A prussian blue nanogold-based biosensor, and a preparation method and application thereof

Abstract

The application belongs to the technical field of biomedical engineering, and relates to detection and analysis of microorganisms, in particular to a biosensor based on Prussian blue nanogold, a preparation method and application, which is composed of a detection probe, a detection capillary and hydrogen peroxide; the detection probe is composed of Prussian blue nanogold and a first recognition molecule, and the first recognition molecule is connected to the surface of the Prussian blue nanogold; the detection capillary is sequentially provided with a detection zone and a measurement zone from one end to the other end, and the inner wall of the detection zone is provided with a second recognition molecule; the hydrogen peroxide is matched with the detection capillary; and the first recognition molecule and the second recognition molecule are both materials capable of specifically combining with target microorganisms. The biosensor based on Prussian blue nanogold can realize rapid, sensitive and semi-quantitative detection of target microorganisms such as salmonella.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical engineering, and relates to detection and analysis of microorganisms, in particular to a biosensor based on Prussian blue nanogold (PB@Au) and a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art that is already known in any jurisdiction.

[0003] The traditional microorganism culture method is a national standard method for detecting Salmonella, which has high accuracy but is time-consuming, laborious and professional, and is not suitable for on-site rapid detection. Immune analysis methods such as enzyme-linked immunoassay and fluorescence immunoassay use antibodies as recognition molecules, which can accurately identify target analytes, but the professional nature and instrument dependence of immune assays make it difficult to use for on-site rapid detection. The immune chromatography method has the advantages of convenient operation, rapid detection, low cost and low requirement for detection personnel, and is widely used in medical diagnosis, food safety, environmental monitoring and other fields. However, the sensitivity and accuracy of the immune chromatography technology still need to be improved due to the non-uniformity of nitrocellulose membrane and the influence of environment, and the quantitative detection method still needs to be explored. SUMMARY

[0004] In order to solve the problems of the prior art, the present application provides a biosensor based on Prussian blue nanogold and a preparation method and application thereof, which can realize rapid, sensitive and semi-quantitative detection of target microorganisms such as Salmonella.

[0005] In order to achieve the above-mentioned purposes, the technical scheme of the present application is as follows:

[0006] On the one hand, a biosensor based on Prussian blue nanogold is composed of a detection probe, a detection capillary and hydrogen peroxide. The detection probe is composed of Prussian blue nanogold and a first recognition molecule, and the first recognition molecule is connected to the surface of the Prussian blue nanogold. The detection capillary is sequentially provided with a detection zone and a measurement zone from one end to the other end, and the second recognition molecule is fixed on the inner wall of the detection zone. The hydrogen peroxide is matched with the detection capillary.

[0007] The first recognition molecule and the second recognition molecule are both materials capable of specifically binding to target microorganisms.

[0008] The biosensor provided by the application can realize specific binding of the first recognition molecule and the second recognition molecule to the target microorganism, so that the detection probe is fixed on the detection area of the detection capillary, at this time, hydrogen peroxide is added to the detection capillary, the hydrogen peroxide is catalytically decomposed into O2 by the Prussian blue nanogold, and the catalysis and the expansion gas are accelerated under the cooperation of the photothermal effect of the Prussian blue nanogold under laser irradiation, so that the generated gas drives the movement of the hydrogen peroxide liquid column in the capillary. By establishing a standard curve of the movement distance (ΔL) of the hydrogen peroxide liquid column and the number of target microorganisms, the purpose of quantitative detection of target microorganisms is achieved.

[0009] The first recognition molecule can be an antibody, an aptamer or the like, and the second recognition molecule can be an antibody, an aptamer or the like. When the first recognition molecule is an antibody, the detection probe is an immunological probe; and when the second recognition molecule is an antibody, the detection capillary is an immunological capillary.

[0010] In another aspect, a preparation method of a biosensor based on Prussian blue nanogold includes preparation of a detection probe and preparation of a detection capillary.

[0011] Preparation of the detection probe: an alkaline compound is added to a solution of Prussian blue nanogold and mixed uniformly, and then an antibody of the target microorganism is added and mixed, and the detection probe is obtained.

[0012] Preparation of the detection capillary: the inner surface of a glass capillary is modified with amino groups, an antibody solution of the target microorganism is injected into the detection area of the amino-modified glass capillary, and the detection capillary is obtained after incubation.

[0013] In a third aspect, the biosensor based on Prussian blue nanogold is applied to detection of target microorganisms.

[0014] In a fourth aspect, a detection method of Salmonella is provided, the biosensor based on Prussian blue nanogold is provided, and the target microorganism is Salmonella; and the method includes the following steps:

[0015] (1) mixing a detection probe solution with a solution to be detected containing Salmonella, and incubating;

[0016] (2) adding the solution obtained in step (1) to the detection area of a detection capillary, incubating, and removing the solution after incubation;

[0017] (3) injecting hydrogen peroxide into the detection area of the detection capillary obtained in step (2), irradiating the detection area, and recording the movement distance ΔL of the hydrogen peroxide liquid column;

[0018] (4) detecting Salmonella according to the ΔL obtained in step (3).

[0019] In a fifth aspect, a detection kit of Salmonella comprises the above-mentioned Prussian blue nanogold-based biosensor, a buffer and a light source.

[0020] The present application has the following advantages:

[0021] (1) The present application utilizes the catalase-like activity of Prussian blue nanogold to catalyze H2O2 to generate O2, and accelerates the catalysis and expands the gas volume by virtue of the photothermal conversion capability of Prussian blue nanogold, converts the volume signal generated in the detection into the moving distance signal (ΔL) of the capillary liquid column, establishes the standard curve of ΔL and the concentration of bacteria, and realizes sensitive and rapid detection of Salmonella typhimurium.

[0022] (2) The present application utilizes Prussian blue nanogold as a labeling material for a capillary detection method to construct a detection probe, and utilizes the catalytic activity of the probe to hydrogen peroxide instead of the traditional enzyme-catalyzed reaction of biological macromolecules, which greatly improves the stability of the enzyme, reduces the detection cost, and improves the detection sensitivity.

[0023] (3) The present application establishes an immune capillary rapid detection method based on the excellent photothermal conversion performance of Prussian blue nanogold, realizes rapid, sensitive and semi-quantitative detection of the test substance according to the standard curve of the moving distance of the liquid column converted from the gas volume after Prussian blue nanogold is combined with the immune capillary and the concentration of pathogenic bacteria, and has high detection sensitivity. The entire detection time is about 30 min, which is significantly lower than the detection process of enzyme-linked immunoassay (about 3 h). BRIEF DESCRIPTION OF DRAWINGS

[0024] The drawings accompanying the specification of the present application form a part thereof, serve to provide further understanding of the present application, and together with the description of the exemplary embodiments of the present application and the explanation thereof serve to explain the present application, and do not constitute an improper limitation of the present application.

[0025] Figure 1 FIG. 1 is a schematic diagram of the detection principle in the embodiments of the present application; A is a preparation process diagram of the immune probe and the immune capillary, and B is a process diagram of detecting Salmonella typhimurium by using the immune probe and the immune capillary.

[0026] Figure 2 FIG. 3 is a columnar diagram of the K3[Fe(CN)6] concentration optimization of the preparation process of Prussian blue (PB) in the embodiments of the present application.

[0027] Figure 3 FIG. 4 is a diagram of the PB concentration optimization of the preparation process of PB@Au and a stability characterization diagram of PB@Au composite material at room temperature for 12 h in the embodiments of the present application.

[0028] Figure 4UV-Vis absorption spectra of (A) PB and PB@Au in the embodiments of the present application; inset: images of PB and PB@Au solution; (B) hydrated particle size of PB and PB@Au; (C) Zeta potential of PB and PB@Au; (D) TEM image of PB; (E) TEM image of PB@Au; (F) XPS spectrum of PB; (G) XPS spectrum of PB@Au; (H) photothermal conversion efficiency of PB and PB@Au.

[0029] Figure 5 PB@Au concentration optimization chart for capillary connection in the embodiments of the present application.

[0030] Figure 6 PB@Au catalytic H2O2 concentration optimization chart in the embodiments of the present application.

[0031] Figure 7 Optimization selection chart of different laser irradiation modes in the embodiments of the present application.

[0032] Figure 8 Laser irradiation power optimization chart in the embodiments of the present application.

[0033] Figure 9 Laser irradiation time optimization chart in the embodiments of the present application.

[0034] Figure 10 PB@Au sealing liquid concentration optimization chart in the embodiments of the present application.

[0035] Figure 11 (A) Color change chart of PB@Au added with different concentrations of NaOH and (B) PB@Au added NaOH concentration optimization chart in the embodiments of the present application.

[0036] Figure 12 PB@Au connection antibody concentration optimization chart in the embodiments of the present application.

[0037] Figure 13 Capillary sealing liquid concentration optimization chart in the embodiments of the present application.

[0038] Figure 14 Capillary coating antibody concentration optimization chart in the embodiments of the present application.

[0039] Figure 15 Detection chart of different concentrations of Salmonella typhimurium in the embodiments of the present application.

[0040] Figure 16 Method specificity investigation chart in the embodiments of the present application; ****, P<0.0001.

[0041] Figure 17 Method repeatability investigation chart in the embodiments of the present application.

[0042] Figure 18 Figure 1 is a graph for investigating the 37℃ storage stability in the embodiment of the present application.

[0043] Figure 19 Figure 2 is a graph for detecting Salmonella typhi in the actual sample in the embodiment of the present application. DETAILED DESCRIPTION

[0044] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0045] It is to be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0046] In order to provide a new method for quantitatively detecting microorganisms such as Salmonella, the present application provides a Prussian blue nanogold-based biosensor and a preparation method and application thereof.

[0047] In a typical embodiment of the present application, a Prussian blue nanogold-based biosensor is provided, which comprises a detection probe, a detection capillary and hydrogen peroxide; the detection probe is composed of Prussian blue nanogold and a first recognition molecule, the first recognition molecule is connected to the surface of the Prussian blue nanogold to recognize the target; the detection capillary is sequentially provided with a detection zone and a measurement zone from one end to the other end, and a second recognition molecule is fixed on the inner wall of the detection zone; the hydrogen peroxide cooperates with the detection capillary.

[0048] The first recognition molecule and the second recognition molecule are both materials capable of specifically binding to the target microorganism.

[0049] The first recognition molecule can be an antibody, an aptamer, etc., and the second recognition molecule can be an antibody, an aptamer, etc. When the first recognition molecule is an antibody, the detection probe is an immunological probe; when the second recognition molecule is an antibody, the detection capillary is an immunological capillary. In some embodiments, the first recognition molecule is an antibody, and the detection probe is an immunological probe; the second recognition molecule is an antibody, and the detection capillary is an immunological capillary.

[0050] In one or more embodiments, the inner wall of the detection zone of the immunological capillary is modified with an amino group, and then the amino group is connected to the antibody.

[0051] In one or more embodiments, the immunocapillary detection zone is blocked by bovine serum albumin (BSA) from non-specific sites.

[0052] In one or more embodiments, the immunoprobe surface is blocked by BSA from non-specific sites.

[0053] Another embodiment of the present application provides a preparation method of a Prussian blue nanogold-based biosensor, comprising preparation of a detection probe and preparation of a detection capillary.

[0054] Preparation of the detection probe: adding an alkaline compound to a Prussian blue nanogold solution and mixing uniformly, and then adding a target microorganism antibody and mixing, to obtain the detection probe.

[0055] Preparation of the detection capillary: performing amino modification on the inner surface of a glass capillary, injecting a target microorganism antibody solution into a detection zone of the amino-modified glass capillary, and incubating to obtain the detection capillary.

[0056] The alkaline compound is, for example, potassium hydroxide, sodium hydroxide, etc.

[0057] In some embodiments, in the preparation process of the detection probe, the temperature for mixing the target microorganism antibody is 35-40°C.

[0058] In some embodiments, in the preparation process of the detection probe, the concentration of the Prussian blue nanogold solution is 0.6-1.0 mg·mL -1 . Preferably, 0.75-0.85 mg·mL -1 .

[0059] In some embodiments, when the concentration of the Prussian blue nanogold solution is 0.75-0.85 mg·mL -1 , the amount of the Prussian blue nanogold solution added is 9.5-10.5 μL, and the amount of the alkaline compound solution added is 2.3-2.7 μL, the concentration of the alkaline compound solution is 0-0.05 mol·L -1 . Preferably, 0-0.02 mol·L -1 , and further preferably, 0.005-0.015 mol·L -1 . The effect is better under this condition.

[0060] In some embodiments, when the concentration of the Prussian blue nanogold solution is 0.75-0.85 mg·mL -1 , the amount of the Prussian blue nanogold solution added is 9.5-10.5 μL, and the amount of the target microorganism antibody solution added is 3.5-4.5 μL, the concentration of the target microorganism antibody solution is not less than 25 μg·mL -1 . Preferably, not less than 75 μg·mL -1. Further preferably, the concentration of the BSA solution is 75-125 μg·mL -1 . The effect is better under this condition.

[0061] In some embodiments, during the preparation of the detection probe, after the target microorganism antibody is mixed, BSA is added for mixing.

[0062] In some embodiments, when the concentration of the Prussian blue nanogold solution is 0.75-0.85 mg·mL -1 , the amount of the Prussian blue nanogold solution added is 9.5-10.5 μL, the concentration of the target microorganism antibody solution is 70-80 μg·mL -1 , the amount of the target microorganism antibody solution added is 3.5-4.5 μL, and the amount of the BSA solution added is 7.5-8.5 μL, the concentration of the BSA solution is not less than 2% (mass percentage). Preferably, the concentration of the BSA solution is not less than 6%. The effect is better under this condition. Further preferably, the concentration of the BSA solution is 6-8%.

[0063] In some embodiments, the preparation method of the Prussian blue nanogold is as follows: a Prussian blue solution and a chloroauric acid solution are mixed, heated to boiling, then a sodium citrate solution is added, stirred and kept boiling, and then cooled to obtain the Prussian blue nanogold.

[0064] In one or more embodiments, when 155-165 μL of a chloroauric acid (HAuCl4) solution with a concentration of 0.5-1.5% (w / v) is added, and the amount of the Prussian blue solution added is 15-16 mL, the concentration of the Prussian blue solution is 0.08-0.16 mg·mL -1 . Preferably, the concentration of the Prussian blue solution is 0.10-0.14 mg·mL -1 . Further preferably, the concentration of the Prussian blue solution is 0.11-0.13 mg·mL -1 .

[0065] In one or more embodiments, the preparation method of the Prussian blue is as follows: polyvinylpyrrolidone and potassium ferricyanide are dissolved in water to obtain a precursor solution, hydrochloric acid is added to the precursor solution, heated to 160-180 °C for reaction, and then cooled to obtain the Prussian blue. The concentration of the potassium ferricyanide in the precursor solution is 0.005-0.03 mol·L -1 . When the concentration of the potassium ferricyanide in the precursor solution is 0.005-0.006 mol·L -1 , the catalytic activity of the Prussian blue is higher.

[0066] In some embodiments, the inner surface of the glass capillary tube is modified with amino groups by using 3-aminopropyltriethoxysilane (APTES). Specifically, the capillary tube is immersed in a methanol solution of APTES for 9-15 h, washed and dried.

[0067] In some embodiments, when 2.0-3.0 μL of the target microorganism antibody solution is injected into the detection zone of the amino-modified glass capillary, the concentration of the target microorganism antibody solution is not less than 70 μg·mL -1 . Preferably, 75-800 μg·mL -1 . Further preferably, 100-800 μg·mL -1 . Still further preferably, 400-600 μg·mL -1 .

[0068] In some embodiments, after the target microorganism antibody solution is injected into the detection zone of the amino-modified glass capillary, BSA solution is added for blocking treatment after incubation.

[0069] In one or more embodiments, 2.0-3.0 μL of the target microorganism antibody solution is injected into the detection zone of the amino-modified glass capillary, the concentration of the target microorganism antibody solution is not less than 70 μg·mL -1 , incubation is performed at 35-40°C, then washing is performed, and blocking treatment is performed using BSA solution with a concentration of not less than 2% (mass percentage). Preferably, the concentration of the BSA solution is not less than 4%, and further preferably, 4-8%.

[0070] In a third aspect, the application provides use of the above-mentioned Prussian blue nanogold-based biosensor in detection of target microorganisms.

[0071] Specifically, the target microorganism is Salmonella. More specifically, the target microorganism is Salmonella typhimurium.

[0072] In a fourth aspect, the application provides a method for detecting Salmonella, which comprises the following steps:

[0073] (1) mixing a detection probe solution with a solution to be tested containing Salmonella, and performing incubation;

[0074] (2) adding the solution obtained in step (1) to the detection zone of a detection capillary, performing incubation, and removing the solution after incubation;

[0075] (3) injecting hydrogen peroxide into the detection zone of the detection capillary obtained in step (2), irradiating the detection zone, and recording the moving distance ΔL of the hydrogen peroxide liquid column;

[0076] (4) detecting Salmonella according to ΔL obtained in step (3).

[0077] In some embodiments, the irradiation is laser irradiation. Preferably, the power of the laser irradiation is 1-4 W, and more preferably, 3-4 W. The laser irradiation time is not less than 80 s, and preferably, 80-120 s.

[0078] In a fifth aspect, a detection kit of Salmonella comprises the above-mentioned Prussian blue nanogold-based biosensor, a buffer, and a light source.

[0079] Specifically, the buffer is a PBS buffer.

[0080] Specifically, the light source is a laser.

[0081] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific embodiments.

[0082] Embodiments

[0083] This embodiment constructs a Prussian blue nanogold-based biosensor based on the double-antibody sandwich principle, and establishes a capillary gas-driven analysis method for rapid detection of Salmonella typhimurium using the biosensor. The biosensor utilizes the peroxidase-like activity and photothermal effect of the prepared Prussian blue nanogold (PB@Au) nanocomposite to improve the sensitivity of detecting Salmonella typhimurium. In the construction process of the biosensor, Salmonella typhimurium antibodies are connected to the surface of PB@Au nanoparticles, and the active sites of unbound antibodies are blocked by bovine serum albumin (BSA) to obtain a PB@Au immunoprobe for detection. The glass capillary modified by 3-aminopropyltriethoxysilane (APTES) has abundant amino groups, which is easy to bind with Salmonella typhimurium antibodies. After the capillary binds with the antibodies, BSA is used for blocking to obtain an immunocapillary. According to the double-antibody sandwich principle, the immunoprobe is fixed on the immunocapillary through Salmonella typhimurium, and the added H2O2 in the capillary is catalytically decomposed into O2 by the PB@Au of the immunoprobe, and under laser irradiation, the photothermal effect of PB@Au is utilized to accelerate the catalysis and expansion of the gas, and the generated gas pushes the H2O2 liquid column to move in the capillary. By establishing the standard curve of the moving distance (ΔL) of the H2O2 liquid column and the number of bacteria, the purpose of quantitative detection of Salmonella typhimurium is achieved, as shown in Figure 1 .

[0084] (1) Preparation of PB nanomaterials

[0085] Take 1.5 g of polyvinylpyrrolidone (PVP) and 0.033 g of potassium ferricyanide (K3[Fe(CN)6]) solid powder, dissolve in 20 mL of ultrapure water, add 800 μL of concentrated hydrochloric acid (HCl) and continue stirring for 30 min. The above mixed solution is transferred to the reaction kettle, heated at 170℃ for 1 h. After taking out and cooling to room temperature, centrifugation at 12000 rpm for 15 min, remove the supernatant, wash and redissolve with ultrapure water, anhydrous ethanol and ultrapure water respectively, finally centrifugation at 12000 rpm for 15 min and redissolve with ultrapure water, PB nanomaterials are obtained and stored at 4℃ for use.

[0086] Optimization of potassium ferricyanide concentration

[0087] K3[Fe(CN)6] is one of the important raw materials for synthesizing PB, and in the synthesis of PB,

[0088] The lower the concentration of K3[Fe(CN)6], the smaller the size of the synthesized PB. Due to the surface effect of nanomaterials, the smaller the size of PB, the greater the surface energy it possesses, and the stronger the catalytic performance. In order to find PB with higher catalytic activity and improve the detection sensitivity, it is necessary to optimize the concentration of K3[Fe(CN)6] in the synthesis of PB.

[0089] Take 0.033 g, 0.066 g, 0.132 g and 0.198 g of K3[Fe(CN)6] respectively and dissolve in 20 mL of ultrapure water to prepare K3[Fe(CN)6] solution with concentrations of 0.005 mol·L -1 , 0.01 mol·L -1 , 0.02 mol·L -1 and 0.03 mol·L -1 for PB synthesis, and in the capillary, the moving distance of H2O2 is used as an indicator to explore the catalytic ability of each group of PB. The results are shown in Figure 2 , with the increase of K3[Fe(CN)6] concentration in the preparation process, the moving distance of H2O2 decreases, and the PB prepared by 0.005 mol·L -1 K3[Fe(CN)6] makes the H2O2 liquid column move the longest distance, which proves that the PB prepared under this condition has the highest catalytic activity, so the optimal concentration of K3[Fe(CN)6] is 0.005 mol·L -1 .

[0090] (2) Preparation of PB@Au composite material

[0091] First, immerse a 50 mL beaker in piranha solution for more than 15 min, and rinse with a large amount of ultrapure water until it is neutral. Dilute the PB solution to 0.12 mg·mL -1, 15.84 mL of PB solution was taken in a beaker, 160 μL of 1% (w / v) HAuCl4 solution was added, and the solution was heated and stirred on an electric heating plate until boiling. After 5 min of boiling, 340 μL of 1% (w / v) sodium citrate solution was quickly added, and the stirring and boiling were continued for another 5 min. After the electric heating plate was turned off, the solution was cooled, centrifuged at 10,000 rpm for 10 min, and the supernatant was removed. The solution was washed twice with ultrapure water and then redissolved to the original volume. Finally, the solution was centrifuged at 10,000 rpm for 10 min, redissolved with ultrapure water, and stored at 4°C for later use.

[0092] Optimization of the concentration of Prussian blue in the preparation of the composite material

[0093] PB has good catalytic performance, but the lack of sufficient active sites on its surface makes it difficult to connect antibodies. Gold nanoparticles can be connected to antibodies not only through electrostatic force but also through Au-NH2 covalent bonds formed with the amino groups of the antibodies. To improve the connection efficiency of antibodies and nanomaterials, PB@Au composite materials were prepared, and the ratio of PB to gold nanoparticles was optimized to improve the catalytic efficiency of the composite materials.

[0094] In the preparation of PB@Au composite materials, the concentrations of HAuCl4 and sodium citrate solutions were fixed, and the process of synthesizing gold nanoparticles was kept consistent. The concentration of PB was adjusted to change the ratio of PB to gold nanoparticles. PB solutions were diluted to 0.04 mg·mL -1 , 0.08 mg·mL -1 , 0.12 mg·mL -1 , and 0.16 mg·mL -1 , respectively, and mixed with HAuCl4 solution. After heating to boiling, sodium citrate solution was added to obtain different PB@Au composite materials. The composite materials were connected to capillaries, and H2O2 was injected to explore the catalytic efficiency of PB@Au composite materials prepared at different concentrations of PB. The results are shown in Figure 3 In a liquid environment, sodium citrate provides OH - ions, which can decompose PB into Fe(OH)3, losing catalytic activity. Therefore, the PB@Au solution prepared with 0.04 mg·mL -1 PB became colorless and could not catalyze the decomposition of H2O2 to move the liquid column. In the other three groups of concentrations, the PB@Au prepared with 0.12 mg·mL -1 PB had the largest moving distance after catalyzing H2O2, and as Figure 3The stability of PB@Au was the best after 12 h storage at room temperature, as shown in FIG. 1. The absolute value of the Zeta potential of the composite material was increased due to the gold complexed in the preparation process of PB, which made the composite material more stable. Meanwhile, the catalytic ability of the composite material to H2O2 was improved due to the catalytic activity of the nano-gold. However, too many ions in the solution would cause the aggregation of the composite material and affect its catalytic performance. Therefore, the optimal PB concentration for preparing PB@Au was 0.12 mg·mL -1 .

[0095] (3) Measurement of the light-heat conversion efficiency of PB@Au composite material

[0096] The PB@Au solution was diluted to a certain concentration, and the absorbance value at 808 nm wavelength was measured at this concentration, denoted as A λ . 200 μL of the PB@Au solution at this concentration and ultrapure water were added to different enzyme mark holes, respectively, and the temperatures of the PB@Au solution and ultrapure water at this time were read by a thermal imager and denoted as T 0,mix and T 0,H2O , respectively. The PB@Au solution and ultrapure water were continuously irradiated with a laser with a power of I = 0.4 W and a wavelength of 808 nm, and after the temperature no longer increased, the temperatures of the PB@Au solution and ultrapure water at this time were read by a thermal imager and denoted as T mix and T H2O , respectively. The temperature change values of the PB@Au solution and ultrapure water after laser irradiation were calculated as ΔT max,mix = T mix -T 0,mix and ΔT max,H2O = T H2O -T 0,H2O , respectively. Starting from the time when the laser stopped irradiation, the difference between the temperature of the PB@Au solution and the ambient temperature was calculated every 10 s, and θ = ΔT max,mix - ΔT’ was introduced. The linear regression equation was plotted with -Lnθ as the abscissa and the time t when the laser stopped irradiation as the ordinate, and the slope of the calculated linear regression equation was denoted as θ’. The above data and the specific heat capacity C of water and the mass m of water were substituted into formula 1, and the light-heat conversion efficiency η of PB@Au at this concentration was obtained.

[0097]

[0098] (4) Characterization of PB@Au composite material

[0099] In order to analyze the physical properties of PB and PB@Au composite material and prove the successful synthesis of the material, PB and PB@Au nanomaterials were characterized by ultraviolet-visible light spectrophotometry, dynamic light scattering (DLS), Zeta potential measurement, transmission electron microscopy (TEM) photography, and X-ray photoelectron spectroscopy (XPS) analysis.

[0100] Figure 4 The ultraviolet-visible light absorption spectrum of A shows that the synthesized PB and PB@Au nanomaterials have maximum absorption peaks at wavelengths of 764 nm and 736 nm, respectively, proving the presence of PB, and the maximum absorption peak of the synthesized PB@Au composite material is about 28 nm red-shifted from that of PB, and the color change of the composite material after synthesis in the illustration proves the successful synthesis of the PB@Au composite material.

[0101] Figure 4 The dynamic light scattering of B shows that the hydrated particle size of the synthesized PB@Au composite material is about 75 nm larger than that of the PB nanoparticles, proving the successful compounding of the PB nanoparticles and the Au nanoparticles.

[0102] Figure 4 The Zeta potential of the PB@Au composite material in C is about 2 mV lower than that of PB, and the increase in the absolute value of the Zeta potential proves the successful compounding of the material.

[0103] In Figure 4 D and Figure 4 E, the transmission electron microscope images of PB and PB@Au show that the PB nanoparticles are cubic and about 200 nm long, and the size of the nanomaterials is uniform and has good dispersibility; the PB@Au nanoparticles are formed by wrapping the PB nanoparticles with many Au nanoparticles, and the Au nanoparticles are uniformly dispersed on the surface of the PB nanoparticles.

[0104] In Figure 4 F and Figure 4 G, the XPS energy spectrum of PB and PB@Au shows that the peaks of Fe elements and the peaks of Fe elements and Au elements appear, respectively, proving the synthesis of PB and the successful compounding of PB@Au.

[0105] Figure 5 H is the photothermal conversion efficiency of PB and PB@Au, and through calculation, the photothermal performance of the nanomaterials is improved from 19.5% of PB to 31.1% after compounding, proving that the synthesized composite material has stronger photothermal conversion capacity than PB.

[0106] (5) Optimization of the concentration of the capillary-connected composite material

[0107] In order to determine the maximum amount of the capillary-connected composite material and reduce the waste of materials and antibodies, the concentration of the material connected to the capillary was optimized. The PB@Au solution was diluted to 0.6 mg·mL -1 , 0.7 mg·mL -1 , 0.8 mg·mL -1 , 0.9 mg·mL -1 and 1 mg·mL -1and the moving distance AL of the H2O2 liquid column in each group of capillary was calculated after the injection of H2O2 solution and catalysis. The results are shown in Figure 6 As shown in Fig. 4, with the increase of the concentration of PB@Au solution, the moving distance of H2O2 liquid column increased constantly, and when the concentration of PB@Au solution rose to 0.8 mg·mL -1 After that, the moving distance of H2O2 liquid column almost did not change, which proved that the capillary reached the saturation state of connecting PB@Au composite material through the force of covalent bond and electrostatic bond, so 0.8 mg·mL -1 was selected as the optimal concentration of PB@Au solution for connecting capillary.

[0108] (6) Optimization of catalytic and photothermal performance of composite material

[0109] ①Optimization of PB@Au catalytic hydrogen peroxide concentration

[0110] PB@Au with peroxidase-like activity can decompose H2O2 to produce O2, which promotes the movement of H2O2 liquid column in the capillary. The length of the moving distance of the liquid column in the same time proves the strength of the catalytic ability of PB@Au composite material. If the concentration of H2O2 is too low, the detection sensitivity will be reduced; if the concentration of H2O2 is too high, its spontaneous decomposition may cause the background signal of detection to be too high, so it is necessary to optimize the concentration of catalytic H2O2. The ultrapure water and H2O2 solution with concentrations of 10%, 20% and 30% were injected into the capillary combined with the same concentration of PB@Au, and the moving distance AL of the liquid column was recorded after the catalysis of H2O2. As shown in Fig. 5, with the increase of the concentration of H2O2, the catalytic rate of nanomaterials also increased, making the moving distance of the liquid column in the capillary longer. When the concentration of H2O2 rose to 30%, the AL of the liquid column in the capillary was the largest, so 30% was selected as the optimal concentration of H2O2 for catalytic reaction. Figure 7

[0111] ②Selection of laser irradiation mode

[0112] ​H2O2 is catalyzed by PB@Au to produce O2 gas, causing the H2O2 liquid column to move in the capillary. The PB@Au connected in the capillary has excellent light-heat conversion performance and can produce heat under laser irradiation, promote catalysis and make the gas expand, expand the gas volume, and amplify the detection signal again. In order to determine the amplification ability of the detection signal of the catalysis and light-heat performance of the PB@Au composite material, and analyze the strength of the detection signal amplification ability of different catalysis combined with light-heat methods, a suitable irradiation method needs to be selected. Four groups of capillaries connected with PB@Au are selected, and only one group of capillary that needs to be irradiated with laser is injected with 10 μL of ultrapure water in the detection area, and the remaining three groups of capillaries are injected with 10 μL of H2O2 solution with a concentration of 30% in the detection area. The initial position L0 of each group of liquid columns is recorded. Only one group of capillary that needs to be catalyzed, the position L1 of the liquid column after 5 min of catalysis is recorded, and the moving distance ΔL1 of the liquid column is calculated as ΔL1=L1-L0; only one group of capillary that needs to be irradiated with laser, the position L2 of the liquid column after 80 s of laser irradiation is recorded, and the moving distance ΔL2 of the liquid column is calculated as ΔL2=L2-L0; one group of capillary that needs to be catalyzed first and then irradiated with laser, first make H2O2 catalyze in the capillary for 5 min, then irradiate with laser in the detection area for 80 s, record the position L3 of the liquid column after irradiation, and calculate the moving distance ΔL3 of the liquid column as ΔL3=L3-L0; one group of capillary that needs to be catalyzed and irradiated with laser at the same time, use 3 W laser to continuously irradiate the H2O2 liquid column in the detection area of the capillary for 5 min, record the position L4 of the liquid column, and calculate the moving distance ΔL4 of the liquid column as ΔL4=L4-L0. Compare the moving distances of the liquid columns under the four different irradiation methods, and the results are shown in Figure 8 As compared with catalysis and laser irradiation alone, the combination of catalysis and light-heat significantly improves the moving distance of the liquid column; the heat generated by the PB@Au composite material increases the speed of H2O2 catalytic decomposition when the laser irradiation is performed at the same time, resulting in the longest moving distance of the liquid column and the shortest detection time of the capillary that first catalyzes and then irradiates. Therefore, the method of simultaneous catalysis and laser irradiation is selected for subsequent detection of Salmonella typhimurium.

[0113] ③Optimization of laser irradiation power

[0114] The irradiation power of the laser directly affects the moving distance of the H2O2 liquid column in the capillary. If the power is too low, it will affect the sensitivity of the detection; if the power is too high, it will cause unnecessary energy loss, and long-time irradiation may even cause the laser to overheat and be damaged, so the power of the laser irradiation needs to be optimized. The H2O2 in the detection area of the capillary is irradiated with 0 W, 1 W, 2 W, 3 W and 4 W laser respectively, and the ΔL of the H2O2 liquid column under different power laser irradiation is observed and calculated. The results are shown in Figure 9As shown, when the laser irradiation power is 0 W, it indicates that H2O2 is only subjected to the catalysis of PB@Au connected in the capillary. With the increase of laser irradiation power, more heat is converted by PB@Au, which promotes the catalysis of PB@Au on H2O2, so that the moving distance of H2O2 liquid column is longer. When the laser power rises to 3 W, due to the too fast catalytic reaction rate, the H2O2 liquid column stays in the capillary detection area for a short time, and the amount of gas produced by catalytic decomposition is small,

[0115] The ΔL growth trend of H2O2 liquid column becomes slow, so 3 W is selected as the optimal power of capillary laser irradiation.

[0116] (4) Optimization of laser irradiation time

[0117] The moving distance of H2O2 liquid column in the capillary continuously increases with the continuous irradiation of laser. When the gas produced by catalysis pushes the H2O2 liquid column to move away from the detection area, and the temperature of PB@Au after laser irradiation reaches dynamic equilibrium, the ΔL of the liquid column no longer changes. In order to ensure the sensitivity of detection and avoid unnecessary energy loss, the time of laser irradiation needs to be optimized. The capillary detection area is continuously irradiated by 3 W laser, and the position L of H2O2 liquid column is observed and recorded every certain time. The moving distance ΔL of H2O2 liquid column in the capillary is calculated as ΔL=L-L0. The results are shown in Figure 10 As shown, with the continuous irradiation of laser, the temperature rise caused by the photothermal effect of PB@Au accelerates the decomposition of H2O2 to produce gas, so that the moving distance of H2O2 liquid column continuously increases. With the H2O2 liquid column being gradually pushed away from the capillary detection area by the produced gas, the amount of catalytic gas production gradually decreases and the temperature of nanomaterial gradually tends to be stable, so that the growth trend of H2O2 liquid column becomes slow. When the laser irradiation time reaches 80 s, the H2O2 liquid column completely leaves the detection area and no longer produces gas by catalytic decomposition, and the temperature reaches dynamic equilibrium, so that the ΔL of H2O2 liquid column almost no longer increases, therefore, 80 s is selected as the optimal time of laser irradiation.

[0118] (7) Preparation of PB@Au immunoprobes

[0119] The PB@Au solution is diluted to 0.8 mg·mL -1 , 10 μL of PB@Au solution is taken, 2.5 μL of 0.01 mol·L -1 sodium hydroxide (NaOH) solution is added, and mixed uniformly. 4 μL of PB@Au solution mixed with NaOH is taken, and 4 μL of 75 μg·mL -1Salmonella typhimurium antibody, 37 ℃ oscillation combined with 1.5h. Then add 8 μL concentration of 6 % BSA solution, 37 ℃ oscillation combined with 1.5h. The above mixed solution 10000 rpm centrifugation 10 min, remove the supernatant and washed with phosphate buffer solution (PBS) to the original volume, centrifugation again, remove the supernatant and dissolved in PBS to 4 μL to obtain PB@Au immune probe.

[0120] ①PB@Au composite material blocking solution concentration optimization

[0121] In order to prevent the PB@Au composite material through Au-NH2 bond and electrostatic binding force non specific binding of Salmonella typhimurium, also need to optimize the blocking solution concentration of PB@Au. PB@Au solution was mixed with ultrapure water and 2 %, 4 %, 6 % and 8 % BSA solution, 37 ℃ blocking 1.5h after centrifugation and combined with Salmonella typhimurium solution, then injected into the immunocapillary, the detection of Salmonella. Results as shown in Figure 11 , with the increase of BSA concentration of PB@Au composite material blocking, the moving distance of H2O2 liquid column in capillary is constantly declining, when the BSA concentration rises to 6 %, the H2O2 liquid column ΔL almost no longer change, represent the PB@Au composite material is completely blocked by BSA, therefore, 6 % as the optimal blocking solution concentration of PB@Au composite material.

[0122] ②PB@Au composite material connected antibody pH optimization

[0123] The pH of the solution affects the connection effect of antibody and nanocomposite material, the antibody usually in weak alkaline environment and nanomaterial achieve the best connection effect, but in alkaline conditions, PB is easy to decompose into iron hydroxide precipitate, make catalytic and photothermal ability reduce, and in the process of PB decomposition, OH- in the environment is constantly consumed, resulting in the solution pH almost no change before PB is decomposed, can't through the determination and adjustment of pH optimization antibody and PB@Au composite material connection effect. Therefore, select to add different concentrations of NaOH solution to PB@Au solution for optimization.

[0124] First take the concentration of 0.8 mg·mL -1 PB@Au solution, according to the volume ratio of material: NaOH = 4:1, to the PB@Au solution added concentration of 0 ~ 0.09 mol·L -1 NaOH solution. Results as shown in Figure 11 A, with the increase of the concentration of NaOH solution added, PB@Au solution showed blue to green and then yellow color change, prove the generation of Fe(OH)3 and the consumption of PB in the solution. Subsequently selected NaOH solution concentration gradient is 0 mol·L -10.01 mol·L -1 0.02 mol·L -1 0.03 mol·L -1 0.04 mol·L 1 and 0.05 mol·L -1 The results are shown in Figure 12 B. The added NaOH first improved the effect of PB@Au connecting antibody, but with the increase of NaOH concentration, the decomposition of PB@Au composite material gradually reduced the number of capillary connecting immune probes, and the moving distance of H2O2 liquid column showed a trend of first rising and then falling. When the concentration of NaOH was 0.01 mol·L -1 , the ΔL of H2O2 liquid column was the largest, so 0.01 mol·L -1 was selected as the optimal concentration of NaOH added to the PB@Au solution.

[0125] ③PB@Au composite material connecting antibody concentration optimization

[0126] The same as the purpose of optimizing the antibody concentration of capillary coating, the antibody concentration connected by PB@Au composite material also needs to be optimized. Salmonella typhi antibody with concentrations of 25 μg·mL -1 , 50 μg·mL -1 , 75 μg·mL -1 , 100 μg·mL -1 and 125 μg·mL -1 were combined with PB@Au composite material, and another group was added with ultrapure water as a blank control. After BSA blocking, the detection was carried out. The results are shown in Figure 13 . With the increase of antibody concentration connected by PB@Au, the moving distance of H2O2 liquid column also continuously increased, and the upward trend basically stopped after the antibody concentration increased to 75 μg·mL -1 , at which time the connection of antibody and capillary reached saturation state. Therefore, 75 μg·mL -1 was selected as the optimal concentration of salmonella typhi antibody connected by PB@Au.

[0127] (8) Modification of capillary

[0128] First, the capillary tubes were thoroughly cleaned and dried. They were then immersed in a piranha solution and sonicated for 1 hour. Afterward, they were rinsed with a large amount of ultrapure water until neutral and placed in an oven at 110°C for 1.5 hours to dry. After drying, the capillary tubes were cooled to room temperature and immersed in a 20% APTES methanol solution for 12 hours. Afterward, the residual APTES solution was rinsed with anhydrous methanol, and the capillary tubes were placed in a fume hood for 15 minutes to allow the methanol to evaporate. Finally, the capillary tubes were dried in an oven at 50°C for 30 minutes to obtain APTES-modified capillary tubes, which were stored in a nitrogen atmosphere at 4°C until use.

[0129] (9) Preparation of immunocapillaries

[0130] 2.5 μL of a concentration of 400 μg·mL -1 The Salmonella typhimurium antibody was injected into one end of a capillary tube, and this location was marked as the detection zone. The tube was coated at 37°C for 1.5 hours and then immersed in PBS solution for three cycles of rotation and washing. The antibody-coated capillary tube was then immersed in 4% BSA for 1.5 hours at 37°C, followed by three cycles of rotation and washing in PBS solution to obtain an immunocapillary tube coated with Salmonella typhimurium antibody. This was then stored at 4°C for later use.

[0131] ① Optimization of capillary sealing solution concentration

[0132] To prevent non-specific adsorption of Salmonella typhimurium to the active sites within the capillary tubes, which could lead to false positives, the capillaries needed to be blocked with BSA. The concentration of the blocking solution was optimized to achieve the best blocking effect. The modified capillaries were immersed in ultrapure water and 2%, 4%, 6%, and 8% BSA solutions, respectively. After blocking, Salmonella typhimurium was detected. The results are as follows: Figure 14 As shown, with the increase of BSA concentration in the capillary blocking solution, the unbound antibody active sites in the capillary are gradually blocked by the blocking solution, causing the movement distance of the H2O2 column in the capillary to continuously decrease. When the BSA concentration increases to 4%, the remaining active sites in the capillary are completely blocked by BSA, making the ΔL of the H2O2 column almost unchanged. Therefore, 4% is selected as the optimal blocking solution concentration for the capillary.

[0133] ② Optimization of capillary coating antibody concentration

[0134] Antibodies, as specific recognition molecules for Salmonella typhimurium, have their concentration on capillary tubes directly affecting detection efficiency. Insufficient antibody concentration reduces detection sensitivity, while excessive concentration leads to waste and increased costs. Therefore, optimizing the antibody concentration for capillary tube attachment is necessary.

[0135] Inject 2.5 μL of a solution with a concentration of 100 μg·mL into one end of the capillary tube. -1 200 μg·mL-1 400 μg / mL -1 600 μg / mL -1 and 800 μg / mL -1 of Salmonella typhi antibody, another group was added with ultrapure water as a blank control, and BSA was blocked before detecting Salmonella typhi. The results are shown in Figure 15 As the concentration of antibody coated on the capillary increased, the AL of H2O2 liquid column was constantly rising, and the rising trend basically stopped when the antibody concentration reached 400 μg / mL -1 , at which time the connection of antibody and capillary reached a saturated state. Therefore, 400 μg / mL -1 was selected as the optimal concentration of Salmonella typhi antibody coated on the capillary.

[0136] (10) Detection of Salmonella typhi

[0137] PB@Au immune probe solution was added to the sample solution containing 10 2 CFU / mL -1 , 10 4 CFU / mL -1 , 10 6 CFU / mL -1 and 10 8 CFU / mL -1 of Salmonella typhi, and placed in a bacterial incubator at 37°C for 30 min. The above mixed solution was injected into the detection area of the capillary, and placed in a bacterial incubator at 37°C for 10 min. After the end, the capillary was immersed in PBS solution and shaken up and down for 3 times. 10 μL of 30% H2O2 was injected into the detection area of the capillary, and the capillary was closed at one end, and the initial position L0 of the H2O2 liquid column was recorded. After irradiating the capillary detection area with a laser with a wavelength of 808 nm and a power of 3 W for 5 min, the laser was turned off. The position of the H2O2 liquid column gradually moved with the gas generated in the detection area, and the position L of the H2O2 liquid column after the laser was turned off was recorded. The moving distance AL of the H2O2 liquid column was calculated as AL=L-L0. The results are shown in Figure 16 As the concentration of the detected bacteria increased, more PB@Au was captured by the immune capillary to Salmonella typhi and connected to the capillary, making the AL of the H2O2 liquid column constantly larger. By establishing a standard curve of the number of Salmonella typhi and AL, the regression equation between AL and bacterial concentration was Y=0.07667X-0.041, R 2 =0.9425, and the detection limit was calculated to be about 37 CFU / mL -1 .

[0138] (11) Analysis of detection performance

[0139] ① Detection specificity analysis

[0140] To verify the established detection method can specifically recognize Salmonella typhimurium, the specificity of the detection method was analyzed, four common foodborne pathogens: Escherichia coli, Salmonella enteritidis, Listeria and Staphylococcus aureus were selected as non-target bacteria for detection. The concentration of the four non-target bacteria for detection was diluted to 10 7 CFU·mL -1 , the concentration of the target bacteria Salmonella typhimurium was diluted to 10 6 CFU·mL -1 . The results are shown in Figure 17 , because of the specific recognition of the antibody to Salmonella typhimurium, only the capillary group for detecting Salmonella typhimurium has a significant ΔL, the remaining four groups of capillary groups for detecting Escherichia coli, Salmonella enteritidis, Listeria and Staphylococcus aureus have no significant ΔL, proving that the established detection method has good specificity.

[0141] ② Detection repeatability analysis

[0142] To analyze the repeatability of the detection method, five groups of parallel experiments were conducted on Salmonella typhimurium with a concentration of 10 8 CFU·mL -1 . The results are shown in Figure 18 , the parallel detection results of the five groups of Salmonella typhimurium have no significant difference. Through calculation, the coefficients of variation (CV) of the detection results within and between groups were 3.74% and 6.08% respectively, proving that the established detection method can meet the repeatability requirements of rapid detection.

[0143] ③ Immune capillary stability analysis

[0144] To analyze the influence of antibody inactivation and other reasons on the detection efficiency during storage, the stability of the detection method was analyzed. 37℃ was selected as the storage temperature, and 0, 1, 2, 3 days were selected as the storage time to investigate the stability of the immune capillary. The results are shown in Figure 19 , with the extension of storage time, the gradual inactivation of the antibody makes the detection efficiency decrease, after 3 days of storage at 37℃, the final detection efficiency is retained at 82%, indicating that the established detection method has good storage stability.

[0145] (12) Detection of Salmonella typhimurium in actual samples

[0146] To analyze the feasibility of the detection method in actual samples, three kinds of actual samples susceptible to Salmonella typhimurium infection: tap water, milk and grape juice were selected for bacterial addition recovery rate determination. Different concentrations of Salmonella typhimurium (10 4 CFU·mL-1 , 10 6 CFU·mL -1 and 10 8 CFU·mL -1 ) were determined. The results are shown in Table 2. The detection efficiency of the established detection method in three actual samples was different from that in PBS solution due to the influence of the complex matrix such as ions, proteins in milk, fat, and pH in grape juice. The calculated recovery of Salmonella typhimurium in the actual sample was between 86.6% and 113.6%, indicating that the detection method has good feasibility and applicability in the detection of actual samples. ​

[0147] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. The present application can be variously changed and altered for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. A biosensor based on Prussian blue gold nanoparticles, characterized in that, It consists of a detection probe, a detection capillary, and hydrogen peroxide; the detection probe is composed of Prussian blue gold nanoparticles and a first recognition molecule, the first recognition molecule being attached to the surface of the Prussian blue gold nanoparticles; the detection capillary is provided with a detection area and a measurement area from one end to the other, and a second recognition molecule is fixed on the inner wall of the detection area; hydrogen peroxide works in conjunction with the detection capillary. Both the first and second recognition molecules are materials capable of specifically binding to the target microorganism; The first recognition molecule is an antibody; the second recognition molecule is an antibody; and the detection capillary is an immunocapillary. The inner wall of the immunocapillary detection area is modified with amino groups, and then linked to antibodies via amino groups. The Prussian blue nanogold catalyzes the production of O2 from H2O2 and accelerates catalysis and expands the gas volume by utilizing its photothermal conversion capability, converting the detected volume signal into a capillary liquid column movement distance signal.

2. The Prussian blue gold nanoparticle-based biosensor as described in claim 1, characterized in that, The detection probe is an immune probe.

3. The Prussian blue gold nanoparticle-based biosensor as described in claim 2, characterized in that, The immunocapillary detection zone uses BSA to block non-specific sites. Alternatively, non-specific sites on the surface of the immune probe can be blocked by BSA.

4. The method for preparing a Prussian blue gold nanoparticle-based biosensor as described in any one of claims 1-3, characterized in that, This includes the preparation of detection probes and the preparation of detection capillaries; Preparation of detection probe: Add an alkaline compound to a solution of Prussian blue gold nanoparticles and mix thoroughly, then add the target microbial antibody and mix again to obtain the probe. Preparation of the detection capillary: The inner surface of the glass capillary is modified with amino groups, and the target microbial antibody solution is injected into the detection area of ​​the amino-modified glass capillary. After incubation, the detection capillary is obtained.

5. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 4, characterized in that, During the preparation of the detection probe, the temperature at which the target microbial antibody is added and mixed is 35~40℃; Alternatively, during the preparation of the detection probe, the concentration of the Prussian blue gold nanoparticle solution is 0.6–1.0 mg / mL. -1 .

6. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 5, characterized in that, The concentration of Prussian blue gold nanoparticle solution is 0.75~0.85 mg·mL. -1 .

7. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 6, characterized in that, When the concentration of Prussian blue gold nanoparticle solution is 0.75~0.85 mg·mL -1 When the amount of Prussian blue gold nanoparticle solution added is 9.5~10.5 μL and the amount of alkaline compound solution added is 2.3~2.7 μL, the concentration of alkaline compound solution is 0~0.05 mol·L⁻¹. -1 .

8. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 7, characterized in that, The concentration of the alkaline compound solution is 0~0.02 mol·L⁻¹ -1 .

9. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 8, characterized in that, The concentration of the alkaline compound solution is 0.005~0.015 mol·L⁻¹ -1 .

10. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 6, characterized in that, When the concentration of Prussian blue gold nanoparticle solution is 0.75~0.85 mg·mL -1 When the amount of Prussian blue gold nanoparticle solution added is 9.5~10.5 μL and the amount of target microbial antibody solution added is 3.5~4.5 μL, the concentration of the target microbial antibody solution is not less than 25 μg·mL. -1 .

11. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 10, characterized in that, The concentration of the target microbial antibody solution shall not be less than 75 μg·mL -1 .

12. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 11, characterized in that, The concentration of the target microbial antibody solution is 75–125 μg / mL. -1 .

13. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 5, characterized in that, During the preparation of the detection probe, after mixing with the target microbial antibody, BSA is added and mixed again.

14. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 13, characterized in that, When the concentration of Prussian blue gold nanoparticle solution is 0.75~0.85 mg·mL -1 The amount of Prussian blue gold nanoparticle solution added was 9.5~10.5 μL, and the concentration of the target microbial antibody solution was 70~80 μg·mL. -1 When the amount of target microbial antibody solution added is 3.5~4.5 μL and the amount of BSA solution added is 7.5~8.5 μL, the concentration of BSA solution is not less than 2% by mass.

15. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 14, characterized in that, The concentration of the BSA solution shall not be less than 6%.

16. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 15, characterized in that, The concentration of the BSA solution is 6-8%.

17. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 5, characterized in that, The method for preparing Prussian blue nanogold is as follows: Prussian blue solution and chloroauric acid solution are mixed, heated to boiling, sodium citrate solution is added, stirred and kept boiling, and then cooled to obtain the nanogold.

18. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 17, characterized in that, When 155–165 μL of chloroauric acid solution with a concentration of 0.5–1.5% w / v is added, and the volume of Prussian blue solution added is 15–16 mL, the concentration of Prussian blue solution is 0.08–0.16 mg·mL. -1 .

19. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 17, characterized in that, The concentration of Prussian blue solution is 0.10–0.14 mg / mL. -1 .

20. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 17, characterized in that, The concentration of Prussian blue solution is 0.11–0.13 mg / mL. -1 .

21. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 17, characterized in that, The preparation method of Prussian blue is as follows: polyvinylpyrrolidone and potassium ferricyanide are dissolved in water and mixed to obtain a precursor solution. Hydrochloric acid is added to the precursor solution, and the mixture is heated to 160~180℃ to carry out the reaction. The result is Prussian blue.

22. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 21, characterized in that the precursor... The concentration of potassium ferricyanide in the solution was 0.005–0.03 mol·L⁻¹. -1 .

23. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 22, characterized in that, The concentration of potassium ferricyanide in the precursor solution was 0.005–0.006 mol·L⁻¹. -1 .

24. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 5, characterized in that, Amine modification of the inner surface of glass capillary was performed using APTES.

25. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 24, characterized in that, Immerse the capillary tube in a methanol solution of APTES for 9-15 hours, then wash and dry.

26. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 25, characterized in that, When injecting 2.0–3.0 μL of the target microbial antibody solution into the detection area of ​​an amino-modified glass capillary, the concentration of the target microbial antibody solution should not be less than 70 μg·mL. -1 .

27. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 26, characterized in that, The concentration of the target microbial antibody solution is 75~800 μg·mL -1 .

28. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 27, characterized in that, The concentration of the target microbial antibody solution is 100~800 μg·mL -1 .

29. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 28, characterized in that, The concentration of the target microbial antibody solution is 400~600 μg·mL -1 .

30. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 25, characterized in that, The target microbial antibody solution was injected into the detection area of ​​an amino-modified glass capillary. After incubation, BSA solution was added for blocking.

31. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 30, characterized in that, 2.0–3.0 μL of the target microbial antibody solution was injected into the detection zone of the amino-modified glass capillary. The concentration of the target microbial antibody solution was not less than 70 μg·mL. -1 Incubate at 35~40℃, then clean and block with a BSA solution containing not less than 2% by mass.

32. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 31, characterized in that, The concentration of the BSA solution shall not be less than 4%.

33. The method for preparing a biosensor based on Prussian blue gold nanoparticles as described in claim 32, characterized in that, The concentration of the BSA solution is 4-8%.

34. The application of a Prussian blue gold nanoparticle-based biosensor as described in any one of claims 1-3 or a Prussian blue gold nanoparticle-based biosensor obtained by any one of the preparation methods described in claims 4-33 in the detection of target microorganisms.

35. The application as described in claim 34, characterized in that, The target microorganism is Salmonella.

36. The application as described in claim 35, characterized in that, The target microorganism is Salmonella typhimurium.

37. A method for detecting Salmonella, characterized in that, Provide a Prussian blue gold nanoparticle-based biosensor as described in any one of claims 1-3 or a Prussian blue gold nanoparticle-based biosensor obtained by the preparation method described in any one of claims 4-33, wherein the target microorganism is Salmonella; comprising the following steps: (1) Mix the detection probe solution with the solution containing Salmonella to be tested and incubate it; (2) Add the solution obtained in step (1) to the detection area of ​​the detection capillary, incubate, and remove the solution after incubation; (3) Inject hydrogen peroxide into the detection area of ​​the detection capillary obtained in step (2), irradiate the detection area with light, and record the moving distance ΔL of the hydrogen peroxide liquid column; (4) Detect Salmonella based on ΔL obtained in step (3).

38. The method for detecting Salmonella as described in claim 37, characterized in that, The illumination is laser irradiation.

39. The method for detecting Salmonella as described in claim 38, characterized in that, The laser irradiation power is 1~4 W; the laser irradiation time is not less than 80 s.

40. The method for detecting Salmonella as described in claim 39, characterized in that, The laser irradiation power is 3~4 W; the laser irradiation time is 80~120 s.

41. A Salmonella detection kit, characterized in that, The invention includes the Prussian blue gold nanoparticle-based biosensor as described in any one of claims 1-3, or the Prussian blue gold nanoparticle-based biosensor obtained by the preparation method described in any one of claims 4-33, along with a buffer solution and a light source.

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