CN115060889B - Fenvalerate molecular imprinting electrostatic spinning chromatography test strip, preparation method and detection method thereof - Google Patents
Fenvalerate molecular imprinting electrostatic spinning chromatography test strip, preparation method and detection method thereof Download PDFInfo
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Abstract
The invention discloses a fenvalerate molecular imprinting electrostatic spinning chromatographic test strip, a preparation method and a detection method thereof, wherein the preparation method comprises the steps of coupling a fenvalerate with a mouse IgG to prepare an FH-IgG conjugate; labeling time-resolved fluorescence microsphere Eu on FH-IgG conjugate to form FH-IgG-Eu complex; preparing a molecularly imprinted polymer; preparing a molecularly imprinted electrospun membrane based on the molecularly imprinted polymer; and (5) constructing a chromatographic test strip. The detection method has the advantages of being quick, simple, portable and the like, and can be used for realizing qualitative and quantitative detection of fenvalerate residues in actual samples. The invention solves the defects of fluorescent agglomeration and easy quenching and natural fluorescent interference of fluorescein isothiocyanate caused by cutting NC film, improves the stability and recognition performance of T line, and provides a new thought for fluorescent immunochromatography test paper technology based on novel bionic recognition material.
Description
Technical Field
The invention belongs to the technical field of food safety detection, and particularly relates to a fenvalerate molecular imprinting electrostatic spinning chromatographic test strip, a preparation method and a detection method thereof.
Background
Fenvalerate is a broad-spectrum, efficient, low-residue, medium-toxicity and biodegradable pyrethroid pesticide, and is widely used in fruits and vegetables. After entering the natural environment, the pyrethroid circulates in solid, liquid and gas, enters organisms through biological chains, and causes health risks to the organisms, wherein the concentration and the detection rate of fenvalerate are highest in crops. At present, the fenvalerate detection technology mainly comprises a confirmation technology and a rapid detection technology, but the technologies generally have the defects of expensive instruments, long analysis time, difficult antibody preparation and the like.
The immunochromatography technology is a detection and analysis method combining the immunological technology and the chromatographic technology, uses colloidal gold and fluorescent substances as markers, and is widely used in various fields as a rapid detection test strip. Because of the characteristics of high detection speed, strong specificity and the like, the immunochromatography technology does not need a professional instrument to judge results and does not need professional personnel to operate, and is widely applied to monitoring agricultural products, and market supervision personnel and common consumers can also detect pesticide residues in the agricultural products on site. However, the conventional immunochromatography requires the use of antibodies, while pesticides are small molecular substances, so that the preparation of antibodies is difficult, the conditions for preserving the antibodies are severe, and the defects of sacrificing animals and the like are also required.
The molecularly imprinted polymer can be used as a specific bionic recognition material to replace antibodies to overcome the defects. So far, the molecular imprinting bionic fluorescence immunochromatography test strip is only a triazophos competitive fluorescein isothiocyanate test strip established in the laboratory Hong Saihui. The test strip has the defects that fluorescein is easy to quench, and the result accuracy is reduced due to poor chromatographic effect and error easily caused by the phenomenon of fluorescein agglomeration caused by cutting of two sides of a T line.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a fenvalerate molecular imprinting electrostatic spinning chromatographic test strip, a preparation method and a detection method thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
the preparation method of the fenvalerate molecular imprinting electrostatic spinning chromatographic test strip comprises the following steps:
s1, coupling a mouse IgG with fenvalerate to prepare an FH-IgG conjugate;
s2, marking time-resolved fluorescence microsphere Eu on the FH-IgG conjugate to form FH-IgG-Eu complex;
s3, preparing a molecularly imprinted polymer;
s4, preparing a molecular imprinting electrostatic spinning membrane based on the molecular imprinting polymer;
s5, constructing a chromatographic test strip:
s5-1, spraying the FH-IgG-Eu compound on a fluorescent pad at a speed of 0.5 mu L/mm, and drying in a baking oven at 37 ℃ for later use;
s5-2, treating the sample pad with the sample pad treatment liquid for 10min, and drying in a drying oven at 37 ℃ for later use;
s5-3, sticking an NC film on a back plate of the test strip, drawing goat anti-mouse IgG at the C line position of the NC film, and drying;
s5-4, adhering an electrostatic spinning fiber membrane to a transparent adhesive tape, cutting an electrostatic spinning membrane area into thin strips with the width of 1.5-2.5mm and the length of 3.5-4.5mm, and adhering the thin strips at the position, which is 5mm away from a C line, of the NC membrane to serve as a T line;
s5-5, sequentially sticking a sample pad, a fluorescent pad and water absorbing paper on the upper side and the lower side of the NC film, covering each pad and the NC film with 1mm, drying at 37 ℃, and storing in a dryer at room temperature.
Further, the specific method of step S1 comprises the following sub-steps:
s1-1, weighing 5-20mg of fenvalerate in a 5mL glass bottle, and dropwise adding 0.1mL of anhydrous DMF to completely dissolve the fenvalerate;
s1-2, respectively weighing 3-8mgNHS and 5-10mgDCC in two centrifuge tubes, and respectively adding 0.1mL and 0.3mL of anhydrous DMF to completely dissolve;
s1-3, transferring the solutions in the two centrifuge tubes to the glass bottle, and magnetically stirring the solution at room temperature overnight to obtain activated fenvalerate;
s1-4, centrifuging the activated fenvalerate at 4 ℃ for 1min, and taking a supernatant;
s1-5, slowly dripping the supernatant into 1.5mL of PBS solution dissolved in 2-8mg of mouse IgG, and stirring for 3h at room temperature;
s1-6, transferring the obtained solution into a dialysis bag, dialyzing in PBS solution of 0.01moL/L at 4 ℃ until the dialysate is clear and free of turbidity, and obtaining the FH-IgG conjugate;
s1-7, freeze-drying the FH-IgG conjugate for 24 hours, and then freeze-preserving at-20 ℃ for later use.
Further, the specific method of step S2 comprises the following sub-steps:
s2-1, sucking 100 mu L of time-resolved fluorescent microspheres, and adding the time-resolved fluorescent microspheres into 900 mu L of marking buffer;
s2-2, respectively weighing NHS and EDC, and dissolving into 20mg/mL by using a marking buffer solution;
s2-3, sucking 5-20 mu LNHS, adding into the time-resolved fluorescence microsphere after S2-1 cleaning, rapidly mixing, sucking 2-10 mu L EDC, adding into the mixture, rapidly mixing, oscillating and incubating for 20min at room temperature;
s2-4, centrifuging the solution at 9000rpm/min for 15min, removing the supernatant, re-suspending the fluorescent microspheres by using 1mL of buffer marking solution for precipitation, and repeating the process once to obtain the re-suspended time-resolved fluorescent microspheres;
s2-5, weighing 10-50mg of BSA, completely dissolving with 0.05M PBS, preparing 20mg/mL of BSA, and preparing a blocking solution for later use;
s2-6, taking 0.2-1mgFH-IgG conjugate in a 2mL centrifuge tube, adding the resuspended time-resolved fluorescence microsphere, quickly mixing, mixing at room temperature, and incubating for 2h; adding 100 mu L of the sealing liquid, uniformly mixing at room temperature, and incubating for 1h;
s2-7, centrifuging the obtained solution at 9000rpm/min for 15min, removing the supernatant, re-suspending the fluorescent microspheres by using 1mL of diluent for precipitation, repeating twice to remove unbound FH-IgG conjugate, obtaining the FH-IgG-Eu complex, and refrigerating at 4 ℃ for later use.
Further, the specific method of step S3 comprises the following sub-steps:
s3-1, taking a mixture as a virtual double-template molecule, placing the mixture into a 150mL round bottom flask, adding acetonitrile for dissolution, adding 0.6mol/L acrylamide after the template molecule is completely dissolved, and placing the mixture on a shaking table for prepolymerization for 4 hours at room temperature, wherein the mol ratio of the 0.15mol/L diphenyl ether-biphenyl eutectic to the first ethyl chrysanthemate is 1:2;
s3-2, adding 3mol/L dimethyl glycol acrylate and 20-40mg azodiisobutyronitrile into the solution after the prepolymerization, carrying out ultrasonic treatment until the azodiisobutyronitrile is completely dissolved, filling nitrogen to remove oxygen for 15min, sealing, putting into a water bath kettle at 60 ℃ for oscillation, 120rpm/min, and reacting for 24h;
s3-3, filtering to remove supernatant after the reaction is finished, flushing the precipitate with methanol for 2-3 times, and drying at 60 ℃; eluting the template molecules with acetic acid-methanol solution in a Soxhlet extractor until the template molecules cannot be detected by an ultraviolet spectrophotometer, thereby obtaining the molecularly imprinted polymer.
Further, the specific method of step S4 comprises the following sub-steps:
s4-1, preparing an electrostatic spinning solution: weighing CA solid, dissolving in 8-20mL of acetone-methanol, and completely dissolving in water bath at 50deg.C; weighing the imprinted polymer, dispersing the imprinted polymer into 1mL of methanol, and magnetically stirring the imprinted polymer at room temperature after ultrasonic treatment for 30min until the polymer is uniformly dispersed in the methanol; adding the molecularly imprinted polymer methanol dispersion liquid into a CA-acetone methanol solution, and magnetically stirring the mixture in a water bath at 50 ℃ until a uniform spinning dispersion system is formed;
s4-2, preparing an electrostatic spinning film: before spinning starts, checking whether grounding is good, recording temperature and humidity, setting the flow rate of a micro-flow pump to be 50 mu L/min, spinning under the conditions that the spinning voltage is 12.0kV and the spinning distance is 15cm, using tin foil to replace a collector, collecting a molecularly imprinted electrostatic spinning film, conveniently lifting and taking down, applying, putting the spun electrostatic spinning film into an oven, and drying at 37 ℃ for later use.
The fenvalerate molecular imprinting electrostatic spinning chromatographic test strip prepared by the preparation method is provided.
Providing a detection method of a fenvalerate molecular imprinting electrostatic spinning chromatographic test strip, dropwise adding 100 mu L of fenvalerate standard solution or sample extracting solution into a sample hole of the test strip, reacting for 15min in a dark environment, and performing fluorescence detection; and (5) reading the T/C value, and calculating the fenvalerate content through a standard curve.
Further, the fenvalerate standard solution or the sample extracting solution is diluted by using 0.02M PB+20%TWEEN-20 buffer solution, and the organic solvent is added into the sample hole after the concentration is below 5 percent.
The beneficial effects of the invention are as follows:
1. the fenvalerate molecularly imprinted polymer provided by the invention has higher selectivity, can solve the problem of template leakage by adopting a virtual template, and can be used for replacing a biological antibody in an immunochromatography technology; the preparation method is prepared by a chemical method, has higher mechanical stability and long service life, can be suitable for a severe environment, and overcomes the defects of long preparation period, easy inactivation, animal sacrifice and the like of the traditional biological antibody.
2. The invention utilizes the advantages of time-resolved fluorescence microsphere Eu, namely strong fluorescence and long decay time, can eliminate natural fluorescence interference and improve the sensitivity of the method. The preparation method comprises the steps of preparing a fenvalerate-IgG-Eu compound, combining a molecular imprinting electrostatic spinning fluorescent immunochromatography test strip detection technology, developing a nano-membrane chromatography test strip for specifically recognizing fenvalerate, establishing a detection method for quick response of fenvalerate pesticides, and finally forming linear correlation (y= -0.3028x+1.1434, R) of the test strip to fenvalerate within the range of 50-1000 mug/L 2 = 0.9761), the detection limit is 50 μg/L, and the detection time is only 15min.
3. The detection method provided by the invention has the advantages of being quick, simple, portable, suitable for on-site quick detection and the like, and is expected to realize qualitative and quantitative detection of fenvalerate residues in actual samples in the future. The invention solves the defects of fluorescent agglomeration and easy quenching and natural fluorescent interference of fluorescein isothiocyanate caused by cutting NC film, improves the stability and recognition performance of T line, and provides a new thought for fluorescent immunochromatography test paper technology based on novel bionic recognition material.
Drawings
FIG. 1 is a schematic diagram of a fenvalerate molecularly imprinted electrospun chromatographic test strip and a detection scheme thereof provided in the example;
FIG. 2 is a standard graph of a fenvalerate molecularly imprinted electrostatic spinning chromatographic test strip provided in the example; wherein the abscissa represents the fenvalerate concentration and the ordinate represents the T/C value.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.
Example 1
A preparation method of a fenvalerate molecular imprinting electrostatic spinning chromatographic test strip comprises the following steps:
preparation of FH-IgG conjugate:
(1) Weighing 5-20mg fenvalerate in a 5mL glass bottle (marked as bottle 1);
(2) Respectively weighing 3-8mgNHS and 5-10mgDCC in a centrifuge tube (respectively marked as No. 2 bottle and No. 3 bottle);
(3) 0.5mL of anhydrous DMF is respectively added into the three bottles in a dropwise manner, and 0.1mL, 0.1mL and 0.3mL are respectively added into the three bottles in a dropwise manner, so that the three bottles are completely dissolved;
(4) Transferring the solutions in the No. 2 and No. 3 bottles into a No. 1 bottle, magnetically stirring at room temperature overnight, and activating fenvalerate;
(5) Centrifuging activated fenvalerate at 4deg.C for 1min to obtain supernatant;
(6) Slowly dripping all the supernatant into 1.5mL PBS solution dissolved in 2-8mg mouse IgG, and stirring for 3h at room temperature;
(7) All the obtained solution is transferred into a dialysis bag, and dialyzed in PBS solution (pH=7.4) with the concentration of 0.01moL/L at the temperature of 4 ℃ until the dialysate is clear and has no turbidity, so that FH-IgG is obtained;
(8) The conjugate was lyophilized in a freeze dryer for 24h and then stored frozen at-20℃until use.
2. Time-resolved fluorescence microsphere Eu label FH-IgG conjugate (FH-IgG-Eu):
(1) 100. Mu.L of time-resolved fluorescent microspheres were pipetted into 900. Mu.L of labeling buffer (50 mM MES, pH=6.0).
(2) A certain amount of NHS and EDC are weighed and dissolved into 20mg/mL by using a marking buffer solution respectively, and the mixture is prepared on site.
(3) Adding 5-20 mu LNHS into the washed fluorescent microsphere, and rapidly and uniformly mixing. Then sucking 2-10 mu LEDC, adding into fluorescent microsphere, and mixing. Incubate with shaking at room temperature for 20min.
(4) Centrifuging the solution at 9000rpm/min for 15min, removing the supernatant, re-suspending the fluorescent microspheres by using 1mL of buffer marking solution for precipitation, and repeating the process once to obtain re-suspended fluorescent microspheres for later use.
(5) 10-50mg BSA was weighed, dissolved completely in 0.05M PBS, and 20mg/mL BSA was prepared and used as a blocking solution.
(6) Taking 0.2-1mgFH-IgG conjugate in a 2mL centrifuge tube, adding activated fluorescent microspheres, quickly mixing, mixing at room temperature, incubating for 2h, adding 100 mu L of the sealing solution, mixing at room temperature, and incubating for 1h;
(7) The solution obtained above was centrifuged at 9000rpm/min for 15min, the supernatant was removed, the pellet was resuspended in 1mL of diluent (50 mm pbs, ph=7.4), the procedure was repeated twice to remove unbound FH-IgG conjugate, to obtain FH-IgG-Eu complex, and refrigerated at 4 ℃ for further use.
3. Preparation of molecularly imprinted polymer:
placing 0.15mol/L diphenyl ether-biphenyl eutectic and first ethyl chrysanthemate (the mol ratio of the diphenyl ether-biphenyl eutectic to the first ethyl chrysanthemate is 1:2) into a 150mL round-bottom flask, adding a certain amount of acetonitrile for dissolution, adding 0.6mol/L acrylamide (AA) after template molecules are completely dissolved, and placing the mixture on a shaking table for prepolymerization at room temperature for 4h; adding 3mol/L dimethyl ethylene glycol acrylate (EGDMA) and 20-40mg Azodiisobutyronitrile (AIBN), performing ultrasonic treatment until the azodiisobutyronitrile is completely dissolved, filling nitrogen to remove oxygen for 15min, sealing, and then placing into a water bath kettle at 60 ℃ for oscillating (120 rpm/min) reaction for 24h. After the reaction, filtering to remove the supernatant, washing the precipitate with methanol for 2-3 times, and drying at 60 ℃. The template molecules were eluted with acetic acid-methanol (2:8, v/v) solution in a Soxhlet extractor until no template molecules were detected by UV spectrophotometry. Obtaining the molecularly imprinted polymer.
4. Preparation of a molecular imprinting electrostatic spinning membrane:
(1) Preparing an electrostatic spinning solution: weighing 5-10mg of CA solid, dissolving into 8-20mL of acetone-methanol, and completely dissolving under the water bath condition of 50 ℃. Then 20-30mg of molecularly imprinted polymer is weighed and dispersed into 1mL of methanol, after 30min of ultrasound, the mixture is magnetically stirred at room temperature until the polymer is uniformly dispersed in the methanol. Finally, 10-20mL of the molecularly imprinted polymer methanol dispersion is added to 2-5mL of CA-acetone methanol solution, and the mixture is magnetically stirred in a water bath at 50 ℃ until a uniform spinning dispersion system is formed.
(2) Preparation of an electrostatic spinning film: the self-made electrostatic spinning device in a laboratory comprises an automatic micro-flow pump, a 5ml syringe, a height adjusting frame, a spray needle (20G), a receiving plate and a high-voltage power supply. Before spinning starts, checking whether grounding is good, recording temperature and humidity, setting the flow rate of a micro-flow pump to be 50 mu L/min, spinning under the conditions that the spinning voltage is 12.0kV and the spinning distance is 15cm, using tin foil to replace a collector, collecting a molecularly imprinted electrostatic spinning film, conveniently lifting and taking down, applying, putting the spun electrostatic spinning film into an oven, and drying at 37 ℃ for later use.
5. And (3) constructing a chromatographic test strip:
spraying FH-IgG-Eu compound on the fluorescent pad at a speed of 0.5 mu L/mm by using an XYZ three-dimensional film-drawing metal spraying instrument, and then putting the fluorescent pad into a baking oven at 37 ℃ for drying for later use; treating the sample pad with sample pad treating liquid for 10min, and drying in a 37 ℃ oven for later use; after an NC film is attached to a test strip backboard, a drawing film instrument is used for drawing goat anti-mouse IgG at the position of a C line of the NC film, the NC film is put into an oven for drying for 15 hours, then the test strip is taken out, an electrostatic spinning fiber film is taken off from aluminum foil paper and is adhered to a transparent adhesive tape, a region of the transparent adhesive tape with the electrostatic spinning film is cut into thin strips with the width of about 2mm and the length of about 4mm, the region with the spinning film is adhered to the position of the NC film, which is 5mm away from the C line, as a T line, then a sample pad, a fluorescent pad and water absorbing paper are sequentially adhered to the upper side and the lower side of the NC film, each pad and the NC film are covered by 1mm, and the test strip is assembled.
Example 2
The detection method of the fenvalerate molecular imprinting electrostatic spinning chromatographic test strip prepared in the embodiment 1 specifically comprises the steps of dripping 100 mu L of fenvalerate standard solution or sample extracting solution (diluted by 0.02M PB+20%TWEEN-20 buffer solution, ensuring that the organic solvent is below 5 percent, dripping the organic solvent into the sample pad) into a sample pad of the test strip, reacting for 15 minutes in a dark environment, and then performing fluorescence detection.
And (3) reading the T/C value by using a handheld fluorescence immunoassay analyzer, and calculating the fenvalerate content by using a standard curve. As shown in fig. 2.
According to the characteristic that the pyrethrin pesticide is specifically adsorbed by the pyrethrin pesticide molecularly imprinted polymer, the detection principle of the molecularly imprinted electrostatic spinning test strip is adopted. As shown in FIG. 1, an electrostatic spinning film containing a fenvalerate pesticide molecularly imprinted polymer is taken as a T line, goat anti-mouse IgG is taken as a C line, an FH-IgG-Eu compound is sprayed on a fluorescent pad, when a sample liquid containing fenvalerate is dripped on the sample pad, the sample liquid dissolves a marker together to flow to the T line through the fluorescent pad, and the marker and the dripped fenvalerate generate a situation of competing with the binding site of the molecularly imprinted polymer due to the fact that the marker also contains a fenvalerate structure, so that the fluorescence intensity of the T line forms an inverse relation with the concentration of the fenvalerate, and excessive marker flows to the C line to combine with a secondary antibody and then fluorescence is generated as a quality control condition. Finally, T, C and T/C values were read using an immunofluorescence reader. And (5) carrying out qualitative and quantitative measurement according to the fluorescence intensity of the T line and the T/C value.
The fenvalerate molecularly imprinted polymer provided by the invention has higher selectivity, takes diphenyl ether-biphenyl eutectic and first ethyl chrysanthemate as virtual templates, effectively solves the problem of leakage of the traditional molecularly imprinted polymer templates, and can be applied to immunochromatography instead of biological antibodies; the preparation method is prepared by a chemical method, has higher mechanical stability and long service life, can be suitable for a severe environment, and overcomes the defects of long preparation period, easy inactivation, animal sacrifice and the like of the traditional biological antibody.
The invention utilizes the advantages of time-resolved fluorescence microsphere Eu, namely strong fluorescence and long decay time, can eliminate natural fluorescence interference and improve the sensitivity of the method. The fenvalerate-IgG-Eu complex is prepared, and a chromatographic test strip technology is combined to develop a molecular imprinting nano-film chromatographic test strip for specifically recognizing fenvalerate, so as to establish the detection of the quick response of fenvalerate pesticideMeasuring method, and finally preparing test strip with linear correlation (y= -0.3028x+1.1434, R) to fenvalerate in 50-1000 mug/L range 2 = 0.9761), the detection limit is 50 μg/kg, and the detection time is only 15min.
The detection method provided by the invention has the advantages of being quick, simple, portable, suitable for on-site quick detection and the like, and is expected to realize qualitative and quantitative detection of fenvalerate residues in actual samples in the future. The invention solves the defects of fluorescent agglomeration and easy quenching and natural fluorescent interference of fluorescein isothiocyanate caused by cutting NC film, improves the stability and recognition performance of T line, and provides a new thought for fluorescent immunochromatography test paper technology based on novel bionic recognition material.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (6)
1. The preparation method of the fenvalerate molecular imprinting electrostatic spinning chromatographic test strip is characterized by comprising the following steps of:
s1, coupling a mouse IgG with fenvalerate to prepare an FH-IgG conjugate;
s2, marking time-resolved fluorescence microsphere Eu on the FH-IgG conjugate to form FH-IgG-Eu complex;
s3, preparing a molecularly imprinted polymer;
s4, preparing a molecular imprinting electrostatic spinning membrane based on the molecular imprinting polymer;
s5, constructing a chromatographic test strip:
s5-1, spraying the FH-IgG-Eu compound on a fluorescent pad at a speed of 0.5 mu L/mm, and putting the fluorescent pad into a baking oven at 37 ℃ for drying after finishing;
s5-2, treating the sample pad with the sample pad treatment liquid for 10min, and drying in a drying oven at 37 ℃ for later use;
s5-3, sticking an NC film on a back plate of the test strip, drawing goat anti-mouse IgG at the C line position of the NC film, and drying;
s5-4, adhering an electrostatic spinning fiber membrane to a transparent adhesive tape, cutting an electrostatic spinning membrane area into thin strips with the width of 1.5-2.5mm and the length of 3.5-4.5mm, and adhering the thin strips at the position, which is 5mm away from a C line, of the NC membrane to serve as a T line;
s5-5, sequentially sticking a sample pad, a fluorescent pad and water absorbing paper on the upper side and the lower side of the NC film, covering each pad and the NC film with 1mm, drying at 37 ℃, and storing in a dryer at room temperature;
the specific method of the step S2 comprises the following substeps:
s2-1, sucking 100 mu L of time-resolved fluorescent microspheres, and adding the time-resolved fluorescent microspheres into 900 mu L of marking buffer solution;
s2-2, respectively weighing NHS and EDC, and dissolving into 20mg/mL by using a marking buffer solution;
s2-3, sucking 5-20 mu LNHS, adding the mixture into the time-resolved fluorescence microsphere cleaned by the S2-1, rapidly mixing, sucking 2-10 mu L EDC, adding the mixture, rapidly mixing, oscillating and incubating for 20min at room temperature;
s2-4, centrifuging the solution at 9000rpm/min for 15min, removing the supernatant, re-suspending the fluorescent microspheres by using 1mL buffer marking solution for precipitation, and repeating the process once to obtain the re-suspended time-resolved fluorescent microspheres;
s2-5, weighing 10-50mg of BSA, completely dissolving with 0.05M PBS, preparing 20mg/mL of BSA, and preparing a blocking solution for later use;
s2-6, taking 0.2-1mgFH-IgG conjugate in a 2mL centrifuge tube, adding the resuspended time-resolved fluorescence microsphere, quickly mixing, mixing at room temperature, and incubating for 2 hours; adding 100 mu L of the sealing liquid, uniformly mixing at room temperature, and incubating for 1h;
s2-7, centrifuging the obtained solution at 9000rpm/min for 15min, removing the supernatant, re-suspending the fluorescent microspheres by using 1mL of diluent for precipitation, repeating the steps twice to remove unbound FH-IgG conjugate, obtaining FH-IgG-Eu complex, and refrigerating at 4 ℃ for later use;
the specific method of the step S3 comprises the following substeps:
s3-1, placing 0.15mol/L diphenyl ether-biphenyl eutectic and first ethyl chrysanthemate in a molar ratio of 1:2 in a 150mL round bottom flask, adding acetonitrile for dissolution, adding 0.6mol/L acrylamide after template molecules are completely dissolved, and placing on a shaking table for prepolymerization for 4 hours at room temperature;
s3-2, adding 3mol/L dimethyl glycol acrylate and 20-40mg azodiisobutyronitrile into the pre-polymerized solution, performing ultrasonic treatment until the solution is completely dissolved, filling nitrogen to remove oxygen for 15min, sealing, putting into a water bath kettle at 60 ℃ for oscillation, and reacting at 120rpm/min for 24h;
s3-3, filtering to remove supernatant after the reaction is finished, flushing the precipitate with methanol for 2-3 times, and drying at 60 ℃; eluting the template molecules with acetic acid-methanol solution in a Soxhlet extractor until the template molecules cannot be detected by an ultraviolet spectrophotometer, thereby obtaining the molecularly imprinted polymer.
2. The preparation method of the fenvalerate molecularly imprinted electrostatic spinning chromatographic test strip according to claim 1, wherein the specific method of the step S1 comprises the following sub-steps:
s1-1, weighing 5-20mg of fenvalerate in a 5mL glass bottle, and dropwise adding 0.1mL of anhydrous DMF to completely dissolve the fenvalerate;
s1-2, respectively weighing 3-8mgNHS and 5-10mgDCC in two centrifuge tubes, and respectively adding 0.1mL and 0.3mL of anhydrous DMF to completely dissolve;
s1-3, transferring the solutions in the two centrifuge tubes to the glass bottle, and magnetically stirring the solution at room temperature overnight to obtain activated fenvalerate;
s1-4, centrifuging the activated fenvalerate at 4 ℃ for 1min, and taking a supernatant;
s1-5, slowly dripping the supernatant into 1.5mL of PBS solution dissolved in 2-8mg of mouse IgG, and stirring for 3h at room temperature;
s1-6, transferring the obtained solution into a dialysis bag, dialyzing in PBS solution of 0.01moL/L at 4 ℃ until the dialysate is clear and free of turbidity, and obtaining the FH-IgG conjugate;
s1-7, freeze-drying the FH-IgG conjugate for 24 hours, and then freeze-preserving at-20 ℃ for later use.
3. The preparation method of the fenvalerate molecularly imprinted electrostatic spinning chromatographic test strip according to claim 1, wherein the specific method of the step S4 comprises the following sub-steps:
s4-1, preparing an electrostatic spinning solution: weighing CA solid, dissolving in 8-20mL acetone methanol, and completely dissolving in 50deg.C water bath; weighing the imprinted polymer, dispersing the imprinted polymer into 1mL of methanol, and magnetically stirring the imprinted polymer at room temperature after ultrasonic treatment for 30min until the polymer is uniformly dispersed in the methanol; adding the molecularly imprinted polymer methanol dispersion liquid into a CA-acetone methanol solution, and magnetically stirring the mixture in a water bath at 50 ℃ until a uniform spinning dispersion system is formed;
s4-2, preparing an electrostatic spinning film: before spinning starts, checking whether grounding is good, recording temperature and humidity, setting the flow rate of a micro-flow pump to be 50 mu L/min, spinning under the conditions that the spinning voltage is 12.0kV and the spinning distance is 15cm, collecting a molecularly imprinted electrostatic spinning film by using tinfoil instead of a collector, conveniently lifting and taking down the film, applying the film, putting the spun electrostatic spinning film into a drying oven, and drying at 37 ℃ for later use.
4. A fenvalerate molecularly imprinted electrospun chromatography test strip prepared according to the preparation method of any one of claims 1-3.
5. A method for detecting a fenvalerate molecular imprinting electrostatic spinning chromatographic test strip according to claim 4, which is characterized in that 100 μl of fenvalerate standard solution or sample extracting solution is dripped into a sample hole of the test strip, and fluorescent detection is carried out after reaction for 15min in a dark environment; and (5) reading the T/C value, and calculating the fenvalerate content through a standard curve.
6. The method for detecting the fenvalerate molecular imprinting electrostatic spinning chromatographic test strip according to claim 5, wherein the fenvalerate standard solution or the sample extracting solution is diluted by 0.02M PB+20%TWEEN-20 buffer solution, and the organic solvent is added into the sample hole after the organic solvent is below 5%.
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