A photothermal lateral flow assay device for simultaneously detecting multiple pathogenic bacteria and application thereof

Abstract

The application discloses a photothermal lateral flow analysis device for simultaneously detecting multiple pathogenic bacteria and application thereof. The device comprises a test strip, n photothermal signal probes, an 808nm plane laser light source and a thermal detection device, the n photothermal signal probes are prepared by respectively adsorbing n pathogenic bacteria antibodies, i.e. first primary antibodies, on the surface of CuS@SnS2-Au composite, and n is greater than or equal to 2; the second primary antibodies of the n pathogenic bacteria to be detected are sequentially fixed on the detection area of the test strip, and the second primary antibodies of the n pathogenic bacteria to be detected are sequentially fixed on the detection area of the test strip. The application steps are as follows: the n photothermal signal probes, the sample liquid to be detected and the buffer are mixed, the sample pad end of the test strip is inserted into the mixed solution for chromatography, the whole detection area is irradiated by the 808nm plane laser, and the photothermal temperature of the detection area is collected by a thermal imaging or temperature measuring device; standard curves are established according to the temperature and the concentration of the n pathogenic bacteria to be detected, and photothermal qualitative and quantitative analysis of the n pathogenic bacteria to be detected is carried out.

Description

Technical Field

[0001] This invention relates to a photothermal lateral flow analysis device and its application for simultaneously detecting multiple pathogenic bacteria, belonging to the field of food analysis and testing technology. Background Technology

[0002] Related surveys show that diseases caused by pathogenic bacteria are the most important factor endangering food safety. Common pathogens include Salmonella Typhimurium, Escherichia coli Hemorrhagicum, Campylobacter jejuni, and Listeria monocytogenes. Due to their rapid transmission, once an outbreak occurs, it is difficult to control effectively. Conventional methods for detecting pathogens are time-consuming. Because various pathogens share similar ecological conditions for growth, one pathogen can contaminate multiple agricultural products and foods. Furthermore, given dietary diversity, food is often contaminated by coexisting pathogens, posing a greater threat to humans. Therefore, there is an urgent need to develop high-throughput rapid detection methods for pathogens.

[0003] Sideflow chromatography is widely used for the detection of pathogenic bacteria due to its fast detection performance, low cost, and good sensitivity. In the past 5-10 years, a signal amplification strategy based on current sideflow analysis technology has emerged. This strategy mainly relies on laser-excited plasma nanomaterials. When the excitation light resonates with the surface plasmon resonance of the nanoparticles, the particles absorb the light energy and release it as heat, achieving a signal amplification effect. Small changes in analyte concentration can be detected through temperature variations, resulting in a low background signal and high sensitivity. It has been successfully applied to the detection of various target analytes.

[0004] Compared to colorimetric and fluorescence lateral flow analysis techniques, photothermal lateral flow analysis has higher sensitivity and signal-to-noise ratio, and has received increasing attention in recent years. Zhang et al. developed an ultrasensitive photothermal lateral flow immunoassay method based on multifunctional photothermal nanoparticles Fe3O4@CuS for the quantitative detection of Escherichia coli O157:H7 (Food Chemistry 2020, 332, 127398). Zhang et al. developed a photothermal lateral flow immunoassay method for detecting Salmonella Typhimurium (STY) using PB@Au as the photothermal material (Food Chemistry 2022, 385, 132649).

[0005] However, existing photothermal lateral flow analyses cannot achieve simultaneous detection of multiple analytes because they all use point lasers as light sources, which can only illuminate one detection site on the test strip at a time. Even if multiple analytes are placed in the detection area, each area can only be illuminated and its temperature measured sequentially. Multiple analytes can only be detected sequentially, and the total analysis time is the sum of the individual analysis times. Therefore, achieving simultaneous detection of multiple analytes in photothermal lateral flow analysis remains a challenge and bottleneck in the field. Summary of the Invention

[0006] Technical issues: Existing photothermal lateral flow analysis of pathogenic bacteria has the following limitations: 1. The light source is a point laser, with a small spot area (5*8mm). 2 First, each detection can only detect a single target object; second, the data acquisition and analysis of one target object requires 10-20 minutes of photothermal sideflow analysis, and if there are three target objects, the analysis time will be 30-60 minutes.

[0007] Technical solution: On the one hand, this application provides a photothermal lateral flow analysis method for simultaneously detecting multiple pathogenic bacteria. The method utilizes a photothermal lateral flow analysis device to detect multiple pathogenic bacteria, and includes the following steps:

[0008] (1) Construct a photothermal lateral flow analysis device for simultaneous detection of multiple pathogens, specifically comprising four parts: preparation of test strips, preparation of n kinds of photothermal signal probes, construction of an 808nm surface laser source and thermal detection device. The test strip includes a base plate, on which a sample pad, an NC membrane, and an absorption pad are sequentially overlapped and pasted in a horizontal direction. The NC membrane includes a detection area and a quality control area, i.e., the C area. The detection area of ​​the test strip includes n independent T areas, each T area having a different secondary primary antibody (mAbn') of the pathogen to be tested, denoted as T. n The test strip has a C region with n≥2; the C region is fixed with a corresponding secondary antibody; the photothermal signal probe is made of CuS@SnS2-Au composite material; the 808nm surface laser source is formed by expanding an 808nm point laser through a surface diverging laser beam expander, and the spot size can be adjusted to simultaneously cover all T regions. n district;

[0009] (2) Mix n kinds of photothermal signal probes, the sample solution to be tested, and the buffer solution to form a mixture. Insert the sample pad end of the test strip into the mixture for chromatography.

[0010] (3) Irradiate the detection area with an 808nm surface laser to simultaneously excite all T n n photothermal signal probes in the area, using thermal imaging or temperature measurement equipment to simultaneously collect T n The photothermal temperatures of n photothermal signal probes in the region are analyzed qualitatively and / or quantitatively.

[0011] As an optional implementation method, the qualitative analysis refers to, based on T n Whether the corresponding T zone in the region appears dark purple or whether there are changes in light, heat, and temperature determines whether the sample solution contains the corresponding pathogenic bacteria; the quantitative analysis refers to establishing standard curves based on light, heat, temperature, and the concentration of n pathogenic bacteria to be tested, and performing quantitative analysis of n target substances.

[0012] As an optional implementation, in step (3), each detection area, i.e., T n The changes in light, heat, and temperature in the area are positively correlated with the content of the corresponding pathogenic bacteria to be tested, specifically:

[0013] When the sample does not contain pathogenic bacteria, T n The corresponding T zone in the region does not show color and its light and heat temperature is close to room temperature;

[0014] When the sample contains pathogenic bacteria, T n The corresponding T region appears dark purple and its photothermal temperature increases;

[0015] The quality control area, which serves as a reference for verifying the validity of the test strip results, always appears dark purple.

[0016] As an optional implementation method, the analytical method of this application is applicable to detecting whether food contains multiple pathogenic bacteria. The food mentioned in this application includes, for example, poultry, livestock, eggs, dairy products, and grains.

[0017] As an optional implementation, the pathogenic bacteria include, but are not limited to, at least two of the following: Salmonella Typhimurium, Listeria monocytogenes, Escherichia coli hemorrhagicum, Bacillus cereus, and Staphylococcus aureus.

[0018] As an optional implementation, the antibody against the pathogen to be tested is either the first primary antibody or the second primary antibody. These are different primary antibodies, and the sources of the primary antibodies include, but are not limited to, mice, rats, and rabbits.

[0019] As an optional implementation, the secondary antibody includes, but is not limited to, sheep anti-mouse secondary antibody, rabbit anti-mouse secondary antibody, sheep anti-rabbit secondary antibody, and donkey anti-rabbit secondary antibody.

[0020] On the other hand, a method for preparing a photothermal lateral flow analysis device for simultaneously detecting multiple pathogenic bacteria is provided, wherein the preparation method of the device includes:

[0021] (1) Preparation of signal probes: CuS@SnS2-Au composite material was reacted with n antibodies against pathogenic bacteria to be tested, i.e., the primary antibody mAbs. n The complex is then combined with bovine serum albumin to form a conjugate, thus yielding the signal probe CuS@SnS2-Au-mAb. n The signal probe is stored in a sealed container in the form of solution or lyophilized powder for later use.

[0022] (2) Constructing the test strip: T n In the test zone T, n types of primary and secondary antibodies mAbn' against the pathogens to be tested are fixed sequentially. In the test zone C, secondary antibodies against the primary antibody source are added by dripping or spraying. After drying, the mixture is stored in a vacuum bag for later use.

[0023] As an optional implementation, the photothermal signal probe is a CuS@SnS2-Au composite material reacted with n antibodies against the pathogenic bacteria to be tested, i.e., the primary antibody mAb. n The complex formed after compounding with bovine serum albumin is CuS@SnS2-Au-mAb. n .

[0024] As an optional implementation method, the specific method for preparing CuS@SnS2-Au composite material is as follows: dilute the prepared CuS@SnS2 nanocrystal dispersion, take the diluted CuS@SnS2 solution into a centrifuge tube, add trisodium citrate solution, mix well, add HAuCl4 solution, add ultrapure water, and then shake the reaction at room temperature to obtain CuS@SnS2-Au composite material, which is then refrigerated for later use.

[0025] As an optional implementation, the signal probe is prepared as CuS@SnS2-Au-mAb. n The specific method is as follows: Take CuS@SnS2-Au composite material into a centrifuge tube, add a weak alkaline solution to adjust the pH of the system to 6-7.5, shake to mix, and then add the first primary antibody mAb of the pathogen to be tested. n After shaking and reacting at room temperature for 30–60 min, bovine serum albumin (BSA) was added for blocking. The mixture was then shaken and reacted at room temperature for 1–1.5 h. After centrifugation, the supernatant was removed, and the remaining material was reconstituted in buffer to obtain CuS@SnS2-Au-mAb. n Store in the refrigerator for later use.

[0026] As an optional implementation, the buffer solution includes any one of phosphate buffer, borate buffer, and carbonate buffer.

[0027] As an optional implementation, the T n In the test zone T, n types of primary antibody mAbn' for the target pathogens are sequentially fixed, including the sequential dripping or spraying of the primary antibody mAbn' for the target pathogens in the T zone. Specifically: T n Add or spray 5-20mM PBS solution containing 0.5-10mg / mL of another antibody (mAbn') of the pathogen to be tested in the respective areas.

[0028] As an optional implementation, the fixation of the secondary antibody in region C includes dripping or spraying a secondary antibody against the primary antibody source, i.e., anti-primary antibody, into region C. Specifically, this involves dripping or spraying a 5-20 mM PBS solution containing 0.05-10 mg / mL of secondary antibody into region C.

[0029] As an alternative implementation, the running buffer is a phosphate or citrate solution containing sucrose, BSA, Tween.

[0030] On the other hand, the present invention provides a photothermal lateral flow analysis device for simultaneously detecting multiple pathogenic bacteria. The photothermal lateral flow analysis device comprises four parts: a test strip, n kinds of photothermal signal probes, an 808nm surface laser source, and a thermal detection device.

[0031] The test strip body includes a base plate, on which a sample pad, an NC membrane, and an absorbent pad are sequentially and overlapping in a horizontal direction. The nitrocellulose membrane (NC membrane) is used to separate and detect analytes from other substances in the sample. The sample pad is used for sample loading, and the absorbent pad is used to absorb excess liquid. The PVC base plate provides physical support for the test strip. The NC membrane includes a detection area, i.e., T... n The quality control area, also known as area C, has n≥2; where T n The region includes n independent T regions, with one T region set at intervals of 3-5 mm. Each T region is fixed with a different secondary primary antibody mAbn' for the pathogen to be tested. The C region is fixed with a secondary antibody, which is a secondary antibody against the source of the primary antibody, i.e., anti-primary antibody.

[0032] The photothermal signal probe is a CuS@SnS2-Au composite material, which is reacted with n antibodies against pathogenic bacteria to be tested, namely the primary antibody (mAb). n The complex formed by combining CuS@SnS2 with bovine serum albumin is called CuS@SnS2-Au-mAb. n In this application, the photothermal signal probe is used independently of the photothermal test strip structure. After chromatographic migration, the signal probe flows through the detection area and is detected by the corresponding T. n The second primary antibody mAbn' capture in the region;

[0033] The 808nm surface laser is formed by expanding an 808nm point laser beam using a surface-diverging laser beam expander. The surface-diverging laser beam expander is used as an optical accessory for the 808nm point laser, and can expand a 5*8mm beam. 2 A small spot size can be magnified 1.5-30 times, and a surface laser can simultaneously irradiate n T-regions, where n≥2. When the magnification factor is 2.5 times, a 5*8mm area can be magnified. 2 The original light spot was enlarged to 12.5*20mm. 2 The laser can simultaneously irradiate all T-regions;

[0034] The thermal detection device refers to thermal imaging or temperature measurement equipment.

[0035] As an optional implementation, the thermal imaging or temperature measurement device includes, but is not limited to, any one of the following: mobile phone infrared thermal imaging analysis accessories, infrared thermal imagers, handheld infrared thermal imaging analyzers, or infrared thermal imaging temperature guns. The thermal imaging or temperature measurement device acquires photothermal imaging photos and outputs and displays them through a connected smart display terminal.

[0036] As an optional implementation, the smart display terminal includes a computer or a smartphone.

[0037] The detection principle of the photothermal lateral flow analyzer for simultaneous detection of multiple pathogenic bacteria in this invention is explained below, taking *Salmonella Typhimurium* (STY), *Listeria monocytogenes* (LM), and *Escherichia coli* (EHEC) as examples: When the sample does not contain any of the three pathogenic bacteria, CuS@SnS2-Au-mAb n Since no signal probe is captured in any of the three T regions, there is no accumulation in these regions, resulting in no color development. Simultaneous irradiation of the three T regions with an 808nm surface laser does not raise the photothermal temperature, remaining close to room temperature. When only STY is present in the sample, it first binds to its corresponding Bi2Se3@FeSe2-Au-mAb1 complex. Upon migrating forward through the corresponding T1 region on the NC film, it is captured, forming a sandwich complex. The T1 region displays a bright dark purple color, while the T2 and T3 regions do not capture the signal probe. Simultaneous irradiation of the three T regions with an 808nm line laser significantly raises the photothermal temperature of the T1 region, while the T2 and T3 regions do not, remaining close to room temperature. When both STY and LM are present in the sample, the T1 and T2 regions show significant photothermal increases, while the T3 region does not, remaining close to room temperature; and so on. n The degree of temperature change in the region is directly proportional to the content of pathogenic bacteria in the sample. Excess free CuS@SnS2-Au-mAb n The complex binds to the secondary antibody on the control area (C area), always displaying a bright dark purple color, serving as a reference for verifying the validity of the test strip results.

[0038] Beneficial effects:

[0039] The detection method provided by this invention can simultaneously complete the photothermal quantitative detection of multiple pathogens within 20 minutes in a single chromatography step, with higher sensitivity. Moreover, it expands the detection throughput without increasing the detection time compared to existing photothermal sideflow analysis methods. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the test strip structure of the present invention;

[0041] Figure 2 This describes the response of the test strip of the present invention to negative and positive samples;

[0042] Figure 3This describes the specific response of the test strip detection area and probes in this invention. Detailed Implementation

[0043] The present invention can be better understood from the following embodiments. However, the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0044] The following embodiments of this application disclose the preparation and application of a photothermal lateral flow analysis device for simultaneously detecting multiple pathogenic bacteria, including the following steps:

[0045] (1) Preparation of photothermal signal probe: CuS@SnS2-Au composite material was compounded with antibodies against n pathogenic bacteria to be tested, and then mixed with bovine serum albumin to prepare CuS@SnS2-Au-mAb. n That is, a photothermal signal probe;

[0046] (2) Assemble the test strip: In the detection area (T n In the quality control area (C area), another antibody, namely the secondary antibody mAbn', is fixed sequentially for n types of pathogenic bacteria to be tested.

[0047] (3) Mix n kinds of photothermal signal probes, the sample solution to be tested, and the buffer solution, and then insert the sample pad end of the test strip into the mixture for chromatography.

[0048] (4) Irradiate the detection area with an 808nm surface laser, collect the temperature with thermal imaging or temperature measurement equipment, establish standard curves based on the temperature and the concentration of n pathogenic bacteria to be tested, and perform qualitative and / or quantitative analysis of n analytes.

[0049] This invention enables simultaneous photothermal qualitative and / or quantitative analysis of multiple pathogenic bacteria. Based on the number of T-regions, the magnification factor of the surface-diverging laser beam expander in the surface laser can be adjusted to regulate the surface laser irradiation area, ensuring the spot area meets the coverage requirements of n T-regions. The following embodiments use STY, LM, and EHEC as examples of pathogenic bacteria to provide a thorough and detailed explanation of the preparation and application methods of the entire device.

[0050] Example 1:

[0051] The preparation of a photothermal lateral flow analyzer for the simultaneous detection of three pathogenic bacteria, STY, LM, and EHEC, and its detection of negative and positive samples are described below.

[0052] 1. Preparation of test paper materials

[0053] 1.1 Preparation of CuS@SnS2-Au composite material

[0054] Dilute the CuS@SnS2 dispersion with deionized water to a volume fraction of 1%. Take the diluted CuS@SnS2 solution into a centrifuge tube, add ultrapure water, trisodium citrate solution (0.3 mol / L), and HAuCl4 solution (m / v = 1%). Then immediately place it on a vortex shaker and shake for 7 min at room temperature to obtain CuS@SnS2-Au, a composite nanomaterial of CuS@SnS2 nanocrystals and AuNPs. Store it in a refrigerator at 4℃ for later use.

[0055] 1.2 Preparation of photothermal signal probe CuS@SnS 2- Au-mAb n

[0056] Take 1 mL of CuS@SnS2-Au into three centrifuge tubes respectively, add 0.2 mol / L K2CO3 solution to adjust the pH of the system to 6.5, 6.5 and 6 respectively, shake to mix well, then add 5 μL of 1 mg / mL LTY-mAb, 3 μL of LM-mAb and 1 μL of EHEC-mAb respectively, mix well and react at room temperature (150 rpm) for 45 min. After the reaction was completed, 100 μL of BSA (m / m = 10%) was added for blocking for 1 h (150 rpm). After centrifugation at 12000 r / min for 30 min, the supernatant was removed and then redissolved in 100 μL of buffer (20 mmol / L Na3PO4, 5% BSA, 0.25% Tween-20, 10% sucrose) to obtain the photothermal probes CuS@SnS2-Au-mAb1, CuS@SnS2-Au-mAb2, and CuS@SnS2-Au-mAb3 (mAb1, mAb2, mAb3 correspond to STY-mAb, LM-mAb, and EHEC-mAb, respectively, and are denoted as T1, T2, and T3). The probes were stored in a refrigerator at 4℃ for later use.

[0057] 1.3 Detection Area (T) n Preparation of solution in the zone

[0058] STY-mAb1', LM-mAb2', and EHEC-mAb3' were diluted with 10 mM PBS solution (pH 7.4) to 0.4 mg / mL, 0.6 mg / mL, and 0.2 mg / mL, respectively, corresponding to solutions in the T1, T2, and T3 regions.

[0059] 1.4 Preparation of solutions for the quality control zone (Zone C)

[0060] The goat anti-mouse secondary antibody was diluted to 0.2 mg / mL with 10 mM PBS solution (pH 7.4).

[0061] 2. Preparation of test strips

[0062] according to Figure 1 The membrane assembly method involves attaching the NC membrane to the center of a PVC base plate. The sample pad and absorbent pad overlap the left and right ends of the NC membrane, respectively, covering it by approximately 2mm. The assembled card is then cut into 3mm wide strips to obtain blank test strips. These are then used in... Figure 1 China T n Add 0.5 μL of T to zone C and zone C at 5 mm intervals. n Prepare zone A solution and 0.5 μL zone C solution. Place the spotted test strips in an oven and dry at 37°C for 60 min, then store in a vacuum bag for later use.

[0063] 3. Drawing working curves

[0064] Prepare 10 mL of 1×10 using acetonitrile. 6 CFU / mL STY, LM, and EHEC standard solutions were prepared by diluting the STY standard solution to a concentration of 0.5 × 10⁻⁶ with 10 mM PBS solution (pH 7.4). 3 CFU / mL, 2.5×10 3 CFU / mL, 5×10 3 CFU / mL, 25×10 3 CFU / mL, 50×10 3 CFU / mL, 250×10 3 CFU / mL, 500

[0065] ×10 3 CFU / mL, 650×10 3 CFU / mL, 800×10 3 CFU / mL.

[0066] Similarly, the LM standard solution was diluted to a concentration of 0.2 × 10⁻⁶. 3 CFU / mL, 0.4×10 3 CFU / mL, 2×10 3 CFU / mL, 5×10 3 CFU / mL, 10×10 3 CFU / mL, 50×10 3 CFU / mL, 100×10 3 CFU / mL, 250×10 3 CFU / mL, 400×10 3 CFU / mL is prepared as the test solution.

[0067] Similarly, the EHEC standard solution was diluted to a concentration of 0.075 × 10⁻⁶. 3 CFU / mL, 0.15×10 3CFU / mL, 0.3×10 3 CFU / mL, 1.5×10 3 CFU / mL, 3×10 3 CFU / mL, 7.5×10 3 CFU / mL, 15

[0068] ×10 3 CFU / mL, 22.5×10 3 CFU / mL, 30×10 3 CFU / mL is prepared as the test solution.

[0069] Take 24 μL of each of the three test solutions, totaling 72 μL, and place them in centrifuge tubes. Then, take 3 μL of each of the three photothermal probes, totaling 9 μL, and add them to the centrifuge tubes. Next, add 9 μL of running buffer (10 mM PBS solution containing 5% sucrose, 1% BSA, 1% Tween-20, pH 7.4) to the centrifuge tubes and mix for 10 min. Then, insert the test strip into the centrifuge tubes for chromatography. After chromatography, use an 808 nm surface laser (power 1.96 W / cm²) to perform the chromatography. 2 Irradiate for 5 minutes, and use infrared thermal imaging accessories to monitor the temperature changes in detection areas T1, T2, and T3.

[0070] The results show that... Figure 2 Positive test strip T n The area shows a distinct dark purple hue, indicating high light and heat temperature. Negative test strip T n The area does not show color, and the light, heat, and temperature are close to room temperature.

[0071] The operating curves of STY, LM, and EHEC are as follows: Figure 2 As shown in (C), (D), and (E).

[0072] Example 2:

[0073] Specificity verification of a photothermal lateral flow analyzer for simultaneously detecting three pathogenic bacteria (STY, LM, and EHEC) against multiple pathogens includes the following steps:

[0074] 1. Preparation of test paper materials

[0075] Same as Example 1

[0076] 2. Preparation of test strips

[0077] Same as Example 1

[0078] 3. Sample pretreatment

[0079] Use 10mM PBS solution (pH 7.4) as the negative test solution.

[0080] 4. Sample testing

[0081] Add 72 μL of test solutions containing STY pathogens, LM pathogens, and EHEC pathogens (labeled T1, T2, and T3, respectively) to three centrifuge tubes, respectively. Then add 3 μL of CuS@SnS2-Au-mAb1, 3 μL of CuS@SnS2-Au-mAb2, and 3 μL of CuS@SnS2-Au-mAb3 to each tube. Next, add 9 μL of running buffer (10 mM PBS solution containing 5% sucrose, 1% BSA, 1% Tween-20, pH 7.4), mix, and incubate for 10 min. Then insert the test strip into the centrifuge tube for chromatography. After the test strip chromatography is complete, use an 808 nm laser (power 1.96 W / cm²) equipped with a surface-diverging laser beam expander. 2 Irradiate for 5 minutes, and monitor the detection area T using infrared thermal imaging accessories. n The temperature changes were shown in Table 1.

[0082] As shown in Table 1, the CuS@SnS2-Au-mAb1 probe showed color development and a high photothermal temperature in the STY detection region, while the CuS@SnS2-Au-mAb2 probe did not show color development in the LM detection region, and the CuS@SnS2-Au-mAb3 probe did not show color development in the EHEC detection region, with photothermal temperatures close to room temperature. The CuS@SnS2-Au-mAb2 probe showed color development and a high photothermal temperature in the LM detection region, while the CuS@SnS2-Au-mAb1 probe showed color development and a high photothermal temperature in the EHEC detection region. In the STY detection region, neither the CuS@SnS2-Au-mAb3 probe nor the CuS@SnS2-Au-mAb3 probe showed color development in the EHEC detection region, and the photothermal temperature was close to room temperature. In the EHEC detection region, the CuS@SnS2-Au-mAb3 probe showed color development and the photothermal temperature was relatively high. In the STY detection region, neither the CuS@SnS2-Au-mAb1 probe nor the CuS@SnS2-Au-mAb2 probe showed color development in the LM detection region, and the photothermal temperature was close to room temperature. The probes showed good specificity recognition results.

[0083] Table 1. Specificity verification results of the photothermal sideflow analysis device for various pathogens.

[0084]

[0085] (+: deep purple, high light and heat temperature; —: colorless, light and heat temperature close to room temperature)

[0086] Example 3:

[0087] A photothermal lateral flow analyzer for the simultaneous detection of multiple pathogenic bacteria is applied to actual sample analysis, comprising the following steps:

[0088] 1. Preparation of test paper materials

[0089] Same as Example 1

[0090] 2. Preparation of test strips

[0091] Same as Example 1

[0092] 3. Sample pretreatment

[0093] Take 5g of food sample, 0.5g of NaCl, and mix with 20mL of 84% acetonitrile aqueous solution. Sonicate for 20min, centrifuge for 10min, filter the supernatant through a 0.22μm filter membrane and treat with nitrogen blowing. Redissolve the sample in 10mM PBS (pH 7.4). Prepare 10mL of 1×10⁻⁶ acetonitrile solution. 6 CFU / mL STY, LM, EHEC, BC, and SA standard solutions were used. The STY standard solution was diluted to a concentration of 5 × 10⁻⁶ using actual sample solutions. 3 CFU / mL, 50×10 3 CFU / mL, 500×10 3 CFU / mL.

[0094] Similarly, the LM standard solution was diluted to a concentration of 1×10⁻⁶. 3 CFU / mL, 10×10 3 CFU / mL, 100×10 3 CFU / mL is prepared as the test solution.

[0095] Similarly, the EHEC standard solution was diluted to a concentration of 0.5 × 10⁻⁶. 3 CFU / mL, 5×10 3 CFU / mL, 15×10 3 CFU / mL is prepared as the test solution.

[0096] Similarly, dilute the BC and SA standard solutions to 100 × 10⁻⁶. 3 CFU / mL is prepared as the test solution.

[0097] 4. Sample testing

[0098] Add 72 μL of the actual sample solution to each of three centrifuge tubes, followed by 9 μL each of CuS@SnS2-Au-mAb1, CuS@SnS2-Au-mAb2, and CuS@SnS2-Au-mAb3 (denoted as T1, T2, and T3, respectively). Then add 9 μL of running buffer (10 mM PBS solution containing 5% sucrose, 1% BSA, 1% Tween-20, pH 7.4), mix, and incubate for 10 min. Then insert the test strip into the centrifuge tube for chromatography. After the test strip chromatography is complete, use an 808 nm laser (power 1.96 W / cm²) equipped with a surface-diverging laser beam expander. 2 Irradiate for 5 minutes, and monitor the detection area T using infrared thermal imaging accessories.n Temperature changes.

[0099] For samples containing STY, LM, and EHEC respectively, the detection areas of the test strips all showed a deep dark purple color and a high photothermal temperature; for samples containing SA and BC respectively, the corresponding detection areas of the test strips showed a lighter color and a lower photothermal temperature, close to room temperature; the photothermal sideflow analysis device showed good accuracy in detecting actual samples. The detection limits for the three pathogenic bacteria STY, LM, and EHEC were 0.53 × 10⁻⁶. 3 CFU / mL, 0.32×10 3 CFU / mL, 0.25×10 3 CFU / mL.

[0100] Table 2. Detection results of the photothermal lateral flow analyzer for various pathogenic bacteria applied to actual sample analysis.

[0101]

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A photothermal lateral flow analysis method for simultaneous detection of multiple pathogenic bacteria, characterized in that, The method for simultaneously detecting multiple pathogens using a photothermal lateral flow analyzer includes the following steps: (1) Construct a photothermal lateral flow analysis device for simultaneous detection of multiple pathogens, specifically including four parts: preparation of test strips, preparation of n kinds of photothermal signal probes, construction of an 808 nm surface laser source and thermal detection device. The test strip includes a base plate, on which a sample pad, an NC membrane, and an absorption pad are sequentially overlapped and pasted in the horizontal direction. The NC membrane includes a detection area and a quality control area, i.e., the C area. The detection area of ​​the test strip includes n independent T areas, and each T area is fixed with a different secondary primary antibody of the pathogen to be tested, i.e., mAbn', denoted as T. n The test strip has a C region with n≥2; the C region is fixed with a corresponding secondary antibody; the photothermal signal probe is made of CuS@SnS2-Au composite material; the 808 nm surface laser source is formed by expanding an 808 nm point laser through a surface diverging laser beam expander, and the spot size can be adjusted to simultaneously cover all T regions. n district; (2) Mix n kinds of photothermal signal probes, the sample solution to be tested, and the buffer solution to form a mixture. Insert the sample pad end of the test strip into the mixture for chromatography. (3) n photothermal signal probes migrate through the detection area due to tomography and are detected by the corresponding T n Region capture; the detection region is irradiated with an 808nm surface laser to simultaneously excite all T... n n photothermal signal probes in the area, using thermal imaging or temperature measurement equipment to simultaneously collect T n The photothermal temperatures of n photothermal signal probes in the region are analyzed qualitatively and / or quantitatively.

2. The method according to claim 1, characterized in that, In step (3), each detection area, i.e., T n The changes in light, heat, and temperature in the area are positively correlated with the content of the corresponding pathogenic bacteria to be tested, specifically: When the sample does not contain pathogenic bacteria, T n The corresponding T zone in the region does not show color and its light and heat temperature is close to room temperature; When the sample contains pathogenic bacteria, T n The corresponding T region appears dark purple and its photothermal temperature increases; The quality control area, which serves as a reference for verifying the validity of the test strip results, always appears dark purple.

3. The method according to claim 1, characterized in that, The pathogenic bacteria include, but are not limited to, at least two of the following: Salmonella Typhimurium, Listeria monocytogenes, Escherichia coli hemorrhagicum, Bacillus cereus, and Staphylococcus aureus.

4. The method according to claim 1, characterized in that, The signal probe is composed of CuS@SnS2-Au composite material and n antibodies against the target pathogens, i.e., the primary antibody mAb. n The complex formed after compounding with bovine serum albumin is CuS@SnS2-Au-mAb. n .

5. The method according to claim 4, characterized in that, The preparation method of the CuS@SnS2-Au composite material includes: diluting the CuS@SnS2 nanocrystal dispersion, taking the diluted CuS@SnS2 solution into a centrifuge tube, adding trisodium citrate solution, mixing well, adding HAuCl4 solution, adding ultrapure water, and then shaking the reaction at room temperature to obtain the CuS@SnS2-Au composite material, which is then refrigerated for later use.

6. The method according to claim 4, characterized in that, The signal probe CuS@SnS2-Au-mAb n The preparation method includes: placing CuS@SnS2-Au composite material into a centrifuge tube, adding a weakly alkaline solution to adjust the pH of the system to 6-7.5, shaking to mix, and then adding the primary antibody (first antibody) of the pathogenic bacteria to be tested. After shaking and reacting at room temperature for 30-60 min, bovine serum albumin (BSA) is added, and the mixture is shaken and reacted at room temperature for 1-1.5 h. After centrifugation, the supernatant is removed, and the remaining substances are reconstituted in buffer solution to obtain CuS@SnS2-Au-mAb. n That is, n kinds of photothermal signal probes; the signal probes are stored in a sealed container in solution or lyophilized powder form for later use.

7. The method according to claim 1, characterized in that, The test strip detection area is respectively dripped or sprayed with 5-20 mM PBS solution containing 0.5-10 mg / mL of the secondary primary antibody mAbn' of the pathogen to be tested; the C area is dripped or sprayed with 5-20 mM PBS solution containing 0.05-10 mg / mL of secondary antibody.

8. A photothermal lateral flow analysis device for simultaneously detecting multiple pathogenic bacteria, characterized in that, The device comprises four parts: a test strip, n kinds of photothermal signal probes, an 808 nm surface laser source, and a thermal detection device. The test strip includes a base plate, on which a sample pad, an NC membrane, and an absorbent pad are sequentially overlapped and pasted in a horizontal direction. The NC membrane includes a detection area, i.e., T. n The quality control area, also known as area C, has n≥2, where T n The region includes n independent T regions, each T region is fixed with a different secondary primary antibody against the pathogen to be tested, namely mAbn', and the C region is fixed with a secondary antibody; The 808 nm surface laser source is formed by using an 808 nm point laser through a surface diverging laser beam expander, and the spot size can be adjusted to simultaneously cover all T. n district; The thermal detection device refers to a thermal imaging or temperature measurement device; The photothermal signal probe is a CuS@SnS2-Au composite material, which is reacted with n antibodies against pathogenic bacteria (i.e., primary antibody mAbs). n The complex formed after compounding with bovine serum albumin is CuS@SnS2-Au-mAb. n .

9. The apparatus according to claim 8, characterized in that, The thermal imaging or temperature measurement device includes, but is not limited to, any one of the following: mobile phone infrared thermal imaging analysis accessories, infrared thermal imager, handheld infrared thermal imaging analyzer, or infrared thermal imaging temperature gun. The thermal imaging or temperature measurement device acquires photothermal imaging photos and outputs and displays them through a connected smart display terminal, which includes a computer or smartphone.

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