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A novel FRET biosensor based on four-way branch migration HCR for Vibrio parahaemolyticus detection
Sensors & Actuators: B. Chemical 296 (2019) 126577
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A novel FRET biosensor based on four-way branch migration HCR for Vibrio parahaemolyticus detection Dongxia Rena, Chengjun Suna,b, Zhijun Huangc, Zewei Luoc, Chen Zhoua, Yongxin Lia,b,
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a
West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu 610041, China Provincial Key Laboratory of Food Safety Monitoring and Risk Assessment of Sichuan, Chengdu 610041, China c College of Life Science, Sichuan University, Chengdu 610047, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Vibrio parahaemolyticus Fluorescence resonance energy transfer Four-way branch migration hybridization chain reaction Biosensor
A versatile and powerful fluorescence resonance energy transfer (FRET) biosensor based on four-way branch migration hybridization chain reaction (HCR) was proposed for the detection of Vibrio parahaemolyticus (V. parahaemolyticus). In the system, two partly complementary hairpin structures of H1 and H2, and an assistant DNA strand (R) were designed. H1 was labeled with carboxyfluorescein (FAM) as fluorophore donor, and H2 was labeled with tetramethylrhodamine (TAMRA) as fluorophore acceptor. When target DNA was presented in the system, it would firstly bind with the assistant DNA to form a short double-strand DNA (dsDNA), and subsequently trigger the four-way branch migration HCR, which would produce long nicked dsDNA concatamer and bring a good deal of FAM and TAMRA in close proximity, so that dramatic FRET signals could be achieved. With the assistance of well-designed four-way branch migration HCR circuits, this FRET biosensor exhibited a superior detection capability, which could detect 0.067 nM target DNA and as low as 10 CFU mL−1 of V. parahaemolyticus. To our knowledge, it is the first time that a four-way branch migration HCR strategy for signal amplification has been adapted to the FRET biosensor, which might show great potential in food safety and clinical diagnosis.
1. Introduction Pathogenic bacteria are the predominant cause of foodborne illnesses which have a serious impact on human health. Vibrio parahaemolyticus (V. parahaemolyticus), a gram-negative bacterium, is one of the important pathogenic bacteria. It is commonly found in seawater and marine products, especially bivalve molluscan shellfish. People who take the foods contaminated with V. parahaemolyticus may suffer from acute gastroenteritis, characterized by nausea, diarrhea, crampy abdominal pain, vomiting, headache and low-grade fever [1,2]. In recent years, the outbreaks caused by V. parahaemolyticus have occurred frequently around the world, especially in coastal areas [3,4]. Therefore, it is greatly significant to develop rapid and sensitive detection method of V. parahaemolyticus for food safety. Classical methods for the detection of V. parahaemolyticus are on the basis of bacterial culture and biochemical identification, which require both time and complicated operation [5]. In recent years, with the advantages of simple device, flexible design, rapid detection and low cost, biosensors play an increasingly crucial role in bacteria detection; therefore they have attracted much research interest. Moreover, a series
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of signal amplification strategies have also been developed to improve the sensitivity of biosensors, including nanomaterials, enzymatic reaction, DNA nanotechnology and so on [6–11]. Among them, enzymebased DNA amplification techniques, such as polymerase chain reaction (PCR) [12,13], linked enzyme chain reaction (LCR) [14], and rolling circle amplification (RCA) [15], are rapid and sensitive; however, they need expensive and unstable enzymes, and require rigorous reaction conditions. As a contrast, hybridization chain reaction (HCR) possesses such prominent highlights as enzyme-free and isothermal conditions. Traditional HCR was introduced by Dirk and Pierce in 2004, and has been applied to the amplified detection of various DNA, ions, cells, etc [16–21]. However, the three-way HCR exposed more single-stranded DNA (ssDNA), which would lead to an increased possibility of base mismatch. In consideration of this fact, a novel HCR strategy named four-way branch migration HCR was put forward by Pierce in 2007 [22]. In this design, the target strand A and the assistant strand R are polymerized primarily to form the A•R duplex, which can dramatically decrease the possibility of nonspecific initiation. At the same time, fourway branch migration HCR is able to further relax the requirements of the sequence design [22–24].
Corresponding author at: West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu 610041, China. E-mail address: [email protected] (Y. Li).
https://doi.org/10.1016/j.snb.2019.05.054 Received 26 October 2018; Received in revised form 14 May 2019; Accepted 15 May 2019 Available online 15 May 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
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2.3. Atomic force microscopy (AFM) imaging
A variety of sensing strategies were reported for detecting target bacterial DNA, such as electrochemical biosensors, fluorescent biosensors, colorimetric biosensors, and surface plasmon resonance (SPR) sensors [25–28]. Among them, fluorescent biosensors based on fluorescence resonance energy transfer (FRET) have been proved a particularly useful approach due to the advantages of high sensitivity and selectivity, and avoidance of wash steps. Compared with common fluorescence sensors, FRET allows to simultaneously record two emission intensities at different wavelengths, which can weaken the interference of other uncontrollable factors and minimize the experimental errors, thereby achieving more accurate results [29–31]. Recently, the integration of FRET with the isothermal amplification strategies has been reported to improve the detection sensitivity. Huang et al. [32] visualized tumor-related mRNA in single cells and tissue sections sensitively by a FRET-based HCR strategy. Although three hairpin probes were employed, it was actually still the conventional HCR. Quan et al. [33] established a sensitive biosensor based on FRET and two-layer nucleic acid circuits of catalyzed hairpin assembly (CHA) and HCR for the detection of nucleic acid, which could yield 50,000-fold signal amplification. However, this system involves four hairpins and some of them with big loops, which are prone to nonspecific reactions, and also lead to a complicated and demanding design. Herein, we have, for the first time, demonstrated a FRET biosensor based on four-way branch migration HCR for the detection of V. parahaemolyticus. Different from traditional HCR based biosensors, in the proposed biosensor the target DNA was firstly recognized and captured by complementary DNA, and then the sticky ends of the formed A•R duplex will further specifically recognize both ends of H1, which has fewer off-pathway base pairings and greatly improves the detection specificity. By four-way branch migration HCR and FRET dual signal amplification strategy, each copy of the target can form long nicked dsDNA that contains a large number of repeated FRET units, thereby amplifying the fluorescent signal powerfully. In addition, the proposed strategy also possesses the advantages of relatively simple sequence design requirement and avoidance of wash steps
The synthesized oligomers were prepared into a stock solution of 100 μM in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and stored at -20 ℃. The stock solutions were diluted to the working solutions with 1 × SPSC (1 M NaCl, 50 mM Na2HPO4, pH 7.5) with suitable concentration, and stored at 4 ℃. The hairpin H1, H2, and the mixture of target strand A and assistant strand R were respectively denatured at 95 ℃ for 5 min, and then cooled down slowly to room temperature. In HCR reaction, 2.5 μL H1 (4 μM) and 2.5 μL H2 (4 μM) were added 2.5 μL A•R mixture with 2.4 μM A and 4 μM R, followed by suitable amount of 1 × SPSC to obtain a final volume of 40 μL. The mixture was incubated at 30 ℃ for 90 min. After 20 μL MgCl2 (5 mM) was added on the freshly dissociated mica surface for 15 min, the mica was cleaned three times with ultra-pure water to remove the excess MgCl2, and then dried with nitrogen. 15 μL of the HCR product and blank sample were slowly dropped onto the surface of the treated mica, respectively. 10 min later, the mica surface was rinsed 3 times with ultrapure water to remove excess mixture, and then dried in the air. The prepared sample was imaged by AFM (Bruker dimension icon, USA). 2.4. Fluorescence analysis
2. Experimental section
In the reaction system, 2.5 μL H1 (4 μM) and 2.5 μL H2 (4 μM) were added 2.5 μL A•R mixture with a series of concentration of A and a fixed concentration of R at 4 μM, to obtain a total volume of 100 μL in 1 × SPSC. After incubated at 30 ℃ for 90 min, the fluorescence measurements were performed at room temperature by using a LS 55 Fluorescence Spectrometer (PerkinElmer, USA), and the emission spectra of 510–650 nm was recorded with the excitation wavelength of 498 nm. The slit width of excitation and emission was set at 5 nm. The ratios of normalized fluorescence intensities ((I0D525/I0A588)/(ID525/ IA588)) for the experimental and the blank groups were calculated to quantify the target DNA, in which the ID525/IA588 referred to the ratio of fluorescence intensity at a wavelength of 525 nm to fluorescence intensity at 588 nm in the experimental group, and the ratio of I0D525/I0A588 to the blank group.
2.1. Reagents and materials
3. Results and discussion
The oligonucleotides presented in Table S1 were obtained from Sangon Biotech. Co., Ltd. (Shanghai, China). The 100 bp DNA marker, 25 mM MgCl2, dNTP Mixture (each 2.5 mM), TaKaRa Taq (5 units μL−1) and 10 × PCR buffer were purchased from Takara (Dalian, China). Disodium hydrogen phosphate (Na2HPO4), sodium chloride (NaCl), potassium chloride (KCl), potassium dihydrogen phosphate (KH2PO4) and tris (hydroxymethyl) aminomethane (Tris) were bought from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). The chemicals mentioned above were all analytical reagents, and all the solutions were prepared with ultrapure water from a Millipore Milli-Q water purification system.
3.1. Mechanism for the proposed strategy The principle of the proposed biosensor was shown in Scheme 1. In this design, the cascade fluorescent signal amplification was achieved based on FRET and four-way branch migration HCR. There were four DNA fragments in this sensing system: the target strand named A, the assistant strand named R, and the hairpins of H1 and H2. R was partly complementary with A, which had 3 nucleotide (nt) sticky end at 5′ end and 3′ end, respectively. In addition to the complementary sequences with R, A had 6 nt sticky end at 3′ end. Each hairpin probe of H1 and H2 had a stem of 12 base pairs with a 6 nt loop. H1 had 6 nt sticky end at 5′ end and 3 nt sticky end at 3′ end, while H2 had 3 nt sticky end at 5′ end and 6 nt sticky end at 3′ end. H1 was labeled with carboxyfluorescein (FAM) as fluorophore donor, and its partly complementary hairpin H2 was labeled with tetramethylrhodamine (TAMRA) as fluorophore acceptor. The absorption peak of FAM was 498 nm, and emission peak was 525 nm. TAMRA’s emission intensity at 588 nm could be enhanced when FRET occurred. When target DNA (A) presented in the system, it would hybridize with R to form A•R duplex with single-stranded overhangs, which were located at 3′ end of A with 6 nt, 3′ and 5′ end of R with 3 nt, respectively. The sticky ends of A•R duplex would bind with H1 and unfold its hairpin structure according to the principle of complementary base pairing. The newly exposed hairpin domain of H1 served as the toeholds and bound with 3′ end of H2, and the overhang at 3′ end of R bound with 5′ end of H2. Thus, the hairpin structure of H2
2.2. Bacterial culture V. parahaemolyticus (ATCC17802), kindly provided by Chengdu Center for Disease Control and Prevention, were incubated in LuriaBertani (LB) broth at 37 ℃ for 24 h with shaking. The bacteria were counted by plating 1 mL of various 10-fold dilutions in sterilized saline onto nutritional agar containing 3% NaCl. After incubated at 37 ℃ for 24 h, the culture colonies were counted to estimate the number of bacteria. At the same time, the genomic DNA was extracted from 1.0 mL the culture of each dilution by using bacterial genomic DNA extraction kit (Tiangen Biochemical Technology, Beijing).
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Scheme 1. Schematic illustration of the FRET sensor based on four-way branch migration HCR for detecting the specific DNA of Vibrio parahaemolyticus.
was opened, and the exposed sticky ends were identical to the original A•R duplex, beginning the next fuel cycle, which could form a long nicked dsDNA and bring FAM and TAMRA in close proximity so that FRET occurred. As four-way branch migration HCR was triggered and propagated, each copy of the target could form long dsDNA with a large number of repeated FRET units, thereby amplifying the fluorescent signal powerfully. On the contrary, if there was no target DNA (A) in the system, the hairpin probes of H1 and H2 were stable, and the assistant DNA strand R could hardly open them; thus, the FRET phenomenon could not occur, and only quite weak background fluorescence was obtained.
3.2. Feasibility of the cascade sensing system To investigate the feasibility of four-way branch migration HCR reaction, polyacrylamide gel electrophoresis (PAGE) was employed to represent the results with or without target DNA. As shown in Fig. S1, only the band of H1 or H2 was observed in lane 2 (H1) and lane 3 (H2). Besides the mixture of H1 and H2, another slight band of R/H1/H2 complex was found in lane 4, indicating that four-way branch migration HCR could hardly occur without the target DNA. However, with the increase of target DNA concentration, it could be observed that the bands of H1, H2 and R gradually became weak in lane 5–8 (A•R/H1/ H2), indicating that the monomers of H1, H2 and R were constantly consumed and four-way branch migration HCR was triggered. Meanwhile, it also illustrated that higher target DNA concentration led to increased monomers consumption, meaning that the results of fourway branch migration HCR was related to the concentration of target DNA. This result was in agreement with the work reported by Weihong Tan [24]. The probable reason may be that higher concentration of target DNA leads to more formation of A•R duplex, thereby producing more active sticky end to initiate four-way branch migration HCR. At the same time, the HCR products were characterized by atomic force microscopy (shown in Fig. S2). The AFM image showed that there were a lot of linear nanowires generated in four-way branch migration HCR and adsorbed on the surface of mica (Fig. S2B and Fig. S2D) compared with the experimental results without the target (Fig. S2A and Fig. S2C), further indicating the feasibility of four-way branch migration HCR. After that, we verified the feasibility of the strategy combining FRET with four-way branch migration HCR. The fluorescence emission spectra of the proposed biosensor with the excitation wavelength at 498 nm were displayed in Fig. 1. There was a strong emission peak at 525 nm for H1 labeled with FAM, while a weak emission peak at
Fig. 1. Florescent spectrogram of H1, H2, the mixture of R, H1 and H2, and the mixture of A•R, H1 and H2 at an excitation wavelength of 498 nm. The concentration of R, H1 (H2) and T are 100 nM, 100 nM and 60 nM, respectively.
588 nm for H2 labeled with TAMRA. As for the mixture of R, H1, and H2, the fluorescence intensity of FAM at 525 nm slightly decreased, and that of TAMRA at 588 nm mildly increased. It indicated that four-way branch migration HCR hardly occurred in the absence of target DNA, thus only a very small amount of FRET units generated. When target DNA was added into the system, the fluorescence intensity of 525 nm dramatically decreased, while that of 588 nm greatly increased. It demonstrated that a large number of FRET units were generated since the target DNA (A) hybridized with the assistant strand (R), and then triggered four-way branch migration HCR to form long dsDNA concatemer, which brought a good deal of FAM and TAMRA in close proximity. The fluorescence results were consistent with the PAGE results, further proving that the proposed sensing strategy was feasible and could offer significant signal amplification for the detection of target DNA.
3.3. Optimization of the experimental conditions 3.3.1. Optimization of the probes The sequence of R, H1 and H2 have a great impact not only on the triggering efficiency of the target DNA, but also the background signal intensity of the four-way branch migration HCR reaction, which further affect the detection performance of the proposed biosensor. Referring to the reported literature [22], three sets of the probes sequences were 3
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Fig. 2. (A) The normalized fluorescent response of the proposed sensor with various concentrations of target DNA. (B) Calibration plot for the sensing strategy with different concentrations of target DNA.
the formation of hydrogen bonds, thus decreased the yields of A•R duplex, which had an adverse effects on the initiation of the four-way branch migration HCR.
designed according to complementary base pairing with the aids of NUPACK software (Table S1). Fig. S3 showed that there were large amount of hairpin probes left in lane 2, 9 and 11, which demonstrated low reaction efficiency of the probe pairs of H3 and H4, H5 and H6, H7 and H8. With combination of Fig. S1, H1 and H2 were selected as the probes in the subsequent experiments, with high reaction efficiency and low background.
3.3.5. Optimization of the reaction time The reaction time is a critical factor influencing the sensor sensitivity. To achieve optimized reaction time, we investigated the fluorescence responses of different reaction time from 0 to 180 min. As shown in Fig. S5B, an obvious increase of the ratio of normalized fluorescent intensity was obtained within 90 min, and then the increase rate slowed down, indicating that the hybridization chain reaction tended to be complete when the reaction time was 90 min. As a contrast, the response of the blank still increased after 90 min, and the reason was that the spontaneous nonspecific binding would be intense over time, which would make the detection specificity and sensitivity lower. It is not true that prolonged time may facilitate to react completely; however, prolonged time is disadvantageous for the performance of the sensing system. Thus, 90 min was selected as the reaction time.
3.3.2. Optimization of NaCl concentration It is generally acknowledged that the concentration of salt plays an important role in DNA self-assembly reaction including four-way branch migration HCR. The phosphate groups on DNA chain are negatively charged, which leads to the intramolecular electrostatic repulsion in dsDNA and the decreased efficiency of DNA self-assembly reaction. After adding salt ions, the positive ions can block the negative charges of the phosphate groups and decrease the electrostatic repulsion in dsDNA, thereby improving the DNA self-assembly efficiency. Therefore, to obtain good performance of the sensing strategy, we optimized the concentrations of NaCl in SPSC buffer. As shown in Fig. S4A, the ratio of normalized fluorescent intensity was enhanced with the increase of NaCl concentration in the range of 0.4 M to 1 M. While, if NaCl concentration exceeded 1 M, the ratio of normalized fluorescent intensity was reduced. Thus, 1 M NaCl in SPSC buffer was selected in subsequent experiments.
3.4. Analytical performance of the developed sensing system 3.4.1. Sensitivity The sensitivity of the proposed sensing system was assessed by measuring the fluorescence responses with different concentrations of target DNA under the optimal conditions (shown in Fig. 2). As expected, the rising speed of the normalized fluorescent intensity gradually decreased with the increasing concentration of target DNA from 0 nM to 100 nM (Fig. 2A). This further proved that the sensing strategy was feasible, and it could be utilized to detect the target sequence. In the range of 0.2–60 nM, a good linear relationship was shown between the signal responses and target DNA concentrations (Fig. 2B). The regression equation for the method was y = 0.0082x+1.065 (R = 0.9965), with a detection limit of 0.067 nM (defined as 3σ/K, where σ is the standard deviation of the blank, and K is the slope of the calibration curve). Compared with the three-way HCR combined with FRET with a detection limit of 0.7 nM reported in the previous literature [29], the proposed strategy showed an order of magnitude improvement in sensitivity. This is probably because single stranded DNA always has secondary structure at or near room temperature, which can interfere with the hybridization reaction at the kinetic and equilibrium thermodynamics level. The double stranded DNA of A•R duplex discourages off-pathways that result in either kinetic traps or spurious interactions [34]. Moreover, there are more toeholds of the A•R, and more assembly sites are formed to open H1 and H2, so that it would be more effective to trigger four-way branch migration HCR.
3.3.3. Optimization of the reaction temperature The temperature would affect the binding efficiency and biosensor sensitivity since the free energy of combination gradually decreased with the temperature increasing. Therefore, we examined the responses from 4 ℃ to 50 ℃. As shown in Fig. S4B, the ratio of normalized fluorescent intensity reached a plateau at 30 ℃. The reason was that when the temperature was low, the free energy of combination was relatively high, and the hairpin probes were too stable to cross open, thus resulted in a low binding efficiency and weak fluorescent signal. However, the hairpin probes showed poor thermal stability with low free energy of combination at a relatively high temperature, which led to strong background signal. Therefore, 30 ℃ was selected as the reaction temperature. 3.3.4. Optimization of the reaction pH pH has an influence on hydrogen bonding, thereby affecting the stability of dsDNA and the performance of the sensing system. As shown in Fig. S5A, the ratio of normalized fluorescent intensity increased quickly with the increase of pH when the PH value was below 7.5, and after that it slowly decreased. The maximal fluorescent response was obtained at pH 7.5. Thus pH 7.5 was chosen in subsequent experiments. The reason might be that the acid or alkaline environment weakened 4
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Table 1 Recovery of Target Assay with Different Concentrations Spiked into Real Sample (n = 3). Sample
Spiked (nM)
Found (nM)
Recovery (%)
RSD (%)
Sample 1 Sample 2 Sample 3
1.00 20.00 60.00
1.09 18.82 59.54
109.93 94.10 99.23
5.51 5.58 7.15
*Sample 1, Sample 2 and Sample 3 were three parallel samples of a negative sample.
effect on the hybridization yield. 3.5. Application in real samples
Fig. 3. The net normalized fluorescent signals (△ Normalized intensity ratio) of the proposed sensor in this study and the sensor based on conventional HCR for different DNA oligonucleotides. (a) Single-base deleted target DNA; (b) Single-base inserted target DNA; (c) Single-base mismatched target DNA; (d) Specific DNA for Staphylococcus aureus; (e) Specific DNA for Listeria monocytogenes; (f) Specific DNA for Vibrio cholerae; (g) Target DNA. The concentration of R, H1 (H2) are both 100 nM. And the concentration of others are 60 nM, respectively.
To investigate the actual application potential of the strategy proposed in this work, the proposed strategy was further applied to the detection of PCR product of V. parahaemolyticus gyrB gene. The cultured V. parahaemolyticus was diluted to obtain serial concentrations from 0 to 106 CFU mL−1 and genomic DNA was extracted from each dilution according to the bacterial genomic DNA extraction kit. Then, the extracted DNA of V. parahaemolyticus was firstly denatured at 95 ℃ for 10 min, followed by 35 cycles of 95 ℃ for 30 s, 60 ℃ for 15 s, 72 ℃ for 10 s, and finally 5 min of extension. The PCR product and R were heated at 95 ℃ for 6 min, and immediately cooled down in ice bath to obtain the A•R duplex, and then detected by PAGE and the proposed biosensor, respectively. As shown in Fig. 4, the product band for 100 CFU mL-1 was hardly observed on polyacrylamide gel electrophoresis; while, the signal responses of the real samples distinguished from that of the blank sample were obvious, and as low as 10 CFU mL−1 V. parahaemolyticus could be detected in this sensing system. The sensitivity of the proposed strategy based on four-way branch migration HCR and FRET was compared with that of other strategies for the detection of V. parahaemolyticus or other bacterial DNA reported recently (Table S2). The results showed that the detection limit of this proposed method was better or comparable to some other strategies. This was attributed to the use of the cascade signal amplification strategy of four-way branch migration HCR and FRET. On the other hand, our sensing strategy for V. parahaemolyticus detection could greatly shorten the analysis time and simplify the detection procedure. Furthermore, the recovery experiments were performed by standard addition method. As depicted in Table 1, the recoveries were in the range of 94.10%–109.9%, which illustrated that the newly developed sensor could detect V. parahaemolyticus with high accuracy. Therefore, the results mentioned above elucidated that the proposed sensing
3.4.2. Selectivity The specificity of the proposed strategy for V. parahaemolyticus detection was also evaluated. We detected the target sequence, the specific sequences of staphylococcus aureus, listeria monocytogenes and Vibrio cholerae, as well as mutant target molecules including insertion, deletion and mismatch. As shown in Fig. 3, a very little response was obtained for other bacterial sequences and the mutant target molecules, which was significantly lower than that of the target sequence. The results demonstrated that the strategy we put forward possessed high specificity in the discrimination of target sequence from other sequences. At the same time, a FRET biosensor based on conventional HCR for V. parahaemolyticus detection was designed, and its specificity was compared with that of this proposed biosensor. The results (shown in Fig. 3) indicated that both biosensors based on four-way branch migration HCR and the conventional HCR could specifically detect V. parahaemolyticus, but for mutant target molecules of mismatch, insertion or deletion, the specificity of the former biosensor was better than the latter. It was attributed to the fact that target DNA firstly bound with the designed assistant R by complementary base pairing to form double-stranded structures, and then reacted with H1 to initiate the four-way branch migration reaction; while, the base mismatches could cause slight thermodynamic changes that had disproportionately large
Fig. 4. (A) The gel electrophoresis images of the PCR products for different concentrations of Vibrio parahaemolyticus. (1–6: serial dilution of Vibrio parahaemolyticus, 101–106 CFU mL−1, 7: marker). (B) The experimental results of the DNA biosensor. 5
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strategy could be potentially applied to the detection of V. parahaemolyticus in real samples.
[12]
4. Conclusions [13]
In conclusion, a novel enzyme-free signal amplification strategy based on FRET and four-way branch migration HCR was proposed and successfully applied to the specific detection of V. parahaemolyticus. The target DNA firstly bound to the assistant strand, and then triggered four-way branch migration HCR to form long nicked dsDNA with a large number of FRET signals, thus powerfully enhanced the detection sensitivity. It integrated the advantageous features of four-way branch migration HCR with FRET, and could work as a versatile sensor for V. parahaemolyticus detection with simple operation and short analysis time. Moreover, this method showed an excellent detection capability, whose specificity and sensitivity were even better than some reported amplification methods. Meanwhile, four-way branch migration HCR dramatically improved the specificity of the sensor due to the decreased base mismatch possibility. Further research should focus on the construction of aptasensors for intact target bacterial cells by using the proposed strategy to reduce the whole detection time, and on the extension of application scope to other pathogenic bacteria and viruses. It has great potential for the application of pathogens detection in clinical infection and food safety.
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[19]
[20]
[21]
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Acknowledgements
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This work was supported by China Postdoctoral Science Foundation (2017T100703), Project of Sichuan Provincial Science and Technology Department (2018HH0147), and Technology Innovation Research and Development Project of Chengdu Science and Technology Bureau (2018-YF05-01201-SN).
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Appendix A. Supplementary data
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Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.05.054.
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Dongxia Ren graduated from West China School of Public Health, Sichuan University and obtained the BS degree in 2016. Currently, she is a postgraduate student in West China School of Public Health, Sichuan University. Her research interest is focused on the novel biosensors based on four-way branch migration HCR. Chengjun Sun is a professor in West China School of Public Health, Sichuan University, Chengdu, PR China. He is mainly interested in the research of gas chromatography, high performance liquid chromatography, capillary electrophoresis and their application in food analysis, as well as study of adulterants in various foods, especially in functional foods. Zhijun Huang received his BS degree in biotechnology from Guangxi University, and received his MS degree in microbiology from Graduate University of Chinese Academy of Sciences. Now, he is a PhD student in the Research Center of Analytical Instrumentation,
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University in 2014. Presently, she is a PhD student in West China School of Public Health, Sichuan University after two year’s MS course. She is focused on the development of new biosensors based on fiber optic surface plasmon resonance (FOSPR) and their application in food analysis in West China School of Public Health, Sichuan University, Chengdu, PR China.
college of life science, Sichuan University. His current research interests focus on the development of novel biosensors. Zewei Luo received his BS degree in bioengineering from Huaihua University, Huaihua, China, in 2013. He presently is a PhD student in the Research Center of Analytical Instrumentation, college of life science, Sichuan University after three year’s MS course. His current research interest is focused on the development of new biosensors based on fiber optic surface plasmon resonance (FOSPR) and their application in detection of small molecules and cancer cells.
Yongxin Li is an associate professor in West China School of Public Health, Sichuan University, China. She is mainly engaged in the study of capillary electrophoresis, microfluidic chip and their application in public health, particularly in foodborne-pathogenic bacteria, viruses and chemical detection, and the development of novel biosensors for various applications.
Chen Zhou received her BS degree from West China School of Public Health, Sichuan
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Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A novel FRET biosensor based on four-way branch migration HCR for Vibrio parahaemolyticus detection Dongxia Rena, Chengjun Suna,b, Zhijun Huangc, Zewei Luoc, Chen Zhoua, Yongxin Lia,b,
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a
West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu 610041, China Provincial Key Laboratory of Food Safety Monitoring and Risk Assessment of Sichuan, Chengdu 610041, China c College of Life Science, Sichuan University, Chengdu 610047, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Vibrio parahaemolyticus Fluorescence resonance energy transfer Four-way branch migration hybridization chain reaction Biosensor
A versatile and powerful fluorescence resonance energy transfer (FRET) biosensor based on four-way branch migration hybridization chain reaction (HCR) was proposed for the detection of Vibrio parahaemolyticus (V. parahaemolyticus). In the system, two partly complementary hairpin structures of H1 and H2, and an assistant DNA strand (R) were designed. H1 was labeled with carboxyfluorescein (FAM) as fluorophore donor, and H2 was labeled with tetramethylrhodamine (TAMRA) as fluorophore acceptor. When target DNA was presented in the system, it would firstly bind with the assistant DNA to form a short double-strand DNA (dsDNA), and subsequently trigger the four-way branch migration HCR, which would produce long nicked dsDNA concatamer and bring a good deal of FAM and TAMRA in close proximity, so that dramatic FRET signals could be achieved. With the assistance of well-designed four-way branch migration HCR circuits, this FRET biosensor exhibited a superior detection capability, which could detect 0.067 nM target DNA and as low as 10 CFU mL−1 of V. parahaemolyticus. To our knowledge, it is the first time that a four-way branch migration HCR strategy for signal amplification has been adapted to the FRET biosensor, which might show great potential in food safety and clinical diagnosis.
1. Introduction Pathogenic bacteria are the predominant cause of foodborne illnesses which have a serious impact on human health. Vibrio parahaemolyticus (V. parahaemolyticus), a gram-negative bacterium, is one of the important pathogenic bacteria. It is commonly found in seawater and marine products, especially bivalve molluscan shellfish. People who take the foods contaminated with V. parahaemolyticus may suffer from acute gastroenteritis, characterized by nausea, diarrhea, crampy abdominal pain, vomiting, headache and low-grade fever [1,2]. In recent years, the outbreaks caused by V. parahaemolyticus have occurred frequently around the world, especially in coastal areas [3,4]. Therefore, it is greatly significant to develop rapid and sensitive detection method of V. parahaemolyticus for food safety. Classical methods for the detection of V. parahaemolyticus are on the basis of bacterial culture and biochemical identification, which require both time and complicated operation [5]. In recent years, with the advantages of simple device, flexible design, rapid detection and low cost, biosensors play an increasingly crucial role in bacteria detection; therefore they have attracted much research interest. Moreover, a series
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of signal amplification strategies have also been developed to improve the sensitivity of biosensors, including nanomaterials, enzymatic reaction, DNA nanotechnology and so on [6–11]. Among them, enzymebased DNA amplification techniques, such as polymerase chain reaction (PCR) [12,13], linked enzyme chain reaction (LCR) [14], and rolling circle amplification (RCA) [15], are rapid and sensitive; however, they need expensive and unstable enzymes, and require rigorous reaction conditions. As a contrast, hybridization chain reaction (HCR) possesses such prominent highlights as enzyme-free and isothermal conditions. Traditional HCR was introduced by Dirk and Pierce in 2004, and has been applied to the amplified detection of various DNA, ions, cells, etc [16–21]. However, the three-way HCR exposed more single-stranded DNA (ssDNA), which would lead to an increased possibility of base mismatch. In consideration of this fact, a novel HCR strategy named four-way branch migration HCR was put forward by Pierce in 2007 [22]. In this design, the target strand A and the assistant strand R are polymerized primarily to form the A•R duplex, which can dramatically decrease the possibility of nonspecific initiation. At the same time, fourway branch migration HCR is able to further relax the requirements of the sequence design [22–24].
Corresponding author at: West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu 610041, China. E-mail address: [email protected] (Y. Li).
https://doi.org/10.1016/j.snb.2019.05.054 Received 26 October 2018; Received in revised form 14 May 2019; Accepted 15 May 2019 Available online 15 May 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
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2.3. Atomic force microscopy (AFM) imaging
A variety of sensing strategies were reported for detecting target bacterial DNA, such as electrochemical biosensors, fluorescent biosensors, colorimetric biosensors, and surface plasmon resonance (SPR) sensors [25–28]. Among them, fluorescent biosensors based on fluorescence resonance energy transfer (FRET) have been proved a particularly useful approach due to the advantages of high sensitivity and selectivity, and avoidance of wash steps. Compared with common fluorescence sensors, FRET allows to simultaneously record two emission intensities at different wavelengths, which can weaken the interference of other uncontrollable factors and minimize the experimental errors, thereby achieving more accurate results [29–31]. Recently, the integration of FRET with the isothermal amplification strategies has been reported to improve the detection sensitivity. Huang et al. [32] visualized tumor-related mRNA in single cells and tissue sections sensitively by a FRET-based HCR strategy. Although three hairpin probes were employed, it was actually still the conventional HCR. Quan et al. [33] established a sensitive biosensor based on FRET and two-layer nucleic acid circuits of catalyzed hairpin assembly (CHA) and HCR for the detection of nucleic acid, which could yield 50,000-fold signal amplification. However, this system involves four hairpins and some of them with big loops, which are prone to nonspecific reactions, and also lead to a complicated and demanding design. Herein, we have, for the first time, demonstrated a FRET biosensor based on four-way branch migration HCR for the detection of V. parahaemolyticus. Different from traditional HCR based biosensors, in the proposed biosensor the target DNA was firstly recognized and captured by complementary DNA, and then the sticky ends of the formed A•R duplex will further specifically recognize both ends of H1, which has fewer off-pathway base pairings and greatly improves the detection specificity. By four-way branch migration HCR and FRET dual signal amplification strategy, each copy of the target can form long nicked dsDNA that contains a large number of repeated FRET units, thereby amplifying the fluorescent signal powerfully. In addition, the proposed strategy also possesses the advantages of relatively simple sequence design requirement and avoidance of wash steps
The synthesized oligomers were prepared into a stock solution of 100 μM in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and stored at -20 ℃. The stock solutions were diluted to the working solutions with 1 × SPSC (1 M NaCl, 50 mM Na2HPO4, pH 7.5) with suitable concentration, and stored at 4 ℃. The hairpin H1, H2, and the mixture of target strand A and assistant strand R were respectively denatured at 95 ℃ for 5 min, and then cooled down slowly to room temperature. In HCR reaction, 2.5 μL H1 (4 μM) and 2.5 μL H2 (4 μM) were added 2.5 μL A•R mixture with 2.4 μM A and 4 μM R, followed by suitable amount of 1 × SPSC to obtain a final volume of 40 μL. The mixture was incubated at 30 ℃ for 90 min. After 20 μL MgCl2 (5 mM) was added on the freshly dissociated mica surface for 15 min, the mica was cleaned three times with ultra-pure water to remove the excess MgCl2, and then dried with nitrogen. 15 μL of the HCR product and blank sample were slowly dropped onto the surface of the treated mica, respectively. 10 min later, the mica surface was rinsed 3 times with ultrapure water to remove excess mixture, and then dried in the air. The prepared sample was imaged by AFM (Bruker dimension icon, USA). 2.4. Fluorescence analysis
2. Experimental section
In the reaction system, 2.5 μL H1 (4 μM) and 2.5 μL H2 (4 μM) were added 2.5 μL A•R mixture with a series of concentration of A and a fixed concentration of R at 4 μM, to obtain a total volume of 100 μL in 1 × SPSC. After incubated at 30 ℃ for 90 min, the fluorescence measurements were performed at room temperature by using a LS 55 Fluorescence Spectrometer (PerkinElmer, USA), and the emission spectra of 510–650 nm was recorded with the excitation wavelength of 498 nm. The slit width of excitation and emission was set at 5 nm. The ratios of normalized fluorescence intensities ((I0D525/I0A588)/(ID525/ IA588)) for the experimental and the blank groups were calculated to quantify the target DNA, in which the ID525/IA588 referred to the ratio of fluorescence intensity at a wavelength of 525 nm to fluorescence intensity at 588 nm in the experimental group, and the ratio of I0D525/I0A588 to the blank group.
2.1. Reagents and materials
3. Results and discussion
The oligonucleotides presented in Table S1 were obtained from Sangon Biotech. Co., Ltd. (Shanghai, China). The 100 bp DNA marker, 25 mM MgCl2, dNTP Mixture (each 2.5 mM), TaKaRa Taq (5 units μL−1) and 10 × PCR buffer were purchased from Takara (Dalian, China). Disodium hydrogen phosphate (Na2HPO4), sodium chloride (NaCl), potassium chloride (KCl), potassium dihydrogen phosphate (KH2PO4) and tris (hydroxymethyl) aminomethane (Tris) were bought from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). The chemicals mentioned above were all analytical reagents, and all the solutions were prepared with ultrapure water from a Millipore Milli-Q water purification system.
3.1. Mechanism for the proposed strategy The principle of the proposed biosensor was shown in Scheme 1. In this design, the cascade fluorescent signal amplification was achieved based on FRET and four-way branch migration HCR. There were four DNA fragments in this sensing system: the target strand named A, the assistant strand named R, and the hairpins of H1 and H2. R was partly complementary with A, which had 3 nucleotide (nt) sticky end at 5′ end and 3′ end, respectively. In addition to the complementary sequences with R, A had 6 nt sticky end at 3′ end. Each hairpin probe of H1 and H2 had a stem of 12 base pairs with a 6 nt loop. H1 had 6 nt sticky end at 5′ end and 3 nt sticky end at 3′ end, while H2 had 3 nt sticky end at 5′ end and 6 nt sticky end at 3′ end. H1 was labeled with carboxyfluorescein (FAM) as fluorophore donor, and its partly complementary hairpin H2 was labeled with tetramethylrhodamine (TAMRA) as fluorophore acceptor. The absorption peak of FAM was 498 nm, and emission peak was 525 nm. TAMRA’s emission intensity at 588 nm could be enhanced when FRET occurred. When target DNA (A) presented in the system, it would hybridize with R to form A•R duplex with single-stranded overhangs, which were located at 3′ end of A with 6 nt, 3′ and 5′ end of R with 3 nt, respectively. The sticky ends of A•R duplex would bind with H1 and unfold its hairpin structure according to the principle of complementary base pairing. The newly exposed hairpin domain of H1 served as the toeholds and bound with 3′ end of H2, and the overhang at 3′ end of R bound with 5′ end of H2. Thus, the hairpin structure of H2
2.2. Bacterial culture V. parahaemolyticus (ATCC17802), kindly provided by Chengdu Center for Disease Control and Prevention, were incubated in LuriaBertani (LB) broth at 37 ℃ for 24 h with shaking. The bacteria were counted by plating 1 mL of various 10-fold dilutions in sterilized saline onto nutritional agar containing 3% NaCl. After incubated at 37 ℃ for 24 h, the culture colonies were counted to estimate the number of bacteria. At the same time, the genomic DNA was extracted from 1.0 mL the culture of each dilution by using bacterial genomic DNA extraction kit (Tiangen Biochemical Technology, Beijing).
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Scheme 1. Schematic illustration of the FRET sensor based on four-way branch migration HCR for detecting the specific DNA of Vibrio parahaemolyticus.
was opened, and the exposed sticky ends were identical to the original A•R duplex, beginning the next fuel cycle, which could form a long nicked dsDNA and bring FAM and TAMRA in close proximity so that FRET occurred. As four-way branch migration HCR was triggered and propagated, each copy of the target could form long dsDNA with a large number of repeated FRET units, thereby amplifying the fluorescent signal powerfully. On the contrary, if there was no target DNA (A) in the system, the hairpin probes of H1 and H2 were stable, and the assistant DNA strand R could hardly open them; thus, the FRET phenomenon could not occur, and only quite weak background fluorescence was obtained.
3.2. Feasibility of the cascade sensing system To investigate the feasibility of four-way branch migration HCR reaction, polyacrylamide gel electrophoresis (PAGE) was employed to represent the results with or without target DNA. As shown in Fig. S1, only the band of H1 or H2 was observed in lane 2 (H1) and lane 3 (H2). Besides the mixture of H1 and H2, another slight band of R/H1/H2 complex was found in lane 4, indicating that four-way branch migration HCR could hardly occur without the target DNA. However, with the increase of target DNA concentration, it could be observed that the bands of H1, H2 and R gradually became weak in lane 5–8 (A•R/H1/ H2), indicating that the monomers of H1, H2 and R were constantly consumed and four-way branch migration HCR was triggered. Meanwhile, it also illustrated that higher target DNA concentration led to increased monomers consumption, meaning that the results of fourway branch migration HCR was related to the concentration of target DNA. This result was in agreement with the work reported by Weihong Tan [24]. The probable reason may be that higher concentration of target DNA leads to more formation of A•R duplex, thereby producing more active sticky end to initiate four-way branch migration HCR. At the same time, the HCR products were characterized by atomic force microscopy (shown in Fig. S2). The AFM image showed that there were a lot of linear nanowires generated in four-way branch migration HCR and adsorbed on the surface of mica (Fig. S2B and Fig. S2D) compared with the experimental results without the target (Fig. S2A and Fig. S2C), further indicating the feasibility of four-way branch migration HCR. After that, we verified the feasibility of the strategy combining FRET with four-way branch migration HCR. The fluorescence emission spectra of the proposed biosensor with the excitation wavelength at 498 nm were displayed in Fig. 1. There was a strong emission peak at 525 nm for H1 labeled with FAM, while a weak emission peak at
Fig. 1. Florescent spectrogram of H1, H2, the mixture of R, H1 and H2, and the mixture of A•R, H1 and H2 at an excitation wavelength of 498 nm. The concentration of R, H1 (H2) and T are 100 nM, 100 nM and 60 nM, respectively.
588 nm for H2 labeled with TAMRA. As for the mixture of R, H1, and H2, the fluorescence intensity of FAM at 525 nm slightly decreased, and that of TAMRA at 588 nm mildly increased. It indicated that four-way branch migration HCR hardly occurred in the absence of target DNA, thus only a very small amount of FRET units generated. When target DNA was added into the system, the fluorescence intensity of 525 nm dramatically decreased, while that of 588 nm greatly increased. It demonstrated that a large number of FRET units were generated since the target DNA (A) hybridized with the assistant strand (R), and then triggered four-way branch migration HCR to form long dsDNA concatemer, which brought a good deal of FAM and TAMRA in close proximity. The fluorescence results were consistent with the PAGE results, further proving that the proposed sensing strategy was feasible and could offer significant signal amplification for the detection of target DNA.
3.3. Optimization of the experimental conditions 3.3.1. Optimization of the probes The sequence of R, H1 and H2 have a great impact not only on the triggering efficiency of the target DNA, but also the background signal intensity of the four-way branch migration HCR reaction, which further affect the detection performance of the proposed biosensor. Referring to the reported literature [22], three sets of the probes sequences were 3
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Fig. 2. (A) The normalized fluorescent response of the proposed sensor with various concentrations of target DNA. (B) Calibration plot for the sensing strategy with different concentrations of target DNA.
the formation of hydrogen bonds, thus decreased the yields of A•R duplex, which had an adverse effects on the initiation of the four-way branch migration HCR.
designed according to complementary base pairing with the aids of NUPACK software (Table S1). Fig. S3 showed that there were large amount of hairpin probes left in lane 2, 9 and 11, which demonstrated low reaction efficiency of the probe pairs of H3 and H4, H5 and H6, H7 and H8. With combination of Fig. S1, H1 and H2 were selected as the probes in the subsequent experiments, with high reaction efficiency and low background.
3.3.5. Optimization of the reaction time The reaction time is a critical factor influencing the sensor sensitivity. To achieve optimized reaction time, we investigated the fluorescence responses of different reaction time from 0 to 180 min. As shown in Fig. S5B, an obvious increase of the ratio of normalized fluorescent intensity was obtained within 90 min, and then the increase rate slowed down, indicating that the hybridization chain reaction tended to be complete when the reaction time was 90 min. As a contrast, the response of the blank still increased after 90 min, and the reason was that the spontaneous nonspecific binding would be intense over time, which would make the detection specificity and sensitivity lower. It is not true that prolonged time may facilitate to react completely; however, prolonged time is disadvantageous for the performance of the sensing system. Thus, 90 min was selected as the reaction time.
3.3.2. Optimization of NaCl concentration It is generally acknowledged that the concentration of salt plays an important role in DNA self-assembly reaction including four-way branch migration HCR. The phosphate groups on DNA chain are negatively charged, which leads to the intramolecular electrostatic repulsion in dsDNA and the decreased efficiency of DNA self-assembly reaction. After adding salt ions, the positive ions can block the negative charges of the phosphate groups and decrease the electrostatic repulsion in dsDNA, thereby improving the DNA self-assembly efficiency. Therefore, to obtain good performance of the sensing strategy, we optimized the concentrations of NaCl in SPSC buffer. As shown in Fig. S4A, the ratio of normalized fluorescent intensity was enhanced with the increase of NaCl concentration in the range of 0.4 M to 1 M. While, if NaCl concentration exceeded 1 M, the ratio of normalized fluorescent intensity was reduced. Thus, 1 M NaCl in SPSC buffer was selected in subsequent experiments.
3.4. Analytical performance of the developed sensing system 3.4.1. Sensitivity The sensitivity of the proposed sensing system was assessed by measuring the fluorescence responses with different concentrations of target DNA under the optimal conditions (shown in Fig. 2). As expected, the rising speed of the normalized fluorescent intensity gradually decreased with the increasing concentration of target DNA from 0 nM to 100 nM (Fig. 2A). This further proved that the sensing strategy was feasible, and it could be utilized to detect the target sequence. In the range of 0.2–60 nM, a good linear relationship was shown between the signal responses and target DNA concentrations (Fig. 2B). The regression equation for the method was y = 0.0082x+1.065 (R = 0.9965), with a detection limit of 0.067 nM (defined as 3σ/K, where σ is the standard deviation of the blank, and K is the slope of the calibration curve). Compared with the three-way HCR combined with FRET with a detection limit of 0.7 nM reported in the previous literature [29], the proposed strategy showed an order of magnitude improvement in sensitivity. This is probably because single stranded DNA always has secondary structure at or near room temperature, which can interfere with the hybridization reaction at the kinetic and equilibrium thermodynamics level. The double stranded DNA of A•R duplex discourages off-pathways that result in either kinetic traps or spurious interactions [34]. Moreover, there are more toeholds of the A•R, and more assembly sites are formed to open H1 and H2, so that it would be more effective to trigger four-way branch migration HCR.
3.3.3. Optimization of the reaction temperature The temperature would affect the binding efficiency and biosensor sensitivity since the free energy of combination gradually decreased with the temperature increasing. Therefore, we examined the responses from 4 ℃ to 50 ℃. As shown in Fig. S4B, the ratio of normalized fluorescent intensity reached a plateau at 30 ℃. The reason was that when the temperature was low, the free energy of combination was relatively high, and the hairpin probes were too stable to cross open, thus resulted in a low binding efficiency and weak fluorescent signal. However, the hairpin probes showed poor thermal stability with low free energy of combination at a relatively high temperature, which led to strong background signal. Therefore, 30 ℃ was selected as the reaction temperature. 3.3.4. Optimization of the reaction pH pH has an influence on hydrogen bonding, thereby affecting the stability of dsDNA and the performance of the sensing system. As shown in Fig. S5A, the ratio of normalized fluorescent intensity increased quickly with the increase of pH when the PH value was below 7.5, and after that it slowly decreased. The maximal fluorescent response was obtained at pH 7.5. Thus pH 7.5 was chosen in subsequent experiments. The reason might be that the acid or alkaline environment weakened 4
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Table 1 Recovery of Target Assay with Different Concentrations Spiked into Real Sample (n = 3). Sample
Spiked (nM)
Found (nM)
Recovery (%)
RSD (%)
Sample 1 Sample 2 Sample 3
1.00 20.00 60.00
1.09 18.82 59.54
109.93 94.10 99.23
5.51 5.58 7.15
*Sample 1, Sample 2 and Sample 3 were three parallel samples of a negative sample.
effect on the hybridization yield. 3.5. Application in real samples
Fig. 3. The net normalized fluorescent signals (△ Normalized intensity ratio) of the proposed sensor in this study and the sensor based on conventional HCR for different DNA oligonucleotides. (a) Single-base deleted target DNA; (b) Single-base inserted target DNA; (c) Single-base mismatched target DNA; (d) Specific DNA for Staphylococcus aureus; (e) Specific DNA for Listeria monocytogenes; (f) Specific DNA for Vibrio cholerae; (g) Target DNA. The concentration of R, H1 (H2) are both 100 nM. And the concentration of others are 60 nM, respectively.
To investigate the actual application potential of the strategy proposed in this work, the proposed strategy was further applied to the detection of PCR product of V. parahaemolyticus gyrB gene. The cultured V. parahaemolyticus was diluted to obtain serial concentrations from 0 to 106 CFU mL−1 and genomic DNA was extracted from each dilution according to the bacterial genomic DNA extraction kit. Then, the extracted DNA of V. parahaemolyticus was firstly denatured at 95 ℃ for 10 min, followed by 35 cycles of 95 ℃ for 30 s, 60 ℃ for 15 s, 72 ℃ for 10 s, and finally 5 min of extension. The PCR product and R were heated at 95 ℃ for 6 min, and immediately cooled down in ice bath to obtain the A•R duplex, and then detected by PAGE and the proposed biosensor, respectively. As shown in Fig. 4, the product band for 100 CFU mL-1 was hardly observed on polyacrylamide gel electrophoresis; while, the signal responses of the real samples distinguished from that of the blank sample were obvious, and as low as 10 CFU mL−1 V. parahaemolyticus could be detected in this sensing system. The sensitivity of the proposed strategy based on four-way branch migration HCR and FRET was compared with that of other strategies for the detection of V. parahaemolyticus or other bacterial DNA reported recently (Table S2). The results showed that the detection limit of this proposed method was better or comparable to some other strategies. This was attributed to the use of the cascade signal amplification strategy of four-way branch migration HCR and FRET. On the other hand, our sensing strategy for V. parahaemolyticus detection could greatly shorten the analysis time and simplify the detection procedure. Furthermore, the recovery experiments were performed by standard addition method. As depicted in Table 1, the recoveries were in the range of 94.10%–109.9%, which illustrated that the newly developed sensor could detect V. parahaemolyticus with high accuracy. Therefore, the results mentioned above elucidated that the proposed sensing
3.4.2. Selectivity The specificity of the proposed strategy for V. parahaemolyticus detection was also evaluated. We detected the target sequence, the specific sequences of staphylococcus aureus, listeria monocytogenes and Vibrio cholerae, as well as mutant target molecules including insertion, deletion and mismatch. As shown in Fig. 3, a very little response was obtained for other bacterial sequences and the mutant target molecules, which was significantly lower than that of the target sequence. The results demonstrated that the strategy we put forward possessed high specificity in the discrimination of target sequence from other sequences. At the same time, a FRET biosensor based on conventional HCR for V. parahaemolyticus detection was designed, and its specificity was compared with that of this proposed biosensor. The results (shown in Fig. 3) indicated that both biosensors based on four-way branch migration HCR and the conventional HCR could specifically detect V. parahaemolyticus, but for mutant target molecules of mismatch, insertion or deletion, the specificity of the former biosensor was better than the latter. It was attributed to the fact that target DNA firstly bound with the designed assistant R by complementary base pairing to form double-stranded structures, and then reacted with H1 to initiate the four-way branch migration reaction; while, the base mismatches could cause slight thermodynamic changes that had disproportionately large
Fig. 4. (A) The gel electrophoresis images of the PCR products for different concentrations of Vibrio parahaemolyticus. (1–6: serial dilution of Vibrio parahaemolyticus, 101–106 CFU mL−1, 7: marker). (B) The experimental results of the DNA biosensor. 5
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strategy could be potentially applied to the detection of V. parahaemolyticus in real samples.
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4. Conclusions [13]
In conclusion, a novel enzyme-free signal amplification strategy based on FRET and four-way branch migration HCR was proposed and successfully applied to the specific detection of V. parahaemolyticus. The target DNA firstly bound to the assistant strand, and then triggered four-way branch migration HCR to form long nicked dsDNA with a large number of FRET signals, thus powerfully enhanced the detection sensitivity. It integrated the advantageous features of four-way branch migration HCR with FRET, and could work as a versatile sensor for V. parahaemolyticus detection with simple operation and short analysis time. Moreover, this method showed an excellent detection capability, whose specificity and sensitivity were even better than some reported amplification methods. Meanwhile, four-way branch migration HCR dramatically improved the specificity of the sensor due to the decreased base mismatch possibility. Further research should focus on the construction of aptasensors for intact target bacterial cells by using the proposed strategy to reduce the whole detection time, and on the extension of application scope to other pathogenic bacteria and viruses. It has great potential for the application of pathogens detection in clinical infection and food safety.
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Acknowledgements
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This work was supported by China Postdoctoral Science Foundation (2017T100703), Project of Sichuan Provincial Science and Technology Department (2018HH0147), and Technology Innovation Research and Development Project of Chengdu Science and Technology Bureau (2018-YF05-01201-SN).
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Appendix A. Supplementary data
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Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.05.054.
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Dongxia Ren graduated from West China School of Public Health, Sichuan University and obtained the BS degree in 2016. Currently, she is a postgraduate student in West China School of Public Health, Sichuan University. Her research interest is focused on the novel biosensors based on four-way branch migration HCR. Chengjun Sun is a professor in West China School of Public Health, Sichuan University, Chengdu, PR China. He is mainly interested in the research of gas chromatography, high performance liquid chromatography, capillary electrophoresis and their application in food analysis, as well as study of adulterants in various foods, especially in functional foods. Zhijun Huang received his BS degree in biotechnology from Guangxi University, and received his MS degree in microbiology from Graduate University of Chinese Academy of Sciences. Now, he is a PhD student in the Research Center of Analytical Instrumentation,
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University in 2014. Presently, she is a PhD student in West China School of Public Health, Sichuan University after two year’s MS course. She is focused on the development of new biosensors based on fiber optic surface plasmon resonance (FOSPR) and their application in food analysis in West China School of Public Health, Sichuan University, Chengdu, PR China.
college of life science, Sichuan University. His current research interests focus on the development of novel biosensors. Zewei Luo received his BS degree in bioengineering from Huaihua University, Huaihua, China, in 2013. He presently is a PhD student in the Research Center of Analytical Instrumentation, college of life science, Sichuan University after three year’s MS course. His current research interest is focused on the development of new biosensors based on fiber optic surface plasmon resonance (FOSPR) and their application in detection of small molecules and cancer cells.
Yongxin Li is an associate professor in West China School of Public Health, Sichuan University, China. She is mainly engaged in the study of capillary electrophoresis, microfluidic chip and their application in public health, particularly in foodborne-pathogenic bacteria, viruses and chemical detection, and the development of novel biosensors for various applications.
Chen Zhou received her BS degree from West China School of Public Health, Sichuan
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