CN114414636A

CN114414636A - Electrochemical biosensor composition, working solution, electrochemical biosensor and application thereof

Info

Publication number: CN114414636A
Application number: CN202111671238.3A
Authority: CN
Inventors: 万家余; 卜胜君; 刘文森; 李忠义; 郝镯
Original assignee: Military Veterinary Research Institute Academy Of Military Medical Sciences
Current assignee: Military Veterinary Research Institute Academy Of Military Medical Sciences
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2022-04-29

Abstract

The invention relates to the field of electrochemical biosensors, in particular to a CPf1 CRISPR system from a bacterium in the family Musaceae, which is combined with a hybrid chain reaction to develop an electrochemical biosensor for detecting pathogenic bacteria salmonella typhimurium. The autonomous cross-opening of the functional DNA hairpin structure of the HCR creates a double-stranded DNA strand of polymer consisting of numerous single-stranded DNAs that initiate the trans-cleavage activity of CRISPR-Cas12a to indiscriminately cleave the DNA hairpin loop of the MB modified on the gold electrode surface. This process will result in a change in the electron transfer of the electrochemical tag. The polymer double stranded DNA of HCR is immobilized on DBs by Salmonella typhimurium aptamers. The established method can selectively and sensitively quantify the Salmonella typhimurium in the sample, and the detection limit is 20 CFU/mL.

Description

Electrochemical biosensor composition, working solution, electrochemical biosensor and application thereof

Technical Field

The invention relates to the field of electrochemical biosensors, in particular to an electrochemical biosensor composition, a working solution, an electrochemical biosensor and application thereof.

Background

Food safety issues due to food-borne pathogens are of increasing concern worldwide. Of these, Salmonella typhimurium (Salmonella typhimurium) is one of the major food-borne pathogens, which threatens human health mainly by eating contaminated food. The existing gold standard method for detecting salmonella typhimurium mainly comprises a culture method, an enzyme-linked immunosorbent assay (ELISA), a Polymerase Chain Reaction (PCR) and the like. However, these methods have a number of disadvantages. For example, culturing is time consuming, typically requiring 2-3 days. PCR typically requires complex DNA extraction procedures. ELISA lacks sufficient sensitivity. Moreover, these methods require highly trained technicians and expensive equipment to perform. Therefore, there is a need to develop a sensitive and simple method for detecting salmonella typhimurium to ensure food safety.

The CRISPR-Cas12a system is commonly used as an RNA-guided endonuclease. Cas12a with trans activity triggered by dsDNA or ssDNA can cleave nearby non-specific ssDNA. Based on non-specific cleavage of the labeled nucleic acid reporter, the CRISPR-Cas12a system, along with the trans-activity, can detect the nucleic acid target. For non-nucleic acid detection, a non-nucleic acid target is transduced into a Cas12 a-gRNA binary complex of DNA or RNA. The excellent properties of CRISPR-Cas12a are of great interest in the development of next generation biosensors for diagnosis, including severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), influenza a and b viruses, african swine fever virus. To detect low abundance targets, it is often necessary to combine the side effects of Cas12a with isothermal amplification using various nucleic acid amplification strategies such as Recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling circle amplification, and catalytic hairpin assembly, creating a rapid and specific detection platform.

The most widely used hybrid strand reaction (HCR) is a simple, enzyme-free, robust and efficient isothermal amplification of nucleic acids; long, linear duplex concatemers are formed by the cross-opening and self-assembly of two DNA hairpins initiated by the target probe. In addition, the amplification of the non-target HCR is a probe amplification technology; cross contamination and false positive results often occurring in LAMP, RPA and PCR are effectively reduced. To date, HCRs have been explored for the detection of various targets, such as nucleic acids, proteins, and pathogens. The use of HCR in biosensors has undergone significant development over the past few decades due to the specific use of the formed duplex in biosensors. Binding of Cas12a to HCR provides a method of detecting a target. Xing et al are based on HCR and CRISPR-Cas12 a. An aptamer-HCR-CRISPR-Cas 12a fluorescence readout method was developed for direct detection of tumor-derived extracellular vesicle proteins. Kachwala combines HCR with CRISPR-Cas12a and develops a detection method based on gel electrophoresis. Conserved ssDNA and dsDNA regions of the tobacco circovirus and hepatitis B virus genomes were detected, respectively. Cas12 can recognize the target dsDNA and initiate the trans-cleavage event under the guidance of crRNA, but it requires a Protospacer Adjacent Motif (PAM) sequence adjacent to the target dsDNA, which may limit its broader HCR application.

Disclosure of Invention

In view of this, the present invention labels the 3' ends of the two hairpins of the HCR, which forms a polymeric dsDNA strand carrying multiple recognition target sequences that activate the Cas12-crRNA complex.

In order to achieve the above object, the present invention provides the following technical solutions:

in a first aspect, the invention provides an electrochemical biosensing composition comprising streptavidin DBs, an aptamer, a linker probe, H1 hairpin of HCR, H2 hairpin of HCR, Cas12a, and crRNA.

In a second aspect, the invention also provides the application of the electrochemical biosensor composition in preparing a working solution of an electrochemical biosensor or an electrochemical biosensor.

In a third aspect, the invention also provides a working solution of the electrochemical biosensor, which comprises the electrochemical biosensor composition and acceptable auxiliary materials or auxiliary agents.

In a fourth aspect, the invention also provides application of the working solution of the electrochemical biosensor in preparation of the electrochemical biosensor or a kit.

In a fifth aspect, the present invention also provides an electrochemical biosensor, comprising the electrochemical biosensor composition or a working fluid of the electrochemical biosensor, and an electrode system.

In some embodiments of the invention, the electrode system comprises a reference electrode, an auxiliary electrode, and a working electrode;

the reference electrode comprises an Ag/AgCl electrode; and/or

The auxiliary electrode comprises a platinum wire; and/or

The working electrode comprises a bare gold electrode GE modified by an MB probe.

In a sixth aspect, the invention further provides an application of the electrochemical biosensor in preparing a device for detecting pathogenic bacteria or food safety detection.

In the present invention, HCR-based CRISPR-Cas12a was introduced into an electrochemical biosensor platform for sensitive and specific detection of salmonella typhimurium (as shown in figure 1). Thus, a practical or deployable point-of-care detection system is provided. Non-specific ssDNA reporter [ Methylene Blue (MB) tag modified on hairpin dna (hpdna) ], linked by disulfide bonding at the electrode sensor surface and using the MB electrochemical tag moiety for signal transduction to obtain current signals. Thus, the electron transfer process between the Au electrode and the redox active species on the ssDNA can be initiated and transduced electrochemically. The autonomous cross-opening of the functional DNA hairpin structure of HCR is used to generate a DNA double helix structure composed of numerous single-stranded DNAs. These single-stranded DNAs can enable the formation of Cas12 a-crRNA-target DNA ternary complexes, thereby initiating the trans-cleavage activity of CRISPR-Cas12 a. CRISPR-Cas12a cleaved random single stranded DNA labeling MB electrochemical tags at the electrode surface (MB reporter) and resulted in changes in electrochemical tag electron transfer. The polymer double-stranded DNA of HCR is immobilized on DBs by a linker probe. Initially, the linker probe is locked by the salmonella typhimurium aptamer. The aptamer is a single-stranded oligonucleotide sequence of a target molecule, and can be efficiently and specifically bound to the target molecule. Aptamer shedding, HCR binding site exposure. In the presence of the target, the polymer double stranded DNA of HCR is ligated through the linker of DBs, activating Cas12a trans-cleavage activity and removing the MB reporter from the Gold Electrode (GE) surface, thereby significantly reducing the MB transduction signal. When the target was absent, no Cas12a trans-cleavage activity was observed and the polymer double stranded DNA of the HCR was released from the DB, thereby retaining the MB probe on the GE surface. The electrochemical signal output based on the presence/absence of Salmonella typhimurium is shown in FIG. 1. Based on HCR and CRISPR-Cas12a, we developed a universal electrochemical biosensor for detection of pathogenic bacteria.

In some embodiments of the invention, the pathogen includes, but is not limited to, salmonella typhimurium.

In a seventh aspect, the invention further provides a method for detecting pathogenic bacteria, wherein a sample to be detected is taken to be mixed with the electrochemical biosensor composition or the working solution of the electrochemical biosensor, and the mixture is placed on a working electrode of the electrochemical biosensor for detection.

In some embodiments of the invention, the optimal conditions for the CRISPR-Cas12a system are 0.3 μ M Cas12a and 1 μ M crRNA.

In some embodiments of the invention, the optimal digestion time of the Lba Cas12a is 60 min.

In some embodiments of the invention, the optimal concentration of aptamer is 4 μ M.

In some embodiments of the invention, the HCR incubation time is 60 minutes.

In some embodiments of the invention, DB is used in an amount of 5 μ L.

In some embodiments of the invention, the aptamer is incubated at a temperature of 37 ℃.

The invention successfully constructs a rapid and sensitive electrochemical biosensor based on pathogenic bacterium aptamers Cas12a-crRNA and HCR, and the electrochemical biosensor is used for detecting pathogenic bacteria. The result shows that the method realizes high specificity and extremely high sensitivity among different bacterial strains. LOD as low as 20 CFU/mL. By varying the aptamer, the method can be used with other contemplated bacteria. The established method can be applied to the field diagnosis of pathogenic bacteria using specific aptamers and introduction of isothermal amplification HCR method and Cas12 a-crRNA.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a schematic diagram of an electrochemical biosensor for detecting Salmonella typhimurium;

FIG. 2 shows a polyacrylamide gel electrophoresis image; wherein, M: 2000bp DNAmarker; the concentrations of H1 and H2 in lanes 1-9 were both 1. mu.M, and the concentrations of the starting sequence T were 0.01. mu.M, 0.03. mu.M, 0.05. mu.M, 0.1. mu.M, 0.3. mu.M, 0.5. mu.M, 0.7. mu.M, 0.9. mu.M, respectively;

FIG. 3 shows EIS (A) and CV (B) response plots for stepwise modification of the electrodes; wherein, (a) a bare Au electrode, (b) an MB-DNA/MCH/target modified by the electrode, (c) an MB-DNA modified by the electrode, and (d) an MB-DNA/MCH modified by the electrode;

inset in the upper right corner of fig. 3A: a Randles equivalent circuit for fitting impedance data; rct, CPE, Zw, and Rs represent charge transfer resistance, constant phase capacitance element, Warburg resistance, and diffusion resistance of electrolyte, respectively; using a solution containing 5mM [ Fe (CN)₆]^3-/4-And 0.1M KCl in buffer for EIS and CV; the EIS measurement frequency range is 0.1-100kHz, and the amplitude is 5 mV; the potential range of CV measurement is-0.2V to 0.6V, and the scanning rate is 50 mV/s;

FIG. 4 shows the actual curve (red) and the fitted curve (green) of curves a to d in FIG. 3A;

FIG. 5 shows DPV responses corresponding to (a) GE/MB/MCH/Cas12a/crRNA, (b) GE/MB/MCH/target/Cas 12a, (c) GE/MB/MCH/target/crRNA, and (d) GE/MB/MCH/target/Cas 12 a/crRNA;

fig. 6 shows optimization of CRISPR-Cas12a system conditions; wherein, (a) crRNA concentration; (B) cas12a concentration; (C) incubation time of Cas12 a;

FIG. 7 shows the optimization experiment conditions; wherein, (A) AP concentration; (B) dynabeads (dbs) dose; (C) HCR incubation time; (D) experiment incubation temperature;

FIG. 8 shows sensitivity analysis of targets; wherein FIG. 8A shows a DPV plot at different Salmonella typhimurium concentrations; FIG. 8B shows the linear interval of DPV current and log of Salmonella typhimurium; PBS buffer (1mM) was used as a negative control; error bars represent the standard deviation of at least three replicates;

FIG. 9 shows a specificity assay; wherein, FIG. 9A shows a histogram, FIG. 9B shows an electrochemical DPV response curve, the target bacteria concentration is 10⁸CFU/mL; PBS buffer (1mM) was used as a negative control.

Detailed Description

The invention discloses an electrochemical biosensor composition, a working solution, an electrochemical biosensor and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

In the present invention, HCR-based CRISPR-Cas12a was introduced into an electrochemical biosensor platform for sensitive and specific detection of salmonella typhimurium (as shown in figure 1). Thus, a practical or deployable point-of-care detection system is provided. Non-specific ssDNA reporter [ Methylene Blue (MB) tag modified on hairpin dna (hpdna) ] signal was acquired using MB electrochemical tag for signal transduction and thiol moieties attached to the sensor surface. Thus, the electron transfer process between the Au electrode and the redox active species on the ssDNA can be initiated and transduced electrochemically. The autonomous cross-opening of the functional DNA hairpin structure of HCR is used to generate a DNA double helix composed of numerous single-stranded DNAs. These single-stranded DNAs can enable the formation of Cas12 a-crRNA-target DNA ternary complexes, thereby initiating the trans-cleavage activity of CRISPR-Cas12 a. CRISPR-Cas12a cleaved random single stranded DNA labeling MB electrochemical tags at the electrode surface (MB reporter) and resulted in changes in electrochemical tag electron transfer. The polymer double-stranded DNA of HCR is immobilized on DBs by a linker probe. Initially, the linker probe is locked by a Salmonella typhimurium aptamer. Aptamers are single-stranded oligonucleotide sequences of target molecules that can bind to the target molecules with high efficiency and specificity. Aptamer shedding, HCR binding site exposure. In the presence of the target, the polymer double stranded DNA of HCR was ligated by the linker of DBs, activating Cas12a trans-cleavage activity and removing the MB reporter from the Gold Electrode (GE) surface, thereby significantly reducing MB transduction signal. When the target was absent, no Cas12a trans-cleavage activity was observed and the polymer double stranded DNA of the HCR was released from the DB, thereby retaining the MB probe on the GE surface. The electrochemical signal output based on the presence/absence of Salmonella typhimurium is shown in FIG. 1. Based on HCR and CRISPR-Cas12a, we developed a universal electrochemical biosensor for detection of pathogenic bacteria.

Apparatus and materials

EnGen Lba Cas12a and 10 XNE buffer 2.1(0.5M NaCl, 0.1M Tris-HCl, 0.1M MgCl)₂And 1mg/mL BSA, pH 7.9) was supplied by New England Biolabs (Mass.). Tris (2-carboxythroughout) -phosphine hydrochloride (TCEP) and all the oligonucleotides used in this study (table 1) were purified and synthesized by Sangon Biotechnology co.ltd (shanghai, china). DNase/RNase-free deionized water and RNase inhibitor were purchased from Tiangen Biotechnology (Beijing) Ltd. Dynabeads (DBs) M-270Streptavidin and SYBR Safe DNA gel dyes were purchased from Saimer Feishel technologies, Inc. (Norway). Furthermore, 20-bp DNA Ladder (Dye Plus) was purchased from Takara Biotech (Chinese Union). Additionally, 6-mercapto-1-hexanol (MCH) and 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid were purchased from Sigma-Aldrich (st. louis, MO, USA). The aptamer is ssDNA and can bind with high affinity to Salmonella typhimurium. Salmonella typhimurium (CICC 21484), Escherichia coli (CICC 10389), Listeria monocytogenes (CICC 21633), Staphylococcus aureus (CICC 21600), and Vibrio paraacidophilus (CICC21617) strains were purchased from the China center for biological Collection, China. All reagents were diluted with DNase/RNase free deionized water throughout the study.

TABLE 1 oligonucleotide sequences

The bold sequence represents the T7 promoter. The underlined sequence represents the activation sequence of the Lba Cas12 a. Initiator (Trigger): an initiator that initiates hybridization of H1 and H2 to produce a DNA duplex of HCR; h1: a hairpin probe for HCR; h2: another hairpin probe of HCR; linker (Linker): the probe is used for immobilizing HCR products on magnetic beads; crRNA: cas12a leader sequence, which can recognize the target dsDNA and initiate the trans-cleavage event; aptamer: the single-stranded oligonucleotide sequence of the target molecule can efficiently and specifically combine with the salmonella typhimurium; MB probe: an electrochemical tag for signal transduction.

In the present invention, an electrochemical biosensor for detecting the pathogenic bacterium salmonella typhimurium was developed in combination with a Hybrid Chain Reaction (HCR) in a CRISPR system where the Lba Cas12a (Cpf1) is derived from a bacterium of the family lachnospiraceae. The autonomous cross-opening of the functional DNA hairpin structure of the HCR creates a double-stranded DNA strand of polymer consisting of numerous single-stranded DNAs that initiate the trans-cleavage activity of CRISPR-Cas12a to indiscriminately cleave the DNA hairpin loop of the MB modified on the gold electrode surface. This process will result in a change in the electron transfer of the electrochemical tag. The polymer double stranded DNA of HCR is immobilized on Dynabeads (DBs) by Salmonella typhimurium aptamers. The established method can selectively and sensitively quantify the Salmonella typhimurium in the sample, and the detection limit is 20 CFU/mL. Our studies provide new insights for exploring pathogenic bacteria detection methods based on CRISPR-Cas12a and HCR.

In the electrochemical biosensor composition, the working solution, the electrochemical biosensor and the application thereof, all the raw materials and reagents are commercially available.

The invention is further illustrated by the following examples:

EXAMPLE 1 preparation of biosensor

Preparation of MB probe-modified biosensing electrode

Immersing bare Gold Electrode (GE) in piranha solution (H)₂SO₄/H₂O₂3:1) for 20 minutes to remove impurities and then scrubbed with absorbent cotton balls soaked in deionized water. The electrode was polished with 0.05 μ M alumina powder to a smooth surface and then scrubbed with a cotton wool swab. Thiolated MB probe was dissolved in 1mM Phosphate Buffered Saline (PBS) solution (containing 14mM NaCl, 0.3mM KCl, and 10mM TCEP, pH 7.4). Then, 10 μ L of 0.3 μ M MB probe was added to the pre-treated electrode surface and incubated overnight at room temperature. In addition, 10. mu.L MCH (1. mu.M) was added dropwise to the GE surface and incubated at 37 ℃ for 1 hour. Finally, the unbound material on the electrode surface was removed with ultrapure water for electrochemical measurements and subsequent experiments.

Amplification of ssDNA by HCR

Corresponding stock solutions (100. mu.M) were treated with HEPES buffer (20mM HEPES, 8mM MgCl)₂.6H₂O, 20mM NaCl, 10mM KCL, pH 7.4) to prepare the desired concentration of oligonucleotide. For amplified ssDNA analysis, different concentrations of T were incubated with H1 (1. mu.M) and H2 (1. mu.M) in HEPES buffer (30. mu.L) for 1 hour at 37 ℃ and then stored at 4 ℃ for later use.

Target Capture procedure

(1) And 4 mu L of modified streptavidin DBs are taken and washed by deionized water for 3 times and then added into a 200 mu L PCR tube. Next, the treated L-P solution [ probe linker (0.5. mu.M) and aptamer (4. mu.M) mixed in equal volumes ] was incubated at 90 ℃ for 3 minutes, immediately transferred to an ice bath and incubated at 37 ℃ for 60 minutes.

(2) The probe not bound to DBs was washed with HEPES buffer pH 7.4, and 50. mu.L of Salmonella typhimurium and 50. mu.L of HEPES buffer were added as negative controls, and incubated at 37 ℃ for 60 minutes.

(3) DBs were washed with HEPES buffer pH 7.4, pretreated HCR product was added and incubated at 37 ℃ for 60 minutes.

(4) DBs were washed again with HEPES buffer pH 7.4, resuspended in 4. mu.L of HEPES buffer, and stored at 4 ℃ until use.

Preparation steps of CRISPR-Cas12a restriction enzyme system

A mixed solution containing 4 μ L Cas12a (0.3 μ M), 4 μ L crRNA (1 μ M), 1 μ L RNase inhibitor (10U) and 4 μ L of the solution resuspended in the last step 2.4- (4) was left at room temperature for 13 minutes to bind the Cas12a protein, crRNA and ssDNA fragments on the HCR product to enhance activation of the CRISPR-Cas12a digestive system.

Electrochemical measurement procedure

Electrochemical measurements were performed on a CHI660E electrochemical workstation (shanghai chenhua instruments ltd, china), including Electrochemical Impedance Spectroscopy (EIS), Cyclic Voltammetry (CV), and Differential Pulse Voltammetry (DPV). The electrochemical workstation adopts a classical three-electrode system, a platinum wire as an auxiliary electrode, GE as a working electrode and an Ag/AgCl electrode as a reference electrode. EIS and CV measurements in the presence of 5mM [ Fe (CN)₆]^3-/4-And 0.1M KCl in buffer. The frequency range of EIS measurement is 0.1-100kHz, and the amplitude is 5 mV. The potential range for CV measurement was-0.2V to 0.6V, and the scan rate was 50 mV/s. DPV measurements were made in 2 XPBS solution (2.5mM MgCl)₂50mM NaCl, pH 7.4); the amplitude and pulse period were 50mV and 0.5s, respectively.

Polyacrylamide gel electrophoresis procedure

Formation of HCR product was checked using polyacrylamide gel electrophoresis (PAGE) using a 12% polyacrylamide gel. Electrophoresis was performed in 1 XTBE buffer (89mM Tris, 89mM boric acid and 2mM EDTA, pH 8.3) at a constant voltage of 200V for 45 minutes before staining with SYBR Safe DNA gel dye. Finally, visual analysis was performed in a gel imaging system.

EXAMPLE 2 actual sample determination

Different Salmonella typhimurium densities (10)⁴、10⁵And 10⁶CFU/mL) was added to the sterilized milk sample for recovery analysis. HEPES buffer was used as negative control.

Effect example 1 characterization of HCR formation

The reaction efficiency of HCR is closely related to the sensitivity of the assay. Thus, PAGE assays were performed to verify the formation of HCR product (figure 2). The concentration of initiator is related to the average molecular weight of the polymer produced by the HCR. Too much initiator (Trigger) may result in hybridization between H1 and H2, which may not form a sufficiently long DNA polymer chain. Therefore, different concentrations of trigger are used to optimize the optimal performance of the HCR system. As shown in fig. 2, line 1 has little high band polymer when the Trigger concentration is low. This finding indicates that no HCR occurs due to the lack of Trigger. As the Trigger concentration was increased from 0.03 to 0.3. mu.M, the efficiency of HCR increased and the number of hairpin probes at residues H1 and H2 decreased. When the concentration of Trigger was further increased from 0.3 to 1 μ M, the molecular weight of the HCR product decreased and the polymer began to disperse. This may be detrimental to efficient signal amplification. Therefore, a Trigger having a Trigger concentration of 0.3. mu.M was determined for the following experiment.

Effect example 2 electrochemical characterization of biosensor

Use 5mM [ Fe (CN)₆]^3-/4-Solutions EIS and CV were measured as electrochemical probes containing 100mM KCl to study biosensor configuration. As shown in fig. 3 and table 2, the response of the charge transfer resistance (Rct) was significantly increased (curve a, Rct ═ 200.7 Ω) (GE/MB, curve c, Rct ═ 20720 Ω) after modification with MB, and the conductivity of the modified electrode was decreased. It demonstrates the successful modification of the MB probe on GE by an Au-S covalent bond. After MCH was fixed on the GE/MB electrode, the resistance increased slightly (curve d, Rct 23710 Ω). This indicates that MCH blocks unbound sites of the electrode and reduces electron transfer capability. However, the presence of the target initiated the HCR/CRISPR approach, leading to cleavage of the MB probe by activating the Cas12a system. Therefore, after the MB probe on the GE was withdrawn, the resistance was significantly decreased (curve b, Rct 5740. omega.). At the same time, the CV measurement is consistent with the EIS measurement. The CV and EIS results show that this technique has great potential for monitoring the electrode surface at every step of biosensor fabrication.

TABLE 2 fitting values of electrochemical impedance spectroscopy equivalent circuit elements

And (3) Rct: a charge transfer resistance; rs: ohmic resistance of the electrolyte; CPE: constant phase element (Yo and n represent non-zero real part and constant phase element, respectively); zw: warburg impedance.

Effect example 3 feasibility of electrochemical biosensor

DPV signals were measured to verify the feasibility of the HCR-CRISPR/Cas12a method for detecting Salmonella typhimurium. Since the HCR-CRISPR/Cas12a method was developed based on the Cas12a system, the DPV current signal was used to estimate the current signal. Upon assembly of the HCR product in the presence of salmonella typhimurium of interest, the CRISPR-Cas12a system is directly activated, resulting in MB probe excision on the electrode, with a significant decrease in current signal (fig. 5, curve d.) the trans-cleavage activity of Cas12a is inhibitory in the absence of any elements (e.g., crRNA, target and Cas12a) (fig. 5, curves a, b and c). Thus, the MB probe on the GE was retained and the DPV current signal did not change significantly.

Effect example 4 optimization of experiment conditions

In HCR-CRISPR, the efficient trans-cleavage activity of the CRISPR-Cas12a system plays an important role, and the sensitivity of the whole experiment is influenced. Therefore, we first optimized experimental conditions, such as Cas12a concentration and amount of crRNA. As shown in fig. 6, when the Cas12a concentration is 0.3 μ M and the crRNA concentration is 1 μ M, a higher rate of enzymatic cleavage reaction is obtained (fig. 6A and 6B). Therefore, 0.3 μ M Cas12a and 1 μ McrRNA were chosen as the optimal conditions for the CRISPR-Cas12a system. In addition, the current signal of DPV was used to monitor the restriction time of the Lba Cas12 a. As shown in fig. 6C, as the digestion time of the Lba Cas12a increased from 5 minutes to 60 minutes, the current signal increased and then became stable. Therefore, 60min was selected as the optimal digestion time for LbaCas12a for subsequent experiments.

We also optimized some other important reaction conditions to achieve better biosensor performance in terms of aptamer concentration, HCR incubation time and DB usage (figure 7). The amount of aptamer is important to the overall experiment as an important component of identifying the target. High concentrations of aptamers cause steric hindrance, while low concentrations affect sensitivity. As shown in FIG. 7A, the peak current increases from 2 μ M to 4 μ M and then begins to decrease. Therefore, an aptamer concentration of 4 μ M was selected as the optimal concentration for subsequent experiments. The incubation time for detection of HCR was then 5 to 75 minutes. As shown in fig. 7D, the current signal value gradually increased as the HCR reaction time increased from 5 minutes to 30 minutes, and then reached a maximum value within 60 minutes. Therefore, an HCR incubation time of 60 minutes was chosen for the experiments. After that, we optimize the amount of DB. When DB was 5 μ L, the optimum DPV signal was monitored (fig. 7B). Therefore, 5 μ L DB was applied throughout the subsequent experiments. Finally, the influence of temperature as a key factor influencing the activity of the aptamer on the experiment is researched; the results are shown in FIG. 7C. The reaction temperature was increased in the range of 15-37 deg.C, reached a maximum at 37 deg.C, and then decreased as the incubation temperature increased. This result indicates that high temperature affects the binding efficiency of aptamers. Thus, an incubation temperature of 37 ℃ was used in this experiment.

Effect example 5 sensitivity

The sensitivity of the biosensor was studied by measuring the DPV response of ten-fold serial dilutions of salmonella typhimurium solutions under optimal optimized conditions. As shown in fig. 8, by a power supply at 10-10⁸Increasing the target bacterial concentration in the CFU/mL range achieves a gradual decrease in DPV response. The target bacteria of Salmonella typhimurium can be quantified in the concentration range of 104-108 CFU/mL. The correlation equation is Y-0.1027X +0.03242, R²0.947, where Y is the DPV current signal and X is the number of Salmonella typhimurium. The limit of detection was calculated from the 3 theta/slope (theta is three times the standard deviation) to be 20 CFU/mL. Compared with other reported CRISPR and electrochemical bacterial detection methods, as shown in table 3. The results show that the limit of detection (LOD) is comparable or better than most other detection methods.

TABLE 3

10^4	10^5	10^6	10^7	10^8
					0.4842550	0.4722820	0.5936760	0.7105950	0.8753647
0.5428390	0.5226520	0.6022490	0.7675610	0.9083730
					0.4256710	0.5730220	0.5851020	0.8245260	0.8423564

Effect example 6 specificity

To study specificity, we evaluated the detection method for different food-borne pathogens; the results are shown as the DPV response in fig. 9. Other non-target bacteria, including negative controls such as E.coli, Listeria monocytogenes, Staphylococcus aureus, and Vibrio parahaemolyticus, have low current responses, demonstrating the specificity of this method of differentiation.

TABLE 4

Effect example 7 analysis of actual samples

According to the Chinese national Standard protocol (GB 4789), the culture of Salmonella typhimurium was diluted to 10⁴、10⁵And 10⁶CFU/mL, followed by the addition of a 10-fold diluted milk sample (25mL milk was mixed with 225mL PBS buffer for recovery measurements. As shown in Table 5, the recovery of the milk sample was 97.5% -114.7% with a relative standard deviation of 1.7% to 16.8%.

TABLE 5 detection of Salmonella typhimurium in milk samples using the developed method

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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Claims

1. An electrochemical biosensing composition comprising streptavidin DBs, aptamers, linker probes, H1 hairpin of HCR, H2 hairpin of HCR, Cas12a, and crRNA.

2. Use of the electrochemical biosensor composition according to claim 1 for preparing a working fluid for electrochemical biosensors or electrochemical biosensors.

3. The working solution of an electrochemical biosensor, comprising the electrochemical biosensor composition according to claim 1 and an acceptable adjuvant or auxiliary agent.

4. Use of a working solution of an electrochemical biosensor according to claim 3 for the preparation of an electrochemical biosensor or kit.

5. An electrochemical biosensor comprising the electrochemical biosensor composition according to claim 1 or the working solution of the electrochemical biosensor according to claim 3, and an electrode system.

6. The electrochemical biosensor of claim 5, wherein the electrode system comprises a reference electrode, an auxiliary electrode, and a working electrode;

the reference electrode comprises an Ag/AgCl electrode; and/or

The auxiliary electrode comprises a platinum wire; and/or

7. Use of an electrochemical biosensor as claimed in claim 5 or 6 in the manufacture of a device for detecting pathogenic bacteria or food safety.

8. The use of claim 7, wherein the pathogenic bacteria include, but are not limited to, Salmonella typhimurium.

9. A method for detecting pathogenic bacteria, comprising the steps of mixing a sample to be detected with the electrochemical biosensor composition according to claim 1 or the working solution of the electrochemical biosensor according to claim 3, and placing the mixture on the working electrode of the electrochemical biosensor according to claim 5 or 6 for detection.

10. The assay of claim 9 wherein the pathogenic bacteria include but are not limited to salmonella typhimurium.