Nano enzyme, preparation method thereof, kit and detection method of kit

Abstract

The invention discloses a nano enzyme, a preparation method thereof, a kit and a detection method of the kit. The nano-enzyme comprises a dendritic polymer, a food-borne pathogen aptamer and nano-particles, wherein the food-borne pathogen aptamer and the nano-particles are loaded on the dendritic polymer; the dendritic polymer is a dendritic polymer of which the surface is subjected to RCA reaction, and the surface of the dendritic polymer is provided with a multi-copy repeatedly-serially-connected aptamer chain capable of specifically capturing food-borne pathogens and a multi-copy repeatedly-serially-connected nano-particle repetitive hybridization binding site, wherein the aptamer chain and the multi-copy repeatedly-serially-connected nano-particle repetitive hybridization binding site are in multi-copy repeatedly-serially-connected aptamer chain. According to the kit, due to the quadruple amplification mechanism, the detection signal is remarkably enhanced, the detection sensitivity is improved, the detection range is expanded, and the kit is suitable for detecting the food-borne pathogens in a real sample.

Description

Technical Field

[0001] The present invention relates to the field of enzymes, particularly to nanozymes, their preparation methods, kits, and detection methods of the kits. Background Art

[0002] Foodborne pathogens pose a significant threat to human health, resulting in serious socio-economic losses.

[0003] Traditional plate culture techniques are considered the gold standard for detecting foodborne pathogens, but traditional plate culture techniques are labor-intensive and time-consuming, usually taking several days to obtain accurate results. To address these issues, researchers have developed alternative methods such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), enabling faster detection. However, the ELISA method has low sensitivity, and the detection limit range is usually 10 3 to 10 4 cells / mL, which is often difficult for detecting pathogens with low infection doses, such as Salmonella typhimurium (S.T.). Although the PCR method has high sensitivity, it relies on complex sample pretreatment steps, such as bacterial enrichment and DNA extraction, which significantly prolongs the detection time and depends on dedicated laboratories and professional operators, limiting its practical application. Summary of the Invention

[0004] According to various embodiments of the present application, a nanozyme is provided.

[0005] A nanozyme includes a dendritic polymer, a foodborne pathogen aptamer, and nanoparticles, wherein the foodborne pathogen aptamer and the nanoparticles are loaded on the dendritic polymer; the dendritic polymer is a dendritic polymer with RCA reaction on its surface, and there are multi-copy repeated tandem aptamer chains that can specifically capture foodborne pathogens and multi-copy repeated tandem nanoparticle hybridization binding sites on the surface of the dendritic polymer.

[0006] Compared with the prior art, the dendritic polymer with RCA reaction on its surface in the present application can provide more binding sites for nanoparticles, making the signal amplification effect more significant. In addition, the nanoparticles in the present application can bind with aptamers to form stable complexes, thereby generating stronger signals during the detection process, and further making the nanozyme of the present application have the advantages of high detection sensitivity, high stability, and wide applicability.

[0007] In some embodiments, the foodborne pathogen aptamer is selected from Salmonella typhimurium-specific aptamers, and the sequence of the Salmonella typhimurium-specific aptamer includes SEQ ID NO: 1.

[0008] In some embodiments, the dendrimer is selected from one or more mixtures of PAMAM dendrimers, poly(propyleneimine) dendrimers, triazine dendrimers, phosphorus dendrimers, and polyether dendrimers.

[0009] In some embodiments, the nanoparticles are selected from one of gold nanoparticles, cobalt ferrite oxide nanoparticles, platinum nanoparticles, palladium nanoparticles, platinum-palladium alloy nanoparticles and their composites or mixtures of multiple ones and their composites.

[0010] The present application also provides a method for preparing a nanozyme, comprising the following steps:

[0011] Conjugate and couple the nanoparticles with ssDNA to synthesize an ssDNA-nanoparticle complex;

[0012] Mix a primer and a padlock probe, and obtain a ligation product through hybridization and ligation reactions. The ligation product includes the complementary sequence of the foodborne pathogen aptamer and the ssDNA sequence;

[0013] Conjugate the ligation product with the dendrimer to synthesize a dendrimer-ligation product complex;

[0014] Perform RCA amplification reaction on the dendrimer-ligation product complex to obtain a dendrimer-RCA complex; and,

[0015] Hybridize the ssDNA-nanoparticle complex with the dendrimer-RCA complex to synthesize the nanozyme.

[0016] In some embodiments, the nanoparticles are gold nanoparticles, the dendrimer is a 6.5-generation polyamidoamine dendrimer, the nanozyme is a G6.5-RCA-AuNPs nanozyme, and the method for preparing the G6.5-RCA-AuNPs nanozyme comprises the following steps:

[0017] Conjugate gold nanoparticles with the ssDNA to synthesize an ssDNA-AuNPs complex;

[0018] Mix a primer and a padlock probe, and obtain a ligation product through hybridization and ligation reactions. The ligation product includes the complementary sequence of the Salmonella typhimurium-specific aptamer and the ssDNA sequence; the sequence of the Salmonella typhimurium-specific aptamer includes SEQ ID NO: 1, the sequence of the primer includes SEQ ID NO: 2, the sequence of the padlock probe includes SEQ ID NO: 3, and the sequence of the ssDNA includes SEQ ID NO: 4;

[0019] Conjugate the said ligation product with the 6.5th generation polyamidoamine dendrimer to synthesize a G6.5-ligation product complex;

[0020] Perform RCA amplification reaction on the G6.5-ligation product complex to obtain a G6.5-RCA complex;

[0021] Hybridize the ssDNA-AuNPs complex with the G6.5-RCA complex to synthesize a nanozyme.

[0022] In some embodiments, the synthesis of the G6.5-RCA complex comprises the following steps:

[0023] S1: Take the 6.5th generation polyamidoamine dendrimer and the said ligation product and react them in a buffer solution;

[0024] S2: Filter to obtain the G6.5-ligation product complex;

[0025] S3: React the G6.5-ligation product complex in an RCA reaction mixture;

[0026] S4: Terminate the reaction and perform ultrafiltration to obtain the G6.5-RCA complex.

[0027] This application provides a kit, comprising the above-mentioned nanozyme, magnetic nanoparticle-antibody complex, manganese dioxide and glucose; the kit is used for detecting the foodborne pathogen corresponding to the foodborne pathogen aptamer.

[0028] This application provides a detection method for detecting foodborne pathogens based on the kit. Using the above-mentioned kit, it comprises the following steps:

[0029] Mix the magnetic nanoparticle-antibody complex, the nanozyme and the sample to be detected to synthesize a nanozyme-foodborne pathogen-magnetic nanoparticle sandwich complex;

[0030] Separate the nanozyme-foodborne pathogen-magnetic nanoparticle sandwich complex by magnetic separation, and wash it with a buffer solution to obtain a precipitate, which comprises the nanozyme-foodborne pathogen-magnetic nanoparticle sandwich complex; and,

[0031] Resuspend the precipitate in a reaction solution with a buffer solution, the reaction solution comprises glucose and manganese dioxide, incubate for 25 min - 35 min, then add TMB for color development reaction, and then add a termination solution to terminate the reaction, and measure the absorbance value.

[0032] In some embodiments, the kit is used for detecting Salmonella typhimurium, the nanozyme is G6.5-RCA-AuNPs nanozyme, the foodborne pathogen is Salmonella typhimurium, and the nanozyme-foodborne pathogen-magnetic nanoparticle complex is G6.5-RCA-AuNPs nanozyme-Salmonella typhimurium-magnetic nanoparticle sandwich complex.

[0033] The preparation method of the nanozyme in this application has the following beneficial effects.

[0034] (1) Through the RCA technology, after binding the ligation product with the dendritic polymer and then amplifying, a large amount of single-stranded DNA can be generated. This process significantly increases the copy number of the aptamer, thus greatly enhancing the intensity of the detection signal. Compared with traditional signal amplification methods, the RCA technology can rapidly produce a large number of repeated single-stranded DNAs in a short time, enabling the nanozyme to detect the target pathogen even at extremely low concentrations. Meanwhile, in the preparation method of the nanozyme described in this application, nanoparticles are used as signal carriers. After binding with the dendritic polymer, the stability and intensity of the signal are further enhanced. The multi-branched structure of the dendritic polymer provides a large number of binding sites for the nanoparticles, making the signal amplification effect more significant. In addition, in the preparation method of the nanozyme described in this application, the binding of the nanoparticles with the aptamer further improves the detection sensitivity. The size and surface properties of the nanoparticles enable them to efficiently bind with the aptamer to form a stable complex, thereby generating a stronger signal during the detection process. Therefore, the nanozyme prepared by using the preparation method of the nanozyme in this application has high detection sensitivity.

[0035] (2) The preparation method of the nanozyme in this application combines nanoparticles with dendritic polymers through a series of efficient chemical reactions and hybridization steps to form a nanozyme with high activity. The entire preparation process is simple to operate and takes a short time, and the synthesis of the nanozyme can be completed in a short time. The materials used in this method are relatively low in price, with less consumption and controllable costs. At the same time, in this application, by optimizing the reaction conditions, the efficient utilization of materials is ensured, and waste is reduced.

[0036] (3) In the preparation method of this application, the dendritic polymer used has good chemical stability and biocompatibility. During the preparation process, the dendritic polymer can stably load the aptamer and nanoparticles, ensuring the stability of the nanozyme during storage and use. In addition, the preparation process of this method has a high degree of repeatability. Through standardized operation steps and strict control of reaction conditions, the performance of the nanozyme prepared each time is consistent, which can ensure the reliability and repeatability of the detection results.

[0037] (4) In the method for preparing the nanozyme in this application, different nanoparticles can be selected as signal carriers. Different nanoparticles have different optical, electrical, and catalytic properties, and appropriate nanoparticles can be selected according to detection requirements to achieve multiple detection modes. In addition, in the method for preparing the nanozyme described in this application, different types of aptamer sequences can be selected. This makes the nanozyme prepared by this method have broad application prospects in the field of food safety detection.

[0038] (5) The materials used in this method are all biocompatible materials, such as dendrimers and nanoparticles, which are non-toxic to the human body and the environment. At the same time, the preparation process does not involve toxic and harmful chemical reagents and complex organic synthesis steps, which conforms to the concept of green chemistry. Description of the Drawings

[0039] Figure 1 Schematic diagram of the sensitive detection of Salmonella typhimurium (S.T.) bacteria in some embodiments;

[0040] Figure 2A Schematic diagram of the TEM image of AuNPs in some embodiments;

[0041] Figure 2B Schematic diagram of the particle size analysis of AuNPs and ssDNA-AuNP conjugates by DLS in some embodiments;

[0042] Figure 2C Schematic diagram of the results obtained by comparing the physical adsorption of AuNPs, ssDNA-AuNPs, FAM-ssDNA on AuNPs, and the fluorescence intensity (F.I.) of FAM-ssDNA-AuNPs in some embodiments;

[0043] Figure 2D Schematic diagram of the results obtained by UV-Vis spectral analysis of ssDNA, AuNPs, and ssDNA-AuNP conjugates in some embodiments;

[0044] Figure 2E Schematic diagram of the results of 8% polyacrylamide gel electrophoresis of RCA products in some embodiments.

[0045] Figure 2F Schematic diagram of the results of comparing the fluorescence intensity (F.I.) of G6.5, G6.5-ligation products, and G6.5-RCA products stained with SYBR Gold in some embodiments;

[0046] Figure 2G Schematic diagram of the results of particle size analysis of G6.5, G6.5-ligation products, and G6.5-RCA products by DLS in some embodiments;

[0047] Figure 3A Schematic diagram of the results of ultraviolet-visible spectroscopy comparison of G6.5-RCA complex, ssDNA-AuNPs complex and G6.5-RCA-AuNP nanozyme in some embodiments;

[0048] Figure 3B Schematic diagram of the results of particle size analysis of G6.5-RCA complex and G6.5-RCA-AuNP nanozyme using DLS in some embodiments;

[0049] Figure 3C Schematic diagram of the results of transmission electron microscopy (TEM) analysis of G6.5-RCA-AuNP nanozyme in some embodiments;

[0050] Figure 3D Schematic diagram of the results of transmission electron microscopy (TEM) analysis of G6.5-RCA-AuNP nanozyme in some embodiments;

[0051] Figure 3E Schematic diagram of the untreated control group in the TEM image of Salmonella typhimurium (S.T.) cells in some embodiments;

[0052] Figure 3F Schematic diagram of bacteria conjugated with MNP-Ab in the TEM image of Salmonella typhimurium (S.T.) cells in some embodiments;

[0053] Figure 3G Schematic diagram of bacteria conjugated with G6.5-RCA-AuNPs in the TEM image of Salmonella typhimurium (S.T.) cells in some embodiments;

[0054] Figure 3H Schematic diagram of bacteria conjugated with both MNP-Ab and G6.5-RCA-AuNPs in the TEM image of Salmonella typhimurium (S.T.) cells in some embodiments;

[0055] Figure 4A Schematic diagram of the results of TMB absorbance after the reaction of manganese dioxide nanosheets with different solutions (prepared in sodium acetate buffer (pH 5.0), different solution systems are labeled on the X-axis) in some embodiments;

[0056] Figure 4B Schematic diagram of the results of analyzing the changes of manganese dioxide nanosheets before and after cascade enzymatic reaction by ultraviolet-visible spectroscopy (UV-vis) in some embodiments;

[0057] Figure 4CSchematic diagram of the results of analyzing the changes in manganese dioxide nanosheets before and after cascade enzymatic reactions by dynamic light scattering (DLS) in some embodiments;

[0058] Figure 4D Schematic diagram of a transmission electron microscope (TEM) image of manganese dioxide nanosheets before decomposition in some embodiments;

[0059] Figure 4E Schematic diagram of a transmission electron microscope (TEM) image of manganese dioxide nanosheets after decomposition in some embodiments;

[0060] Figure 5 Schematic diagram of the detection performance results of various enzymatic sensors for S. T. bacteria in a wide concentration range in some embodiments;

[0061] Figures 6A-1 to 6A-4 Schematic diagram of the effects of different sample treatment strategies on improving detection performance in some embodiments;

[0062] Figure 6B Schematic diagram of the detection specificity results of G6.5-RCA-AuNP nanozyme in some embodiments;

[0063] Figure 6C Schematic diagram of the repeatability and precision results of G6.5-RCA-AuNP nanozyme in whole milk and beef samples with different concentrations of S. T. cells added in some embodiments. Detailed implementation manners

[0064] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0065] Unless otherwise defined, all technical and scientific terms used in the specification of the present application have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs. The terms used in the specification of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application.

[0066] Ⅰ Definitions

[0067] 6.5-generation polyamidoamine dendrimer: G6.5, a PAMAM dendrimer with a generation number of 6.5.

[0068] Gold nanoparticles (AuNP or AuNPs): Tiny particles of gold with a diameter in the range of 10 nm - 60 nm.

[0069] RCA: Rolling Circle Amplification is a method for generating long single-stranded DNA molecules from circular templates, and the generated DNA molecules contain hundreds or thousands of tandem repeats.

[0070] G6.5-RCA-AuNPs nanozyme: A nanozyme synthesized from 6.5-generation polyamidoamine dendrimers, RCA products, and AuNPs.

[0071] Magnetic nanoparticles-antibody (MNPs-Ab): That is, the MNP-Ab conjugate, magnetic nanoparticles conjugated with foodborne pathogen antibodies.

[0072] ssDNA: Single-stranded DNA. The single-stranded DNA in this application is used to form complementary sequences with the sequences at the multiple-copy repeat tandem nanoparticle hybridization sites in the RCA products.

[0073] Hybridization: DNA hybridization. DNA molecules with complementary base sequences can interact through hydrogen bonding between base pairs and form stable double-stranded regions.

[0074] Sandwich mode: That is, the sandwich sandwich mode, specifically forming a structure of nanozyme-bacteria-magnetic nanoparticles.

[0075] Sandwich complex: A complex with a structure of nanozyme-bacteria-magnetic nanoparticles.

[0076] Multiple-copy repeat tandem aptamer chains that can specifically capture foodborne pathogens: Repeated tandem single-stranded DNA. That is, this single-stranded DNA is an aptamer that can specifically capture foodborne pathogens.

[0077] Multiple-copy repeat tandem nanoparticle hybridization sites: Repeated tandem single-stranded DNA. Each hybridization site can specifically hybridize and bind to nanoparticles. For example, the multiple-copy repeat tandem nanoparticle hybridization sites can bind nanoparticles to the dendrimer by hybridizing with the ssDNA probes on the nanoparticles.

[0078] S.T.: That is, Salmonella typhimurium.

[0079] S.T.-specific aptamer: Salmonella typhimurium-specific aptamer.

[0080] Positive sample: A food sample containing S.T. cells.

[0081] Negative sample: A food sample that does not contain S.T. cells.

[0082] Multi-arm G6.5-COOH molecule: A 6.5-generation polyamidoamine dendrimer with carboxyl groups on its surface.

[0083] ssDNA-AuNPs complex: The hybridization product of gold nanoparticles and ssDNA probes, also known as ssDNA-AuNPs conjugate.

[0084] Ⅱ Kit and the principle of kit use

[0085] The present application provides a kit, which works through a series of enzymatic reactions triggered by the interaction of synthetic nanoenzymes, magnetic nanoparticles-antibodies, glucose, and MnO2 nanosheets.

[0086] In some embodiments, the nanoenzyme includes dendrimers, foodborne pathogen aptamers, and nanoparticles. The foodborne pathogen aptamers and nanoparticles are loaded on the dendrimers. The dendrimer is a dendrimer with RCA reaction on its surface. There are multi-copy repeated tandem aptamer chains that can specifically capture foodborne pathogens and multi-copy repeated tandem nanoparticle hybridization binding sites on the surface of the dendrimer.

[0087] In some embodiments, the nanoparticles are different types of nanoparticles.

[0088] In some embodiments, the aptamers are different types of foodborne pathogen aptamers.

[0089] Specifically, taking the detection of S.T. as an example, that is, the foodborne pathogen is S.T., and among them, the nanoparticles are gold nanoparticles. The principle of the kit for detecting S.T. is as follows: Through the RCA reaction, repetitive multi-copy repeated tandem aptamer chains that can specifically capture S.T. and multi-copy repeated tandem gold nanoparticle hybridization binding sites are generated on the surface of the 6.5th generation polyamidoamine dendrimer, and then hybridized with the ssDNA-AuNP probe to synthesize G6.5-RCA-AuNPs nanoenzyme ( Figure 1 Figure A in). The detection of S.T. bacteria in food samples is achieved by a sandwich mode, in which S.T. cells are combined with G6.5-RCA-AuNPs nanoenzyme and MNPs-Ab labeled with anti-S.T. antibody. The obtained sandwich complex is magnetically separated to remove the food matrix, and then reacted with glucose and MnO2 nanosheets to initiate a series of enzymatic reactions ( Figure 1 Figure B). In positive samples, the AuNP on the captured S.T. cells catalyzes the reaction with glucose to generate H2O2 and gluconic acid, further triggering the decomposition of MnO2, thereby inhibiting the color reaction with the TMB substrate; conversely, negative samples produce obvious colorimetric signals ( Figure 1 Figure C in).

[0090] Among them, the principle of the enzymatic reaction is as follows: AuNP catalyzes the reaction with glucose to generate H2O2 and gluconic acid. The generated H2O2 and gluconic acid catalyze the decomposition of MnO2, and the decomposition of MnO2 generates manganese ions.

[0091] Among them, in the positive sample, manganese ions will not catalyze the oxidation of TMB, so no color will appear. In the negative sample, since there is no AuNP, it will not catalyze the reaction of glucose to generate H2O2 and gluconic acid. Therefore, MnO2 will not be catalytically degraded. Thus, it catalyzes the oxidation of TMB and shows color. Therefore, it can be judged whether Salmonella typhimurium exists in the food according to the final color development result.

[0092] Ⅲ Specific Embodiments

[0093] In order to more clearly illustrate the method provided by the present invention, it is described in detail through the following embodiments. The raw materials used in the experimental part in the following embodiments are as follows.

[0094] N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), phi29 DNA polymerase, phi29 DNA polymerase reaction buffer (containing 330 mM Tris-acetate, pH 7.9, 100 mM magnesium acetate, 660 mM potassium acetate, 1% Tween 20, 10 mM dithiothreitol), T4 DNA ligase, T4 ligase buffer (containing 400 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP, pH 7.8), and deoxynucleoside triphosphate (dNTP) solution mixture were all purchased from ThermoFisher Scientific (Shanghai, China). Gold chloride (HAuCl4), poly(amidoamine) dendrimer generation 6.5 (G6.5-COOH), Ultrafiltration tubes (3 kDa & 100 kDa MWCO) and trisodium citrate were purchased from Sigma-Aldrich (Shanghai, China). Potassium permanganate, 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.0), phosphate buffered saline (PBS, 0.01 M, pH 7.4), phosphate buffer (PB, pH 8.0), acetate buffer (pH 3.5), sodium chloride, 3,3',5,5'-tetramethylbenzidine (TMB), sodium dodecyl sulfate (SDS), and dimethyl sulfoxide (DMSO) were all purchased from Aladdin (Shanghai, China). Carboxylated magnetic nanoparticles (MNPs, d = 180 nm) were purchased from Allrun Nano Science & Technology (Shanghai, China). Antibodies (Ab) specific to Salmonella typhimurium (S.T. ATCC 14028) were purchased from Meridian Bioscience (Cincinnati, USA). Nutrient agar and LB broth were purchased from Aoboxing Bio-tech (Beijing, China). Bacterial strains including S.T. ATCC 14028, Vibrio parahaemolyticus ATCC 17802, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Salmonella Enteritidis CVCC 1806 were provided by Dr. Lin's team at Xianghu Laboratory (Hangzhou, China). Custom DNA oligonucleotides were synthesized by Sangon Biotech (Shanghai, China).

[0095] Some of the sequences involved in this application are shown in Table 1 below:

[0096] Table 1: Sequences involved in this application

[0097]

[0098] The analytical methods involved in this application include:

[0099] Absorption spectrum and fluorescence intensity measurement: Absorption spectrum and fluorescence intensity measurement were performed using a BioTek Synergy H1 multimode microplate reader (Agilent Technologies, Santa Clara, California, USA). The absorption spectrum was recorded at 1 nm intervals within the detection range. When measuring fluorescence intensity, the excitation / emission wavelengths were set as follows: fluorescein (FAM) 459 / 520 nm, fluorescein isothiocyanate (FITC) 459 / 519 nm, and SYBR TM Gold-labeled samples 495 / 537 nm.

[0100] Dynamic light scattering measurement: The dynamic light scattering (DLS) experiment was carried out using a nanoparticle analyzer (Zetasizer Nano ZS90, Malvern, UK) to determine the particle size distribution of the samples. Each test consisted of 20 scans, and each scan lasted for 20 seconds. The scattering angle was set at 90°, and the experimental temperature was 25 °C. The average value of all scan data was taken to ensure the accuracy and consistency of the results.

[0101] Transmission electron microscope (TEM) analysis: The morphological characteristics of the samples were observed by a FEITALOS F200X transmission electron microscope (ThermoFisher Scientific, Waltham, Massachusetts, USA). When preparing the samples, the samples were evenly mixed, a drop of the sample was dropped onto a copper grid, and then dried under an infrared lamp. Finally, the samples were loaded onto the microscope for observation.

[0102] Polyacrylamide gel electrophoresis: The polyacrylamide gel electrophoresis experiment was carried out using an 8% polyacrylamide gel at a voltage of 55 V in TBE buffer for 75 minutes. After electrophoresis, the gel was immersed in TBE buffer containing 1×SYBRGold dye for staining for 10 minutes, and then rinsed with deionized water. The stained gel was imaged by a ChemiDocXRS+ imaging system (Bio-Rad, Hercules, California, USA).

[0103] Specific disassembly and explanation of the specification drawings in this application.

[0104] Figure 1 In Figure A, it is a schematic diagram of the synthesis process of G6.5-RCA-AuNP nanozyme; Figure 1 In Figure B, it is a schematic diagram of the sample preparation process using a two-pot treatment process; Figure 1 Figure C is a schematic diagram of the principle of cascade enzymatic reaction for signal amplification.

[0105] Figures 2A - 2G Characterization of ssDNA-AuNP conjugate and G6.5-RCA product. Among them, in Figures 2A - 2G The error bars represent the standard deviation (n = 3). The student's t-test (two-tailed) was used to determine statistical significance: ***p < 0.001.

[0106] Figures 3A - 3H Schematic diagram of the characterization of G6.5-RCA-AuNP nanozyme and the bacterial binding ability of the synthesized MNP-Ab conjugate and G6.5-RCA-AuNP nanozyme.Figure 3A UV-Vis spectra comparison of G6.5-RCA products, ssDNA-AuNPs, and G6.5-RCA-AuNPs nanozymes. Figure 3B Particle size analysis of G6.5-RCA products and G6.5-RCA-AuNPs nanozymes using DLS. Figure 3C -E) Transmission electron microscopy (TEM) analysis of G6.5-RCA-AuNPs nanozymes: Figure 3C Stitched TEM image showing a linear structure composed of densely packed AuNPs with a scale bar of 1 μm; Figure 3D Elemental mapping analysis to confirm the presence of gold and phosphorus elements. Figure 3E TEM image of the untreated control group; Figure 3F TEM image of bacteria conjugated with MNP-Ab; Figure 3G TEM image of bacteria conjugated with G6.5-RCA-AuNPs; Figure 3H TEM image of bacteria conjugated with both MNP-Ab and G6.5-RCA-AuNPs.

[0107] Figures 4A - 4E Schematic diagram for the verification of the cascade enzymatic reaction; Figure 4A TMB absorbance of manganese dioxide nanosheets after reaction with different solutions (prepared in sodium acetate buffer (pH 5.0), different solution systems are labeled on the x-axis). Error bars represent standard deviation (n = 3). Student's t-test (two-tailed) was used to determine statistical significance: ***p < 0.001. Figure 4B and Figure 4C Analysis of the changes in manganese dioxide nanosheets before and after the cascade enzymatic reaction by ultraviolet-visible spectroscopy (UV-vis) and dynamic light scattering (DLS), respectively. Figure 4D and Figure 4E Transmission electron microscopy (TEM) images of manganese dioxide nanosheets before and after decomposition, showing structural changes.

[0108] Figure 5 In Figure A, the performance of Aptamer-AuNP, G6.5-Aptamer-AuNP, RCA-AuNP, and G6.5-RCA-AuNP kits, whose structures are described in the legend. Figure 5 In Figure B, the performance of the GOx-MS-Ab kit with microspheres (MSs) containing glucose oxidase (GOx) and S.T.-specific antibody (Ab). Figure 5 In Figure C, the performance of the HRP-Ab kit with HRP-labeled S.T.-specific antibody. Figure 5The illustrations in panels B and C show the structure of the kit and the reaction principle. “BK” indicates the background signal measured at a cell concentration of 0.

[0109] Figure 6A-1 for “three-pot process A”, in which ssDNA-AuNP hybridization was performed in a separate step; Figure 6A-2 is the “three-pot process B”, in which the MnO2 decomposition is carried out in a separate step; Figure 6A-3 It is a "two-pot process" and is the scheme used in the preparation of the kit of this application; Figure 6A-4 is the signal change, which represents the difference between the background signal and the detection signal I0-I under different concentrations of ST cells under different sample processing strategies. Figure 6B To demonstrate the detection specificity of the G6.5-RCA-AuNP kit, the target STATCC14028 cells and non-target Vibrio parahaemolyticus ATCC17802, Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, and Salmonella Enteritidis CVCC1806 were used for detection tests. The concentration of all microorganisms was 10 4 CFU / mL. Figure 6C To test the repeatability and precision of the G6.5-RCA-AuNP kit in whole milk and beef samples supplemented with different concentrations of ST cells. Error bars represent standard deviation (n=3). Statistical significance was determined by Student's t-test (two-tailed): ***p<0.001.

[0110] The Chinese and English translations in the attached drawings are as follows.

[0111] Synthesis of RCA-G6.5-AuNPs (Synthesis of RCA-G6.5-AuNP), Key Features (Key Features), Multiple branches (Multiple branches), Hybridization sites (Hybridization sites), Aptamers and AuNPs conjugation (Aptamer and gold nanoparticle binding), RCA·reactions (Rolling circle amplification reaction), Repeated S.T.aptamers and AuNPs hybridization sites (Repeated S.T. aptamer and gold nanoparticle hybridization sites), Loading with abundant AuNPs (Loading with a large number of gold nanoparticles), Providing the first dual-signal amplification (Providing the first dual-signal amplification), Buffer exchange (Buffer exchange), Biorecognition & separation (Biorecognition and separation), Two-pot treatments (Two-step treatment), Ligation product (Ligation product), G6.5-ligation product (G6.5 ligation product), G6.5-RCA conjugate (G6.5-RCA conjugate), ssDNA-AuNPs (Single-stranded DNA-AuNP), MNPs-Ab (Magnetic nanoparticle-antibody), Glucose (Glucose), MnO2 nanosheet (Manganese dioxide nanosheet), Decomposed MnO2 nanosheet (Decomposed manganese dioxide nanosheet), Providing the second dual-signal amplification (Providing the second dual-signal amplification), Cascade enzymatic reactions (Cascade enzyme reaction), H2O2 & Gluconic acid (Hydrogen peroxide and gluconic acid), Synergistic reactions (Synergistic reaction), TMB (3,3′,5,5′-Tetramethylbenzidine), Positive (Positive), Negative (Negative), Absorbance (Absorbance), Wavelength (nm) (Wavelength (nanometers)), Intensity (Percent) (Intensity (percentage)), Size (d.nm) (Size (diameter, nanometers)), Physical adsorption (Physical adsorption), G6.5-RCA product (G6.5-Rolling circle amplification product), Copper grid, An AuNPs string, Darkfield Image, Au analysis, P analysis, Merged (image), CAT (Catalase), Glucose+MnO2 (control), Glucose+AuNPs+MnO2, Bacterial concentrations.

[0112] Preparation Example

[0113] Preparation Example 1

[0114] Synthesis of AuNPs: In a conical flask, 1 mL of 1 wt% chloroauric acid solution (dissolved in deionized water) was added to 99 mL of deionized water, and the mixture was stirred and heated until the solution boiled. With stirring and heating maintained, 2 mL of 1% (w / w) sodium citrate solution (dissolved in deionized water) was added. The color of the solution changed from light yellow to blue-gray within 5 minutes and then to wine red within the next 5 minutes, indicating the formation of AuNPs. After continuing to stir for 10 minutes (without heating), it was cooled to below 25 °C, and finally the volume of the solution was adjusted to 100 mL with deionized water.

[0115] In some embodiments, the weight percentage of the chloroauric acid solution can be any value within the range of 0.8 wt% - 1.2 wt%. For example, the weight percentage of the chloroauric acid solution can be 0.8 wt%, 0.9 wt%, 1 wt%, 1.1 wt% or 1.2 wt%.

[0116] In some embodiments, the concentration of the sodium citrate solution can be any value within the range of 0.8 - 1.2%. For example, the concentration of the sodium citrate solution can be 0.8%, 0.9%, 1%, 1.1% or 1.2%.

[0117] Preparation Example 2

[0118] Preparation of magnetic nanoparticle-antibody: It was prepared by the following steps: Take 2 mg of carboxylated magnetic nanoparticles (MNPs), wash them three times with phosphate buffer (PB, 0.01 M, pH 6.0), and then resuspend them in 2 mL of phosphate buffer. To activate the carboxyl groups, 14.4 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 16.3 mg of NHS (N-hydroxysuccinimide) were added to the magnetic nanoparticle suspension, and incubated at 25 °C for 1 hour. Then, the magnetic nanoparticles were washed three times with phosphate buffer to remove the excess reagents, and 0.5 mg of antibody (Ab) was added, and allowed to react with the activated magnetic nanoparticles for 2 hours. After the reaction, the magnetic nanoparticle-antibody conjugate was blocked with 1% (mass / volume) bovine serum albumin (BSA) for 45 minutes to prevent non-specific binding. Subsequently, the conjugate was washed three times with a washing solution composed of phosphate buffered saline (PBS, 0.01 M, pH 7.4) and 0.05% Tween 20 to remove the unbound antibody. Finally, the magnetic nanoparticle-antibody was resuspended in 1 mL of phosphate buffered saline (PBS, 0.01 M, pH 7.4) and stored at 4 °C to obtain the magnetic nanoparticle-antibody.

[0119] Preparation Example 3

[0120] Preparation of manganese dioxide: It was specifically prepared by the following steps: Mix 10 mL of 0.01 M potassium permanganate (KMnO4) solution with 10 mL of 0.1 M 2-(morpholino)ethanesulfonic acid (MES, pH 6.0) buffer solution, and ultrasonically treat for 5 minutes to obtain a black-brown colloid. Subsequently, the synthesized black-brown colloid was centrifuged at 8500 rpm for 10 min and washed five times with ultrapure water to finally obtain MnO2 nanosheets. Finally, the MnO2 nanosheets were resuspended in water to obtain a MnO2 nanosheet solution with a concentration of 1 mg / mL.

[0121] Example

[0122] Example 1: A method for preparing a nanozyme specifically includes the following steps.

[0123] Synthesis of ssDNA-AuNPs: The newly prepared AuNPs were conjugated with ssDNA (SEQ ID NO: 4, ssDNA) to synthesize ssDNA-AuNPs. The specific operation is as follows: 6 μL (1 mM) of disulfide-functionalized ssDNA was treated with 122.4 mM of newly prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP–HCl) at 25 °C for 2 hours to obtain -SH (mercapto)-functionalized ssDNA. The -SH (mercapto)-functionalized ssDNA was added to a conjugation solution containing 2.9 mL of AuNPs and 30 μL of 1 M Na2HPO4 solution (containing 1% w / w SDS), and the reaction was carried out at 25 °C in the dark for 1 hour to obtain a reaction solution. Then, a salt aging solution (containing 2 M NaCl, 0.01 M Na2HPO4, and 0.01% w / w SDS) was gradually added to the reaction solution to increase the NaCl concentration to 0.1 M. After sonication for 10 seconds, the mixture was incubated at 25 °C for 20 minutes. The salt aging step was repeated seven times, with the NaCl concentration increased by 0.1 M each time until the final concentration reached 0.7 M to obtain a reaction mixture. Subsequently, the reaction mixture was stored at 25 °C for 12 hours and then centrifuged (13,500×g) for 20 minutes to remove the supernatant. The precipitate (i.e., ssDNA-AuNPs) was collected, resuspended in 200 μL of PBS (0.01 M, pH 7.4), and stored at 4 °C to obtain the ssDNA-AuNPs solution.

[0124] Among them, the AuNPs were from Preparation Example 1.

[0125] The samples of Preparation Example 1 and Example 1 were analyzed by transmission electron microscopy (TEM), dynamic light scattering (DLS), ultraviolet-visible (UV-vis) spectroscopy, and fluorescence labeling method. The results are as Figures 2A - 2G shown. Figure 2A The TEM images shown indicate that the synthesized AuNPs were mainly uniform spherical particles with an average diameter of 16.2 ± 1.4 nm (n = 100), indicating a high synthesis quality. In addition, Figure 2B the DLS analysis in [[ ]] shows that ssDNA-AuNP exhibited a larger particle size compared to unmodified AuNPs (35.71 nm and 21.96 nm, respectively), which was due to the successful conjugation of ssDNA on the surface of AuNP. This was further confirmed by conjugating fluorescently labeled ssDNA (FAM-ssDNA) with AuNPs, Figure 2C showing that the fluorescence intensity of the FAM-ssDNA-AuNP conjugate was significantly (p < 0.001) higher than that of its physically adsorbed control (non-fluorescent ssDNA-AuNP conjugate) and unmodified AuNPs. Figure 2DThe UV-vis spectrum shown provides additional evidence for the successful synthesis of ssDNA-AuNPs, as it contains an absorption peak specific to ssDNA at 260 nm, and there is a 5 nm red shift in the absorption peak compared to unmodified AuNPs (from 519 nm to 524 nm). Additionally, the number of ssDNA on each AuNP was calculated to be 63.4 ± 3.7 (n = 3). It can be confirmed that ssDNA-AuNP was successfully synthesized in Preparation Example 1.

[0126] In some embodiments, AuNP can be equivalently replaced with CoFe2O4 NPs (cobalt ferrite oxide nanoparticles), PtNPs (platinum nanoparticles), PdNPs (palladium nanoparticles), Pt-Pd Alloy NPs (platinum-palladium alloy nanoparticles), etc., which are nanoparticles capable of catalyzing glucose to form gluconic acid and hydrogen peroxide.

[0127] Synthesis of the ligation product: Mix 1 μL of primer (SEQ ID NO: 2, 100 μM), 1 μL of padlock probe (SEQ ID NO: 4, 100 μM), and 86 μL of nuclease-free water, incubate at 95 °C for 10 minutes, after ice bath for 1 minute, incubate at 37 °C for 30 minutes to hybridize the primer with the padlock probe. Then, add 10 μL of T4 ligation buffer (10×) and 2 μL of T4 DNA ligase (5 U / μL), and incubate at 25 °C for 2 hours. Subsequently, heat at 65 °C for 10 minutes to terminate the reaction and inactivate the T4 DNA ligase.

[0128] Among them, the primer is a 5'-end NH2-capped primer, so that the synthesized ligation product is also NH2-capped at the 5'-end.

[0129] Synthesis of the G6.5-RCA complex: Conjugate the synthesized 5'-end NH2-capped ligation product with the multi-armed G6.5-COOH molecule to synthesize the G6.5-RCA complex. Specifically, it includes the following steps: React in a 0.1 M 2-(morpholino)ethanesulfonic acid (MES) buffer (pH 6.0) with a total volume of 200 μL containing 10 μM ligation product, 20 nM G6.5-COOH, 10 mM NHS (N-hydroxysuccinimide), and 10 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) at 25 °C for 60 minutes. Subsequently, use The solution was treated with an ultrafiltration tube (100 kDa) to remove unconjugated linkers, resulting in a G6.5-linked product complex. Subsequently, an RCA reaction was carried out on the surface of the G6.5 molecule. The G6.5-linked product complex was incubated in an RCA reaction mixture containing 1× phi29 buffer, 0.4 mM dNTPs (deoxynucleotide triphosphates), and 0.1 U / μL phi29 polymerase. The total volume of the reaction system was 400 μL, the reaction temperature was 37 °C, and the reaction time was 2 hours. Subsequently, the reaction was terminated by heating at 65 °C for 10 minutes to inactivate the phi29 polymerase. Subsequently, ultrafiltration was performed to obtain the purified G6.5-RCA complex.

[0130] In some embodiments, the functional groups on the linked product react with the functional groups on the dendrimer to synthesize a dendrimer with the linked product. For example, in this application, NH2 is introduced at the 5' end of the linked product through a 5'-end amino-capped primer. The 6.5th generation polyamidoamine dendrimer with a carboxyl group on the surface is selected, and the 6.5th generation polyamidoamine dendrimer with the linked product is synthesized through the condensation reaction of amino and carboxyl groups. In some other embodiments, the primer may carry functional groups such as carboxyl groups and phosphate groups, and the dendrimer may carry functional groups such as amino groups, which are not limited herein.

[0131] In some embodiments, the reaction time of the multi-arm G6.5-COOH molecule and the synthesized 5'-end NH2-capped linked product in the buffer can be between 50 min and 70 min, such as 50 min, 55 min, 60 min, 65 min, or 70 min.

[0132] In some embodiments, the reaction time of the G6.5-linked product complex in the RCA reaction mixture can be between 30 min and 180 h, such as 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, 120 min, 135 min, 150 min, 165 min, or 180 min.

[0133] In the RCA amplification reaction, the utilization rate of the dendrimer was maximized by precisely controlling the reaction time and temperature.

[0134] To verify the successful synthesis of the G6.5-RCA complex, gel electrophoresis was first used to confirm the success of the RCA reaction. As Figure 2EAs shown, the primer and the padlock probe showed distinct bands at approximately 35 bp and 55 bp, respectively. After hybridization, the hybridization product presented a band at approximately 90 bp. The RCA product, due to its high molecular weight, had an extremely low mobility and remained in the gel wells, indicating the successful progress of the RCA reaction. The successful implementation of the RCA reaction on the surface of G6.5 molecules was further confirmed by fluorescence analysis and DLS analysis. As Figure 2F shown, the fluorescence intensity of the G6.5-RCA product stained with SYBR Gold was significantly (p<0.001) higher than that of G6.5 and the G6.5-ligation product stained with SYBR Gold, indicating that more nucleic acid sequences were generated on the surface of G6.5, thus confirming the successful progress of the RCA reaction on G6.5 molecules. Figure 2G The DLS results in

[0135] further supported this, as the G6.5-RCA product showed a larger molecular size (1007.32 nm) compared to the G6.5-ligation product (15.64 nm) and the G6.5 molecule (9.87 nm). In addition, we also estimated the number of ligation products conjugated to each G6.5 molecule. The results showed that each G6.5 molecule could bind a maximum of 411±21 ligation products, indicating that G6.5 molecules facilitated the conjugation of multiple ligation products, thus promoting the formation of multi-branched RCA products.

[0136] Synthesis of G6.5-RCA-AuNPs nanozyme: The G6.5-RCA-AuNPs nanozyme was synthesized by hybridizing ssDNA-AuNPs with the G6.5-RCA product. Specifically, 100 μL of the prepared ssDNA-AuNPs was mixed with 400 μL of the synthesized G6.5-RCA complex and incubated at 25 °C for 30 minutes to allow complementary base pairing between the ssDNA-AuNPs and the RCA product. Subsequently, it was centrifuged (10000×g) for 15 minutes to remove the unbound ssDNA-AuNPs. The precipitate (i.e., G6.5-RCA-AuNPs) was collected and resuspended in 100 μL of PBS (0.01 M, pH 7.4) to obtain the G6.5-RCA-AuNPs nanozyme.

[0137] To confirm the successful synthesis of G6.5-RCA-AuNPs nanozyme, ultraviolet-visible (UV-vis) spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM) were used to analyze the relevant samples. As Figure 3A shown, compared with the G6.5-RCA complex, G6.5-RCA-AuNPs nanozyme showed a significant increase in absorbance at 535 nm, and there was an 11-nm red shift in the absorbance peak compared with the ssDNA-AuNPs complex (whose absorbance peak was at 524 nm). These spectral changes strongly indicated the successful synthesis of G6.5-RCA-AuNPs nanozyme. In addition, Figure 3B the DLS results shown in Figure 3C also provided additional evidence that G6.5-RCA-AuNPs nanozyme had a larger molecular size than the G6.5-RCA complex (1309.55 nm and 1007.32 nm, respectively), indicating the successful conjugation of the G6.5-RCA complex with AuNPs. Additionally, we also analyzed G6.5-RCA-AuNPs nanozyme using TEM and observed a linear structure composed of high-density AuNPs as shown in Figure 3D . Further observation of this structure revealed that AuNPs were bound to a fine filamentous shadow structure (as indicated by the arrow in

[0138] We speculated that these AuNP linear structures were due to the successful attachment of AuNP probes to the long-chain ssDNA products generated by RCA. To verify this hypothesis, we performed elemental mapping analysis and detected gold and phosphorus elements (the latter originating from the DNA structure), providing supportive evidence for our interpretation. In summary, all these results supported the conclusion that G6.5-RCA-AuNPs nanozyme had been successfully synthesized.

[0139] Furthermore, to confirm the ability of the synthesized MNP-Ab conjugate and G6.5-RCA-AuNPs nanozyme to bind to S.T. cells, TEM was used to analyze S.T. cells treated differently. As Figure 3E shown, untreated cells were used as a control and showed no nanoparticle attachment. In contrast, cell samples treated with MNPs-Ab or G6.5-RCA-AuNPs nanozyme were as shown in Figure 3F and 3GAs shown, significant attachment of MNPs or AuNPs to the cell surface was presented. It is worth noting that when the cells were treated with MNPs-Ab and G6.5-RCA-AuNPs nanozymes simultaneously, nanoparticles of G6.5-RCA-AuNPs nanozyme and MNPs-Ab could be clearly observed on the surface of S.T. cells (as Figure 3H shown). These results indicate that both MNPs-Ab and G6.5-RCA-AuNPs nanozymes show high biocompatibility to S.T. cells, and there is minimal interference in their binding to each other when they are applied simultaneously, thus enabling efficient biological recognition of target cells.

[0140] Preparation of the kit:

[0141] Take G6.5-RCA-AuNPs nanozyme, magnetic nanoparticle-antibody, manganese dioxide, glucose and chromogenic substance to form the kit.

[0142] Among them, the G6.5-RCA-AuNPs nanozyme is from Example 1, the magnetic nanoparticle-antibody is from Preparation Example 2, the manganese dioxide is from Preparation Example 3, and the chromogenic substance is selected as TMB.

[0143] In some embodiments, the kit is composed of 0.5 mL of G6.5-RCA-AuNPs nanozyme, 0.5 mL of magnetic nanoparticle-antibody, 5 mL of manganese dioxide solution, 5 mL of glucose solution and 15 mL of chromogenic substance solution.

[0144] In some embodiments, the sequences of the primers and padlock probes can be designed according to the foodborne pathogens to be detected, so that subsequent RCA synthesizes multi-copy repeated tandem aptamer chains that can specifically capture foodborne pathogens and multi-copy repeated tandem nanoparticle hybridization sites. The foodborne pathogens can be Salmonella typhimurium, Staphylococcus aureus, Vibrio cholerae, Escherichia coli, etc. In this example, the sequences of the primers and padlock probes are designed for detecting Salmonella typhimurium.

[0145] Example 2: A detection method for detecting foodborne pathogens based on a kit, comprising the following steps.

[0146] Mix 20 μL of the synthesized MNPs-Ab with 20 μL of G6.5-RCA-AuNPs nanozyme and 1 mL of the sample to be detected, and incubate at 25 °C for 20 minutes. Subsequently, obtain the G6.5-RCA-AuNPs nanozyme-S.T.-magnetic nanoparticle sandwich complex by magnetic separation, and rinse with phosphate buffered saline (PBS, pH 7.4) to remove the unbound G6.5-RCA-AuNPs nanozyme and MNPs-Ab. Then, resuspend the precipitate containing the G6.5-RCA-AuNPs nanozyme-S.T.-magnetic nanoparticle sandwich complex in 100 μL of the reaction solution, which contains 100 mM sodium acetate buffer (pH 5.0), 20 mM glucose, and 0.02 mg / mL manganese dioxide nanosheets. Generate H2O2 and gluconic acid through the reaction of AuNP-catalyzed with glucose. The generated H2O2 and gluconic acid catalyze the decomposition of MnO2 to generate manganese ions. After incubating at 25 °C for 30 minutes, add 100 μL of TMB substrate (color-developing substance) for a color reaction for 10 min. The absorbance of the final solution is measured at a wavelength of 370 nm using a BioTek SynergyH1 multimode microplate reader.

[0147] Among them, the kit is selected from Example 1.

[0148] Among them, the sample to be detected contains S.T. ATCC 14028.

[0149] In some embodiments, the absorbance of the solution can be measured at wavelengths such as 370 nm, 640 nm, etc. In this example, the absorbance is selected to be measured at 370 nm.

[0150] In some embodiments, the TMB substrate can be replaced with an equal amount of o-phenylenediamine (OPD), 3-amino-9-ethylcarbazole (AEC), or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS).

[0151] Comparative Example

[0152] Comparative Example 1, a detection method for foodborne pathogens based on a kit, which is different from Example 2 in that the G6.5-RCA-AuNPs nanozyme is replaced with an equal amount of Aptamer-AuNP nanozyme.

[0153] Comparative Example 2, a detection method for foodborne pathogens based on a kit, which is different from Example 2 in that the G6.5-RCA-AuNPs nanozyme is replaced with an equal amount of G6.5-Aptamer-AuNP nanozyme.

[0154] Comparative Example 3, a detection method for foodborne pathogens based on a kit, which is different from Example 2 in that the G6.5-RCA-AuNPs nanozyme is replaced with an equal amount of RCA-AuNP nanozyme.

[0155] Comparative Example 4, a detection method for foodborne pathogens based on a kit, which is different from Example 2 in that the G6.5-RCA-AuNPs nanozyme is replaced with an equal amount of GOx-MS-Ab nanozyme.

[0156] Comparative Example 5, a detection method for foodborne pathogens based on a kit, which is different from Example 2 in that the G6.5-RCA-AuNPs nanozyme is replaced with an equal amount of HRP-Ab nanozyme.

[0157] Comparative Example 6, a detection method for foodborne pathogens based on a kit, which is different from Example 2 in that, as Figure 6A-1 shown, specifically includes the following steps: Mix 20 μL of the synthesized MNPs-Ab with 20 μL of the G6.5-RCA complex and 1 mL of the sample to be tested, and incubate at 25 °C for 20 minutes to allow the MNPs-Ab and the G6.5-RCA complex to bind to bacterial cells. Subsequently, add 80 μL of the synthesized AuNPs-ssDNA complex and continue the reaction for 20 minutes. Subsequently, magnetically separate the bacterial sandwich complex and thoroughly wash it with phosphate-buffered saline (PBS, pH 7.4). Then, resuspend the obtained precipitate in 100 μL of the reaction solution, which contains 100 mM sodium acetate buffer (pH 5.0), 20 mM glucose, and 0.02 mg / mL manganese dioxide nanosheets, to initiate the cascade enzymatic reaction. After incubating at 25 °C for 30 minutes, add 100 μL of the TMB substrate for a colorimetric reaction. The absorbance of the final solution is measured at a wavelength of 370 nm using a BioTek Synergy H1 multimode microplate reader.

[0158] Among them, the AuNPs-ssDNA complex is from Preparation Example 1, and the G6.5-RCA complex is from Example 1.

[0159] Comparative Example 7, a detection method for foodborne pathogens based on a kit, which is different from Example 2 in that, as Figure 6A-2As shown in the figure, it specifically includes the following steps: Mix 20 μL of the synthesized MNPs-Ab with 20 μL of the G6.5-RCA-AuNPs complex and 1 mL of the sample to be tested, and incubate at 25 °C for 20 minutes to allow them to bind to bacterial cells. Subsequently, magnetically separate the bacterial sandwich complex and thoroughly rinse it with phosphate buffered saline (PBS, pH 7.4). Then, resuspend the obtained precipitate in 100 μL of the reaction solution, which contains 100 mM sodium acetate buffer (pH 5.0) and 20 mM glucose. After incubating at 25 °C for 30 minutes, perform magnetic separation again to extract the supernatant. Subsequently, react the supernatant with 0.02 mg / mL manganese dioxide nanosheets for 30 minutes, and then add 100 μL of the TMB substrate for color development reaction. The absorbance of the final solution is measured at a wavelength of 370 nm using a BioTek SynergyH1 multimode microplate reader.

[0160] Comparative Example 8, a detection method for foodborne pathogens based on a kit, which is different from Example 2 in that S.T. ATCC14028 in the sample to be tested is replaced with an equal amount of Vibrio parahaemolyticus ATCC17802.

[0161] Comparative Example 9, a detection method for foodborne pathogens based on a kit, which is different from Example 2 in that S.T. ATCC14028 in the sample to be tested is replaced with an equal amount of Vibrio parahaemolyticus ATCC17802.

[0162] Comparative Example 10, a detection method for foodborne pathogens based on a kit, which is different from Example 2 in that S.T. ATCC14028 in the sample to be tested is replaced with an equal amount of Staphylococcus aureus ATCC25923.

[0163] Comparative Example 11, a detection method for foodborne pathogens based on a kit, which is different from Example 2 in that S.T. ATCC14028 in the sample to be tested is replaced with an equal amount of Escherichia coli ATCC25922.

[0164] Comparative Example 12, a detection method for foodborne pathogens based on a kit, which is different from Example 2 in that S.T. ATCC14028 in the sample to be tested is replaced with an equal amount of Salmonella Enteritidis CVCC18062.

[0165] Performance detection experiment

[0166] Test experiment 1:

[0167] For Example 2, Example 1 and Comparative Examples 1-5, the detection method steps of Example 2 were used for detection under different concentrations of S.T bacterial samples, and the results are as follows Figure 5As shown in Figure 5 As shown in Figure A in the middle, in the present application kit, at all tested bacterial concentrations, compared with the Aptamer-AuNP kit, stronger detection signals were exhibited. This improvement is attributed to the 6.5-generation polyamidoamine dendrimers with multi-site binding, which can perform multiple bindings with Aptamer-AuNPs, thereby increasing the AuNP loading and achieving multivalent binding to enhance the binding affinity with bacteria. These factors result in a higher AuNP loading on the bacterial surface, while the Aptamer-AuNP kit only allows single AuNP binding and monovalent binding. Further, compared with the G6.5-Aptamer-AuNP kit, the RCA-AuNP kit exhibited stronger detection signals, indicating that the RCA technology can achieve a higher AuNP loading capacity on the surface of S.T. cells compared to only using G6.5. This improvement is due to the fact that the RCA process generates repetitive aptamers and multiple hybridization sites for AuNP probes, thereby further promoting the multiple binding of AuNPs and multivalent binding with bacteria. In addition, the kit of the present application exhibited the highest detection signal among all tested kits, indicating the highest AuNP loading on the bacterial surface. This excellent performance highlights the full utilization of the advantages of G6.5 and RCA, and the double enhancement of AuNP loading through their synergistic effect to amplify the detection signal. In addition, the kit of the present application rapidly consumed MnO2 nanosheets and reached a stable signal at an S.T. concentration of 1.2×10 6 CFU / mL, further demonstrating the high sensitivity and effectiveness of this double amplification strategy.

[0168] In addition, the kit of the present application exhibited excellent linear correlation, and its linear equation is y = -0.3029x + 1.9946 (R 2= 0.9965), where X represents the bacterial concentration and Y represents the signal response value. Moreover, compared with other kits studied, the slope of its response curve indicates that this kit has higher detection sensitivity. In addition, the limit of detection (LOD) of each kit, that is, the lowest target concentration at least three standard deviations higher than the background signal, was evaluated. The evaluation method was cited from the literature Shrivastava, A. and V. B. Gupta, Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chron. Young Sci, 2011.2(1): p. 21-25. The results show that the LOD of the kit of the present application is 5 CFU / mL, which is about 8 times lower than that of the RCA-AuNP kit (41 CFU / mL), 127 times lower than that of the G6.5-Aptamer-AuNP kit (637 CFU / mL), and 12,684 times lower than that of the Aptamer-AuNP kit (63,421 CFU / mL). These results highlight the effectiveness of the dual amplification strategy in improving detection sensitivity and limit of detection.

[0169] In addition, to further demonstrate the excellent detection performance of our kit, we compared the detection results of the kit of the present application with those of the GOx-MS-Ab kit (including microspheres (MSs) loaded with glucose oxidase (GOx) and S. T. specific antibody) and the traditional HRP-Ab kit (including HRP-labeled S. T. specific antibody). The structures and reaction principles of these kits are shown respectively in Figure 5 Figure B in Figure 5 and Figure 5 Figure C in. As shown in Figure 5 Figure B, although the GOx-MS-Ab kit also adopts a signal amplification strategy similar to that of the kit of the present application, involving a cascade enzymatic reaction based on glucose and MnO2, its detection sensitivity is low, and the LOD is 83 CFU / mL, higher than that of the kit of the present application. These results indicate that the catalytic ability of the GOx-MS-Ab kit is not as good as that of the kit of the present application. Furthermore, it shows that the catalytic efficiency of the gold nanoparticles in the kit of the present application is higher than that of GOx.

[0170] In addition, as shown in Figure 5 Figure C, the traditional HRP-Ab kit relies on signal amplification based on HRP and H2O2 (see Figure 5As shown in Figure C of the Chinese patent, it does not incorporate any of the quadruple signal amplification mechanisms developed in our research, such as the combined action of G6.5 and RCA and the synergistic effect of H2O2 and gluconic acid. The results show that compared with the kit of the present application, the detection sensitivity of the traditional HRP-Ab kit is reduced by 21 times, the limit of detection (LOD) is increased by 3,569 times, and the detection range is narrow (from 10 3 to 10 7 CFU / mL). These results further highlight the effectiveness of our quadruple signal amplification method in enhancing detection performance.

[0171] Test Experiment 2:

[0172] The steps of the detection method of Example 2 were used to detect Example 2, Comparative Example 6, and Comparative Example 7 under different concentrations of S.T. bacteria, and the signal changes representing the difference between the background signal and the detection signal (I0-I) were compared.

[0173] The results are as Figure 6A-4 shown. At all tested cell concentrations of the kits of Example 2 and Comparative Example 1, the signal changes were significantly greater than those of Comparative Example 2 (the signal change of Comparative Example 2 was p<0.001). This improvement was due to the participation of MnO2 in the catalytic reaction of AuNPs and glucose in Example 2 and Comparative Example 1, which promoted the consumption of H2O2 and gluconic acid, thus improving the reaction kinetics. It should be noted that although there was no significant difference in the signal changes between Example 2 and Comparative Example 1 (the signal change p>0.05), the standard deviation of Comparative Example 1 was larger, indicating poor stability, which was due to the errors introduced by the additional experimental steps. In addition, Example 2 had a significant advantage in the overall reaction time, only requiring 50 minutes, while Comparative Example 1 required 70 minutes and Comparative Example 2 required 80 minutes. In summary, Example 2 showed superiority in simplicity, reaction time, and stability, giving it great advantages in our detection system.

[0174] Test Experiment 3:

[0175] The steps of Example 1 and Comparative Examples 8-12 were detected using the steps of the detection method of Example 2. The results are as Figure 6BIt is shown that the absorbance of the target S.T. cells is significantly lower than that of non-target strains, indicating that the detection system has excellent specificity for S.T. ATCC14028. And it shows that the selected Salmonella typhimurium aptamer has extremely high affinity and specificity, and can specifically recognize and bind to the target pathogen. By precisely designing the aptamer sequence (such as SEQ ID NO: 1), the high specificity of the detection is ensured. In addition, in order to further evaluate the detection performance of the kit in actual food samples, the kits of the present application were used to detect whole milk and beef samples spiked with S.T. at different concentrations. Each spiked sample was tested in triplicate. Figure 6C It is shown that after spiking S.T. into milk (Whole milk) and beef (Beef) samples, in the concentration range of 6×10 1 to 6×10 3 CFU / mL, the recovery rates were between 93.27% and 107.31%, and the relative standard deviations (RSD) were all less than 10%. These results indicate that the kits of the present application have excellent reproducibility and accuracy in detecting S.T. in actual food samples.

[0176] Test experiment 4:

[0177] The results of detecting the samples in Example 1 by using the detection method of Example 2 were compared with the nanozyme colorimetric kits for S.T. detection in the related art. The comparison results are shown in Table 2.

[0178] Table 2 Comparison results of nanozyme colorimetric kits for S.T. detection.

[0179]

[0180]

[0181] From the experimental results in Table 2 above, we can find that the kit of the present application adopts a quadruple signal amplification strategy combined with a simple two-pot sample treatment method, which can sensitively detect S.T. bacteria. The first two amplification stages are promoted by G6.5-RCA-AuNPs nanozymes. In the first stage, the 6.5th generation polyamidoamine dendrimer provides multiple binding sites, prompting multiple RCA products to be connected to the 6.5th generation polyamidoamine dendrimer; in the second stage, the RCA products generate repetitive hybridization sites for AuNP binding, thus achieving a high loading of AuNPs. These two factors significantly enhance the overall AuNP loading, thereby amplifying the detection signal. The subsequent dual amplification stage is driven by the synergistic reaction of H2O2 and gluconic acid with MnO2 nanosheets, further enhancing the signal. This quadruple amplification cascade increases the detection sensitivity by 21 times, and the limit of detection (LOD) is reduced by 3,569 times compared with the traditional HRP kit. The two-pot sample treatment design simplifies the detection process and shortens the detection time to 50 minutes. The kit shows a wide linear detection range (10–10 6 CFU / mL) and a low detection limit (5 CFU / mL) in the detection of S.T. In the detection of actual food samples, the detection recoveries (93.27%–107.31%) and detection precisions (RSD < 10%) of this kit in milk and beef samples are excellent. These results highlight the practical application potential of the kit in food safety monitoring and pathogen detection.

[0182] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0183] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A nanozyme, characterized in that: It comprises a dendritic polymer, a foodborne pathogen aptamer and nanoparticles, wherein the foodborne pathogen aptamer and the nanoparticles are loaded on the dendritic polymer; the dendritic polymer is a dendritic polymer on the surface of which RCA reaction is performed, and the surface of the dendritic polymer has multiple copies of aptamer chains that are repeatedly connected in series and can specifically capture foodborne pathogens, and multiple copies of nanoparticles that are repeatedly connected in series and have repeated hybridization binding sites.

2. The nanozyme according to claim 1, characterized in that The foodborne pathogen aptamer is selected from a Salmonella typhimurium-specific aptamer, and the sequence of the Salmonella typhimurium-specific aptamer includes SEQ ID NO:

1.

3. The nanozyme according to claim 1, characterized in that The dendrimer is selected from a mixture of one or more of PAMAM dendrimers, poly(propyleneimine) dendrimers, triazine dendrimers, phosphorus dendrimers and polyether dendrimers.

4. The nanozyme according to claim 1, characterized in that The nanoparticles are selected from one of nano-gold particles, cobalt iron oxide nanoparticles, platinum nanoparticles, palladium nanoparticles, platinum-palladium alloy nanoparticles, and composites thereof or a mixture of multiple nanoparticles and composites thereof.

5. A method for preparing the nanozyme according to any one of claims 1 to 4, characterized in that: The following steps are involved: Conjugating the nanoparticles with ssDNA to form ssDNA-nanoparticle complexes; Mixing the primer with the padlock probe, and obtaining a ligation product through hybridization and ligation reaction, wherein the ligation product includes the complementary sequence of the foodborne pathogen aptamer and the ssDNA sequence; conjugating the ligation product with the dendritic polymer to synthesize a dendritic polymer-ligation product complex; performing RCA amplification reaction on the dendrimer-ligation product complex to obtain a dendrimer-RCA complex; and, The ssDNA-nanoparticle complex is hybridized with the dendrimer-RCA complex to synthesize the nanozyme.

6. The method for preparing the nanozyme according to claim 5, characterized in that: The nanoparticles are nanogold particles, the dendritic polymer is a 6.5-generation polyamidoamine dendritic molecule, the nanozyme is a G6.5-RCA-AuNPs nanozyme, and the preparation method of the G6.5-RCA-AuNPs nanozyme comprises the following steps: conjugating the gold nanoparticles with the ssDNA to synthesize a ssDNA-AuNPs complex; Mixing the primer and the padlock probe, and obtaining a ligation product by hybridization and ligation reaction, wherein the ligation product includes a complementary sequence of a Salmonella typhimurium-specific aptamer and a ssDNA sequence; the sequence of the Salmonella typhimurium-specific aptamer includes SEQ ID NO: 1, the sequence of the primer includes SEQ ID NO: 2, the sequence of the padlock probe includes SEQ ID NO: 3, and the sequence of the ssDNA includes SEQ ID NO: 4; Conjugating the ligation product with the 6.5-generation polyamidoamine dendrimer to synthesize a G6.5-ligation product complex; The G6.5-ligation product complex is subjected to RCA amplification reaction to obtain a G6.5-RCA complex; The ssDNA-AuNPs complex was hybridized with the G6.5-RCA complex to synthesize the nanozyme.

7. The method for preparing nanozyme according to claim 6, characterized in that: The synthesis of the G6.5-RCA complex comprises the following steps: S1: taking a 6.5-generation polyamidoamine dendrimer and the connection product to react in a buffer solution; S2: Filtration to obtain the G6.5-ligation product complex; S3: reacting the G6.5-ligation product complex in an RCA reaction mixture; S4: Terminate the reaction and ultrafilter to obtain the G6.5-RCA complex.

8. A kit, characterized in that: The kit comprises the nanozyme, magnetic nanoparticle-antibody complex, manganese dioxide and glucose as described in any one of claims 1 to 4; the kit is used to detect foodborne pathogens corresponding to the foodborne pathogen aptamers.

9. A method for detecting foodborne pathogens based on a kit, characterized in that: The kit according to claim 8 comprises the following steps: The magnetic nanoparticle-antibody complex, the nanozyme and a sample to be detected are mixed to synthesize a nanozyme-foodborne pathogen-magnetic nanoparticle sandwich complex; Separating the nanozyme-foodborne pathogen-magnetic nanoparticle sandwich complex by a magnetic separation method, and washing with a buffer to obtain a precipitate, wherein the precipitate includes the nanozyme-foodborne pathogen-magnetic nanoparticle sandwich complex; and, The precipitate is resuspended in a reaction solution with a buffer solution, wherein the reaction solution includes glucose and manganese dioxide. After incubation for 25 min-35 min, TMB is added for color development reaction, and then a stop solution is added to stop the reaction, and the absorbance value is measured.

10. A method for detecting foodborne pathogens based on the kit according to claim 9, characterized in that: The kit is used to detect Salmonella typhimurium, the nanozyme is G6.5-RCA-AuNPs nanozyme, the foodborne pathogen is Salmonella typhimurium, and the nanozyme-foodborne pathogen-magnetic nanoparticle complex is a G6.5-RCA-AuNPs nanozyme-Salmonella typhimurium-magnetic nanoparticle sandwich complex.

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