Fluorescent kit for rapidly detecting vanillin in edible oil as well as preparation and application of fluorescent kit

Abstract

The invention belongs to the field of immunodetection, and particularly discloses a fluorescent kit for rapidly detecting vanillin in edible oil as well as preparation and application of the fluorescent kit. Comprising a fluorescent test strip and a reaction microwell plate, the fluorescent test strip comprises a bottom plate, a water absorption pad, a detection pad and a sample pad are sequentially adhered to the bottom plate from top to bottom, adjacent pads are overlapped and connected at joints, a nitrocellulose membrane is used as a base pad of the detection pad, a quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom, and the quality control line is connected with the detection line. The quality control line is coated with a goat anti-mouse second antibody, the detection line is coated with a vanillin complete antigen, and the vanillin complete antigen is vanillin-bovine serum albumin (VAN-BSA); the reaction microwell plate is internally provided with a freeze-dried fluorescently-labeled vanillin-resistant monoclonal antibody, and the vanillin-resistant monoclonal antibody is generated by a hybridoma cell strain VAND8 of which the preservation number is CCTCC (China Center For Type Culture Collection) NO.C 2023186. The method can be used for detecting the content of vanillin in edible oil, and has the characteristics of rapidness in detection, simplicity in operation and high sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of immunoassay, and specifically relates to a fluorescent kit for rapidly detecting vanillin in edible oil, and its preparation and application. Background Art

[0002] Vanillin is a common food additive as a flavor. Although it is not toxic, excessive consumption can also harm human health. In severe cases, it may cause headaches, nausea, vomiting, breathing difficulties and even liver and kidney damage. Therefore, my country's "National Food Safety Standard for the Use of Food Additives" (GB2760-2014) clearly stipulates that food flavors and spices shall not be added to pasteurized milk, sterilized milk, fermented milk, and cream, and no edible spices shall be added to infant formula for 0 to 6 months. The maximum amount of vanillin used in formula for older infants and toddlers is 5mg / 100mL, and the maximum amount of vanillin used in cereal complementary foods for infants and young children is 7mg / 100g. Therefore, the use of vanillin in food must be strictly regulated, and dairy products, baked goods, beverages and edible oils are the focus of vanillin content monitoring.

[0003] Existing literature reports that the detection methods of vanillin in food include high performance liquid chromatography, high performance liquid-tandem mass spectrometry, gas chromatography, gas chromatography-tandem mass spectrometry, spectrophotometry, etc. These methods are highly sensitive and have accurate test results, but the required instruments and equipment are expensive and the sample pretreatment process is cumbersome. Immunoassay is a qualitative and quantitative detection of ultra-trace residues using the specific binding reaction of antigens and antibodies and the biological, physical or chemical amplification of markers on antibodies and antigens. Fluorescent test strips often use fluorescent markers (such as quantum dots, time-resolved fluorescence, upconversion nanoparticles, etc.) as probes, which have the characteristics of good fluorescence stability, high brightness and low photobleaching, can achieve high signal-to-noise ratio, and improve sensitivity. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a fluorescent kit for rapid detection of vanillin in edible oil, preparation and application thereof, in view of the deficiency of the existing technology in the field of rapid detection of vanillin. The kit can be used to detect the content of vanillin in edible oil, and has the characteristics of rapid detection, simple operation and high sensitivity.

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

[0006] A fluorescence kit for rapid detection of vanillin in edible oil comprises a fluorescence test strip and a reaction microwell plate, wherein the fluorescence test strip comprises a bottom plate, a water-absorbing pad, a detection pad and a sample pad are sequentially attached to the bottom plate from top to bottom, and adjacent pads are overlapped and connected at the connection point, the detection pad uses a nitrocellulose membrane as a base pad, a quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom, the quality control line is coated with a sheep anti-mouse secondary antibody, and the detection line is coated with a complete vanillin antigen, wherein the complete vanillin antigen is vanillin-bovine serum albumin (VAN-BSA); the reaction microwell plate contains a freeze-dried fluorescently labeled anti-vanillin monoclonal antibody, wherein the anti-vanillin monoclonal antibody is produced by a hybridoma cell line VAND8 with a deposit number of CCTCC NO.C 2023186, and the structural formula of vanillin-bovine serum albumin (VAN-BSA) is as follows:

[0007]

[0008] The above-mentioned hybridoma cell line VAND8 was deposited in the China Center for Type Culture Collection (CCTCC) on September 20, 2023. The deposit address is Wuhan University, Wuhan, China. The deposit number is CCTCC NO.C 2023186, and the classification name is mouse hybridoma cell VAND8, Hybridoma cell line VAND8. According to the above scheme, the specific preparation steps of the vanillin complete antigen are: weigh vanillin amine hydrochloride and react with maleic anhydride at room temperature under the catalysis of triethylamine to obtain a hapten: (2Z)-4-[(4-hydroxy-3-methoxybenzyl)amino]-4-oxobut-2-enoic acid ((2Z)-4-[(4-hydroxy-3-methoxybenzyl)amino]-4-oxobut-2-enoic acid), with a molecular formula of C 12 H 13 NO5, with a relative molecular mass of 251.2; the active ester method was used to synthesize the complete vanillin antigen VAN-BSA: the above hapten was subjected to oximation reaction with NH2OH, and then reacted with N-hydroxysuccinimide (NHS) in the dark under the action of dicyclohexylcarbodiimide (DCC), and the supernatant active ester solution was coupled with BSA to obtain the complete antigen VAN-BSA.

[0009] According to the above scheme, the absorbent pad is 20-25mm long and 3-5mm wide; the detection pad is 25-30mm long and 3-5mm wide; the sample pad is 20-25mm long and 3-5mm wide, and the overlapping length of adjacent pads is 1-2mm.

[0010] According to the above solution, the absorbent pad is absorbent paper.

[0011] According to the above scheme, the distance between the detection line on the detection pad and the upper edge of the nitrocellulose membrane is 10-20 mm, and the distance between the quality control line and the detection line is 5-15 mm.

[0012] According to the above scheme, the coating amount of vanillin complete antigen (VAN-BSA) required for each centimeter of the test line on the test pad is 100-250 ng; the coating amount of goat anti-mouse secondary antibody required for each centimeter of the quality control line is 50-300 ng.

[0013] According to the above scheme, the fluorescently labeled anti-vanillin monoclonal antibody is prepared according to the following method: the fluorescent labeling reagent is activated and mixed with the anti-vanillin monoclonal antibody, and shaken overnight to obtain the target product fluorescently labeled anti-vanillin monoclonal antibody. The fluorescent labeling material can be quantum dots, time-resolved microspheres, etc. with fluorescent labeling effects. The specific fluorescent labeling material used can be europium oxide latex or cadmium sulfide / cadmium telluride red light quantum dots modified silica microspheres, etc. The particle size of the fluorescent labeling material is 100-400nm.

[0014] According to the above scheme, the fluorescently labeled anti-vanillin monoclonal antibody is immobilized in the wells of the reaction microplate by freeze-drying process.

[0015] The method for preparing the fluorescent kit for rapid detection of vanillin as described above comprises the following steps:

[0016] Preparation of fluorescent test strips:

[0017] (1) Preparation of absorbent pad

[0018] Cut absorbent paper into absorbent pads;

[0019] (2) Preparation of detection pad

[0020] Test line coating:

[0021] Vanillin complete antigen (VAN-BSA) is prepared into a coating solution of 0.2-0.5 mg / mL, and the coating solution is sprayed on the position 10-20 mm from the upper edge of the nitrocellulose membrane by line spraying. The coating amount of vanillin complete antigen required per centimeter of the test line is 100-250 ng, and then dried at 37-42°C for 60-120 minutes;

[0022] Coating of quality control line:

[0023] Prepare the goat anti-mouse secondary antibody into a coating solution of 0.1-0.5 mg / mL, and spray the coating solution on the position 5-15 mm from the upper edge of the nitrocellulose membrane by line spraying. The coating amount of the goat anti-mouse secondary antibody required on the detection line is 50-300 ng, and then dry it at 37-42 ° C for 60-120 min;

[0024] (3) Preparation of sample pad

[0025] Soak the glass fiber mat in the blocking solution, take it out, dry it at 37-42°C for 10-16 hours to obtain a sample mat, and then store it in a desiccator at room temperature;

[0026] (4) Assembly of test strips

[0027] The absorbent pad, the detection pad and the sample pad are sequentially attached to the bottom plate from top to bottom, and the adjacent pads are overlapped and connected at the connection points to obtain a high-sensitivity immunochromatographic test strip for rapid detection of vanillin;

[0028] Preparation of reaction microplates

[0029] Dilute the fluorescently labeled anti-vanillin monoclonal antibody 100-500 times with diluent, and dispense it into the wells of the reaction microplate. The volume of each well is 100-200 μL. Place it in a vacuum freeze dryer for freeze drying. After completion, cover it with a moisture-proof cover to obtain the reaction microplate.

[0030] According to the above scheme, the coating solution is prepared according to the following method:

[0031] The coating solution for coating the antigen comprises: 0.5-1% bovine serum albumin, 1-2.5% sucrose, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, and water is added to make the volume to 100mL;

[0032] The sealing liquid composition of the glass fiber mat is: 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, 2% bovine serum albumin, 0.1% Triton 100, 0.3% polyvinylpyrrolidone (PVPK-30), 2.5% sucrose, 0.02% sodium azide;

[0033] The reaction microwell diluent is: 0.5-2% bovine serum albumin, 1-2.5% sucrose, 0.5-2% polyvinylpyrrolidone (PVPK-30), 0.5-1.5% Tween 20, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, and water is added to make the volume to 100mL.

[0034] Application of the above-mentioned fluorescent test strip for rapid detection of vanillin in the quantitative detection of vanillin:

[0035] After adding the sample solution to be tested into the wells of the reaction microplate and mixing, insert the fluorescent test paper strip into the wells of the reaction microplate so that a portion of the sample pad is immersed in the liquid of the reaction microplate. After a period of reaction, the test is performed using a time-resolved fluorescence tester to obtain the ratio of the time-resolved fluorescence intensity of the detection line on the fluorescent test paper strip to the time-resolved fluorescence intensity of the quality control line; based on the relationship curve of the ratio of the time-resolved fluorescence intensity of the detection line of the fluorescent test paper strip to the time-resolved fluorescence intensity of the quality control line and the vanillin concentration obtained in advance, the content of vanillin in the sample solution to be tested is obtained, and finally the content of vanillin in the sample to be tested is obtained by conversion.

[0036] According to the above scheme, the relationship curve between the ratio of the time-resolved fluorescence intensity of the detection line of the fluorescent test strip and the time-resolved fluorescence intensity of the quality control line and the concentration of vanillin is obtained by the following method:

[0037] (1) preparing a series of vanillin standard solutions with different concentrations;

[0038] (2) adding appropriate amounts of the above-mentioned vanillin standard solutions of various concentrations into the wells of the reaction microplate, mixing, inserting the fluorescent test strips into the wells of the reaction microplate, mixing, inserting the fluorescent test strips into the wells of the reaction microplate, reacting for a period of time, and using a time-resolved fluorescence immunoassay to obtain the time-resolved fluorescence intensity values ​​of the test line and the quality control line on each fluorescent test strip, thereby obtaining the ratio of the time-resolved fluorescence intensity of the test line of each fluorescent test strip to the time-resolved fluorescence intensity of the quality control line;

[0039] (3) The relationship curve between the ratio of the time-resolved fluorescence intensity of the detection line of the fluorescent test strip and the time-resolved fluorescence intensity of the quality control line and the concentration of vanillin was obtained by fitting.

[0040] The working principle of the fluorescent kit for rapid detection of vanillin is as follows: it is based on the specific binding of fluorescently labeled anti-vanillin monoclonal antibodies to the vanillin complete antigen coated on the detection line to generate a fluorescent signal, and when vanillin exists in the sample to be tested, the vanillin will compete with the vanillin complete antigen for binding to the anti-vanillin monoclonal antibody, so that the fluorescently labeled anti-vanillin monoclonal antibodies bound to the vanillin complete antigen are reduced, thereby the fluorescence intensity decreases with the increase of the vanillin content in the sample, and then based on the relationship between the ratio (T / C) of the fluorescence intensity of the detection line of the fluorescent test strip and the fluorescence intensity of the quality control line and the vanillin content, rapid and accurate detection of vanillin is achieved.

[0041] This patent is based on the high sensitivity and good specificity of anti-vanillin monoclonal antibody (its 50% inhibition concentration IC 50The cross-reaction rate with vanillin structural analogues was 22.1 μg / mL, the cross-reaction rate with ethyl vanillin was low or even no cross-reaction, the cross-reaction rate with ethyl vanillin was 21.65%, and the cross-reaction rate with methyl vanillin was less than 1%). A fluorescent kit for rapid detection of vanillin in edible oil was constructed, which has the characteristics of simple operation, wide application range and high sensitivity.

[0042] The beneficial effects of the present invention are:

[0043] (1) Detection of vanillin content. The fluorescent kit for rapid detection of vanillin provided by the present invention is used to detect the content of vanillin in edible oil, and has great practical application value.

[0044] (2) The sample pretreatment method is simple. The edible oil samples can be tested after extraction and concentration. The pretreatment time is 15 minutes. The entire sample pretreatment process is simple and fast.

[0045] (3) Simple operation. When using the immunochromatographic test strip for rapid detection of vanillin content, it is only necessary to add the sample extract dropwise onto the sample pad of the test strip. This is a one-step operation, does not require professional personnel, and is simple and convenient to operate.

[0046] (4) High sensitivity. The fluorescent kit for rapid detection of vanillin provided by the present invention is used for vanillin detection, and has high detection sensitivity of 0.8 μg / mL, which can meet the needs of vanillin detection in edible oil. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The structure schematic diagram of the fluorescent test strip for rapid detection of vanillin of the present invention is shown in the figure: 1 is a water absorption pad; 2 is a detection pad; 3 is a sample pad; 4 is a quality control line; 5 is a detection line; and 6 is a reaction microwell. Figure 2 The standard curve in Example 2 is obtained by using the logarithm of the vanillin concentration measured by the fluorescent test strip as the horizontal axis and the T / C signal value of the test strip as the vertical axis. Figure 3 The standard curve in Example 3 is the logarithm of the vanillin concentration measured by the fluorescent test strip as the horizontal axis and the T / C signal value of the test strip as the vertical axis. DETAILED DESCRIPTION

[0048] Example 1: Obtaining anti-vanillin monoclonal antibodies

[0049] The obtained hybridoma cell line VAND8 was injected into BALB / c mice treated with Freund's incomplete adjuvant in advance, and the ascites of the mice was collected, and then the ascites was purified to obtain anti-vanillin monoclonal antibodies. The specific operation is: the mouse ascites is filtered with double-layer filter paper, the filtered ascites is centrifuged at 4°C, 12000r / min for more than 15min, the supernatant is aspirated, the supernatant is mixed with 3 times the volume of acetate buffer, and the pH is adjusted to 4.5-4.8 with 2mol / L HCl, and then octanoic acid is slowly added while stirring. The volume of octanoic acid required for each milliliter of ascites is 33μL. After adding, stir at room temperature for 30min and stand at 10°C for more than 4h. After standing, centrifuge at 4°C, 12000r / min for 30min, discard the precipitate and take the supernatant to filter with double-layer filter paper, and the filtered antibody filtrate is concentrated by ultrafiltration membrane in an ice bath. After determining the IgG content of the concentrate, the concentrate is freeze-dried in a vacuum and the lyophilized powder is collected to obtain anti-vanillin monoclonal antibody powder.

[0050] The acetate buffer solution is formulated as follows: 0.29 g sodium acetate, 0.141 mL acetic acid, and pure water to 100 mL;

[0051] The 0.01 mol / L phosphate buffer solution is 0.8 g sodium chloride, 0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g potassium chloride, and 0.02 g potassium hydrogen phosphate, and water is added to make the volume to 1000 mL.

[0052] The titer of VAND8 mouse ascites antibody measured by conventional non-competitive enzyme-linked immunosorbent assay (ELISA) was 3.2×10 5 , i.e. mouse ascites antibody dilution 3.2×10 5 The results of the solution assay were positive. The sensitivity of the vanillin was 22.1 μg / mL using the conventional indirect competitive ELISA method, the cross-reaction rate with ethyl vanillin was 21.65%, and the cross-reaction rate with methyl vanillin was less than 1%.

[0053] The subtype of the anti-vanillin monoclonal antibody secreted by the hybridoma cell line VAND8 was identified as IgG2a using a commercially available subtype identification kit.

[0054] Screening of hybridoma cell line VAND8

[0055] 1. Antigen synthesis and animal immunization

[0056] The complete antigen synthesis was performed using vanillin ammonium salt, and the specific synthesis steps were as follows: 1 g (about 0.005 mol) of vanillin amine hydrochloride was weighed and dissolved in 10 mL of tetrahydrofuran, and 0.6 g (about 0.006 mol) of triethylamine was added dropwise with stirring, and stirred at room temperature for 45 min. 0.15 g (0.0015 mol) of maleic anhydride was weighed and added to the above reaction solution, and the reaction was stirred at room temperature for 7 to 8 hours, and the raw material point disappeared when monitored by TLC (developing solvent is petroleum ether: ethyl acetate = 2:1). After the reaction solution was dried by rotary evaporation at 40°C, 30 mL of water and 50 mL of ethyl acetate were added to the reaction bottle, and the mixture was magnetically stirred at room temperature for about 10 min. The mixture was allowed to stand for 15 min until the solution was separated into two layers. The ethyl acetate layer was rotary evaporated and dried to obtain a crude white powder of vanillin hapten. The crude product was recrystallized from 10 mL of anhydrous ethanol, i.e., the artificial vanillin hapten (2Z)-4-[(4-hydroxy-3-methoxybenzyl)amino]-4-oxobut-2-enoic acid, with a molecular formula of C 12 H 13 NO5, relative molecular mass is 251.2.

[0057] Weigh 200 mg of VAN hapten powder (about 0.8 mmol) into a reaction bottle, add 5 mL of DMF to dissolve, weigh 35 mg (about 0.5 mmol) of hydroxylamine hydrochloride into the reaction bottle. Reflux in an oil bath for 20 min, then cool for use. Weigh 120 mg (about 1 mmol) of NHS into the reaction bottle, stir at room temperature for 1 h, weigh 200 mg (about 1 mmol) of DCC in 2 mL of DMF, add the DCC / DMF solution dropwise to the above reaction bottle, stir at room temperature in the dark for 6 h, and stand at 4 ° C in the dark overnight. Centrifuge at 8000 r / min for 5 min, take the supernatant active ester solution and add it dropwise to 30 mL of 7 mg / mL BSA solution for reaction, and the reaction buffer is 0.05 mol / L pH 9.6 carbonate buffer. The reaction is stirred at room temperature in the dark for 6 h under magnetic stirring. The reaction solution was placed in a dialysis bag and dialyzed in 0.01 mol / L pH 7.4 PBS at 4°C with stirring, and the dialysis solution was replaced every 4 hours for a total of 48 hours. After the dialysis, the vanillin artificial complete antigen immune antigen VAN-BSA was obtained.

[0058] Three 6-week-old female BALB / c mice were purchased and immunized with complete antigen VAN-BSA. For the first immunization, the complete antigen VAN-BSA was mixed with an equal amount of Freund's complete adjuvant and emulsified, and then injected subcutaneously at multiple points on the back of the mouse's neck. The second immunization was performed 3 weeks after the first immunization, using Freund's incomplete adjuvant and an equal volume of complete antigen VAN-BSA emulsified and injected subcutaneously at multiple points on the back of the mouse's neck. The third immunization was 2 weeks apart from the second immunization, and the immunization method and dose were the same as the second. The fourth immunization was performed 2 weeks after the third immunization, and the immunization method and immunization dose were the same as the second immunization, and each immunization dose was 100 μg per mouse. One week after the third immunization, blood was collected from the tail vein, serum was separated, and the mouse serum antibody titer was monitored by indirect ELISA. One week after the fourth immunization, blood was collected from the tail vein and serum was separated. The indirect ELISA method was used to monitor the mouse serum antibody titer, and the indirect competitive ELISA method was used to determine the mouse serum sensitivity. Mice corresponding to sera with relatively high titer and sensitivity were selected for the final booster immunization. The immunization dose was twice that of the previous one. The VAN-BSA antigen was diluted to 500 μg / mL with 0.01 mol / L PBS, and 0.2 mL was injected intraperitoneally into each mouse.

[0059] 2. Cell Fusion

[0060] (1) Preparation of myeloma cells (SP2 / 0)

[0061] First, take out the SP2 / 0 myeloma cells from liquid nitrogen for resuscitation. Quickly place the cryopreserved tube containing SP2 / 0 myeloma cells in a 37°C water bath and shake it continuously to thaw it quickly. Then take it to the clean bench and pour it into a 50mL centrifuge tube containing 15mL PRMI-1640 basal culture medium. Gently mix it and centrifuge it at 1000r / min for 5 minutes, and discard the supernatant. Then resuspend the myeloma cells with 30mL RPMI-1640 complete culture medium and transfer them to a cell culture bottle for culture. Culture in a 37°C 5% CO2 constant temperature incubator for 3 to 5 days. When the myeloma cells almost fill the entire culture bottle and are in good condition, the myeloma cells can be collected into a centrifuge tube, centrifuged at 1000r / min for 5 minutes, and then resuspend the cells with 10mL RPMI-1640 basal culture medium and count them. The number of SP2 / 0 myeloma cells is approximately 2 to 5×10 7 indivual.

[0062] (2) Preparation of immune spleen cells

[0063] After the boosted immunization mice were killed by dislocation, they were soaked in 75% alcohol for 5 minutes. The mice were moved to the clean bench, with the abdomen facing up, and the limbs were fixed on the plastic foam board covered with sterilized newspapers with syringe needles. The mouse peritoneum was cut open with medical scissors, and the mouse spleen was peeled off and removed with tweezers. The separated spleen was placed in a plate with RPMI-1640 base culture medium added in advance, a 2mL syringe was taken, and the spleen was evenly pierced with a syringe needle, and then gently ground with the inner core of the syringe to release the spleen cells from the spleen outer membrane. The fat and connective tissue were filtered through a 300-mesh screen, and the RPMI-1640 base culture medium with dispersed spleen cells was filtered into a centrifuge tube, centrifuged at 1000r / min for 5min, and the supernatant was discarded. Resuspended in 40mL RPMI-1640 base culture medium and centrifuged again at 1000r / min for 5min, repeated three times, and the red blood cells and connective tissue in the cells were washed away. Finally, the spleen cells were resuspended in 10 mL RPMI-1640 medium and counted. The number of spleen cells was about 2 to 5 × 10 8 indivual.

[0064] (3) Cell fusion

[0065] Mix the above myeloma cells and spleen cells at a ratio of 1:5-10, centrifuge at 1000r / min for 5 minutes, pour out the supernatant gently, turn it upside down on a square absorbent paper to remove the liquid around the tube mouth, and then gently insert the strip of absorbent paper into the tube to absorb the remaining liquid on the tube wall and the bottom of the tube. Immerse the bottom of the centrifuge tube in a 37℃ water bath, incubate for 1 minute, and slowly add 1mL 1500PEG to the mixed cells, stirring gently while adding to disperse the cell clusters. The centrifuge tube is placed in a 37℃ water bath environment during the whole process. After adding PEG, continue to gently shake the centrifuge tube for 1 minute. Then slowly add 1mL of RPMI-1640 basal medium incubated at 37℃ within 1min, stirring while adding. Then slowly add 3mL of RPMI-1640 basal medium incubated at 37℃ within 3min, stirring while adding. Continue to slowly add 15mL of RPMI-1640 basal medium incubated at 37℃, stirring gently while adding. After adding, cover the lid, gently invert and mix several times, and finally put it in a 37℃ incubator and let it stand for 5min. Centrifuge at 1000r / min for 5min, pour out the supernatant, resuspend with 15mL RPMI-1640 basal culture solution, centrifuge again at 1000r / min for 5min, pour out the supernatant and wash off the PEG. Finally, gently resuspend the fused cells in 150mL of HAT medium. Add the HAT medium containing fused cells to a 96-well cell culture plate, 150μL per well, and place it in a 37℃ 5% CO2 constant temperature incubator for culture.

[0066] 3. Screening and cloning of cell lines

[0067] Around the 12th day after cell fusion, when the cell colonies grow to occupy 1 / 2 of the area at the bottom of the well and the culture fluid turns yellow, antibody detection can be performed. The culture wells where hybridoma cells grow are screened using the ELISA method. The screening is carried out in two steps. The first step is to use the indirect ELISA method to screen the positive wells that are resistant to vanillin but not to the carrier protein BSA. The second step is to use the indirect competitive ELISA method to detect the positive wells screened in the first step, using vanillin as the competitor, and selecting wells with higher absorbance and sensitivity (higher absorbance means that the final measured value of the well with a competitor of 0 is higher, that is, the negative control well, and higher sensitivity means that the competitor concentration when the inhibition rate is 50%, that is, the IC 50 The value was small), and the limiting dilution method was used for cloning. The same two-step method was used for detection about 10 days after cloning. After repeating the cloning 2-3 times, the hybridoma cell line VAND8 was obtained.

[0068] 4. Determination of variable region sequence of anti-vanillin monoclonal antibody:

[0069] (1) Cultivate hybridomas. After cell recovery, the hybridoma cell line VAND8 was cultured until the cell number expanded to about 1x10 7When the cells were centrifuged at 1000r / min for 5min, they were collected. (2) Extracting cellular RNA. Under a clean bench environment, 1mL of Trizol reagent was added to the centrifuged cells, allowed to stand for 5min, 2mL of chloroform was added, and the mixture was shaken vigorously for 15s. The mixture was allowed to stand for 3min at room temperature, and centrifuged at 12000r / min for 15min. The upper aqueous layer was transferred to a new EP tube, and 0.5mL of isopropanol was added. The mixture was allowed to stand for 10min at room temperature. Centrifuged at 12000r / min for 10min. The supernatant was discarded, 1mL of 75% ethanol was added, and the mixture was centrifuged at 7500r / min for 5min. The precipitate was dried and 50μL of double distilled water was added. The purity was identified and quantified by agarose electrophoresis, and the mixture was stored at -70℃ for later use. (3) Reverse transcription was used to prepare cDNA. Total cell RNA 1μL, RNaseFree ddH2 0 6μL, oligo dT Primer 0.5μL, PRIME Script RTEnzyme Mix 10.5μL, 5xPrime Script Buffer 2μL, mix well, 37℃15min, 85℃, 5s. (4) Amplify cDNA. Use mouse IgG VH VL primer library to amplify the above cDNA respectively. 5x Prime Star Buffer 10μL, dNTP 4pL, cDNA1μL, upstream primer 1pL, downstream primer 1μL, PrimeSTAR 0.5μL, add water to 50μL, and perform PCR reaction according to the following reaction conditions: incubate at 94℃ for 5min, denature at 94℃ for 45s, anneal at 63℃ for 45s, extend at 72℃ for 1min, and extend at 72℃ for 10min after 30 cycles. (5) Agarose gel electrophoresis and gel recovery. The PCR products were subjected to agarose gel electrophoresis, and the electrophoresis results were observed. The amplified products with a molecular weight of 250-350 bp were sent for sequencing. The sequencing was performed by Wuhan Dai'an Technology Co., Ltd.

[0070] The heavy chain sequencing results are:

[0071] GAGGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAA

[0072] CTCTCCTGTGCAGCCTCT GGATTCATTTTCAGTACTTATTAC ATGTCTTGGGTTCGCCAG

[0073] ACTCCAGAGAAGAGGCTGGAGTTGGTCGCAACC ATTCATGGTAATGGTAATAGCATC TA

[0074] CTATCTAGACAGTGTGAAGGGTCGATTCGCCATCTCCAGAGACAATGCCAAGAACACT

[0075] CTGTACCTGCAGATGAGCAGTCTGAAGTCTGAGGACACAGCCCTGTATTAC TGTGTAAG

[0076] ACATGATGGTTATTACGTGGACCATGCTATGGACTGC TGGGGTCAGGGAACCTCAGTCACCGTCTCCTCA, as shown in SEQ ID No.1.

[0077] Heavy chain variable region sequence:

[0078] EVKLVESGGGLVKPGGSLKLSCAAS GFIFSTYY MSWVRQTPEKRLELVAT IHGNGNSI YY

[0079] DSVKGRFAISRDNAKNTLYLQMSSLKSEDTALYY CVRHDGYYVDHAMDC WGQGTSVTV SS, as shown in SEQ ID No. 3, wherein:

[0080] CDR1:GFIFSTYY

[0081] CDR2:IHGNGNSI

[0082] CDR3:VRHDGYYVDHAMDC

[0083] The light chain sequencing results are:

[0084] GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTC

[0085] CATCTCTTGCAGATCTAGT CAGAGGATTGTACATGTTAATGGAAACACCTAT TTAGATTG

[0086] GTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTAATCTAC AAAGTTTCC AACCGA

[0087] TTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGGACAGATTTCACACTCA

[0088] AGATCAGCAGAGTGGCGGCTGAGGATCTGGGAGTTTATTACTGC TTTCAAGGTTCACATGTTCCTCGG ACGTTCGGTGGAGGCACCAGGCTGGAAATGAAA, the light chain variable region sequence as shown in SEQ ID No.2:

[0089] DVLMTQTPLSLPVSLGDQASISCRSS QRIVHVNGNTY LDWYLQKPGQSPKLLIY KVS NRFS GVPDRFSGSGSGTDFTLKISRVAAEDLGVYYC FQGSHVPRT FGGGTRLEMK, as shown in SEQ ID No. 4, wherein:

[0090] CDR1:QRIVHVNGNTY

[0091] CDR2:KVS

[0092] CDR3:FQGSHVPRT

[0093] The obtained gene sequence results showed that the heavy chain variable region encoding gene sequence was 363 bp long, and the sequence was shown in SEQ ID NO: 1. According to the obtained gene sequence, it was deduced that the heavy chain variable region encoded by the gene sequence consisted of 121 amino acids, and the sequence was shown in SEQ ID NO: 3. The light chain variable region encoding gene sequence was 336 bp long, and the sequence was shown in SEQ ID NO: 2. According to the obtained gene sequence, it was deduced that the light chain variable region encoded by the gene sequence consisted of 112 amino acids, and the sequence was shown in SEQ ID NO: 4.

[0094] Example 2: A method for preparing a fluorescent test strip (europium oxide fluorescence) for rapid detection of vanillin, comprising the following steps:

[0095] (1) Preparation of absorbent pad

[0096] Cut the absorbent paper into 20mm*4.5mm width to make an absorbent pad;

[0097] (2) Preparation of detection pad

[0098] Test line coating:

[0099] Vanillin-bovine serum albumin (VAN-BSA) was prepared into a coating solution of 0.25 mg / mL, and the coating solution was horizontally coated on the nitrocellulose membrane by line spraying (speed: 1 μL / cm) at a position 20 mm away from the upper edge of the nitrocellulose membrane to obtain a test line. The coating amount of vanillin-bovine serum albumin required per centimeter of the test line was 112.5 ng, and then dried at 37°C for 90 minutes;

[0100] Coating of quality control line:

[0101] Prepare a 0.5 mg / mL coating solution of goat anti-mouse secondary antibody; coat the coating solution horizontally on the nitrocellulose membrane at a distance of 10 mm from the detection line by line spraying (speed: 0.6 μL / cm) to obtain a quality control line. The coating amount of goat anti-mouse secondary antibody required per cm of the quality control line is 135 ng, and then dry at 37°C for 90 minutes;

[0102] (3) Preparation of sample pad:

[0103] Soak the glass fiber membrane in the blocking solution, take it out, dry it at 37°C for 12 hours to obtain the sample pad, and then store it in a desiccator at room temperature;

[0104] (4) Assembly of test strips

[0105] 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, thereby obtaining a high-sensitivity immunochromatographic test strip for rapid detection of vanillin;

[0106] The coating buffer used in the coating solution for coating the antigen is: 1 g bovine serum albumin, 1% sucrose, 0.8 g sodium chloride, 0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g potassium chloride, 0.02 g potassium dihydrogen phosphate, and water is added to make up to 100 mL;

[0107] (5) Anti-vanillin monoclonal antibody coupled with europium oxide fluorescent latex:

[0108] Take 800μL of 0.2mol / L pH8.18 boric acid buffer, add 200μL of europium oxide fluorescent latex, disperse by ultrasound, add 40μL of 15mg / mL freshly prepared EDC solution, and shake for 15min. Then centrifuge at 12000r / min, 10℃ for 10min, discard the supernatant, remove EDC, etc., add 15μL of boric acid buffer containing 1mg / mL vanillin antibody, mix well, and shake on a shaker at 250r / min and 4℃ for 2 hours. Take out, centrifuge at 12000r / min, 10℃ for 10min, pour out the supernatant, add 1mL of 0.5% BSA boric acid buffer, shake and mix well, then put it in a shaker, shake at 250r / min, 20℃ for 1h, take it out and put it in a 4℃ refrigerator for use.

[0109] The fluorescently labeled anti-vanillin monoclonal antibody was diluted 300 times with a sustained-release solution, wherein the diluent is: 0.5% bovine serum albumin, 2% sucrose, 0.5% polyvinylpyrrolidone (PVPK-30), 0.5% Tween 20, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, and water was added to make the volume to 100mL; 200μL of the diluted fluorescently labeled anti-vanillin monoclonal antibody per well was added to the enzyme-labeled well of the reaction microplate, and the plate was placed in a vacuum freeze dryer and frozen at -40°C, and a moisture-proof cover was applied to obtain a sample reaction microplate.

[0110] (6) Edible oil sample pretreatment

[0111] Weigh 10g of oil sample and place it in a 50mL centrifuge tube. Add 10mL of methanol, shake vigorously for 20min, centrifuge at 6000rpm for 5min, collect the methanol layer and place it in another 50mL centrifuge tube. Add 10mL of methanol to the sample tube again, shake vigorously for 20min, centrifuge at 6000rpm for 5min, collect the methanol layer, and combine the two extracts. Blow the methanol until it is almost dry, and add 0.5mL of 20% methanol solution to re-dissolve.

[0112] (7) Establishment of the relationship curve between the ratio of the fluorescence intensity of the test line of the fluorescent test strip to the fluorescence intensity of the quality control line (T / C) and the concentration of vanillin:

[0113] 1. Pre-treat the edible oil samples that tested negative for vanillin by liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and spike vanillin to standard solutions with concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 10 μg / mL, 5 μg / mL, and 2 μg / mL.

[0114] 2. Take 100 μL of each of the above-mentioned vanillin standard solutions of each concentration, add them to the wells of the sample reaction microwells, mix them, insert the fluorescent test strips, react at 37°C for 7 minutes, use absorbent paper to absorb the residual liquid on the sample pad, and immediately detect with a time-resolved fluorescence immunoassay analyzer (excitation wavelength: 365 nm, measurement wavelength: 615 nm) to obtain the fluorescence intensity values ​​at the test line (T) and the quality control line (C) on each fluorescent test strip, thereby obtaining the ratio of the fluorescence intensity of the test line of each fluorescent test strip to the fluorescence intensity of the quality control line (T / C);

[0115] 3. Vanillin standard was added to the blank edible oil sample at concentrations of 50 μg / mL, 25 μg / mL, 12 μg / mL, 5 μg / mL, 2.5 μg / mL, 1 μg / mL, and 0.5 μg / mL. The logarithm of the vanillin concentration was measured with a fluorescent test strip as the abscissa and the T / C signal value of the test strip as the ordinate. The recovery rate of this method was between 82.7% and 119.3%, and the correlation coefficient of the standard curve was 0.9796 (R 2 )(See Figure 2 ).

[0116] 4. Select 5 portions of soybean oil, peanut oil, sesame oil, rapeseed oil and corn oil purchased from the market, extract and purify them according to the above pretreatment steps, and substitute them into the standard curve to calculate the vanillin content. The vanillin contents are: not detected, not detected, 2.4 μg / mL, 1.3 μg / mL, not detected.

[0117] Example 3: A method for preparing a fluorescent test strip (SiO2-CdTe / CdS fluorescent quantum dots) for rapid detection of vanillin, comprising the following steps:

[0118] (1) Preparation of absorbent pad

[0119] Cut the absorbent paper into 20mm*4mm width to make an absorbent pad;

[0120] (2) Preparation of detection pad

[0121] Test line coating:

[0122] Vanillin-bovine serum albumin was prepared into a coating solution of 0.4 mg / mL, and the coating solution was horizontally coated on the nitrocellulose membrane by line spraying (speed: 0.8 μL / cm) at a position 15 mm away from the upper edge of the nitrocellulose membrane to obtain a test line. The coating amount of vanillin-bovine serum albumin required per centimeter of the test line was 128 ng, and then dried at 37°C for 90 minutes;

[0123] Coating of quality control line:

[0124] Prepare a coating solution of 0.25 mg / mL of goat anti-mouse secondary antibody; coat the coating solution horizontally on the nitrocellulose membrane at a distance of 10 mm from the detection line by line spraying (speed: 1 μL / cm) to obtain a quality control line. The coating amount of goat anti-mouse secondary antibody required per cm of the quality control line is 100 ng, and then dry at 37°C for 90 minutes;

[0125] (3) Preparation of sample pad:

[0126] Soak the glass fiber membrane in the blocking solution, take it out, dry it at 37°C for 12 hours to obtain the sample pad, and then store it in a desiccator at room temperature;

[0127] (4) Assembly of test strips

[0128] 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, thereby obtaining a high-sensitivity immunochromatographic test strip for rapid detection of vanillin;

[0129] The coating buffer used in the coating solution for coating the antigen is: 1.5 g bovine serum albumin, 2% sucrose, 0.8 g sodium chloride, 0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g potassium chloride, 0.02 g potassium dihydrogen phosphate, and water is added to make up to 100 mL;

[0130] (5) Method for coupling anti-vanillin monoclonal antibody with fluorescent quantum dots:

[0131] Take 900uL of 0.05mmol MES buffer (pH 6.0), add 100uL SiO2-CdTe / CdS, disperse by ultrasonic, add 60uL of freshly prepared 20mg / ml NHS and EDC mixed solution, react at room temperature for 15min, centrifuge at 17000rpm for 20min, remove the supernatant, add 100μL 1mg / mL vanillin antibody in MES buffer, shake at 250r / min at 4℃ for 10 hours, centrifuge at 12000r / mn at 10℃ for 10min, discard the supernatant, add 1mL 0.5% BSAMES buffer, shake and mix well for use.

[0132] The fluorescently labeled anti-vanillin monoclonal antibody is diluted 500 times with a slow-release solution, wherein the diluent is: 1% bovine serum albumin, 2% sucrose, 1% polyvinylpyrrolidone (PVPK-30), 0.5% Tween 20, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, and water is added to make the volume 100mL; 300μL of the diluted fluorescently labeled anti-vanillin monoclonal antibody per well is added to the enzyme-labeled well, and the well is placed in a vacuum freeze dryer and frozen at -40°C, and a moisture-proof cover is applied to obtain a sample reaction microwell.

[0133] (6) Edible oil sample pretreatment

[0134] Weigh 10g of oil sample and place it in a 50mL centrifuge tube. Add 10mL of methanol, shake vigorously for 20min, centrifuge at 6000rpm for 5min, collect the methanol layer and place it in another 50mL centrifuge tube. Add 10mL of methanol to the sample tube again, shake vigorously for 20min, centrifuge at 6000rpm for 5min, collect the methanol layer, and combine the two extracts. Take 5mL of methanol extract and blow it to near dryness with nitrogen, then add 0.2mL of 20% methanol aqueous solution to re-dissolve.

[0135] (7) Establishment of the relationship curve between the ratio of the fluorescence intensity of the test line of the fluorescent test strip to the fluorescence intensity of the quality control line (T / C) and the concentration of vanillin:

[0136] 1. Pre-treat the edible oil samples that tested negative for vanillin by liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and spike vanillin to standard solutions with concentrations of 50 μg / mL, 25 μg / mL, 12 μg / mL, 5 μg / mL, 2.5 μg / mL, 1 μg / mL, and 0.5 μg / mL.

[0137] 2. Take 100 μL of each of the above-mentioned vanillin standard solutions of each concentration, add them to the sample reaction cup, mix them, insert the fluorescent test strip, react at 37°C for 7 minutes, use absorbent paper to absorb the residual liquid on the sample pad, and use a fluorescence reader to detect the fluorescence intensity values ​​at the test line (T) and the quality control line (C) on each fluorescent test strip, thereby obtaining the ratio of the fluorescence intensity of the test line of each fluorescent test strip to the fluorescence intensity of the quality control line (T / C);

[0138] 3. Vanillin standard was added to the blank edible oil sample at concentrations of 50 μg / mL, 25 μg / mL, 12 μg / mL, 5 μg / mL, 2.5 μg / mL, 1 μg / mL, and 0.5 μg / mL. The logarithm of the vanillin concentration was measured with a fluorescent test strip as the abscissa and the T / C signal value of the test strip as the ordinate. The recovery rate of this method was between 86.1% and 115.3%, and the correlation coefficient of the standard curve was 0.9961 (R 2 ), see Figure (3).

[0139] 4. Select 3 portions of soybean oil, peanut oil and sesame oil purchased from the market, extract and purify them according to the above pretreatment steps, and bring them into the standard curve to calculate their vanillin contents. The vanillin contents were: not detected, not detected, and 2.4 μg / mL, respectively.

[0140] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. A fluorescent kit for rapid detection of vanillin in edible oil, characterized in that ::Including a fluorescent test strip and a reaction microplate, the fluorescent test strip includes a bottom plate, the bottom plate 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 quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom, the quality control line is coated with a sheep anti-mouse secondary antibody, and the detection line is coated with a vanillin complete antigen, and the vanillin complete antigen is vanillin-bovine serum albumin (VAN-BSA); the reaction microplate contains a freeze-dried fluorescently labeled anti-vanillin monoclonal antibody, and the anti-vanillin monoclonal antibody is produced by a hybridoma cell line VAND8 with a deposit number of CCTCC NO.C 2023186, and the structural formula of vanillin-bovine serum albumin (VAN-BSA) is as follows:

2. The fluorescence kit according to claim 1, characterized in that: The absorbent pad is 20-25mm long and 3-5mm wide; the detection pad is 25-30mm long and 3-5mm wide; the sample pad is 20-25mm long and 3-5mm wide, and the overlapping length of adjacent pads is 1-2mm; the distance between the detection line on the detection pad and the upper edge of the nitrocellulose membrane is 10-20mm, and the distance between the quality control line and the detection line is 5-15mm.

3. The fluorescence kit according to claim 1, characterized in that: The coating amount of vanillin complete antigen (VAN-BSA) required for each centimeter of the detection line on the detection pad is 100-250 ng; the coating amount of goat anti-mouse secondary antibody required for each centimeter of the quality control line is 50-300 ng.

4. The fluorescence kit according to claim 1, characterized in that: The fluorescently labeled anti-vanillin monoclonal antibody is prepared according to the following method: activating the fluorescent labeling reagent and mixing it with the anti-vanillin monoclonal antibody, shaking overnight, and obtaining the target product, the fluorescently labeled anti-vanillin monoclonal antibody.

5. The fluorescence kit according to claim 1, characterized in that: The fluorescent labeling material is a quantum dot or a time-resolved microsphere having a fluorescent labeling effect, and the particle size of the fluorescent labeling material is 100-400 nm.

6. The fluorescent kit according to claim 1, characterized in that: The fluorescently labeled anti-vanillin monoclonal antibody was immobilized in the wells of the reaction microplate by freeze-drying process.

7. The method for preparing the fluorescent kit for rapid detection of vanillin according to claim 1, characterized in that: The following steps are involved: Preparation of fluorescent test strips: (1) Preparation of absorbent pad Cut absorbent paper into absorbent pads; (2) Preparation of detection pad Test line coating: Vanillin complete antigen (VAN-BSA) is prepared into a coating solution of 0.2-0.5 mg / mL, and the coating solution is sprayed on the position 10-20 mm from the upper edge of the nitrocellulose membrane by line spraying. The coating amount of vanillin complete antigen required per centimeter of the test line is 100-250 ng, and then dried at 37-42°C for 60-120 minutes; Coating of quality control line: Prepare the goat anti-mouse secondary antibody into a coating solution of 0.1-0.5 mg / mL, and spray the coating solution on the position 5-15 mm from the upper edge of the nitrocellulose membrane by line spraying. The coating amount of the goat anti-mouse secondary antibody required on the detection line is 50-300 ng, and then dry it at 37-42 ° C for 60-120 min; (3) Preparation of sample pad Soak the glass fiber mat in the blocking solution, take it out, dry it at 37-42°C for 10-16 hours to obtain a sample mat, and then store it in a desiccator at room temperature; (4) Assembly of test strips The absorbent pad, the detection pad and the sample pad are sequentially attached to the bottom plate from top to bottom, and the adjacent pads are overlapped and connected at the connection points to obtain a high-sensitivity immunochromatographic test strip for rapid detection of vanillin; Preparation of reaction microplate: Dilute the fluorescently labeled anti-vanillin monoclonal antibody 100-500 times with diluent, dispense into the wells, with a volume of 100-200 μL per well, put into a vacuum freeze dryer for freeze drying, and cover with a moisture-proof cover to obtain a reaction microplate.

8. Use of the fluorescent test strip for rapid detection of vanillin according to claim 1 in the quantitative detection of vanillin, characterized in that: After adding the sample solution to be tested into the wells of the reaction microplate and mixing, insert the fluorescent test paper strip into the wells of the reaction microplate so that a portion of the sample pad is immersed in the liquid of the reaction microplate. After a period of reaction, the test is performed using a time-resolved fluorescence tester to obtain the ratio of the time-resolved fluorescence intensity of the detection line on the fluorescent test paper strip to the time-resolved fluorescence intensity of the quality control line; based on the relationship curve of the ratio of the time-resolved fluorescence intensity of the detection line of the fluorescent test paper strip to the time-resolved fluorescence intensity of the quality control line and the vanillin concentration obtained in advance, the content of vanillin in the sample solution to be tested is obtained, and finally the content of vanillin in the sample to be tested is obtained by conversion.

9. The use according to claim 8, characterized in that: The relationship curve between the ratio of the time-resolved fluorescence intensity of the detection line of the fluorescent test strip and the time-resolved fluorescence intensity of the quality control line and the concentration of vanillin is obtained by the following method: (1) preparing a series of vanillin standard solutions with different concentrations; (2) adding appropriate amounts of the above-mentioned vanillin standard solutions of various concentrations into the wells of the reaction microplate, mixing, inserting the fluorescent test strips into the wells of the reaction microplate, mixing, inserting the fluorescent test strips into the wells of the reaction microplate, reacting for a period of time, and using a time-resolved fluorescence immunoassay to obtain the time-resolved fluorescence intensity values ​​of the test line and the quality control line on each fluorescent test strip, thereby obtaining the ratio of the time-resolved fluorescence intensity of the test line of each fluorescent test strip to the time-resolved fluorescence intensity of the quality control line; (3) The relationship curve between the ratio of the time-resolved fluorescence intensity of the detection line of the fluorescent test strip and the time-resolved fluorescence intensity of the quality control line and the concentration of vanillin was obtained by fitting.

Images