An electrochemical sensor based on synergistic mediation of tdt enzyme and cbago and application thereof in detection of cobra venom protein

Abstract

The application discloses an electrochemical sensor based on TdT enzyme and CbAgo synergistically mediated and application thereof in detection of cobra venom protein, and belongs to the technical field of electrochemical biosensors.The application constructs a protein-nucleic acid conversion technology mediated by TdT enzyme and CbAgo protein through magnetic bead enrichment, CbAgo protein cleavage and TdT extension reaction, converts the detection target from cobra venom protein into a nucleic acid product, and then utilizes the nucleic acid product to activate a Cas12a system to cleave a probe P combined on a gold electrode, and finally converts into an electrochemical signal, so that efficient and accurate detection of the cobra venom protein is realized.The electrochemical biosensor has high sensitivity and good specificity, and has extremely high application value in accurate identification and quantitative analysis of the cobra venom protein.

Description

Technical Field

[0001] This invention relates to the field of electrochemical biosensor technology, and in particular to an electrochemical sensor based on the synergistic mediation of TdT enzyme and CbAgo and its application in the detection of cobra venom proteins. Background Technology

[0002] In the fields of snake venom biology and clinical medicine, cobra venom is complex, containing various protein components such as neurotoxins, cytotoxins, and cardiotoxins. Its toxicological mechanisms vary, leading to severe symptoms and high mortality rates. Currently, clinical treatment for cobra bites heavily relies on the timely administration of specific antivenom serum. However, existing diagnostic methods, primarily based on clinical symptoms and limited immunological testing, have significant technical limitations.

[0003] Therefore, developing a technology that can detect and identify cobra venom proteins with high specificity, high sensitivity, and rapid and convenient detection is of urgent clinical need and significant industrial value for achieving accurate medical diagnosis of cobra bites, guiding the rational use of effective antitoxin serum, improving the success rate of critical poisoning treatment, and promoting innovative biomedical research and development based on cobra venom proteins. Summary of the Invention

[0004] The purpose of this invention is to provide an electrochemical sensor based on the synergistic mediation of TdT enzyme and CbAgo and its application in the detection of cobra venom proteins, thereby addressing the problems existing in the prior art. This invention constructs a protein-nucleic acid conversion technology synergistically mediated by TdT enzyme and CbAgo protein through magnetic bead enrichment, CbAgo protein cleavage, and TdT extension reaction. This converts the target cobra venom protein into a nucleic acid product, which is then used to activate the Cas12a system to cleave the probe P bound to the gold electrode, ultimately converting it into an electrochemical signal. This achieves efficient and accurate detection of cobra venom proteins.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides an electrochemical sensor based on the synergistic mediation of TdT enzyme and CbAgo, the electrochemical sensor comprising a cobra venom protein enrichment system, a CbAgo protein reaction system, a TdT reaction system, a Cas12a reaction system, and a working electrode;

[0007] The cobra venom protein enrichment system includes magnetic beads coupled with a primary antibody against cobra venom protein and probe 1 coupled with a secondary antibody against cobra venom protein.

[0008] The CbAgo protein reaction system includes CbAgo protein, probe 2, and CbAgo reaction buffer.

[0009] The TdT reaction system includes TdT enzyme, TdT buffer, and dATP;

[0010] The Cas12a reaction system includes a reaction buffer, Cas12a protein, and crRNA;

[0011] The working electrode is a gold electrode with a probe P bonded to its surface;

[0012] The nucleotide sequence of probe 1 is shown in SEQ ID NO.1; the nucleotide sequence of probe 2 is shown in SEQ ID NO.2; the nucleotide sequence of probe P is shown in SEQ ID NO.3; and the nucleotide sequence of crRNA is shown in SEQ ID NO.4.

[0013] Furthermore, the amino acid sequence of the CbAgo protein has the NCBI accession number WP_058142162.1.

[0014] Furthermore, the method for preparing the working electrode includes the following steps:

[0015] a. The surface of the gold electrode is cleaned and activated to obtain the GE electrode;

[0016] b. Add a mixed solution containing streptavidin, ruthenium pyridine and chitosan to the surface of the GE electrode and incubate at 37°C for 50-80 min;

[0017] c. Place the biotin-modified probe P on the surface of the GE electrode after sealing in step b to obtain the GE electrode modified with probe P;

[0018] d. Block the non-specific active sites on the GE electrode modified with probe P using a blocking solution to obtain the working electrode.

[0019] The present invention also provides the application of the above-mentioned electrochemical sensor in the preparation of products for detecting cobra venom proteins.

[0020] The present invention also provides a kit for detecting cobra venom proteins, the kit comprising the aforementioned electrochemical sensor.

[0021] The present invention also provides the application of the above-mentioned electrochemical sensor in the detection of cobra venom protein concentration for non-diagnostic purposes.

[0022] The present invention also provides a method for detecting cobra venom protein concentration for non-diagnostic purposes, using the above-mentioned electrochemical sensor, comprising the following steps:

[0023] S1. Add the magnetic beads conjugated with the primary antibody against cobra venom protein to the sample to be tested to obtain a complex that captures snake venom protein; assemble the complex that captures snake venom protein with probe 1 conjugated with the secondary antibody against cobra venom protein to obtain a protein-nucleic acid magnetic nanocomposite.

[0024] S2. The CbAgo protein reaction system and the TdT reaction system are mixed and added to the protein-nucleic acid magnetic nanocomposite to carry out CbAgo protein cleavage and TdT extension cycle reaction to obtain TdT extension product;

[0025] S3. Add the TdT extension product to the Cas12a reaction system to carry out the Cas12a reaction and obtain the Cas12a system product;

[0026] S4. Add the product of the Cas12a system to the surface of the working electrode, incubate, and then perform electrochemical detection; calculate the concentration of snake venom protein in the sample to be tested according to the standard curve.

[0027] Further, in step S2, the CbAgo protein reaction system comprises: 2 μL of CbAgo protein at a concentration of 100 μg / mL, 2 μL of probe 2 at a concentration of 10 μM, 1 μL of CbAgo reaction buffer at a concentration of 10 mM, and 5 μL of nuclease-free water; the CbAgo reaction buffer contains manganese chloride at a final concentration of 8 mM.

[0028] The TdT reaction system consists of: 1 μL TdT buffer, 2 μL 10 mM dATP, 0.5 μL TdT enzyme, and 6.5 μL nuclease-free water.

[0029] The conditions for the CbAgo protein cleavage and TdT extension cycle reaction were: first react at 37°C for 1 h, then react at 80°C for 10 min.

[0030] Further, in step S3, the system for the Cas12a reaction is: 5 μL of TdT extension product, 1 μL of reaction buffer, 1 μL of Cas12a protein at a concentration of 1 μM, 0.5 μL of crRNA at a concentration of 2.5 μM, and 2.5 μL of nuclease-free water;

[0031] The conditions for the Cas12a reaction were: reaction at 37°C for 30 min.

[0032] Further, in step S4, the incubation conditions are: incubation at 37°C for 15-20 minutes;

[0033] The electrochemical detection conditions are as follows: detection is performed in a solution of 0.1 M PBS, 10 mM K2S2O8, pH=7.4, with a scan potential range of -1.6V to 0V and a voltage of 600V.

[0034] The present invention discloses the following technical effects:

[0035] 1. This invention establishes an electrochemical sensor based on a protein-nucleic acid conversion technology synergistically mediated by TdT enzyme and CbAgo protein. First, cobra venom protein is enriched using magnetic beads modified with a protein primary antibody. Then, it is assembled with a cobra venom protein secondary antibody and signal transduction unit probe 1 (Ab2-ssDNA). Probe 1 (ssDNA) is cleaved by CbAgo protein and its corresponding phosphorylated guide DNA (probe 2), converting the target cobra venom protein into the cleaved nucleic acid (i.e., the protein-nucleic acid conversion product). The biomarker nucleic acid conversion product is then treated using a TdT reaction system to prepare a TdT extension product. The TdT extension product then binds to the corresponding phosphorylated guide DNA, activating CbAgo protein for cleavage. This cyclic cleavage process generates a large number of TdT extension products. The TdT extension products are then treated using a Cas12a reaction system to prepare the Cas12a system product. Based on this, the present invention introduces a gold electrode modified with probe P, and the probe P bound on the gold electrode is cleaved by the product of the Cas12a system. Finally, the concentration of snake venom protein can be detected by electrochemical treatment.

[0036] 2. The cobra venom protein secondary antibody and signal transduction unit probe 1 (Ab2-ssDNA) in this invention have multiple components. When they bind to cobra venom protein and are cleaved by CbAgo protein, multiple protein-nucleic acid conversion products are formed, resulting in a primary cascade amplification effect. The marker nucleic acid conversion products are treated with TdT enzyme to form TdT extension products. The TdT extension products then bind to the corresponding phosphorylated guide DNA, activating CbAgo protein for cleavage. Through this cyclic cleavage, a large number of TdT extension products are formed. The Cas12a reaction system then reacts with the TdT extension products to generate the Cas12a system product. The Cas12a system product cleaves the probe P bound to the gold electrode, again achieving a cascade amplification effect, thereby significantly improving the detection sensitivity of cobra venom protein.

[0037] 3. The electrochemical sensor of this invention exhibits excellent sensitivity and specificity, and a good linear calibration curve was established in the concentration range of 0.000005 ng / mL to 50 ng / mL. 2With a concentration of 0.998 and a detection limit of 2.79 fg / mL, it exhibits high sensitivity and can be used for the precise analysis of cobra venom protein samples. It has extremely high application value in the accurate identification and quantitative analysis of cobra venom proteins. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 This is a schematic diagram illustrating the principle of the cobra venom protein detection method of the present invention;

[0040] Figure 2 This is a schematic diagram (A) showing the structure of the CbAgo protein in this invention and its expression process (B).

[0041] Figure 3 The results of SDS-PAGE analysis (A) and Western Blot (B) of the preliminary expression of CbAgo protein in this invention are shown below; in A, lane M: Protein Marker; lane 1: uninduced whole cells; lanes 2-6: induced whole cells; in B, lane Protein: induced whole cells.

[0042] Figure 4 This document presents the results of experimental condition optimization (A) and solubility analysis (B) during CbAgo protein expression in this invention. In A, lane M represents the Protein Marker; lane 1 represents whole cells induced with 0.2 mM IPTG at 15°C; lane 2 represents whole cells induced with 1.0 mM IPTG at 15°C; lane 3 represents whole cells induced with 0.2 mM IPTG at 37°C; lane 4 represents whole cells induced with 1.0 mM IPTG at 37°C; lane 5 represents uninduced whole cells; lane 6 represents the precipitate after induction with 1.0 mM IPTG at 37°C; lane 7 represents the supernatant after induction with 1.0 mM IPTG at 37°C; lane 8 represents the precipitate after induction with 0.2 mM IPTG at 37°C; lane 9 represents the supernatant after induction with 0.2 mM IPTG at 37°C; lane 10 represents the precipitate after induction with 1.0 mM IPTG at 15°C; and lane 11 represents the precipitate after induction with 1.0 mM IPTG at 15°C. Supernatant after IPTG induction; Lane 12: Precipitate after 0.2mM IPTG induction at 15℃; Lane 13: Supernatant after 0.2mM IPTG induction at 15℃; In B, Lane 1: Precipitate after disruption; Lane 2: Supernatant after disruption; Lane 3: Eluent; Lane 4: Washing sample; Lane 5: Elution sample;

[0043] Figure 5 This is the final expression and purification result of the CbAgo protein in this invention;

[0044] Figure 6 This is a schematic diagram illustrating the design principle of the single-stranded DNA substrate (probe 1) used for cyclic "extension-cutting" amplification in this invention.

[0045] Figure 7 The image shows the size detection results (A) of the magnetic beads after being conjugated with cobra venom protein primary antibody and (B) of the assembled composite material in this invention.

[0046] Figure 8 This is a graph showing the verification results of the TdT enzyme extension activity in this invention; where lane N is the control group and lane P is the experimental group.

[0047] Figure 9 This is a diagram showing the verification results of the CbAgo protein-TdT-Cas12a reaction system under fluorescence conditions in this invention;

[0048] Figure 10 The images show the EIS results (A) and CV results (B) of the electrochemical biosensor in this invention.

[0049] Figure 11 This is a graph showing the ECL results of the electrochemical biosensor used in this invention for detecting snake venom proteins.

[0050] Figure 12 The image shows the detection results of different concentrations of snake venom protein by the electrochemiluminescence sensor in this invention.

[0051] Figure 13 This is a graph showing the relationship between ECL intensity and the logarithm of the target snake venom protein in this invention;

[0052] Figure 14 This is a graph showing the specific detection results of the electrochemical biosensor of the present invention. Detailed Implementation

[0053] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0054] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0055] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0056] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0057] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0058] A schematic diagram illustrating the detection principle of the electrochemical sensor for snake venom proteins in this invention is shown below. Figure 1 As shown, the working principle is as follows: In the presence of cobra venom protein, cobra venom protein is enriched and captured using magnetic beads (MNPs-Ab1) modified with a protein primary antibody. Then, it is assembled with a cobra venom protein secondary antibody and signal transduction unit probe 1 (Ab2-ssDNA). Probe 1 (ssDNA) is cleaved by CbAgo protein and the corresponding phosphorylated guide DNA (probe 2), converting the target cobra venom protein into the cleaved nucleic acid (i.e., protein-nucleic acid conversion product). The protein-nucleic acid conversion product is processed using a TdT reaction system to obtain a TdT extension product. The TdT extension product then binds to the corresponding phosphorylated guide DNA (probe 2), activating CbAgo protein for cleavage. Through this cyclic cleavage, a large number of TdT extension products are formed. The TdT extension products are then reacted using a Cas12a reaction system to prepare the Cas12a system product. The Cas12a system product is used to cleave probe P bound to the gold electrode. Finally, the concentration of cobra venom protein can be detected through electrochemical treatment.

[0059] In the CbAgo-TdT-Cas12a reaction system, the extension reaction involving the TdT enzyme requires an oligonucleotide consisting of at least three nucleotides as a primer. The TdT enzyme can only function after the CbAgo protein cleaves the signal transduction unit (probe 1). The Cas12a protein, guided by crRNA, cleaves ssDNA with a PAM sequence. With the help of the PAM sequence, the trans-cleavage activity of the Cas12a protein is activated, thereby cleaving any surrounding ssDNA.

[0060] The electrochemical sensor designed in this invention utilizes the TdT extended A base sequence and the designed corresponding phosphorylation guide DNA to achieve cyclic cleavage. In the presence of universal crRNA, it activates the non-specific ssDNA cleavage effect of the CRISPR / Cas12a system, significantly improving the sensitivity of cobra venom protein detection. Streptavidin (SAV) and ruthenium pyridine [dispersed in 0.5% (w / w) chitosan solution] were added to the surface of the GE working electrode. Non-specific active sites were blocked with BSA, and the bioprobe P (Fc-ssDNA) was immobilized on the electrode through the interaction of streptavidin (SAV) and biotin. Only in the presence of cobra venom protein could the CbAgo protein cleave the relevant sequence on the signal transduction unit, thereby enabling the TdT enzyme to extend the relevant base sequence to achieve cyclic cleavage and activate the cleavage activity of Cas12a protein. After being dropped onto the electrode surface, the activated Cas12a protein cleaves probe P (Fc-ssDNA), resulting in the recovery of the electrochemiluminescence signal. Conversely, in the absence of the target protein (cobra venom protein), the Cas12a protein could not be activated, and when added to the electrode surface, it could not cleave probe P (Fc-ssDNA), and the electrochemiluminescence signal could not be recovered.

[0061] The experimental materials (reagents and instruments) involved in the following embodiments of the present invention are as follows:

[0062] Terminal deoxynucleotidyl transferase (TdT enzyme) and EnGen® Lba Cas12a protein were purchased from New England Biolabs (Beijing, China). K3Fe[CN]6 and K4Fe[CN]2 were supplied by Shanghai Guoyao Chemical Reagent Co., Ltd., China. Gold electrodes, platinum wire electrodes, and reference electrodes were purchased from Shanghai Chenhua Instrument Co., Ltd. Chloroauric acid (HAuCl4·3H2O), imidazole, and tetramethylethylenediamine (TEMED) were purchased from Shanghai Aladdin Reagent Co., Ltd. N,N'-Methylenebisacrylamide (Acr-Bis), ammonium persulfate (APS), bovine serum albumin (BSA), 20×PBS (pH=7.2-7.6), DNA marker (25-500bp), 5×TBE buffer (pH=8.0), chitosan, deoxyadenine triphosphate (dATP), trisodium citrate, streptavidin (SAV), manganese chloride (MnCl2), Tris-NaCl, isopropyl-β-D-thiogalactoside (IPTG), glycerol, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), and benzyl sulfonyl fluoride (PMSF) were all purchased from Shanghai Sangon Biotech Co., Ltd., China. Disulfitol (DTT) and sodium chloride (NaCl) were purchased from China National Pharmaceutical Group Corporation. SYBR Green I (10000×) nucleic acid dye and 6× DNA loading buffer were purchased from Beijing Solarbio Science & Technology Co., Ltd. Magnetic beads were purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd. The primary antibody (bs-0763R) and secondary antibody (bs-0295G) against cobra venom protein were both purchased from Beijing Bio-Sens Biotechnology Co., Ltd.

[0063] The oligonucleotides and biological probe P purified by HPLC were synthesized by Shanghai Sangon Biotech Co., Ltd., and the RNA probe (crRNA) was purchased from General Biotech Co., Ltd. Specific information is shown in Table 1.

[0064] Table 1 Sequence Information

[0065]

[0066] The main experimental instruments and equipment are shown in Table 2.

[0067] Table 2 Main Experimental Instruments and Equipment

[0068]

[0069] Example 1: Preparation of CbAgo protein

[0070] The CbAgo protein consists of a bilobed structure composed of a MID-PIWI domain and an NPAZ domain. This structure enables it to perform site-specific cleavage of nucleic acid substrates at room temperature, guided by guide DNA (gDNA). The structure of the CbAgo protein is as follows: Figure 2 As shown in Figure A.

[0071] The CbAgo protein used in this invention is prepared according to... Figure 2 The preparation process shown in Figure B involves the following steps: The CbAgo gene (WP_058142162.1) is cloned into the pET-28a expression vector and transformed into *Escherichia coli* Rosetta (DE3) cells. The transformed cells are cultured in LB medium at 37°C. Single clones are selected and inoculated into LB medium, and cultured at 37°C until the bacterial OD reaches 100%. 600 The concentration was 0.6-0.8, and IPTG was added to a final concentration of 0.5 mM. After incubation at 37℃ for 4 h, the cells were centrifuged, and samples were collected and analyzed by SDS-PAGE and Western Blot. The results are as follows: Figure 3 As shown in Figures A and B, SDS-PAGE and Western Blot analyses indicate that CbAgo protein was expressed after IPTG induction.

[0072] To improve the expression yield of CbAgo protein, this invention optimized the expression conditions and performed solubility analysis. Optimized conditions included induction with 0.2 mM IPTG at 15°C, induction with 1.0 mM IPTG at 15°C, induction with 0.2 mM IPTG at 37°C, and induction with 1.0 mM IPTG at 37°C. The induced bacteria were lysed, and the precipitate and supernatant were separated by centrifugation and analyzed using SDS-PAGE. The results are as follows: Figure 4 As shown in Figure A, the expression efficiency of CbAgo protein in samples, supernatants, and precipitates induced by IPTG at 15℃ and 37℃ is presented. The results indicate that the protein expression efficiency in the supernatant was highest after induction with 1.0 mM IPTG at 15℃. Therefore, large-scale expression of the protein under these conditions is recommended. Figure 4 As shown in Figure B, the results indicate that the CbAgo protein is a soluble protein.

[0073] After optimizing the optimal conditions for CbAgo protein expression, the expression and purification were carried out according to the optimized method, and the results are as follows. Figure 5 As shown, high-purity CbAgo protein was finally obtained.

[0074] Example 2: Design of a single-stranded DNA substrate (probe 1) for cyclic extension-cutting amplification

[0075] To achieve the TdT / CbAgo-mediated cyclic "extension-cutting" process, probe 1 (SEQ ID NO. 1) was designed in this embodiment. For example... Figure 6As shown, a single-stranded DNA (ssDNA) substrate was engineered to include an adenine (A)-rich region, and the cleavage site of the CbAgo protein was located within this region. This design allows TdT to regenerate a new cleavage substrate after each CbAgo-mediated cleavage, thus enabling a continuous amplification cycle.

[0076] Example 3: Construction and Detection Performance Verification of Electrochemical Biosensors

[0077] 1. Enrichment verification of cobra venom proteins

[0078] Magnetic beads were conjugated with cobra venom protein primary antibody (Ab1) to obtain magnetic bead-antibody 1 complex (MNPs-Ab1); the particle size of MNPs-Ab1 was detected, and the results are as follows. Figure 7 As shown in Figure A, after the magnetic beads are coupled with the capture antibody (Ab1), their hydrodynamic diameter increases from 154 nm to 183.34 nm, confirming the successful formation of MNPs-Ab1.

[0079] 0.1 mg of MNPs-Ab1 was added to 5 mL of cobra venom protein solution (200 μg / mL) and incubated for 60 min to capture the cobra venom protein, obtaining magnetic beads for capturing the venom protein. The magnetic beads containing the captured venom protein were then mixed with cobra venom protein secondary antibody and signal transduction unit probe 1 (Ab2-ssDNA) at a 1:1 ratio and assembled over 60 min. The protein-nucleic acid magnetic nanocomposite was obtained by magnetic separation. The particle size of the obtained protein-nucleic acid magnetic nanocomposite was analyzed, and the results are as follows: Figure 7 As shown in Figure B, the average particle size further increased to 221.89 nm, indicating that cobra venom proteins were effectively captured and enriched, laying a solid foundation for the subsequent TdT / CbAgo-mediated signal transduction process.

[0080] 2. Validation of the CbAgo-TdT-Cas12a reaction system

[0081] (1) Construction of the CbAgo-TdT-Cas12a reaction system

[0082] a. Preparation of TdT extension products (TdT / CbAgo-mediated protein-nucleic acid conversion process):

[0083] A CbAgo protein reaction system was prepared, comprising: 2 μL of 100 μg / mL CbAgo protein, 2 μL of 10 μM phosphorylated guide DNA (probe 2, SEQ ID NO. 2), 1 μL of 10 mM Tris-HCl buffer (containing 8 mM MnCl2), and 5 μL of nuclease-free water. After mixing all components, the mixture was incubated at 37°C for 15 min.

[0084] Prepare a TdT reaction system comprising: 1 μL 10×TdT buffer, 2 μL dATP (10 mM), 0.5 μL TdT enzyme, and 6.5 μL nuclease-free water. Mix all the components together.

[0085] The 10 μL CbAgo protein reaction system and the 10 μL TdT reaction system were mixed and added to a centrifuge tube containing the protein-nucleic acid magnetic nanocomposite obtained in "1. Enrichment Verification of Cobra Venom Protein". The mixture was first reacted at 37°C for 1 h, and the supernatant was collected by magnetic separation. Then, the mixture was reacted at 80°C for 10 min to inactivate any residual enzymes. The TdT-extended nucleic acid product was obtained by 12% polyacrylamide electrophoresis.

[0086] b. Preparation of products in the Cas12a system:

[0087] The TdT extension product was added to the Cas12a reaction system and incubated at 37°C for 30 min. The Cas12a reaction system consisted of: 5 μL of TdT extension product, 1 μL of 10×NEB buffer 2.1, 1 μL of Cas12a protein at a concentration of 1 μM, 0.5 μL of crRNA (SEQ ID NO.4) at a concentration of 2.5 μM, and 2.5 μL of nuclease-free water.

[0088] To facilitate subsequent validation experiments, 2 μL of a 5 μM probe with the sequence BHQ-TCTCTGAA-FAM was added to the above system. The Cas12a product was obtained through fluorescence analysis. In actual detection, this probe is not required.

[0089] (2) Verification of TdT enzyme activity

[0090] After cleaning and assembling the gel preparation equipment and checking for leaks, prepare a 12% PAGE gel. Take a clean 20 mL centrifuge tube, add 6 mL of 30% N,N'-methylenebisacrylamide (Acr-Bis), 5.9 mL of ultrapure water, 3 mL of 5×TBE buffer, and 110 μL of 10% ammonium persulfate (APS), and vortex to mix. Add 10 μL of TEMED, gently invert to mix, and quickly add the gel solution to the assembled gel preparation glass plate. Insert a comb and let it stand until the gel solidifies.

[0091] Pour an appropriate amount of 1×TBE buffer solution into the solidified gel vertical electrophoresis tank; the sample loading groups include DNAMaker (25-500 bp), control group, and experimental group. Among them, the control group is the TdT reaction system without TdT enzyme, and the experimental group is the TdT reaction system with TdT enzyme.

[0092] Before loading the sample, add 2 μL of 6× DNA loading buffer and 2 μL of SYBR Green I (100×) nucleic acid dye, mix and then load the sample; set the voltage to 80V and the time to 90min. After the experiment, use a gel imaging system to observe and save the results.

[0093] The results are as follows Figure 8 As shown, lane N is the control group and lane P is the experimental group. It can be seen that after cobra venom protein is enriched by magnetic beads and cleaved by CbAgo protein, the protein nucleic acid product will only be extended in the presence of TdT enzyme.

[0094] (3) Fluorescence verification

[0095] Set up the following groups:

[0096] Group A: No target analyte (snake venom protein); protein-nucleic acid magnetic nanocomposites were prepared according to "1. Enrichment verification of cobra venom protein"; CbAgo protein reaction system was added only to the protein-nucleic acid magnetic nanocomposites to perform CbAgo protein cleavage;

[0097] Group b: No target analyte (snake venom protein); protein-nucleic acid magnetic nanocomposites were prepared according to "1. Enrichment verification of cobra venom protein"; TdT extension was carried out by adding only TdT reaction system to the protein-nucleic acid magnetic nanocomposites;

[0098] Group C: No target analyte (snake venom protein); protein-nucleic acid magnetic nanocomposites were prepared according to "1. Enrichment verification of cobra venom protein"; CbAgo protein reaction system and TdT reaction system were added to the protein-nucleic acid magnetic nanocomposites to perform CbAgo protein cleavage and TdT extension;

[0099] Group d: Target analyte (snake venom protein); Prepare protein-nucleic acid magnetic nanocomposites according to "1. Enrichment verification of cobra venom protein"; Add only CbAgo protein reaction system to the protein-nucleic acid magnetic nanocomposites to perform CbAgo protein cleavage;

[0100] Group e: Target analyte (snake venom protein); Prepare protein-nucleic acid magnetic nanocomposites according to "1. Enrichment verification of cobra venom protein"; Add only TdT reaction system to the protein-nucleic acid magnetic nanocomposites to extend TdT;

[0101] Group f: Target analyte (snake venom protein); Prepare protein-nucleic acid magnetic nanocomposites according to "1. Enrichment verification of cobra venom protein"; Add CbAgo protein reaction system and TdT reaction system to the protein-nucleic acid magnetic nanocomposites to perform CbAgo protein cleavage and TdT extension;

[0102] After the above groups were processed, the Cas12a reaction was carried out in step b of “(1) Construction of CbAgo-TdT-Cas12a reaction system”.

[0103] After the reaction was completed, the fluorescence signal of each group was detected, and the results are as follows: Figure 9 As shown, in the absence of cobra venom proteins, neither CbAgo alone, nor TdT alone, nor a combination of both, activated the Cas12a system, producing only very weak background fluorescence (curve ac). In stark contrast, the fluorescence signal significantly increased after the introduction of the target analyte. However, CbAgo alone (curve d) or TdT alone (curve e) induced only limited signal enhancement, while the simultaneous presence of both (curve f) produced the highest fluorescence output intensity. These results indicate that the TdT / CbAgo-mediated protein-nucleic acid conversion system possesses synergistic amplification capabilities.

[0104] 3. Fabrication and Detection Performance Verification of Electrochemical Biosensors

[0105] (1) Preparation of electrochemical biosensors

[0106] a. Cleaning and activation treatment of the gold electrode (GE): First, a 3 mm diameter gold electrode was polished with 0.05 μm alumina for 5 min to obtain a mirror-like surface. After polishing, the gold electrode was immersed in a piranha solution (H₂SO₄ / H₂O₂, volume ratio 3:1) for 30 min. Subsequently, the electrode was thoroughly rinsed with ultrapure water and ultrasonically treated in ethanol for 5 min to remove impurities. Next, the electrode was activated by scanning the potential in 0.5 M sulfuric acid solution at a scan rate of 4 V / s in the range of -0.35 V to 1.5 V until a stable cyclic voltammetric peak was obtained. Then, the electrode was scanned in a mixed solution of potassium ferrocyanide and potassium ferrocyanide at a scan rate of 0.05 V / s in the potential range of -0.2 V to 0.6 V. A peak potential difference of approximately 100 mV indicated successful electrode polishing. Finally, the electrode was rinsed with ultrapure water and dried under a nitrogen flow.

[0107] b. Add 10 μL of a solution containing 2 μL of streptavidin (SAV) at a concentration of 1 mg / mL and 2 μL of ruthenium pyridine (Ru(bpy)3). 2+ A mixture of 1 mg / mL chitosan and 6 μL chitosan solution [0.5% (w / w)] was added to the surface of the gold electrode and incubated at 37°C for 60 min.

[0108] c. Add 5 μL of biotin-modified bioprobe P (Biotin-ssDNA-Fc) at a concentration of 500 nM. Through the specific binding of biotin and streptavidin, bioprobe P (SEQ ID NO.3) is bound to the surface of the gold electrode. Then, the surface of the gold electrode is repeatedly rinsed with ultrapure water and dried with nitrogen to complete the surface modification of the electrode.

[0109] d. Block non-specific active sites with 1% BSA blocking solution at 37°C for 1 h. Then, repeatedly rinse the gold electrode surface with ultrapure water and dry it with nitrogen. The working electrode is then obtained.

[0110] e. Immerse the working electrode in 10 μL of the Cas12a system product and incubate at 37°C for 15-20 min to obtain an electrochemical biosensor for detecting target biomarkers.

[0111] (2) Verification of electrochemical signal response of electrochemical biosensor

[0112] The electrochemical detection conditions are: at 5 mM [Fe(CN)6] 3− / 4− Cyclic voltammetry (CV) scans were performed in the solution from -0.2 V to 0.6 V at a scan rate of 50 mV / s; in 1 mM [Fe(CN)6] 3− / 4−Electrochemical impedance spectroscopy (EIS) scans were performed in the solution at a frequency of 0.1 Hz to 10 kHz at 0.2 V; electrochemiluminescence signals were detected in a solution of 0.1 M PBS, 10 mM K2S2O8, pH=7.4, with a scan potential range of -1.6 V to 0 V and a voltage set to 600 V.

[0113] According to the above electrochemical detection conditions, the electrodes / electrochemical biosensors prepared in steps a, b, c, d, and e of "(1) Preparation of Electrochemical Biosensors" were detected, and the results are as follows. Figure 10 As shown.

[0114] like Figure 10 As shown in Figure A, the Nyquist plot of EIS reflects the modification of the electrode at various stages. Initially, the bare electrode exhibits an almost linear plot (curve a), indicating excellent conductivity and virtually no hindrance to electron transfer. When ruthenium terpyridine [Ru(bpy)3] is used... 2+ After modification with streptavidin (SAV) to form an ECL interface (curve b), the charge transfer resistance (Rct) increased, mainly due to the electrostatic repulsion generated by the negatively charged amino acid residues in SAV. Subsequent immobilization of biotinylated single-stranded DNA-ferrocene (Biotin-ssDNA-Fc) led to a significant increase in Rct (curve c), due to the negatively charged phosphate backbone of the DNA. Further blocking with bovine serum albumin (BSA) resulted in another increase in Rct (curve d), as unoccupied electrode sites were passivated. When the working electrode was exposed to an activated CRISPR / Cas12a system, the immobilized probe underwent extensive cleavage, leading to a significant decrease in Rct (curve e). These EIS results collectively confirm the efficient stepwise construction and functionalization of the ECL sensing interface. Figure 10 The CV results shown in Figure B exhibit a similar trend, further validating the stepwise assembly process.

[0115] Furthermore, the following groups are designed:

[0116] Group a: Bare electrodes prepared according to step a in “(1) Preparation of electrochemical biosensors”;

[0117] Group b: Electrodes prepared sequentially according to steps a and b in “(1) Preparation of electrochemical biosensors”;

[0118] Group c: Electrodes prepared sequentially according to steps ad in “(1) Preparation of electrochemical biosensors”;

[0119] Group d: No target substance (snake venom protein); electrochemical biosensors prepared sequentially according to step ae in “(1) Preparation of electrochemical biosensors”;

[0120] Group e: Target substance (snake venom protein); Electrochemical biosensor prepared in sequence according to step ae in “(1) Preparation of electrochemical biosensor”;

[0121] Group f: The target substance (snake venom protein) is present; the reaction system without adding CbAgo protein is omitted, the CbAgo protein cleavage step is omitted, TdT extension is performed directly, and then the electrochemical biosensor is prepared according to the steps ae in "(1) Preparation of electrochemical biosensor";

[0122] Group g: The target substance (snake venom protein) is present; no TdT reaction system is added, the TdT extension step is omitted, only CbAgo protein is cleaved, and then the electrochemical biosensor is prepared according to the steps ae in "(1) Preparation of electrochemical biosensor".

[0123] According to the above electrochemical detection conditions, the above-mentioned group ag was tested respectively, and the results are as follows. Figure 11 As shown.

[0124] The feasibility of the biosensor of the present invention was further confirmed by evaluating the ECL response of the biosensor under different assembly and activation conditions. Figure 11 The results show that the ECL emission of the bare electrode is negligible (curve a), while that of the Ru(bpy)3 electrode is negligible. 2+ The modified electrode produced a strong signal (curve b), confirming Ru(bpy)3 2+ It is an essential luminescent material for ECL production. When Biotin-ssDNA-Fc is immobilized on the electrode surface, due to the energy transfer quenching effect of ferrocene (Fc), Ru(bpy)3... 2+ The ECL intensity was significantly reduced (curve c), exhibiting a "shutdown" signal state. In the absence of cobra venom protein, Cas12a remained inactive, and the Biotin-ssDNA-Fc probe remained on the electrode surface, maintaining low ECL output (curve d). Conversely, in the presence of cobra venom protein, activation of the CRISPR / Cas12a system induced extensive trans-cleavage of the surface-bound Biotin-ssDNA-Fc, effectively restoring the strong ECL signal (curve e). Notably, the ECL enhancement achieved through the synergistic TdT / CbAgo-mediated protein-nucleic acid conversion system (curve e) was significantly greater than the enhancements obtained by using TdT or CbAgo alone (curves f and g), indicating that the synergistic "extension-cleavage" cycle effectively amplified the nucleic acid signal. These results collectively demonstrate the successful fabrication of this electrochemical biosensor.

[0125] Example 4 Sensitivity Detection

[0126] Under optimal experimental conditions, cobra venom proteins with concentrations ranging from 0.000005 ng / mL to 50 ng / mL were enriched with magnetic beads, cleaved with CbAgo protein, extended using a TdT-Cas12a reaction system, and activated according to the method described in Example 3. Finally, the proteins were added to the working electrode surface for cleavage and incubation before measurement, with the measurement conditions consistent with Example 3. Each group underwent at least three repeated measurements, and the electrochemiluminescence intensity of different concentrations of cobra venom proteins was recorded to establish a standard curve.

[0127] The results are as follows Figure 12 As shown, the concentrations of cobra venom protein from bottom to top are 0 ng / mL (curve a), 0.000005 ng / mL (curve b), 0.00005 ng / mL (curve c), 0.00001 ng / mL (curve d), 0.001 ng / mL (curve e), 0.05 ng / mL (curve f), 0.1 ng / mL (curve g), 5 ng / mL (curve h), and 50 ng / mL (curve i). With the continuous increase of cobra venom protein concentration, the electrochemiluminescence signal of the sensor also continuously increases, showing the same trend. After fitting the logarithms of different concentrations of cobra venom protein, the results are as follows... Figure 13 As shown, the linear regression equation is Y = 2052.7lgX + 7140.4 (where Y represents peak intensity and X is the concentration of cobra venom protein), R 2 =0.998, and the detection limit is 2.79 fg / mL.

[0128] Example 5 Specificity Detection

[0129] Under optimal experimental conditions, specificity experiments were conducted using bovine serum albumin (BSA), human serum albumin (HSA), snake venom protein (Cob), BSA+Cob (1:1 mixture, final concentration 200 μg / mL), and HSA+Cob (1:1 mixture, final concentration 200 μg / mL) as detection samples, with nuclease-free water (Blank) as a blank control. Following the method in Example 3, the protein-nucleic acid conversion was performed via magnetic bead enrichment, TdT / CbAgo-mediated protein-nucleic acid conversion, Cas12a reaction, activation, and finally, the sample was added to the working electrode surface for cleavage and incubation before measurement. The measurement conditions were consistent with those in Example 3. Each group underwent at least three repeated measurements.

[0130] The results are as follows Figure 14As shown, the ECL intensities of BSA and HSA are not significantly different from those of the blank control, indicating that the biosensor of the present invention does not respond to BSA and HSA. When BSA and HSA are mixed with snake venom protein respectively, their ECL intensities are also not significantly different from those of snake venom protein, indicating that the presence of BSA and HSA does not affect the detection of snake venom protein.

[0131] The above results show that the biosensor constructed in this invention has high specificity for the detection of snake venom proteins and high anti-interference characteristics.

[0132] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An electrochemical sensor based on the synergistic mediation of TdT enzyme and CbAgo, characterized in that, The electrochemical sensor includes a cobra venom protein enrichment system, a CbAgo protein reaction system, a TdT reaction system, a Cas12a reaction system, and a working electrode; The cobra venom protein enrichment system includes magnetic beads coupled with a primary antibody against cobra venom protein and probe 1 coupled with a secondary antibody against cobra venom protein. The CbAgo protein reaction system includes CbAgo protein, probe 2, and CbAgo reaction buffer; the amino acid sequence of the CbAgo protein has the NCBI accession number WP_058142162.

1. The TdT reaction system includes TdT enzyme, TdT buffer, and dATP; The Cas12a reaction system includes a reaction buffer, Cas12a protein, and crRNA; The working electrode is a gold electrode with a probe P bonded to its surface; The nucleotide sequence of probe 1 is shown in SEQ ID NO.1; the nucleotide sequence of probe 2 is shown in SEQ ID NO.2; the nucleotide sequence of probe P is shown in SEQ ID NO.3; and the nucleotide sequence of crRNA is shown in SEQ ID NO.

4. The method of using the electrochemical sensor includes the following steps: S1. Add the magnetic beads conjugated with the primary antibody against cobra venom protein to the sample to be tested to obtain a complex that captures snake venom protein; assemble the complex that captures snake venom protein with probe 1 conjugated with the secondary antibody against cobra venom protein to obtain a protein-nucleic acid magnetic nanocomposite. S2. The CbAgo protein reaction system and the TdT reaction system are mixed and added to the protein-nucleic acid magnetic nanocomposite to carry out CbAgo protein cleavage and TdT extension cycle reaction to obtain TdT extension product; S3. Add the TdT extension product to the Cas12a reaction system to carry out the Cas12a reaction and obtain the Cas12a system product; S4. The product of the Cas12a system is added to the surface of the working electrode, incubated, and then electrochemically detected. Calculate the concentration of snake venom protein in the sample based on the standard curve.

2. The electrochemical sensor according to claim 1, characterized in that, The method for preparing the working electrode includes the following steps: a. The surface of the gold electrode is cleaned and activated to obtain the GE electrode; b. Add a mixed solution containing streptavidin, ruthenium pyridine and chitosan to the surface of the GE electrode and incubate at 37°C for 50-80 min; c. Place the biotin-modified probe P on the surface of the GE electrode after sealing in step b to obtain the GE electrode modified with probe P; d. Block the non-specific active sites on the GE electrode modified with probe P using a blocking solution to obtain the working electrode.

3. The application of the electrochemical sensor according to claim 1 or 2 in the preparation of products for detecting cobra venom proteins.

4. A kit for detecting cobra venom proteins, characterized in that, The kit contains the electrochemical sensor as described in claim 1 or 2.

5. The application of the electrochemical sensor according to claim 1 or 2 in the detection of cobra venom protein concentration for non-diagnostic purposes.

6. A method for detecting the concentration of cobra venom proteins for non-diagnostic purposes, characterized in that, Detection using the electrochemical sensor according to claim 1 or 2 includes the following steps: S1. Add the magnetic beads conjugated with the primary antibody against cobra venom protein to the sample to be tested to obtain a complex that captures snake venom protein; assemble the complex that captures snake venom protein with probe 1 conjugated with the secondary antibody against cobra venom protein to obtain a protein-nucleic acid magnetic nanocomposite. S2. The CbAgo protein reaction system and the TdT reaction system are mixed and added to the protein-nucleic acid magnetic nanocomposite to carry out CbAgo protein cleavage and TdT extension cycle reaction to obtain TdT extension product; S3. Add the TdT extension product to the Cas12a reaction system to carry out the Cas12a reaction and obtain the Cas12a system product; S4. The product of the Cas12a system is added to the surface of the working electrode, incubated, and then electrochemically detected. Calculate the concentration of snake venom protein in the sample based on the standard curve.

7. The method according to claim 6, characterized in that, In step S2, the CbAgo protein reaction system consists of: 2 μL of CbAgo protein at a concentration of 100 μg / mL, 2 μL of probe 2 at a concentration of 10 μM, 1 μL of CbAgo reaction buffer at a concentration of 10 mM, and 5 μL of nuclease-free water; the CbAgo reaction buffer contains manganese chloride at a final concentration of 8 mM. The TdT reaction system consists of: 1 μL TdT buffer, 2 μL 10 mM dATP, 0.5 μL TdT enzyme, and 6.5 μL nuclease-free water. The conditions for the CbAgo protein cleavage and TdT extension cycle reaction were: first react at 37°C for 1 h, then react at 80°C for 10 min.

8. The method according to claim 6, characterized in that, In step S3, the system for the Cas12a reaction is: 5 μL of TdT extension product, 1 μL of reaction buffer, 1 μL of Cas12a protein at a concentration of 1 μM, 0.5 μL of crRNA at a concentration of 2.5 μM, and 2.5 μL of nuclease-free water; The conditions for the Cas12a reaction were: reaction at 37°C for 30 min.

9. The method according to claim 6, characterized in that, In step S4, the incubation conditions are: incubation at 37°C for 15-20 minutes; The electrochemical detection conditions are as follows: detection is performed in a solution of 0.1 M PBS, 10 mM K2S2O8, pH=7.4, with a scan potential range of -1.6V to 0V and a voltage of 600V.

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