A photothermal lateral flow assay device for simultaneously detecting multiple sulfonamide antibiotic resistance genes and application thereof

Abstract

The application discloses a photothermal lateral flow analysis device for simultaneously detecting multiple sulfonamide antibiotic resistance genes and application, the device comprises a test strip, n photothermal capture probes, a surface laser light source and a thermal detection device, n photothermal capture probes are prepared by respectively adsorbing n resistance gene capture sequences and quality control probe complementary sequences to be detected on the surface of Fe3O4-PDA-Au composite material, and n is greater than or equal to 2; n detection zones on the test strip are sequentially fixed with n detection probes of the resistance genes to be detected, and a quality control probe is fixed on a quality control zone. The n photothermal capture probes, a sample liquid to be detected and a buffer are mixed, the sample pad end of the test strip is inserted into the mixed solution for chromatography, the whole detection zone is irradiated by the surface laser, and the photothermal temperature of the detection zone is collected by a thermal imaging or temperature measuring device, working curves are established according to the temperature and the concentration of the n resistance genes to be detected, and photothermal qualitative and quantitative analysis of the n target substances is carried out.

Description

Technical Field

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

[0002] Antibiotics, used as medicines and feed additives for a long time, have had a significant impact on human society. The long-term overuse of antibiotics in livestock farming has led to the induction of antibiotic-resistant bacteria in the intestines of farmed animals. Antibiotics then enter the environment through daily life, medical treatment, and livestock farming activities, inducing antibiotic resistance in environmental microorganisms. During this process, antibiotic resistance genes continuously accumulate. Furthermore, antibiotic resistance genes can transfer between non-pathogens, pathogens, and even distantly related organisms, and migrate and spread within the environment. Therefore, antibiotic resistance genes not only pollute the environment but also threaten human health. Thus, detecting antibiotic resistance genes is crucial for protecting the ecological environment and human health.

[0003] Sideflow chromatography test strips are characterized by short detection time, good selectivity, low cost, small sample volume requirements, ease of large-scale production, and stable long-term storage. They are widely used in the design of portable analytical sensors and are increasingly being applied in the field of nucleic acid detection. Sideflow analysis technology based on photothermal effect signal amplification relies on laser-excited plasma nanomaterials. When the excitation light matches the surface plasma resonance frequency of the nanoparticles, the particles absorb light energy and release it as heat, resulting in a temperature change and signal amplification. This method, which reflects analyte concentration changes through temperature variations, has a low background signal and high sensitivity, and has been successfully applied to the detection of various targets, including nucleic acids.

[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 for the quantitative detection of ochratoxin A (OTA) based on multifunctional photothermal nanoparticles Fe3O4@Au (Talanta 2021,222,121478). Zhang et al. developed a photothermal lateral flow immunoassay method for the detection of deoxynivalenol (DON) using flower-like gold nanoparticles deposited as manganese dioxide nanocarriers as photothermal materials (Food Chemistry 2021,341,128231).

[0005] However, existing photothermal lateral flow assays cannot achieve simultaneous detection of multiple analytes because they all use point lasers as the light source, 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 must be irradiated 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. Furthermore, it is difficult to control the excitation conditions uniformly when conducting batch tests. Therefore, achieving simultaneous detection of multiple analytes in photothermal lateral flow assays remains a challenge and bottleneck in the field. Summary of the Invention

[0006] Technical issues: Existing nucleic acid photothermal test strips have the following limitations: 1. The light source is a point laser, resulting in a concentrated light spot with a small area (5*8mm). 2 First, it can only illuminate a limited area on the test strip, and each test can only detect a single target in a single sample. Second, the data collection and analysis of a single target takes 10-20 minutes, and the analysis time for multiple targets is the sum of the above times, so the detection efficiency needs to be improved.

[0007] Improving analytical throughput and detection efficiency are crucial issues in the development of timely detection technologies and represent a major direction for current analytical chemistry development. Exploring and developing photothermal lateral flow analytical methods for the simultaneous detection of multiple sulfonamide antibiotic resistance genes from a technical or instrumental perspective has significant scientific and practical implications.

[0008] Technical solution:

[0009] On the one hand, the present invention provides a method for simultaneously detecting multiple sulfonamide antibiotic resistance genes, the specific steps of which include:

[0010] (1) A photothermal lateral flow analysis device for simultaneously detecting multiple sulfonamide antibiotic resistance genes was constructed, specifically comprising four parts: preparing test strips, preparing n kinds of photothermal capture probes, constructing an 808nm 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 absorption pad are sequentially overlapped in the horizontal direction. The NC membrane includes a detection area and a quality control area, i.e., area C. The detection area of ​​the test strip includes n independent T areas, each T area having a different sulfonamide antibiotic resistance gene detection probe fixed thereon, denoted as T0. n The test strip has a C region with n≥2; a corresponding quality control probe is fixed in region C; the photothermal capture probe is made of Fe3O4-PDA-Au composite material with complementary sequences and capture sequences of the quality control probe; 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;

[0011] (2) Mix n kinds of photothermal capture probes, the sample solution to be tested and the buffer solution to form a mixture, and insert the sample pad end of the test strip into the mixture for chromatography.

[0012] (3) n photothermal capture probes migrate through the detection area due to chromatography and are captured by their corresponding T n In the detection zone, photothermal capture probes that have already bound to the corresponding resistance gene in the sample will not be bound by the detection probes in the T zone, but will flow directly through the detection zone and be bound in the quality control zone. Photothermal capture probes that have not bound to the resistance gene in the sample will be bound by the detection probes in the T zone. The detection zone is irradiated with an 808nm surface laser, and the photothermal temperature of the n photothermal capture probes in the detection zone is collected by thermal imaging or temperature measurement equipment for qualitative and / or quantitative analysis.

[0013] As an optional implementation method, the qualitative analysis refers to, based on T n Whether the corresponding T region in the region appears dark purple or whether there are changes in photothermal temperature determines whether the sample solution contains sulfonamide antibiotic resistance genes; the quantitative analysis refers to establishing working curves based on photothermal temperature and the concentration of n sulfonamide antibiotic resistance genes to perform quantitative analysis of n target substances.

[0014] In this application, the photothermal temperature change in each detection zone is negatively correlated with the content of the corresponding sulfonamide antibiotic resistance gene, specifically:

[0015] When the sample does not contain the sulfonamide antibiotic resistance gene to be tested, T n The area appears deep purple and has a high light and heat temperature;

[0016] When a sample contains a sulfonamide antibiotic resistance gene, the corresponding T... n The region exhibits a light, dark purple or even colorless appearance and has low light and heat temperature.

[0017] The quality control area, which serves as a reference for verifying the validity of the test strip results, always appears dark purple.

[0018] As an optional implementation, the sulfonamide antibiotic resistance gene to be tested is a gene fragment that can make bacteria resistant to antibiotics and is free in the environment or present in cells, including but not limited to at least two of the sul1 gene, sul2 gene, and sul3 gene.

[0019] As an optional implementation, the photothermal capture probe is a complex of Fe3O4-PDA-Au photothermal nanomaterial and complementary sequences of the capture sequence and the quality control probe, specifically a quality control probe complementary sequence-Fe3O4-PDA-Au composite material-capture sequence. This is a complex formed by the simultaneous adsorption of the quality control probe complementary sequence and one of n capture sequences by the Fe3O4-PDA-Au photothermal nanomaterial. The number of photothermal capture probes corresponds to the number of genes to be tested. The capture sequence is a nucleic acid sequence partially complementary to the sulfonamide antibiotic resistance gene to be tested, and the quality control probe complementary sequence is a nucleic acid sequence completely complementary to the quality control probe. The PDA is polydopamine.

[0020] As an optional implementation, the detection sequence for the sulfonamide antibiotic resistance gene is a streptavidin-modified single-stranded nucleic acid that is partially identical to the target gene and completely complementary to the capture sequence. Streptavidin endows the detection sequence with the ability to be immobilized on an NC membrane, and the detection probe is used to bind to the photothermal capture probe.

[0021] As an optional implementation, the quality control probe for the sulfonamide antibiotic resistance gene to be tested is a mixture of streptavidin and a quality control probe coupled together. The streptavidin gives the quality control probe the ability to be immobilized on the NC membrane, and the quality control probe is used to capture excess photothermal capture probes.

[0022] As an optional implementation, the sulfonamide antibiotic resistance gene capture sequence to be tested is a single-stranded nucleic acid sequence complementary to the sulfonamide antibiotic resistance gene to be tested.

[0023] As an optional implementation, the detection probe for the sulfonamide antibiotic resistance gene is a segment of nucleic acid sequence identical to the sulfonamide antibiotic resistance gene to be tested.

[0024] As an optional implementation, the quality control probe complement and the quality control probe are a pair of complementary nucleic acid sequences that are unrelated to the nucleic acid sequence of the sulfonamide antibiotic resistance gene to be tested.

[0025] The photothermal detection device includes an 808nm excitation source and a surface-diverging laser beam expander. The surface-diverging laser beam expander used in this application serves as an optical accessory for the 808nm excitation source, reducing the spot size from the original 5*8mm. 2 The laser spot area is enlarged by 1.5 to 30 times, thereby expanding the laser coverage area and enabling photothermal lateral flow chromatography determination of multiple targets.

[0026] As an optional implementation, the detection device of the photothermal lateral flow analysis apparatus for multiple targets is implemented by combining an 808nm excitation source with a surface-diverging laser beam expander. Specifically, a surface-diverging laser beam expander is added between the laser source and the lateral flow chromatography test paper, with the expander positioned close to the laser probe. This optical accessory can expand the excitation area of ​​the point source by 1.5-30 times. As an example, when the expansion factor is 2.5 times, a 5*8mm... 2 The original light spot was enlarged to 12.5*20mm. 2 At this time, the light spot can cover 3 T regions, and 808nm laser excitation is performed on these 3 regions simultaneously, which provides support for expanding the area of ​​the laser source in this invention.

[0027] As an optional implementation, the detection principle of the photothermal side-flow chromatography device is as follows: Fe3O4-PDA-Au composite nanoparticles have photothermal conversion capabilities. Under the excitation of an 808nm laser, a local surface plasmon resonance effect is induced. A portion of this effect causes the Fe3O4-PDA-Au composite material to heat up through non-radiative attenuation. The temperature change of the composite material is linearly related to the concentration of the target analyte within a certain range, thereby achieving quantitative detection.

[0028] On the other hand, a method for preparing a photothermal lateral flow chromatography device for detecting sulfonamide antibiotic resistance genes is provided, wherein the preparation method of the device includes:

[0029] (1) Preparation of n photothermal capture probes: One Fe3O4-PDA-Au composite nanoparticle is used to adsorb a capture sequence of a sulfonamide antibiotic resistance gene to be tested and a complementary sequence of a quality control probe, respectively, thus preparing a photothermal capture probe of the sulfonamide antibiotic resistance gene to be tested. The probe is stored in a sealed container in solution for later use. The n photothermal capture probes of the sulfonamide antibiotic resistance gene to be tested can be prepared by repeating the process n times using the n capture sequences of the sulfonamide antibiotic resistance gene to be tested.

[0030] (2) Constructing the test strip: T n n different types of sulfonamide antibiotic resistance gene detection probes were sequentially fixed in zone C, and quality control probes were fixed in zone C. After drying, they were stored in a vacuum bag for later use.

[0031] As an optional implementation method, the specific method for preparing the Fe3O4-PDA-Au composite material is as follows: dilute the Fe3O4-PDA nanocrystal dispersion, take the diluted Fe3O4-PDA 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 the Fe3O4-PDA-Au composite material, which is then refrigerated for later use.

[0032] As an optional implementation, the specific method for preparing the photothermal capture probe, i.e., the quality control probe complementary sequence-Fe3O4-PDA-Au composite material-capture sequence, is as follows: Place the Fe3O4-PDA-Au composite material in a centrifuge tube, add a weakly alkaline buffer to adjust the pH of the system to 6-7.5, vortex to mix, and then add equal concentrations of the quality control probe complementary sequence and one capture sequence. After vortexing and reacting at room temperature for 30-60 min, add salmon sperm DNA for blocking, and vortex and react at room temperature for 1-1.5 h. After centrifugation, remove the supernatant, and then reconstitute the remaining material in the buffer to obtain the quality control probe complementary sequence-Fe3O4-PDA-Au composite material-capture sequence, which is then refrigerated for later use. Photothermal capture probes for all n required test genes are prepared using the same method.

[0033] As an optional implementation, the buffer solution includes any one of phosphate buffer, borate buffer, and carbonate buffer.

[0034] As an optional implementation, the detection probe is a nucleic acid sequence that is partially identical to the gene to be tested and completely complementary to the capture sequence, and the number of detection probes is consistent with the number of genes to be tested; the quality control probe is a nucleic acid sequence that is unrelated to the sulfonamide antibiotic resistance gene to be tested.

[0035] As an optional implementation, both the detection probe and the control probe are biotin-modified or contain oligonucleotides to enable them to be immobilized on the test strip.

[0036] When the detection probe and the quality control probe are modified with biotin, the n T regions of the test strip, i.e., T... n n different sulfonamide antibiotic resistance gene detection probes are sequentially fixed in region C; quality control probes are fixed in region C.

[0037] As an optional implementation, the T n The test region T contains n different sulfonamide antibiotic resistance gene detection probes, which are sequentially immobilized. This includes sequentially adding or spraying streptavidin and buffer solutions containing the n different sulfonamide antibiotic resistance gene detection probes to the test region T. n Add or spray buffer solutions containing 0.5-5 mg / mL streptavidin and 0.1-2 mg / mL of the sulfonamide antibiotic resistance gene detection probe to the respective areas.

[0038] As an optional implementation, the immobilization of the quality control probe in region C includes adding or spraying a buffer solution containing streptavidin and the quality control probe in region C, specifically: adding or spraying a buffer solution containing 0.05-2.5 mg / mL streptavidin and 0.2-5 mg / mL quality control probe in region C.

[0039] On the one hand, this application provides a photothermal lateral flow chromatography device for simultaneously detecting multiple sulfonamide antibiotic resistance genes. The photothermal lateral flow chromatography device comprises four parts: a test strip, n kinds of photothermal capture probes, an 808nm surface laser light source, and a thermal detection device.

[0040] The test strip body includes a base plate, on which a sample pad, an NC membrane, and an absorbent pad are sequentially overlapped and pasted horizontally. The NC membrane is used to separate and detect analytes from other substances in the sample; the sample pad is used for sample loading; the absorbent pad absorbs excess liquid and provides propulsion for chromatography; and the base plate provides physical support for the test strip. The NC membrane includes a detection zone, 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 a interval of 3-5 mm. Each T region is fixed with a different detection probe for the resistance gene of the sulfonamide antibiotic to be tested; a quality control probe is fixed on the C region.

[0041] The 808nm surface laser source is formed by expanding an 808nm point laser beam through 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 surface laser beam. 2 The small spot size can be magnified 1.5-30 times. A surface laser source 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 It can simultaneously irradiate 3 T zones.

[0042] As an optional implementation, the photothermal capture probe comprises a Fe3O4-PDA-Au composite material, a capture sequence, and a quality control probe complementary sequence, i.e., a mixture of quality control probe complementary sequence-Fe3O4-PDA-Au composite material-target complementary sequence. The photothermal capture probe can specifically recognize the corresponding sulfonamide antibiotic resistance gene and can also be captured by the quality control probe on the quality control region; the PDA is polydopamine.

[0043] The thermal detection device refers to a thermal imaging or temperature measurement device, including but not limited to 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, including a computer or smartphone.

[0044] The detection principle of the photothermal lateral flow analysis device for detecting sulfonamide antibiotic resistance genes in this invention is explained below, taking three sulfonamide antibiotic resistance genes, sul1, sul2, and sul3, as examples: When the sample does not contain any of the three sulfonamide antibiotic resistance genes, the photothermal capture probe is activated by the corresponding three detection regions (T) on the NC membrane. n The samples were sequentially captured (regions), all exhibiting a deep dark purple color. Simultaneous irradiation of the three T regions with an 808nm surface laser resulted in a high photothermal temperature. When the sample contained one or more of the three sulfonamide antibiotic resistance genes, during incubation with the photothermal capture probe, the sulfonamide antibiotic resistance genes in the sample bound to a partially complementary sequence of the quality control probe—a Fe3O4-PDA-Au composite material—a capture sequence conjugate, competitively inhibiting the T regions. n The combination of the on-region detection probe and the photothermal capture probe leads to T n The reduced binding of the region to the quality control probe complementary sequence-Fe3O4-PDA-Au composite material-capture sequence conjugate results in a weaker purple color or even colorless appearance, and a lower photothermal temperature under 808nm surface laser irradiation. n The photothermal temperature change value of the zone is inversely proportional to the content of sulfonamide antibiotic resistance genes in the sample. Excessive photothermal capture probes will bind to the control probes on the control zone (Zone C), always showing a deep dark purple color, serving as a reference for verifying the validity of the test strip results.

[0045] Beneficial effects:

[0046] The detection method provided by this invention can simultaneously perform photothermal quantitative detection of multiple sulfonamide antibiotic resistance genes within 20 minutes in a single chromatography step, with higher sensitivity. Moreover, it expands the detection throughput compared to existing photothermal sideflow analysis methods without increasing the detection time. Attached Figure Description

[0047] Figure 1 This is a schematic diagram illustrating the structure and principle of the test strip of the present invention;

[0048] Figure 2 This is a schematic diagram of the detection device of the present invention;

[0049] Figure 3 This document describes the response of the test strip of the present invention to negative and positive samples, as well as its operating curve.

[0050] Figure 4 This shows the detection area and specificity testing of the test strip of the present invention. Detailed Implementation

[0051] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that 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.

[0052] The following embodiments of this application disclose the preparation and application of a photothermal lateral flow analysis device for simultaneously detecting multiple sulfonamide antibiotic resistance genes, including the following steps:

[0053] (1) Preparation of photothermal capture probes: n Fe3O4-PDA-Au composite nanoparticles adsorbed n corresponding sulfonamide antibiotic resistance gene capture sequences and quality control probe complementary sequences on their surfaces, and mixed with salmon sperm DNA to block excess binding sites, thus obtaining n photothermal capture probes.

[0054] (2) Assemble the test strip: In T n n different sulfonamide antibiotic resistance gene detection probes are sequentially fixed in the test area, and quality control probes are fixed in the quality control area;

[0055] (3) Mix n kinds of photothermal capture 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.

[0056] (4) Irradiate T with an 808nm surface laser n In the zone, temperature is collected using thermal imaging or temperature measurement equipment. Working curves are established based on the temperature and the concentration of resistance genes of n sulfonamide antibiotics to be tested, and quantitative analysis of n target substances is performed.

[0057] This invention enables simultaneous photothermal quantitative analysis of multiple sulfonamide antibiotic resistance genes. Based on the number of T-regions, the magnification 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 examples use the sul1, sul2, and sul3 genes as examples of sulfonamide antibiotic resistance genes to provide a thorough and detailed explanation of the preparation and application methods of the entire device.

[0058] Example 1:

[0059] The preparation and plotting of the working curve of a photothermal lateral flow analyzer for simultaneously detecting multiple sulfonamide antibiotic resistance genes are as follows:

[0060] 1. Preparation of test paper materials

[0061] 1.1 Preparation of Fe3O4-PDA nanocrystals

[0062] Under nitrogen protection, FeCl3·6H2O (0.8 g) and FeCl2·4H2O (0.45 g) were dissolved in 10 mL of deionized water, with 2.5 mL of ammonia (28% w / v) added. The resulting solutions were stirred at room temperature for 10 min. Then, 2.2 g of sodium citrate was added to the mixture under constant mechanical stirring at 90 °C, producing Fe3O4 nanoparticles coated with citrate. The solution was yellowish-brown and collected using a permanent magnet. 3 mg of dopamine hydrochloride was added to 25 mL of 10 mM PBS (pH 8.5) containing 2.5 mg of Fe3O4 nanoparticles. After shaking at room temperature for 4 h, Fe3O4-PDA nanoparticles were obtained by centrifugation and washed three times with water. Relatively purified Fe3O4-PDA nanoparticles were obtained and stored at 4 °C for later use.

[0063] 1.2 Preparation of Fe3O4-PDA-Au composite materials

[0064] Dilute the Fe3O4-PDA dispersion with deionized water to a volume fraction of 1-2.5%. Take the diluted Fe3O4-PDA solution into a centrifuge tube, add ultrapure water, trisodium citrate solution (0.3 mol / L), and HAuCl4 solution (m / v = 1%), and then immediately place it on a vortex mixer and shake it at room temperature for 10 min to obtain the Fe3O4-PDA-Au composite nanomaterial. Store it in a refrigerator at 4℃ for later use.

[0065] 1.3Fe3O4-PDA-Au adsorbs and binds to the complementary and trap sequences of the quality control probe.

[0066] Take 1 mL of Fe3O4-PDA-Au composite material solution 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, vortex to mix, then add 2.5 μL of 1 mg / mL sul1 capture sequence, sul2 capture sequence, sul3 capture sequence and 8 μL of 1 mg / mL quality control probe complementary sequence respectively, mix well and react at 37 degrees Celsius (150 rpm) for 0.5-1 h. The capture sequence of the sul1 gene is: 5'-SH-(CH2)6-GCATAGCGCTGG-3'; the capture sequence of the sul2 gene is: 5'-SH-(CH2)6-GCCTCGCGCCGA-3'; the capture sequence of the sul3 gene is: 5'-SH-(CH2)6-ACACCAGCCTCA-3'; the quality control probe complementary sequence is: 5'-SH-(CH2)6-AAAAAAAAAA-3'. After the reaction, 100 μL of salmon sperm DNA was added for blocking for 0.5–1 h (150 rpm). After centrifugation at 12,000 r / min for 30 min, the supernatant was removed and the sample was redissolved in 100 μL of buffer (20 mmol / L Na3PO4, 5% BSA, 0.25% Tween-20, 10% sucrose) to obtain three photothermal capture probes for the sulfonamide antibiotic resistance genes to be tested, which were labeled as T1, T2, and T3, respectively, and stored in a 4°C refrigerator for later use.

[0067] 1.4 Detection Area (T) n Preparation of solutions for the quality control zone (C zone) and the quality control zone (C zone)

[0068] The biotin-modified sul1, sul2, sul3, and control probes were diluted to 1 mM using 1×TBE solution. The detection probe for the sul1 gene was 5'-biotin-GCATAGCGCTGGGTT-3'; the detection probe for the sul2 gene was 5'-biotin-GCCTCGCGCCGATCT-3'; the detection probe for the sul3 gene was 5'-biotin-ACACCAGCCTCAACT-3'; and the control probe was 5'-biotin-TTTTTTTTTT-3'. Add 300 μL PBS and 600 μL 1.67 mg / mL streptavidin to 100 μL of sul1 detection probe solution, sul2 detection probe solution, sul3 detection probe solution, and quality control probe solution, respectively, and incubate at room temperature for 1 h. Transfer the alkaline solution to an ultrafiltration tube with a molecular weight cutoff of 25 Kd, centrifuge at 8000 rpm for 10 min to remove unbound DNA. Wash the conjugate three times with PBS, and finally adjust the volume to 1 mL to obtain the detection zone (T). nThe solutions are divided into the control zone (C zone) and the quality control zone (C zone).

[0069] 2. Preparation of test strips

[0070] according to Figure 1 The membrane assembly method involves attaching the NC membrane to the center of the base plate, with the sample pad and absorbent pad overlapping the left and right ends of the NC membrane, respectively, covering it by approximately 2mm. The assembled test strip is then cut into 3mm wide strips to obtain blank test strips. n different detection zone solutions are applied to the detection zone T at one end near the sample pad. n Zone C: Apply the control zone solution to one end near the absorbent pad. Place the spotted test strip in an oven and dry at 37°C for 60 minutes. Store in a vacuum bag for later use.

[0071] 3. Sample pretreatment

[0072] Use 10 mM PBS solution (pH 7.4) as the negative test solution. Prepare 1 mg / mL standard solutions of sul1, sul2, and sul3 genes using PBS solution (pH 7.4). Dilute the standard solutions of the antibiotic resistance genes to be tested to concentrations of 0.2 ng / mL, 1 ng / mL, 5 ng / mL, 25 ng / mL, and 125 ng / mL using 10 mM PBS solution (pH 7.4).

[0073] 4. Drawing the working curve

[0074] Take 20 μL of each of the above-mentioned serially diluted antibiotic resistance gene assays, for a total of 60 μL, and place them in centrifuge tubes. Then, take 5 μL of each of the three photothermal probes, for a total of 15 μL, and add them to the centrifuge tubes. Add 15 μL of running buffer to the centrifuge tubes and mix for 10 min. Then, insert the test strip into the centrifuge tubes for chromatography. After chromatography, use... Figure 2 The detection method shown uses an 808nm surface laser (power 1.96W / cm²). 2 Irradiate for 5 minutes, and use infrared thermal imaging accessories to monitor the temperature changes in detection areas T1, T2, and T3.

[0075] According to the detection area T n The temperature change, i.e., the photothermal temperature ΔT, was used to linearly fit the standard concentration with the photothermal temperature ΔT, and the working curve was calculated. The obtained standard photothermal temperature ΔT data are recorded in Table 1, and the results show... Figure 3 (A), (B), Negative test strip T n The area all showed a distinct dark purple color, indicating high light and heat temperature; the positive test strip T n The area appears light purple or even colorless, and has a low temperature due to light and heat.

[0076] The working curves of the sul1, sul2, and sul3 genes are as follows: Figure 3 As shown in (C), (D), and (E).

[0077] The working curve of the sul1 gene is: Y1 = -3.11 × X1 + 16.23, where Y1 is the photothermal temperature of the T1 region (in °C) and X1 is lg (sul1 gene concentration) (in ng / mL);

[0078] The working curve of the sul2 gene is: Y2=-3.45×X2+16.89, where Y2 is the photothermal temperature of the T2 region (in °C) and X2 is lg (sul2 gene concentration) (in ng / mL);

[0079] The working curve of the sul3 gene is: Y3 = -3.13 × X3 + 16.01, where Y3 is the photothermal temperature of the T3 region (in °C) and X3 is lg (sul3 gene concentration) (in ng / mL).

[0080] Table 1 - Photothermal Temperatures Corresponding to Standard Concentration Samples

[0081]

[0082] Example 2:

[0083] A photothermal lateral flow analysis device for simultaneously detecting multiple sulfonamide antibiotic resistance genes is used to verify the specificity of multiple sulfonamide antibiotic resistance genes, comprising the following steps:

[0084] 1. Preparation of test paper materials

[0085] Same as Example 1

[0086] 2. Preparation of test strips

[0087] Same as Example 1

[0088] 3. Sample pretreatment

[0089] Use 10mM PBS solution (pH 7.4) as the negative test solution.

[0090] 4. Specificity verification

[0091] Add 72 μL of negative test solution to each of three centrifuge tubes, then add 9 μL of sul1 photothermal capture probe, sul2 photothermal capture probe, and sul3 photothermal capture probe (denoted as T1, T2, and T3, respectively). 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, irradiate with an 808 nm laser equipped with a planar diverging laser beam expander for 5 min, and monitor the detection area T using an infrared thermal imaging accessory. n Temperature changes, results show as follows Figure 4 .

[0092] like Figure 4 As shown, the detection areas of the sul1 photothermal capture probe chromatography strip, except for the sul1 detection area, are all relatively light and have a low photothermal temperature; the detection areas of the sul2 photothermal capture probe chromatography strip, except for the sul2 detection area, are all relatively light and have a low photothermal temperature; the detection areas of the sul3 photothermal capture probe chromatography strip, except for the sul3 detection area, are all relatively light and have a low photothermal temperature; the specific recognition results between the probes are good.

[0093] Example 3:

[0094] A photothermal lateral flow analyzer for simultaneously detecting multiple sulfonamide antibiotic resistance genes was applied to actual sample analysis, comprising the following steps:

[0095] 1. Preparation of test paper materials

[0096] Same as Example 1

[0097] 2. Preparation of test strips

[0098] Same as Example 1

[0099] 3. Sample pretreatment

[0100] Take 1-5 ml (less than 1.0 × 10⁻⁶) of culture medium containing methicillin-resistant Staphylococcus aureus (containing the sul1, sul2, and sul3 genes). 9 Centrifuge the bacteria (10000 rpm, 11500 × g) for 1 min and discard the culture medium. Extract total genomic DNA from the colonies using a commercial DNA extraction kit and use it as the test solution.

[0101] 4. Drawing the working curve

[0102] Same as Example 1

[0103] 5. Sample testing

[0104] Add 72 μL of the sample solution to each centrifuge tube, then add 9 μL of each of the following photothermal capture probes: sul1, sul2, and sul3. 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 planar diverging laser beam expander. 2 Irradiate for 5 minutes, and monitor the detection area T using infrared thermal imaging accessories. n Temperature changes.

[0105] 6. Data Processing

[0106] The test results are as follows.

[0107] (1) The three detection areas of the test strip of the sample are light in color. The qualitative analysis results can be obtained first, indicating that it contains three sulfonamide antibiotic resistance genes: sul1, sul2, and sul3.

[0108] (2) Furthermore, by substituting the photothermal temperature change data into the working curve obtained in step 4 above, quantitative analysis can be further performed to determine the specific concentrations of the three sulfonamide antibiotic resistance genes, sul1, sul2, and sul3. The specific data are as follows:

[0109] The photothermal temperature value ΔT of the sample measured in region T1 is 10.53. Substituting... Figure 3 The concentration of the sul1 gene in the sample can be calculated to be 70.98 ng / mL from the working curve shown in (C);

[0110] The photothermal temperature value ΔT of the sample measured in region T2 is 13.04. Substituting... Figure 3 The concentration of the sul2 gene in the sample can be calculated to be 12.52 ng / mL from the working curve shown in (D);

[0111] The photothermal temperature value ΔT of the sample measured in region T3 is 12.98. Substituting... Figure 3 The concentration of the sul3 gene in the sample can be calculated to be 12.77 ng / mL from the working curve shown in (E).

[0112] The results of this embodiment show that the test sample containing the unknown component contains 70.98 ng / mL sul1 gene, 12.52 ng / mL sul2 gene, and 12.77 ng / mL sul3 gene.

[0113] 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 simultaneously detecting multiple sulfonamide antibiotic resistance genes, characterized in that, The method for simultaneously detecting multiple sulfonamide antibiotic resistance genes using a photothermal lateral flow analyzer includes the following steps: (1) Construct a photothermal lateral flow analysis device for the simultaneous detection of multiple sulfonamide antibiotic resistance genes, specifically comprising four parts: preparation of test strips, preparation of n kinds of photothermal capture probes, construction of an 808nm surface laser source, and thermal detection devices. 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., area C. The test strip comprises n independent T regions, each T region having a detection probe for a different sulfonamide antibiotic resistance gene immobilized on it, denoted as T. n For a region n≥2, A quality control probe is fixed in area C of the test strip; The photothermal capture probe is a complex of Fe3O4-PDA-Au photothermal nanomaterials and a capture sequence and a quality control probe complementary sequence; the photothermal capture probe is a quality control probe complementary sequence-Fe3O4-PDA-Au composite material-capture sequence, which is a complex formed by Fe3O4-PDA-Au photothermal nanomaterials simultaneously adsorbing a quality control probe complementary sequence and one of n capture sequences; the capture sequence is a nucleic acid sequence partially complementary to the sulfonamide antibiotic resistance gene to be tested, and the quality control probe complementary sequence is a nucleic acid sequence completely complementary to the quality control probe; The 808nm surface laser source is formed by expanding an 808nm point laser using a surface-diverging laser beam expander. The surface-diverging laser beam expander serves as an optical accessory for the 808nm point laser, enlarging a small spot size by 1.5 to 30 times. By adjusting the spot size, it can simultaneously cover all T... n district; The thermal detection device includes thermal imaging or temperature measurement equipment, which includes, but is not limited to, any one of 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, which includes a computer or a smartphone. (2) Mix n kinds of photothermal capture probes, the sample solution to be tested, and the buffer solution to form a mixture, and insert the sample pad end of the test strip into the mixture for chromatography; (3) n photothermal capture probes migrate through the detection area due to chromatography 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 capture probes in the region, using thermal imaging or temperature measurement equipment to simultaneously collect T n The photothermal temperatures of n photothermal capture probes in the region are analyzed qualitatively and / or quantitatively.

2. The method according to claim 1, characterized in that, In step (3), the detection area is T n The changes in light, heat, and temperature in the region were negatively correlated with the content of the corresponding sulfonamide antibiotic resistance genes, specifically: When the sample does not contain the sulfonamide antibiotic resistance gene, T n The corresponding T region in the area appears dark purple and has a high light and heat temperature; When a sample contains a gene for resistance to a certain sulfonamide antibiotic, T n The corresponding T region is light purple or even colorless, and has a low light and heat temperature. 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 sulfonamide antibiotic resistance genes to be tested are gene fragments that can make bacteria resistant to antibiotics and are free in the environment or exist in cells, including but not limited to at least two of the sul1, sul2, and sul3 genes.

4. The method according to claim 1, characterized in that, The preparation method of the Fe3O4-PDA-Au composite material includes: diluting the Fe3O4-PDA nanocrystal dispersion, taking the diluted Fe3O4-PDA 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 Fe3O4-PDA-Au composite material, which is then refrigerated for later use.

5. The method according to claim 1, characterized in that, The specific method for preparing the photothermal capture probe, namely the quality control probe complementary sequence-Fe3O4-PDA-Au composite material-capture sequence, is as follows: Fe3O4-PDA-Au composite material is placed in a centrifuge tube, and a weakly alkaline buffer solution is added to adjust the pH of the system to 6-7.

5. After vortexing and mixing, equal concentrations of the quality control probe complementary sequence and one capture sequence are added. After vortexing and reacting at room temperature for 30-60 min, salmon sperm DNA is added for blocking. The reaction is carried out 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 the quality control probe complementary sequence-Fe3O4-PDA-Au composite material-capture sequence, which is then refrigerated for later use.

6. The method according to claim 1, characterized in that, The detection probe is a nucleic acid sequence that is partially identical to the gene to be tested and completely complementary to the capture sequence, and the number of detection probes is consistent with the number of genes to be tested; the quality control probe is a nucleic acid sequence that is unrelated to the sulfonamide antibiotic resistance gene to be tested.

7. The method according to claim 1, characterized in that, Both the detection probe and the quality control probe are biotin-modified or contain oligonucleotides to enable them to be immobilized on the test strip. When the detection probe and the quality control probe are modified with biotin, the n T regions of the test strip, i.e., T... n Streptavidin solution was added or sprayed onto each zone, followed by a detection probe solution with a concentration of 0.1-2 mg / mL; buffer solution containing streptavidin and a quality control probe of 0.2-5 mg / mL was added or sprayed onto zone C.

8. A photothermal lateral flow analysis device for simultaneously detecting multiple sulfonamide antibiotic resistance genes, characterized in that, The device comprises four parts: a test strip, n types of photothermal capture probes, an 808nm 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 immobilized with a different detection probe for the sulfonamide antibiotic resistance gene to be tested, and the C region is immobilized with a quality control probe; The 808nm surface laser source is formed by expanding an 808nm point laser using a surface-diverging laser beam expander. The surface-diverging laser beam expander serves as an optical accessory for the 808nm point laser, enlarging a small spot size by 1.5 to 30 times. By adjusting the spot size, it can simultaneously cover all T... n district; The thermal detection device refers to a thermal imaging or temperature measurement device; The photothermal capture probe is a complex formed by Fe3O4-PDA-Au composite nanoparticles, a quality control probe complementary sequence, and a capture sequence of one of the n sulfonamide antibiotic resistance genes to be tested, namely, quality control probe complementary sequence-Fe3O4-PDA-Au composite material-capture sequence, wherein the PDA is polydopamine.

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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