Preparation and detection method of fluorescence immunochromatography detection card for lead and cadmium in tobacco

Abstract

The invention belongs to the technical field of immunodetection, and discloses a preparation and detection method of a fluorescence immunochromatography detection card for lead and cadmium in tobacco. The method comprises the following steps: preparing a horseradish peroxidase solution; preparing an immobilized enzyme-MOFs (Metal-Organic Frameworks) compound; preparing a labeled antigen; drawing a T line at one end, close to the sample adding end, of the NC membrane, and drawing a C line at one end, away from the sample adding end, of the NC membrane; mixing the immobilized enzyme-MOFs compound, a labeled antigen and a sample diluent according to a volume ratio of 2: 3: 5, uniformly spraying the mixture on a glass fiber membrane to serve as a conjugate pad, adhering absorbent paper to the other end of the NC membrane, covering a PVC upper cover, and pressing to complete the assembly of the detection card; according to the invention, the interference of short-life background fluorescence in a tobacco sample is effectively reduced, and the detection accuracy is greatly improved; the two heavy metal elements of lead and cadmium can be detected at the same time, and time and detection cost can be saved.

Description

Technical Field

[0001] The invention relates to the technical field of immunoassay detection, and more specifically to the preparation and detection method of a fluorescent immunochromatographic detection card for lead and cadmium in tobacco. Background Art

[0002] With the development of the tobacco industry and people's increasing attention to health, the problem of heavy metal pollution in tobacco has received more and more attention. During the growth process, tobacco is prone to absorb heavy metal elements from the soil, water and atmospheric environment, among which lead and cadmium are the two most common and harmful heavy metals.

[0003] The Chinese patent application with publication number CN116047061A discloses a time-resolved fluorescent immunochromatographic test strip for detecting heavy metal cadmium ions and its preparation method and application, including: preparing europium time-resolved fluorescent microspheres-heavy metal cadmium monoclonal antibody immune complex; preparing a binding pad sprayed with europium time-resolved fluorescent microspheres-heavy metal cadmium monoclonal antibody immune complex; coating the heavy metal cadmium antigen on the T line, coating the sheep anti-mouse secondary antibody on the C line, and after drying, pasting it together with absorbent paper, sample pad, and the binding pad to assemble a time-resolved fluorescent immunochromatographic test strip for detecting heavy metal cadmium ions. The preparation method of the time-resolved fluorescent immunochromatographic test strip for detecting heavy metal cadmium ions constructed by the invention is simple. By establishing the relationship between the concentration of the substance to be tested and the fluorescence intensity, it can realize the detection of cadmium ions in food, and the detection speed is fast and the sensitivity is high. It is suitable for on-site detection in farmland, supermarkets, homes, etc., and can be specifically applied to the detection of cadmium ions in tea, which has important practical significance and application value in tea quality control.

[0004] Although the above method can meet most scenarios, research and practical application of the above method and existing technology have found that the above method and existing technology have at least the following defects:

[0005] Time-resolved fluorescent immunochromatographic test strips are mainly used to detect cadmium ions and cannot provide detection information for lead elements. They mainly rely on the natural reaction process between the cadmium monoclonal antibody immune complex and the antigen on the test strip, and there are no additional complexes to assist in accelerating the reaction or enhancing the signal. As a result, when detecting low-concentration cadmium ions, the reaction speed is slow, the signal is weak, and the sensitivity is relatively low, which cannot meet the needs of accurate detection of trace cadmium pollution in tobacco.

[0006] In view of this, the present invention proposes a preparation and detection method of a fluorescent immunochromatographic detection card for lead and cadmium in tobacco to solve the above problems. Summary of the invention

[0007] In order to overcome the above-mentioned defects of the prior art, the present invention provides the following technical solution: a method for preparing a fluorescent immunochromatographic detection card for lead and cadmium in tobacco, comprising the following steps:

[0008] 5-20 g of MOFs powder was added to a 0.5-2 mg / mL horseradish peroxidase solution, stirred at 4°C, and incubated for 2-4 h. The immobilized enzyme-MOFs complex was collected by centrifugation, and washed repeatedly with a PBS buffer having a pH of 7.0-7.4 and a concentration of 0.01-0.1 mol / L. The immobilized enzyme-MOFs complex was resuspended in a PBS buffer having a pH of 7.0-7.4 and a concentration of 0.01-0.1 mol / L, and stored at 4°C for later use.

[0009] The lead antigen is coupled with the time-resolved fluorescent microspheres to prepare a first labeled antigen; the cadmium antigen is coupled with the time-resolved fluorescent microspheres to prepare a second labeled antigen; the first labeled antigen and the second labeled antigen are mixed in a volume ratio of 1:1 to obtain a labeled antigen;

[0010] NC membranes with different pore sizes and surface chemical properties were pasted in the middle of the PVC bottom plate, a T line was drawn on the end of the NC membranes with different pore sizes and surface chemical properties close to the sample application end, lead and cadmium specific antibodies were evenly spotted on the T line at a rate of 0.5-1.5 μL and a spacing of 3-5 mm using a micro-spotter, and dried; a C line was drawn on the end of the NC membranes with different pore sizes and surface chemical properties away from the sample application end, goat anti-mouse IgG antibodies were evenly spotted at a rate of 0.5-1.5 μL and a spacing of 3-5 mm, and dried for 30-60 minutes;

[0011] The immobilized enzyme-MOFs complex, labeled antigen and sample diluent were mixed in a volume ratio of 2:3:5 and evenly sprayed on the glass fiber membrane as a binding pad. The absorbent paper was pasted on the other end of the NC membrane with different pore sizes and surface chemical properties, covered with a PVC top cover and pressed tightly to complete the assembly of the test card.

[0012] Furthermore, the preparation method of the MOFs powder includes:

[0013] Dissolving dried zinc nitrate in N,N-dimethylformamide, placing on a magnetic stirrer and stirring until completely dissolved, to prepare a first mixed solution with a concentration of 0.1-0.3 mol / L;

[0014] Dissolving the recrystallized phthalic acid in N,N-dimethylformamide, placing it on a magnetic stirrer and stirring it until it is completely dissolved, to prepare a first composite solution with a concentration of 0.05-0.15 mol / L;

[0015] The first mixed solution and the first composite solution are transferred to a stainless steel reactor lined with polytetrafluoroethylene in a molar ratio of 1:1-2, and the filling degree of the reactor is controlled to be 60%-80%; the reactor is sealed and placed in an oven at a temperature of 120-150° C. for reaction for 12-36 hours to form MOFs crystals;

[0016] The oven was turned off and the reactor was allowed to cool naturally to room temperature. The reaction liquid in the reactor was filtered through filter paper, and the solid product on the filter paper was collected to preliminarily separate the MOFs crystals from the reaction solvent. The collected solid product was repeatedly washed with DMF for 3-5 times. The washed MOFs crystals were then transferred to a Soxhlet extractor and extracted for 24-48 hours using anhydrous ethanol as an extractant. The extracted MOFs crystals were placed in a vacuum drying oven and dried at 80-100°C for 10-20 hours to obtain MOFs powder.

[0017] Furthermore, the method for preparing the labeled antigen comprises:

[0018] Step 1, at 20-60° C., the blank microspheres are dispersed in a 0.01-0.1 mol / L terbium chloride solution, and the stirring speed is controlled to be 100-500 rpm for magnetic stirring. After reacting for 12-48 hours, the solution is centrifuged at 5000-10000 rpm for 5-15 minutes and the supernatant is discarded to obtain a microsphere precipitate, and the microsphere precipitate is washed with an ethanol solution with a volume fraction of 70%-95% to obtain a time-resolved fluorescent microsphere;

[0019] Step 2, activating the time-resolved fluorescent microspheres to obtain activated time-resolved fluorescent microspheres, and modifying the activated time-resolved fluorescent microspheres based on polyethylene glycol derivatives to obtain modified time-resolved fluorescent microspheres;

[0020] Step 3, coupling the lead antigen solution and the modified time-resolved fluorescent microspheres at a molar ratio of 5-15:1 to obtain a first coupling solution; coupling the cadmium antigen solution and the modified time-resolved fluorescent microspheres at a molar ratio of 5-15:1 to obtain a second coupling solution;

[0021] Step 4, respectively adding BSA blocking solution to the first coupling solution and the second coupling solution to block the vacant sites on the surface of the modified time-resolved fluorescent microspheres to obtain the first labeled antigen and the second labeled antigen;

[0022] Step 5: Mix the first labeled antigen and the second labeled antigen in a volume ratio of 1:1 to obtain a labeled antigen.

[0023] Furthermore, the method for obtaining the modified time-resolved fluorescent microspheres comprises:

[0024] Step 2.1, suspending the time-resolved fluorescent microspheres with a volume of 1-10 mL and a concentration of 5 mg / mL in a PBS buffer with a volume of 10-50 mL and a concentration of 0.01-0.1 mol / L, wherein the PBS buffer contains EDC and NHS, and the concentrations of EDC and NHS are both 1-10 mM; stirring the reaction at a stirring speed of 200-400 rpm at room temperature for 30-60 minutes, and then centrifuging at a speed of 5000-10000 rpm for 5-15 minutes to obtain a centrifuge, and removing the supernatant of the centrifuge; washing the precipitate with PBS buffer 2-3 times; after each washing, centrifuging at a speed of 5000-10000 rpm for 5-15 minutes to obtain the precipitate of the last centrifugation as the activated time-resolved fluorescent microspheres;

[0025] Step 2.2, resuspending the activated time-resolved fluorescent microspheres in a PBS buffer with a volume of 10-50 mL and a concentration of 0.01-0.1 mol / L, wherein the PBS buffer contains NHS-PEG, and the concentration of NHS-PEG is 1-10 mM, and stirring at a stirring speed of 100-300 rpm at room temperature for 2-24 h to obtain a modified solution;

[0026] Step 2.3, precipitate the modified solution by centrifugation at 5000-10000 rpm for 5-15 min, remove the supernatant to obtain the precipitate, wash the precipitate 3-5 times with 10-50 mL of PBS buffer at a concentration of 0.01-0.1 mol / L, and centrifuge at 5000-10000 rpm for 5-15 min after each wash, and obtain the precipitate from the last centrifugation as the modified time-resolved fluorescent microspheres.

[0027] Further, the method for obtaining the NC membrane with different pore sizes and surface chemical properties comprises:

[0028] Slowly add 5-15g of cellulose nitrate with a purity greater than 95% and a molecular weight of 80000-150000Da into a mixed solvent with a mass fraction of 10%-15% and a volume of 50-150mL, and stir and dissolve at a stirring speed of 200-400rpm at room temperature until a uniform and transparent mixed solution is formed; wherein the mixed solvent is a mixed solvent of acetone and DMF in a volume ratio of 3-5:1;

[0029] Add 0.3-0.9 g PEG2000, 0.5-1.5 g PEG6000, and 0.2-0.6 g PEG20000 to the mixed solution respectively, control the stirring speed to 200-400 rpm and continue stirring for 2-4 h to obtain a second mixed solution;

[0030] Slowly drop 0.15-0.45 g of 3-aminopropyltriethoxysilane into the second mixed solution, and stir at a stirring speed of 200-400 rpm for 1-2 h to obtain a third mixed solution;

[0031] Add 0.015-0.045 g of sodium dodecyl sulfate to the third mixed solution and stir for 30-60 min to obtain a prepared solution;

[0032] Pour the prepared solution onto a clean glass plate placed horizontally, and use a scraper to scrape the film evenly at a speed of 5-10 cm / s and a gap of 100-200 μm to obtain a liquid film;

[0033] Immediately transfer the liquid film and the glass plate to an environmental chamber with a temperature of 25-35°C and a humidity of 40%-60% and place them for 12-24 hours until the liquid film is completely dry;

[0034] After the liquid film is dried, it is peeled off from the glass plate and rinsed with deionized water 2-3 times, each time for 3-5 minutes, to obtain a thin film;

[0035] Soak the film in anhydrous ethanol for 1-2 hours, then take it out and place it in a fume hood to dry;

[0036] The dried film is dried in a vacuum oven at 40-50° C. for 4-6 h to obtain a first NC film;

[0037] Soaking the first NC film in anhydrous ethanol for 10-30 minutes to obtain a pretreated first NC film;

[0038] Slowly add 3-aminopropyltriethoxysilane with a volume of 10-50 mL and a concentration of 0.5-1.5% into anhydrous ethanol, and stir at a stirring speed of 100-300 rpm for 10-30 min at room temperature to obtain an APTES solution;

[0039] The pretreated first NC membrane is immersed in an APTES solution at 20-40° C. for 1-6 hours and then taken out to obtain a second NC membrane;

[0040] The second NC membrane was taken out from the APTES solution and rinsed with anhydrous ethanol for 3-5 times to obtain a third NC membrane;

[0041] After the third NC membrane is immersed in PBS buffer at room temperature for 10-30 minutes, it is rinsed with deionized water 2-3 times to obtain a fourth NC membrane;

[0042] The fourth NC membrane is dried in a vacuum oven at 40-60° C. for 2-6 hours; and the dried product is cut to obtain NC membranes with different pore sizes and surface chemical properties.

[0043] The detection method of the fluorescent immunochromatographic detection card for lead and cadmium in tobacco comprises the following steps:

[0044] Take 5-10g of tobacco sample, cut it into pieces with a particle size of 1-3mm, put it into a container containing 10-15mL of aminotrimethylenephosphonic acid with a concentration of 0.02-0.08mol / L, and connect the container to the inlet of the micro-nano extraction device;

[0045] Turn on the ultrasonic generator, set the ultrasonic frequency to 20-50kHz, the power to 100-300W, and extract for 5-10 minutes;

[0046] After the extraction is completed, a 0.01-0.1 mol / L hydrochloric acid solution is used as an eluent to elute the lead and cadmium ions adsorbed on the extraction material, and the eluate is collected as a sample to be tested for subsequent immunoassay;

[0047] 100-200 μL of the sample to be tested is dripped into the sample well of the test card prepared by the preparation method of the fluorescent immunochromatographic test card for lead and cadmium in tobacco, and incubated for 10-15 minutes;

[0048] Insert the test card into the matching time-resolved fluorescence immunoassay analyzer, read the signal intensity values ​​of the T line and the C line, and process the signal intensity values ​​to obtain the standard concentration of lead and cadmium in tobacco.

[0049] Furthermore, the method of processing the signal intensity value to obtain the standard concentration of lead and cadmium in tobacco includes:

[0050] Preset wavelength and ,in, is the emission wavelength of the labeled antigen; is the reference wavelength;

[0051] Before inserting the test card and adding the sample to be tested, start the background monitoring program of the detection instrument; the instrument collects the fluorescence signal intensity at the T line and C line position at a preset frequency, continuously collects for 10-20 seconds, and obtains a set of background signal intensity data, which is recorded as ,in , indicating the emission wavelength of the selected antigen or reference wavelength , Indicates the number of collections;

[0052] Calculate the average value of the background signal intensity at two wavelengths respectively and standard deviation ;in, is the total number of collections, As wavelength The initial background subtraction value and As wavelength The initial background subtraction value is set at ,in is a constant.

[0053] Furthermore, a pre-prepared standard sample extract with known lead and cadmium concentrations is added dropwise to the test card, and the card is inserted into a time-resolved fluorescence immunoassay analyzer to obtain the fluorescence at the wavelength. and The real-time signal strength values ​​of the lower T line and C line are recorded as , , and ,in, The wavelength of the T line Real-time signal strength under The wavelength of the T line Real-time signal strength under The wavelength of the C line Real-time signal strength under The wavelength of the C line Real-time signal strength under For the standard samples; at the same time, the background signal intensity at this time is collected ;

[0054] Calculate the wavelength of the T line and The signal strength ratio under , and the C line at wavelength and The signal strength ratio under ;

[0055] For each standard sample, calculate its corresponding lead-cadmium concentration ratio ,in and Respectively The concentrations of lead and cadmium in the standard samples;

[0056] Establishing T-line correction coefficients using least squares fitting and C-line correction factor Ratio to concentration The linear relationship is:

[0057] For T-Line: , solve the coefficients by the least squares method and the intercept ;

[0058] For line C: , solve the coefficients in the same way and the intercept ;

[0059] Furthermore, the total signal strength of the T line and the C line at two wavelengths is collected at a preset time interval. , , and ,in, The wavelength of the T line Total signal strength under The wavelength of the T line Total signal strength under The wavelength of the C line Total signal strength under The wavelength of the C line The total signal strength under the condition; at the same time, the background signal strength at this time is collected ,in, , corresponding to the wavelength The background signal intensity at wavelength The background signal intensity under

[0060] like If the confidence interval is within the range, use As a background subtracted value, calculate the net signal intensity:

[0061] ;

[0062] ;

[0063] ;

[0064] ;

[0065] in, The wavelength of the T line The net signal strength under The wavelength of the T line The net signal strength under The wavelength of the C line The net signal strength under The wavelength of the C line The net signal strength under

[0066] like If the confidence interval is exceeded, the background average is recalculated. ,in, is the number of times the background signal is re-collected and used As the new background subtraction value, the net signal intensity is updated;

[0067] Calculate the effective signal strength of the T line after compensation and C line effective signal strength :

[0068] ;

[0069] ;

[0070] in, To estimate the ratio of lead and cadmium concentrations in real-time detection samples;

[0071] Using the standard curve established in advance through standard samples, combined with the effective signal intensity of the T line after compensation , calculate the concentration of lead and cadmium in the sample to be tested by interpolation or regression equation.

[0072] Furthermore, the method of calculating the concentration of lead and cadmium in the sample to be tested by interpolation or regression equation includes:

[0073] Collect the effective signal intensity of the T line after compensation on the standard curve The coordinates of two adjacent data points and , where the effective signal strength of the T line after compensation is lie in and The concentration of lead and cadmium in the sample to be tested is calculated by the following formula:

[0074] ,

[0075] in, is the concentration of lead and cadmium in the sample to be tested.

[0076] The technical effects and advantages of the preparation and detection method of the fluorescent immunochromatographic detection card for lead and cadmium in tobacco provided by the present invention are as follows:

[0077] In the preparation process of the present invention, horseradish peroxidase is combined with MOFs powder to form an immobilized enzyme-MOFs complex, which helps to enhance the stability and efficiency of the immune response; combined with rare earth elements, the reaction with blank microspheres under specific conditions forms microspheres with long fluorescence decay time, which can effectively avoid the interference of short-lived background fluorescence in tobacco samples, and by setting a suitable delay time to collect signals, the detection signal-to-noise ratio and accuracy are greatly improved, and the reliability of lead and cadmium detection in tobacco is enhanced. The time-resolved fluorescent microspheres are activated and modified with polyethylene glycol derivatives to have better water solubility and biocompatibility, reduce nonspecific adsorption, and thus improve the accuracy and sensitivity of the detection. By preparing NC membranes with different pore sizes and surface chemical properties and applying them to the test card, the migration speed of substances in tobacco samples and the impurity retention effect can be effectively regulated to ensure that the antigen-antibody reaction is sufficient and not interfered with, and the antibodies and antigens can be firmly fixed, thereby enhancing the repeatability and stability of the test card. During the detection process, the unique sample pre-treatment method combined with the precise signal intensity processing algorithm can effectively extract lead and cadmium ions in tobacco, accurately deduct background signals and compensate for effective signals, and accurately derive the concentration of lead and cadmium in tobacco through standard curves and a variety of calculation methods, thereby achieving efficient, accurate and rapid detection of lead and cadmium in tobacco, and providing strong technical support for the monitoring of heavy metal pollution in tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 This is a schematic diagram of a fluorescent immunochromatographic detection card for lead and cadmium in tobacco according to Example 1 of the present invention;

[0079] Figure 2 This is a schematic diagram of the cross-sectional structure of a fluorescent immunochromatographic detection card for lead and cadmium in tobacco according to Example 1 of the present invention;

[0080] Figure 3 Schematic diagram of a standard curve of concentration and fluorescence intensity of Example 2 of the present invention;

[0081] Figure 4 This is a schematic diagram of the change of the T-line fluorescence value with the addition amount of horseradish peroxidase powder in Example 3 of the present invention;

[0082] Figure 5 This is a schematic diagram showing the change of the T-line fluorescence value with the addition amount of SM-PEG24 in Example 4 of the present invention;

[0083] Figure 6 This is a schematic diagram of the test results of the test card of Example 5 of the present invention;

[0084] Figure 7 This is a schematic diagram showing the change of the T-line fluorescence value with the coupling molar ratio in Example 6 of the present invention;

[0085] Figure 8This is a schematic diagram of the change of the T-line fluorescence value with the concentration of aminotrimethylenephosphonic acid in Example 7 of the present invention;

[0086] Fig. 9 This is a schematic diagram of the false positive rate results of the test card in Example 8 of the present invention;

[0087] Fig.10 This is a schematic diagram of the false positive rate results of the test card in Example 9 of the present invention;

[0088] Fig.11 Schematic diagram of the test results of the test card according to Example 10 of the present invention.

[0089] The meaning of the accompanying symbols: 1. Quality control line; 2. Detection line; 3. Sample well; 4. Sample pad; 5. Binding pad; 6. NC membrane; 7. PVC top cover; 8. Absorbent paper; 9. PVC bottom plate. DETAILED DESCRIPTION

[0090] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0091] Example 1

[0092] See also Figure 1-Figure 2 As shown, the preparation method of the fluorescent immunochromatographic detection card for lead and cadmium in tobacco in this embodiment comprises the following steps:

[0093] Weigh 25-100 mg of horseradish peroxidase powder and slowly add it to 50 mL of pre-prepared PBS buffer, wherein the pH of the PBS buffer is 7.0-7.4 and the concentration is 0.01-0.1 mol / L. Use a glass rod to gently stir at 4°C until the mixture is evenly mixed to obtain a horseradish peroxidase solution with a concentration of 0.5-2 mg / mL.

[0094] Take 5-20g of MOFs powder and add it to the horseradish peroxidase solution, stir it gently with a glass rod at 4°C, and incubate it for 2h-4h, collect the immobilized enzyme-MOFs complex by centrifugation, wash it repeatedly with PBS buffer with a pH of 7.0-7.4 and a concentration of 0.01-0.1mol / L, resuspend the immobilized enzyme-MOFs complex in PBS buffer with a pH of 7.0-7.4 and a concentration of 0.01-0.1mol / L, and store it at 4°C for later use;

[0095] The preparation method of MOFs powder includes:

[0096] Dissolving dried zinc nitrate in N,N-dimethylformamide, placing on a magnetic stirrer and stirring until completely dissolved, to prepare a first mixed solution with a concentration of 0.1-0.3 mol / L;

[0097] Dissolving the recrystallized phthalic acid in N,N-dimethylformamide, placing it on a magnetic stirrer and stirring it until it is completely dissolved, to prepare a first composite solution with a concentration of 0.05-0.15 mol / L;

[0098] The first mixed solution and the first composite solution are transferred to a stainless steel reactor lined with polytetrafluoroethylene in a molar ratio of 1:1-2, and the filling degree of the reactor is controlled to be 60%-80%; the reactor is sealed and placed in an oven at a temperature of 120-150° C. for reaction for 12-36 hours to form MOFs crystals;

[0099] The oven was turned off and the reactor was allowed to cool naturally to room temperature. The reaction liquid in the reactor was filtered through filter paper, and the solid product on the filter paper was collected to preliminarily separate the MOFs crystals from the reaction solvent. The collected solid product was repeatedly washed with DMF for 3-5 times. The washed MOFs crystals were then transferred to a Soxhlet extractor and extracted for 24-48 hours using anhydrous ethanol as an extractant. The extracted MOFs crystals were placed in a vacuum drying oven and dried at 80-100°C for 10-20 hours to obtain MOFs powder.

[0100] The lead antigen is coupled with the time-resolved fluorescent microspheres to prepare a first labeled antigen; the cadmium antigen is coupled with the time-resolved fluorescent microspheres to prepare a second labeled antigen; the first labeled antigen and the second labeled antigen are mixed in a volume ratio of 1:1 to obtain a labeled antigen;

[0101] The preparation method of labeled antigen is as follows:

[0102] At 20-60°C, the blank microspheres were dispersed in a 0.01 mol / L-0.1 mol / L terbium chloride solution, and the stirring speed was controlled to be 100-500 rpm for magnetic stirring. After reacting for 12-48 hours, the microspheres were centrifuged at 5000-10000 rpm for 5-15 minutes, and the supernatant was discarded to obtain microsphere precipitates. The microsphere precipitates were washed with an ethanol solution with a volume fraction of 70%-95% to obtain time-resolved fluorescent microspheres. The blank microspheres were dispersed in the terbium chloride solution, and the microspheres were subjected to physical adsorption, chemical adsorption, and fluorescence. By means of chemical bonding or electrostatic action, terbium chloride solution can be adsorbed or combined on the surface of the microspheres to form a coating layer doped with rare earth elements, thereby obtaining fluorescent microspheres doped with rare earth elements. Based on this, microspheres with long-life fluorescence characteristics can be selected. Their fluorescence decay time is significantly prolonged compared to ordinary fluorescent substances, and they can effectively avoid the interference of short-life background fluorescence in tobacco samples. During detection, a suitable delay time can be set to collect signals. After the short-life background fluorescence subsides, the fluorescence emitted by the microspheres can be detected, which greatly improves the signal-to-noise ratio.

[0103] Activating the time-resolved fluorescent microspheres to obtain activated time-resolved fluorescent microspheres, and modifying the activated time-resolved fluorescent microspheres based on polyethylene glycol derivatives to obtain modified time-resolved fluorescent microspheres;

[0104] The method for obtaining the modified time-resolved fluorescent microspheres includes:

[0105] The time-resolved fluorescent microspheres with a volume of 1-10 mL and a concentration of 5 mg / mL are suspended in a PBS buffer with a volume of 10-50 mL and a concentration of 0.01-0.1 mol / L, wherein the PBS buffer contains EDC and NHS, and the concentrations of EDC and NHS are both 1-10 mM; the stirring speed is controlled at 200-400 rpm for stirring reaction at room temperature for 30-60 min, and then the centrifugal solution is obtained by centrifugation at a speed of 5000-10000 rpm for 5-15 min, and the supernatant of the centrifugal solution is removed; the precipitate is washed 2-3 times with PBS buffer; after each washing, the precipitate is centrifuged at a speed of 5000-10000 rpm for 5-15 min to obtain the precipitate of the last centrifugation as the activated time-resolved fluorescent microspheres;

[0106] The activated time-resolved fluorescent microspheres are resuspended in a PBS buffer with a volume of 10-50 mL and a concentration of 0.01-0.1 mol / L, wherein the PBS buffer contains NHS-PEG, and the concentration of NHS-PEG is 1-10 mM, and the stirring speed is controlled at 100-300 rpm at room temperature for 2-24 hours to obtain a modified solution;

[0107] The modified solution is precipitated by centrifugation at 5000-10000rpm for 5-15min, the supernatant is removed to obtain the precipitate, and the precipitate is washed 3-5 times with a PBS buffer solution with a volume of 10-50mL and a concentration of 0.01-0.1mol / L, and after each washing, the precipitate is centrifuged at 5000-10000rpm for 5-15min to obtain the precipitate of the last centrifugation as the modified time-resolved fluorescent microsphere. Modifying the surface of the time-resolved fluorescent microsphere can make the time-resolved fluorescent microsphere have better water solubility and biocompatibility, and reduce nonspecific adsorption.

[0108] The lead antigen is added to the modified time-resolved fluorescent microsphere solution at a molar ratio of 5-15:1 to the modified time-resolved fluorescent microsphere solution, and the mixture is mixed. The centrifuge tube is placed in a constant temperature water bath at 25-30° C. and stirred continuously for 1-2 hours.

[0109] Add 1-3% BSA blocking solution to block the free sites on the surface of the modified time-resolved fluorescent microspheres to obtain the first labeled antigen;

[0110] The second labeled antigen was prepared according to the same steps as the first labeled antigen.

[0111] The first labeled antigen and the second labeled antigen were mixed at a volume ratio of 1:1 to obtain a labeled antigen with a concentration of 0.1-0.5 μg / mL.

[0112] Paste the NC membrane 6 with different pore sizes and surface chemical properties to the middle position of the PVC bottom plate 9, draw a T line at one end of the NC membrane 6 with different pore sizes and surface chemical properties close to the sample application end, and use a micro-spotter to evenly spot lead and cadmium specific antibodies on the T line at a distance of 0.5-1.5 μL and 3-5 mm, and dry in the air; draw a C line at one end of the NC membrane 6 with different pore sizes and surface chemical properties away from the sample application end, and evenly spot goat anti-mouse IgG antibodies at a distance of 0.5-1.5 μL and 3-5 mm, and dry in the air for 30-60 minutes;

[0113] Methods for obtaining NC membranes 6 with different pore sizes and surface chemistries include:

[0114] Slowly add 5-15g of cellulose nitrate with a purity greater than 95% and a molecular weight of 80000-150000Da into a mixed solvent with a mass fraction of 10%-15% and a volume of 50-150mL, and stir and dissolve at a stirring speed of 200-400rpm at room temperature until a uniform and transparent mixed solution is formed; wherein the mixed solvent is a mixed solvent of acetone and DMF in a volume ratio of 3-5:1;

[0115] Add 0.3-0.9 g PEG2000, 0.5-1.5 g PEG6000, and 0.2-0.6 g PEG20000 to the mixed solution respectively, control the stirring speed to 200-400 rpm and continue stirring for 2-4 h to obtain a second mixed solution;

[0116] Slowly drop 0.15-0.45 g of 3-aminopropyltriethoxysilane into the second mixed solution, and stir at a stirring speed of 200-400 rpm for 1-2 h to obtain a third mixed solution;

[0117] Add 0.015-0.045 g of sodium dodecyl sulfate to the third mixed solution and stir for 30-60 min to obtain a prepared solution;

[0118] Slowly pour the prepared solution onto a clean glass plate placed horizontally, and use a scraper to evenly scrape the film at a speed of 5-10 cm / s and a gap of 100-200 μm to obtain a liquid film;

[0119] Immediately transfer the liquid film and the glass plate to an environmental chamber with a temperature of 25-35°C and a humidity of 40%-60% and place them for 12-24 hours until the liquid film is completely dry;

[0120] After the liquid film is dried, it is peeled off from the glass plate and rinsed with deionized water 2-3 times, each time for 3-5 minutes, to obtain a thin film;

[0121] Soak the film in anhydrous ethanol for 1-2 hours, then take it out and place it in a fume hood to dry;

[0122] The dried film is dried in a vacuum oven at 40-50° C. for 4-6 h to obtain a first NC film;

[0123] Soaking the first NC film in anhydrous ethanol for 10-30 minutes to obtain a pretreated first NC film;

[0124] Slowly add 3-aminopropyltriethoxysilane with a volume of 10-50 mL and a concentration of 0.5-1.5% into anhydrous ethanol, and stir at a stirring speed of 100-300 rpm for 10-30 min at room temperature to obtain an APTES solution;

[0125] The pretreated first NC membrane is immersed in an APTES solution at 20-40° C. for 1-6 hours and then taken out to obtain a second NC membrane;

[0126] The second NC membrane was taken out from the APTES solution and rinsed with anhydrous ethanol for 3-5 times to obtain a third NC membrane;

[0127] After the third NC membrane is immersed in PBS buffer at room temperature for 10-30 minutes, it is rinsed with deionized water 2-3 times to obtain a fourth NC membrane;

[0128] The fourth NC membrane is dried in a vacuum oven at 40-60°C for 2-6 hours; and the dried product is cut to obtain NC membranes 6 with different pore sizes and surface chemical properties. The chromatographic membranes with different pore sizes and surface chemical properties can prevent the passage of impurity particles in the tobacco sample while ensuring that the antibody and antigen can effectively bind and migrate, thereby reducing false positive results. In addition, the introduction of special functional groups, amino groups, on the surface of the chromatographic membrane can fix antibodies or other capture reagents, improve the fixation efficiency and stability of the antibodies, and thus enhance the repeatability and accuracy of the test card.

[0129] Methods for obtaining lead and cadmium specific antibodies include:

[0130] Primary immunization: Mix the first labeled antigen and Freund's complete adjuvant in a volume ratio of 1:1, place in an ice bath, and fully emulsify using an emulsifier until a first oil-in-water emulsion is formed; select 6-8 week old BALB / c mice, and inject the mice with a dose of 100-200 μL of the first oil-in-water emulsion; in the same way, inject the mice with a dose of 100-200 μL of the second oil-in-water emulsion prepared by the second labeled antigen and Freund's complete adjuvant;

[0131] The first booster immunization is carried out 2-3 weeks after the initial immunization, and the operation is the same as the initial immunization process.

[0132] The corresponding groups of mice were injected subcutaneously, and the injection dose for each mouse could be appropriately increased to 200-300 μL; thereafter, the booster immunization step was repeated every 2-3 weeks, and generally 3-4 booster immunizations were performed to enhance the immune response of the mice and encourage them to produce high-affinity and high-specificity antibodies.

[0133] The spleen cells of successfully immunized mice were fused with myeloma cells, and the cell lines that stably secreted lead and cadmium-specific monoclonal antibodies were obtained through hybridoma screening technology. The lead-specific antibodies and cadmium-specific antibodies were prepared by the ascites method or cell culture method, and purified for later use.

[0134] The immobilized enzyme-MOFs complex, the labeled antigen and the sample diluent are mixed in a volume ratio of 2:3:5 and then evenly sprayed on the glass fiber membrane as a binding pad 5. One end of the NC membrane 6 with different pore sizes and surface chemical properties dotted with the quality control line 1 and the detection line 2, that is, the C line and the T line is adhered to the binding pad 5, and the other end of the binding pad 5 is adhered to the sample pad 4. The absorbent paper 8 is adhered to the other end of the NC membrane 6 with different pore sizes and surface chemical properties, and after drying, it is adhered on the top of the PVC bottom plate 9, covered with the PVC upper cover 7, and pressed to complete the assembly of the test card. The specific method of pasting the test card can be referred to Figure 2 The appearance of the test card can be referred to Figure 1 There is a detection window on the top of the test card. In the detection window, the top is the quality control line 1, that is, the C line, and the bottom is the detection line 2, that is, the T line. Below the detection window is the sample hole 3, which is used to add the test sample.

[0135] Example 2

[0136] The detection method of the fluorescent immunochromatographic detection card for lead and cadmium in tobacco in this embodiment comprises the following steps:

[0137] Take 5-10g of tobacco sample, cut it into pieces with a particle size of 1-3mm, put it into a container containing 10-15mL of aminotrimethylenephosphonic acid with a concentration of 0.02-0.08mol / L, and connect the container to the inlet of the micro-nano extraction device;

[0138] Turn on the ultrasonic generator, set the ultrasonic frequency to 20-50kHz, the power to 100-300W, and extract for 5-10 minutes;

[0139] After the extraction is completed, a 0.01-0.1 mol / L hydrochloric acid solution is used as an eluent to elute the lead and cadmium ions adsorbed on the extraction material, and the eluate is collected as a sample to be tested for subsequent immunoassay;

[0140] 100-200 μL of the sample to be tested is dripped into the sample loading hole of the test card prepared by the preparation method of the fluorescent immunochromatographic test card for lead and cadmium in tobacco in Example 1, and incubated for 10-15 minutes;

[0141] Insert the test card into the matching time-resolved fluorescence immunoassay analyzer, read the signal intensity values ​​of the T line and the C line, and process the signal intensity values ​​to obtain the standard concentration of lead and cadmium in tobacco.

[0142] You can also prepare a series of samples with different concentrations, follow the above test steps, and draw a standard curve with the concentration of the standard solution of the sample to be tested as the horizontal axis and the ratio of the signal intensity of the T line to the C line as the vertical axis; for details, please refer to Figure 3 , the standard curve equation was obtained by linear regression analysis.

[0143] The method for processing the signal intensity value to obtain the standard concentration of lead and cadmium in tobacco comprises:

[0144] Preset wavelength and ,in, is the emission wavelength of the labeled antigen; is the reference wavelength;

[0145] Before inserting the test card and adding the sample to be tested, start the background monitoring program of the detection instrument; the instrument collects the fluorescence signal intensity at the T line and C line position at a preset frequency, continuously collects for 10-20 seconds, and obtains a set of background signal intensity data, which is recorded as ,in , indicating the emission wavelength of the selected antigen or reference wavelength Indicates the number of collections;

[0146] Calculate the average value of the background signal intensity at two wavelengths respectively and standard deviation ;in, is the total number of collections, As wavelength The initial background subtraction value and As wavelength The initial background subtraction value is set at ,in is a constant;

[0147] Add the pre-prepared standard sample extract with known lead and cadmium concentrations to the test card and insert it into the time-resolved fluorescence immunoassay analyzer to obtain the and The real-time signal strength values ​​of the lower T line and C line are recorded as , , and ,in, The wavelength of the T line Real-time signal strength under The wavelength of the T line Real-time signal strength under The wavelength of the C line Real-time signal strength under The wavelength of the C line Real-time signal strength under For the standard samples; at the same time, the background signal intensity at this time is collected ;

[0148] Calculate the wavelength of the T line and The signal strength ratio under , and the C line at wavelength and The signal strength ratio under ;

[0149] For each standard sample, calculate its corresponding lead-cadmium concentration ratio ,in and Respectively The concentrations of lead and cadmium in the standard samples;

[0150] Establishing T-line correction coefficients using least squares fitting and C-line correction factor Ratio to concentration The linear relationship is:

[0151] For T-Line: , solve the coefficients by the least squares method and the intercept ;

[0152] For line C: , solve the coefficients in the same way and the intercept ;

[0153] Collect the total signal intensity of T-line and C-line at two wavelengths at preset time intervals , , and ,in, The wavelength of the T line Total signal strength under The wavelength of the T line Total signal strength under The wavelength of the C line Total signal strength under The wavelength of the C line The total signal strength under the condition; at the same time, the background signal strength at this time is collected ,in, , corresponding to the wavelength The background signal intensity at wavelength The background signal intensity under

[0154] like If the confidence interval is within the range, use As a background subtracted value, calculate the net signal intensity:

[0155] ;

[0156] ;

[0157] ;

[0158] ;

[0159] in, The wavelength of the T line The net signal strength under The wavelength of the T line The net signal strength under The wavelength of the C line The net signal strength under The wavelength of the C line The net signal strength under

[0160] like If the confidence interval is exceeded, the background average is recalculated. ,in, is the number of times the background signal is re-collected and used As the new background subtraction value, the net signal intensity is updated;

[0161] Calculate the effective signal strength of the T line after compensation and C line effective signal strength :

[0162] ;

[0163] ;

[0164] in, To estimate the ratio of lead and cadmium concentrations in real-time detection samples;

[0165] Using the standard curve established in advance through standard samples, combined with the effective signal intensity of the T line after compensation , calculate the concentration of lead and cadmium in the sample to be tested by interpolation or regression equation;

[0166] Methods for calculating the concentration of lead and cadmium in the sample to be tested by interpolation or regression equation include:

[0167] Collect the effective signal intensity of the T line after compensation on the standard curve The coordinates of two adjacent data points and , where the effective signal strength of the T line after compensation is lie in and The concentration of lead and cadmium in the sample to be tested is calculated by the following formula:

[0168]

[0169] in, is the concentration of lead and cadmium in the sample to be tested.

[0170] Example 3

[0171] Optimization of enzyme loading in immobilized enzyme-MOFs composites:

[0172] In the process of preparing the immobilized enzyme-MOFs complex, the concentration of the horseradish peroxidase solution affects the amount of HRP enzyme fixed per gram of MOFs powder, that is, the enzyme loading; too high or too low an enzyme loading will affect the accuracy of subsequent detection of heavy metals lead and cadmium; therefore, the amount of horseradish peroxidase powder added is explored. The preparation method of the immobilized enzyme-MOFs complex is the same as that in Example 1, with the only difference being that the amount of horseradish peroxidase powder added is 10, 25, 50, 75, 100, 115 and 150 μL, respectively, and the corresponding T-line fluorescence values ​​are compared. The relationship between the amount of horseradish peroxidase powder added and the fluorescence value is shown in the figure. Figure 4 As shown, the best effect was achieved when the addition amount of horseradish peroxidase powder in the immobilized enzyme-MOFs composite was 75 mg.

[0173] Example 4

[0174] Optimization of activator concentration:

[0175] In the process of time-resolved fluorescent microsphere labeling of antibodies, the carboxyl groups on the surface of the fluorescent microspheres must first be activated. The quality of the activated microspheres is crucial to the subsequent labeling process. The amount of activator used determines the degree of activation of the carboxyl groups on the surface of the microspheres. After screening out the optimal activation conditions, the amount of activator added is explored. The preparation method of carbendazim antibody-fluorescent microsphere marker is the same as that in Example 1, with the only difference being that SM-PEG24 is added to the diluted microspheres at a molar ratio of 1:1, 1:3, 1:5, 1:7, 1:10, and 1:13 to the microspheres, and the corresponding T-line fluorescence values ​​are compared. The relationship between the amount of SM-PEG24 added and the fluorescence value is shown in the figure. Figure 5 As shown, the best effect was achieved when 6 μL of SM-PEG24 was added to the labeled antigen.

[0176] Example 5

[0177] Optimization of modification of time-resolved fluorescent microspheres:

[0178] In the process of labeling antibodies with time-resolved fluorescent microspheres, the surface of the time-resolved fluorescent microspheres is modified to make them more water-soluble and biocompatible, reduce nonspecific adsorption, protect the fluorescence properties of quantum dots, and enhance stability in the immunochromatographic system. The preparation method of the carbendazim antibody-fluorescent microsphere marker is the same as that in Example 1, and the detection method is the same as that in Example 2, except that the time-resolved fluorescent microspheres are respectively activated, activated + modified, and modified, and left to stand for 30 minutes after a positive result is detected. The samples containing heavy metals lead and cadmium are tested with test paper from the same batch. The results under ultraviolet light are as follows: Figure 6 As shown, all three groups presented positive results, but the control group with only activation showed a positive result and then continued to be placed for a period of time, and then the color became lighter; the experimental group with activation + modification showed a positive result and then continued to be placed for a period of time, and the color remained unchanged; the positive result of the control group with only modification was lighter in color during the test, and after being placed for a period of time, the color also became lighter, indicating that the modification of the time-resolved fluorescent microspheres in the present invention can protect the fluorescent properties of quantum dots and enhance the stability in the immunochromatography system.

[0179] Example 6

[0180] Optimization of the coupling molar ratio of antigen to time-resolved fluorescent microspheres:

[0181] In the process of microsphere labeling antigen, the molar ratio of lead-cadmium antigen solution to microsphere solution is crucial to the subsequent labeling process. The molar ratio can ensure that there are enough antigens to react with the active groups on the surface of the microspheres. If the antigen ratio is too low, the active groups on the surface of the microspheres cannot fully react, resulting in low coupling efficiency and insufficient activity of the labeled antigen; if the antigen ratio is too high, the antigen molecules are too much bound to the surface of the microspheres, which may cause steric hindrance. This will hinder the specific binding of antigen and antibody and affect the accuracy of detection. After screening out the better antigen labeling conditions, the coupling molar ratio is explored. The preparation method of the labeled antigen is the same as that in Example 1, the only difference is that the coupling molar ratio is 1:1, 3:1, 5:1, 7:1, 10:1, 15:1 and 18:1, and the corresponding T-line fluorescence values ​​are compared. The relationship between the coupling molar ratio and the fluorescence value is shown in the following figure. Figure 7 As shown, the best effect was achieved when the coupling molar ratio of the labeled antigen was 10:1.

[0182] Example 7

[0183] Optimization of extract concentration:

[0184] During the processing of tobacco samples, the concentration of the extract affects the extraction efficiency of lead and cadmium ions, and further affects the efficiency of subsequent detection of heavy metal lead and cadmium content in tobacco samples, so the concentration of the extract is further explored. Aminotrimethylenephosphonic acid was selected as the extract, and the method for detecting heavy metal lead and cadmium in tobacco samples was the same as that in Example 2, with the only difference being that the concentration of aminotrimethylenephosphonic acid was 0.01 mol / L, 0.02 mol / L, 0.04 mol / L, 0.06 mol / L, 0.08 mol / L and 0.1 mol / L; the corresponding T-line fluorescence values ​​were compared. The relationship between the concentration of aminotrimethylenephosphonic acid and the fluorescence value is shown in the figure. Figure 8 As shown, the best effect is achieved when the concentration of aminotrimethylenephosphonic acid is 0.06 mol / L.

[0185] Example 8

[0186] Optimization of pore size distribution of NC membranes:

[0187] During the detection process of the chromatography test card, the pore size distribution of the NC membrane affects the detection results of tobacco samples. Appropriate pore size distribution helps the effective migration of lead and cadmium ions and labeled antigens in tobacco samples on the NC membrane. If the pore size is too large, the sample solution may pass through the NC membrane quickly, resulting in too short a binding time between the antigen and the antibody on the membrane, affecting the sensitivity and accuracy of the detection. For example, when the pore size exceeds a certain threshold, some of the labeled antigens that have not reacted fully may directly pass through the T line and the C line, causing a deviation in the detection signal. The preparation method of NC membrane is the same as that of Example 1, except that the NC membrane is prepared by only PEG2000, only PEG6000, only PEG20000, a mixture of PEG2000 and PEG6000, a mixture of PEG6000 and PEG20000, a mixture of PEG2000 and PEG20000, and a mixture of PEG2000, PEG6000 and PEG20000, respectively; the detection method is the same as that of Example 2; the tobacco samples are tested the same number of times, such as 30 times, and are marked as group 1, group 2, group 3, group 4, group 5, group 6 and group 7 in sequence; the false positive rate of all the test results is counted, that is, the percentage of the identification results of tobacco samples not containing lead and cadmium that are identified as positive in the total number of tests, as shown in FIG. Fig. 9 As shown in the figure, it can be seen that group 7, that is, the NC membrane prepared by mixing PEG2000, PEG6000 and PEG20000, has the best detection effect.

[0188] Example 9

[0189] Optimization of chemical properties of NC membranes:

[0190] During the detection process of the chromatographic test card, the chemical properties of the NC membrane determine its ability to fix antibodies and antigens. If functional groups are introduced into the NC membrane, the functional groups can form stable chemical bonds or strong physical adsorption with the antibody or antigen molecules, making the fixation of antibodies and antigens on the NC membrane more firm and uniform. The preparation method of the NC membrane is the same as that in Example 1, except that the NC membrane is prepared by soaking in APTES solution and not soaking in APTES solution respectively; the detection method is the same as that in Example 2; the tobacco samples are tested the same number of times, such as 100 times, and are marked as Group 1 and Group 2 in turn, and all the test results are counted, as shown in detail. Fig.10 As shown in the figure, it can be seen that group 2, that is, the NC membrane prepared by soaking in APTES solution, has the best detection effect.

[0191] Example 10

[0192] Specificity Verification:

[0193] Several common heavy metal elements, such as chromium, nickel, lead and cadmium, were selected for specificity testing. Heavy metal element standards were prepared at a concentration of 2000 μg / mL. Test strips from the same batch were used to conduct interference tests on the above heavy metal element standards. Standards that did not contain heavy metal elements were used as negative controls. The test results are shown in Table 1. Chromium and nickel tests showed negative results. The T values ​​for lead and cadmium tests were significantly lower than those for chromium and nickel, and the C values ​​were equivalent. The T / C value can represent the ratio of the fluorescence intensity of the test line to the quality control line, and is used for the quantification of the corresponding antigen in the sample. The results under ultraviolet light are as follows: Fig.11 As shown, the standard containing lead and cadmium showed positive results, and the others were negative, indicating that the present invention has good specificity for the heavy metal elements lead and cadmium, and has no cross reaction with other heavy metal elements.

[0194] Table 1 Specific detection data of different heavy metal elements

[0195] Heavy Metal Elements Mean T value Mean T value T / C value Blank control 15364 969 15.86 chromium 15343 975 15.74 nickel 14572 986 14.78 lead 2453 996 2.46 cadmium 2448 994 2.46

[0196] As can be seen from Table 1, the detection card provided by the present invention is highly specific and has accurate measurement, which can effectively avoid interference from other substances and ensure that the test results accurately reflect the actual situation of lead and cadmium in the sample; and it is easy to operate, and can quickly perform preliminary detection of lead and cadmium content in a large number of tobacco samples, timely discover excessive heavy metal problems, improve work efficiency, and ensure the quality of tobacco raw materials.

[0197] In summary, the present invention marks antigens on the surface of fluorescent microspheres, and amplifies the binding signals of antigens and antibodies by detecting the fluorescence value of the microspheres. This method has significant advantages in detecting low concentrations of antigens or antibodies; compared with the traditional method of directly detecting antigen-antibody binding, it can detect target substances at lower concentrations, and can detect antigens or antibodies at the nanogram or even picogram level. The time-resolved fluorescent immunochromatographic detection card for quantitatively detecting heavy metal lead and cadmium in tobacco samples established in this way has a higher detection accuracy and better effect; and the present invention uses aminotrimethylenephosphonic acid with a concentration of 0.06 mol / L as an extracting solution to pre-treat tobacco samples, and combined with ultrasonic extraction, it can efficiently extract heavy metal ions in tobacco, provide accurate samples for subsequent detection, and help to more accurately evaluate the heavy metal content in tobacco, and ensure the quality and safety of tobacco products.

[0198] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

[0199] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a fluorescent immunochromatographic detection card for lead and cadmium in tobacco, characterized in that: The steps include: 5-20 g of MOFs powder was added to a 0.5-2 mg / mL horseradish peroxidase solution, stirred at 4°C, and incubated for 2-4 h. The immobilized enzyme-MOFs complex was collected by centrifugation, and washed repeatedly with a PBS buffer having a pH of 7.0-7.4 and a concentration of 0.01-0.1 mol / L. The immobilized enzyme-MOFs complex was resuspended in a PBS buffer having a pH of 7.0-7.4 and a concentration of 0.01-0.1 mol / L, and stored at 4°C for later use. The lead antigen is coupled with the time-resolved fluorescent microspheres to prepare a first labeled antigen; the cadmium antigen is coupled with the time-resolved fluorescent microspheres to prepare a second labeled antigen; the first labeled antigen and the second labeled antigen are mixed in a volume ratio of 1:1 to obtain a labeled antigen; NC membranes with different pore sizes and surface chemical properties are pasted to the middle position of the PVC bottom plate, a T line is drawn on one end of the NC membranes with different pore sizes and surface chemical properties close to the sample application end, and lead-cadmium-specific antibodies obtained by mixing lead-specific monoclonal antibodies and cadmium-specific monoclonal antibodies in a volume ratio of 1:1 are evenly spotted on the T line at a volume of 0.5-1.5 μL and a spacing of 3-5 mm using a micro-spotter, and then dried; a C line is drawn on one end of the NC membranes with different pore sizes and surface chemical properties away from the sample application end, and goat anti-mouse IgG antibodies are evenly spotted at a volume of 0.5-1.5 μL and a spacing of 3-5 mm, and then dried for 30-60 minutes; The immobilized enzyme-MOFs complex, labeled antigen and sample diluent were mixed in a volume ratio of 2:3:5 and evenly sprayed on the glass fiber membrane as a binding pad. The absorbent paper was pasted on the other end of the NC membrane with different pore sizes and surface chemical properties, covered with a PVC top cover and pressed tightly to complete the assembly of the test card.

2. The method for preparing the fluorescent immunochromatographic detection card for lead and cadmium in tobacco according to claim 1, characterized in that: The preparation method of the MOFs powder comprises: Dissolving dried zinc nitrate in N,N-dimethylformamide, placing on a magnetic stirrer and stirring until completely dissolved, to prepare a first mixed solution with a concentration of 0.1-0.3 mol / L; Dissolving the recrystallized phthalic acid in N,N-dimethylformamide, placing it on a magnetic stirrer and stirring it until it is completely dissolved, to prepare a first composite solution with a concentration of 0.05-0.15 mol / L; The first mixed solution and the first composite solution are transferred to a stainless steel reactor lined with polytetrafluoroethylene in a molar ratio of 1:1-2, and the filling degree of the reactor is controlled to be 60%-80%; the reactor is sealed and placed in an oven at a temperature of 120-150° C. for reaction for 12-36 hours to form MOFs crystals; The oven was turned off and the reactor was allowed to cool naturally to room temperature. The reaction liquid in the reactor was filtered through filter paper, and the solid product on the filter paper was collected to preliminarily separate the MOFs crystals from the reaction solvent. The collected solid product was repeatedly washed with DMF for 3-5 times. The washed MOFs crystals were then transferred to a Soxhlet extractor and extracted for 24-48 hours using anhydrous ethanol as an extractant. The extracted MOFs crystals were placed in a vacuum drying oven and dried at 80-100°C for 10-20 hours to obtain MOFs powder.

3. The method for preparing the fluorescent immunochromatographic detection card for lead and cadmium in tobacco according to claim 2, characterized in that: The method for preparing the labeled antigen comprises: Step 1, at 20-60° C., the blank microspheres are dispersed in a 0.01-0.1 mol / L terbium chloride solution, and the stirring speed is controlled to be 100-500 rpm for magnetic stirring. After reacting for 12-48 hours, the solution is centrifuged at 5000-10000 rpm for 5-15 minutes and the supernatant is discarded to obtain a microsphere precipitate, and the microsphere precipitate is washed with an ethanol solution with a volume fraction of 70%-95% to obtain a time-resolved fluorescent microsphere; Step 2, activating the time-resolved fluorescent microspheres to obtain activated time-resolved fluorescent microspheres, and modifying the activated time-resolved fluorescent microspheres based on polyethylene glycol derivatives to obtain modified time-resolved fluorescent microspheres; Step 3, coupling the lead antigen solution and the modified time-resolved fluorescent microspheres at a molar ratio of 5-15:1 to obtain a first coupling solution; coupling the cadmium antigen solution and the modified time-resolved fluorescent microspheres at a molar ratio of 5-15:1 to obtain a second coupling solution; Step 4, respectively adding BSA blocking solution to the first coupling solution and the second coupling solution to block the vacant sites on the surface of the modified time-resolved fluorescent microspheres to obtain the first labeled antigen and the second labeled antigen; Step 5: Mix the first labeled antigen and the second labeled antigen in a volume ratio of 1:1 to obtain a labeled antigen.

4. The method for preparing the fluorescent immunochromatographic detection card for lead and cadmium in tobacco according to claim 3, characterized in that: The method for obtaining the modified time-resolved fluorescent microspheres comprises: Step 2.1, suspending the time-resolved fluorescent microspheres with a volume of 1-10 mL and a concentration of 5 mg / mL in a PBS buffer with a volume of 10-50 mL and a concentration of 0.01-0.1 mol / L, wherein the PBS buffer contains EDC and NHS, and the concentrations of EDC and NHS are both 1-10 mM; stirring the reaction at a stirring speed of 200-400 rpm at room temperature for 30-60 minutes, and then centrifuging at a speed of 5000-10000 rpm for 5-15 minutes to obtain a centrifuge, and removing the supernatant of the centrifuge; washing the precipitate with PBS buffer 2-3 times; after each washing, centrifuging at a speed of 5000-10000 rpm for 5-15 minutes to obtain the precipitate of the last centrifugation as the activated time-resolved fluorescent microspheres; Step 2.2, resuspending the activated time-resolved fluorescent microspheres in a PBS buffer with a volume of 10-50 mL and a concentration of 0.01-0.1 mol / L, wherein the PBS buffer contains NHS-PEG, and the concentration of NHS-PEG is 1-10 mM, and stirring at a stirring speed of 100-300 rpm at room temperature for 2-24 h to obtain a modified solution; Step 2.3, precipitate the modified solution by centrifugation at 5000-10000 rpm for 5-15 min, remove the supernatant to obtain the precipitate, wash the precipitate 3-5 times with 10-50 mL of PBS buffer at a concentration of 0.01-0.1 mol / L, and centrifuge at 5000-10000 rpm for 5-15 min after each wash, and obtain the precipitate from the last centrifugation as the modified time-resolved fluorescent microspheres.

5. The method for preparing the fluorescent immunochromatographic detection card for lead and cadmium in tobacco according to claim 4, characterized in that: The method for obtaining the NC membranes having different pore sizes and surface chemical properties comprises: Slowly add 5-15g of cellulose nitrate with a purity greater than 95% and a molecular weight of 80000-150000Da into a mixed solvent with a mass fraction of 10%-15% and a volume of 50-150mL, and stir and dissolve at a stirring speed of 200-400rpm at room temperature until a uniform and transparent mixed solution is formed; wherein the mixed solvent is a mixed solvent of acetone and DMF in a volume ratio of 3-5:1; Add 0.3-0.9 g PEG2000, 0.5-1.5 g PEG6000, and 0.2-0.6 g PEG20000 to the mixed solution respectively, control the stirring speed to 200-400 rpm and continue stirring for 2-4 h to obtain a second mixed solution; Slowly drop 0.15-0.45 g of 3-aminopropyltriethoxysilane into the second mixed solution, and stir at a stirring speed of 200-400 rpm for 1-2 h to obtain a third mixed solution; Add 0.015-0.045 g of sodium dodecyl sulfate to the third mixed solution and stir for 30-60 min to obtain a prepared solution; Pour the prepared solution onto a clean glass plate placed horizontally, and use a scraper to scrape the film evenly at a speed of 5-10 cm / s and a gap of 100-200 μm to obtain a liquid film; Immediately transfer the liquid film and the glass plate to an environmental chamber with a temperature of 25-35°C and a humidity of 40%-60% and place them for 12-24 hours until the liquid film is completely dry; After the liquid film is dried, it is peeled off from the glass plate and rinsed with deionized water 2-3 times, each time for 3-5 minutes, to obtain a thin film; Soak the film in anhydrous ethanol for 1-2 hours, then take it out and place it in a fume hood to dry; The dried film is dried in a vacuum oven at 40-50° C. for 4-6 h to obtain a first NC film; Soaking the first NC film in anhydrous ethanol for 10-30 minutes to obtain a pretreated first NC film; Slowly add 3-aminopropyltriethoxysilane with a volume of 10-50 mL and a concentration of 0.5-1.5% into anhydrous ethanol, and stir at a stirring speed of 100-300 rpm for 10-30 min at room temperature to obtain an APTES solution; The pretreated first NC membrane is immersed in an APTES solution at 20-40° C. for 1-6 hours and then taken out to obtain a second NC membrane; The second NC membrane was taken out from the APTES solution and rinsed with anhydrous ethanol for 3-5 times to obtain a third NC membrane; After the third NC membrane is immersed in PBS buffer at room temperature for 10-30 minutes, it is rinsed with deionized water 2-3 times to obtain a fourth NC membrane; The fourth NC membrane is dried in a vacuum oven at 40-60° C. for 2-6 hours; and the dried product is cut to obtain NC membranes with different pore sizes and surface chemical properties.

6. A method for detecting lead and cadmium in tobacco using a fluorescent immunochromatographic detection card, characterized in that: The detection card prepared by the preparation method of the fluorescent immunochromatographic detection card for lead and cadmium in tobacco according to any one of claims 1 to 5 comprises the following steps: Take 5-10g of tobacco sample, cut it into pieces with a particle size of 1-3mm, put it into a container containing 10-15mL of aminotrimethylenephosphonic acid with a concentration of 0.02-0.08mol / L, and connect the container to the inlet of the micro-nano extraction device; Turn on the ultrasonic generator, set the ultrasonic frequency to 20-50kHz, the power to 100-300W, and extract for 5-10 minutes; After the extraction is completed, a 0.01-0.1 mol / L hydrochloric acid solution is used as an eluent to elute the lead and cadmium ions adsorbed on the extraction material, and the eluate is collected as a sample to be tested for subsequent immunoassay; 100-200 μL of the sample to be tested is dripped into the sample well of the detection card prepared by the method for preparing the fluorescent immunochromatographic detection card for lead and cadmium in tobacco according to any one of claims 1 to 5, and incubated for 10-15 minutes; Insert the test card into the matching time-resolved fluorescence immunoassay analyzer, read the signal intensity values ​​of the T line and the C line, and process the signal intensity values ​​to obtain the standard concentration of lead and cadmium in tobacco.

7. The method for detecting the fluorescent immunochromatographic detection card for lead and cadmium in tobacco according to claim 6, characterized in that: The method of processing the signal intensity value to obtain the standard concentration of lead and cadmium in tobacco comprises: Preset wavelength and ,in, is the emission wavelength of the labeled antigen; is the reference wavelength; Before inserting the test card and adding the sample to be tested, start the background monitoring program of the detection instrument; the instrument collects the fluorescence signal intensity at the T line and C line position at a preset frequency, continuously collects for 10-20 seconds, and obtains a set of background signal intensity data, which is recorded as ,in , indicating the emission wavelength of the selected antigen or reference wavelength , Indicates the number of collections; Calculate the average value of the background signal intensity at two wavelengths respectively and standard deviation ;in, is the total number of collections, As wavelength The initial background subtraction value and As wavelength The initial background subtraction value is set at ,in is a constant.

8. The method for detecting the fluorescent immunochromatographic detection card for lead and cadmium in tobacco according to claim 7, characterized in that: The standard sample extract with known lead and cadmium concentrations prepared in advance is added dropwise to the test card, and the card is inserted into a time-resolved fluorescence immunoassay analyzer to obtain the fluorescence at the wavelength. and The real-time signal strength values ​​of the lower T line and C line are recorded as , , and ,in, The wavelength of the T line Real-time signal strength under The wavelength of the T line Real-time signal strength under The wavelength of the C line Real-time signal strength under The wavelength of the C line Real-time signal strength under For the standard samples; at the same time, the background signal intensity at this time is collected ; Calculate the wavelength of the T line and The signal intensity ratio under the condition of and For each standard sample, calculate the corresponding lead and cadmium concentration ratio; The T-line correction coefficient was established by fitting the signal intensity ratio using the least squares method. and C-line correction factor Ratio to concentration The linear relationship between the T line and the C line is calculated and the coefficients and intercepts corresponding to the T line and the C line are obtained respectively.

9. The method for detecting the fluorescent immunochromatographic detection card for lead and cadmium in tobacco according to claim 8, characterized in that: The total signal intensity of the T line and the C line at two wavelengths is collected at a preset time interval , , and ,in, The wavelength of the T line Total signal strength under The wavelength of the T line Total signal strength under The wavelength of the C line Total signal strength under The wavelength of the C line The total signal strength under the condition; at the same time, the background signal strength at this time is collected ,in, , corresponding to the wavelength The background signal intensity at wavelength The background signal intensity under like If the confidence interval is within the range, use As the background-subtracted value, the net signal intensity was calculated by the difference between the total signal intensity and the background-subtracted value; like If it is outside the confidence interval, the net signal intensity is updated by recollecting the background signal and calculating the obtained background average as the new background subtraction value; Calculate the effective signal strength of the T line and the C line after compensation; the effective signal strength is calculated by the T line at the wavelength The net signal intensity under the T line is multiplied by the adjustment factor at wavelength The net signal intensity results under the difference calculation is obtained, where the adjustment factor is the T line at the wavelength The net signal strength and coefficient under The ratio of the product of the coefficient, the estimated ratio of the lead and cadmium concentrations in the real-time detection samples and the intercept; The concentration of lead and cadmium in the sample to be tested is calculated by using the standard curve established in advance using the standard sample and the effective signal intensity of the T line after compensation through interpolation or regression equation.

10. The method for detecting the fluorescent immunochromatographic detection card for lead and cadmium in tobacco according to claim 9, characterized in that: Methods for calculating the concentration of lead and cadmium in the sample to be tested by interpolation or regression equation include: Collect the effective signal intensity of the T line after compensation on the standard curve The coordinates of two adjacent data points and , where the effective signal strength of the T line after compensation is lie in and The concentration of lead and cadmium in the sample to be tested is calculated by multiplying the ratio of the difference between the horizontal coordinates and the vertical coordinates of two adjacent data points by the difference between the effective signal intensity and the vertical coordinate of one of the data point coordinates, and then adding the ratio to the horizontal coordinate of the corresponding data point coordinates.

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