Immunodetection kit based on bimetallic nano-elastic biological probe as well as preparation method and application of immunodetection kit

Abstract

The invention discloses an immunodetection kit based on a bimetal nano-bomb biological probe, and relates to the technical field of gene detection, the immunodetection kit comprises a test strip and an immunoprobe, the immunoprobe is a suspension of a bimetal nano-bomb and a monoclonal antibody, and the mixing ratio of the bimetal nano-bomb to the monoclonal antibody is 0.1-0.3 mg: 5 [mu] g. The bimetal nano-bomb has a symmetrical hollow nano-bomb structure, so that the bimetal nano-bomb has excellent colorimetric and photo-thermal properties, and the detection sensitivity of salmonella typhimurium is greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to an immunoassay kit based on a bimetallic nano-bullet bioprobe, and a preparation method and application thereof. Background Art

[0002] Infectious diseases caused by foodborne pathogens continue to pose a major threat to public health. Common foodborne bacteria, such as Salmonella Typhimurium ( S. typhimurium ) and Escherichia coli ( E.coil ), which is transmitted through contaminated water and food (including meat and vegetables). Sensitive and accurate identification and detection of low infectious doses of Salmonella Typhimurium in potentially contaminated food is of great significance for early prevention, diagnosis and control of foodborne disease infections and outbreaks. Therefore, it is of great significance to establish a method for rapid and sensitive detection of Salmonella Typhimurium in food.

[0003] There are currently a variety of known methods for detecting Salmonella Typhimurium, including the gold standard traditional culture method, enzyme-linked immunosorbent assay, polymerase chain reaction (PCR), aptamer detection technology, etc. Although these technologies are used as gold standards or recommended detection methods, in order to meet the needs of on-site detection of Salmonella Typhimurium, many methods for pathogen detection have been developed in recent years, such as lateral flow immunochromatography (LFIA), optical biosensors, surface-enhanced Raman scattering biosensors, and electrochemical biosensors. Among these methods, LFIA has been successfully used for on-site monitoring of foodborne pathogens due to its advantages such as rapidity, naked eye readout, simple operation, low cost, one-step analysis, and portability. However, the traditional single-color labeled LFIA has the problems of blurred color change and limited single-mode reading, which makes it difficult to meet the needs of high-performance detection.

[0004] In recent years, dual-mode LFIAs for Salmonella have been developed, including colorimetric / fluorescence, colorimetric / photothermal, colorimetric / surface enhanced Raman scattering (SERS) and colorimetric / enzyme activity, providing more sensitive quantitative analysis results. Among them, dual-signal LFIA based on photothermal materials seems to have greater potential for field application due to its more cost-effectiveness and portable instrument dependence. The development of materials with better colorimetric and photothermal performance is an urgent problem to be solved by technicians in this field. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an immunoassay kit based on a bimetallic nanobullet biological probe to address the deficiencies in the prior art, and to develop an immunoassay probe based on a bimetallic nanobullet. The bimetallic nanobullet has a symmetrical hollow nanobullet structure, which gives it excellent colorimetric and photothermal properties. It is used to detect Salmonella typhimurium with high detection sensitivity and stable and reliable results.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: An immunoassay kit based on a bimetallic nanobullet bioprobe comprises a test strip and an immunoprobe, wherein the immunoprobe is a suspension of a bimetallic nanobullet and a monoclonal antibody, and the mixing ratio of the bimetallic nanobullet to the monoclonal antibody is 0.1-0.3 mg:5 μg; The bimetallic nanobullet was prepared by the following steps: 10 mL of deionized water and 18 mg of Cl3H 18 N6Ru, 30 mg K2TeO3 and 325 mg PVP were mixed, and the mixture was ultrasonicated for 2 min; 10 mg NH3·H2O and 5 mL N2H4·H2O were added to the above solution in sequence; the solution was hydrothermally treated at 180 °C in an autoclave for 3 h, washed several times, and the final product was dried in a vacuum drying oven overnight to obtain the product.

[0007] Furthermore, the immune probe is a suspension formed by mixing the bimetallic nanoprojectile and the Salmonella typhimurium monoclonal antibody in 200 μL PBS at a mixing ratio of 0.3 mg:5 μg.

[0008] Furthermore, the average diameter of the bimetallic nanobullet is 23.05±3.25 nm, and the length is 139.09±14.37 nm.

[0009] Furthermore, the immune probe is prepared by the following steps: 1 mL of a 0.1-0.3 mg mL -1 The RuTe2 solution was mixed with 5 μg of Salmonella typhimurium monoclonal antibody and incubated at room temperature for 90 min. Unbound sites were blocked with BSA at a final concentration of 1%. After incubation for 90 min, RuTe2-mAb was centrifuged at 11,000 rpm for 10 min, resuspended in 200 μL 10 mM pH = 7.4 PBS, and stored at 4 °C.

[0010] Furthermore, the test strip consists of four parts: a backing plate, a nitrocellulose membrane, an absorption pad, and a sample pad, wherein the absorption pad and the sample pad are respectively placed on both sides of the nitrocellulose membrane, and a detection line (T line) and a quality control line (C line) are horizontally arranged along the upper edge of the non-covered surface of the nitrocellulose membrane.

[0011] Furthermore, 1 mg mL -1 Anti-Salmonella typhimurium antibodies and 2 mg mL -1 Sheep anti-mouse immunoglobulin was used at 1 μL cm -1 The streaking rate is coated on the nitrocellulose membrane, 1-2 mm from the beginning and the end is removed, and the evenly streaked part is taken as the test line and the control line.

[0012] The invention discloses an application of a kit for detecting Salmonella typhimurium.

[0013] Furthermore, the immune probe and the sample to be tested were mixed in a volume ratio of 8:92 and then dropped onto the test strip, and qualitative detection was performed based on the intensity of the T line on the test strip. The T line was irradiated with near-infrared light to obtain the photothermal result of the test strip, and the photothermal data was recorded using a handheld thermal imager to obtain the quantitative result.

[0014] Furthermore, using 808 nm, 2.0 W·cm -2 Laser irradiation of T line was performed for 60 s.

[0015] The beneficial effects of the present invention are: 1. The present invention discloses an immunoassay kit based on a bimetallic nanobullet biological probe, wherein a test strip and an immunoprobe are used together, wherein the immunoprobe is a bimetallic nanobullet loaded with a Salmonella typhimurium monoclonal antibody, and the bimetallic nanobullet RuTe2 in the present application is used as a signal label by Cl3H 18 N6Ru and K2TeO3 are synthesized by hydrothermal reaction. Their special structure gives them excellent performance, which is specifically manifested in the following aspects: (1) RuTe2 is a symmetrical, hollow nano-bullet structure. The hollow refraction effect and the symmetrical structure with strong absorption resonance can greatly increase the photothermal effect, making it have an outstanding photothermal signal. (2) The aspect ratio is ≈6:1, and the specific surface area is large; (3) RuTe2 has a bright black color. After loading the antibody, Ru-S bonds are formed inside. A large number of antibodies are strongly conjugated on its surface, which is conducive to coupling and tracing antibodies. (4) The internal atomic arrangement of RuTe2 is complex, containing a large number of under-coordinated atoms and disorder, which is conducive to the generation of more robust active sites, thereby generating a wider light response. Therefore, the bimetallic nanobullet prepared in the present application greatly improves the colorimetric and photothermal performance of the signal label based on its amorphous active sites, hollow morphology excitation reflection enhancement, symmetrical structure strong absorption resonance, and large specific surface area. Therefore, it can exhibit excellent visual qualitative detection capabilities even under low antibody input conditions; under near-infrared light stimulation, it can more sensitively capture photothermal signals to achieve quantitative detection. Its colorimetric and photothermal linear detection limits in Salmonella typhimurium are 15.85 times and 39.17 times that of AuNPs-LFIA, respectively. The colorimetric and photothermal sensitivities are greatly improved, and the detection stability and accuracy are high.

[0016] 2. The present application utilizes a sandwich immunoassay for the determination of Salmonella typhimurium, i.e., the target molecule in the sample is recognized by the antibody and the antibody sandwiched with the monoclonal antibody loaded with the signal tag bimetallic nanobullet RuTe2 fixed on the test line. That is, the sample solution containing the target molecule is pre-mixed with the immune probe and then dripped onto the sample pad of the test strip. Due to the action of capillary force, the mixed solution will move along the test strip toward the absorbent pad. When moving to the test line, the target molecule in the mixed solution will bind to the immobilized antibody. Therefore, the color depth of the test line is proportional to the content of the target molecule. When the sample contains more target molecules, the fixed antibody binds to more probes and the test line is stronger; on the contrary, the target molecule content decreases and the color of the test line is lighter; and when the number of target molecules is too small, the fixed antibody can hardly bind to the immune probe, and the test line is almost invisible at this time. In addition, a quality control line is set to verify whether the test strip is working properly. Regardless of whether the target molecule exists, the sheep anti-mouse antibody on the quality control line can bind closely to the monoclonal antibody, thereby reflecting the antibody activity and whether the test strip is working properly. Therefore, in the present invention, when the sample contains Salmonella typhimurium (positive), the test strip presents two black-brown strips, and the depth of the test strip detection line changes with the trend of the content of Salmonella typhimurium; when Salmonella typhimurium is not present (negative), the test strip only has the quality control line. In addition, since the depth of the detection line is determined by the enriched signal tag content, RuTe2 nanorods have excellent photothermal conversion properties. Under the stimulation of near-infrared light, they can convert invisible quantitative signals into photothermal signals that can be captured by thermal imagers, which are reflected in the form of temperature to achieve quantitative detection of the test strip, and can realize dual signal conduction modes, namely, naked eye rapid qualitative analysis and photothermal quantitative analysis cross-validation dual-mode analysis, which has strong reliability and can meet the qualitative or quantitative needs of users in different scenarios.

[0017] 3. The kit of this application has a highly specific detection reaction to Salmonella typhimurium. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the schematic diagram of the synthesis of bimetallic nanoprojectiles (A), the morphology and finite element analysis properties of bimetallic nanoprojectiles (B), the structure of the test strip (C), and the schematic diagram of colorimetry and photothermal analysis; Figure 2 SEM images (A) and (B), finite element analysis (C) and potential diagram (D) of the bimetallic nanoprojectile; Figure 3 The photothermal diagram of RuTe2 with different concentrations (A), the temperature curve of RuTe2 with different powers (B), the temperature curve of RuTe2 with different concentrations (C), the photothermal stability curve (D) and the photothermal conversion efficiency curve (E); Figure 4 To evaluate the performance of the immunochromatographic test strip of the present invention; Figure 5 It is the specific detection image of the immunochromatographic test strip; Figure 6 These are the spike recovery experiment diagrams for three samples in colorimetric mode and photothermal mode. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0020] An immunoassay kit based on a bimetallic nano-projectile bioprobe includes a test strip and an immunoprobe for use together, wherein the test strip consists of a backing plate, a nitrocellulose membrane, an absorption pad, and a sample pad. The absorption pad and the sample pad are respectively placed on both sides of the nitrocellulose membrane, and a detection line (T line) and a quality control line (C line) are arranged horizontally on the upper edge of the non-covered surface of the nitrocellulose membrane. For the specific structure, see Figure 1 (C).

[0021] The preparation method of the test strip comprises the following steps: 1) Preparation of absorbent pad: The absorbent pad is cut from absorbent paper and has a size of 12mm*18mm (length*width).

[0022] 2) Preparation of test line and quality control line on nitrocellulose membrane: 1 mg·mL -1 Anti-Salmonella typhimurium antibody (2E6) and 2 mg mL -1 Sheep anti-mouse immunoglobulin was used at 1 μL cm -1 The streaking rate is coated on the nitrocellulose membrane, and the first and last 1-2 mm are removed. The evenly streaked part is taken as the test line (T line) and the control line (C line), avoiding folding, wrinkling and contamination.

[0023] 3) Preparation of sample pad: The sample pad was cut from a glass fiber membrane with a size of 12mm*24mm (length*width). The sample pad was pretreated before assembly, that is, the sample pad was completely soaked in a blocking solution (the blocking solution composition was: 2% BSA), dried at 37°C for about 5h, and wrapped in plastic wrap at 4°C for later use; 4) Assembly of test strips: First, stick the scribed nitrocellulose membrane flatly on the middle area of ​​the backing plate, stick the sample pad on the lower end, press the nitrocellulose membrane 1-3mm, stick the absorption pad on the upper end, press the nitrocellulose membrane 1-3mm, put it into the strip cutter after compacting the upper and lower parts, and cut it at a width of 3mm to obtain the immunochromatographic test strip for rapid detection of Salmonella typhimurium.

[0024] The immune probe (RuTe2-mAb) is a suspension formed by bimetallic nanoprojectiles loaded with Salmonella typhimurium monoclonal antibodies and resuspended in PBS, wherein the mixing ratio of bimetallic nanoprojectiles to monoclonal antibodies is 0.1-0.3 mg: 5 μg. The monoclonal antibodies are prepared by the following steps: 1) Salmonella typhimurium was cultured in liquid medium at 37°C for 24 h, inactivated with 0.5% formalin (w / v) for 24 h, and washed with PBS by centrifugation (5000 rpm, 10 min). After that, the bacterial concentration was adjusted to 10 8 CFU / mL; 2) Salmonella typhimurium was injected into mice multiple times to induce the production of antiserum against Salmonella typhimurium, and then the spleen cells of the mice were taken for cell fusion with SP2 / 0 myeloma cells. Indirect ELISA and indirect competitive ELISA were used to screen the hybridoma cells that could produce the target antibodies, and the cells were expanded for monoclonal culture; the obtained cells were injected into the peritoneal cavity of female Balb / c mice, and the mice were injected with 0.5 mL of Freund's incomplete adjuvant one week in advance; a few days later, the ascites was collected, and the antibodies were purified by the caprylic acid-ammonium sulfate method and lyophilized; a portion of the antibodies were added at a concentration of 1 mg / mL -1 The mixture was dissolved in 10 mM phosphate buffered saline (PBS, pH 7.4) for later use to obtain a Salmonella typhimurium monoclonal antibody (mAb); The bimetallic nanoprojectile (RuTe2) was prepared by the following steps (see schematic diagram for details). Figure 1 A): Mix 10 mL of deionized water with 18 mg of Cl3H 18 N6Ru, 30mgK2TeO3 and 325mgPVP were mixed, and the mixture was ultrasonicated for 2min; 10mgNH3·H2O and 5mLN2H4·H2O were added to the above solution in sequence; the solution was hydrothermally treated at 180°C in an autoclave for 3h, washed several times and the final product was dried in a vacuum oven to obtain black bimetallic nanoprojectiles (RuTe2) with an average diameter of 23.05±3.25nm and a length of 139.09±14.37nm.

[0025] The specific preparation method of the immune probe is: mix 1 mL of a certain concentration of RuTe2 solution with 5 μg of Salmonella typhimurium monoclonal antibody (mAb), incubate at room temperature for 90 minutes, block the unbound sites with BSA at a final concentration of 1%, incubate for 90 minutes, centrifuge at 11,000 rpm for 10 minutes, resuspend in 200 μL PBS (10 mM pH = 7.4), and obtain the immune probe RuTe2-mAb, which is stored at 4°C.

[0026] Examples 1-6 The composition and process of the immunoassay kits of Examples 1-6 are all based on the above method, except that in the process of preparing the immunoprobe, the concentrations of the RuTe2 solution are 0, 0.1, 0.15, 0.2, 0.25, and 0.3 mg·mL, respectively. -1 (corresponding to Examples 1-6, respectively), and detecting the RuTe2 solutions with different concentrations under 808nm laser (2W·cm -2 ) Temperature changes under irradiation.

[0027] 1. Performance Characterization The SEM image of RuTe2 prepared in this application is shown in Figure 2 (A) and 2 (B), it is obvious that the morphology of RuTe2 is a hollow symmetrical "nanobullet" structure. Based on the unique hollow amorphous structure and symmetrical elastic morphology of the material itself, we infer that the photothermal superiority of RuTe2 is mainly due to the following reasons: (1) The heat generation of semiconductor materials depends on the non-radiative recombination formation of electron-hole pairs and the modification of the electronic structure by crystal defects, which will promote energy transfer; (2) Near-infrared lasers with hollow structures will produce multiple light reflections, which will enhance energy conversion through complex light-matter interactions; (3) High symmetry exhibits strong absorption resonance.

[0028] Moreover, the aspect ratio of RuTe2 is about 6:1, and its specific surface area is large, which is helpful for coupling and tracing antibodies.

[0029] To further understand the photothermal imaging capability of bimetallic RuTe2, a theoretical simulation of its photothermal power density was performed using finite element analysis (see Figure 2 C). This figure simulates an 808nm laser incident along the Z axis as a near-infrared light source based on the finite-difference time-domain method (FDTD), and discusses the electric field on the xy cross section. It can be seen intuitively from the figure that there is a significant difference in the effect of RuTe2. The electric field is enhanced by an order of magnitude, and the near-field electromagnetic field of RuTe2 (|E| max 2 =3100) are much higher than RuNPs (|E| max 2 =13) and TeNPs (|E| max 2 =20). Due to the solid structure of RuNPs and the symmetrical structure of TeNPs, the different structural advantages of RuTe2, which is hollow inside and rough outside, are fully utilized. This fully proves that the morphology of nanoparticles is an important factor affecting their optical properties, which in turn makes RuTe2 have a higher local electric field intensity and thus has an excellent photothermal signal.

[0030] Figure 2D shows the potential of RuTe2 and RuTe2-mAb (Example 6). It can be seen that after conjugation with the antibody, the zeta value of RuTe2 dropped from -3.87 mV to -11.77 mV, which confirms the successful electrostatic adsorption of the antibody on the RuTe2 surface. The main reason is that during this process, a large amount of antibody was strongly conjugated on the RuTe2 surface due to the formation of Ru-S bonds.

[0031] 2. Photothermal performance testing Different RuTe2 concentrations in test examples 1-6: 0, 0.1, 0.15, 0.2, 0.25, 0.3 mg·mL -1 (0~300μg·mL -1 ) and the relationship between the photothermal performance, see 3A and 3C for the results. The RuTe2 concentration of 300 μg·mL -1 , power are 0.5, 1, 1.5, 2Wcm -2 The relationship between power and photothermal performance under irradiation, the results are in 3B.

[0032] The research concentration was 0.3 mg mL -1 The RuTe2 cycle effect and photothermal conversion capacity were investigated by turning off the laser when RuTe2 reached its peak temperature, allowing it to cool naturally for 10 minutes, repeating this process four times, using a thermal imaging camera to record the temperature at different time points and draw relevant curves, and calculating the photothermal conversion efficiency (η) based on the cooling curve theory.

[0033]

[0034] Where h is the heat transfer coefficient, S is the specific surface area of ​​the container, T max and T surr are the maximum temperature and ambient temperature of RuTe2 dispersion, Q dis is the heat change of the reagent blank (J*S -1 ), I is the laser power (W), A λ is the absorbance of RuTe2 aqueous solution at 808nm wavelength. h and S can be obtained according to the cooling stage.

[0035]

[0036] where τ s is the time constant of the system (dimensionless), obtained from the linear relationship between t and ln(θ), θ is the thermal drive constant (dimensionless), m i and C p,i Represents the specific heat capacity and mass of the solvent (water).

[0037] Evaluation results: First, record the RuTe2 concentrations (0~300μg·mL-1 ) at 808 nm laser (2 W cm -2 ) The temperature change under irradiation, the temperature of the material is positively correlated with the increase of concentration. Figure 3 A and 3C, where the temperature of RuTe2 increased significantly to 116 °C within 300 s, while the temperature change of pure water blank under the same conditions was negligible. Therefore, in Example 6, the concentration of RuTe2 was 0.3 mg mL -1 As the optimal choice, the mixing ratio of the bimetallic nanoprojectile to the monoclonal antibody in the immune probe is 0.3 mg:5 μg. Figure 3 D shows the RuTe2 concentration is 300 μg mL -1 Changes in photothermal cycle reliability. After four cycles of laser on and off, the photothermal effect of the material remained essentially unchanged. Figure 3 B shows that power is positively correlated with material temperature rise.

[0038] Due to the material advantages brought by the stacking effect, the nanostructure of this material has good thermal stability and thus has strong light-to-heat conversion capability. The above formula shows that the light-to-heat conversion efficiency of RuTe2 is as high as 60.66% ( Figure 3 E), indicating that the RuTe2 prepared in this application has excellent light conversion performance.

[0039] 3. Application Test 1 The performance of RuTe2-LFIA was evaluated using Salmonella typhimurium standards at dilutions of 0, 10 2 , 10 3 , 5×10 3 , 10 4 , 10 5 , 5×10 5 , 10 6 , 5×10 6 and 10 7 cfu·mL -1 8 μL of RuTe2-mAb immune probe (Example 6) and 92 μL of the above-mentioned standard Salmonella typhimurium liquid of different concentrations were mixed, incubated for 5 minutes, and then placed on the test paper sample pad for detection, and nano-gold was used as a control.

[0040] After 12 minutes of reaction, the test strip was photographed with a smartphone, and then the colorimetric results were obtained based on the Image-J test data. A straight line was fitted using the logarithmic value of the typhoid concentration as the horizontal axis (X) and the gray value of the T line as the vertical axis (Y). -2) irradiate T line for 60s to obtain the photothermal results of the test paper. A handheld thermal imager was used to record the photothermal data, including real-time temperature monitoring and photo capture for quantitative analysis.

[0041] See 4 for the test results. Figure 4 AThe first group is the colorimetric image of RuTe2-mAb, the second group is the photothermal image of RuTe2-mAb, and the third group is the colorimetric image of the control group AuNPs-mAb (RuTe2 was replaced by an equal amount of AuNPs). Monitoring the color intensity of the T line with the naked eye can be used to perform qualitative detection of CM-LFIA. Figure 4 A shows the range of 0-10 7 cfu·mL -1 The RuTe2-LFIA and AuNPs-LFIA images of Salmonella typhimurium taken with a smartphone within a concentration range. The RuTe2-LFIA of the present application has advantages over AuNPs in both colorimetry and photothermal signals. As the concentration of the target decreases, the amount of nanoprobes captured on the T line continues to decrease and the color becomes lighter. The bands of RuTe2-CM-LFIA are clearer and wider than those of AuNPs-LFIA. The visual detection limits (vLOD) are 5×10 3 cfu·mL -1 and 10 5 cfu·mL -1 .

[0042] When the LFIA test strips were used for quantitative analysis, the correlation curve between the concentration of Salmonella typhimurium and the T line signal was good ( Figure 4 B). The T-line regression equations are y=2641.4lg(x)-12018.23 (AuNPs-LFIA), y=12.32lg(x)-36.43 (RuTe2-CM-LFIA), and y=26.59lg(x)-67.97 (RuTe2-PT-LFIA). It is worth noting that the strong colorimetric signal and intrinsic photothermal effect of RuTe2 expand the sensitivity range of the target analytes. Figure 4 C shows that the LOD values ​​of RuTe2-LFIA for Salmonella typhi are 2237.92 cfu·mL -1 (RuTe2-CM-LFIA, colorimetric) and 905.69 cfu·mL -1 (RuTe2-PT-LFIA, photothermal). These values ​​are higher than those of AuNPs-LFIA (35477.01 cfu·mL -1 ) are 15.85 times and 39.17 times lower respectively.

[0043] 4. Specificity evaluation test The kit constructed in Example 6 was used to detect the presence of Salmonella typhimurium ( S.typhimurium)ATCC43174, Salmonella london ( S.London )ATCC8389, Salmonella Hadda ( S. Hadar) , Salmonella Enteritidis ( S. enteritidis )ATCC13076, Escherichia coli O157:H7 ( E.coliO157:H7 )ATCC43889, Enterobacter sakazakii ( E.sakazakii )ATCC29544, Staphylococcus aureus ( S. staphylococcus )ATCC29544, Vibrio parahaemolyticus ( V. parahaemolyticus )ATCC178022, Shigella ( S.flexneri )CICC21534, to evaluate the specificity of the prepared test strips to measure their anti-interference ability (the concentration of the detected bacteria was 10 6 cfu·mL -1 , usage 92μL, immunoprobe usage 8μL).

[0044] Test results: Figure 5 Compared with the strong signals generated by the analytes, the signals generated by various microorganisms of the prepared test strips were more similar to the background signals of the blank samples, showing a highly specific detection reaction for Salmonella typhimurium.

[0045] V. Application Test 2 The kit prepared in Example 6 was used for the detection of Salmonella Typhimurium in actual samples, that is, the detection of Salmonella Typhimurium in food samples. All actual samples (water, eggs and lettuce) were not pretreated, and no Salmonella Typhimurium was found to be present in the samples according to the national standard method (GB4789.4-2016).

[0046] When testing actual samples, 8 μL of RuTe2-mAb immune probe and 92 μL of the above-mentioned standard samples of different concentrations were mixed and then tested.

[0047] Test results such as Figure 6 As shown in the figure, the average spike recovery of RuTe2-CM-LFIA was between 89.13% and 116.84%, with a CV of less than 3.57%, while the average spike recovery of RuTe2-PT-LFIA was between 89.32% and 110.44%, with a CV of less than 2.53%. In the actual application of detecting Salmonella typhimurium, the combination of dual detection modes not only inherits each other's complementary advantages, but also provides more flexible and convincing results.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Other modifications or equivalent substitutions made to the technical solution of the present invention by ordinary technicians in the field should be included in the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solution of the present invention.

Claims

1. An immunoassay kit based on a bimetallic nanobullet bioprobe, characterized in that: It includes a test strip and an immune probe, wherein the immune probe is a suspension of a bimetallic nanobullet and a monoclonal antibody, and the mixing ratio of the bimetallic nanobullet to the monoclonal antibody is 0.1-0.3 mg: 5 μg; The bimetallic nanobullet was prepared by the following steps: 10 mL of deionized water and 18 mg of Cl3H 18 N6Ru, 30 mgK2TeO3 and 325 mg PVP were mixed and the mixture was sonicated; 10 mgNH3·H2O and 5 mLN2H4·H2O were added to the above solution in sequence; the solution was hydrothermally treated at 180 °C for 3 h in an autoclave, the product was washed several times and dried to obtain the product.

2. The immunoassay kit based on the bimetallic nanobullet bioprobe according to claim 1, characterized in that: The immune probe is a suspension formed by dispersing the bimetallic nanoprojectile and the monoclonal antibody of Salmonella typhimurium in 200 μL PBS at a mixing ratio of 0.3 mg:5 μg.

3. The immunoassay kit based on the bimetallic nanobullet bioprobe according to claim 1, characterized in that: The bimetallic nanobullet has an average diameter of 23.05±3.25 nm and a length of 139.09±14.37 nm.

4. The immunoassay kit based on the bimetallic nanobullet bioprobe according to claim 1, characterized in that: The immunoprobe is prepared by the following steps: 1 mL of a 0.1-0.3 mg mL -1 The RuTe2 solution was mixed with 5 μg of Salmonella typhimurium monoclonal antibody and incubated at room temperature for 90 min. Unbound sites were blocked with BSA at a final concentration of 1%. After incubation for 90 min, the obtained RuTe2-mAb was centrifuged and resuspended in 200 μL 10 mM pH = 7.4 PBS and stored at 4 °C.

5. The immunoassay kit based on the bimetallic nanobullet bioprobe according to claim 1, characterized in that: The test strip consists of four parts: a backing plate, a nitrocellulose membrane, an absorption pad, and a sample pad. The absorption pad and the sample pad are respectively placed on both sides of the nitrocellulose membrane, and a detection line (T line) and a quality control line (C line) are horizontally arranged along the upper edge of the non-covered surface of the nitrocellulose membrane.

6. The immunoassay kit based on the bimetallic nanobullet bioprobe according to claim 5, characterized in that: 1 mg·mL -1 Anti-Salmonella typhimurium antibodies and 2 mg mL -1 Sheep anti-mouse immunoglobulin was used at 1 μL cm -1 The streaking rate is coated on the nitrocellulose membrane, 1-2 mm from the beginning and the end is removed, and the evenly streaked part is taken as the test line and the control line.

7. Use of the kit according to claim 1 in detecting Salmonella typhimurium.

8. The use according to claim 7, characterized in that: After mixing the immune probe and the sample to be tested in a volume ratio of 8:92, add it dropwise onto the test strip and perform qualitative detection based on the intensity of the T line on the test strip. Irradiate the T line with near-infrared light to obtain the photothermal result of the test strip, and use a handheld thermal imager to record the photothermal data to obtain the quantitative result.

9. The use according to claim 7, characterized in that: Using 808 nm, 2.0 W cm -2 Laser irradiation T line for 60 s.

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