Fluorescent kit for rapid detection of vanillin in edible oil, preparation and application thereof

Abstract

The application belongs to the field of immune detection, and specifically discloses a fluorescent kit for rapidly detecting vanillin in edible oil, preparation and application thereof. The fluorescent kit comprises a fluorescent test strip and a reaction microplate. The fluorescent test strip comprises a bottom plate, and the bottom plate is sequentially pasted with a water absorption pad, a detection pad and a sample pad from top to bottom. Adjacent pads are connected by overlapping at the connection. The detection pad takes nitrocellulose membrane as a base pad. The nitrocellulose membrane is provided with a quality control line and a detection line from top to bottom. The quality control line is coated with a goat anti-mouse secondary antibody, and the detection line is coated with vanillin complete antigen. The vanillin complete antigen is vanillin-bovine serum albumin (VAN-BSA). The reaction microplate is provided with freeze-dried fluorescently labeled anti-vanillin monoclonal antibody. The anti-vanillin monoclonal antibody is produced by a hybridoma cell strain VAND8 with a preservation number of CCTCC NO.C 2023186. The fluorescent kit can be used for detecting the content of vanillin in edible oil, and has the characteristics of rapid detection, simple operation and high sensitivity.

Description

Technical Field

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

[0002] Vanillin is a common food additive as a flavoring. Although it is not toxic, excessive consumption can also harm human health. Severe cases 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) explicitly 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-based 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 key areas where vanillin content monitoring is required.

[0003] Existing literature reports that the detection methods of vanillin in food include high performance liquid chromatography, high performance liquid chromatography-tandem mass spectrometry, gas chromatography, gas chromatography-tandem mass spectrometry, spectrophotometry, etc. These methods are highly sensitive and have accurate detection results, but the required instruments and equipment are expensive and the sample pretreatment process is cumbersome. Immunoassay is a method of qualitative and quantitative detection of ultra-trace residues by utilizing 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 a high signal-to-noise ratio, and improve sensitivity. Summary of the Invention

[0004] The present invention addresses the technical problem of the lack of on-site rapid detection technology for vanillin in existing technologies. The present invention provides a fluorescent kit for rapid detection of vanillin in edible oils, its preparation, and its application. The kit can be used to detect the vanillin content in edible oils, and features 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 oils comprises a fluorescent test strip and a reaction microplate. The fluorescent test strip comprises a base plate, on which a water-absorbing pad, a detection pad, and a sample pad are sequentially attached from top to bottom, with adjacent pads overlapping and connected at the joints. The detection pad is based on a nitrocellulose membrane, on which a quality control line and a detection line are arranged from top to bottom. The quality control line is coated with a goat anti-mouse secondary antibody, and the detection line is coated with a complete vanillin antigen, which is vanillin-bovine serum albumin (VAN-BSA). The reaction microplate contains a freeze-dried fluorescently labeled anti-vanillin monoclonal antibody produced by the hybridoma cell line VAND8 with a deposit number of CCTCC NO.C 2023186. 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.

[0009] According to the above scheme, the specific preparation steps of the vanillin complete antigen are as follows: vanillin amine hydrochloride is weighed and reacted 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 vanillin complete antigen VAN-BSA was synthesized by the active ester method: the above-mentioned hapten was subjected to an 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.

[0010] 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.

[0011] According to the above solution, the absorbent pad is absorbent paper.

[0012] According to the above solution, 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.

[0013] 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.

[0014] According to the above scheme, the fluorescently labeled anti-vanillin monoclonal antibody is prepared by the following method: activating a fluorescent labeling reagent, mixing it with the anti-vanillin monoclonal antibody, and shaking overnight to obtain the target fluorescently labeled anti-vanillin monoclonal antibody. The fluorescent labeling material can be quantum dots or time-resolved microspheres with fluorescent labeling effects. Specifically, the fluorescent labeling material used can be europium oxide latex or cadmium sulfide / cadmium telluride red quantum dot-modified silica microspheres. The particle size of the fluorescent labeling material is 100-400 nm.

[0015] According to the above scheme, the fluorescently labeled anti-vanillin monoclonal antibody was immobilized in the wells of the reaction microplate using a freeze-drying process.

[0016] The method for preparing the fluorescent kit for rapid detection of vanillin as described above comprises the following steps:

[0017] Preparation of fluorescent test strips:

[0018] (1) Preparation of absorbent pad

[0019] Cut absorbent paper into absorbent pads;

[0020] (2) Preparation of detection pad

[0021] Test line coating:

[0022] Prepare a 0.2-0.5 mg / mL coating solution of vanillin complete antigen (VAN-BSA) and spray the coating solution onto a nitrocellulose membrane 10-20 mm from the top edge using a line spray method. The required coating amount of vanillin complete antigen per centimeter of the test line is 100-250 ng. Then dry at 37-42°C for 60-120 minutes.

[0023] Coating of quality control line:

[0024] Prepare a 0.1-0.5 mg / mL coating solution of goat anti-mouse secondary antibody and spray the coating solution onto the nitrocellulose membrane 5-15 mm from the top edge using a line spray method. The coating amount of goat anti-mouse secondary antibody required on the detection line is 50-300 ng. Then dry it at 37-42°C for 60-120 min.

[0025] (3) Preparation of sample pad

[0026] Soak the glass fiber mat in the blocking solution, take it out, dry it at 37-42°C for 10-16 hours to obtain the sample mat, and then store it in a desiccator at room temperature;

[0027] (4) Assembly of test strips

[0028] 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 joints to obtain a high-sensitivity immunochromatographic test strip for rapid detection of vanillin;

[0029] Preparation of reaction microplates

[0030] Dilute the fluorescently labeled anti-vanillin monoclonal antibody 100-500 times with diluent, and dispense it into the wells of a reaction microplate. The volume of each well is 100-200 μL. Place the well in a vacuum freeze dryer for freeze drying. After completion, cover it with a moisture-proof cover to obtain a reaction microplate.

[0031] According to the above scheme, the coating solution is prepared according to the following method:

[0032] 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 up to 100mL;

[0033] The sealing solution of the glass fiber mat comprises: 0.8 g sodium chloride, 0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g potassium chloride, 0.02 g potassium dihydrogen phosphate, 2% bovine serum albumin, 0.1% Triton 100, 0.3% polyvinylpyrrolidone (PVPK-30), 2.5% sucrose, and 0.02% sodium azide;

[0034] 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 up to 100mL.

[0035] Application of the above-mentioned fluorescent test strip for rapid detection of vanillin in the quantitative detection of vanillin:

[0036] After adding the sample solution to be tested into the wells of the reaction microplate and mixing, a fluorescent test strip is inserted 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, detection is performed using a time-resolved fluorescence tester to obtain a ratio of the time-resolved fluorescence intensity of the test line on the fluorescent test strip to the time-resolved fluorescence intensity of the quality control line; based on a pre-obtained relationship curve between the ratio of the time-resolved fluorescence intensity of the test line on the fluorescent test strip to the time-resolved fluorescence intensity of the quality control line and the vanillin concentration, 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 through conversion.

[0037] 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 to the time-resolved fluorescence intensity of the quality control line and the vanillin concentration is obtained by the following method:

[0038] (1) preparing a series of vanillin standard solutions;

[0039] (2) adding appropriate amounts of the above-mentioned vanillin standard solutions of each concentration into the wells of the reaction microplate, mixing them evenly, inserting the fluorescent test strips into the wells of the reaction microplate, mixing them evenly, inserting the fluorescent test strips into the wells of the reaction microplate, reacting for a period of time, and detecting the time-resolved fluorescence intensity values ​​of the test line and the quality control line on each fluorescent test strip using a time-resolved fluorescence immunoassay, 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;

[0040] (3) The relationship curve between the ratio of the time-resolved fluorescence intensity of the detection line of the fluorescent test strip to the time-resolved fluorescence intensity of the quality control line and the concentration of vanillin was obtained by fitting.

[0041] The working principle of the fluorescent kit for rapid vanillin detection 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 test line to generate a fluorescent signal. When vanillin is present in the sample to be tested, the vanillin will compete with the vanillin complete antigen for binding to the anti-vanillin monoclonal antibody, resulting in a decrease in the number of fluorescently labeled anti-vanillin monoclonal antibodies bound to the vanillin complete antigen. As a result, the fluorescence intensity decreases as the vanillin content in the sample increases. Based on the relationship between the ratio of the fluorescence intensity of the test line to the fluorescence intensity of the quality control line (T / C) on the fluorescent test strip and the vanillin content, rapid and accurate detection of vanillin is achieved.

[0042] This patent is based on the highly sensitive and specific anti-vanillin monoclonal antibody produced by the hybridoma cell line VAND8 with the deposit number CCTCC NO.C 2023186 (its 50% inhibitory concentration IC 50The concentration of vanillin in edible oil was 22.1 μg / mL, with low or even no cross-reaction rate with vanillin structural analogs, 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.

[0043] The beneficial effects of the present invention are:

[0044] (1) Detection of vanillin content. The fluorescent kit for rapid detection of vanillin provided by the present invention is used to detect the vanillin content in edible oils, and has great practical application value.

[0045] (2) The sample pretreatment method is simple. The edible oil sample can be tested after extraction and concentration. The pretreatment time is 15 minutes. The entire sample pretreatment process is simple and fast.

[0046] (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 to the sample pad of the test strip. This is a one-step operation that does not require professional personnel and is simple and convenient to operate.

[0047] (4) High sensitivity. The fluorescent kit for rapid detection of vanillin provided by the present invention is used for vanillin detection, and has a high detection sensitivity of 0.8 μg / mL, which can meet the needs of vanillin detection in edible oils. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a schematic diagram of the structure of a fluorescent test strip for rapid detection of vanillin according to the present invention. In the figure: 1 is an absorbent 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.

[0049] 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.

[0050] Figure 3 The standard curve in Example 3 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. DETAILED DESCRIPTION

[0051] Example 1: Acquisition of anti-vanillin monoclonal antibodies

[0052] The obtained hybridoma cell strain VAND8 is injected into BALB / c mice which are pretreated with Freund's incomplete adjuvant, and the ascites of the mice is collected, and then the anti-vanillin monoclonal antibody is purified from the ascites. The specific operation is as follows: the mouse ascites is filtered with double-layer filter paper, the filtered ascites is centrifuged at 12000 r / min for more than 15 min at 4°C, the supernatant is taken, the supernatant is mixed with 3 times volume of acetate buffer, then the pH is adjusted to 4.5-4.8 with 2 mol / L HCl, and then n-octanoic acid is slowly added while stirring, the volume of n-octanoic acid required per milliliter of ascites is 33 μL, after the addition, the solution is stirred at room temperature for 30 min, and then is placed at 10°C for more than 4 h. After standing, the solution is centrifuged at 12000 r / min for 30 min at 4°C, the precipitate is discarded, and the supernatant is filtered with double-layer filter paper, and then the filtered antibody solution is concentrated by ultrafiltration membrane in ice bath. After the IgG content of the concentrated solution is determined, the concentrated solution is frozen and vacuum dried to collect the freeze-dried powder, and then the anti-vanillin monoclonal antibody powder is obtained.

[0053] The formula of the acetate buffer is as follows: 0.29 g of sodium acetate is added into 0.141 mL of acetic acid, and then the volume is made up to 100 mL with pure water.

[0054] The 0.01 mol / L phosphate buffer is as follows: 0.8 g of sodium chloride, 0.29 g of dodecahydrate disodium hydrogen phosphate, 0.02 g of potassium chloride, 0.02 g of potassium hydrogen phosphate, and water are added to make the volume 1000 mL.

[0055] The titer of the mouse ascites antibody of VAND8 is 3.2 x 10 5 , i.e. the solution of the mouse ascites antibody diluted 3.2 x 10 5 times is determined to be positive. The sensitivity of the anti-vanillin monoclonal antibody to vanillin is 22.1 μg / mL by the conventional indirect competitive ELISA method, the cross-reactivity with ethyl vanillin is 21.65%, and the cross-reactivity with methyl vanillin is less than 1%.

[0056] The subtype of the anti-vanillin monoclonal antibody secreted by the hybridoma cell strain VAND8 is IgG2a by using the commercially available subtype identification kit.

[0057] Screening of the hybridoma cell strain VAND8

[0058] 1. Antigen synthesis and animal immunization

[0059] Complete antigen synthesis was performed using vanillin ammonium salt. The specific synthesis steps are as follows: Weigh 1g (approximately 0.005mol) of vanillin amine hydrochloride and dissolve it in 10mL of tetrahydrofuran. Stir and dropwise add 0.6g (approximately 0.006mol) of triethylamine. Stir at room temperature for 45 minutes. Weigh 0.15g (0.0015mol) of maleic anhydride and add it to the reaction mixture. Stir at room temperature for 7-8 hours. TLC (developing solvent: petroleum ether:ethyl acetate = 2:1) indicates the disappearance of the starting material spot. The reaction solution was rotary evaporated to dryness at 40°C, and then 30 mL of water and 50 mL of ethyl acetate were added to the reaction flask. The mixture was magnetically stirred at room temperature for about 10 minutes, and allowed to stand for 15 minutes until the solution separated into two layers. The ethyl acetate layer was rotary evaporated to dryness to obtain a crude white powder of vanillin hapten. The crude product was recrystallized from 10 mL of anhydrous ethanol, namely, the vanillin artificial 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.

[0060] Weigh 200 mg of VAN hapten powder (approximately 0.8 mmol) into a reaction flask and dissolve it in 5 mL of DMF. Weigh 35 mg (approximately 0.5 mmol) of hydroxylamine hydrochloride into the flask. Reflux the mixture in an oil bath for 20 minutes, then cool it for later use. Separately weigh 120 mg (approximately 1 mmol) of NHS and add it to the flask. Stir and react at room temperature for 1 hour. Weigh 200 mg (approximately 1 mmol) of DCC in 2 mL of DMF and add the DCC / DMF solution dropwise to the reaction flask. Stir at room temperature in the dark for 6 hours and then stand at 4°C in the dark overnight. Centrifuge at 8000 rpm for 5 minutes. The supernatant active ester solution is added dropwise to 30 mL of 7 mg / mL BSA solution in 0.05 mol / L carbonate buffer (pH 9.6). The reaction is allowed to proceed at room temperature in the dark for 6 hours 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. The dialysis solution was changed every 4 hours for a total of 48 hours. After the dialysis, the vanillin artificial complete antigen immune antigen VAN-BSA was obtained.

[0061] Purchase 3 six-week-old female BALB / c mice, immunize with complete antigen VAN-BSA. The first immunization emulsifies the complete antigen VAN-BSA with an equal amount of Freund's complete adjuvant, then subcutaneously injects multiple points on the back of the neck of the mouse. The second immunization is performed 3 weeks after the first immunization, emulsifies the complete antigen VAN-BSA with an equal volume of Freund's incomplete adjuvant, and subcutaneously injects multiple points on the back of the neck of the mouse. The third immunization is performed 2 weeks after the second immunization, and the immunization method and dose are the same as the second. The fourth immunization is performed 2 weeks after the third immunization, and the immunization method and dose are the same as the second. The dose of each immunization is 100 μg per mouse. One week after the third immunization, blood is collected from the tail vein, and serum is separated. The antibody titer of the mouse serum is monitored by indirect ELISA. One week after the fourth immunization, blood is collected from the tail vein, and serum is separated. The antibody titer of the mouse serum is monitored by indirect ELISA, and the sensitivity of the mouse serum is determined by indirect competitive ELISA. The mouse corresponding to the serum with relatively high titer and sensitivity is selected for the last boost, and the immunization dose is twice that of the previous one. VAN-BSA antigen is diluted with 0.01 mol / L PBS to 500 μg / mL, and 0.2 mL is injected intraperitoneally per mouse.

[0062] 2. Cell fusion

[0063] (1) Preparation of myeloma cells (SP2 / 0)

[0064] First, take the SP2 / 0 myeloma cells out of liquid nitrogen for recovery. Place the cryovial containing the frozen SP2 / 0 myeloma cells in a 37°C water bath and shake constantly to quickly thaw, then take it to the clean bench and pour it into a 50 mL centrifuge tube containing 15 mL of RPMI-1640 basic culture solution. Mix gently and centrifuge at 1000 r / min for 5 min, then discard the supernatant. Then resuspend the myeloma cells with 30 mL of RPMI-1640 complete medium and transfer them to a cell culture bottle for culture. Culture in a 37°C 5% CO2 incubator for 3-5 days. When the myeloma cells almost cover the entire culture bottle and are in good condition, collect them into a centrifuge tube, centrifuge at 1000 r / min for 5 min, then resuspend the cells with 10 mL of RPMI-1640 basic culture solution and count them. The number of SP2 / 0 myeloma cells is about 2-5 x 10 7

[0065] (2) Preparation of immunized spleen cells

[0066] ​After boosting, mice were sacrificed by dislocation and soaked in 75% alcohol for 5 minutes. The mice were placed in a laminar flow hood, facing upwards, with their limbs secured to a plastic foam board covered in sterile newspaper using syringe needles. The mouse peritoneum was cut open with medical scissors, and the spleen was removed using forceps. The isolated spleen was placed in a dish pre-filled with RPMI-1640-based culture medium. A 2 mL syringe was used to evenly puncture the spleen with the needle. The spleen was then gently triturated with the syringe plunger to release splenocytes from the outer membrane. Fat and connective tissue were removed by filtering through a 300-mesh sieve. The RPMI-1640-based culture medium containing the dispersed splenocytes was filtered into a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded. The cells were resuspended in 40 mL of RPMI-1640-based culture medium and centrifuged again at 1000 rpm for 5 minutes. This process was repeated three times to wash away red blood cells and connective tissue. 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.

[0067] (3) Cell fusion

[0068] Mix the above-mentioned myeloma cells and spleen cells at a ratio of 1:5-10 and centrifuge at 1000 rpm for 5 minutes. Gently discard the supernatant and place the tube upside down on a square of absorbent paper to remove any liquid around the tube opening. Gently insert a strip of absorbent paper into the tube to remove any remaining liquid from the tube walls and bottom. Immerse the bottom of the centrifuge tube in a 37°C water bath and incubate for 1 minute. Slowly add 1 mL of 1500 PEG to the mixed cells, gently stirring to disperse cell clumps. Keep the centrifuge tube in a 37°C water bath throughout the process. Continue to gently shake the centrifuge tube for 1 minute after adding PEG. Then, slowly add 1 mL of RPMI-1640 basal medium incubated at 37°C over 1 minute while stirring. Then, slowly add 3 mL of RPMI-1640 basal medium incubated at 37°C over 3 minutes while stirring. Continue to slowly add 15 mL of RPMI-1640 basal medium incubated at 37°C while stirring. After additions are complete, cover the plate and gently invert several times to mix thoroughly. Finally, place the plate in a 37°C incubator and let it rest for 5 minutes. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in 15 mL of RPMI-1640 basal medium, and centrifuge again at 1000 rpm for 5 minutes. Discard the supernatant to remove the PEG. Finally, gently resuspend the fused cells in 150 mL of HAT medium. Add 150 μL of HAT medium containing the fused cells to a 96-well cell culture plate and incubate in a 37°C, 5% CO2 incubator.

[0069] 3. Screening and cloning of cell lines

[0070] Around day 12 after cell fusion, when the cell colonies have grown to occupy 1 / 2 of the area at the bottom of the well and the culture medium 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 zero competitor, i.e., the negative control well, is higher, and higher sensitivity means that the competitor concentration at which the inhibition rate is 50%, i.e., the IC 50 The value was small), and the hybridoma cell line VAND8 was obtained by limiting dilution method 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.

[0071] 4. Sequence determination of the variable region of anti-vanillin monoclonal antibody:

[0072] (1) Hybridoma culture. After cell recovery, the hybridoma cell line VAND8 was cultured and the cell number was expanded to about 1x10 7(1) Cell collection. After 1000 r / min centrifugation for 5 min, the cells were collected. (2) Extraction of cell RNA. Under the environment of a clean bench, 1 mL of Trizol reagent was added to the centrifuged cells, and the mixture was allowed to stand for 5 min. Then, 2 mL of chloroform was added, and the mixture was shaken for 15 s. The mixture was allowed to stand at room temperature for 3 min, and then centrifuged at 12000 r / min for 15 min. The upper water sample layer was transferred to a new EP tube, 0.5 mL of isopropanol was added, and the mixture was allowed to stand at room temperature for 10 min. The mixture was centrifuged at 12000 r / min for 10 min. The supernatant was discarded, 1 mL of 75% ethanol was added, and the mixture was centrifuged at 7500 r / min for 5 min. The precipitate was dried, and 50 μL of double-distilled water was added. The purity was identified by agarose electrophoresis, and the quantity was quantified. The sample was stored at -70 °C for later use. (3) Preparation of cDNA by reverse transcription. 1 μL of total RNA, 6 μL of RNase-free ddH2O, 0.5 μL of oligo dT Primer, 10.5 μL of PRIME Script RT Enzyme Mix, and 2 μL of 5x Prime Script Buffer were mixed, and the mixture was incubated at 37 °C for 15 min and at 85 °C for 5 s. (4) Amplification of cDNA. The above cDNA was amplified using a mouse IgG VH VL primer library. 10 μL of 5x Prime Star Buffer, 4 pL of dNTP, 1 μL of cDNA, 1 pL of upstream primer, 1 μL of downstream primer, and 0.5 μL of PrimeSTAR were added to a total volume of 50 μL. PCR reaction was performed under the following reaction conditions: incubation at 94 °C for 5 min, denaturation at 94 °C for 45 s, annealing at 63 °C for 45 s, extension at 72 °C for 1 min, 30 cycles, and extension at 72 °C for 10 min. (5) Agarose gel electrophoresis and gel recovery. The PCR product was subjected to agarose gel electrophoresis, and the electrophoresis results were observed. The amplified product with a molecular weight of 250-350 bp was sent for sequencing. Sequencing was performed by Wuhan Dai'an Technology Co., Ltd.

[0073] The heavy chain sequencing results are as follows:

[0074] GAGGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAA

[0075] CTCTCCTGTGCAGCCTCT GGATTCATTTTCAGTACTTATTAC ATGTCTTGGGTTCGCCAG

[0076] ACTCCAGAGAAGAGGCTGGAGTTGGTCGCAACC ATTCATGGTAATGGTAATAGCATC TA

[0077] CTATCTAGACAGTGTGAAGGGTCGATTCGCCATCTCCAGAGACAATGCCAAGAACACT

[0078] CTGTACCTGCAGATGAGCAGTCTGAAGTCTGAGGACACAGCCCTGTATTAC TGTGTAAG

[0079] ACATGATGGTTATTACGTGGACCATGCTATGGACTGC TGGGGTCAGGGAACCTCAGTCACCGTCTCCTCA, as shown in SEQ ID No.1.

[0080] Heavy chain variable region sequence:

[0081] EVKLVESGGGLVKPGGSLKLSCAAS GFIFSTYY MSWVRQTPEKRLELVAT IHGNGNSI YYL

[0082] DSVKGRFAISRDNAKNTLYLQMSSLKSEDTALYY CVRHDGYYVDHAMDC WGQGTSVTV SS, as shown in SEQ ID No. 3, wherein:

[0083] CDR1:GFIFSTYY

[0084] CDR2:IHGNGNSI

[0085] CDR3:VRHDGYYVDHAMDC

[0086] The light chain sequencing results are:

[0087] GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTC

[0088] CATCTCTTGCAGATCTAGT CAGAGGATTGTACATGTTAATGGAAACACCTAT TTAGATTG

[0089] GTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTAATCTAC AAAGTTTCC AACCGA

[0090] TTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGGACAGATTTCACACTCA

[0091] AGATCAGCAGAGTGGCGGCTGAGGATCTGGGAGTTTATTACTGC TTTCAAGGTTCACATGTTCCTCGG ACGTTCGGTGGAGGCACCAGGCTGGAAATGAAA, the light chain variable region sequence shown in SEQ ID No. 2:

[0092] DVLMTQTPLSLPVSLGDQASISCRSS QRIVHVNGNTY LDWYLQKPGQSPKLLIY KVS NRFS GVPDRFSGSGSGTDFTLKISRVAAEDLGVYYC FQGSHVPRT FGGGTRLEMK, as shown in SEQ ID No. 4, wherein:

[0093] CDR1:QRIVHVNGNTY

[0094] CDR2:KVS

[0095] CDR3:FQGSHVPRT

[0096] The resulting gene sequence encoding the heavy chain variable region is 363 bp long, as shown in SEQ ID NO: 1. Based on the obtained gene sequence, it was deduced that the heavy chain variable region encoded by this gene sequence consists of 121 amino acids, as shown in SEQ ID NO: 3. The light chain variable region encoding gene sequence is 336 bp long, as shown in SEQ ID NO: 2. Based on the obtained gene sequence, it was deduced that the light chain variable region encoded by this gene sequence consists of 112 amino acids, as shown in SEQ ID NO: 4.

[0097] Example 2: A method for preparing a fluorescent test strip (europium oxide fluorescence) for rapid detection of vanillin, comprising the following steps:

[0098] (1) Preparation of absorbent pad

[0099] Cut the absorbent paper into 20mm*4.5mm width to make the absorbent pad;

[0100] (2) Preparation of detection pad

[0101] Test line coating:

[0102] A 0.25 mg / mL coating solution of vanillin-bovine serum albumin (VAN-BSA) was prepared and applied horizontally to the nitrocellulose membrane at a distance of 20 mm from the top edge of the membrane using a line spray method (speed: 1 μL / cm) to form a test line. The required coating amount of vanillin-bovine serum albumin per cm of the test line was 112.5 ng. The membrane was then dried at 37°C for 90 minutes.

[0103] Coating of quality control line:

[0104] Prepare a 0.5 mg / mL coating solution of goat anti-mouse secondary antibody. Apply the coating solution horizontally to a nitrocellulose membrane at a distance of 10 mm from the test line using a line spray (speed: 0.6 μL / cm) to create a control line. The coating amount required for each cm of the control line is 135 ng. Dry the membrane at 37°C for 90 minutes.

[0105] (3) Preparation of sample pad:

[0106] 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;

[0107] (4) Assembly of test strips

[0108] On one side of the cardboard, a water-absorbing pad, a detection pad, and a sample pad are sequentially attached from top to bottom, with adjacent pads overlapping at the joints with an overlap length of 2 mm to obtain a high-sensitivity immunochromatographic test strip for rapid detection of vanillin;

[0109] The coating buffer used in the coating solution for antigen coating was: 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 was added to make up to 100 mL.

[0110] (5) Anti-vanillin monoclonal antibody coupled with europium oxide fluorescent latex:

[0111] To 800 μL of 0.2 mol / L borate buffer (pH 8.18), add 200 μL of europium oxide fluorescent latex and disperse by sonication. Add 40 μL of 15 mg / mL freshly prepared EDC solution and shake for 15 minutes. Centrifuge at 12,000 rpm at 10°C for 10 minutes. Discard the supernatant to remove EDC and other substances. Add 15 μL of borate buffer containing 1 mg / mL vanillin antibody and mix thoroughly. Shake at 250 rpm at 4°C for 2 hours. Remove the tube and centrifuge at 12,000 rpm at 10°C for 10 minutes. Discard the supernatant and add 1 mL of 0.5% BSA in borate buffer. Shake thoroughly. Shake at 250 rpm at 20°C for 1 hour. Remove the tube and refrigerate at 4°C until ready for use.

[0112] The fluorescently labeled anti-vanillin monoclonal antibody was diluted 300-fold with a sustained-release solution containing: 0.5% bovine serum albumin, 2% sucrose, 0.5% polyvinylpyrrolidone (PVPK-30), 0.5% Tween 20, 0.8 g sodium chloride, 0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g potassium chloride, and 0.02 g potassium dihydrogen phosphate, and the volume was adjusted to 100 mL with water. 200 μL of the diluted fluorescently labeled anti-vanillin monoclonal antibody per well was added to the enzyme-labeled wells of the reaction microplate, and the plate was placed in a vacuum freeze dryer and frozen at -40°C. After covering with a moisture-proof cover, the sample reaction microplate was obtained.

[0113] (6) Pretreatment of edible oil samples

[0114] Weigh 10g of oil sample into a 50mL centrifuge tube. Add 10mL of methanol, shake vigorously for 20 minutes, and centrifuge at 6000rpm for 5 minutes. Collect the methanol layer and place it in another 50mL centrifuge tube. Add another 10mL of methanol to the sample tube, shake vigorously for 20 minutes, and centrifuge at 6000rpm for 5 minutes. Collect the methanol layer and combine the two extracts. Drain the methanol until nearly dry and reconstitute it with 0.5mL of 20% methanol solution.

[0115] (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:

[0116] 1. Pre-treat edible oil samples that tested negative for vanillin by liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and spike vanillin into standard solutions with concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 10 μg / mL, 5 μg / mL, and 2 μg / mL.

[0117] 2. Take 100 μL of each of the above-mentioned vanillin standard solutions at each concentration and add them to the sample reaction microwells. Mix thoroughly and insert a fluorescent test strip. Incubate at 37°C for 7 minutes. Blot any remaining liquid on the sample pad with absorbent paper and immediately measure the sample using a time-resolved fluorescence immunoassay (excitation wavelength: 365 nm, measurement wavelength: 615 nm). Obtain the fluorescence intensity values ​​at the test line (T) and the control line (C) on each fluorescent test strip. From this, calculate the ratio of the fluorescence intensity of the test line to the fluorescence intensity of the control line (T / C) on each fluorescent test strip.

[0118] 3. Vanillin standard was added to edible oil blank samples 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 ).

[0119] 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 their vanillin content. The vanillin contents were: not detected, not detected, 2.4 μg / mL, 1.3 μg / mL, not detected, respectively.

[0120] 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:

[0121] (1) Preparation of absorbent pad

[0122] Cut the absorbent paper into 20mm*4mm width to make an absorbent pad;

[0123] (2) Preparation of detection pad

[0124] Test line coating:

[0125] A 0.4 mg / mL coating solution of vanillin-bovine serum albumin was prepared and applied horizontally to the nitrocellulose membrane at a distance of 15 mm from the top edge of the membrane using a line spray method (speed: 0.8 μL / cm) to form a test line. The required coating amount of vanillin-bovine serum albumin per cm of the test line was 128 ng. The membrane was then dried at 37°C for 90 minutes.

[0126] Coating of quality control line:

[0127] Prepare a 0.25 mg / mL coating solution of goat anti-mouse secondary antibody. Apply the coating solution horizontally to a nitrocellulose membrane 10 mm from the test line using a line spray (speed: 1 μL / cm) to create a control line. The required coating amount of goat anti-mouse secondary antibody per cm of the control line is 100 ng. Dry the membrane at 37°C for 90 minutes.

[0128] (3) Preparation of sample pad:

[0129] 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;

[0130] (4) Assembly of test strip

[0131] The water absorption pad, the detection pad and the sample pad are sequentially pasted on one side of the paperboard from top to bottom, and adjacent pads are connected by overlapping at the connection part with an overlapping length of 2 mm, thereby obtaining the high-sensitivity immunochromatographic test strip for rapidly detecting vanillin.

[0132] The coating buffer used in the coating solution of the coating antigen is: 1.5 g of bovine serum albumin, 2% sucrose, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate, and water to make up to 100 mL.

[0133] (5) Method for coupling anti-vanillin monoclonal antibody and fluorescent quantum dots

[0134] Take 0.05 mmol MES buffer (pH 6.0) 900 uL, add 100 uL SiO2-CdTe / CdS, ultrasonic dispersion, add 60 uL of 20 mg / ml NHS and EDC mixed solution prepared in advance, react at room temperature for 15 min, centrifuge at 17000 rpm for 20 min to remove the supernatant, add 100 uL of 1 mg / mL vanillin antibody in MES buffer, shake at 250 r / min for 10 hours at 4°C, centrifuge at 12000 r / mn for 10 min to discard the supernatant, add 1 mL of 0.5% BSA MES buffer, shake to mix, and reserve.

[0135] After diluting the fluorescently labeled anti-vanillin monoclonal antibody 500 times with the slow-release solution, the diluent is: 1% bovine serum albumin, 2% sucrose, 1% polyvinylpyrrolidone (PVPK-30), 0.5% Tween 20, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate, and water to make up to 100 mL; 300 uL of the diluted fluorescently labeled anti-vanillin monoclonal antibody is added to the enzyme-labeled hole, and the sample reaction micropore is obtained after being frozen in a vacuum freeze dryer at -40°C and covered with a moisture-proof cover.

[0136] (6) Pretreatment of edible oil sample

[0137] Weigh 10 g of oil sample into a 50 mL centrifuge tube, add 10 mL of methanol, shake vigorously for 20 min, centrifuge at 6000 rpm for 5 min, collect the methanol layer in another 50 mL centrifuge tube. Add 10 mL of methanol to the sample tube, shake vigorously for 20 min, centrifuge at 6000 rpm for 5 min, collect the methanol layer, and combine the two extraction solutions. Remove 5 mL of methanol extract and nitrogen blow to near dryness, add 0.2 mL of 20% methanol aqueous solution to dissolve.

[0138] (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:

[0139] 1. Pre-treat edible oil samples that tested negative for vanillin by liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and spike vanillin into 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.

[0140] 2. Take 100 μL of each of the above-mentioned vanillin standard solutions and add them to the sample reaction cups. Mix well and insert fluorescent test strips. Incubate at 37°C for 7 minutes. Use absorbent paper to absorb any residual liquid on the sample pad. Use a fluorescence reader to measure the fluorescence intensity at the test line (T) and the control line (C) on each fluorescent test strip. Calculate the ratio of the fluorescence intensity of the test line to the fluorescence intensity of the control line (T / C) on each fluorescent test strip.

[0141] 3. Vanillin standard was added to edible oil blank samples 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).

[0142] 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 use the standard curve to calculate their vanillin content. The vanillin contents were: not detected, not detected, and 2.4 μg / mL, respectively.

[0143] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all such 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 oils, characterized by: The invention comprises a fluorescent test strip and a reaction microplate. The fluorescent test strip comprises a bottom plate. The bottom plate is sequentially attached with a water-absorbing pad, a detection pad and a sample pad from top to bottom. Adjacent pads are overlapped and connected at the connection point. The detection pad is based on a nitrocellulose membrane. 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 goat anti-mouse secondary antibody, and the detection line is coated with a complete vanillin antigen. The complete vanillin antigen is vanillin-bovine serum albumin. The reaction microplate contains a freeze-dried fluorescent-labeled anti-vanillin monoclonal antibody. The anti-vanillin monoclonal antibody is produced by the hybridoma cell line VAND8 with a deposit number of CCTCC NO.C 2023186. The structural formula of vanillin-bovine serum albumin is as follows: 。 2. The fluorescence kit according to claim 1, wherein: The absorbent pad is 20-25 mm long and 3-5 mm wide; the detection pad is 25-30 mm long and 3-5 mm wide; the sample pad is 20-25 mm long and 3-5 mm wide, and the overlapping length of adjacent pads is 1-2 mm; 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.

3. The fluorescence kit according to claim 1, wherein: The coating amount of vanillin complete antigen 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.

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

5. The fluorescence kit according to claim 1, wherein: The fluorescent labeling material is quantum dots or time-resolved microspheres with a fluorescent labeling effect, and the particle size of the fluorescent labeling material is 100-400 nm.

6. The fluorescence kit according to claim 1, wherein: The fluorescently labeled anti-vanillin monoclonal antibody was immobilized in the wells of the reaction microplate using a freeze-drying process.

7. The method for preparing the fluorescent kit according to claim 1, wherein: The following steps are involved: Preparation of fluorescent test strips: (1) Preparation of absorbent pad Cut absorbent paper into absorbent pads; (2) Preparation of test pad Test line coating: Prepare a 0.2-0.5 mg / mL coating solution of vanillin complete antigen and line-spray the solution onto a nitrocellulose membrane 10-20 mm from the top edge. The required coating amount of vanillin complete antigen per cm of the test line is 100-250 ng. Dry the membrane at 37-42°C for 60-120 min. Coating of quality control line: Prepare a 0.1-0.5 mg / mL coating solution of goat anti-mouse secondary antibody and apply it to the nitrocellulose membrane 5-15 mm from the top edge using a line spray method. The required coating amount of goat anti-mouse secondary antibody on the detection line is 50-300 ng. 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℃ for 10-16 hours to obtain the sample mat, and then store it in a desiccator at room temperature; (4) Assembly of test strips 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 joints 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, and dispense 100-200 µL per well. Place the well in a vacuum freeze dryer for freeze drying. After completion, cover with a moisture-proof cover to obtain a reaction microplate.

8. Use of the fluorescent kit 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, a fluorescent test strip is inserted 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, detection is performed using a time-resolved fluorescence tester to obtain a ratio of the time-resolved fluorescence intensity of the test line on the fluorescent test strip to the time-resolved fluorescence intensity of the quality control line; based on a pre-obtained relationship curve between the ratio of the time-resolved fluorescence intensity of the test line on the fluorescent test strip to the time-resolved fluorescence intensity of the quality control line and the vanillin concentration, 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 through 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 to the time-resolved fluorescence intensity of the quality control line and the vanillin concentration is obtained by the following method: (1) Prepare a series of vanillin standard solutions; (2) Add appropriate amounts of vanillin standard solutions of various concentrations to the wells of the reaction microplate, mix well, insert the fluorescent test strips into the wells of the reaction microplate, mix well, insert the fluorescent test strips into the wells of the reaction microplate, react for a period of time, and use 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 to the time-resolved fluorescence intensity of the quality control line and the concentration of vanillin was obtained by fitting.

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