaction product with urease in zone 2 to zone 3. The
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Sensors and Actuators: B. Chemical 331 (2021) 129422
color changed in zone 3. This method resulted in a limit of detection of
104 CFU/mL, providing specific and sensitive detection of H. pylori in
stool samples. These findings laid the foundation for aptazymes -based
paper devices that can be applied to the detection of bacteria in complex
samples. The advantage of this kind of chemical reaction to obtain the
signal is that there is no background interference, but the use of pH
changes to obtain the signal requires control of the initial pH value and
the use of very low-level buffers, both of which may cause problems with
detection limit and background signals.
Zheng et al. studied a DNA sensor system that used aptazyme as the
recognition element and DNA-templated silver nanoclusters (DNA-
AgNCs) as the fluorescence signal readout element for E. coli detection
[67]. As shown in Fig. 4C, this design mainly includes three parts:
magnetic nanoparticles, aptazyme, and acetylcholinesterase (ATchE).
DNA was first hybridized with ATchE to aptazyme and then connected to
streptavidin magnetic beads. Aptazyme was subsequently activated in
the presence of E. coli lysates. After magnetic separation, the remaining
fragments of aptazymes with ATchE were transferred to a solution
containing DNA-AgNCs to catalyze the hydrolysis of acetylthiocholine to
produce thiocholine, which could react with DNA-AgNCs via Ag-S
bonds, offering significant enhancement of the fluorescence of
DNA-AgNCs and providing an ultrasensitive detection limit of 60
CFU/mL. The results suggested that the introduced aptazyme-based
method could be applied to the analysis of E. coli in environmental
samples.
Generally, these biosensors reported limits of detection of 10-105
CFUs/mL. This shows that aptazymes-based sensors can be used for the
detection of bacteria in complex matrices. Based on the same principle,
different bacteria-specific aptazymes can be used to design sensors and
apply them to actual sample detection.
3.3. Aptazymes and rolling circle amplification reaction
RCA is a simple and efficient nucleic acid amplification method since
it occurs at a constant temperature [68]. The cleavage fragments of
aptazymes can generate a long-chain DNA via RCA [69,70], and the
bacteria can be detected by adding SBYE gold and metal nanoclusters to
the amplified DNA sequences. Further improving performance figures of
X. Ma et al. Sensors and Actuators: B. Chemical 331 (2021) 129422

Fig. 4. (A) Schematic diagram of aptazyme-based colorimetric sensor for detecting E. coli. (B) Schematic diagram of H. pylori detection. Reprinted with permission
from Ref [59,64]. Copyright (2014/2019) Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (C) Schematic representation of DNA-templated fluorescent silver
nanoclusters-based sensing system for pathogenic bacterial detection. Reprinted with permission from Ref [67]. Copyright (2018) Elsevier B.V.

merit such as a limit of detection. Combined with aptazyme and RCA,
Liu et al. developed a strategy for E. coli detection [71]. As shown in
Fig. 5A, the design included the following parts: (1) Three kinds of DNA:
a DNA primer (DP), a circular DNA template (CDT) containing the
antisense sequence of the aptazyme (RCD), and an RNA-containing DNA
sequence (RDS) as the substrate of an RCD, wherein the 5′ portion
sequence is the same as DP. (2) Two complexes. DP and CDT (Complex I)
induced the RCA reaction, producing many long RCA products con­
taining repetitive RCD units. An RDS/CDT hybrid (Complex II) was
added, and the 3′ end of RDS was combined with aptazyme. E. coli ly­
sates could active aptazyme for cleavage reaction, producing the hybrid
of CDT with the 5′ cleavage fragment. Trimming of unpaired nucleotides
from the cleavage fragment by Polf29 produced more Complex I, which
was feedback into the RCA process to amplify the DNA. Fluorescence

Fig. 5. Schematic of aptazyme-based rolling circle amplification technology used for bacteria detection. (A) Aptazymes feedback amplification for E. coli bacteria.
Reprinted with permission from Ref [71]. Copyright (2017) Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (B) Magnetic aptazymes-CuNCs fluorescent biosensor
for the detection of E. coli. Reprinted with permission from Ref [57]. Copyright (2020) Elsevier B.V.

5
X. Ma et al.
measurement was carried out in the presence of SYBR Gold. This method
can detect 10 CFU/mL using a 60 min reaction time. Compared with
DNAzyme without signal amplification, this method improved 100-fold
in the limit [56].
Zhou et al. developed a fluorescent biosensor coupled with magnetic
beads, aptazymes, and copper nanoclusters to detect E. coli O157:H7
[57]. As shown in Fig. 5B, aptazymes were connected to streptavidin
magnetic beads and activated by E. coli lysates for a cleavage reaction.
The remaining fragments of aptazymes cleavage induced RCA reaction.
In the presence of copper sulfate and sodium ascorbate, Cu2+ can bind to
specific sites on bases and generate copper nanoclusters (CuNCs). Under
the irradiation of ultraviolet light, CuNCs could emit red light to realize
the whole detection process, and the detection limit was as low as 1.57
CFU/mL in 1.5 h. This sensor showed better practicability for E. coli
detection in both pure drinking water and apple juice.
The described biosensor uses RCA to amplify partial fragments of
aptazymes to amplify the detection signal, thereby converting the
detection of bacteria into the detection of RCA products. Different from
the detection method that directly uses a fluorescent label and a
quenching group to modify DNA, this nucleic acid signal amplification
method can improve detection sensitivity. Hence, it has great applica­
tion value and potential in pathogenic bacteria detection.
3.4. Aptazymes and hybrid chain reaction
Aptazyme-integrated plasmonic nanosensors (DIPNs) were used by
the Yu group to detect a model bacteria E. coli [72]. In their design, the
aptazyme was attached to streptavidin magnetic beads and can be
activated for a cleavage reaction in the presence of E. coli lysates. After
magnetic separation, the aptazyme residual after cleavage attached to
the magnetic beads initiated the processes of hybridization chain reac­
tion (HCR) between two hairpin oligonucleotides (H1 and H2). Through
the biotin-streptavidin interaction, multiple glucose oxidase (GOx) units
were fixed on the nicked sites of the DNA double helix. They were
well-mixed with silver triangular nanoplates, and GOx could convert
glucose into H2O2 and glucose acid. The former could effectively etch
the blue silver triangle nanoplates into smaller spherical silver nano­
particles, causing rapid silver prism color fading of the blue solution,
thus achieving the purpose of detecting E. coli (Fig. 6). This
DNAzyme-integrated plasmonic nanosensor system can detect 50
CFU/mL E. coli with naked eyes. The potential targets that exist in the
sample matrix (for example the river water, apple juice, silk milk, etc.)
could be detected. This method is 1–100 fold more sensitive than the
previous colorimetric detection, which is very important for the
Fig. 6. Schematic diagram of aptazyme-integrated plasmonic nanosensors (DIPNs) for bacteria detection. Reprinted with permission from [72]. Copyright (2016)
Elsevier B.V.
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Sensors and Actuators: B. Chemical 331 (2021) 129422
detection of pathogenic bacteria [56,73].
4. DNAzymes act as a signaling element
PMD can catalyze the oxidation of hydrogen peroxide that can be
used to visually detect pathogenic bacteria by a simple colorimetric
experiment. The less background signal is an advantage of pathogenic
bacteria detection. Several cost-effective and sensitive sensing methods
for different targets were developed. Guo et al. designed an electro­
chemical biosensor based on the coupling of RCA and PMD to detect
E. coli [74]. As shown in Fig. 7A, this sensor mainly includes five parts:
(1) The anti-E. coli polyclonal antibody was immobilized on the elec­
trode surface via the assembly of cysteamine and the bifunctional linker
of glutaraldehyde. (2) In the presence of E. coli, through specific
recognition, the E. coli was captured on the Au electrode surface. (3)A
primer-probe designed to contain the anti-E. coli aptamer binds to E. coli.
(4) Adding the circular probe designed with two G4 units started the
RCA reaction and generates repetitive G4 DNA molecules. (5) After
adding heme to the DNA sequence, the G4 unit is bound to the heme
molecule and folds into an active HRP mimicking the DNAzyme struc­
ture. A dramatically strong current signal was achieved owing to the
electro-reduction of the H2O2 reaction catalyzed by PMD. Under optimal
conditions, the proposed biosensor exhibits ultrahigh sensitivity toward
E. coli with a detection limit of 8 CFU/mL. This method can directly
detect E. coli in the spiked milk sample without any pretreatment, which
showed higher specificity and sensitivity.
Hui et al. designed a paper-based sensor platform to detect a DNA
aptamer for glutamate dehydrogenase (GDH, a marker for C. difficile)
[75]. As shown in Fig. 7B, the paper sensor consisted of two pieces of
paper. The first paper was configured to be a molecular
recognition-fluorescence readout zone (Zone 1), so a fluorescently
labeled DNA aptamer for GDH mixed with graphene oxide (GO) was
printed onto a nitrocellulose membrane in Zone 1. RCA reagents, a
circular DNA template, and hemin in pullulan solution were printed on
the second piece of paper, and a DNA amplification colorimetric readout
zone (Zone 2) with a bridge was used to transfer the reaction mixture
from Zone 1 to Zone 2. The test sample was added to Zone 1, where,
upon target binding, the aptamer desorbed from GO, producing an
initial fluorescence signal, and migrated to Zone 2, triggering the RCA
reaction. The RCA products contained repetitive units of a PMD, PW17,
which generated a colorimetric signal in the presence of H2O2, and the
detection limit was 3 nm for colorimetric readouts by the GDH sensor.
DNA aptamers or DNAzymes can be linked-to RCA for signal generation,
which can be read in an equipment-free manner. This method of
X. Ma et al. Sensors and Actuators: B. Chemical 331 (2021) 129422

Fig. 7. (A) Illustration of the label-free electrochemical assay for highly sensitive detection of E. coli based on RCA and DNAzymes amplification. Reprinted with
permission from Ref [74]. Copyright (2015) Elsevier B.V. (B) Design of the dual output paper-based biosensor. Reprinted with permission from Ref [75]. Copyright
(2018) Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (C) Schematic illustration of the oPAD working principle. Reprinted with permission from Ref [73]. D) A
Multi-component All-DNA Biosensing System Controlled by DNAzyme for E. coli detection. Reprinted with permission from Ref [76]. Copyright (2020) Wiley-VCH
Verlag GmbH & Co. KGaA, Weinheim.

detecting bacteria by bacterial markers is very novel and can be used to
design more sensors for detecting pathogenic bacteria.
Sun et al. designed a paper origami device (oPAD) that combined a
DNAzyme and RCA to detect E. coli [73]. This design included the
following parts: (1) Panel A: An absorbent pad for sample purification
and washing. (2) Panel B, (2) Pane B: Whatman chromatography for cell
lysis. (3) Panel C, A circular DNA template containing anti-EC1 for RCA
reaction. The obtained RCA products were then incubated to
self-assemble into 3D EC1 to print on Panel C. After immersion into the
blocking buffer, the obtained bioactive paper was dried at room tem­
perature. The fluorogenic RNA substrate was then mixed with pullulan
and printed onto Panel C. (4) Panel D, RCA reagents, a circular DNA
template (CDT2), and hemin in pullulan solution was printed on Panel
D. Panel B was first folded onto Panel A to enable the on-paper protein
extraction from E. coli. When Panel B was in contact with Panel C, a
cleavage reaction occurred in Panel C, and the cleaved DNA fragments
could flow to Panel D as a template for RCA. The RCA product contained
PW17, a PMD, and generated a colorimetric signal by oxidizing the
chromogenic substrate TMB in the presence of hemin for detection
(Fig. 7C). Detection of ~103 CFU/mL in 35 min was reported, proving
the feasibility of the sensor. An amplified colorimetric response can be
produced using DNAzyme-initiated RCA. The sample can be easily
detected without equipment.
A Multi-component all-DNA catalytic hairpin amplification system
for detection of E. coli was reported by Zhou et al. [76]. As shown in
Fig. 7D, this method was a sensor detection system integrating
DNAzyme-mediated RNA cleavage (DRC), assembly-mediated strand
release (ASR), catalytic hairpin assembly (CHA), and a split G4 reas­
sembly (SGR) for detecting bacteria. Without the target, the DNA sub­
strate containing RNA could combine with specially designed DNA to
form a 4 WJ structure, release DNA5, which entered the CHA process
and bound to a tail-containing hairpin-shaped DNA molecule named H1.
After adding another tail-containing hairpin-shaped DNA molecule
named H2, which could produce an H1/H2 complex. The release of
DNA5 could initiate another H1/H2 binding reaction. The production of
H1/H2 increased with time vis the cyclic CHA process. The sequence of
H1 and H2 was designed so that the DNA part of the H1/H2 complex
interacts, and the resulting G4 could bind to protoporphyrin IX, thereby
enhancing fluorescence. In the presence of the target, the DNA substrate
containing RNA was cleaved, so that the entire reaction was terminated
during the DRC process. This method used a catalytic hairpin amplifi­
cation system to generate a detection limit of 50 CFU/mL. Compared
with DNAzyme without signal amplification, this method of combining
DNAzyme with isothermal amplification technology (RCA, CHA) has the
ability to generate large amounts of DNA amplicons relatively quickly at
a constant temperature and achieve sensitive detection of a broad se­
lection of targets, and the detection time and selectivity are also very
similar [77].
5. Conclusion and future prospects
In recent years, DNAzymes have been widely used in pathogenic
bacterial detection and achieved considerable results [78,79]. DNA­
zymes can target chemicals such as metal ions, proteins, and other
macromolecules, and better DNAzymes are expected to be isolated and
characterized in the future [80–82]. DNAzymes need to recognize a wide
range of bacterial cell mixtures, which is challenging due to the high
catalytic activity and specificity of individual cell species [83,84].
Fortunately, aptazymes’ selection results can be guided by changing the
selection conditions. For example, reverse selection has been used to
reduce the non-specific binding of aptazymes. Properly extending the
negative binding time can effectively remove the binding sequence,
thereby increasing the specificity of aptazymes. During the growth
process of bacteria, exogenous nutrients are used to make new cells.

7
X. Ma et al.
Bacteria produce some organic compounds secreted outside the cell,
such as proteins, nucleic acids, lipids, and polysaccharides containing
potential biomarkers for screening aptazymes. Importantly, instead of
using the entire bacterial cells, the use of CEM and CIM as targets
allowed for excellent specificity, avoiding the nonspecific binding
problems suffered by aptamers [84]. In-depth identification of targets
can fish out the specific target molecules. There have been many reports
predicting the target of aptazymes. In most cases, the target is a protein.
Studies have also shown that the target is a transcription factor. Future
experiments should study these targets in depth while obtaining apta­
zymes. It may be possible to use aptazymes to detect a particular type of
bacteria and convert it into a specific protein. It is hoped that aptazymes
is likely to function more effectively on the diagnosis of medical diseases
[22]. More importantly, the mutation and optimization of aptazymes
found that partial mutations and truncation were meaningful, which
could obtain aptazymes with higher activity and could specifically
detect bacteria [59]. Meanwhile, experiments have proved that the
cis-acting aptazymes could cleave the covalently linked substrate.
However, by separating the substrate sequence from the aptazymes and
converting them into a trans catalyst, aptazymes could still trans-cleave
substrate sequence in a CEM-dependent manner. The successful estab­
lishment of trans-acting aptazymes are important for the detection of
pathogenic bacteria [56]. Considering that the structure prediction may
not specifically reflect the natural folding pattern of aptazymes struc­
ture, point mutation, and sequence truncation can be used to verify the
core binding site of aptazymes.
DNAzymes can also be used as a signaling element, PMD can use a
Horseradish peroxidase (HRP) substrate for signal amplification, which
has recently been described for the detection of Salmonella [85,86],
Vibrio parahaemolyticus [87], H. pylori [88], Listeria monocytogenes
[89–91], and Cronobacter sakazakii [92], etc. In fact, the detection of
bacterial genes is transformed into bacterial detection. DNA extraction
requires a great deal of sample pre-processing, which hinders the rapid
detection of bacteria and is thus unsuitable for simple biosensors.
However, Hui et al. reported a paper sensor that can directly detect
glutamate dehydrogenase (a marker for Clostridium difficile) showing
that the PMD method can be used with unprocessed samples [75].
Further PMD can become a highly sensitive optical (chemiluminescence
or colorimetric) sensing platform via combining with some isothermal
amplification technologies [93].
DNAzymes will have a wider range of applications and broad pros­
pects in the future [69]. First, more detailed studies of the currently
selected aptazymes are needed to understand their target recognition
mechanisms, especially the roles of metal ions and target analytes. The
target analyte is likely to exert an allosteric effect to activate the apta­
zymes, while the catalysis requires a metal ion [94]. Second, expanding
the type of target from bacterial cells to other microorganisms, such as
protozoa and algae, maybe an interesting research direction because
they may produce different, but still specific, CEMs or CIMs [95].
DNAzymes have been effectively used for in situ amplification and
detection of miRNA in living cells [96]. There are also reports that
DNAzymes have made great progress in the field of tumor gene therapy
[97,98]. DNAzymes were designed to cleave specific mRNA and can be
utilized as in vivo therapeutic agents to destroy specific mRNA targets,
thereby down-regulating gene expression, which are a major advance­
ment in the application of DNAzymes in medical testing [99–102].
Third, using DNAzymes for both target recognition and signal genera­
tion might be engineered [79]. Finally, combining nanotechnology and
nanomaterials with DNA can provide further capabilities to concentrate
target analytes and generate enhanced signals [103–105]. The combi­
nation of nanomaterials and DNA has been effectively developed.
DNAzymes can be conjugated to nanomaterials for signal transduction,
which further improves the detection sensitivity of pathogenic bacteria
[80,106]. Technologies such as isothermal amplification technique,
molecularly imprinted polymers, and nanoenzymes can be used to
enhance target recognition and signal generation, and their combination
8
Sensors and Actuators: B. Chemical 331 (2021) 129422
with DNAzymes can further research the detection of pathogenic bac­
terial cells.
Declaration of Competing Interest
The authors confirm that there are no conflicts of interest regarding
this paper.
Acknowledgements
This study was supported by the National Key R&D Program of China
(2018YFC0311106), the Priority Academic Program Development of
Jiangsu Higher Education Institutions (PAPD), and the Key R&D Pro­
gram of Gaoxing Qu Lianyungang Jiangsu (KYCX19-2298, ZD201918).
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Xiaoyi Ma completed a Bachelor of Science at Gansu Agricultural University in 2018. She
is now studying a Master’s degree and working on the application of Functionalized
Nucleic Acid in bacteria detection in Jiangsu Ocean University.
Wen Ding completed a Bachelor of Science at Jiangsu Ocean University in 2020. He will
continue to study for a Master’s degree in Jiangsu Ocean University.
Cang Wang completed a Bachelor of Engineering at Shanxi Agricultural University in
2017. She is now studying a Master’s degree and working on the mechanism of Nano-
Layered Double Hydroxide on bacteria in Jiangsu Ocean University.
Hangjie Wu completed a Bachelor of Engineering at Jiangsu Normal University Kewen
College in 2018. Her research focuses on the screening of nucleic acid aptamers and their
diagnosis and treatment in infected microorganisms in Jiangsu Ocean University.
Xiaopeng Tian completed a Bachelor of Engineering at Longdong University in 2018. His
researches mainly focus on screening and application of Marine dextroglycosidase in
Jiangsu Ocean University.
Mingsheng Lyu received his Ph. D degree in Food Science from the Shanghai Ocean
University in 2016. He is a professor and as a principle investigator at Jiangsu Ocean
University. Recently, he focuses on the faster diagnosis of pathogenic bacteria by func­
tional nucleic acid. Also, his research area is enzymes and their functional hydrolysates.
Shujun Wang received her Ph. D degree in Food Science from the Nanjing Agriculture
University in 2008. She is a professor and as the Dean of Jiangsu Key Laboratory of Marine
Bioresources and Environment and College of Food Science and Technology at Jiangsu
Ocean University. She focuses on enzymes secreted by microorganisms and the diagnosis
of pathogenic bacteria by functional nucleic acid.