Dual-mode test strips, preparation and application thereof

Abstract

The present invention provides a dual-mode test strip, preparation and application thereof. The present invention provides a method for preparing a dual-mode immune test strip, and a highly sensitive detection method for detecting aflatoxin-producing fungi in a sample using the test strip. The present invention has the advantages of simple operation, high sensitivity, high accuracy, fast detection speed, low cost, and no limitation by detection equipment, and can realize rapid detection of aflatoxin-producing fungi in a sample.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a dual-mode test strip, preparation and application thereof. Background Art

[0002] Immunochromatographic test strips have been widely used in food safety, medical disease diagnosis, environmental monitoring and other fields due to their advantages such as easy operation, portability, rapidity and on-site analysis. However, the disadvantages of immunochromatographic test strips such as low sensitivity and poor repeatability have limited their application and promotion in the field of analytical testing to a certain extent. In addition, existing methods usually use a single colorimetric signal or fluorescent signal to quantify the content of the target substance. This single-signal detection method has a single system and is easily interfered by environmental factors, which will affect the accuracy of the experimental results to a certain extent. Therefore, it is urgent to develop dual-modal immunochromatographic test strips with higher sensitivity and reliability to meet the needs of analytical testing.

[0003] Aflatoxins are toxic secondary metabolites secreted by Aspergillus flavus and Aspergillus parasiticus. They are the most toxic and carcinogenic fungal toxins discovered so far. Aflatoxin-producing fungi are widely distributed and have strong infection ability. They have the opportunity to cause pollution in various links such as the growth, harvesting, transportation, storage, and processing of crops. At present, the detection of toxic fungi in food still relies heavily on the traditional microbial morphological characteristics observation method. Due to the wide variety of toxic fungi and the complex morphological characteristics, this detection method is greatly affected by subjective factors and has a high misjudgment rate. At the same time, the detection process is time-consuming and cumbersome to operate, which makes it difficult to meet the needs of fast, efficient, and instant analysis and detection. Enzyme-linked immunosorbent assay (ELISA) has been used to detect aflatoxin-producing Aspergillus flavus (Anal. Chem. 2013, 85, 10992-10999, Scientific reports, 2017, 7(1): 4348, Chinese Agricultural Science, 53(7): 1473-1481), but there are still some shortcomings, such as the catalytic activity of natural enzymes is easily disturbed by the external environment, the stability of natural enzymes is poor, and the detection sensitivity of the method is low, which makes it difficult to meet the requirements of efficient and sensitive real-time detection of aflatoxin-producing fungi. In addition, most of the current immunoassay methods are ELISA methods, and there are no reports on the use of immunochromatographic test strips for aflatoxin-producing fungi. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a dual-mode test strip, preparation method and application thereof.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A dual-modal test strip comprises a chromatographic test strip and a polyclonal antibody reaction reagent marked with a polydopamine-copper composite nanomaterial that can bind to an object to be tested, wherein: the chromatographic test strip comprises a cardboard, one side of the cardboard is sequentially pasted with a water-absorbing pad, a detection pad and a sample pad from top to bottom, and adjacent pads are overlapped and connected at a connection point; the detection pad uses a nitrocellulose membrane as a base pad, and a transverse quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom; the detection line is coated with a nano antibody or a monoclonal antibody that recognizes the object to be tested, and the quality control line is coated with a polyclonal antibody (IgG) that can bind to the polyclonal antibody marked with the polydopamine-copper nanomaterial.

[0007] According to the above scheme, the absorbent pad in the chromatography test strip is 40-45 mm long and 3-5 mm wide; the detection pad is 25-30 mm long and 3-5 mm wide; the sample pad is 12-18 mm long and 2-4 mm wide, and the overlapping length of adjacent pads is 1-3 mm; the distance between the detection line on the detection pad and the upper edge of the sample pad is 5-8 mm, and the distance between the quality control line and the detection line is 5-12 mm.

[0008] According to the above scheme, the coating amount of the nano-antibody or monoclonal antibody of the object to be tested required for each centimeter of the detection line on the detection pad in the chromatography test strip is 150-900 ng; the coating amount of the goat anti-rabbit polyclonal antibody (IgG) required for each centimeter of the quality control line is 150-600 ng.

[0009] As a further improvement of the present invention, the above-mentioned analytes may be pathogenic bacteria such as aflatoxin-producing fungi, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Salmonella typhimurium.

[0010] According to the above scheme, the analyte is aflatoxin-producing fungus; the detection line is coated with antibodies against aflatoxin-producing fungi, and the polyclonal antibody that combines with the antibodies against aflatoxin-producing fungi is a rabbit polyclonal antibody; the antibody that can combine with the polyclonal antibody against aflatoxin-producing fungi is a sheep anti-rabbit polyclonal antibody (IgG).

[0011] According to the above scheme, the nanoantibody that recognizes the aflatoxin-producing fungus to be detected can be obtained by the following detection method: using AFT-YJFZ01 as an immune antigen, immunizing alpacas or Balb / c mice in a conventional manner, and then using known conventional nanoantibodies or mouse monoclonal antibodies to prepare the nanoantibody, as described in Chinese Patent 2021106091381.

[0012] The polyclonal antibody for identifying the aflatoxin-producing fungi to be detected can be obtained by the following detection method: AFT-YJFZ01 is used as an immune antigen, New Zealand white rabbits and other test rabbits are immunized in a conventional manner, and then a rabbit-derived polyclonal antibody for identifying the aflatoxin-producing fungi to be detected is prepared by using a known conventional polyclonal antibody preparation technology scheme. As described in Chinese Patent 2021106091381.

[0013] According to the above scheme, the polyclonal antibody labeled with the polydopamine-copper composite nanomaterial that can bind to the object to be detected is prepared by blocking the polydopamine-copper nanomaterial and the polyclonal antibody that can identify the object to be detected after physical adsorption. The preparation method is as follows: the dopamine-copper nano dispersion and the polyclonal antibody solution that can identify the detection object are mixed and shaken to allow the polydopamine-copper composite material and the polyclonal antibody that can identify the object to be detected to fully bind, then the excess active sites are blocked, and the polyclonal antibody labeled with the polydopamine-copper nanomaterial is obtained after centrifugation.

[0014] As a further improvement of the present invention, the concentration of the polydopamine-copper nano-dispersion is 1.0-3.0 mg / mL; the concentration of the polyclonal antibody solution capable of identifying the object to be detected is 10-1000 μg / mL; the mass ratio of the polydopamine-copper nano-dispersion to the polyclonal antibody solution capable of identifying the object to be detected is 1.5-50:1, and the shaking time is 1-4 hours; the blocking solution is a bovine serum albumin aqueous solution with a mass percentage of 0.5-5.0%, the supernatant is discarded by centrifugation, and the centrifuged precipitate is dispersed in PBS for later use.

[0015] According to the above scheme, the polydopamine-copper nano-dispersion can be prepared by a one-step method. The preparation method is as follows: a certain amount of dopamine hydrochloride is dissolved in a mixed solution of tris(hydroxymethyl)aminomethane buffer and ethanol, and the mixture is stirred at 20-35°C for 0.5-3h; an aqueous solution of copper sulfate is added dropwise, and the mixture is stirred at 20-35°C for 2-6h. After the reaction is completed, polydopamine-copper nanoparticles are obtained by post-treatment. The post-treatment is as follows: the above solution is centrifuged at 4°C for 10-30min, and the precipitate is washed by centrifugation repeatedly with ultrapure water for 2-5 times. The obtained polydopamine-copper nanoparticles are dispersed to obtain a polydopamine-copper nano-dispersion with a concentration of 1.0-3.0mg / mL. In the preparation process: the hydroxyl functional groups on the surface of dopamine react with Cu 2+ Coordination, Cu after coordination 2+ Reduced to Cu by dopamine + Cu 0 of nanoparticles.

[0016] As a further improvement of the present invention, the concentration of the dopamine hydrochloride solution is 0.5-1.5 mg / mL; the concentration of the tris(hydroxymethyl)aminomethane buffer solution is 10 mM, pH 8.5, and the volume ratio of the tris(hydroxymethyl)aminomethane buffer solution to ethanol is 3-5:1; the concentration of the copper sulfate aqueous solution is 10-50 mg / mL, and the molar ratio of the copper sulfate to dopamine hydrochloride is 0.7-9.5:1.

[0017] According to the above scheme, the chromatography test strip also includes a sample diluent and a sample diluent pipette, wherein the sample diluent is a mixture of 1.0-6.0% Tween-20 by volume and 0.5-5.0% BSA aqueous solution by volume.

[0018] According to the above scheme, the preparation method of the dual-modal test strip is as follows:

[0019] (1) Cut absorbent paper into absorbent pads;

[0020] (2) Preparation of detection pad

[0021] 1) Coating of the test line

[0022] A coating solution is prepared by preparing a nano-antibody or monoclonal antibody that recognizes the object to be tested, and a coating solution with a concentration of 0.25-1.5 mg / mL can be prepared using 0.01 mol / L phosphate buffer (PBS); at a position of 5-8 mm along the upper edge of the sample pad, it is horizontally coated on the nitrocellulose membrane by line spraying to obtain a detection line, and the coating amount of the nano-antibody or monoclonal antibody that recognizes the object to be tested per centimeter of the detection line is 150-900 ng, and dried, specifically, it can be dried at 37°C for 1.5 hours;

[0023] 2) Coating of quality control line

[0024] A polyclonal antibody (IgG) that can bind to the polyclonal antibody labeled with the polydopamine-copper nanomaterial is prepared into a coating solution, which can be prepared into a coating solution with a concentration of 0.25-1.0 mg / mL using a 0.01 mol / L PBS solution containing 0.02% (m / v) sodium azide; at a position 5-12 mm away from the detection line, it is horizontally coated on a nitrocellulose membrane by line spraying to obtain a quality control line, and the required coating amount of the polyclonal antibody (IgG) per centimeter of the quality control line is 150-600 ng, and then dried, specifically, it can be dried at 37° C. for 1.5 hours;

[0025] The detection pad can be made of CN95 membrane produced by Bedford Millipore Corporation or IAB120 or IAB135 membrane produced by ToyoRoshi Kaisha, a Japanese company, preferably CN95 membrane;

[0026] (3) Preparation of sample pad

[0027] A nitrocellulose membrane is used as a sample pad, specifically Fusion3 or Fusion5 produced by the Chinese company Jieyi Biotechnology Corporation can be used as the sample pad, with Fusion3 being preferred.

[0028] (4) Assembly of test strips

[0029] On one side of the cardboard, a water-absorbing pad, a detection pad and a sample pad are sequentially pasted from top to bottom, and adjacent pads are overlapped and connected at the joints with an overlap length of 1 to 3 mm to obtain a chromatography test strip.

[0030] According to the above scheme, 0.01 mol / L phosphate buffer (PBS) contains 8 g of sodium chloride, 0.2 g of potassium chloride, 2.9 g of disodium hydrogen phosphate dodecahydrate, and 0.24 g of potassium dihydrogen phosphate per 1 L, and the pH value is adjusted to 7.4 with hydrochloric acid.

[0031] According to the above scheme, the 0.01 mol / L PBS solution containing 0.02% (m:v) sodium azide means that every 50 mL of the 0.01 mol / L PBS solution contains 0.01 g of sodium azide.

[0032] The blocking solution used in the preparation of the chromatography test strip is: 0.5 g of bovine serum albumin and 1.6 g of Tween 20 in every 100 mL of 0.01 mol / L PBS solution.

[0033] Based on the dual-modality detection method of the dual-modality immunoassay test strip, the detection method is as follows:

[0034] Provide a sample solution to be tested, provide a polyclonal antibody solution for identifying the test object labeled with polydopamine-copper nanomaterials, add them into the enzyme-labeled wells respectively, mix them, insert the chromatography test strips, incubate for a period of time, and read the results; and provide a negative control sample group at the same time.

[0035] According to the above scheme, the sample to be tested is one or more of corn, peanuts, and rice.

[0036] According to the above scheme, the incubation process is as follows: the polyclonal antibody reaction reagent labeled with the polydopamine-copper composite material is diluted to a fixed volume with a slow-release solution and added to the enzyme-labeled well, and the sample solution to be tested is added to the enzyme-labeled well containing the detection solution for incubation.

[0037] According to the above scheme, the above sustained-release solution is: each 1 mL of the sustained-release solution contains 16 μL of Tween-20, 5 mg of BSA, and 984 μL of 0.01 mol / L phosphate buffer.

[0038] According to the above scheme, the polydopamine-copper nanomaterial labeled polyclonal antibody reaction reagent sustained-release solution and the test substance solution are mixed at a volume ratio of 4-6.5:1.

[0039] According to the above protocol, incubate at 25-37°C for 5-15 min and read the colorimetric signal directly.

[0040] According to the above method, add signal amplification colorimetric solution to the detection line, place it at 25°C for 15-45s, and read the catalytic amplification colorimetric signal result.

[0041] According to the above scheme, the signal amplification color developing solution is TMB-H 2 O 2 , the concentration of TMB solution is 5-50mmol / L, H 2 O 2 The mass concentration of TMB-H 2 O 2 Dispersed in an acidic buffer solution with a pH of 3.0-6.5.

[0042] According to the above scheme, the acidic buffer solution is one of acetic acid-sodium acetate or citric acid-sodium citrate buffer solutions.

[0043] According to the above method, the test line of the test strip was irradiated with 808nm laser, and the photothermal signal results were recorded using a near-infrared thermal imager.

[0044] According to the above scheme, the laser power is 0.5-2.0W / cm2 and the irradiation time is 2-6min;

[0045] According to the above scheme, the specific calculation method of the aflatoxin-producing fungi content in the sample to be tested is: based on the intensity gray value change rate or temperature change ((II 0 ) / I 0 or ΔT, obtain ΔI (i.e. II 0 ) / I 0 or ΔT (ΔT is the temperature rise value, that is, ΔT 1 -ΔT 0 , T 1 and T 0 The relationship curve between the temperature difference between the experimental group and the blank group after laser irradiation) and the analyte is obtained by fitting, and the relationship curve between the colorimetric intensity change value or temperature change value of the test strip detection line and the concentration of the analyte is obtained. The obtained detection line colorimetric intensity change value or temperature change value of the sample solution to be tested is substituted into the above-mentioned fitting relationship curve to obtain the content of the analyte in the sample solution to be tested, and finally the content of the analyte in the sample to be tested is obtained by conversion.

[0046] The detection principle of the dual-mode test strip of the present invention is:

[0047] The solution of the test object, taking aflatoxin-producing fungi as an example, adds the cell lysate of aflatoxin-producing fungi and the polyclonal antibody solution of aflatoxin-producing fungi labeled with polydopamine-copper composite nanomaterials to the enzyme-labeled wells in turn, inserts the immunochromatographic test strip, and flows toward the absorption pad by capillary force. When the test solution contains the test object aflatoxin-producing fungi, the aflatoxin-producing fungi and the nano-antibodies or monoclonal antibodies that recognize the aflatoxin-producing fungi on the detection line of the immunochromatographic test strip are specifically immune-recognized. At the same time, the aflatoxin-producing fungi can be specifically immune-recognized with the polyclonal antibodies of the aflatoxin-producing fungi labeled with polydopamine-copper nanomaterials, and then a sandwich structure of antibody-aflatoxin-producing fungi-polyclonal antibodies labeled with polydopamine copper is formed on the detection line. The more the content of the target aflatoxin-producing fungi in the test solution, the more sandwich structures are formed, and the darker the color of the detection line is. At the same time, the TMB-H 2 O 2 The deeper the blue color after catalytic amplification, the higher the temperature. On the contrary, when the test solution does not contain the aflatoxin-producing fungi, a sandwich structure cannot be formed, and the test line has no obvious color enhancement. 2 O 2 There is no obvious blue enhancement and no obvious temperature enhancement after the system is catalytically amplified. 2 O 2 The principle of catalytic amplification is: in the presence of TMB-H 2 O 2 In the solution, the copper nanoparticles with catalytic activity centers in the nanoantibodies or monoclonal antibodies of aflatoxin-producing fungi labeled with polydopamine-copper nanomaterials catalyze H 2 O 2 The generated hydroxyl radicals further react with the colorless substrate TMB to form a blue oxidized TMB product. The more analytes there are, the more polydopamine-copper-labeled aflatoxin-producing fungi nanoantibodies or monoclonal antibodies that bind to the analytes, and the more TMB-H 2 O 2 The more blue oxidized TMB is produced, the darker the blue color.

[0048] Beneficial effects of the present invention:

[0049] The present invention provides a dual-mode immune detection test strip. The present invention utilizes a polydopamine-copper composite nanomaterial as a signal conduction medium, can simultaneously obtain colorimetric and photothermal signals for detection, and provides a dual-mode immune detection test strip.

[0050] Compared with the existing methods, the present invention has the following advantages:

[0051] (1) In addition to the traditional direct colorimetric detection signal, the efficient peroxidase mimicking activity of polydopamine-copper composite nanoparticles can also be used to greatly improve the amplification output of the detection signal; and the high photothermal conversion efficiency of polydopamine-copper can be used to provide a new photothermal signal source for detection, thereby enhancing the reliability of the detection method.

[0052] (2) Wide detection range, multiple signal output, and high detection sensitivity. The dual-mode test strip provided by the present invention can be used to determine aflatoxin-producing fungi in the test object. Taking Aspergillus flavus as an example, the detection range is 1ng / mL-100μg / mL, and the minimum detection of Aspergillus flavus is 1ng / mL. This overcomes the shortcomings of the prior art that the sensitivity is low and the detection effect is judged only by the colorimetric signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Schematic diagram of dual-modal colorimetric-photothermal test strip detection. DETAILED DESCRIPTION

[0054] Example 1

[0055] Preparation and detection method of dual-modal aflatoxin-producing fungi test strip, the specific method is:

[0056] 1. Preparation of immunoassay strips

[0057] (1) Preparation of absorbent pad

[0058] The absorbent paper was cut into a size of 45 mm in length and 3 mm in width to prepare an absorbent pad;

[0059] (2) Preparation of detection pad

[0060] Paste the CN95 nitrocellulose membrane with a specification of 25mm in length and 3mm in width to the corresponding position of the cardboard and then spray the test line and quality control line respectively.

[0061] 1) Coating of the test line

[0062] Weigh 1.0 mg of aflatoxin-producing fungus nanoantibody, dissolve it in 1 mL of 0.01 mol / L phosphate buffer solution (PBS) and prepare a coating solution with a concentration of 1 mg / mL; coat it horizontally on a nitrocellulose membrane by line spraying at a position 5 mm away from the upper edge of the sample pad to obtain a detection line, the spraying speed is 0.6 μL / cm, and 600 ng of aflatoxin-producing fungus nanoantibody is sprayed on each centimeter of the detection line, and then dried at 37°C for 1.5 h;

[0063] The PBS formula described above: 8 g sodium chloride, 0.2 g potassium chloride, 2.9 g disodium hydrogen phosphate dodecahydrate, 0.24 g potassium dihydrogen phosphate, after dissolving in 800 mL water, adjust the pH value to 7.4 with hydrochloric acid, make up to 1 L with ultrapure water, filter with 0.22 μm filter membrane, and prepare 0.01 mol / L PBS.

[0064] 2) Coating of quality control line

[0065] Weigh 0.02 g of sodium azide and dissolve it in 100 mL of 0.01 mol / L phosphate buffer (PBS), filter through a 0.22 μm filter membrane, and prepare a 0.01 mol / L PBS solution containing 0.02% (m / v) sodium azide.

[0066] Weigh 1 mg of goat anti-rabbit polyclonal antibody (IgG), dissolve it in 1 mL of 0.01 mol / L PBS solution containing 0.02% (m / v) sodium azide to prepare a coating solution with a concentration of 1 mg / mL; coat it horizontally on a nitrocellulose membrane by line spraying at a distance of 7 mm from the detection line, with a spraying speed of 0.6 μL / cm to obtain a quality control line, spray 600 ng of goat anti-rabbit polyclonal antibody (IgG) per centimeter of the quality control line, and then dry it at 37°C for 1.5 h.

[0067] (3) Assembly of test strips

[0068] On one side of the cardboard, a water-absorbing pad, a detection pad and a sample pad are sequentially pasted from top to bottom, and the adjacent pads are overlapped and connected at the joints, with an overlap length of 2 mm, to obtain a chromatography test strip;

[0069] The sample pads mentioned above are Fusion 3 and Fusion 5 produced by the Chinese company Jieyi Biotechnology Corporation.

[0070] 2. Preparation of polydopamine-copper labeled polyclonal antibody reaction reagent

[0071] (1) Preparation of polydopamine-copper nanomaterials

[0072] 9.368 mg of dopamine hydrochloride was dissolved in 6.4 mL of tris (10 mM, pH 8.5) and 1.6 mL of ethanol mixed solution (v:v = 4:1), and stirred at 1000 rpm at 25 °C for 1 h. Subsequently, 1 mL of 24 mg / mL CuSO was added dropwise to the reaction solution. 4 ·5H 2O, stirred at 1000 rpm at 25°C for 3 hours. After the reaction was completed, the mixed solution was centrifuged at 10000 rpm at 4°C for 15 minutes, and the precipitate was washed three times by centrifugation with ultrapure water, and finally 3.6 mg of polydopamine-copper composite nanomaterials was obtained. Dispersed with 2 mL of ultrapure water, a polydopamine-copper composite nanodispersion with a concentration of 1.8 mg / mL was obtained.

[0073] (2) Preparation of polydopamine-copper labeled polyclonal antibody reaction reagent

[0074] 100 μL of 1.8 mg / mL polydopamine-copper nanoparticle solution was mixed with 500 μL of 100 μg / mL polyclonal antibody of aflatoxin-producing fungi and gently shaken at 100 rpm for 2 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 15 min at 4 °C, the supernatant was discarded, and the resulting precipitate was dissolved with 200 μL of 0.01 mol / L PBS. Then 200 μL of 2% BSA aqueous solution was added and slowly shaken at 100 rpm for 2 h to block the excess active sites. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 15 min at 4 °C, the clear liquid was discarded, and the precipitate was retained. The precipitate was washed by centrifugation with ultrapure water for 3 times, and finally redispersed with 200 μL of 0.01 mol / L PBS solution and placed in a 4 °C refrigerator for standby use.

[0075] Preparation of 2% BSA solution: weigh 0.02 g of bovine serum albumin (BSA) and dissolve it in 1 mL of ultrapure water, filter through a 0.22 μm filter membrane to obtain a 2% BSA aqueous solution.

[0076] The above-mentioned nanoantibodies that recognize the aflatoxin-producing fungi to be detected can be obtained by the following detection method: using AFT-YJFZ01 as an immune antigen, immunizing alpacas or Balb / c mice in a conventional manner, and then using known conventional nanoantibodies or mouse monoclonal antibodies to prepare the nanoantibodies. As described in Chinese Patent 2021106091381.

[0077] The above-mentioned polyclonal antibody for identifying the aflatoxin-producing fungi to be detected can be obtained by the following detection method: AFT-YJFZ01 is used as an immune antigen, and experimental rabbits such as New Zealand white rabbits are immunized in a conventional manner, and then a rabbit-derived polyclonal antibody for identifying the aflatoxin-producing fungi to be detected is prepared by using a known conventional polyclonal antibody preparation technology scheme. As described in Chinese Patent 2021106091381.

[0078] 3. Detection methods

[0079] (1) Preparation of aflatoxin-producing fungal lysate

[0080] 1) Preparation of Aspergillus flavus spore solution

[0081] Taking Aspergillus flavus 3.4408 as an example, Aspergillus flavus 3.4408 was inoculated on a potato dextrose agar (PDA) solid culture medium for activation, and cultured in a dark incubator at 28°C for 7 days. After being covered with yellow-green spores, the spores were taken out, and the spores were rinsed with sterilized 0.1% Tween 80 water to obtain a spore suspension. The spore morphology was observed and counted under a microscope using a hemocytometer.

[0082] The formula of the PDA solid culture medium is as follows: 20.0 g glucose, 20.0 g agar powder, 6.0 g potato extract powder, dissolved in ultrapure water and fixed to 1 L, sterilized under high pressure at 121° C. for 20 min, and set aside.

[0083] 2) Preparation of Aspergillus flavus mycelium lysate

[0084] Add the spore solution from step 1) into 200 mL of liquid Sabouraud medium to ensure that the final spore concentration is 3 × 10 5 cfu / mL. The above culture medium was placed in a constant temperature shaker at 28°C and cultured at 200rpm for 5 days. The mycelium was collected with sterilized gauze and washed with sterile water 3 to 5 times to remove the culture medium during the collection process. The collected mycelium was dried with a freeze dryer, and the dried mycelium was ground into powder with liquid nitrogen and transferred to PBS to ensure that the final concentration of the mycelium was 10mg / mL. The mycelium was homogenized with a high-pressure homogenizer at low temperature. First, homogenize twice at a low pressure of 100bar, and then homogenize twice at a high pressure of 1000bar to finally obtain the aflatoxin mycelium lysate.

[0085] The Sabouraud medium is formulated as follows: 10 g of peptone, 40 g of glucose, and 10.0 g of 3-(N-morpholine)propanesulfonic acid (MOPS), which are dissolved in ultrapure water and then fixed to 1 L, sterilized under high pressure at 121° C. for 20 min, and set aside.

[0086] (2) Multimodal Detection Procedure for Aflatoxin-Producing Fungi

[0087] 1) Colorimetric detection

[0088] The polyclonal antibody labeled with polydopamine-copper was mixed with Aspergillus flavus cell lysate and PBS (v:v=6:1) in the enzyme-labeled well to make 200 μL. Taking Aspergillus flavus 3.4408 as an example, the concentration of Aspergillus flavus cell lysate ranged from 0.01, 0.1, 1, 10, 50, 100, and 1000 μg / mL. A control group without Aspergillus flavus cell lysate was set up simultaneously. After mixing evenly at 25°C, the immunochromatographic test strip was inserted into the enzyme-labeled well reaction solution and reacted at 25°C. The reaction was stopped after 9 minutes. Regardless of positive or negative results, the quality control line showed a gray band. If the gray band of the quality control line disappeared, the test strip was invalid. After drying at 25°C for 1.5 hours, take a picture and observe the results. The color signal intensity used the gray intensity value in Image J software to obtain the quantitative relationship between the gray intensity value and the concentration of Aspergillus flavus cell lysate.

[0089] 2) Catalytically amplified colorimetric detection

[0090] The polyclonal antibody labeled with polydopamine-copper was mixed with Aspergillus flavus cell lysate and PBS (v:v=6:1) in the enzyme-labeled well to make 200 μL. Taking Aspergillus flavus 3.4408 as an example, the concentration of Aspergillus flavus cell lysate ranged from 0.001, 0.01, 0.1, 1, 10, 50, and 100 μg / mL. A control group without Aspergillus flavus cell lysate was set up simultaneously. After mixing evenly at 25°C, the immunochromatographic test strip was inserted into the enzyme-labeled well reaction solution and reacted at 25°C. The reaction was stopped after 9 minutes. After drying at 25°C for 1.5 hours, 1 μL TMB-H was added to the test line. 2 O 2 The color developing solution was catalyzed at 25°C for 30 seconds, and then photographed and recorded to observe the results. Regardless of positive or negative results, the quality control line showed a gray band. If the gray band of the quality control line disappeared, the test strip was invalid. The color signal intensity used the gray intensity value in Image J software to obtain the quantitative relationship between the blue intensity value after catalytic amplification and the concentration of aflatoxin-producing fungi.

[0091] Among them, the above-mentioned TMB-H 2 O 2 The color development solution was prepared as follows: 24 mg of TMB was weighed and then diluted to 1 mL with N,N-dimethylformamide (DMF) solution to prepare a 100 mM TMB color development substrate. Then 3 mL of 25 mM pH 4.0 acetic acid-sodium acetate buffer solution was added, and 1 mL of H 2 O 2 (30%) formed TMB-H 2 O 2 Color developing solution.

[0092] 3) Photothermal detection

[0093] The polyclonal antibody labeled with polydopamine-copper was mixed with Aspergillus flavus lysate and PBS (v:v=6:1) in the enzyme-labeled well to make 200 μL. Taking Aspergillus flavus 3.4408 as an example, the concentration of Aspergillus flavus 3.4408 lysate was set to 0.001, 0.01, 0.1, 1, 10, 50, 100 μg / mL. At the same time, a control group without Aspergillus flavus cell lysate was set. After mixing evenly at 25°C, the immunochromatographic test strip was inserted into the enzyme-labeled well reaction solution, reacted at 25°C, and the reaction was stopped after 9 minutes. After drying at 25°C for 1.5 hours, the detection line on the immune test strip was irradiated with a laser, and the temperature of the detection line was recorded for 3 minutes. The quantitative relationship was obtained by ΔT and the concentration of Aspergillus flavus cell lysate. Regardless of the positive result or the negative result, the quality control line showed a gray band. If the gray band of the quality control line disappeared, the test strip was invalid.

[0094] Where ΔT = T 1 -T 0 , ΔT is the temperature rise, T 1 and T 0 are the temperature differences between the experimental group and the blank group after laser irradiation.

[0095] The laser wavelength is 808nm near-infrared light, and the power is 1.5W / cm 2 .

[0096] Example 2 Application of dual-modal immunoassay test strips, the specific method is as follows:

[0097] (1) Preparation of peanut and corn samples

[0098] Peanuts contaminated with Aspergillus flavus and corn samples spiked with Aspergillus flavus were selected, ground, 10 mg was weighed into a 2 mL centrifuge tube, 1 mL of PBS solution was added, and the mixture was ball milled in a ball mill at 60 Hz for 30 s to obtain a peanut and corn suspension. The mixed suspension was centrifuged at 10000 rpm and 4 °C for 20 min, the supernatant was collected, filtered through a 0.22 μm membrane, the filtrate was collected, diluted 10 times, and a 1 mg / mL solution was prepared for use.

[0099] (2) Detection of aflatoxin-producing fungi in samples

[0100] Peanuts and corns with different concentration gradients were set up, and mixed with polydopamine-copper labeled polyclonal antibody reaction reagent (v:v=6:1) in the enzyme-labeled well to form 200 μL, and a control group without uncontaminated actual samples was set up, and after mixing evenly at 25°C, the immunochromatographic test strip was inserted into the enzyme-labeled well reaction solution, and the reaction was stopped after 9 minutes at 25°C. The content of aflatoxin-producing fungi in peanuts and corns was detected using Example 1.

[0101] Test results: The more contaminated the peanuts and corns are with aflatoxin-producing fungi, the darker the corresponding test strip test line is. 2 O 2 The bluer the color after catalysis, the higher the temperature of the test line. When the concentration of aflatoxin-producing fungi in actual peanuts and corn is lower than 10 ng / mL, conventional visual test lines cannot be used for identification. 2 O 2 After the system is catalytically amplified, the lowest concentration of aflatoxin-producing fungi that can be detected by visual detection is 1 ng / mL. At the same time, the lowest temperature signal value of the detection line can also detect the concentration of aflatoxin-producing fungi as 1 ng / mL. Regardless of positive or negative results, the quality control line shows a gray band. If the gray band of the quality control line disappears, the test strip is invalid.

Claims

1. A dual-modal detection method based on a dual-modal immunoassay test strip, characterized in that: The dual-mode test strip comprises a chromatographic test strip and a polyclonal antibody solution labeled with a polydopamine-copper nanomaterial that can bind to a test object, wherein the test object is aflatoxin-producing fungi, wherein: the chromatographic test strip comprises a cardboard, one side of the cardboard is sequentially pasted with a water-absorbing pad, a detection pad and a sample pad from top to bottom, and adjacent pads are overlapped and connected at the connection point, the detection pad is based on a nitrocellulose membrane, and a horizontal quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom, the detection line is coated with a nano antibody or a monoclonal antibody that recognizes the test object, and the quality control line is coated with a polyclonal antibody that can bind to the polyclonal antibody labeled with the polydopamine-copper nanomaterial; The polydopamine-copper nanomaterial-labeled polyclonal antibody capable of binding to an object to be detected is prepared by blocking the polydopamine-copper nanomaterial and the polyclonal antibody capable of identifying the object to be detected after physical adsorption. The preparation method comprises: mixing and shaking the polydopamine-copper nanomaterial dispersion and the polyclonal antibody solution capable of identifying the object to be detected, so that the polydopamine-copper nanomaterial and the polyclonal antibody capable of identifying the object to be detected are fully combined, and then the excess active sites are blocked, and the polydopamine-copper nanomaterial-labeled polyclonal antibody is obtained after centrifugation. The polydopamine-copper nanomaterial dispersion is prepared by a one-step method, and the preparation method comprises: dissolving a certain amount of dopamine hydrochloride in a mixed solution of tris(hydroxymethylaminomethane) buffer and ethanol for 20-35 min. o C, stirring for 0.5-3 h; adding copper sulfate aqueous solution dropwise, 20-35 o C, stirred for 2-6 h, and after the reaction, post-processed to obtain; The detection method is as follows: providing a sample solution to be tested, adding the sample solution and a polyclonal antibody solution for identifying the test object labeled with polydopamine-copper nanomaterials to the enzyme label wells, mixing, inserting a chromatography test strip, incubating for a period of time, and reading the result; and providing a negative control sample group at the same time; 25-37 o C for 5-15 minutes, and the result is judged by dropping a signal amplification colorimetric solution on the detection line, leaving it for 15-45 seconds, and reading the colorimetric signal result of catalytic amplification; the signal amplification colorimetric solution is TMB-H2O2, the concentration of TMB solution is 5-50 mmol / L, and the mass concentration of H2O2 is 3-8%, and TMB-H2O2 is dispersed in an acidic buffer solution with a pH of 3.0-6.5; or 808nm laser is used to irradiate the detection line of the test strip, and a near-infrared thermal imager is used to record the photothermal signal result.

2. The dual-mode detection method according to claim 1, characterized in that: The sample to be tested is one or more of corn, peanuts and rice; the polyclonal antibody reaction reagent labeled with polydopamine-copper nanomaterial is mixed with the sample solution to be tested at a volume ratio of 4-6.5:

1.

3. The dual-mode detection method according to claim 1, characterized in that: In the chromatographic test strip, the absorbent pad is 40-45 mm long and 3-5 mm wide; the detection pad is 25-30 mm long and 3-5 mm wide; the sample pad is 12-18 mm long and 2-4 mm wide, and the overlapping length of adjacent pads is 1-3 mm; the distance between the detection line on the detection pad and the upper edge of the sample pad is 5-8 mm, and the distance between the quality control line and the detection line is 5-12 mm.

4. The dual-modal detection method according to claim 1, characterized in that: The antibody combined with the nano-antibody or monoclonal antibody identifying the object to be detected on the detection line is a rabbit or mouse polyclonal antibody; the antibody that can be combined with the polyclonal antibody on the quality control line is a goat anti-rabbit or goat anti-mouse polyclonal antibody.

5. The dual-modal detection method according to claim 1, characterized in that: The concentration of the polydopamine-copper nano-dispersion is 1.0-3.0 mg / mL; the concentration of the polyclonal antibody capable of identifying the analyte is 10-1000 μg / mL; the mass ratio of the polydopamine-copper nano-dispersion to the polyclonal antibody solution is 1.5-50:1; the shaking time is 1-4 h; and the blocking solution is a bovine serum albumin aqueous solution with a mass percentage of 0.5-5.0%.

6. The dual-modal detection method according to claim 1, characterized in that: The concentration of the dopamine hydrochloride solution is 0.5-1.5 mg / mL; the concentration of the tris(hydroxymethyl)aminomethane buffer solution is 10 mM, pH 8.5, and the volume ratio of the tris(hydroxymethyl)aminomethane buffer solution to ethanol is 3-5:1; the concentration of the copper sulfate aqueous solution is 10-50 mg / mL, and the molar ratio of the copper sulfate to dopamine hydrochloride is 0.7-9.5:

1.

7. The dual-modal detection method according to claim 1, characterized in that: The acidic buffer solution is one of acetic acid-sodium acetate or citric acid-sodium citrate buffer solutions; Laser power is 0.5-2.0 W / cm 2 , irradiation time is 2-6min; The specific calculation method for the content of aflatoxin-producing fungi in the sample to be tested is as follows: based on the intensity grayscale value change rate or temperature change of the detection line in the test strip with or without the analyte, a relationship curve between the intensity grayscale value change rate or temperature change value of the test strip detection line and the concentration of the analyte is obtained by fitting, and the obtained intensity grayscale value change rate or temperature change value of the detection line of the sample to be tested solution is substituted into the above-mentioned fitted relationship curve to obtain the content of the analyte in the sample to be tested solution, and finally the content of the analyte in the sample to be tested is obtained by conversion.

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