A milk-meat product microbial detection marker composition and a method for preparing the same

Abstract

The application belongs to the field of milk-meat product microbial detection, and provides a milk-meat product microbial detection marker composition and a preparation method thereof. The application takes Prussian blue nanoparticles as an inner core, constructs 3-aminobenzene boronic acid coupled carboxylated silica coated Prussian blue nanoparticles, and is compounded with bovine serum albumin, polysorbate 20, D-(+)-trehalose dihydrate, sodium chloride, potassium chloride, anhydrous disodium hydrogen phosphate and potassium dihydrogen phosphate to form a marker composition. The system takes into account the solid content, low viscosity dispersion stability, average particle size recovery rate after freeze-drying and redissolving, anti-settling property, color development intensity and detection signal-to-noise ratio, solves the problem that the existing system cannot simultaneously take into account high microbial capture efficiency, high detection sensitivity and rapid chromatography migration or microfluidic transmission performance, and is suitable for liquid marker solution, redissolving solution for chromatography test strip marker pad and marker solution for microfluidic chip.

Description

Technical Field

[0001] This invention relates to the field of food microbial detection and labeling materials, specifically to a microbial detection and labeling composition for dairy and meat products and its preparation method. Background Technology

[0002] Dairy and meat products are susceptible to microbial contamination during processing, storage, transportation, and final distribution. Rapid detection systems require stable readouts under complex matrices and field application conditions. Labeling compositions for chromatographic test strip pads and microfluidic chips must not only possess good dispersion stability, suitable average particle size, and narrow distribution, but also ensure rapid chromatographic migration or microfluidic transport performance, high surface recognition site exposure, high microbial capture efficiency, and high detection sensitivity. Simultaneously, the labeled nanoparticles should maintain low viscosity dispersion stability even with increased solid content, and maintain good average particle size recovery, anti-settling properties, color intensity, and signal-to-noise ratio during freeze-drying, storage, transportation, and reconstitution to meet the application requirements for rapid, sensitive, and visual detection of dairy and meat product samples.

[0003] Currently, existing technologies for rapid detection of foodborne pathogens mostly focus on single aspects such as ligand construction, immunochromatographic readout, or optimization of enrichment steps. For example, Chinese patent CN114113592B discloses a side-flow chromatography test strip based on mercaptophenylboronic acid-functionalized gold nanoparticles and its detection method, which focuses on utilizing the interaction between phenylboronic acid and bacterial surface structures to achieve capture and color development. Another example is Chinese patent CN105116146B, which discloses a method for rapid detection of Listeria monocytogenes using nano-immunomagnetic beads combined with colloidal gold chromatography, emphasizing the improvement of rapid screening capabilities through magnetic bead enrichment and colloidal gold chromatography. While these approaches can improve the identification or detection process, they still fall short in comprehensively considering the solid content of labeled particles, low-viscosity dispersion stability, average particle size recovery rate after freeze-drying and rehydration, anti-settling properties, color intensity, and rapid transport performance. Especially under the complex matrix conditions of dairy and meat products, there is often a problem of difficulty in simultaneously optimizing the exposure of recognition sites and the stability of particle structure. Summary of the Invention

[0004] The purpose of this invention is to provide a microbial detection labeling composition for dairy and meat products and its preparation method, which solves the current pain point of labeling nanoparticles in that it is difficult to balance solid content, low viscosity dispersion stability, average particle size recovery rate after freeze-drying and rehydration, anti-settling properties, microbial capture efficiency, detection sensitivity, color intensity and detection signal-to-noise ratio.

[0005] This invention constructs labeled nanoparticles by synergistically integrating a Prussian blue nanoparticle color-developing core, a silica shell interface for stability, and a 3-aminophenylboronic acid recognition unit. Furthermore, the dispersion environment is synergistically regulated by a system of bovine serum albumin, polysorbate 20, D-(+)-trehalose dihydrate, and salts. This achieves a balanced optimization of recognition, transport, color development, and reconstitution stability within the same system, thereby enhancing the comprehensive applicability of microbial detection in dairy and meat products.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A microbial detection labeling composition for dairy and meat products, comprising, based on a total mass of 100 parts by weight: 0.1-5.0 parts of labeled nanoparticles on a dry basis, 0.1-5.0 parts of bovine serum albumin, 0.01-0.5 parts of polysorbate 20, 1.0-10.0 parts of D-(+)-trehalose dihydrate, 0.1-2.0 parts of sodium chloride, 0.005-0.2 parts of potassium chloride, 0.01-0.5 parts of anhydrous disodium hydrogen phosphate, 0.001-0.2 parts of potassium dihydrogen phosphate, with the remainder being deionized water. The deionized water is the amount remaining after adjusting for dry basis mass of the labeled nanoparticles and deducting the water content of the labeled nanoparticle suspension, to bring the total mass to 100 parts by weight, excluding the aforementioned components. The labeled nanoparticles are Prussian blue nanoparticles with a core of Prussian blue nanoparticles and 3-aminophenylboronic acid coupled carboxylated silica-coated Prussian blue nanoparticles. The average particle size of the labeled nanoparticles is 60-150 nm, the silica shell thickness is 8-20 nm, the 3-aminophenylboronic acid grafting amount is 5-80 μmol / g, and the mass fraction of Prussian blue in the labeled nanoparticles is 20-70 wt%. The labeled nanoparticles are prepared sequentially through Prussian blue nanoparticle preparation, silica coating and amination, carboxylation, and 3-aminophenylboronic acid activation coupling. The pH value of the labeled composition is 6.8-7.6.

[0007] Furthermore, the labeled nanoparticles are prepared by the following steps: A1, Disperse the carboxylated silica-coated Prussian blue intermediate in a 2-(N-morpholine)ethanesulfonic acid buffer solution, wherein the concentration of the 2-(N-morpholine)ethanesulfonic acid buffer solution is 10-100 mmol / L and the pH value is 5.0-6.5; A2, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, the molar ratio of the surface carboxyl groups of the carboxylated silica-coated Prussian blue intermediate in step A1, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:(1-5):(1-5), activate at 15-30℃ for 10-60 min; A3, add 3-aminophenylboronic acid, wherein the molar ratio of the carboxyl group on the surface of the intermediate to 3-aminophenylboronic acid is 1:(0.5-3), and react at 15-30℃ for 2-12 hours; A4, washed and resuspended, to obtain the labeled nanoparticles with a 3-aminophenylboronic acid grafting amount of 5-80 μmol / g and residual free 3-aminophenylboronic acid of no more than 0.5 wt% based on the dry weight of the labeled nanoparticles.

[0008] Furthermore, the carboxylated silica-coated Prussian blue intermediate is prepared by the following steps: B1, dispersing the aminated silica-coated Prussian blue intermediate in N,N-dimethylformamide, adding succinic anhydride, wherein the mass ratio of the aminated silica-coated Prussian blue intermediate to succinic anhydride is 1:0.2 to 1:1.5, reacting at 20-35℃ for 2-8 hours; B2 was washed with ethanol and deionized water, 2-5 times each, and then dried to obtain the carboxylated silica-coated Prussian blue intermediate with a carboxyl density of 30-200 μmol / g.

[0009] Furthermore, the aminated silica-coated Prussian blue intermediate is prepared by the following steps: C1, Prussian blue nanoparticles are dispersed in a mixed solution of ethanol and deionized water, tetraethyl orthosilicate is added to make the volume fraction of tetraethyl orthosilicate 1-10 vol%, and ammonia water is added to make the volume fraction of ammonia water 0.5-5 vol%. The reaction is carried out at 20-35℃ for 1-6 h to obtain silica-coated Prussian blue particles. C2, add 3-aminopropyltriethoxysilane to the silica-coated Prussian blue particle dispersion obtained in step C1, so that the volume fraction of 3-aminopropyltriethoxysilane is 0.5-5 vol%, and react at 50-80℃ for 1-6 h. C3, washing and drying, to obtain the aminated silica-coated Prussian blue intermediate with a shell thickness of 5-30 nm and a surface amino density of 50-300 μmol / g.

[0010] Furthermore, the Prussian blue nanoparticles are prepared by the following steps: D1, ferric chloride hexahydrate and potassium ferrocyanide trihydrate are prepared in deionized water, with the molar ratio of ferric ions to ferrocyanide ions being 0.8:1 to 1.2:1. Simultaneously, citrate monohydrate is added to achieve a concentration of 1-20 mmol / L, and polyvinylpyrrolidone K30 is added to achieve a concentration of 0.05-2.0 wt%. D2, stirred at 20-40℃, for 0.5-3 hours; D3, centrifuged and washed 2-5 times with deionized water to obtain the Prussian blue nanoparticles with an average particle size of 20-80 nm and a polydispersity index not higher than 0.30.

[0011] Furthermore, Prussian blue nanoparticles serve as the core of the labeled nanoparticles; the labeled nanoparticles have an average particle size of 60-150 nm, a silica shell thickness of 8-20 nm, a 3-aminophenylboronic acid grafting amount of 5-80 μmol / g, and a Prussian blue mass fraction of 20-70 wt% in the labeled nanoparticles; the pH value of the labeled composition is 6.8-7.6.

[0012] Furthermore, the labeling composition can be used in liquid form and in lyophilized powder form; The liquid application forms include liquid labeling solutions, labeling solutions for microfluidic chips, and labeling solutions for preparing chromatographic test strip labeling pads; The freeze-dried powder storage form is a freeze-dried powder labeling agent obtained by freeze-drying the liquid-use labeling composition; The lyophilized powder labeling agent is reconstituted before use to obtain a reconstituted labeling solution. The reconstituted labeling solution is a labeling composition restored to a liquid form for use and can be used as the liquid labeling solution, the labeling solution for microfluidic chips, or the labeling solution for preparing chromatographic test strip labeling pads. The average particle size recovery rate of the lyophilized powder labeling agent after reconstitution is not less than 85%, and there is no visible sedimentation within 30 minutes after reconstitution. The microorganism to be detected is selected from one or more of Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella.

[0013] Furthermore, the lyophilized powder labeling agent is reconstituted to obtain a reconstituted labeling solution, which is used to label the labeling pad of the chromatography test strip; The labeling process includes applying the reconstituted labeling solution to the labeling pad by impregnation, spotting, or spraying and then drying it, so that the labeled nanoparticles are loaded onto the labeling pad; during detection, the sample to be tested flows through the labeling pad, causing the labeled nanoparticles to redisperse and migrate along the chromatography direction; The average particle size recovery rate of the lyophilized powder labeling agent after reconstitution is calculated as the ratio of the average particle size of the labeled nanoparticles in the reconstituted labeling solution to the average particle size of the labeled nanoparticles in the liquid form of the labeling composition before lyophilization.

[0014] Furthermore, in this invention, liquid labeling solution refers to a labeling composition that comes into direct contact with the sample to be tested, the microorganism to be tested, or the detection reaction system in a liquid state and provides a color signal; Labeling solutions for microfluidic chips refer to liquid labeling compositions that are added to the flow channels or reaction chambers of microfluidic chips and transported with the liquid flow to contact the microorganisms to be detected and provide color signals. Freeze-dried powder labeling agents refer to solid labeling compositions obtained by freeze-drying the liquid-state labeling composition and which can be reconstituted before use; The reconstituted labeling solution refers to a liquid labeling composition obtained by reconstituted the lyophilized powder labeling agent with deionized water to the volume before lyophilization or the solid content of the labeled nanoparticles before lyophilization. The label pad of a chromatography test strip refers to a porous pad material in a chromatography test strip used for preloading and releasing the labeled nanoparticles when the sample liquid flows through it; labeling the label pad of a chromatography test strip refers to applying the reconstituted labeling solution or the labeling composition in liquid form to the label pad and drying it, so that the labeled nanoparticles are loaded onto the label pad.

[0015] Furthermore, when using the lyophilized powder labeling agent for chromatographic test strips, the lyophilized powder labeling agent is first reconstituted with deionized water to the volume before lyophilization or the solid content of the labeled nanoparticles before lyophilization, to obtain a reconstituted labeling solution; then, the reconstituted labeling solution is applied to the labeling pad substrate by impregnation, spotting, or spraying to wet the labeling pad substrate; then, it is dried until there is no flowing liquid, so that the labeled nanoparticles are loaded onto the labeling pad; during use, the sample liquid flows through the labeling pad, and the labeled nanoparticles are released and enter the reaction membrane with the chromatographic flow. The average particle size recovery rate of the lyophilized powder labeling agent after reconstitution is calculated by multiplying 100% by the ratio of the average particle size of the labeled nanoparticles in the reconstituted labeling solution to the average particle size of the labeled nanoparticles in the liquid form of the labeling composition before lyophilization.

[0016] In this invention, the liquid form and the lyophilized powder form are not two independent labeling methods, but rather the same labeling composition existing in different stages of use; wherein, the lyophilized powder labeling agent is used for storage and transportation, the reconstituted labeling solution is used to restore the liquid form for use, and the chromatographic test strip labeling pad is used to carry the labeled nanoparticles in the reconstituted labeling solution and release the labeled nanoparticles during detection.

[0017] As a concept of this invention, it employs a design with Prussian blue nanoparticles as the core, carboxylated silica as the interface coating layer, and further introduces 3-aminophenylboronic acid recognition units. This design primarily enhances the colorimetric performance, dispersion stability, and microbial capture ability in the microbial detection of dairy and meat products. The Prussian blue nanoparticles provide a stable and clear color signal, the silica shell helps maintain the integrity of the particle structure, improves aqueous dispersion, and provides an interface basis for surface functionalization, while 3-aminophenylboronic acid enhances the interaction with microbial surface-related structures. Furthermore, the combination of bovine serum albumin, polysorbate 20, D-(+)-trehalose dihydrate, and salt components to regulate the system's microenvironment allows for the maintenance of low aggregation tendency and good migration, while also considering the average particle size recovery rate after lyophilization and reconstitution, anti-sedimentation properties, and detection signal-to-noise ratio. This improves the applicability of the labeled composition in chromatographic test strips and liquid labeling solutions.

[0018] This invention also discloses a method for preparing a microbial detection marker composition for dairy and meat products, comprising the following steps: S1 provides Prussian blue nanoparticles with an average particle size of 20-80 nm and a polydispersity index of no more than 0.30. S2, the Prussian blue nanoparticles provided by S1 are coated with silica and aminated to obtain aminated silica-coated Prussian blue intermediates with a shell thickness of 5-30 nm and a surface amino density of 50-300 μmol / g. S3, the aminated silica-coated Prussian blue intermediate obtained in S2 is subjected to carboxylation treatment to obtain a carboxylated silica-coated Prussian blue intermediate with a carboxyl density of 30-200 μmol / g; S4, the carboxylated silica-coated Prussian blue intermediate obtained in S3 was activated and coupled to obtain labeled nanoparticles with an average particle size of 60-150 nm and a 3-aminophenylboronic acid grafting amount of 5-80 μmol / g. S5, the labeled nanoparticle suspension obtained in S4 is mixed with bovine serum albumin, polysorbate 20, D-(+)-trehalose dihydrate, sodium chloride, potassium chloride, anhydrous disodium hydrogen phosphate, potassium dihydrogen phosphate and deionized water according to the mass parts of each component mentioned above, and the mixture is made up to 100 parts by mass with deionized water, and the pH value is adjusted to 6.8-7.6 to obtain the labeled composition.

[0019] Furthermore, in step S5, the mixing temperature is 4-25℃ and the mixing time is 15-60 min; Step S5 is followed by a freeze-drying step, which includes: a pre-freezing temperature of -50 to -35°C, a pre-freezing time of 2-8 hours, a main drying time of 12-48 hours, and a residual moisture content of no more than 3.0 wt% after freeze-drying.

[0020] Furthermore, when the liquid labeling composition is measured by mass parts, each component is proportioned according to the mass parts, and deionized water is used to make up the balance of the total mass of the labeling composition to 100 parts by mass based on the proportions.

[0021] Furthermore, the amount of 3-aminophenylboronic acid grafted is expressed per gram of labeled nanoparticles, and the mass fraction of Prussian blue is expressed as the total mass of the labeled nanoparticles.

[0022] Furthermore, the average particle size and polydispersity index of Prussian blue nanoparticles and labeled nanoparticles were determined by dynamic light scattering, and the thickness of the silica shell was determined by transmission electron microscopy.

[0023] Furthermore, the surface amino density, carboxyl density, and 3-aminophenylboronic acid grafting amount were determined using quantitative analysis methods, and the test samples, sample pretreatment, quantitative basis, calculation formulas, and result caliber were clearly defined.

[0024] Furthermore, in step D1, the concentrations of citric acid monohydrate and polyvinylpyrrolidone K30 are expressed as the final concentrations of the reaction solution.

[0025] Furthermore, in step D2, the stirring method is magnetic stirring or mechanical stirring, and the stirring speed is 100-1500 rpm.

[0026] Furthermore, the centrifugation conditions in step D3 include a relative centrifugal force of 5000-15000g, a centrifugation time of 10-60min, and a centrifugation temperature of 4-25℃. The washing conditions include a washing liquid volume of 5-20 times the precipitate volume for each wash.

[0027] Furthermore, in step C1, the solid content of the Prussian blue nanoparticles in the mixed solution of ethanol and deionized water is 0.1-5 wt%, and the composition ratio of the mixed solution of ethanol and deionized water is expressed as a volume ratio. The reaction is carried out under magnetic stirring or mechanical stirring conditions, and the stirring speed is 100-1500 rpm.

[0028] Furthermore, the volume fractions of tetraethyl orthosilicate and ammonia in step C1 are based on the total volume of the mixed solution of ethanol and deionized water, and the volume fraction of 3-aminopropyltriethoxysilane in step C2 is based on the total volume of the reaction system in step C1.

[0029] Furthermore, step C2 is carried out in a reaction system equipped with a reflux condenser or in a closed reaction system.

[0030] Furthermore, in step C3, the washing solvents are ethanol and deionized water, and the washing is performed 2-5 times, with the volume of the washing liquid each time being 5-20 times the volume of the precipitate. The drying conditions include drying temperature, drying time, and vacuum degree.

[0031] Furthermore, in step B1, the solid content of the aminated silica-coated Prussian blue intermediate in N,N-dimethylformamide is 1-20 wt%, and the reaction is carried out under magnetic or mechanical stirring at a speed of 100-1500 rpm.

[0032] Furthermore, the centrifugation conditions in step B2 include a relative centrifugal force of 5000-15000g, a centrifugation time of 10-60min, and a centrifugation temperature of 4-25℃. The volume of each washing liquid is 5-20 times the volume of the precipitate. The total amount of residual small molecules in the intermediate obtained in step B2 is not higher than 0.5wt%. The drying conditions include drying temperature, drying time, and vacuum degree.

[0033] Furthermore, in step A1, the solid content of the carboxylated silica-coated Prussian blue intermediate in the 2-(N-morpholine)ethanesulfonic acid buffer is 0.5-10 wt%.

[0034] Furthermore, in step A2, the amounts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are determined based on the molar number of surface carboxyl groups calculated from the mass of the intermediate in step A1 and the carboxyl group density. In step A3, the amount of 3-aminophenylboronic acid is determined based on the aforementioned molar number of surface carboxyl groups. Steps A2 and A3 are carried out under magnetic stirring or mechanical stirring conditions at a stirring speed of 100-1500 rpm.

[0035] Furthermore, the resuspension medium in step A4 is deionized water or 2-(N-morpholine)ethanesulfonic acid buffer, and the washing is performed 3-8 times, with each washing solution volume being 5-20 times the precipitate volume. The content of residual free 3-aminophenylboronic acid in the labeled nanoparticles obtained in step A4 is not higher than 0.5 wt% based on the dry weight of the labeled nanoparticles, and the solid content of the labeled nanoparticles after resuspension is 0.5-10 wt%.

[0036] Furthermore, in step S5, the pH value is adjusted by adding acid or alkali solution dropwise. The acid solution is hydrochloric acid solution or the alkali solution is sodium hydroxide solution. The concentration of the acid or alkali solution is 0.01-1 mol / L. The mixing is carried out under magnetic stirring or mechanical stirring conditions, and the stirring speed is 100-1000 rpm.

[0037] Furthermore, the reconstituted labeling solution used to prepare the chromatographic test strip labeling pad is a liquid labeling composition obtained by reconstituted the lyophilized powder labeling agent with deionized water to the volume before lyophilization or the solid content of the labeled nanoparticles before lyophilization; the reconstituted labeling solution is used to label the chromatographic test strip labeling pad, and the labeling treatment refers to applying the reconstituted labeling solution to the labeling pad and drying it, so that the labeled nanoparticles are preloaded on the labeling pad.

[0038] Furthermore, the average particle size recovery rate after reconstitution is calculated as the ratio of the average particle size after reconstitution to the average particle size before freeze-drying. The observation conditions for no visible sedimentation within 30 minutes after reconstitution are as follows: add 5 mL of reconstituted sample to a 5 mL glass bottle and let it stand at 25 °C for 30 minutes for observation.

[0039] Furthermore, the freeze-drying step includes a primary drying temperature of -40 to -20°C, a primary drying vacuum of 1-50 Pa, and secondary drying conditions, wherein the secondary drying conditions include a secondary drying temperature of -10 to 25°C, a secondary drying vacuum of 1-20 Pa, and a secondary drying time of 2-12 h.

[0040] As another aspect of this invention, a sequential preparation path is employed, consisting of Prussian blue nanoparticle preparation, silica coating and amination, carboxylation treatment, activation coupling, and final mixing. This path primarily enhances the controllable construction capability of labeled nanoparticles and the consistency of the labeled composition. By progressively limiting the average particle size, shell thickness, surface amino density, carboxyl density, and 3-aminophenylboronic acid grafting amount, this path enables the chromogenic core, interface layer, and recognition unit to achieve orderly matching within the same process chain. Furthermore, by controlling pH, mixing conditions, and lyophilization conditions, it balances reconstitution stability, rapid transport, and final detection sensitivity. This provides a scalable, recombinant preparation basis for microbial detection in dairy and meat products, adaptable to different carrier formats.

[0041] Prussian blue nanoparticles primarily function to generate color signals and establish signal intensity, serving as the core colorimetric unit for visual detection in the labeling system. The 3-aminophenylboronic acid-coupled carboxylated silica coating focuses more on interfacial stability, surface functionalization, and recognition of relevant structures on the microbial surface. The former determines the clarity and readability of the labeling signal, while the latter determines whether the particles can be stably dispersed and effectively approach the microorganisms to be detected. When both coexist synergistically within the same labeled nanoparticle, the silica coating not only buffers the aggregation tendency of Prussian blue nanoparticles in the system but also provides a stable carrier for 3-aminophenylboronic acid, allowing recognition and color development to occur simultaneously during transmission. This, in turn, improves microbial capture efficiency, color intensity, and the detection signal-to-noise ratio.

[0042] Beneficial technical effects 1. By constructing a carboxylated silica coating structure with Prussian blue nanoparticles as the core and 3-aminophenylboronic acid coupled on the outer layer, the labeled nanoparticles can simultaneously possess stable color development ability and functionalizable interface, which is beneficial to maintain a clear detection signal and high surface recognition site exposure in the complex matrix of dairy and meat products.

[0043] 2. By synergistically regulating the microenvironment of the labeled composition through bovine serum albumin, polysorbate 20, D-(+)-trehalose dihydrate and salt components, particle aggregation can be effectively inhibited and low viscosity dispersion stability can be improved. While increasing the solid content, good anti-settling and reconstitution compatibility can be maintained.

[0044] 3. By synergistically limiting the average particle size, silica shell thickness, 3-aminophenylboronic acid grafting amount, Prussian blue mass fraction, and pH value, the labeled nanoparticles can balance color intensity and transport efficiency during rapid chromatographic migration, which is beneficial to improving high microbial capture efficiency and high detection sensitivity.

[0045] 4. By setting the labeling composition into a liquid form for use and a lyophilized powder form for storage, wherein the liquid form includes liquid labeling solution, labeling solution for microfluidic chips, and labeling solution for preparing chromatographic test strip labeling pads; and the lyophilized powder form is a lyophilized powder labeling agent, which is reconstituted before use to obtain a reconstituted labeling solution, the reconstituted labeling solution can be used to label chromatographic test strip labeling pads, which can take into account the convenience of storage and transportation, the average particle size recovery rate after lyophilization and reconstitution, and the detection signal-to-noise ratio in different use scenarios, thereby improving the application flexibility of the system. Attached Figure Description

[0046] Figure 1 The image shows the particle size distribution of DLS before resolution in Examples 1, 8, and 9.

[0047] Figure 2 The image shows the particle size distribution of DLS after resolution in Examples 1, 8, and 9.

[0048] Figure 3 The Z-average retention scatter plots are for Example 1, Comparative Example 8, and Comparative Example 9.

[0049] Figure 4 The PDI scatter plots are for Example 1, Comparative Example 8, and Comparative Example 9.

[0050] Figure 5 The figures are rotational rheological viscosity-shear rate curves for Example 1 and Comparative Example 8.

[0051] Figure 6 The images shown are high-resolution XPS B1s spectra of Example 1, Comparative Example 6, and Comparative Example 10.

[0052] Figure 7 The images show the high-resolution XPS N1s spectra of Example 1, Comparative Example 6, and Comparative Example 10.

[0053] Figure 8 The image shows the B / Si atomic ratio dot plots for Example 1, Comparative Example 6, and Comparative Example 10.

[0054] Figure 9 The image shows the B / N atomic ratio dot plots for Example 1, Comparative Example 6, and Comparative Example 10.

[0055] Figure 10 The graphs show the capture efficiency-bacterial concentration curves of Staphylococcus aureus for Example 1 and Comparative Example 10.

[0056] Figure 11 The graphs show the capture efficiency-bacterial concentration curves of Escherichia coli for Example 1 and Comparative Example 10.

[0057] Figure 12 This is a superimposed image of the particle size distribution of Example 1 and Comparative Example 7.

[0058] Figure 13 The images show two-dimensional window scatter plots of particle size versus shell thickness for Example 1 and Comparative Example 7.

[0059] Figure 14 The above are the net signal-concentration curves of Staphylococcus aureus T-line for Example 1 and Comparative Example 7.

[0060] Figure 15 The T-line net signal-concentration curves for Listeria monocytogenes in Example 1 and Comparative Example 7 are shown.

[0061] Figure 16 Macroscopic optical photograph of the labeled nanoparticle freeze-dried powder prepared in Example 1 of this invention.

[0062] Figure 17 This is a scanning electron microscope image of the labeled nanoparticles prepared in Example 1 of the present invention.

[0063] Figure 18 Transmission electron microscopy (TEM) image of labeled nanoparticles prepared in Example 1 of this invention. Detailed Implementation

[0064] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0065] Example 1 I. Preparation of the labeling composition This embodiment provides a microbial detection labeling composition for dairy and meat products, comprising the following components based on 100 parts by weight of the total labeling composition: The composition consisted of 2.5 parts labeled nanoparticles, 2.5 parts bovine serum albumin, 0.25 parts polysorbate 20, 5.5 parts D-(+)-trehalose dihydrate, 1.0 part sodium chloride, 0.10 parts potassium chloride, 0.25 parts anhydrous disodium hydrogen phosphate, 0.10 parts potassium dihydrogen phosphate, and the remainder was 87.8 parts deionized water.

[0066] The labeled nanoparticles in this embodiment are Prussian blue nanoparticles with a Prussian blue nanoparticle core and 3-aminophenylboronic acid coupled carboxylated silica coated with Prussian blue. The average particle size of the labeled nanoparticles in this embodiment is 105 nm, the silica shell thickness is 14 nm, the 3-aminophenylboronic acid grafting amount is 40 μmol / g, and the mass fraction of Prussian blue in the labeled nanoparticles in this embodiment is 45 wt%. The pH value of the labeling composition in this embodiment is 7.2.

[0067] II. Preparation of labeled nanoparticles The labeled nanoparticles in this embodiment are prepared by the following steps: Step S1: Preparation of Prussian Blue Nanoparticles: Ferric chloride hexahydrate and potassium ferrocyanide trihydrate were prepared in deionized water, with a molar ratio of ferric ions to ferrocyanide ions of 1:1. Citric acid monohydrate was added to a concentration of 10 mmol / L, and polyvinylpyrrolidone K30 was added to a concentration of 1.0 wt%. The reaction was carried out at 30°C with magnetic stirring at 600 rpm for 1.5 h. After the reaction was complete, the nanoparticles were centrifuged at a relative centrifugal force of 8000 g for 30 min at a centrifugation temperature of 15°C. The nanoparticles were washed three times with deionized water, with each wash volume being 10 times the volume of the precipitate, yielding Prussian Blue nanoparticles with an average particle size of 50 nm and a polydispersity index of 0.22.

[0068] Step S2: Preparation of aminated silica-coated Prussian blue intermediate: The Prussian blue nanoparticles obtained in step S1 were dispersed in a mixed solution of ethanol and deionized water (ethanol to deionized water volume ratio of 4:1) to make the solid content of Prussian blue nanoparticles 2.0 wt%. Tetraethyl orthosilicate was added to make the volume fraction of tetraethyl orthosilicate 5 vol%, and ammonia water was added to make the volume fraction of ammonia water 2.5 vol%. The reaction was carried out at 28°C with magnetic stirring at 500 rpm for 3 h to obtain silica-coated Prussian blue particles.

[0069] 3-Aminopropyltriethoxysilane was added to the above silica-coated Prussian blue particle dispersion to make the volume fraction of 3-aminopropyltriethoxysilane 2.5 vol%, and the mixture was reacted at 65°C in a reaction system equipped with a reflux condenser for 3 h.

[0070] After the reaction was complete, the sample was washed three times each with ethanol and deionized water, with each washing solution being 10 times the volume of the precipitate. The sample was then vacuum dried at 60°C for 8 hours (vacuum degree 10 Pa) to obtain an aminated silica-coated Prussian blue intermediate with a shell thickness of 13 nm and a surface amino density of 175 μmol / g.

[0071] Step S3: Preparation of carboxylated silica-coated Prussian blue intermediate: The aminated silica-coated Prussian blue intermediate obtained in step S2 was dispersed in N,N-dimethylformamide to make the solid content 8.0 wt%. Succinic anhydride was added. In this embodiment, the mass ratio of aminated silica-coated Prussian blue intermediate to succinic anhydride was 1:0.8. The reaction was carried out at 28°C with magnetic stirring at 500 rpm for 5 h.

[0072] After the reaction was complete, the sample was centrifuged at a relative centrifugal force of 8000g for 30 min at a centrifugation temperature of 15℃. It was washed three times each with ethanol and deionized water, with each washing liquid volume being 10 times the volume of the precipitate. The sample was then vacuum dried at 60℃ for 8 h (vacuum degree 10 Pa) to obtain a carboxylated silica-coated Prussian blue intermediate with a carboxyl group density of 115 μmol / g and a total residual small molecule content of 0.3 wt%.

[0073] Step S4: Preparation of 3-aminophenylboronic acid coupled-labeled nanoparticles: The carboxylated silica-coated Prussian blue intermediate obtained in step S3 was dispersed in 2-(N-morpholine)ethanesulfonic acid buffer to make the solid content 5.0 wt%. In this example, the concentration of 2-(N-morpholine)ethanesulfonic acid buffer was 55 mmol / L and the pH value was 5.8.

[0074] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added. In this example, the molar ratio of the intermediate surface carboxyl group, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide was 1:3:3. The mixture was activated at 23°C with magnetic stirring at 400 rpm for 35 min.

[0075] 3-Aminophenylboronic acid was added. In this example, the molar ratio of carboxyl groups on the surface of the intermediate to 3-aminophenylboronic acid was 1:1.75. The reaction was carried out at 23°C with magnetic stirring at 400 rpm for 7 hours.

[0076] After the reaction was complete, the sample was centrifuged at a relative centrifugal force of 8000g for 30 min, washed 5 times with deionized water, with each wash volume being 10 times the volume of the precipitate, and then resuspended in deionized water to obtain labeled nanoparticles with a 3-aminophenylboronic acid grafting amount of 40 μmol / g, a residual free 3-aminophenylboronic acid content of 0.3 wt% based on the dry weight of the labeled nanoparticles, and a solid content of 3.0 wt%.

[0077] Step S5: Preparation of the labeling composition: The labeled nanoparticle suspension obtained in step S4 is mixed with bovine serum albumin, polysorbate 20, D-(+)-trehalose dihydrate, sodium chloride, potassium chloride, anhydrous disodium hydrogen phosphate, and potassium dihydrogen phosphate in the above-mentioned mass ratio according to the dry basis mass of the labeled nanoparticles. Deionized water is added to make up to 100 parts by mass. The mixture is stirred magnetically at 500 rpm for 38 min at 15°C. The pH value is adjusted to 7.2 with 0.1 mol / L sodium hydroxide solution to obtain the liquid labeling composition.

[0078] Step S6: Preparation of lyophilized powder labeling agent: The liquid labeling composition obtained in step S5 was pre-frozen at a pre-freezing temperature of -42℃ for 5 hours, then mainly dried at a main drying temperature of -30℃ and a main drying vacuum of 20Pa for 30 hours, followed by secondary drying at a secondary drying temperature of 10℃ and a secondary drying vacuum of 5Pa for 6 hours, to obtain a lyophilized powder labeling agent with a residual moisture content of 1.8wt%. For reconstitution, the lyophilized powder labeling agent was reconstituted with deionized water to the volume before lyophilization or the solid content of the labeled nanoparticles before lyophilization, and shaken for 30 seconds to obtain a reconstituted labeling solution. In this embodiment, the average particle size recovery rate of the lyophilized powder labeling agent after reconstitution was 92%, and 5 mL of the reconstituted labeling solution was placed in a 5 mL glass bottle and allowed to stand at 25℃ for 30 minutes; no visible sedimentation was observed.

[0079] The labeling composition prepared in this embodiment can be used as a liquid labeling solution or a labeling solution for microfluidic chips; it can also be lyophilized and stored as a lyophilized powder labeling agent, and reconstituted before use to obtain a reconstituted labeling solution, which can be used to label the labeling pad of chromatography test strips.

[0080] Example 2 I. Preparation of the labeling composition This embodiment provides a microbial detection labeling composition for dairy and meat products, comprising the following components based on 100 parts by weight of the total labeling composition: The composition consisted of 1.5 parts labeled nanoparticles, 3.5 parts bovine serum albumin, 0.15 parts polysorbate 20, 7.0 parts D-(+)-trehalose dihydrate, 0.6 parts sodium chloride, 0.06 parts potassium chloride, 0.15 parts anhydrous disodium hydrogen phosphate, 0.06 parts potassium dihydrogen phosphate, and the remainder was 86.98 parts deionized water.

[0081] The labeled nanoparticles in this embodiment are Prussian blue nanoparticles with a Prussian blue nanoparticle core and 3-aminophenylboronic acid-coupled carboxylated silica-coated Prussian blue nanoparticles. The average particle size of the labeled nanoparticles in this embodiment is 85 nm, the silica shell thickness is 11 nm, the 3-aminophenylboronic acid grafting amount is 25 μmol / g, and the mass fraction of Prussian blue in the labeled nanoparticles in this embodiment is 55 wt%. The pH value of the labeling composition in this embodiment is 7.0.

[0082] II. Preparation of labeled nanoparticles The labeled nanoparticles in this embodiment are prepared by the following steps: Step S1: Preparation of Prussian Blue Nanoparticles: Ferric chloride hexahydrate and potassium ferrocyanide trihydrate were prepared in deionized water, with a molar ratio of ferric ions to ferrocyanide ions of 0.95:1. Citric acid monohydrate was added to a concentration of 6 mmol / L, and polyvinylpyrrolidone K30 was added to a concentration of 0.6 wt%. The reaction was carried out at 26 °C with magnetic stirring at 500 rpm for 1.0 h. After the reaction was complete, the nanoparticles were centrifuged at a relative centrifugal force of 10000 g for 25 min at a centrifugation temperature of 10 °C. The nanoparticles were washed three times with deionized water, each time with a washing volume eight times the precipitate volume, to obtain Prussian Blue nanoparticles with an average particle size of 38 nm and a polydispersity index of 0.18.

[0083] Step S2: Preparation of aminated silica-coated Prussian blue intermediate: The Prussian blue nanoparticles obtained in step S1 were dispersed in a mixed solution of ethanol and deionized water (ethanol to deionized water volume ratio of 3:1) to make the solid content of Prussian blue nanoparticles 1.5wt%. Tetraethyl orthosilicate was added to make the volume fraction of tetraethyl orthosilicate 3vol%, and ammonia water was added to make the volume fraction of ammonia water 1.5vol%. The reaction was carried out at 24℃ with magnetic stirring at 400rpm for 2h to obtain silica-coated Prussian blue particles.

[0084] 3-Aminopropyltriethoxysilane was added to the above silica-coated Prussian blue particle dispersion to make the volume fraction of 3-aminopropyltriethoxysilane 1.5 vol%, and the mixture was reacted at 58°C in a reaction system equipped with a reflux condenser for 2 h.

[0085] After the reaction was complete, the sample was washed three times each with ethanol and deionized water, with each washing solution being eight times the volume of the precipitate. The sample was then vacuum dried at 55°C for 6 hours (vacuum degree 15 Pa) to obtain an aminated silica-coated Prussian blue intermediate with a shell thickness of 10 nm and a surface amino density of 120 μmol / g.

[0086] Step S3: Preparation of carboxylated silica-coated Prussian blue intermediate: The aminated silica-coated Prussian blue intermediate obtained in step S2 was dispersed in N,N-dimethylformamide to make the solid content 5.0 wt%. Succinic anhydride was added. In this embodiment, the mass ratio of aminated silica-coated Prussian blue intermediate to succinic anhydride was 1:0.5. The reaction was carried out at 24°C with magnetic stirring at 400 rpm for 3 h.

[0087] After the reaction was complete, the sample was centrifuged at a relative centrifugal force of 10000g for 25 min at a centrifugation temperature of 10℃. It was washed three times each with ethanol and deionized water, with each wash volume being 8 times the volume of the precipitate. The sample was then vacuum dried at 55℃ for 6 h (vacuum degree 15Pa) to obtain a carboxylated silica-coated Prussian blue intermediate with a carboxyl group density of 70 μmol / g and a residual small molecule total of 0.2 wt%.

[0088] Step S4: Preparation of 3-aminophenylboronic acid coupled-labeled nanoparticles: The carboxylated silica-coated Prussian blue intermediate obtained in step S3 was dispersed in 2-(N-morpholine)ethanesulfonic acid buffer to make the solid content 3.0 wt%. In this example, the concentration of 2-(N-morpholine)ethanesulfonic acid buffer was 35 mmol / L and the pH value was 5.4.

[0089] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added. In this example, the molar ratio of the intermediate surface carboxyl group, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide was 1:2:2. The mixture was activated at 19°C with magnetic stirring at 300 rpm for 20 min.

[0090] 3-Aminophenylboronic acid was added. In this example, the molar ratio of carboxyl groups on the surface of the intermediate to 3-aminophenylboronic acid was 1:1.0. The mixture was then reacted at 19°C with magnetic stirring at 300 rpm for 4 hours.

[0091] After the reaction was complete, the sample was centrifuged at a relative centrifugal force of 10000g for 25 min, washed 6 times with deionized water, with each wash volume being 8 times the volume of the precipitate, and then resuspended in deionized water to obtain labeled nanoparticles with a 3-aminophenylboronic acid grafting amount of 25 μmol / g, a residual free 3-aminophenylboronic acid content of 0.2 wt% based on the dry weight of the labeled nanoparticles, and a solid content of 2.0 wt%.

[0092] Step S5: Preparation of the labeling composition: The labeled nanoparticle suspension obtained in step S4 is mixed with bovine serum albumin, polysorbate 20, D-(+)-trehalose dihydrate, sodium chloride, potassium chloride, anhydrous disodium hydrogen phosphate, and potassium dihydrogen phosphate in the above-mentioned mass ratio according to the dry basis mass of the labeled nanoparticles. Deionized water is added to make up to 100 parts by mass. The mixture is stirred magnetically at 400 rpm for 25 min at 10°C. The pH value is adjusted to 7.0 with 0.1 mol / L sodium hydroxide solution to obtain the liquid labeling composition.

[0093] Step S6: Preparation of lyophilized powder labeling agent: The liquid labeling composition obtained in step S5 was pre-frozen at a pre-freezing temperature of -45℃ for 3 hours, then mainly dried at a main drying temperature of -35℃ and a main drying vacuum of 25Pa for 20 hours, followed by secondary drying at a secondary drying temperature of 5℃ and a secondary drying vacuum of 8Pa for 4 hours, to obtain a lyophilized powder labeling agent with a residual moisture content of 1.2wt%. For reconstitution, the lyophilized powder labeling agent was reconstituted with deionized water to the volume before lyophilization or the solid content of the labeled nanoparticles before lyophilization, and shaken for 30 seconds to obtain a reconstituted labeling solution. In this embodiment, the average particle size recovery rate of the lyophilized powder labeling agent after reconstitution was 89%, and 5 mL of the reconstituted labeling solution was placed in a 5 mL glass bottle and allowed to stand at 25℃ for 30 minutes; no visible sedimentation was observed.

[0094] The labeling composition prepared in this embodiment can be used as a liquid labeling solution or a labeling solution for microfluidic chips; it can also be lyophilized and stored as a lyophilized powder labeling agent, and reconstituted before use to obtain a reconstituted labeling solution, which can be used to label the labeling pad of chromatography test strips.

[0095] Example 3 I. Preparation of the labeling composition This embodiment provides a microbial detection labeling composition for dairy and meat products, comprising the following components based on 100 parts by weight of the total labeling composition: The composition consisted of 3.5 parts labeled nanoparticles, 1.5 parts bovine serum albumin, 0.35 parts polysorbate 20, 3.5 parts D-(+)-trehalose dihydrate, 1.4 parts sodium chloride, 0.14 parts potassium chloride, 0.35 parts anhydrous disodium hydrogen phosphate, 0.14 parts potassium dihydrogen phosphate, and the remainder was 89.12 parts deionized water.

[0096] The labeled nanoparticles in this embodiment are Prussian blue nanoparticles with a Prussian blue nanoparticle core and 3-aminophenylboronic acid coupled carboxylated silica coated with Prussian blue. The average particle size of the labeled nanoparticles in this embodiment is 125 nm, the silica shell thickness is 17 nm, the 3-aminophenylboronic acid grafting amount is 55 μmol / g, and the mass fraction of Prussian blue in the labeled nanoparticles in this embodiment is 35 wt%. The pH value of the labeling composition in this embodiment is 7.4.

[0097] II. Preparation of labeled nanoparticles The labeled nanoparticles in this embodiment are prepared by the following steps: Step S1: Preparation of Prussian Blue Nanoparticles: Ferric chloride hexahydrate and potassium ferrocyanide trihydrate were prepared in deionized water, with a molar ratio of ferric ions to ferrocyanide ions of 1.05:1. Citric acid monohydrate was added to a concentration of 14 mmol / L, and polyvinylpyrrolidone K30 was added to a concentration of 1.4 wt%. The reaction was carried out at 34 °C with magnetic stirring at 800 rpm for 2.0 h. After the reaction was complete, the nanoparticles were centrifuged at a relative centrifugal force of 7000 g for 35 min at a centrifugation temperature of 20 °C. The nanoparticles were washed four times with deionized water, with each wash volume being 12 times the volume of the precipitate, yielding Prussian Blue nanoparticles with an average particle size of 62 nm and a polydispersity index of 0.26.

[0098] Step S2: Preparation of aminated silica-coated Prussian blue intermediate: The Prussian blue nanoparticles obtained in step S1 were dispersed in a mixed solution of ethanol and deionized water (ethanol to deionized water volume ratio of 5:1) to make the solid content of Prussian blue nanoparticles 3.5wt%. Tetraethyl orthosilicate was added to make the volume fraction of tetraethyl orthosilicate 7vol%, and ammonia water was added to make the volume fraction of ammonia water 3.5vol%. The reaction was carried out at 32℃ with magnetic stirring at 700rpm for 4h to obtain silica-coated Prussian blue particles.

[0099] 3-Aminopropyltriethoxysilane was added to the above silica-coated Prussian blue particle dispersion to make the volume fraction of 3-aminopropyltriethoxysilane 3.5 vol%, and the mixture was reacted at 72°C in a reaction system equipped with a reflux condenser for 4 h.

[0100] After the reaction was complete, the sample was washed four times each with ethanol and deionized water, with each wash volume being 12 times the volume of the precipitate. The sample was then vacuum dried at 65°C for 10 h (vacuum degree 8 Pa) to obtain an aminated silica-coated Prussian blue intermediate with a shell thickness of 16 nm and a surface amino density of 225 μmol / g.

[0101] Step S3: Preparation of carboxylated silica-coated Prussian blue intermediate: The aminated silica-coated Prussian blue intermediate obtained in step S2 was dispersed in N,N-dimethylformamide to make the solid content 12.0 wt%. Succinic anhydride was added. In this embodiment, the mass ratio of aminated silica-coated Prussian blue intermediate to succinic anhydride was 1:1.1. The reaction was carried out at 32°C with magnetic stirring at 700 rpm for 6 h.

[0102] After the reaction was complete, the sample was centrifuged at a relative centrifugal force of 7000g for 35 min at a centrifugation temperature of 20℃. It was washed four times each with ethanol and deionized water, with each wash volume being 12 times the volume of the precipitate. The sample was then vacuum dried at 65℃ for 10 h (vacuum degree 8Pa) to obtain a carboxylated silica-coated Prussian blue intermediate with a carboxyl group density of 155 μmol / g and a total residual small molecule content of 0.4 wt%.

[0103] Step S4: Preparation of 3-aminophenylboronic acid coupled-labeled nanoparticles: The carboxylated silica-coated Prussian blue intermediate obtained in step S3 was dispersed in 2-(N-morpholine)ethanesulfonic acid buffer to make the solid content 7.0 wt%. In this example, the concentration of 2-(N-morpholine)ethanesulfonic acid buffer was 70 mmol / L and the pH value was 6.1.

[0104] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added. In this example, the molar ratio of the intermediate surface carboxyl group, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide was 1:4:4. The mixture was activated at 27°C with magnetic stirring at 600 rpm for 48 min.

[0105] 3-Aminophenylboronic acid was added. In this example, the molar ratio of carboxyl groups on the surface of the intermediate to 3-aminophenylboronic acid was 1:2.3. The mixture was reacted at 27°C with magnetic stirring at 600 rpm for 9 hours.

[0106] After the reaction was complete, the sample was centrifuged at a relative centrifugal force of 7000g for 35 min, washed 5 times with deionized water, with each wash volume being 12 times the volume of the precipitate, and then resuspended in deionized water to obtain labeled nanoparticles with a 3-aminophenylboronic acid grafting amount of 55 μmol / g, a residual free 3-aminophenylboronic acid content of 0.4 wt% based on the dry weight of the labeled nanoparticles, and a solid content of 6.0 wt%.

[0107] Step S5: Preparation of the labeling composition: The labeled nanoparticle suspension obtained in step S4 is mixed with bovine serum albumin, polysorbate 20, D-(+)-trehalose dihydrate, sodium chloride, potassium chloride, anhydrous disodium hydrogen phosphate, and potassium dihydrogen phosphate in the above-mentioned mass ratio according to the dry basis mass of the labeled nanoparticles. Deionized water is added to make up to 100 parts by mass. The mixture is stirred magnetically at 600 rpm for 48 min at 19°C. The pH value is adjusted to 7.4 with 0.1 mol / L sodium hydroxide solution to obtain the liquid labeling composition.

[0108] Step S6: Preparation of lyophilized powder labeling agent: The liquid labeling composition obtained in step S5 was pre-frozen at a pre-freezing temperature of -38℃ for 6 hours, then mainly dried at a main drying temperature of -25℃ and a main drying vacuum of 15Pa for 38 hours, followed by secondary drying at a secondary drying temperature of 18℃ and a secondary drying vacuum of 3Pa for 8 hours, to obtain a lyophilized powder labeling agent with a residual moisture content of 2.4wt%. For reconstitution, the lyophilized powder labeling agent was reconstituted with deionized water to the volume before lyophilization or the solid content of the labeled nanoparticles before lyophilization, and shaken for 30 seconds to obtain a reconstituted labeling solution. In this embodiment, the average particle size recovery rate of the lyophilized powder labeling agent after reconstitution was 94%, and 5 mL of the reconstituted labeling solution was placed in a 5 mL glass bottle and allowed to stand at 25℃ for 30 minutes; no visible sedimentation was observed.

[0109] The labeling composition prepared in this embodiment can be used as a liquid labeling solution or a labeling solution for microfluidic chips; it can also be lyophilized and stored as a lyophilized powder labeling agent, and reconstituted before use to obtain a reconstituted labeling solution, which can be used to label the labeling pad of chromatography test strips.

[0110] Example 4 I. Preparation of the labeling composition This embodiment provides a microbial detection labeling composition for dairy and meat products, comprising the following components based on 100 parts by weight of the total labeling composition: The composition consisted of 4.3 parts labeled nanoparticles, 4.3 parts bovine serum albumin, 0.43 parts polysorbate 20, 8.5 parts D-(+)-trehalose dihydrate, 0.2 parts sodium chloride, 0.018 parts potassium chloride, 0.045 parts anhydrous disodium hydrogen phosphate, 0.018 parts potassium dihydrogen phosphate, and the remainder was 82.189 parts deionized water.

[0111] The labeled nanoparticles in this embodiment are Prussian blue nanoparticles with a core of 3-aminophenylboronic acid coupled carboxylated silica coated with Prussian blue nanoparticles. The average particle size of the labeled nanoparticles in this embodiment is 138 nm, the silica shell thickness is 18 nm, the 3-aminophenylboronic acid grafting amount is 70 μmol / g, and the mass fraction of Prussian blue in the labeled nanoparticles in this embodiment is 25 wt%. The pH value of the labeling composition in this embodiment is 6.9.

[0112] II. Preparation of labeled nanoparticles The labeled nanoparticles in this embodiment are prepared by the following steps: Step S1: Preparation of Prussian Blue Nanoparticles: Ferric chloride hexahydrate and potassium ferrocyanide trihydrate were prepared in deionized water, with a molar ratio of ferric ions to ferrocyanide ions of 0.85:1. Citric acid monohydrate was added to a concentration of 17 mmol / L, and polyvinylpyrrolidone K30 was added to a concentration of 0.15 wt%. The reaction was carried out at 37°C with magnetic stirring at 1200 rpm for 2.6 h. After the reaction was complete, the nanoparticles were centrifuged at a relative centrifugal force of 12000 g for 45 min at a centrifugation temperature of 25°C. The nanoparticles were washed four times with deionized water, with each wash volume being 15 times the volume of the precipitate, yielding Prussian Blue nanoparticles with an average particle size of 73 nm and a polydispersity index of 0.28.

[0113] Step S2: Preparation of aminated silica-coated Prussian blue intermediate: The Prussian blue nanoparticles obtained in step S1 were dispersed in a mixed solution of ethanol and deionized water (ethanol to deionized water volume ratio of 6:1) to make the solid content of Prussian blue nanoparticles 4.2 wt%. Tetraethyl orthosilicate was added to make the volume fraction of tetraethyl orthosilicate 8.5 vol%, and ammonia water was added to make the volume fraction of ammonia water 4.3 vol%. The reaction was carried out at 33°C with magnetic stirring at 1200 rpm for 5.2 h to obtain silica-coated Prussian blue particles.

[0114] 3-Aminopropyltriethoxysilane was added to the above silica-coated Prussian blue particle dispersion to make the volume fraction of 3-aminopropyltriethoxysilane 4.3 vol%, and the mixture was reacted at 75°C in a reaction system equipped with a reflux condenser for 5.2 h.

[0115] After the reaction was complete, the sample was washed five times each with ethanol and deionized water, with each wash volume being 15 times the volume of the precipitate. The sample was then vacuum dried at 70°C for 12 hours (vacuum degree 5 Pa) to obtain an aminated silica-coated Prussian blue intermediate with a shell thickness of 17 nm and a surface amino density of 265 μmol / g.

[0116] Step S3: Preparation of carboxylated silica-coated Prussian blue intermediate: The aminated silica-coated Prussian blue intermediate obtained in step S2 was dispersed in N,N-dimethylformamide to make the solid content 15.0 wt%. Succinic anhydride was added. In this embodiment, the mass ratio of aminated silica-coated Prussian blue intermediate to succinic anhydride was 1:1.35. The reaction was carried out at 33°C with magnetic stirring at 1200 rpm for 7 h.

[0117] After the reaction was complete, the sample was centrifuged at a relative centrifugal force of 12000g for 45 min at a centrifugation temperature of 25℃. It was washed 5 times each with ethanol and deionized water, with each wash volume being 15 times the volume of the precipitate. The sample was then vacuum dried at 70℃ for 12 h (vacuum degree 5 Pa) to obtain a carboxylated silica-coated Prussian blue intermediate with a carboxyl group density of 178 μmol / g and a total residual small molecule content of 0.45 wt%.

[0118] Step S4: Preparation of 3-aminophenylboronic acid coupled-labeled nanoparticles: The carboxylated silica-coated Prussian blue intermediate obtained in step S3 was dispersed in 2-(N-morpholine)ethanesulfonic acid buffer to make the solid content 8.5 wt%. In this example, the concentration of 2-(N-morpholine)ethanesulfonic acid buffer was 85 mmol / L and the pH value was 6.3.

[0119] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added. In this example, the molar ratio of the intermediate surface carboxyl group, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide was 1:4.5:4.5. The mixture was activated at 28°C with magnetic stirring at 800 rpm for 53 min.

[0120] 3-Aminophenylboronic acid was added. In this example, the molar ratio of carboxyl groups on the surface of the intermediate to 3-aminophenylboronic acid was 1:2.7. The reaction was carried out at 28°C with magnetic stirring at 800 rpm for 10.5 h.

[0121] After the reaction was complete, the sample was centrifuged at a relative centrifugal force of 12000g for 45 min, washed 8 times with deionized water, with each wash volume being 15 times the volume of the precipitate, and then resuspended in deionized water to obtain labeled nanoparticles with a 3-aminophenylboronic acid grafting amount of 70 μmol / g, a residual free 3-aminophenylboronic acid content of 0.45 wt% based on the dry weight of the labeled nanoparticles, and a solid content of 8.0 wt%.

[0122] Step S5: Preparation of the labeling composition: The labeled nanoparticle suspension obtained in step S4 is mixed with bovine serum albumin, polysorbate 20, D-(+)-trehalose dihydrate, sodium chloride, potassium chloride, anhydrous disodium hydrogen phosphate, and potassium dihydrogen phosphate in the above-mentioned mass ratio according to the dry basis mass of the labeled nanoparticles. The mixture is then made up to 100 parts by mass with deionized water. The mixture is stirred magnetically at 800 rpm for 53 min at 22°C. The pH value is adjusted to 6.9 with 0.1 mol / L sodium hydroxide solution to obtain the liquid labeling composition.

[0123] Step S6: Preparation of lyophilized powder labeling agent: The liquid labeling composition obtained in step S5 was pre-frozen at a pre-freezing temperature of -37℃ for 7 hours, then mainly dried at a main drying temperature of -22℃ and a main drying vacuum of 10 Pa for 43 hours, followed by secondary drying at a secondary drying temperature of 22℃ and a secondary drying vacuum of 2 Pa for 10 hours, to obtain a lyophilized powder labeling agent with a residual moisture content of 2.7 wt%. For reconstitution, the lyophilized powder labeling agent was reconstituted with deionized water to the volume before lyophilization or the solid content of the labeled nanoparticles before lyophilization, and shaken for 30 seconds to obtain a reconstituted labeling solution. In this embodiment, the average particle size recovery rate of the lyophilized powder labeling agent after reconstitution was 96%, and 5 mL of the reconstituted labeling solution was placed in a 5 mL glass bottle and allowed to stand at 25℃ for 30 minutes; no visible sedimentation was observed.

[0124] The labeling composition prepared in this embodiment can be used as a liquid labeling solution or a labeling solution for microfluidic chips; it can also be lyophilized and stored as a lyophilized powder labeling agent, and reconstituted before use to obtain a reconstituted labeling solution, which can be used to label the labeling pad of chromatography test strips.

[0125] Comparative Example 1: Basically the same as Example 1, except that the amount of labeled nanoparticles used was 0.8 parts, and the amount of deionized water was made up to 100 parts by mass, while other conditions remained unchanged.

[0126] Comparative Example 2: Basically the same as Example 1, except that the amount of bovine serum albumin used was 4.8 parts, and the amount of deionized water was made up to 100 parts by weight, while other conditions remained unchanged.

[0127] Comparative Example 3: It is basically the same as Example 1, except that the amount of D-(+)-trehalose dihydrate is 1.5 parts, and the amount of deionized water is made up to 100 parts by mass. Other conditions remain unchanged.

[0128] Comparative Example 4: Basically the same as Example 1, except that the amount of polysorbate 20 is 0.02 parts, and the amount of deionized water is made up to 100 parts by weight, and other conditions remain unchanged.

[0129] Comparative Example 5: Basically the same as Example 1, except that in step S5, the pH value of the labeled composition was adjusted to 6.4 using 0.1 mol / L hydrochloric acid solution, while other conditions remained unchanged.

[0130] Comparative Example 6: It is basically the same as Example 1, except that in step S4, the molar ratio of carboxyl groups on the surface of the intermediate to 3-aminophenylboronic acid is 1:0.6, and coupling is carried out under the same activation and reaction conditions to obtain labeled nanoparticles with a 3-aminophenylboronic acid grafting amount of 12 μmol / g. Other conditions remain unchanged.

[0131] Comparative Example 7: It is basically the same as Example 1, except that the volume fraction of tetraethyl orthosilicate in step S2 is 1.5 vol%, and labeled nanoparticles with an average particle size of 72 nm and a silica shell thickness of 8 nm are prepared under the same subsequent conditions, with other conditions remaining unchanged.

[0132] Comparative Example 8: Essentially the same as Example 1, except that bovine serum albumin was removed, and only polysorbate 20 and D-(+)-trehalose dihydrate were retained. Deionized water was added to bring the balance to 100 parts by weight. Other conditions remained unchanged. This comparative example was used to verify the synergistic effect of bovine serum albumin with polysorbate 20 and D-(+)-trehalose dihydrate.

[0133] Comparative Example 9: Essentially the same as Example 1, except that D-(+)-trehalose dihydrate was removed, and only bovine serum albumin and polysorbate 20 were retained. Deionized water was added to bring the balance to 100 parts by weight. Other conditions remained unchanged. This comparative example was used to verify the synergistic effect of D-(+)-trehalose dihydrate with bovine serum albumin and polysorbate 20.

[0134] Comparative Example 10: Essentially the same as Example 1, except that in step S4, aniline was used instead of 3-aminophenylboronic acid, resulting in a molar ratio of carboxyl groups to aniline on the intermediate surface of 1:1.75. Activation coupling and post-treatment were performed under the same conditions as in Example 1, with other conditions remaining unchanged. This comparative example was used to verify the synergistic effect of the Prussian blue core, silica shell, and 3-aminophenylboronic acid recognition interface.

[0135] Performance testing: XPS was used to compare labeled nanoparticle samples and verify the surface exposure and interfacial chemical environment of the 3-aminophenylboronic acid recognition sites. After washing, the samples were drop-coated onto conductive silicon wafers and vacuum dried. Broadband and high-resolution spectra were acquired, calibrated with C1s 284.8 eV, with a pass energy of 20 eV and a step size of 0.05 eV. Each sample was tested three times, and the raw CSV was output. The characteristic peaks of B1s, N1s, Si2p, and Fe2p and the changes in atomic ratios were compared. The B / Si and B / N atomic ratios were calculated in detail.

[0136] Dynamic light scattering was used to determine the liquid-labeled composition and verify the equilibrium window of small particle size, narrow distribution, and high loading of labeled nanoparticles. Samples were diluted to a suitable scattering intensity and equilibrated at 25°C for 120 s with a backscattering angle of 173°. Each sample was tested three times consecutively, with at least 12 sub-runs per run. Z-average, PDI, and intensity distribution CSV were output. The colorimetric intensity was expressed as the absorbance A700 at 700 nm for samples with equal solid content, and the correlation between particle size parameters and this A700 value was established.

[0137] The interfacial electrostability of liquid-labeled compositions under low viscosity conditions was evaluated by electrophoretic light scattering determination. Samples were diluted with low ionic strength buffer and electrophoretic mobility distribution was measured at 25°C. Conductivity was controlled within the range of 0.2–2.0 mS / cm. Each sample was tested three times. Zeta potential was calculated from the electrophoretic mobility to determine the electrostatic repulsion ability of the particles. The mobility and zeta potential (CSV) were output, and the absolute values ​​were statistically analyzed.

[0138] Rotational rheometer was used to determine the low viscosity miscibility and compatibility with chromatography / microfluidic transport of liquid-labeled compositions at high solid content, evaluating their suitability. Tests were performed at 25°C using a cone-plate clamp (50 μm gap) for 0.1–1000 s. -1 Gradient scanning, pre-shearing for 30 seconds and balancing for 60 seconds, then acquiring the up and down curves, each test is performed 3 times, and the output is 100 seconds long. -1 Apparent viscosity, thixotropic area, and full curve CSV.

[0139] The reconstitution performance of the lyophilized powder labeling agent was evaluated. The lyophilized powder labeling agent was reconstituted with deionized water to the volume before lyophilization or the solid content of the labeled nanoparticles before lyophilization, and shaken for 30 s to obtain the reconstituted labeling solution. The overall absorbance A700 of the uniformly dispersed sample at 700 nm was measured as the initial value. The DLS particle size was measured 5 min after reconstitution. Then, 5 mL of the sample was placed in a 5 mL glass bottle and allowed to stand at 25 °C for 30 min. The supernatant A700 was measured. Each sample was tested 3 times. The particle size recovery rate was calculated as the average particle size after reconstitution / the average particle size before lyophilization × 100%. The dispersion stability index at 30 min was calculated as the supernatant A700 after standing for 30 min / the initial overall A700 × 100%. The anti-settling ability was evaluated in conjunction with the appearance after standing.

[0140] Labeled nanoparticles were incubated with Staphylococcus aureus and Escherichia coli model samples to evaluate the exposure of surface recognition sites and microbial capture ability. (10) 3 -10 6 Standard bacterial solutions were prepared at CFU / mL and incubated with the labeled system at 25℃ and 200rpm for 10 min before separation. The initial bacterial count and supernatant residual bacterial count were counted using Baird-Parker plates and TBX chromogenic medium, respectively, with 6 replicates per sample. The capture efficiency was calculated as (initial bacterial count - supernatant residual bacterial count) / initial bacterial count × 100%. The microbial capture efficiency in Table 1 is the average capture efficiency obtained by statistically analyzing the two model bacteria at each test concentration.

[0141] The reconstituted solution of the chromatographic test strip marker pad was tested with a series of target bacterial samples at different concentrations to evaluate the band response intensity, detection signal-to-noise ratio, and relative detection capability. Staphylococcus aureus and Listeria monocytogenes were used as target bacteria, at a concentration of 10...1 -10 5 Samples were prepared at CFU / mL. T-line grayscale and background noise were collected 10 min after sample loading. Six replicates were performed for each concentration, and the bacterial count was confirmed using the reference method in parallel. The detection signal-to-noise ratio was calculated as the net signal of the T-line / standard deviation of the background noise. The detection signal-to-noise ratio in Table 1 is the average value obtained from the statistical analysis of each test concentration within the linear working interval. The concentration-signal curve was fitted and the relative detection limit was calculated.

[0142] Figure 1 The image shows the particle size distribution of DLS before reconstitution for Example 1, Comparative Example 8, and Comparative Example 9. The particle size distribution of the three samples before reconstitution was characterized by dynamic light scattering. The results show that the main peak of Example 1 is more concentrated and the distribution is narrower, indicating that its initial dispersion state is more uniform.

[0143] Figure 2 The image shows the particle size distribution of DLS after reconstitution of Example 1, Comparative Example 8, and Comparative Example 9. The particle size distribution of the three samples after reconstitution was characterized by dynamic light scattering. The results show that the position of the main peak of Example 1 changed little after reconstitution, while the comparative example showed obvious shift and broadening, indicating that the reconstitution stability of Example 1 is better.

[0144] Figure 3 The Z-average retention scatter plots for Example 1, Comparative Example 8, and Comparative Example 9 are shown. The average hydrated particle size before and after reconstitution was statistically analyzed using the dynamic light scattering method and repeated comparisons were performed. The results show that the particle size retention of Example 1 is higher before and after reconstitution, indicating that the system is more structurally stable during drying and reconstitution.

[0145] Figure 4 The PDI scatter plots for Example 1, Comparative Example 8, and Comparative Example 9 are shown. The dynamic light scattering method was used to compare the changes in polydispersity index of each sample before and after reconstitution. The results show that the increase in PDI in Example 1 is relatively small, indicating that its particle distribution uniformity can still be well maintained after reconstitution.

[0146] Figure 5 The rotational rheological viscosity-shear rate curves of Example 1 and Comparative Example 8 are shown. The viscosity response and upward and downward hysteresis loops of the samples at different shear rates were compared by rotational rheological testing. The results show that Example 1 has less hysteresis and more stable rheological behavior, indicating that the system has both flowability and structural recovery ability.

[0147] Figure 6 The XPS B1s high-resolution spectra of Example 1, Comparative Example 6, and Comparative Example 10 are shown. The chemical state of boron on the sample surface was characterized by X-ray photoelectron spectroscopy. The results show that Example 1 has a more distinct B1s characteristic peak, indicating that the boric acid recognition group is more fully introduced on the surface.

[0148] Figure 7The XPS N1s high-resolution spectra of Example 1, Comparative Example 6, and Comparative Example 10 are shown. X-ray photoelectron spectroscopy was used to analyze the chemical environment of nitrogen on the sample surface. The results show that the N1s signal of Example 1 matches the target interface structure better, indicating that the surface functional layer is more complete.

[0149] Figure 8 The atomic ratio dot plots of B / Si for Example 1, Comparative Example 6, and Comparative Example 10 are shown. Based on the XPS quantitative results, the atomic ratio of boron to silicon on the sample surface is compared. The results show that the B / Si atomic ratio of Example 1 is the highest, indicating that the surface functional group loading level is higher and the shell surface modification is more effective.

[0150] Figure 9 The atomic ratio dot plots of B / N for Example 1, Comparative Example 6, and Comparative Example 10 are shown. Based on the XPS quantitative results, the atomic ratio of boron to nitrogen on the sample surface is compared. The results show that the B / N atomic ratio of Example 1 is better, indicating that the introduction of boric acid functional groups and the matching between nitrogen-containing interface components are more reasonable.

[0151] Figure 10 The image shows the capture efficiency-bacterial concentration curves of Staphylococcus aureus for Example 1 and Comparative Example 10. The capture efficiency at different initial bacterial concentrations was evaluated using a bacterial capture experiment combined with colony counting. The results show that Example 1 maintained a higher capture rate in all concentration ranges, indicating that it has a stronger ability to identify and enrich the target bacteria.

[0152] Figure 11 The graphs show the capture efficiency-bacterial concentration curves of Escherichia coli in Example 1 and Comparative Example 10. The capture efficiency at different initial bacterial concentrations was evaluated by combining bacterial capture experiments with colony counting. The results show that the overall capture efficiency of Example 1 is higher and the change is more stable, indicating that the interface still has good capture ability for different bacterial systems.

[0153] Figure 12 The particle size distribution of Example 1 and Comparative Example 7 is overlaid. The particle size distribution of the two samples was characterized by dynamic light scattering. The results show that the particle size center of Example 1 is more consistent with the target range and the distribution is more concentrated, indicating that its particle configuration is more conducive to the subsequent detection performance.

[0154] Figure 13 The two-dimensional window scatter plots of particle size and shell thickness for Example 1 and Comparative Example 7 are shown. The structural window was analyzed using single particle size statistics and shell thickness statistics methods. The results show that the particle size and shell thickness distribution of Example 1 are more concentrated in the effective window, indicating that its structural design is more coordinated and reasonable.

[0155] Figure 14The T-line net signal-concentration curves of Staphylococcus aureus in Example 1 and Comparative Example 7 are shown. The signal response intensity at different bacterial concentrations was compared using the T-line grayscale analysis method. The results show that the signal growth in Example 1 is more obvious and the linear relationship is better, indicating that it is more sensitive to the detection response of the target bacteria.

[0156] Figure 15 The T-line net signal-concentration curves of Listeria monocytogenes in Example 1 and Comparative Example 7 are shown. The signal response intensity at different bacterial concentrations was compared using the T-line grayscale analysis method. The results show that Example 1 exhibits a higher net signal in both the low and high concentration ranges, indicating that its detection stability and response capability are superior.

[0157] Figure 16 This is a macroscopic optical photograph of the labeled nanoparticle freeze-dried powder prepared in Example 1 of the present invention. The freeze-dried powder is a light blue, loose powder with a residual moisture content of 1.8 wt%. After reconstitution, it forms a uniform colloidal suspension. After standing for 30 minutes, there is no visible sedimentation. The average particle size recovery rate reaches 92%, which proves that the process parameters of pre-freezing temperature -42℃ and main drying vacuum degree of 20 Pa can effectively maintain the integrity of particle morphology and rapid reconstitution performance.

[0158] Figure 17 The image shown is a scanning electron microscope image of the labeled nanoparticles prepared in Example 1 of this invention. It shows that the freeze-dried sample is in a loosely packed state, with continuous particle surfaces and distinguishable boundaries.

[0159] Figure 18 The image shown is a transmission electron microscope image of the labeled nanoparticles prepared in Example 1 of this invention. It shows that the particles have core-shell appearance characteristics, and the interface between the Prussian blue core and the silica shell is identifiable.

[0160] Table 1 Summary of performance of examples and comparative examples

[0161] Note: Apparent viscosity is 100s. -1 The apparent viscosity was measured at 30 min; the dispersion stability index was calculated based on the A700 retention rate described above; the microbial capture efficiency was the average capture efficiency obtained statistically from the two model bacteria at each test concentration; the color intensity was expressed as the absorbance A700 of the sample with equal solids content at 700 nm; the detection signal-to-noise ratio was the average of the ratio of the net signal of the T line to the standard deviation of the background noise within the linear working interval.

[0162] As can be seen from the performance of the examples and comparative examples in Table 1, Examples 1-4 are generally superior to the comparative examples in key comprehensive indicators such as microbial capture efficiency and detection signal-to-noise ratio, and show better balance among average particle size recovery rate after reconstitution, 30-minute dispersion stability index, and apparent viscosity. Although some comparative examples are close to or higher than some examples in individual reconstitution indicators, their microbial capture efficiency, interfacial stability, or detection signal-to-noise ratio are significantly reduced. Among them, Example 3 shows the highest color intensity and detection signal-to-noise ratio due to the synergistic matching of the amount of labeled nanoparticles, grafting amount, and particle size; Example 4 achieves the best reconstitution recovery rate and anti-settling performance with the synergistic support of high protein / trehalose protection and freeze-drying conditions; Example 2 is more balanced between low viscosity and high interfacial electrical stability; and Example 1 shows a comprehensive balance advantage. Comparative Examples 6, 8, 9, and 10 respectively demonstrate the necessity and synergy of the technical solution of the present invention from three directions: insufficient recognition sites, missing stable units, and non-synergistic substitution.

[0163] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A microbial detection and labeling composition for dairy and meat products, characterized in that, The total mass of the labeled composition is 100 parts by weight, comprising: 0.1-5.0 parts of labeled nanoparticles on a dry basis, 0.1-5.0 parts of bovine serum albumin, 0.01-0.5 parts of polysorbate 20, 1.0-10.0 parts of D-(+)-trehalose dihydrate, 0.1-2.0 parts of sodium chloride, 0.005-0.2 parts of potassium chloride, 0.01-0.5 parts of anhydrous disodium hydrogen phosphate, 0.001-0.2 parts of potassium dihydrogen phosphate, and the remainder being deionized water. The deionized water is the amount remaining after adjusting for dry basis mass of the labeled nanoparticles and deducting the water content of the labeled nanoparticle suspension, to a total mass of 100 parts by weight, excluding the aforementioned components. The labeled nanoparticles are Prussian blue nanoparticles with a core of Prussian blue nanoparticles and 3-aminophenylboronic acid coupled carboxylated silica-coated Prussian blue nanoparticles. The average particle size of the labeled nanoparticles is 60-150 nm, the silica shell thickness is 8-20 nm, the 3-aminophenylboronic acid grafting amount is 5-80 μmol / g, and the mass fraction of Prussian blue in the labeled nanoparticles is 20-70 wt%. The labeled nanoparticles are prepared sequentially through Prussian blue nanoparticle preparation, silica coating and amination, carboxylation, and 3-aminophenylboronic acid activation coupling. The pH value of the labeled composition is 6.8-7.

6.

2. The labeling composition according to claim 1, characterized in that, The labeled nanoparticles are prepared by the following steps: A1, Disperse the carboxylated silica-coated Prussian blue intermediate in a 2-(N-morpholine)ethanesulfonic acid buffer solution, wherein the concentration of the 2-(N-morpholine)ethanesulfonic acid buffer solution is 10-100 mmol / L and the pH value is 5.0-6.5; A2, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, the molar ratio of the surface carboxyl groups of the carboxylated silica-coated Prussian blue intermediate in step A1, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:(1-5):(1-5), activate at 15-30℃ for 10-60 min; A3, add 3-aminophenylboronic acid, wherein the molar ratio of the carboxyl group on the surface of the intermediate to 3-aminophenylboronic acid is 1:(0.5-3), and react at 15-30℃ for 2-12 hours; A4, washed and resuspended, to obtain the labeled nanoparticles with a 3-aminophenylboronic acid grafting amount of 5-80 μmol / g and residual free 3-aminophenylboronic acid of no more than 0.5 wt% based on the dry weight of the labeled nanoparticles.

3. The labeling composition according to claim 2, characterized in that, The carboxylated silica-coated Prussian blue intermediate was prepared by the following steps: B1, dispersing the aminated silica-coated Prussian blue intermediate in N,N-dimethylformamide, adding succinic anhydride, wherein the mass ratio of the aminated silica-coated Prussian blue intermediate to succinic anhydride is 1:0.2 to 1:1.5, reacting at 20-35℃ for 2-8 hours; B2 was washed with ethanol and deionized water, 2-5 times each, and then dried to obtain the carboxylated silica-coated Prussian blue intermediate with a carboxyl density of 30-200 μmol / g.

4. The labeling composition according to claim 3, characterized in that, The aminated silica-coated Prussian blue intermediate was prepared by the following steps: C1, Prussian blue nanoparticles are dispersed in a mixed solution of ethanol and deionized water, tetraethyl orthosilicate is added to make the volume fraction of tetraethyl orthosilicate 1-10 vol%, and ammonia water is added to make the volume fraction of ammonia water 0.5-5 vol%. The reaction is carried out at 20-35℃ for 1-6 h to obtain silica-coated Prussian blue particles. C2, add 3-aminopropyltriethoxysilane to the silica-coated Prussian blue particle dispersion obtained in step C1, so that the volume fraction of 3-aminopropyltriethoxysilane is 0.5-5 vol%, and react at 50-80℃ for 1-6 h. C3, washing and drying, to obtain the aminated silica-coated Prussian blue intermediate with a shell thickness of 5-30 nm and a surface amino density of 50-300 μmol / g.

5. The labeling composition according to claim 4, characterized in that, The Prussian blue nanoparticles were prepared by the following steps: D1, ferric chloride hexahydrate and potassium ferrocyanide trihydrate are prepared in deionized water, with the molar ratio of ferric ions to ferrocyanide ions being 0.8:1 to 1.2:

1. Simultaneously, citrate monohydrate is added to achieve a concentration of 1-20 mmol / L, and polyvinylpyrrolidone K30 is added to achieve a concentration of 0.05-2.0 wt%. D2, stirred at 20-40℃, for 0.5-3 hours; D3, centrifuged and washed 2-5 times with deionized water to obtain the Prussian blue nanoparticles with an average particle size of 20-80 nm and a polydispersity index not higher than 0.

30.

6. The labeling composition according to claim 1, characterized in that, Prussian blue nanoparticles serve as the core of the labeled nanoparticles; the labeled nanoparticles have an average particle size of 60-150 nm, a silica shell thickness of 8-20 nm, a 3-aminophenylboronic acid grafting amount of 5-80 μmol / g, and a Prussian blue mass fraction of 20-70 wt% in the labeled nanoparticles; the pH value of the labeled composition is 6.8-7.

6.

7. The labeling composition according to claim 1, characterized in that, The labeling composition can be used in liquid form and in lyophilized powder form; The liquid application forms include liquid labeling solutions, labeling solutions for microfluidic chips, and labeling solutions for preparing chromatographic test strip labeling pads; The freeze-dried powder storage form is a freeze-dried powder labeling agent obtained by freeze-drying the liquid-use labeling composition; The lyophilized powder labeling agent is reconstituted before use to obtain a reconstituted labeling solution. The reconstituted labeling solution is a labeling composition restored to a liquid form for use and can be used as the liquid labeling solution, the labeling solution for microfluidic chips, or the labeling solution for preparing chromatographic test strip labeling pads. The average particle size recovery rate of the lyophilized powder labeling agent after reconstitution is not less than 85%, and there is no visible sedimentation within 30 minutes after reconstitution; the microorganism to be tested is selected from one or more of Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella.

8. The labeling composition according to claim 7, characterized in that, The labeling solution used to prepare the chromatographic test strip labeling pad is a liquid labeling composition; When the liquid labeling composition is derived from the lyophilized powder labeling agent, the labeling solution used to prepare the chromatographic test strip labeling pad is a reconstituted labeling solution, which is obtained by reconstituted the lyophilized powder labeling agent with deionized water to the volume before lyophilization or the solid content of the labeled nanoparticles before lyophilization. The label pad of the chromatography test strip is a porous pad material used in the chromatography test strip for preloading and releasing the labeled nanoparticles when the sample liquid flows through it; The preparation of the chromatographic test strip labeling pad, that is, the labeling treatment of the chromatographic test strip labeling pad, includes applying the reconstituted labeling solution to the labeling pad by means of impregnation, spotting or spraying and drying, so that the labeling nanoparticles are loaded on the labeling pad.

9. A method for preparing a microbial detection marker composition for dairy and meat products as described in any one of claims 1-8, characterized in that, Includes the following steps: S1 provides Prussian blue nanoparticles with an average particle size of 20-80 nm and a polydispersity index of no more than 0.

30. S2, the Prussian blue nanoparticles provided by S1 are coated with silica and aminated to obtain aminated silica-coated Prussian blue intermediates with a shell thickness of 5-30 nm and a surface amino density of 50-300 μmol / g. S3, the aminated silica-coated Prussian blue intermediate obtained in S2 is subjected to carboxylation treatment to obtain a carboxylated silica-coated Prussian blue intermediate with a carboxyl density of 30-200 μmol / g; S4, the carboxylated silica-coated Prussian blue intermediate obtained in S3 was activated and coupled to obtain labeled nanoparticles with an average particle size of 60-150 nm and a 3-aminophenylboronic acid grafting amount of 5-80 μmol / g. S5, the labeled nanoparticle suspension obtained in S4 is mixed with bovine serum albumin, polysorbate 20, D-(+)-trehalose dihydrate, sodium chloride, potassium chloride, anhydrous disodium hydrogen phosphate, potassium dihydrogen phosphate and deionized water according to the mass parts of each component mentioned above, and the mixture is made up to 100 parts by mass with deionized water, and the pH value is adjusted to 6.8-7.6 to obtain the labeled composition.

10. The preparation method according to claim 9, characterized in that, In step S5, the mixing temperature is 4-25℃ and the mixing time is 15-60 min; Step S5 is followed by a freeze-drying step, which includes: a pre-freezing temperature of -50 to -35°C, a pre-freezing time of 2-8 hours, a main drying time of 12-48 hours, and a residual moisture content of no more than 3.0 wt% after freeze-drying.

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