Bimetal nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a bimetallic nano-enzyme as well as a preparation method and application thereof, and relates to the technical field of biological detection. The bimetallic nano-enzyme is a DNA-Ag / Pt bimetallic nano-enzyme material prepared on the basis of a specific DNA template sequence or containing the specific DNA template sequence, and can be combined with a double-emission ratio type silver nano-cluster to be used for double-signal detection of vibrio parahaemolyticus. The bimetallic nano-enzyme has peroxidase-like activity, the enzyme activity efficiency is adjustable, and the bimetallic nano-enzyme can be used for double-signal detection of vibrio parahaemolyticus. Compared with the prior art, the color development absorbance of the bimetallic nano-enzyme provided by the invention is improved by about 15%, the lowest detection limit of a colorimetric detection method is 10 CFU / mL, and the lowest detection limit of a fluorescence detection method is 1 CFU / mL; the method has no cross reaction on staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, salmonella, vibrio vulnificus and the like, and is suitable for on-site rapid screening, visual interpretation and semi-quantitative detection of scenes such as water samples and marine products.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a bimetallic nanozyme, its preparation method, and its application. Background Technology

[0002] Vibrio parahaemolyticus is a common halophilic foodborne pathogen widely found in marine environments and aquatic products, and is one of the main pathogens causing foodborne illnesses such as acute gastroenteritis. With the increasing consumption of aquatic products, foodborne illnesses caused by Vibrio parahaemolyticus are on the rise, posing a serious threat to public health and safety. Therefore, developing an efficient, sensitive, and convenient method for detecting Vibrio parahaemolyticus is of great significance for the safety supervision of aquatic products and the prevention and control of diseases.

[0003] Currently, routine detection methods for Vibrio parahaemolyticus mainly include plate culture, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA). Among these, plate culture offers high specificity and accuracy, but its cumbersome procedures and long detection cycle make it unsuitable for rapid on-site screening. PCR is highly sensitive but relies on expensive equipment, has stringent environmental requirements, and is prone to contamination. While ELISA offers good specificity, it is sensitive to antibody quality and reaction conditions, making it difficult to widely implement due to limited resources. In summary, these traditional methods still have significant limitations in terms of speed, sensitivity, and portability.

[0004] On the one hand, metal nanoclusters, as a class of nanomaterials with enzyme-like catalytic activity or fluorescence emission properties, have advantages such as low cost, good stability, scalable preparation, and tolerance to complex environments, and have broad application potential in the fields of biosensing, food safety, and environmental monitoring. Chinese invention patent CN114456802A discloses a ratiometric silver nanocluster fluorescent probe and its preparation method and application. The disclosed DNA-silver nanoclusters (DNA-AgNCs nanoclusters) can nucleate on cytosine-rich sequences. Silver nanoclusters prepared using stem-loop structure sequences as templates can undergo secondary structure transformation through CHA amplification, thereby achieving ratiometric fluorescence signal output, possessing self-calibration capabilities and good resistance to background interference. However, in practical applications for the detection of Vibrio parahaemolyticus, DNA-AgNCs nanoclusters are only suitable for semi-quantitative detection. In scenarios where only qualitative detection is required, this detection method cannot quickly provide results, and other colorimetric methods are also difficult to perform both quantitative and qualitative detection simultaneously.

[0005] On the other hand, CRISPR / Cas-based detection strategies for Vibrio parahaemolyticus often employ a single intensity signal (primarily colorimetric), resulting in limited colorimetric differences at low target concentrations, frequently requiring spectrophotometers to obtain stable readings. Furthermore, the colorimetric system is sensitive to temperature, reaction time, and light, easily leading to batch-to-batch fluctuations and inconsistent interpretations. For example, Chinese invention patent CN119753179A discloses a CRISPR-Cas12a sensing system mediated by gold@platinum nanozymes and its application in biodetection. This system is obtained by reacting a gold@platinum-ssDNA-magnetic bead complex with components A and B, wherein the gold@platinum-ssDNA-magnetic bead complex is prepared by linking the gold@platinum nanozyme and magnetic beads via ssDNA linkers. In the detection of Mycobacterium tuberculosis using the gold@platinum nanozyme-mediated CRISPR-Cas12a system, the gold@platinum nanozyme is attached to magnetic beads via single-stranded DNA (ssDNA). As an oxidase-like nanozyme, it induces a color change in 3,3',5,5'-tetramethylbenzidine (TMB). This color change is visually identifiable and exhibits good selectivity, repeatability, anti-interference ability, and accuracy, along with advantages such as immediacy and high sensitivity. However, interpreting the color development during large-scale sample detection presents challenges. Since colorimetric detection systems often rely on a single color change resulting from the substrate-enzyme reaction, this signal output is difficult to produce a sufficiently significant color difference at low target concentrations. This reliance on the experience of the testing personnel and the equipment is high, especially in the detection of actual food and environmental samples where complex backgrounds and significant signal attenuation further increase the uncertainty of the detection.

[0006] Third, single fluorescence intensity readout is also affected by factors such as light source drift, detector gain, and sample self-absorption / turbidity, making it difficult to obtain reliable conclusions in complex food and environmental matrices, thus limiting its application in rapid on-site screening and resource-constrained scenarios.

[0007] Therefore, there is an urgent need to build a visual detection output system with higher signal strength, better stability, and the ability to meet both quantitative and qualitative detection needs in different scenarios, as well as correction capabilities. Summary of the Invention

[0008] To overcome the limitations of existing technologies, such as limited colorimetric intensity of nucleic acid detection, sensitivity to the environment, and difficulty in visually interpreting low-concentration targets, this invention provides a bimetallic nanozyme, its preparation method, and its application, thereby compensating for the shortcomings of existing technologies.

[0009] To achieve the above-mentioned objectives, the present invention employs the technical solution described below.

[0010] As a first aspect of the invention, the present invention provides a method for preparing bimetallic nanozymes, comprising: using DNA with a single-stranded nucleic acid sequence as a DNA template, and performing an in-situ reduction reaction of platinum salt and silver salt using a high-temperature one-step method to obtain DNA-Ag / Pt bimetallic nanozyme material; wherein, the single-stranded nucleic acid sequence is an oligonucleotide sequence containing a specific base, or a variant having ≥90% homology with the oligonucleotide sequence containing the specific base and maintaining equivalent catalytic enhancement function; the single-stranded nucleic acid sequence is shown in SEQ ID No. 1.

[0011] In a preferred embodiment, the single-stranded nucleic acid sequence further includes a complementary sequence.

[0012] In a preferred embodiment, the single-stranded nucleic acid sequence is shown in SEQ ID No. 2, and contains a binding domain complementary to the 5'-GCCTACGCCACTAGCTCCAACTA-3' sequence.

[0013] As a preferred embodiment, the high-temperature one-step method includes: mixing DNA template, platinum salt and silver salt in a sodium citrate buffer system, shaking vigorously, placing it in a metal bath, heating at 90~100℃ for 5~20 min, and then naturally cooling and annealing to obtain DNA-Ag / Pt bimetallic nanozyme.

[0014] As a second aspect of the invention, the present invention provides a bimetallic nanozyme, which is prepared using the preparation method described above.

[0015] As a third aspect of the invention, the present invention provides an application of bimetallic nanozymes prepared by the preparation method described above in the detection of Vibrio parahaemolyticus.

[0016] As a fourth aspect of the invention, the present invention provides a colorimetric / fluorescent detection method for Vibrio parahaemolyticus, the specific steps of which include:

[0017] (1) After extracting the bacterial genome from the sample to be tested, LAMP amplification was performed to obtain the amplification product;

[0018] (2) The amplification product was mixed with Trigger DNA and CRISPR / Cas12a reaction system and subjected to trans-shearing reaction to inactivate the product, resulting in reaction solution 1.

[0019] (3) Add the DNA-Ag / Pt bimetallic nanozyme as described above to the reaction solution 1, heat the reaction and then cool and anneal naturally to obtain reaction solution 2;

[0020] (4) Mix the reaction solution 2 with the color development system, and interpret the results by visually identifying or detecting the absorbance value.

[0021] In a preferred embodiment, step (1) includes amplifying the target gene using LAMP primers.

[0022] Preferably, the LAMP amplification conditions include isothermal incubation at 60-65°C for 30-45 min.

[0023] Preferably, the LAMP primers include at least primer F3, primer B3, primer FIP, and primer BIP; wherein the sequence of primer F3 is as shown in SEQ ID No. 7; the sequence of primer B3 is as shown in SEQ ID No. 8; the sequence of primer FIP is as shown in SEQ ID No. 9; and the sequence of BIP is as shown in SEQ ID No. 10.

[0024] Preferably, the LAMP primers further include primer LF and / or primer LB; wherein the sequence of primer LF is as shown in SEQ ID No. 11; and the sequence of primer LB is as shown in SEQ ID No. 12.

[0025] In a preferred embodiment, in step (2), the CRISPR / Cas12a reaction system includes: Cas12a protein, crRNA and Trigger DNA.

[0026] Preferably, in the CRISPR / Cas12a reaction system, the final concentration of Cas12a protein is 200 nM and the final concentration of crRNA is 200 nM.

[0027] Preferably, the crRNA sequence is as shown in SEQ ID No. 13, and its target site is adjacent to the TTTV PAM site.

[0028] Preferably, the sequence of the trigger DNA is at least partially complementary to the single-stranded nucleic acid sequence of the DNA template.

[0029] Preferably, the sequence of the Trigger DNA is shown in SEQ ID No. 3.

[0030] Preferably, the conditions for the trans-shear reaction include incubation at 37 °C for 20-30 min.

[0031] Preferably, the inactivation includes inactivation at 85 °C for 5 min.

[0032] Preferably, the colorimetric system is a TMB / H2O2 colorimetric reaction solution, which includes a buffer solution, TMB, and H2O2.

[0033] More preferably, the buffer solution is a 0.2 M acetate-sodium acetate buffer solution with a pH value of 3.8 to 4.2.

[0034] Preferably, the final concentration of TMB is 0.02~0.8 mM; and the final concentration of H2O2 is 0~100 mM.

[0035] In a preferred embodiment, in step (4), the result interpretation includes visually identifying or detecting the 652 nm absorbance value to interpret the result. A dark blue color or a relatively high absorbance value indicates that the target bacteria are not present in the sample; a light blue color or a relatively low absorbance value indicates that the target bacteria are detected in the sample.

[0036] As a preferred embodiment, when the target bacteria are not detected in step (4), the sample to be tested is retested. The retesting method includes:

[0037] (1) Add Initiator DNA to the reaction solution 1 and incubate at room temperature to obtain reaction solution 3;

[0038] (2) DNA-AgNCs nanoclusters were added to the reaction solution 3, and hairpin probe H2 was added at the same time. The reaction solution 4 was obtained by incubation at room temperature.

[0039] (3) The results can be interpreted by visual inspection under ultraviolet light or by detecting the fluorescence intensity at emission wavelengths of 520 nm and 602 nm respectively; the fluorescence intensity at emission wavelength of 520 nm is recorded as Fg, and the fluorescence intensity at emission wavelength of 602 nm is recorded as Fr; the fluorescence color changes from orange-red to green, or the Fg / Fr value increases, indicating that the target bacteria are detected in the sample.

[0040] In a preferred embodiment, the sequence of the initiator DNA is complementary to the sequence of the trigger DNA.

[0041] Preferably, the sequence of the initiator DNA is shown in SEQ ID No. 4.

[0042] Preferably, the sequence of the auxiliary hairpin probe H2 is shown in SEQ ID No. 6.

[0043] Preferably, the DNA-AgNCs nanoclusters are obtained by reacting stem-circular DNA strand H1 with silver nitrate and sodium borohydride.

[0044] Specifically, the preparation method of the DNA-AgNCs nanoclusters includes: using stem-circular DNA strand H1 as a template, mixing it with silver nitrate in PB buffer, adding sodium borohydride as a reducing agent, and then carrying out a reduction reaction to prepare the nanoclusters.

[0045] Preferably, the sequence of the stem-circular DNA strand H1 is as shown in SEQ ID No. 5.

[0046] Preferably, the initiator DNA is complementary to the H1 portion of the stem-circular DNA strand.

[0047] For example, the present invention provides a method for preparing the DNA-AgNCs nanoclusters, the specific steps of which include: adding a stem-circular DNA template sequence (SEQ ID No. 5, 100 μM, 10 μL) and AgNO3 (1 mM, 12 μL) to PB buffer (20 mM, 66 μL), mixing well, vortexing thoroughly for 2 min, and reacting in the dark at 4 ℃ for 30 min; then, adding sodium borohydride (1 mM, 12 μL) and vortexing thoroughly for 2 min, and storing at 4 ℃ for 24 h for later use.

[0048] As a fifth aspect of the invention, the present invention provides a kit for detecting Vibrio parahaemolyticus, comprising at least: a bimetallic nanozyme prepared by the preparation method described above, DNA-AgNCs nanoclusters prepared using stem-circular DNA strand H1 as a template, a LAMP primer set, Bst 2.0 DNA polymerase; Cas12a protein and its crRNA, Trigger DNA, Initiator DNA, and TMB / H2O2 colorimetric reaction solution.

[0049] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0050] 1. This invention utilizes the programmable nature of DNA templates to precisely guide the coordination and in-situ reduction of metal ions to construct catalytically active silver / platinum bimetallic nanoclusters, and also to regulate the emission behavior of luminescent silver nanoclusters.

[0051] 2. DNA-Ag / Pt bimetallic nanozymes (DNA-Ag / Pt NCs nanozymes) prepared using DNA as a template have good biocompatibility and stable peroxidase-like activity. By regulating the base composition and spatial configuration of the DNA sequence, the size and electronic structure of the nanozyme can be effectively affected, thereby significantly improving catalytic efficiency and colorimetric signal intensity.

[0052] 3. Compared with traditional materials, DNA-Ag / Pt bimetallic nanozymes and DNA-AgNCs nanoclusters have the characteristics of programmable design and easy integration, making them suitable for constructing multi-signal synergistic molecular diagnostic systems.

[0053] 4. The combination of colorimetric attenuation of DNA-Ag / Pt bimetallic nanozymes and ratiometric fluorescence readout of DNA-AgNCs nanoclusters not only helps to improve the sensitivity and specificity of Vibrio parahaemolyticus detection, but also enables rapid visual qualitative interpretation and semi-quantitative detection without the need for specialized instruments, providing more efficient and reliable technical support for rapid on-site screening in food safety and environmental monitoring. Attached Figure Description

[0054] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0055] Figure 1 This is a schematic diagram of the colorimetric / fluorescence dual-mode detection method for Vibrio parahaemolyticus constructed in this invention.

[0056] Figure 2 This is a transmission electron microscope (TEM) image of the DNA-AgNCs nanoclusters prepared in Example 1 of this invention.

[0057] Figure 3 This is a histogram showing the size distribution of the DNA-AgNCs nanoclusters prepared in Example 1 of this invention.

[0058] Figure 4 This is a comparison of the UV-Vis absorption spectra of DNA-AgNCs nanoclusters prepared under different reaction systems using H1 as a template in Example 1 of this invention.

[0059] Figure 5 This is a dual-emission fluorescence spectrum of the DNA-AgNCs nanoclusters prepared in Example 1 of this invention, wherein the blue and green lines are excitation spectra, and the yellow and red lines are emission spectra.

[0060] Figure 6 This is a transmission electron microscope (TEM) image of the DNA-Ag / Pt bimetallic nanozyme prepared in Example 3 of this invention.

[0061] Figure 7 This is a histogram showing the size distribution of the DNA-Ag / Pt bimetallic nanozyme particles prepared in Example 3 of this invention.

[0062] Figure 8 This is a comparison of the fluorescence emission spectra of the DNA-AgNCs nanoclusters prepared in Example 1 and the DNA-Ag / Pt bimetallic nanozymes prepared in Example 3 of this invention.

[0063] Figure 9The figures show the HAADF diagram and EDS elemental distribution mapping of the DNA-Ag / Pt bimetallic nanozyme prepared in Example 3 of this invention. The figures show the spatial distribution of silver and platinum elements (scale bar = 200 nm).

[0064] Figure 10 This is the energy dispersive X-ray spectrum of the DNA-Ag / Pt bimetallic nanozyme prepared in Example 3 of this invention.

[0065] Figure 11 This is a comparison diagram of the enzyme catalytic activities of different DNA-Ag / Pt bimetallic nanozymes synthesized using different template DNAs in Examples 2, 3 and Comparative Example 1 of the present invention.

[0066] Figure 12 This is a kinetic performance analysis diagram of the DNA-Ag / Pt bimetallic nanozyme prepared in Example 4 of this invention.

[0067] Figure 13 This is a comparison chart showing the feasibility verification of colorimetric signal detection of the DNA-Ag / Pt bimetallic nanozyme prepared in Example 3 of this invention under different colorimetric system conditions.

[0068] Figure 14 This is a comparison diagram showing the feasibility of using the DNA-Ag / Pt bimetallic nanozyme prepared in Example 3 of this invention for colorimetric signal detection under conditions of no target presence and the presence or absence of Cas12a or crRNA in the system.

[0069] Figure 15 This is a CHA amplification reaction of the template sequence of DNA-AgNCs nanoclusters prepared in Example 1 of the present invention and its assembly process under different conditions, as shown in the non-denaturing PAGE images.

[0070] Figure 16 This is the assembly process of the template sequence of the DNA-AgNCs nanocluster prepared in Example 1 of the present invention under the action of the target-triggered CRISPR / Cas12a system in the CHA amplification reaction and its non-denaturing PAGE image.

[0071] Figure 17 This is a dual-emission fluorescence spectrum of the DNA-AgNCs nanoclusters prepared in Example 1 of this invention, mediated by the Cas12a / crRNA reaction in the presence or absence of a target, where NC stands for Negative Control and PC stands for Positive Control.

[0072] Figure 18This is a comparison diagram of the fluorescence intensity and fluorescence ratio (Fg / Fr) of the DNA-AgNCs nanoclusters prepared in Example 1 of this invention under the conditions of presence or absence of a target, and under the presence or absence of Cas12a or crRNA in the system, as well as the color development of the solution under ultraviolet light.

[0073] Figure 19 This is the sensitivity determination result of the colorimetric / fluorescence dual-mode detection method for Vibrio parahaemolyticus constructed in Example 5 of this invention.

[0074] Figure 20 The results of the colorimetric / fluorescence dual-mode detection method for Vibrio parahaemolyticus constructed in Example 5 of this invention are shown by visual observation under different target concentrations and by solution color development under ultraviolet light.

[0075] Figure 21 This is a comparison chart of the specificity determination of the colorimetric / fluorescence dual-mode detection method for Vibrio parahaemolyticus constructed in Example 5 of the present invention, where NTC stands for No Bacteria Template Control.

[0076] Figure 22 These are the UV-Vis absorption spectra of the nanoparticles prepared in Example 3 and Comparative Examples 2-5 of this invention; wherein, 1 mM Ag corresponds to the nanoparticles prepared in Example 2, 2 mM Pt corresponds to the nanoparticles prepared in Example 3, ssDNA-AgPt NCs corresponds to the nanoparticles prepared in Example 3, dsDNA-AgPt NCs corresponds to the nanoparticles prepared in Example 4, and Control corresponds to the nanoparticles prepared in Example 4.

[0077] Figure 23 This is a comparison table of the catalytic activities of nanozymes disclosed in Example 3 of the present invention and in the prior art. Detailed Implementation

[0078] Detailed embodiments of the invention are disclosed herein. It should be understood that the disclosed embodiments are merely exemplary of the invention, which can be embodied in various forms. Therefore, the specific functional details disclosed herein should not be construed as limiting, but rather as the basis for the claims and as intended to teach those skilled in the art to employ the representative basis of the invention in different ways in any suitable detailed embodiments.

[0079] As one aspect of the invention, the present invention first provides a bimetallic nanozyme, a DNA-Ag / Pt bimetallic nanozyme material prepared with a specific single-stranded DNA template sequence (SEQ ID NO:1) or containing the DNA template sequence.

[0080] Preferably, the oligonucleotide sequence of the single-stranded DNA sequence is SEQ ID No. 1, as shown in 5'-CGTCCCCCCCCCACG-3'; or, a variant having ≥90% homology with SEQ ID No. 1 and maintaining equivalent catalytic enhancement function.

[0081] Preferably, the bimetallic nanozyme provided by the present invention is a single-stranded nucleic acid nanozyme with peroxidase-like activity and adjustable enzyme activity.

[0082] In a preferred embodiment, the bimetallic nanozyme contains a template nucleic acid sequence and a complementary sequence, thereby giving the obtained bimetallic nanozyme the characteristic of tunable enzyme activity.

[0083] As a second aspect of the invention, the present invention also provides a method for preparing bimetallic nanozymes, comprising:

[0084] In a preferred embodiment, a single-stranded DNA sequence is used as a template and obtained by in-situ reduction and annealing in a sodium citrate buffer system using a metal salt solution containing silver and platinum salts.

[0085] Preferably, the preparation method is carried out in a sodium citrate buffer system.

[0086] The metal salt solution is a mixed solution of K2PtCl4 and AgNO3.

[0087] See Figure 1 This invention constructs a colorimetric / fluorescence dual-mode detection method for Vibrio parahaemolyticus, and the schematic diagram of the detection principle of this invention is provided. Figure 1 As shown in Figure A, firstly, a DNA-AgNCs nanocluster and a DNA-Ag / Pt bimetallic nanozyme are provided; using a single-stranded nucleic acid sequence as a template, Pt is used in a sodium citrate buffer system. 2+ With Ag + In-situ reduction and annealing (95 °C, 10 min) yielded ssDNA-Ag / Pt bimetallic nanozymes, which were then converted to dsDNA-Ag / Pt bimetallic nanozymes by adding Trigger DNA.

[0088] DNA-AgNCs nanoclusters were prepared by mixing stem-circular DNA strand H1 with silver nitrate in PB buffer, adding sodium borohydride as a reducing agent, and then carrying out a reduction reaction.

[0089] Figure 1 Figure B illustrates the extraction of bacterial genomic DNA and the principle of LAMP isothermal amplification. The fragment containing the Vibrio parahaemolyticus-specific gene (tlh) is amplified by LAMP isothermal amplification, and the amplification product is used as the target.

[0090] Furthermore, such as Figure 1 As shown in Figure C, by combining DNA-Ag / Pt bimetallic nanozymes and DNA-AgNCs nanoclusters, the amplification products are mixed with the CRISPR / Cas12a reaction system. When the CRISPR / Cas12a system recognizes the target, it activates the trans-cleavage reaction to cleave the trigger DNA. That is, if the sample contains the target Vibrio parahaemolyticus, the trans-cleavage reaction will be initiated; if there is no target Vibrio parahaemolyticus, the reaction will not occur.

[0091] This detection method utilizes this difference to modulate the microscopic properties of DNA-Ag / Pt bimetallic nanozymes and DNA-AgNCs nanoclusters, thereby further realizing colorimetric / fluorescence dual-modal signal output.

[0092] The combination of catalytic colorimetric attenuation of DNA-Ag / Pt bimetallic nanozymes and ratiometric fluorescence readout of DNA-AgNCs nanoclusters not only helps to improve the sensitivity and specificity of Vibrio parahaemolyticus detection, but also enables rapid visual qualitative interpretation and semi-quantitative detection without the need for specialized instruments.

[0093] In some specific embodiments, the DNA-Ag / Pt bimetallic nanozyme is obtained by in-situ reduction and annealing of a single-stranded nucleic acid sequence with K2PtCl4 and AgNO3 in a sodium citrate buffer system, using a single-stranded nucleic acid sequence as a template; the single-stranded nucleic acid nanozyme has peroxidase-like activity and the enzyme activity is tunable.

[0094] Preferably, the oligonucleotide sequence is SEQ ID No. 1, as shown in 5'-CGTCCCCCCCCCACG-3'; or a variant having ≥90% homology with it and maintaining equivalent catalytic enhancement function. The metal salt solution is a silver salt and a platinum salt; the preparation method is in-situ reduction and annealing with K2PtCl4 and AgNO3 in a sodium citrate buffer system.

[0095] Preferably, the nucleic acid sequence is an oligonucleotide sequence containing specific bases of the sequence SEQ ID No. 1; the oligonucleotide sequence is SEQ ID No. 2, wherein...

[0096] 5'-GCCTACGCCACTAGCTCCAACTA-3' is a complementary sequence binding domain; the conditions for tunable enzyme activity include: template nucleic acid sequence and complementary sequence.

[0097] In some specific embodiments, the preparation method of bimetallic nanozymes using single-stranded nucleic acid as a template includes the following steps: adding single-stranded nucleic acid template (SEQ ID No. 1), K2PtCl4, and AgNO3 into sodium citrate buffer and mixing well; after vigorous shaking, placing it in a metal bath, heating at 95°C for 10 min, and then naturally cooling and annealing to obtain a DNA-Ag / Pt bimetallic nanozyme working solution with peroxidase-like activity.

[0098] Among them, the bimetallic nanozymes synthesized with SEQ ID No. 1 compared with other sequences in the comparative examples showed an improvement in catalytic efficiency of about 15%.

[0099] In some specific embodiments, the method for detecting Vibrio parahaemolyticus based on colorimetric / fluorescence dual-mode signals of DNA-Ag / Pt bimetallic nanozymes or DNA-AgNCs nanoclusters includes the following steps:

[0100] (1) Based on the thermolabile hemolysin tlh gene of Vibrio parahaemolyticus, six LAMP amplification primers (SEQ ID No. 7–12) were designed. After extracting the bacterial genome from the sample to be tested, the sample was amplified at 60~65 ℃ (preferably 63~65 ℃) for 30~45 min to obtain the amplification product.

[0101] (2) Mixing the amplification product with the CRISPR / Cas reaction system, specifically including:

[0102] A Cas12a protein / crRNA ribonucleoprotein complex was mixed and reacted to enable crRNA to specifically recognize the tlh amplified fragment and activate the non-specific single-stranded DNA cleavage activity (trans cleavage activity) of the Cas12a enzyme to obtain reaction solution 1. In this reaction solution, a pre-placed oligonucleotide sequence Trigger DNA (SEQ ID No. 3) was prepared, and a corresponding crRNA sequence (SEQ ID No. 13) was designed based on the amplified target region. The pre-placed Trigger DNA sequence in the system carried a nucleotide segment (10~40 nt) complementary to SEQ ID No. 2.

[0103] (3) Inactivate the reaction solution obtained in step (2) at 85 °C for 5 min to obtain reaction solution 2;

[0104] (4) Add DNA-Ag / Pt bimetallic nanozyme to reaction solution 2, heat at 95 °C for 2 min, cool naturally and anneal to obtain reaction solution 3;

[0105] (5) Mix the reaction solution 3 obtained in step (4) with the TMB-H2O2 system and interpret the result by visually identifying or detecting the absorbance value at 652nm. If the color is dark blue or the absorbance value is relatively high, it indicates that there are no target bacteria in the sample; if the color is light blue or the absorbance value is relatively low, it indicates that target bacteria are detected in the sample.

[0106] Preferably, the total volume of the CRISPR / Cas12a reaction system of the present invention is 20 µL, including 2 µL of 10×Cas reaction buffer, a final concentration of Cas12a enzyme of 200 nM, a final concentration of crRNA of 200 nM (Cas12a enzyme and crRNA pre-assemble for 10–15 min to form a ribonucleoprotein complex), 0.5–5 µL of LAMP amplification product (equivalent to 25–250 nM target DNA) is added, and trigger DNA with a final concentration of 1000–2000 nM is added. The trigger DNA carries a 10–40 nt nucleotide segment complementary to SEQ ID No. 2; preferably, the segment is 10–35 nt, and most preferably, it is 35 nt.

[0107] Preferably, the colorimetric buffer is a 0.2 M acetate-sodium acetate buffer with a pH of 3.0 to 5.0; preferably, the pH is 4.0; the final concentration of TMB is 0.02 to 0.8 mM, preferably, the final concentration of TMB is 0.8 mM.

[0108] Preferably, the final concentration of H2O2 is 0~100 mM, more preferably, the final concentration of H2O2 is 10~50 mM, and most preferably, the final concentration of H2O2 is 50 mM.

[0109] Preparation of DNA-AgNCs nanoclusters:

[0110] The stem-circular DNA strand H1 was prepared by mixing it with silver nitrate in PB buffer, adding sodium borohydride as a reducing agent, and then performing a reduction reaction. The stem-circular DNA was mixed with AgNO3 in PB buffer solution, vigorously shaken for 2 min, and then incubated at 4 ℃ in the dark for 30 min. Afterwards, sodium borohydride was added, the mixture was vigorously shaken for 2 min, and then stored at 4 ℃ for 24 h before use. The sequence of the stem-circular DNA strand H1 is SEQ ID No. 5: 5'-TAT CCG TCC CCC CCC CAC GGA TAG CCT ACGCCA CTA GCT CCA ACT AGG AGA CCA TGT TAG TTG GAG CTA GTG GCC CCC CTA ATT CCCCC-3'.

[0111] A colorimetric / fluorescence dual-mode detection method for Vibrio parahaemolyticus, comprising the following steps:

[0112] (1) Add the oligonucleotide sequence of Initiator DNA (SEQ ID No. 4) to reaction solution 2 in the above scheme and incubate at room temperature for 10 min;

[0113] (2) Add DNA-AgNCs nanoclusters prepared using sequence SEQ ID No. 5 as a template, and add auxiliary sequence H2 (SEQ ID No. 6) at the same time, and incubate at room temperature for 1 hour;

[0114] (3) The results can be determined by visual identification under ultraviolet light or by detecting the fluorescence intensity at emission wavelengths of 520 nm and 602 nm. The fluorescence color changes from orange-red to green, or the Fg / Fr value increases, indicating that the target bacteria are detected in the sample. The excitation wavelengths are 406 nm and 550 nm, respectively. The fluorescence intensity at emission wavelength of 520 nm is recorded as Fg, and the fluorescence intensity at emission wavelength of 602 nm is recorded as Fr.

[0115] The technical solution of the present invention will be illustrated below through specific embodiments.

[0116] Example 1

[0117] This embodiment provides a method for synthesizing DNA-AgNCs nanoclusters, comprising: using stem-circular DNA strand H1 as a template, mixing it with silver nitrate in PB buffer, adding sodium borohydride as a reducing agent, and then carrying out a reduction reaction to prepare the nanoclusters.

[0118] The specific procedure is as follows: Add the stem-circular DNA template sequence (SEQ ID No. 5, 100 μM, 10 μL) and AgNO3 (1 mM, 12 μL) to PB buffer (20 mM, 66 μL), mix well, vortex thoroughly for 2 min, and incubate at 4°C in the dark for 30 min. Then, add sodium borohydride (1 mM, 12 μL) and vortex thoroughly for 2 min, and store at 4°C for 24 h before use.

[0119] The DNA-AgNCs nanoclusters were characterized by transmission electron microscopy and particle size distribution analysis. The process involved diluting the prepared DNA-AgNCs nanocluster stock solution with water by 10 times, shaking and mixing it, then dropping it onto an ultrathin copper grid, drying it, and observing its morphology under a transmission electron microscope.

[0120] like Figure 2The image shown is a transmission electron microscope (TEM) image of DNA-AgNCs nanoclusters. As can be seen, the synthesized ratiometric silver nanocluster fluorescent probe is spherical and exhibits good dispersibility. Further statistical analysis of particles in typical regions of the TEM image was performed. Figure 3 The histogram of the particle size distribution of DNA-AgNCs nanoclusters shows that the average diameter of the obtained silver nanoclusters is approximately 2.37 ± 0.35 nm, indicating that the nanoclusters have good monodispersity and reproducibility. The main peak particle size is concentrated in the range of about 2 to 3 nm, which is consistent with the typical scale of silver nanoclusters that can produce characteristic fluorescence signals.

[0121] Furthermore, this embodiment also compared different reaction systems, including: a reaction system containing only H1 single-stranded DNA (i.e., stem-circular DNA strand H1), labeled "Only H1"; a reaction system in which H1 single-stranded DNA was added to AgNO3, labeled "H1+AgNO3"; and a reaction system in which NaBH4 was further added to the H1+AgNO3 reaction system, labeled "H1+AgNO3+NaBH4", in which NaBH4 reduced silver ions. The results are as follows. Figure 4 The image shows a comparison of the UV-Vis absorption spectra of DNA-AgNCs nanoclusters prepared under different reaction systems. As can be seen from the image, the Only H1 reaction system containing only H1 single-stranded DNA has almost no significant absorption characteristic curve in the 300~800 nm range; the UV absorption of the H1+AgNO3 reaction system increases slightly, but is still weak; while the H1+AgNO3+NaBH4 reaction system shows a significantly enhanced absorption characteristic compared with other reaction systems, and exhibits multiple fluctuating peaks in the visible light region, indicating that silver nanoclusters with stable optical response are formed after the reduction reaction.

[0122] like Figure 5 As shown, the DNA-AgNCs nanoclusters provided in Example 1 were subjected to excitation-emission spectroscopy to obtain their fluorescence excitation and emission spectra. The optimal excitation wavelengths were determined to be 406 nm and 550 nm, and the optimal emission wavelengths were determined to be 520 nm and 602 nm. In the figure, the blue and green lines are the excitation spectra, and the yellow and red lines are the emission spectra.

[0123] Example 2

[0124] This embodiment provides a method for synthesizing DNA-Ag / Pt bimetallic nanozymes via a one-step high-temperature process, the specific steps of which are as follows:

[0125] The DNA template sequence (DNA-Ag / Pt bimetallic nanozyme template chain 1, SEQ ID No. 1, 100 μM, 100 μL), K2PtCl4 (1 mM, 200 μL) and AgNO3 (1 mM, 100 μL) were added to sodium citrate buffer (10 mM, 600 μL), mixed thoroughly, vortexed for 2 min, placed in a 95℃ metal bath for 10 min, and allowed to cool and anneal naturally to obtain a working solution of DNA-Ag / Pt nanozyme with peroxidase-like activity.

[0126] Example 3

[0127] This embodiment provides a method for synthesizing DNA-Ag / Pt bimetallic nanozymes using DNA-Ag / Pt bimetallic nanozyme template chain 2 (SEQ ID No. 2). The preparation steps are the same as in Example 2, including:

[0128] The DNA template sequence (DNA-Ag / Pt bimetallic nanozyme template chain 2 or ssDNA, SEQ ID No. 2, 100 μM, 100 μL), K2PtCl4 (1 mM, 200 μL) and AgNO3 (1 mM, 100 μL) were added to sodium citrate buffer (10 mM, 600 μL), mixed thoroughly, vortexed for 2 min, placed in a 95℃ metal bath for 10 min, and allowed to cool and anneal naturally to obtain a working solution of DNA-Ag / Pt nanozyme with peroxidase-like activity.

[0129] The DNA-Ag / Pt bimetallic nanozymes were characterized by transmission electron microscopy, energy dispersive spectroscopy (EDS) analysis, and particle size distribution analysis. The process involved diluting the prepared DNA-Ag / Pt bimetallic nanozyme stock solution with water by 10 times, shaking and mixing it, then dropping it onto an ultrathin copper grid, drying it, and observing its morphology under a transmission electron microscope.

[0130] like Figure 6 The image shown is a transmission electron microscope (TEM) image of the DNA-Ag / Pt bimetallic nanozyme synthesized in this embodiment. As can be seen from the image, the DNA-Ag / Pt bimetallic nanozyme particles are uniformly distributed without obvious aggregation, and the small nanoclusters indicate that the DNA-Ag / Pt bimetallic nanozyme was successfully synthesized in this embodiment.

[0131] like Figure 7 The particle size distribution of the DNA-Ag / Pt bimetallic nanozyme particles in a typical field of view was measured and statistically analyzed. The average diameter was found to be approximately 4.8 ± 0.69 nm, indicating that the DNA-Ag / Pt bimetallic nanozyme has good monodispersity.

[0132] like Figure 8The figure shows a comparison of the fluorescence spectra of the DNA-Ag / Pt bimetallic nanozyme prepared in this embodiment and the DNA-AgNCs nanoclusters prepared in Example 1, measured by a fluorescence spectrophotometer. The figure shows that the DNA-AgNCs nanoclusters have a significant fluorescence emission peak at 602 nm, while the DNA-Ag / Pt bimetallic nanozyme does not show a fluorescence emission peak. This indicates that the introduction of platinum ions significantly weakens or essentially quenches the fluorescence emission signal of the DNA-Ag / Pt bimetallic nanozyme.

[0133] To further confirm the elemental composition and spatial distribution of the DNA-Ag / Pt bimetallic nanozyme described in this invention, high-angle annular dark-field imaging (HAADF) and EDS elemental analysis were performed on representative products. The results are as follows: Figure 9 As shown in the figure, the spatial distribution of silver and platinum elements is illustrated. Well-dispersed nanoparticles can be observed within the field of view of a transmission electron microscope. Combined with the corresponding EDS elemental surface scan results, it can be seen that Ag and Pt exhibit highly overlapping distribution signals within the same nanoparticle region, with no obvious spatial separation or large-scale phase stratification observed. At the same time, signals of light elements such as C, N, and P, as well as O element signals, were also detected, where C, N, and P correspond to the organic components of the DNA oligonucleotide template backbone. The results indicate that silver and platinum in the DNA-Ag / Pt bimetallic nanozyme do not form independent particles, but rather are synergistically reduced and constructed into DNA-Ag / Pt bimetallic nanozymes under the confinement of the DNA template.

[0134] like Figure 10 The energy dispersive X-ray spectrum of the DNA-Ag / Pt bimetallic nanozyme shown can simultaneously resolve the characteristic X-ray emission peak signals corresponding to Ag and Pt in the same sample, thus confirming at the composition level that silver and platinum elements coexist in the nanozyme particles.

[0135] Example 4

[0136] This embodiment analyzes the kinetic properties of DNA-Ag / Pt bimetallic nanozymes, specifically including: quantitatively evaluating the peroxidase-like catalytic activity of the DNA-Ag / Pt bimetallic nanozyme obtained in Example 3; and obtaining the Michaelis constant (Km) of the nanozyme by measuring its initial reaction rate with tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). m ) and maximum reaction rate (V max );like Figure 12 The figure shown is a kinetic performance analysis diagram of DNA-Ag / Pt bimetallic nanozymes.

[0137] Figure 12As shown in Figure A, under the condition of a fixed H2O2 concentration of 50 mM, the initial reaction rate was obtained by gradually increasing the TMB concentration (0.02 mM, 0.04 mM, 0.06 mM, 0.1 mM, 0.2 mM, 0.4 mM, and 0.8 mM, respectively), and the kinetic analysis of TMB was obtained; the corresponding Lineweaver–Burk double reciprocal linear fit is shown in Figure A. Figure 12 As shown in Figure B, the Michaelis constant K is obtained. m The maximum reaction rate V is 0.74 mM. max It is 14.26×10 -8 Ms -1 Similarly, in Figure 12 In Figure C, with the TMB concentration fixed at 0.8 mM, the H2O2 concentration was gradually changed (2 mM, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM), and the kinetic analysis of H2O2 was obtained accordingly; the corresponding Lineweaver–Burk double reciprocal linear fit is shown below. Figure 12 As shown in Figure D, the Michaelis constant K is obtained. m The maximum reaction rate V is 18.15 mM. max It is 8.29×10 -8 Ms -1 .

[0138] Analysis of the kinetic properties of DNA-Ag / Pt bimetallic nanozymes revealed that they possess peroxidase-like catalytic activity.

[0139] Furthermore, this invention also compares the DNA-Ag / Pt bimetallic nanozymes provided in the above embodiments with those of DNA-Ag / Pt bimetallic nanozymes and other nanozymes reported in the prior art through kinetic performance (Michaelis constant K). m and maximum reaction rate V max The differences in enzyme activity were assessed, comparing the Vo of the nanozyme prepared in this invention with those of existing HRP enzymes. max Compared to the value (10.0), the DNA-Ag / Pt bimetallic nanozyme exhibited a higher Vc for TMB catalysis. max The value (14.26) indicates that the DNA-Ag / Pt bimetallic nanozyme provides a faster and more efficient reaction rate and stronger colorimetric output. Meanwhile, this V... maxThe value is comparable to HRP, indicating that the peroxidase-like activity of the nanozyme prepared in this invention is sufficient to support highly sensitive colorimetric detection. The above kinetic data confirm that the Ag / Pt bimetallic nanozyme synthesized based on the novel DNA template optimized in this invention is a robust peroxidase mimic material, providing a highly efficient colorimetric signal amplification unit for a dual-mode biosensing platform.

[0140] The bimetallic nanozyme prepared and synthesized in this invention exhibits peroxidase-like catalytic activity. Its enzyme activity can be evaluated by analyzing its kinetic properties, and its activity against K... m and V max Tests were conducted.

[0141] See Figure 23 This table compares the catalytic activity of existing nanozymes with that of Example 3; "Effective reaction rate" in enzyme kinetics refers to the rate of enzyme-catalyzed reaction at practically achievable substrate concentrations (usually far below saturation concentrations), primarily determined by Km (Michaelis constant) and V. max (Maximum reaction rate) is determined by two parameters; the smaller the Km value, the higher the affinity between the enzyme and the substrate, and the higher the reaction rate can be achieved at a lower substrate concentration; V max The higher the value, the stronger the maximum catalytic ability of the enzyme when it is completely saturated with the substrate.

[0142] In colorimetric detection, "stronger output" generally means that, under the same reaction time and enzyme dosage, the product (oxidized TMB) is generated at a faster rate and reaches a higher final concentration, resulting in a more significant change in absorbance (deeper color), a higher signal-to-noise ratio, and higher detection sensitivity. Therefore, a higher Vo max This means that more colored products can be produced per unit of time, thereby achieving faster color changes and stronger final signals.

[0143] Compared with other biological enzymes or nanozymes ( Figure 23 (Serial numbers 1-5) The nanozymes synthesized in Example 3 have high V-resistance to TMB. max The value indicates that at high substrate concentrations, the nanozyme of this invention has a faster effective reaction rate and stronger colorimetric output performance.

[0144] Specifically, the maximum reaction rate of the nanozyme of this invention with the substrate TMB is V. max (TMB) = 14.26 × 10 -8 M·s -1 It is significantly higher than that of previously reported DNA-Ag / Pt nanozymes ( Figure 23 5.42 × 10 (in serial number 5) -8 M·s -1(Approximately 2.63 times higher), therefore, more oxidized TMB (oxTMB) can be generated more quickly within the same reaction time, resulting in a faster effective reaction rate and stronger colorimetric output (Abs). 652nm (The signal amplification is greater, and the color rendering is deeper). Meanwhile, this V... max The (TMB) value is 10.0 × 10⁻⁶ for natural peroxidase HRP. -8 M·s -1 The fact that they are on the same order of magnitude indicates that the peroxidase-like activity of the nanozyme prepared in Example 3 is sufficient to support high-sensitivity colorimetric detection.

[0145] Comparative Example 1

[0146] In this comparative example, SEQ ID Nos. 14-25 were used as template sequences (referred to as A15, T15, C15, G15, E1, E2, E3, E4, E5, E6, E7, and E8, respectively) to replace the DNA template sequence SEQ ID No. 1 in Example 2. DNA-Ag / Pt bimetallic nanozymes were synthesized using the same method as in Example 2, through a one-step high-temperature synthesis method. Specifically, the DNA template sequences (SEQ ID Nos. 14-25, 100 μM, 100 μL), K2PtCl4 (1 mM, 200 μL), and AgNO3 (1 mM, 100 μL) were added to sodium citrate buffer (10 mM, 600 μL), mixed thoroughly, vortexed for 2 min, placed in a 95 ℃ metal bath for 10 min, and then naturally cooled and annealed to obtain DNA-Ag / Pt bimetallic nanozymes with peroxidase-like activity.

[0147] Bimetallic nanozymes were synthesized using oligonucleotide sequences A15, T15, C15, and G15 as template chains to investigate the influence of template chain base composition on the construction process and catalytic performance of bimetallic nanozymes. Figure 11 The results showed that the peroxidase-like activities of the prepared Ag / Pt nanozymes exhibited a clear order: C15 > A15 > G15 > T15, indicating that their catalytic activity is largely regulated by the coordination affinity between nucleotide bases and metal ions. This trend is consistent with the DNA-templated silver nanocluster system, in which cytosine and Ag... + The strong coordination between them can effectively provide metal binding sites and nucleation centers, thereby promoting the nucleation and growth of nanoclusters.

[0148] The DNA template sequence E5 is a cytosine-rich sequence. Further optimization of the cytosine content based on E5 yielded the optimized template sequence E9 (SEQ ID No. 1, sequence 5'-CGTCCCCCCCCCACG-3'). The Ag / Pt nanozyme synthesized using the E9 template is significantly improved compared to the previously reported sequence E1 (see LL. Wu et al., Colorimetric detection of Hg). 2+ Based on inhibiting the peroxidase-like activity of DNA-Ag / Pt nanoclusters, RSC Adv. 6 (2016) 75384-75389. https: / / doi.org / 10.1039 / C6RA12597) showed approximately 15% higher catalytic activity.

[0149] Furthermore, the present invention uses the template sequence E9 as a homologous sequence to synthesize an equivalent template sequence E10 (as shown in SEQ ID No. 2) with ≥90% homology, and uses it as a working template for bimetallic nanozymes. The catalytic results show that the bimetallic nanozymes synthesized using the E10 sequence containing the E9 sequence as a template still have high catalytic activity.

[0150] For detailed results, please refer to... Figure 11 The DNA-Ag / Pt bimetallic nanozymes synthesized using the different DNA template sequences described above catalyze the substrates, resulting in color development using UV-Vis absorption light. 652nm The signal intensity comparison diagram shows that A15, T15, C15, G15, E1, E2, E3, E4, E5, E6, E7, and E8 are DNA-Ag / Pt bimetallic nanozymes synthesized using SEQ ID No. 14-25 as templates, respectively. E9 is a nanozyme synthesized using SEQ ID No. 1 as a template (i.e., Example 2), and E10 contains an equivalent template sequence of E9 (i.e., Example 3). Obviously, the peroxidase-like activity of the nanozyme synthesized using E9 (Example 2) is significantly higher than that of the peroxidase-like activity of the nanozymes synthesized using SEQ ID No. 14-25 as templates.

[0151] Comparative Example 2

[0152] The only difference between this comparative example and Example 3 is that K2PtCl4 was not added during the preparation process; only ssDNA and AgNO3 were added. The other preparation steps were the same, and the resulting product was 1 mM Ag.

[0153] Comparative Example 3

[0154] The only difference between this comparative example and Example 3 is that AgNO3 was not added during the preparation process; only ssDNA and K2PtCl4 were added. The other preparation steps were the same, and the total amount was 2 mM Pt.

[0155] Comparative Example 4

[0156] The only difference between this comparative example and Example 3 is that Trigger DNA, which is complementary to the ssDNA sequence, is added to the DNA-Ag / Pt bimetallic nanozyme working solution prepared in Example 3 at a ratio of 1:1 (molar ratio), and is called dsDNA-AgPt NCs.

[0157] Comparative Example 5

[0158] The only difference between this comparative example and Example 3 is that ssDNA is not added during the preparation process; only AgNO3 and K2PtCl4 are added. The preparation steps are the same, and this is referred to as Control.

[0159] See Figure 22 The images shown are the UV-Vis absorption spectra of the nanoparticles obtained in Example 3 and Comparative Examples 2-5. In the UV-Vis absorption spectra of the nanoparticles obtained in Comparative Examples 2-5, there are no obvious characteristic peaks.

[0160] Example 5

[0161] This embodiment illustrates the specific application of DNA-Ag / Pt bimetallic nanozymes and DNA-AgNCs nanoclusters in the detection of Vibrio parahaemolyticus, specifically including:

[0162] 1. Using the *Vibrio parahaemolyticus* heat-labile hemolytic toxin encoding gene *tlh* as a target gene for detecting *Vibrio parahaemolyticus*, LAMP primers were designed for isothermal amplification, and a CRISPR / Cas12a system was used to achieve non-specific cleavage of the trigger DNA sequence (SEQ ID No. 3) triggered by the trans-cleavage reaction after target recognition. The trigger DNA sequence is complementary to the DNA template strand of the nanozyme (SEQ ID No. 2), and can regulate peroxidase-like catalytic activity by forming a double strand through binding with the DNA-Ag / Pt bimetallic nanozyme synthesized with the template strand. Initiator DNA (SEQ ID No. 4) and trigger DNA are partially complementary sequences in the system, and the initiator DNA can trigger the CHA amplification reaction mediated by the H1 sequence (SEQ ID No. 5) and the H2 sequence (SEQ ID No. 6). By controlling the microscopic distance between the silver nanoclusters at both ends of H1, ratiometric fluorescence signal conversion can be achieved. When the target bacteria are absent from the sample, the Trigger DNA remains intact and binds to the DNA-Ag / Pt bimetallic nanozyme template strand to form a double strand, enhancing its catalytic activity and resulting in a visible deep blue color in the detection solution. Conversely, the Trigger DNA forms a double strand with the Initiator, and the H1 sequence maintains its hairpin structure, causing the solution to display an orange-red composite fluorescence signal under UV light. When the target *Vibrio parahaemolyticus* is detected in the sample, the target gene fragment amplified by LAMP activates the CRISPR / Cas12a trans-cleavage reaction, cleaving the Trigger DNA into oligonucleotides. The DNA-Ag / Pt bimetallic nanozyme remains single-stranded and exhibits relatively low catalytic activity, resulting in a relatively light blue color in the detection solution. Simultaneously, the Initiator DNA initiates the CHA reaction, and H1 forms an H1 / H2 double strand from its hairpin structure. The microscopic distance between the silver nanoclusters at both ends of H1 increases, causing the solution to display a visible green composite fluorescence signal under UV light.

[0163] The specific testing steps are as follows:

[0164] (1) Bacterial culture and DNA preparation: Each bacterial strain was stored in a -80℃ glycerol storage tube, revived, and spread onto solid enrichment medium. The culture was then incubated at 30-37℃ for 18-24 h. Single colonies were picked and inoculated onto liquid enrichment medium, and cultured at 37℃ with shaking until the logarithmic growth phase. 1 mL of bacterial culture was taken, centrifuged, and the supernatant was discarded. DNA was extracted according to the bacterial genomic DNA extraction kit instructions and stored at -20℃ for later use. The concentration series used for sensitivity assessment was calibrated using the plate count method and then diluted with nuclease-free water to obtain a gradient concentration of 10. 8 ~10 0 The test solution with CFU / mL;

[0165] (2) LAMP primer design and optimization: Based on the tlh gene of Vibrio parahaemolyticus as the detection target, specific LAMP primers F3 / B3 (sequences shown in SEQ ID NO:7 and SEQ ID NO:8), FIP / BIP (sequences shown in SEQ ID NO:9 and SEQ ID NO:10), and LF / LB (sequences shown in SEQ ID NO:11 and SEQ ID NO:12) were designed using PrimerExplorer V5 based on its conserved region, and were verified by BLAST to avoid cross-reaction with other Vibrio species;

[0166] (3) Colorimetric detection reaction system: Each 20 μL system contains: 2 μL of 10× Cas reaction buffer (Thermo Fisher Scientific); 200 nM final concentration of Cas12a; 200 nM final concentration of crRNA (SEQ ID NO:13); 2 μL of LAMP product; 2 μM final concentration of Trigger DNA (SEQ ID No.3). The reaction is carried out at 37 ℃ for 30 min, and Cas12a protein is inactivated at 85 ℃ for 5 min. After the reaction, 10 μL of the reaction solution is annealed with 90 μL of nanozyme at 95 ℃ for 10 min, and then the colorimetric solution is added. The reaction is carried out at room temperature for 10~30 min. The color is observed by the naked eye or the Abs is measured. 652nm value;

[0167] (4) Ratio-rate fluorescence detection reaction system: Add 2 μL of 10× Cas reaction buffer, Cas12a to a final concentration of 200 nM, and crRNA (SEQ ID No. 13) to a 20 μL system; add 2 μL of LAMP product; add Trigger DNA (SEQ ID No. 3) to a final concentration of 2 μM; react at 37 ℃ for 30 min; inactivate Cas12a protein at 85 ℃ for 5 min; add Initiator DNA (final concentration 2 μM); incubate at room temperature for 5 min; after the reaction, take 10 μL of the reaction and mix it with silver nanoclusters (10 μL, 10 μM) and H2 (10 μL, 10 μM) for CHA amplification reaction; after reacting at room temperature for 1 h, measure the fluorescence signal.

[0168] 2. Feasibility Experiment:

[0169] Feasibility results of colorimetric signal detection are as follows Figure 14As shown, DNA-Ag / Pt bimetallic nanozymes can catalyze the color development of TMB / H2O2 substrates, and the formation of a double strand by binding with the complementary strand can significantly enhance the catalytic activity of the nanozyme. Figure 14 As shown, the solution changes from deep blue to a light blue that is visible to the naked eye only when the target is present. See [link to CHA amplification and assembly results and feasibility results for ratiometric fluorescence signal detection]. Figures 15-18 .

[0170] The chain assembly process of the CHA system was characterized using non-modifying PAGE gel, such as... Figure 15 The image shows the CHA amplification reaction of the template sequence of the DNA-AgNCs nanoclusters prepared in Example 2 and its assembly process under different conditions, as shown in non-denaturing PAGE images. The results indicate that the initiator DNA can open H1 and further hybridize with H2, while the presence of the trigger DNA inhibits this strand substitution reaction. Figure 16 The image shows the assembly process of the template sequence of the DNA-AgNCs nanoclusters prepared in Example 2 of this invention in the CHA amplification reaction under the action of the target-triggered CRISPR / Cas12a system and its non-denaturing PAGE image. The results show that the initiator DNA can trigger the CHA amplification reaction by cleaving the trigger DNA sequence in the presence of the target.

[0171] The trans-cleavage activity of CRISPR / Cas12a was analyzed using non-denaturing PAGE gels, such as... Figure 17 The image shows the dual emission peak fluorescence spectrum of the DNA-AgNCs nanoclusters prepared in Example 2 mediated by the Cas12a / crRNA reaction in the presence or absence of a target.

[0172] like Figure 17 and Figure 18 The image shows the dual emission peak fluorescence spectrum and dual characteristic emission peak fluorescence intensity comparison diagram of the DNA-AgNCs nanoclusters prepared in Example 2 under the conditions of presence and absence of target, and the presence or absence of Cas12a or crRNA in the system. The results show that CHA amplification reaction can only be carried out when the target, Cas12a and crRNA are present in the system at the same time. The fluorescence signal of the DNA-AgNCs nanoclusters (H1) changes from orange-red to green. However, under the conditions of no target, no Cas12a or crRNA, the color of the fluorescence signal does not change, that is, no CHA amplification reaction occurs.

[0173] 3. Sensitivity determination: Different concentrations of Vibrio parahaemolyticus were used as samples, and the Abs in the solution were detected using an ELISA reader. 652nm The absorbance values ​​were plotted as a curve of Vibrio parahaemolyticus concentration, and the results are as follows: Figure 19 and Figure 20As shown.

[0174] Prepare standard solutions of Vibrio parahaemolyticus with concentration gradients of NTC (No Bacteria Template Control, i.e., 0) and 10⁻⁶. 1 10 2 10 3 10 4 10 5 10 6 10 7 CFU / mL.

[0175] See Figure 19 Figure A shows the sensitivity test results of the colorimetric / fluorescence dual-mode detection method for Vibrio parahaemolyticus constructed using the above-mentioned technical solution against the aforementioned gradient concentrations of Vibrio parahaemolyticus standard solutions. Figure B shows the UV-Vis absorption spectrum response of the established sensing method at different target bacterial concentrations; Figure B shows the standard curve of the colorimetric signal, where Abs... 652 nm Figure C shows the fluorescence spectral response of the established sensing method at different target bacterial concentrations, with the green / red fluorescence signal ratio (Fg / Fr) on the ordinate and the bacterial concentration (CFU / mL) on the abscissa. Figure D shows the standard curve of the fluorescence signal, with the green / red fluorescence signal ratio (Fg / Fr) on the ordinate and the bacterial concentration (CFU / mL) on the abscissa.

[0176] Specifically, Figure 19 As shown in Figures A and B, with increasing Vibrio parahaemolyticus concentration, Abs... 652nm The value decreases linearly, with the linear equation: y = -0.14089x + 1.40863, and the correlation coefficient R0. 2 =0.9938, indicating a good linear relationship, and the calculated detection limit is 10 CFU / mL.

[0177] The fluorescence intensity at emission wavelengths of 520 nm (Fg) and 602 nm (Fr) was measured using a fluorescence spectrophotometer with excitation wavelengths of 406 nm and 550 nm, respectively. Fg / Fr value-Vibrio parahaemolyticus concentration curves were plotted, and the results are as follows: Figure 19 As shown in Figures C and D, the linear equation is y = 0.09357x + 0.31222, with a correlation coefficient RC. 2 =0.998, indicating a good linear relationship and a detection limit of 1 CFU / mL; this demonstrates that the technical solution of this invention can be used for quantitative detection of Vibrio parahaemolyticus at concentrations as low as 1 CFU / mL.

[0178] like Figure 20The colorimetric / fluorescence dual-mode detection method for Vibrio parahaemolyticus constructed in Example 5 shows the visual observation and solution color development under ultraviolet light at different target concentrations. At the standard concentrations of the above gradients, the color development of the solutions is significantly different. That is, using the technical solution of this invention, Vibrio parahaemolyticus can be directly qualitatively detected through colorimetric reactions.

[0179] 4. Specificity assay: Extract 10... 7 CFU / mL Vibrio parahaemolyticus, 10 7 CFU / mL Vibrio vulnificus, 10 7 CFU / mL Staphylococcus aureus, 10 7 CFU / mL Pseudomonas aeruginosa, 10 7 CFU / mL E. coli and 10 7 The genome of Salmonella at CFU / mL was used as the sample to be tested, and the results were as follows: Figure 21 As shown.

[0180] Figure 21 Figure A shows the specificity analysis of the DNA-Ag / Pt bimetallic nanozyme colorimetric detection system, where the concentration of each bacterium is 10. 7 CFU / mL, Abs corresponding to Vibrio parahaemolyticus 652nm The signal value decreased significantly, while other non-target strains showed higher readings, indicating that the system has good specificity for Vibrio parahaemolyticus; Figure B shows the specificity analysis of the DNA-AgNCs nanocluster fluorescence detection system, with each bacterium concentration being 10. 7 The fluorescence ratio signal (Fg / Fr) for Vibrio parahaemolyticus was significantly increased at CFU / mL, while other non-target strains showed a lower ratio, indicating that the system has good specificity for Vibrio parahaemolyticus.

[0181] Clearly, only the Abs in the target group solution 652nm The value is significantly lower and can be used for specific detection of Vibrio parahaemolyticus; the Fg / Fr value of the target group is 0.85 and significantly higher than that of other groups, and the solution color is visible green under ultraviolet light, which can be used for specific detection of Vibrio parahaemolyticus.

[0182] Based on the analysis of the above technical solutions, the present invention can use the bimetallic nanozyme provided by the present invention to prepare a LAMP isothermal amplification kit for qualitative / quantitative detection to achieve specific detection of Vibrio parahaemolyticus; the detection reagents include: DNA-Ag / Pt bimetallic nanozyme, DNA-AgNCs nanoclusters prepared using stem-circular DNA strand H1 as a template, LAMP primer set and Bst2.0 DNA polymerase, Cas12a protein and its crRNA, Trigger DNA, Initiator DNA, and TMB / H2O2 colorimetric reaction solution; the LAMP primer set (shown in SEQ ID NO:7~SEQ ID NO:12), Bst 2.0 DNA polymerase, Cas12a protein and its crRNA, and TMB / H2O2 colorimetric reaction solution can all be common reagents in the prior art, and the present invention does not make specific limitations.

[0183] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims.

Claims

1. A method for preparing a bimetallic nanozyme, characterized in that, include: Using DNA with single-stranded nucleic acid sequences as a DNA template, a high-temperature one-step in-situ reduction reaction of platinum and silver salts was employed to obtain DNA-Ag / Pt bimetallic nanozyme materials; among which, The single-stranded nucleic acid sequence is an oligonucleotide sequence containing a specific base, or a variant that has ≥90% homology with the oligonucleotide sequence containing the specific base and maintains equivalent catalytic enhancement function; The single-stranded nucleic acid sequence is shown in SEQ ID No.

1.

2. The preparation method according to claim 1, characterized in that, The single-stranded nucleic acid sequence also includes a binding domain sequence; The single-stranded nucleic acid sequence is shown in SEQ ID No. 2, and contains a binding domain complementary to the 5'-GCCTACGCCACTAGCTCCAACTA-3' sequence.

3. The preparation method according to any one of claims 1-2, characterized in that, The high-temperature one-step method includes: mixing DNA template, platinum salt and silver salt in a sodium citrate buffer system, shaking and placing it in a metal bath, heating at 90~100℃ for 5~20 min, and then naturally cooling and annealing to obtain DNA-Ag / Pt bimetallic nanozyme.

4. A bimetallic nanozyme, prepared by the preparation method according to any one of claims 1-3.

5. A bimetallic nanozyme prepared by the preparation method according to any one of claims 1-3, or the application of the bimetallic nanozyme according to claim 4 in the detection of Vibrio parahaemolyticus.

6. A colorimetric / fluorescent detection method for Vibrio parahaemolyticus, characterized in that, The specific steps include: (1) After extracting the bacterial genome from the sample to be tested, LAMP amplification was performed to obtain the amplification product; (2) The amplification product was mixed with Trigger DNA and CRISPR / Cas12a reaction system and then subjected to trans-shearing reaction to inactivate the product, resulting in reaction solution 1. (3) Add the bimetallic nanozyme prepared by the preparation method according to any one of claims 1-3, or the bimetallic nanozyme according to claim 4, to the reaction solution 1, heat the reaction and then cool and anneal naturally to obtain the reaction solution 2; (4) Mix the reaction solution 2 with the color development system, and interpret the results by visually identifying or detecting the absorbance value.

7. The colorimetric / fluorescence detection method according to claim 6, characterized in that, In step (1), the LAMP amplification includes amplifying the target gene using LAMP primers; And / or, the conditions for LAMP amplification include isothermal incubation at 60-65°C for 30-45 min; And / or, the LAMP primers include at least primer F3, primer B3, primer FIP, and primer BIP; wherein the sequence of primer F3 is as shown in SEQ ID No. 7; the sequence of primer B3 is as shown in SEQ ID No. 8; the sequence of primer FIP is as shown in SEQ ID No. 9; and the sequence of primer BIP is as shown in SEQ ID No.

10. And / or, in step (2), the CRISPR / Cas12a reaction system includes: Cas12a protein and crRNA; And / or, in the CRISPR / Cas12a reaction system, the final concentration of Cas12a protein is 200 nM, and the final concentration of crRNA is 200 nM; And / or, the sequence of the crRNA is shown in SEQ ID No. 13, and its target site is adjacent to the TTTV PAM site; And / or, the sequence of the trigger DNA is at least partially complementary to the single-stranded nucleic acid sequence of the DNA template; And / or, the sequence of the trigger DNA is shown in SEQ ID No. 3; And / or, the conditions for the trans-shear reaction include: incubation at 37 °C for 20-30 min; And / or, the inactivation includes inactivation at 85 °C for 5 min; And / or, the colorimetric system is a TMB / H2O2 colorimetric reaction solution, which includes buffer solution, TMB and H2O2; And / or, the buffer solution is a 0.2 M acetate-sodium acetate buffer solution with a pH of 3.8 to 4.2; the final concentration of TMB is 0.02 to 0.8 mM; and the final concentration of H2O2 is 0 to 100 mM. And / or, in step (4), the result interpretation includes visually identifying or detecting the 652 nm absorbance value to interpret the result. A dark blue color or a relatively high absorbance value indicates that the target bacteria are not present in the sample; a light blue color or a relatively low absorbance value indicates that the target bacteria are detected in the sample.

8. The colorimetric / fluorescence detection method according to claim 7, characterized in that, The LAMP primers further include primer LF and / or primer LB; wherein the sequence of primer LF is as shown in SEQ ID No. 11; and the sequence of primer LB is as shown in SEQ ID No.

12.

9. The colorimetric / fluorescence detection method according to any one of claims 7-8, characterized in that, When the target bacteria are determined to be absent in step (4), the sample to be tested is retested. The retesting method includes: (1) Add Initiator DNA to the reaction solution 1 and incubate at room temperature to obtain reaction solution 3; (2) DNA-AgNCs nanoclusters were added to the reaction solution 3, and hairpin probe H2 was added at the same time. The reaction solution 4 was obtained by incubation at room temperature. (3) The results can be interpreted by visual inspection under ultraviolet light or by detecting the fluorescence intensity at emission wavelengths of 520 nm and 602 nm respectively; the fluorescence intensity at emission wavelength of 520 nm is recorded as Fg, and the fluorescence intensity at emission wavelength of 602 nm is recorded as Fr; if the fluorescence color changes from orange-red to green, or the Fg / Fr value increases, it indicates that the target bacteria have been detected in the sample.

10. The colorimetric / fluorescence detection method according to claim 9, characterized in that, The sequence of the initiator DNA is complementary to the sequence of the trigger DNA. And / or, the sequence of the initiator DNA is shown in SEQ ID No. 4; And / or, the sequence of the auxiliary hairpin probe H2 is shown in SEQ ID No. 6; And / or, the DNA-AgNCs nanoclusters are obtained by reacting stem-circular DNA strand H1 with silver nitrate and sodium borohydride; And / or, the sequence of the stem-circular DNA strand H1 is as shown in SEQ ID No. 5; And / or, the sequence of the initiator DNA is partially complementary to the sequence of the stem-circular DNA strand H1.

11. A LAMP isothermal amplification kit for detecting Vibrio parahaemolyticus, characterized in that, It includes at least: a bimetallic nanozyme prepared by the preparation method according to any one of claims 1-3, or a bimetallic nanozyme according to claim 4; DNA-AgNCs nanoclusters prepared using stem-circular DNA strand H1 as a template; LAMP primer set and Bst 2.0 DNA polymerase; Cas12a protein and its crRNA; Trigger DNA; Initiator DNA; and TMB / H2O2 colorimetric reaction solution.

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