A salmonella typhimurium aptamer lateral flow chromatography test strip based on triiron tetroxide@colloidal gold composite nanomaterial photothermal effect, preparation method and application

Abstract

The application discloses a kind of salmonella typhimurium aptamer lateral flow chromatography test strip based on ferroferric oxide@colloidal gold composite nanomaterial photo-thermal effect, preparation method and application, belong to lateral flow chromatography test strip technical field, the preparation method method includes preparation probe;The preparation probe includes preparation Fe3O4@Au composite nanomaterial;The preparation Fe3O4@Au composite nanomaterial, under nitrogen protection, FeCl3·6H2O and FeCl2·4H2O are dissolved in deionized water, add ammonia water, stirring at room temperature, add trisodium citrate, with magnet collection, then added to boiling HAuCl4 solution, keep boiling, until the solution color changes to red brown, continue heating, cooling, with magnet collection;The test strip of the application can realize the quantitative detection of salmonella typhimurium.

Description

Technical Field

[0001] This invention relates to the field of lateral flow chromatography test strip technology, specifically to a Salmonella typhimurium aptamer lateral flow chromatography test strip based on the photothermal effect of ferric oxide@colloidal gold composite nanomaterials, its preparation method, and its application. Background Technology

[0002] Salmonella typhimurium is an important foodborne pathogen that causes acute gastroenteritis. This pathogen can cross the intestinal barrier, causing systemic infection, and globally, 150,000 people die annually from Salmonella typhimurium infection. Furthermore, food contamination caused by Salmonella typhimurium leads to lost productivity and food recalls, resulting in severe economic consequences for the food industry. Therefore, developing more sensitive and convenient methods for detecting Salmonella typhimurium in food is of great significance to ensuring food safety.

[0003] In recent years, photothermal test strips have emerged as a novel detection method for detecting pathogenic bacteria in food. Currently, the main nanomaterial used in photothermal immunoassay strips is colloidal gold (GNP) particles. However, the low photothermal conversion efficiency and relatively light color development of GNPs limit their detection sensitivity. Furthermore, most commercially available test strips are immunoassay strips, using antibodies as recognition molecules. Antibodies suffer from drawbacks such as high cost, low stability, and significant batch-to-batch variability.

[0004] Chinese patent CN113881790B discloses a magnetic Fe3O4@aptamer and its application in combination with fluorescent test strips for detecting foodborne pathogens. The preparation method of the magnetic Fe3O4@aptamer involves first preparing Fe3O4 powder, then washing the Fe3O4 powder with ultrapure water and resuspending it in ultrapure water. After adding the aptamer, the mixture is shaken at room temperature for 20-30 minutes, magnetic separation is performed, the supernatant is discarded, and the mixture is washed again with ultrapure water and resuspended in ultrapure water to obtain the final product. This patent utilizes PCR amplification technology and fluorescence signals to improve the sensitivity of detecting foodborne pathogens, making it suitable for detecting pathogens in food samples. However, the photothermal conversion efficiency of the magnetic Fe3O4@aptamer prepared in this patent is low. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a lateral flow chromatography test strip for Salmonella Typhimurium aptamers based on the photothermal effect of iron(III)O4@colloidal gold composite nanomaterials, its preparation method, and its application. The test strip's detection is based on the fact that the Fe3O4@Au content on the T-line of the test strip is proportional to the Salmonella Typhimurium concentration (the higher the Fe3O4@Au content, the higher the Salmonella Typhimurium concentration). By measuring the photothermal effect of the Fe3O4@Au material using a thermal imager, the Salmonella Typhimurium content can be deduced, thereby achieving quantitative detection of Salmonella Typhimurium.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing a Salmonella Typhimurium aptamer lateral flow chromatography test strip based on the photothermal effect of ferric oxide@colloidal gold composite nanomaterials includes: preparing the test strip, preparing the probe, preparing the T-line aptamer solution, preparing the C-line aptamer solution, and streaking.

[0008] To prepare the test strip, a nitrocellulose membrane is attached to a base plate, an absorbent pad is attached above the nitrocellulose membrane, and a sample pad is attached below the nitrocellulose membrane to obtain the test strip.

[0009] In the preparation of the test strip, the absorbent pad covers the nitrocellulose membrane for a length of 2 mm, and the sample pad covers the nitrocellulose membrane for a length of 2 mm.

[0010] The nitrocellulose membrane, model CN90, was purchased from Sartorius.

[0011] The base plate is model DB-6 and was purchased from Shanghai Jieyi Biotechnology Co., Ltd.

[0012] The sample pad was a Fusion3 model, purchased from Shanghai Jieyi Biotechnology Co., Ltd.

[0013] The absorbent pad is model H5073 and was purchased from Shanghai Jieyi Biotechnology Co., Ltd.

[0014] The base plate is 6cm wide, the sample pad is 2.4cm wide, the nitrocellulose membrane is 2.5cm wide, and the absorbent pad is 2cm wide.

[0015] The preparation of the probe includes: preparing Fe3O4@Au composite nanomaterials and synthesizing the probe;

[0016] The preparation of Fe3O4@Au composite nanomaterials involves dissolving FeCl3·6H2O and FeCl2·4H2O in deionized water under nitrogen protection, adding ammonia, stirring at room temperature, adding trisodium citrate at 88-92℃, and collecting the solution with a magnet to obtain citrate-coated Fe3O4•NPS; adding the citrate-coated Fe3O4•NPS to a boiling HAuCl4 solution, maintaining boiling until the solution turns reddish-brown, continuing heating, then stirring and cooling until room temperature, and collecting the solution with a magnet to obtain Fe3O4@Au composite nanomaterials.

[0017] In the preparation of Fe3O4@Au composite nanomaterials, the ratio of FeCl3·6H2O, FeCl2·4H2O, and deionized water is 1.62-1.63g:0.99-1g:18-22mL.

[0018] The ratio of FeCl3·6H2O to ammonia is 1.62-1.63 g: 5-6 mL;

[0019] The mass ratio of FeCl3·6H2O to trisodium citrate is 1.62-1.63:4.3-4.5;

[0020] The volume fraction of the ammonia solution is 27-28%.

[0021] Stirring time at room temperature is 9-11 minutes;

[0022] The volume ratio of citrate-coated Fe3O4•NPS to HAuCl4 solution was 10:18-22.

[0023] The concentration of the HAuCl4 solution is 0.17-0.18 mg / mL;

[0024] Continue heating for 9-11 minutes;

[0025] Furthermore, the obtained Fe3O4@Au composite nanomaterials were stored at 3-5℃ for later use.

[0026] The synthetic probe was prepared by collecting Fe3O4@Au composite nanomaterials with a magnet, diluting them with ultrapure water to obtain diluted composite nanomaterials, sonicating them, and adjusting the pH to 7.8-8.2 to obtain a Fe3O4@Au composite material solution. TCEP was added to a thiol-modified aptamer, and activated at 36-38°C in the dark to obtain an activated mixed aptamer solution. The activated mixed aptamer solution was mixed with the Fe3O4@Au composite material solution and incubated at room temperature until the thiol groups on the aptamer formed gold-sulfur bonds with the Au on the Fe3O4@Au composite material. Sodium chloride solution was then added, and the reaction was carried out at 4°C to obtain a mixed composite solution. The mixed composite solution sample was collected with a magnet and reconstituted with PEG-containing PBS buffer to obtain the Fe3O4@Au aptamer probe.

[0027] In the synthetic probe, the volume ratio of Fe3O4@Au composite nanomaterial to diluted composite nanomaterial is 98-102:98-102;

[0028] The volume ratio of the thiol-modified aptamer to TCEP was 7.4-7.6:1.9-2.1;

[0029] The volume ratio of Fe3O4@Au composite nanomaterials to thiol-modified aptamers is 98-102:7.4-7.6;

[0030] The volume ratio of Fe3O4@Au composite nanomaterials to sodium chloride solution is 98-102:1.9-2.1;

[0031] The volume ratio of the mixed composite liquid sample to the Fe3O4@Au aptamer probe was 98-102:49-51;

[0032] K₂CO₃ is used to adjust the pH to 7.8-8.2;

[0033] The concentration of the thiol-modified aptamer is 19-21 μM;

[0034] The concentration of TCEP is 0.24-0.26%;

[0035] The concentration of the sodium chloride solution is 0.9-1.1 M;

[0036] The PEG in the PEG-containing PBS buffer is PEG-8000;

[0037] The PEG-containing PBS buffer has a PEG concentration of 0.9-1.1% and a pH of 7.3-7.5.

[0038] The ultrasonic treatment time is 18-22 minutes;

[0039] Activation at 36-38℃ in the dark takes 1-1.5 hours.

[0040] The incubation time at room temperature is 3-3.5 hours;

[0041] The reaction time at 4°C is overnight;

[0042] The sequence of the aptamer in the thiol-modified aptamer is shown in SEQ ID No. 1 of the sequence listing;

[0043] The Fe3O4@Au aptamer probe was used to detect the mixture with Salmonella typhimurium solution.

[0044] The preparation of the T-line aptamer solution involves adding streptavidin solution to a biotin-modified aptamer and reacting at room temperature to obtain a T-line aptamer solution with a streptavidin concentration of 6 mg / mL and an aptamer concentration of 110 μM.

[0045] In the preparation of the T-line aptamer solution, the sequence of the aptamer in the biotin-modified aptamer is shown in SEQ ID No. 1 of the sequence listing;

[0046] The reaction time at room temperature is 40-50 minutes;

[0047] The preparation of the C-line aptamer solution involves adding streptavidin solution to cDNA complementary to the aptamer and reacting at room temperature to obtain a C-line aptamer solution with a streptavidin concentration of 1 mg / mL and a cDNA concentration of 30 μM complementary to the aptamer.

[0048] The sequence of the cDNA complementary to the aptamer in the preparation of the C-line aptamer solution is shown in SEQ ID No. 2 of the sequence listing;

[0049] The reaction time at room temperature is 40-50 minutes;

[0050] The line marking process involves using a film marking instrument to draw T-line and C-line aptamer solutions on the test strip, then drying at 37°C and cutting into strips to obtain a Salmonella typhimurium aptamer lateral flow chromatography test strip based on the photothermal effect of iron oxide@colloidal gold composite nanomaterials.

[0051] In the scribed lines, the distance between line T and line C is 3.9-4.1 mm;

[0052] The drying time at 37℃ is 1-1.5 hours;

[0053] The width of the test strip is 3.9-4.1 mm.

[0054] A lateral flow chromatography test strip for Salmonella typhimurium aptamers based on the photothermal effect of iron oxide@colloidal gold composite nanomaterials, prepared by the aforementioned method.

[0055] An application of a Salmonella Typhimurium aptamer lateral flow chromatography test strip prepared by the aforementioned method, based on the photothermal effect of iron(III) oxide@colloidal gold composite nanomaterials, involves mixing 69-71 μL of the test solution with 9.8-10.2 μL of FFe3O4@Au aptamer probe, reacting on a shaker, collecting the solution with a magnet, removing the solution, reconstituted with PBS, and adding the solution to the sample pad area of ​​the test strip. After 9-11 min, 39.5-40.5 μL of a mixture of H2O2 and TMB is added to the nitrocellulose membrane of the test strip. The reaction is observed for 9-11 min, and the color development is observed. After drying, the initial temperature of the T-line is measured using a thermal imager. Then, the strip is irradiated with a laser generator with an irradiation power of 0.9-1.1 W for 9.8-10.2 s, and the final temperature of the T-line is measured using a thermal imager. The temperature difference is calculated, substituted into the standard curve equation, and the concentration of Salmonella Typhimurium in the test solution is calculated.

[0056] In this application, the reaction time of the shaker is 28-32 minutes;

[0057] The H2O2 and TMB mixed liquid is a mixture of H2O2 aqueous solution with a concentration of 29.5-30.5% and TMB colorimetric solution, wherein the volume ratio of H2O2 aqueous solution to TMB colorimetric solution is 7.8-8.2:31-33;

[0058] The temperature difference = final temperature - initial temperature.

[0059] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0060] (1) The Salmonella typhimurium aptamer lateral flow chromatography test strip prepared by the present invention based on the photothermal effect of Fe3O4@Au composite nanomaterial has a stronger thermal effect than Fe3O4 and AuNPs alone.

[0061] (2) The Salmonella Typhimurium aptamer lateral flow chromatography test strip prepared by the present invention, based on the photothermal effect of Fe3O4@Au composite nanomaterials, establishes a standard curve between the temperature rise value and the concentration of Salmonella Typhimurium when using the test strip of the present invention for detection, by utilizing the synergistic photothermal effect of Fe3O4@Au composite nanomaterials. In actual detection, the temperature rise value generated during sample detection can be substituted into the standard curve to obtain the concentration of Salmonella Typhimurium in the sample. Based on the expected good specificity and sensitivity of this method, it can be used for the subsequent detection of actual food samples to meet the basic requirements of food safety detection.

[0062] (3) The Salmonella Typhimurium aptamer lateral flow chromatography test strip prepared by the present invention based on the photothermal effect of Fe3O4@Au composite nanomaterial has good photothermal conversion performance and peroxidase catalytic activity. When applied to test strip detection, it can significantly improve the signal intensity.

[0063] (4) The Salmonella typhimurium aptamer lateral flow chromatography test strip prepared by the present invention based on the photothermal effect of iron oxide@colloidal gold composite nanomaterial has a detection limit of 302 CFU / mL and strong specificity. Attached Figure Description

[0064] Figure 1 This is a comprehensive characterization diagram of the citrate-coated Fe3O4•NPS and Fe3O4@Au composite nanomaterials obtained in Example 1;

[0065] In the picture, Figure 1 A is an electron micrograph of citrate-coated Fe3O4•NPS. Figure 1 B is an electron micrograph of the Fe3O4@Au composite nanomaterial; Figure 1 C represents the particle size distribution of citrate-coated Fe3O4•NPS; Figure 1D is the particle size distribution diagram of the Fe3O4@Au composite nanomaterial; Figure 1 E represents the energy dispersive spectroscopy (EDS) analysis of the Fe3O4@Au composite nanomaterial. Figure 1 F is the EDS elemental analysis diagram of Fe3O4@Au composite nanomaterials;

[0066] Figure 2 This is a schematic diagram of the side-flow chromatography test strip for the Salmonella typhimurium aptamer prepared in Example 1.

[0067] In the picture, Figure 2 A represents the process of preparing the probe. Figure 2 B represents the process of mixing the probe with Salmonella typhimurium. Figure 2 C is a schematic diagram of the detection process and results;

[0068] Figure 2 In C, aptamer 1 is an aptamer that has not been modified with biotin, and aptamer 2 is an aptamer that has been modified with biotin (but does not contain biotin).

[0069] Figure 3 The temperature change curve of the Fe3O4@Au composite nanomaterial in Test Example 1 under laser irradiation;

[0070] Figure 4 The results of visual inspection and photothermal inspection are used to determine the specificity of the test in Example 1.

[0071] In the picture, Figure 4 A represents the visual detection result in the detection of specificity in Test Example 1; Figure 4 B represents the photothermal detection result in the detection specificity test of Test Example 1;

[0072] Figure 5 The results of the photothermal performance test of Fe3O4@Au composite nanomaterial and Fe3O4•NPS coated with citrate in Test Example 2;

[0073] In the picture, Figure 5 A is a comparison of the photothermal properties of Fe3O4@Au composite nanomaterials, citrate-coated Fe3O4•NPS, and H2O in Test Example 2; Figure 5 B is a graph showing the temperature rise of Fe3O4@Au composite nanomaterials at different concentrations; Figure 5 C represents the photothermal cycling curve of the Fe3O4@Au composite nanomaterial. Detailed Implementation

[0074] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0075] Example 1

[0076] A method for preparing a Salmonella Typhimurium aptamer lateral flow chromatography test strip based on the photothermal effect of ferric oxide@colloidal gold composite nanomaterials, specifically as follows:

[0077] 1. Preparation of test strips: Attach the nitrocellulose membrane to the base plate, attach an absorbent pad above the nitrocellulose membrane with a length of 2 mm covering the nitrocellulose membrane, and attach a sample pad below the nitrocellulose membrane with a length of 2 mm covering the nitrocellulose membrane to obtain the test strip;

[0078] The nitrocellulose membrane, model CN90, was purchased from Sartorius.

[0079] The base plate is model DB-6 and was purchased from Shanghai Jieyi Biotechnology Co., Ltd.

[0080] The sample pad was a Fusion3 model, purchased from Shanghai Jieyi Biotechnology Co., Ltd.

[0081] The absorbent pad is model H5073 and was purchased from Shanghai Jieyi Biotechnology Co., Ltd.

[0082] The base plate is 6cm wide, the sample pad is 2.4cm wide, the nitrocellulose membrane is 2.5cm wide, and the absorbent pad is 2cm wide.

[0083] 2. Probe preparation:

[0084] (1) Preparation of Fe3O4@Au composite nanomaterials: Under nitrogen protection, 1.622g FeCl3·6H2O and 0.994g FeCl2·4H2O were dissolved in 20mL of deionized water, and then 5mL of ammonia (volume fraction of 28%) was added. The resulting solution was stirred at room temperature for 10min, and 4.4g of trisodium citrate was added at 90℃. The solution was collected with a magnet to obtain Fe3O4•NPS coated with citrate.

[0085] Take 10 mL of citrate-coated Fe3O4•NPS (since it was not dried after being collected by the magnet, the obtained citrate-coated Fe3O4•NPS was in liquid form) and add it to 20 mL of boiling HAuCl4 solution with a concentration of 0.175 mg / mL. Keep boiling until the solution turns reddish-brown, then continue heating for 10 min. Then continue stirring and cooling until it cools to room temperature. Collect it with a magnet to obtain Fe3O4@Au composite nanomaterials (since it was not dried after being collected by the magnet, the obtained Fe3O4@Au was also in liquid form). Store the obtained Fe3O4@Au composite nanomaterials at 4 °C for later use.

[0086] The obtained citrate-coated Fe3O4•NPS and Fe3O4@Au composite nanomaterials were comprehensively characterized using thermal field emission electron microscopy (FET), observing their size, morphology, and dispersion. The particle size of the Fe3O4@Au composite nanomaterials was analyzed using nanoparticle size and Zeta potential analyzers. The results are shown below. Figure 1 .

[0087] (2) Probe synthesis: Take 100 μL of Fe3O4@Au composite nanomaterial, continue to collect it with a magnet, add ultrapure water to the total volume of 100 μL, sonicate for 20 min, add K2CO3 to adjust the pH to 8, and obtain Fe3O4@Au composite material solution; add 2 μL of 20 μM thiol-modified aptamer (see SEQ ID No. 1 in the sequence listing) to 7.5 μL. 0.25% TCEP was activated at 37°C in the dark for 1 hour to obtain an activated mixed aptamer solution. The activated mixed aptamer solution was mixed with the Fe3O4@Au composite nanomaterial and incubated at room temperature for 3 hours. After the thiol groups on the aptamer formed gold-sulfur bonds with the Au on the Fe3O4@Au composite nanomaterial, 2 μL of 1M sodium chloride solution was added and the reaction was carried out overnight at 4°C. 100 μL of the solution was collected with a magnet and reconstituted with 1% PEG-8000 PBS buffer (pH 7.4) to prepare 50 μL of Fe3O4@Au aptamer probe (the Fe3O4@Au aptamer probe was used to detect the Salmonella typhimurium solution).

[0088] 3. Preparation of T-line aptamer solution: Add streptavidin solution to biotin-modified aptamer (see SEQ ID No. 1 in the sequence listing) and react at room temperature for 40 min to obtain T-line aptamer solution with streptavidin concentration of 6 mg / mL and aptamer concentration of 110 μM;

[0089] 4. Preparation of C-line aptamer solution: Add streptavidin solution to cDNA complementary to the aptamer (see SEQ ID No. 2 in the sequence listing) (no thiolization required), react at room temperature for 40 min, and obtain a C-line aptamer solution with a streptavidin concentration of 1 mg / mL and a cDNA concentration of 30 μM complementary to the aptamer.

[0090] 5. Streaking: Using a streak scribing apparatus, streak the T-line aptamer solution and the C-line aptamer solution onto the test strip to draw the T-line and C-line, ensuring that the distance between the T-line and C-line is 4 mm. Then, dry the entire test strip at 37°C for 1 hour and cut it into 4 mm wide Salmonella Typhimurium aptamer lateral flow chromatography test strips based on the photothermal effect of ferric oxide@colloidal gold composite nanomaterials.

[0091] This embodiment provides a Salmonella Typhimurium aptamer lateral flow chromatography test strip prepared by the aforementioned preparation method.

[0092] In this embodiment, a Salmonella Typhimurium aptamer lateral flow chromatography test strip was prepared. Fe3O4@Au binds to the thiol-modified aptamer chain via gold-sulfur bonds to form a specific detection probe for Salmonella Typhimurium. Excess sites on the probe surface were sealed with polyethylene glycol-8000. A biotin-modified aptamer, linked to streptavidin, was sprayed onto the surface of a nitrocellulose membrane to form the T-line of the test strip.

[0093] Test Example 1

[0094] When the Salmonella Typhimurium aptamer lateral flow chromatography test strip prepared in Example 1 was applied to the detection of Salmonella Typhimurium, the Salmonella Typhimurium aptamer lateral flow chromatography test strip, a laser generator, and a thermal imager were used. The schematic diagram is shown below. Figure 2 In this application, by adjusting the angle between the laser generator probe and the T-line of the side-flow chromatography test strip, the specific wavelength laser emitted by the laser generator is ensured to irradiate the T-line of the side-flow chromatography test strip, thereby achieving photothermal signal conversion of the Fe3O4@Au nanomaterial. A thermal imager is then used to record the temperature change. The detection principle is that after Fe3O4@Au-Apt specifically captures Salmonella typhimurium, it forms a sandwich structure. The catalytic effect of Fe3O4@Au on H2O2 generates reactive oxygen species, which then oxidize the TMB chromogenic solution to generate a chromogenic product, amplifying the chromogenic signal. The dried test strip is then irradiated with a laser, and the thermal imager measures and records the temperature difference before and after irradiation of the T-line of the test strip, performing photothermal detection to improve detection sensitivity and achieve quantitative detection.

[0095] When performing laser irradiation, it is necessary to determine the laser irradiation time. The method used to determine this time is to drop the Fe3O4@Au composite nanomaterial obtained in step (1) of the probe preparation in Example 1 onto the surface of a nitrocellulose membrane. After drying, the membrane is irradiated with a laser, and the temperature change is recorded using a thermal imager. The resulting temperature change curve is shown in [Figure 1]. Figure 3 ,Depend on Figure 3 It can be observed that the temperature rises continuously with the increase of irradiation time, until it gradually stabilizes and reaches its peak after 10 seconds. Therefore, 10 seconds was chosen as the laser irradiation time.

[0096] Before applying the Salmonella Typhimurium aptamer lateral flow chromatography test strip prepared in Example 1, the sensitivity and specificity were tested. The specific testing methods are as follows:

[0097] 1. Detection sensitivity

[0098] 70 μL of a known concentration of Salmonella Typhimurium solution was mixed with 10 μL of the Fe3O4@Au aptamer probe prepared in step (2) of probe preparation in Example 1. The mixture was shaken for 30 min, collected with a magnet, and the solution was removed. The solution was then reconstituted with 80 μL of PBS buffer (pH 7.4) containing 1% polyethylene glycol-8000 to obtain a sample mixture. The sample mixture was added to the sample pad area of ​​the Salmonella Typhimurium aptamer lateral flow chromatography test strip prepared in Example 1. After 10 min, 40 μL of the mixture (8 μL of 30% H2O2 aqueous solution and 32 μL of...) was added... A mixture of TMB colorimetric solutions was added to the nitrocellulose membrane of the Salmonella Typhimurium aptamer lateral flow chromatography test strip prepared in Example 1. The reaction was allowed to proceed for 10 minutes. After drying, the initial temperature of the T-line was measured using a thermal imager. Then, the strip was irradiated with a 1W laser generator for 10 seconds, and the final temperature of the T-line was measured again. The temperature difference ΔT was calculated as ΔT = final temperature - initial temperature. A standard curve was constructed with the logarithm of the Salmonella Typhimurium concentration on the x-axis and ΔT on the y-axis. The standard curve equation was obtained by fitting the curve. The obtained standard curve equation is Y = 1.463X + 0.261, R... 2 =0.998. The detection limit was set to 302 CFU / mL.

[0099] 2. Detection specificity

[0100] The Salmonella typhimurium aptamer lateral flow chromatography test strip prepared in Example 1 showed a bacterial concentration of 10. 7 CFU / mL of Salmonella typhimurium and 10 7 Other foodborne pathogens (Salmonella paratyphi, Listeria, Escherichia coli, Staphylococcus aureus) were tested at CFU / mL, and the visual and photothermal test results are as follows: Figure 4 As shown, only the test strip containing Salmonella typhimurium can achieve color development while significantly enhancing the photothermal signal, proving that the test strip has detection specificity.

[0101] The application method of the Salmonella Typhimurium aptamer lateral flow chromatography test strip prepared in Example 1 is as follows:

[0102] First, mix 70 μL of the test solution with 10 μL of the Fe3O4@Au aptamer probe obtained in step (2) of probe preparation in Example 1. React on a shaker for 30 min. Then, collect the solution with a magnet and remove it. Redissolve the solution with PBS and add it to the sample pad area of ​​the test strip. After 10 min, slowly add 40 μL of H2O2 and TMB mixture (8 μL of 30% H2O2 aqueous solution and 32 μL of TMB colorimetric solution) to the nitrocellulose membrane of the test strip. React for 10 min and observe the color development. After drying, measure the initial temperature of the T line with a thermal imager. Then, irradiate with a laser generator with an irradiation power of 1W for 10 s and measure the final temperature of the T line with a thermal imager. Calculate the temperature difference ΔT, ΔT = final temperature - initial temperature. Substitute into the standard curve equation to calculate the concentration of Salmonella typhimurium in the test solution.

[0103] Test Example 2

[0104] Take 2 μL of Fe3O4@Au composite nanomaterials and Fe3O4•NPS obtained in step (1) of the probe preparation in Example 1, and place them on nitrocellulose membranes respectively. After drying, irradiate the materials with a laser and measure the temperature with a thermal imager. When the temperature reaches the highest value, turn off the laser and record the temperature drop curve with a thermal imager until the material cools down to the lowest temperature. The comparison results of the obtained photothermal performance curves are shown in the figure. Figure 5 A. Repeat the above operation five times, and record the photothermal cycling curve of the Fe3O4@Au composite nanomaterial obtained in Example 1. The results are shown in [the table below]. Figure 5 C; Continue to test the temperature rise of the Fe3O4@Au composite nanomaterials obtained in Example 1 at different concentrations using the above method. The test results are shown in […]. Figure 5 B.

Claims

1. A method for preparing a Salmonella Typhimurium aptamer lateral flow chromatography test strip based on the photothermal effect of iron(II,III) oxide@colloidal gold composite nanomaterials, characterized in that, include: Prepare test strips, prepare probes, prepare T-line aptamer solution, prepare C-line aptamer solution, and apply lines; The preparation of the probe includes: preparing Fe3O4@Au composite nanomaterials and synthesizing the probe; The preparation of Fe3O4@Au composite nanomaterials involves dissolving FeCl3·6H2O and FeCl2·4H2O in deionized water under nitrogen protection, adding ammonia, stirring at room temperature, adding trisodium citrate at 88-92℃, and collecting the solution with a magnet to obtain citrate-coated Fe3O4•NPS; adding the citrate-coated Fe3O4•NPS to a boiling HAuCl4 solution, maintaining boiling until it turns reddish-brown, continuing heating, cooling to room temperature, and collecting the solution with a magnet to obtain Fe3O4@Au composite nanomaterials. In the preparation of Fe3O4@Au composite nanomaterials, the ratio of FeCl3·6H2O, FeCl2·4H2O, and deionized water is 1.62-1.63g:0.99-1g:18-22mL. The ratio of FeCl3·6H2O to ammonia is 1.62-1.63 g: 5-6 mL; The mass ratio of FeCl3·6H2O to trisodium citrate is 1.62-1.63:4.3-4.5; The volume ratio of citrate-coated Fe3O4•NPS to HAuCl4 solution was 10:18-22. The synthesized probe was prepared by collecting the Fe3O4@Au composite nanomaterials with a magnet, diluting them with ultrapure water to obtain diluted composite nanomaterials, sonicating them, and adjusting the pH to 7.8-8.2 to obtain a Fe3O4@Au composite material solution. TCEP was added to the thiol-modified aptamer, and activated at 36-38°C in the dark to obtain an activated mixed aptamer solution. The activated mixed aptamer solution was mixed with the Fe3O4@Au composite material solution and incubated at room temperature until the thiol groups on the aptamer formed gold-sulfur bonds with the Au on the Fe3O4@Au composite material. Sodium chloride solution was then added, and the reaction was carried out at 4°C to obtain a mixed composite solution. The mixed composite solution sample was collected with a magnet and reconstituted with PEG-containing PBS buffer to obtain the Fe3O4@Au aptamer probe. In the synthetic probe, the sequence of the aptamer in the thiol-modified aptamer is shown in SEQ ID No. 1 of the sequence listing; The preparation of the T-line aptamer solution involves adding streptavidin solution to a biotin-modified aptamer and reacting at room temperature to obtain the T-line aptamer solution. In the preparation of the T-line aptamer solution, the sequence of the aptamer in the biotin-modified aptamer is shown in SEQ ID No. 1 of the sequence listing.

2. The method for preparing the Salmonella Typhimurium aptamer lateral flow chromatography test strip based on the photothermal effect of iron(II,III) oxide@colloidal gold composite nanomaterials according to claim 1, characterized in that, To prepare the test strip, a nitrocellulose membrane is attached to a base plate, an absorbent pad is attached above the nitrocellulose membrane, and a sample pad is attached below the nitrocellulose membrane to obtain the test strip. In the preparation of the test strip, the absorbent pad covers the nitrocellulose membrane for a length of 2 mm, and the sample pad covers the nitrocellulose membrane for a length of 2 mm. The nitrocellulose membrane, model CN90, was purchased from Sartorius. The base plate is model DB-6 and was purchased from Shanghai Jieyi Biotechnology Co., Ltd. The sample pad was a Fusion3 model, purchased from Shanghai Jieyi Biotechnology Co., Ltd. The absorbent pad is model H5073 and was purchased from Shanghai Jieyi Biotechnology Co., Ltd. The base plate is 6cm wide, the sample pad is 2.4cm wide, the nitrocellulose membrane is 2.5cm wide, and the absorbent pad is 2cm wide.

3. The method for preparing the Salmonella Typhimurium aptamer lateral flow chromatography test strip based on the photothermal effect of iron(II,III) oxide@colloidal gold composite nanomaterials according to claim 1, characterized in that, In the preparation of Fe3O4@Au composite nanomaterials, the volume fraction of ammonia water is 27-28%. Stirring time at room temperature is 9-11 minutes; The concentration of the HAuCl4 solution is 0.17-0.18 mg / mL; Continue heating for 9-11 minutes.

4. The method for preparing the Salmonella Typhimurium aptamer lateral flow chromatography test strip based on the photothermal effect of iron(II,III) oxide@colloidal gold composite nanomaterials according to claim 1, characterized in that, In the synthetic probe, the volume ratio of Fe3O4@Au composite nanomaterial to diluted composite nanomaterial is 98-102:98-102; The volume ratio of the thiol-modified aptamer to TCEP was 7.4-7.6:1.9-2.1; The volume ratio of Fe3O4@Au composite nanomaterials to thiol-modified aptamers is 98-102:7.4-7.6; The volume ratio of Fe3O4@Au composite nanomaterials to sodium chloride solution is 98-102:1.9-2.1; The volume ratio of the mixed composite liquid sample to the Fe3O4@Au aptamer probe was 98-102:49-51; K₂CO₃ is used to adjust the pH to 7.8-8.2; The concentration of the thiol-modified aptamer is 19-21 μM; The concentration of TCEP is 0.24-0.26%; The concentration of the sodium chloride solution is 0.9-1.1 M; The PEG in the PEG-containing PBS buffer is PEG-8000; The PEG-containing PBS buffer has a PEG concentration of 0.9-1.1% and a pH of 7.3-7.

5. The ultrasonic treatment time is 18-22 minutes; Activation at 36-38℃ in the dark takes 1-1.5 hours. The incubation time at room temperature is 3-3.5 hours; The reaction time at 4°C is overnight; The Fe3O4@Au aptamer probe was used to detect the mixing of Salmonella typhimurium solution.

5. The method for preparing the Salmonella Typhimurium aptamer lateral flow chromatography test strip based on the photothermal effect of iron(II,III) oxide@colloidal gold composite nanomaterials according to claim 1, characterized in that, In the preparation of the T-line aptamer solution, the reaction time at room temperature is 40-50 min; The concentration of streptavidin in the T-line aptamer solution was 6 mg / mL, and the concentration of the aptamer was 110 μM.

6. The method for preparing the Salmonella Typhimurium aptamer lateral flow chromatography test strip based on the photothermal effect of iron(II,III) oxide@colloidal gold composite nanomaterials according to claim 1, characterized in that, The preparation of the C-line aptamer solution involves adding streptavidin solution to cDNA complementary to the aptamer and reacting at room temperature to obtain the C-line aptamer solution. The sequence of the cDNA complementary to the aptamer in the preparation of the C-line aptamer solution is shown in SEQ ID No. 2 of the sequence listing; The reaction time at room temperature is 40-50 minutes; The concentration of streptavidin in the C-line aptamer solution was 1 mg / mL, and the concentration of cDNA complementary to the aptamer was 30 μM.

7. The method for preparing the Salmonella Typhimurium aptamer lateral flow chromatography test strip based on the photothermal effect of iron(II,III) oxide@colloidal gold composite nanomaterials according to claim 1, characterized in that, The line marking process involves using a film marking instrument to draw T-line and C-line aptamer solutions on the test strip, then drying at 37°C and cutting into strips to obtain a Salmonella typhimurium aptamer lateral flow chromatography test strip based on the photothermal effect of iron oxide@colloidal gold composite nanomaterials. In the scribed lines, the distance between line T and line C is 3.9-4.1 mm; The drying time at 37℃ is 1-1.5 hours; The width of the test strip is 3.9-4.1 mm.

8. A Salmonella typhimurium aptamer lateral flow chromatography test strip based on the photothermal effect of iron(II,III) oxide@colloidal gold composite nanomaterials, prepared by the preparation method according to any one of claims 1-7.

9. The application of a Salmonella Typhimurium aptamer lateral flow chromatography test strip based on the photothermal effect of iron(III) oxide@colloidal gold composite nanomaterials, prepared by the preparation method according to any one of claims 1-7, characterized in that, Mix 69-71 μL of the test solution with 9.8-10.2 μL of Fe3O4@Au aptamer probe, react on a shaker, collect the solution with a magnet and remove it, redissolve with PBS, add the solution to the sample pad area of ​​the test strip, and after 9-11 min, add 39.5-40.5 μL of H2O2 and TMB mixture to the nitrocellulose membrane of the test strip, react for 9-11 min and observe the color development, dry and measure the initial temperature of the T line with a thermal imager, then irradiate with a laser generator with an irradiation power of 0.9-1.1W for 9.8-10.2 s, measure the final temperature of the T line with a thermal imager, calculate the temperature difference, substitute it into the standard curve equation, and calculate the concentration of Salmonella typhimurium in the test solution.

10. The application of the Salmonella Typhimurium aptamer lateral flow chromatography test strip based on the photothermal effect of iron(II,III) oxide@colloidal gold composite nanomaterials according to claim 9, characterized in that, In this application, the reaction time of the shaker is 28-32 minutes; The H2O2 and TMB mixed liquid is a mixture of H2O2 aqueous solution with a concentration of 29.5-30.5% and TMB colorimetric solution, wherein the volume ratio of H2O2 aqueous solution to TMB colorimetric solution is 7.8-8.2:31-33.

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