CN102890155B - Fluorescent test strip based on resonance energy transfer, and preparation method and application for fluorescent test strip - Google Patents

Abstract

The invention discloses a fluorescent test strip based on resonance energy transfer, and a preparation method and application for the fluorescent test strip. The fluorescent test strip comprises a bottom liner, a sample absorption mat, a combining mat, a chromatography membrane and a water-absorbent mat, wherein the sample absorption mat, the combining mat, the chromatography membrane and the water-absorbent mat are sequentially connected with one another closely and are attached to the bottom liner; antibodies marked by fluorescent receptors are attached to the combining mat; a detection line and a calibration line are arranged on the chromatography membrane; the detection line is close to the combining mat; the calibration line is close to the water-absorbent mat; antigens marked by fluorescent substances are attached to the detection line; proteins marked by fluorescent substances are attached to the calibration line; the antigens marked by the fluorescent substances and the antibodies marked by the fluorescent receptors can be combined by specificity of antigen and antibody reaction; and the proteins marked by the fluorescent substances do not react with the antibodies marked by the fluorescent receptors. The fluorescent test strip has the advantages of safety in operation, simplicity, convenience, sensitivity, low cost, speediness and the like, is wide in application range, and can be used for single-item detection or quick detection items such as multiple-item detection.

Description

Fluorescence test strip based on energy resonance transfer and its preparation method and application

technical field

The invention relates to the field of fluorescence immunochromatography detection, belongs to a kind of dry chemical detection method, and particularly relates to a fluorescent test strip based on energy resonance transfer, its preparation method and application.

Background technique

Immunological detection methods are a series of experimental methods designed to detect antigens, antibodies, immune cells and their secreted cytokines and small molecules by applying immunological theory. In immunological detection, radioimmunoassay, enzyme-linked immunosorbent assay, immunoelectrochemical analysis technology, colloidal gold immunochromatography and other methods are mainly used at home and abroad to achieve quantitative or qualitative detection. But these methods all have different degrees of disadvantages:

①Radioimmunoassay and enzyme-linked immunosorbent assay have high sensitivity, but they need large-scale supporting equipment, have certain requirements for the detection environment, and the detection time is long, so they are not suitable for promotion at the grassroots level.

②Colloidal gold immunochromatographic test strips have the characteristics of low cost, fast, efficient, and high-throughput detection. After years of research, colloidal gold immunochromatographic test strips have a very mature process in the selection of raw materials, the release and running of colloidal gold particles, and the stability of test results. However, due to the limited signal amplification of colloidal gold particles, the sensitivity of the colloidal gold immunochromatographic test strip is not high, and the colloidal gold immunochromatographic test strip is difficult to achieve quantitative detection.

③Compared with colloidal gold immunochromatography test strips, fluorescent immunochromatography test strips have high sensitivity and can achieve the purpose of accurate detection. The tracers of fluorescent immunochromatographic test strips include single fluorescent dyes, fluorescent latex microspheres, lanthanides, quantum dots, etc. The signal amplification ability of a single fluorescent dye is limited, and fluorescence quenching is prone to occur under light conditions, which affects the sensitivity and stability of the test strip. Lanthanides have relatively good stability, but the quantum yield is low, which affects the sensitivity of the test strip. Quantum dots have the characteristics of wide excitation spectrum, narrow emission spectrum, large Stokes shift and high stability, but quantum dots also have some shortcomings: quantum dots have medium brightness and high price, which affect the sensitivity of test strips and increase The cost of research and development and production of test strips, and quantum dots contain heavy metal elements, which have certain toxicity. Fluorescent latex microspheres have the advantages of strong fluorescent signal, high stability, and easy protein labeling. They are a good tracer for immunochromatographic test strips. However, fluorescent latex microspheres also have disadvantages: Compared with colloidal gold particles, fluorescent latex microspheres have higher production costs and are more difficult to produce. Compared with colloidal gold immunochromatography test strips, immunochromatography based on fluorescent latex microspheres The research on the release and running of the test strips and the stability of the results is not very mature. According to the different detection objects and antigens and antibodies used, test strips have different requirements for the particle size and fluorescence spectrum of fluorescent latex microspheres, and except for some large foreign companies, it is difficult for domestic fluorescent latex microsphere production companies to provide various Fluorescence Spectrum and Particle Size of Fluorescent Microspheres.

Therefore, further research is still needed in order to obtain a method with high sensitivity, simple operation, rapidity, low cost and quantitative detection.

Contents of the invention

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and provide a fluorescent test strip based on energy resonance transfer.

Another object of the present invention is to provide a method for preparing the above-mentioned fluorescent test strip.

Another object of the present invention is to provide the application of the above fluorescent test strip.

The object of the present invention is achieved through the following technical solutions: a fluorescent test strip based on energy resonance transfer, comprising a substrate, a sample absorption pad, a binding pad, a chromatographic membrane and a water absorption pad; the sample absorption pad, a binding pad, a layer The analysis membrane and the water-absorbing pad are closely connected in turn and attached to the substrate; wherein, the binding pad is attached with a fluorescent receptor-labeled antibody; a detection line and a calibration line are set on the chromatographic membrane, and the detection line is close to the binding pad, and the calibration line is close to the water-absorbing pad. ; There is an antigen labeled with a fluorescent substance attached to the detection line, and a protein labeled with a fluorescent substance is attached to the calibration line; the antigen in the antigen labeled with a fluorescent substance and the antibody in the antibody labeled with a fluorescent receptor can be specifically combined through an antigen-antibody reaction, The protein in the fluorescent substance-labeled protein does not react with the antibody in the fluorescent receptor-labeled antibody;

The fluorescent test strip based on energy resonance transfer also includes a plastic shell, the plastic shell wraps the bottom liner and is attached to the bottom liner and closely connects the sample absorption pad, the binding pad, the chromatographic membrane and the water absorption pad in sequence; wherein, the plastic There are sampling holes and observation holes on the housing, the sampling holes are located at the position of the sample absorption pad, and the observation holes are located in the detection line and calibration line area; the function of the plastic housing is to prevent the fluorescent test strips based on energy resonance transfer from being polluted and Portable and easy to use;

The bottom liner is an impermeable material, preferably polyvinyl chloride (PVC);

The material of the sample absorption pad is preferably glass fiber;

The material of the bonding pad is preferably a polyester film or a glass cellulose film;

The material of the chromatographic membrane is preferably a nitrocellulose membrane;

The material of the absorbent pad is preferably absorbent paper;

The fluorescent substance is preferably a fluorescent dye, fluorescent latex microspheres, lanthanides, quantum dots, fluorescent polymers or fluorescent proteins; more preferably fluorescent latex microspheres;

The fluorescent acceptor is preferably colloidal gold nanoparticles, silver nanoparticles or carbon nanoparticles; more preferably colloidal gold nanoparticles or graphene can quench the fluorescent signal on the detection line;

The antigen is a complete antigen, preferably a complete antigen Cr-iEDTA-BSA or a complete antigen CP-AMOZ-BSA;

The antibody is preferably an anti-Cr-EDTA antibody or an anti-furaltadone metabolite antibody;

The protein is a substance that does not react with antigens or antibodies, preferably bovine serum albumin or ovalbumin;

The fluorescent receptor-labeled antibody is preferably prepared by the following method: mixing the fluorescent receptor and the antibody, stirring evenly; adding bovine serum albumin to block;

The fluorescent receptor-labeled antibody is more preferably prepared by the following method:

① Adjust the pH of the fluorescent receptor substance to 7.8-8.2, add the antibody while stirring, and stir to react;

② Add bovine serum albumin and stir for reaction; centrifuge, and the precipitate is fluorescent receptor-labeled antibody;

The antibody described in step ① is an anti-Cr-EDTA antibody or an anti-furaltadone metabolite antibody;

The time of the stirring reaction described in step 1. and step 2. is 25～35min;

The fluorescent substance-labeled antigen is preferably prepared by the following method: through the action of N-hydroxysuccinimide and carbodiimide, the antigen is coupled to the fluorescent substance;

The antigen labeled with a fluorescent substance is more preferably prepared by the following method:

(1) Mix the fluorescent substance, N-hydroxysuccinimide and carbodiimide and react; centrifuge to collect the precipitate, disperse it with triple distilled water and mix it;

(2) Then add the antigen, mix well and react in the dark;

(3) Add bovine serum albumin and react after mixing; collect the precipitate after centrifugation to obtain the antigen labeled with fluorescent substances; among them, fluorescent latex microspheres, N-hydroxysuccinimide, carbodiimide, antigen and bovine The ratio of serum albumin is 90-100:4:6:0.3:10 by weight;

The fluorescent substance described in step (1) is preferably fluorescent latex microspheres;

The reaction conditions described in step (1) are preferably 20-30°C for 30 minutes;

The conditions for the light-shielding reaction described in step (2) are preferably 20-30°C for 120 minutes;

The antigen described in step (2) is preferably the complete antigen Cr-iEDTA-BSA or the complete antigen CP-AMOZ-BSA;

The reaction conditions described in step (3) are preferably 20-30°C for 120 minutes;

The fluorescent substance-labeled protein is preferably prepared by the following method: through the action of N-hydroxysuccinimide and carbodiimide, bovine serum albumin is coupled to the fluorescent substance;

The protein labeled with fluorescent substance is more preferably prepared by the following method:

(I) Mix the fluorescent substance, N-hydroxysuccinimide and carbodiimide and react; centrifuge to collect the precipitate, disperse it with triple distilled water and mix it;

(II) Then add protein, mix and react; collect the precipitate after centrifugation to obtain fluorescent substance-labeled protein; among them, fluorescent latex microspheres, N-hydroxysuccinimide, carbodiimide and protein in a mass ratio of 90 ~100:4:6:10 ratio;

The protein is preferably bovine serum albumin or ovalbumin;

The fluorescent substance described in step (I) is preferably fluorescent latex microspheres;

The reaction conditions described in the step (I) are preferably 20-30°C for 30 minutes;

The reaction conditions described in step (II) are preferably 20-30°C for 120 minutes;

The preparation method of the fluorescent test strip based on energy resonance transfer comprises the following steps:

(1) Disperse the fluorescent receptor-labeled antibody on the binding pad;

(2) Attach the fluorescent substance-labeled antigen to the chromatographic membrane in a linear manner to form a detection line; attach the fluorescent substance-labeled protein to the chromatographic membrane in a linear manner to form a calibration line; the detection line and the calibration line are parallel relation;

(3) Lap the sample absorbent pad, the conjugation pad obtained in step (1), the chromatographic membrane obtained in step (2) and the water-absorbent pad successively on the backing, where the detection line on the chromatographic membrane is close to the conjugation pad, The calibration line is far away from the binding pad and close to the absorbent pad; obtain a fluorescent test strip based on energy resonance transfer;

Step (1) is preferably: spray the fluorescent receptor-labeled antibody onto the binding pad with a gold sprayer;

The amount of the fluorescent receptor-labeled antibody on the binding pad is preferably 1.2 μl/cm;

The fluorescent substance-labeled antigen described in step (2) is preferably wrapped on the chromatographic membrane by a gold-spraying film device;

The fluorescent substance-labeled protein described in step (2) is preferably wrapped on the chromatographic membrane by a gold-spraying film scriber;

The application of the fluorescent test strip based on energy resonance transfer in the detection field.

The principle of the present invention: Fluorescence resonance energy transfer (FRET) is an energy transfer phenomenon that occurs between two fluorescent substances or between a fluorescent substance and an energy acceptor that are very close to each other. When the emission spectrum of the donor fluorescent substance overlaps with the absorption spectrum of the acceptor, and the distance between the two molecules is within a certain range, a non-radioactive energy transfer will occur, that is, the FRET phenomenon, so that the fluorescence intensity of the donor is higher than that of the acceptor. It is much lower or disappears by itself. Colloidal gold particles, graphene, nano-carbon particles and other materials have a wide absorption spectrum and are ideal FRET acceptors. The invention utilizes fluorescence energy resonance transfer (FERT) between fluorescent substances and corresponding fluorescent receptors with light-absorbing properties at specific wavelengths to prepare high-sensitivity, low-cost, stable and quantitatively detectable immunochromatographic test strips. On the nitrocellulose membrane of the test strip, there are two lines that can emit fluorescence under the excitation of the excitation light source, the detection line and the calibration line. When there is no detection substance in the sample, the fluorescent receptor-labeled antibody binds to the fluorescently-labeled antigen on the detection line, making the fluorescence intensity on the detection line weaken or disappear. When there is a test substance in the sample, the test substance and the antigen on the detection line compete for the binding of the fluorescent receptor-labeled antibody, so that the fluorescent receptor-labeled antibody bound to the detection line decreases, so that the fluorescence on the test line appears. The fluorescence intensity on the detection line increases as the concentration of the test substance increases within a certain range. However, the fluorescence intensity on the calibration line does not change during detection.

Compared with the prior art, the present invention has the following advantages and effects:

(1) Compared with radioimmunotechnology and ELISA, the fluorescent test strip based on energy resonance transfer of the present invention has the advantages of safe operation (no radioactive contamination), simplicity, low cost, and rapidity. Compared with colloidal gold immunochromatographic test strips, it has the characteristics of high sensitivity and quantitative detection. Compared with fluorescent immunochromatographic test strips, it has the following advantages:

① The fluorescent receptors used in the present invention are gold nanoparticles, silver nanoparticles, carbon nanoparticles, etc., and the preparation of these particles is relatively simple, and the production cost is low.

②The fluorescent substance used in the present invention is combined with the nitrocellulose membrane detection line and the calibration line, so the requirements for the quantum yield, particle size, and production process of the fluorescent substance are reduced.

③ The fluorescent substance used in the present invention is fixed on the detection line and the calibration line of the nitrocellulose membrane, so the use of fluorescent substances is reduced in the development and production process of the test strip, and the cost of research and development and production is saved.

④Compared with fluorescent test strips based on fluorescent latex microspheres, the immunofluorescence energy resonance transfer test strip of the present invention is more mature in terms of particle release, running board and stability of test strip results.

(2) The immunofluorescence energy resonance transfer test strip described in the present invention uses the competition method, has a wide range of applications, and can be used for rapid detection items such as single detection or multiple detections in one detection; the detection matrix can be whole blood, serum, saliva, Urine, food samples, water samples, etc.; detection objects can be toxins in food, antibiotics, small molecules related to diseases, harmful substances in the environment, or some substances such as drugs that are banned from production and use by the state.

Description of drawings

Fig. 1 is the schematic diagram of the fluorescent test paper strip based on energy resonance transfer provided by the present invention; Wherein: 11-substrate, 12-sample absorption pad, 13-binding pad, 14-chromatographic membrane, 15-absorbent pad, 16 - detection line, 17 - calibration line, 18 - fluorescent receptor-labeled antibody.

Fig. 2 is the schematic sectional view of the fluorescent test strip camera used in the present invention, wherein: 1-housing; 2-LED lamp radiator; 3-camera; 4-LED light source; 5-for filtering the LED light source A filter for the emission wavelength; 6-a filter for filtering the emission wavelength of the fluorescent substance; 7-a sampling tank.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1

The preparation and application of the fluorescent test strip based on energy resonance transfer for rapid detection of trivalent chromium ions comprises the following steps:

(1) Preparation of fluorescently labeled complete antigen: Add 100 μl fluorescent latex microspheres (PS-G0200-010, Suzhou Zhaokang Biotechnology Co., Ltd.) into a 1.5ml EP tube, then add 400 μl triple distilled water, mix well and ultrasonicate (36000Hz) for 3 minutes; then add 40 μl of NHS (N-hydroxysuccinimide) at a concentration of 100 mg/ml and 60 μl of EDC (carbodiimide) at a concentration of 100 mg/ml, mix well and react at room temperature for 30 minutes; Centrifuge at 10000×g for two minutes, collect the precipitate after centrifugation and disperse and mix with 500 μl triple distilled water; add the complete antigen Cr-iEDTA-BSA with a concentration of ³⁰ mg/ml (Yan Lu et al. Preparation and application, Journal of Cellular and Molecular Immunology, 2011, 27 (4): 422-424) 10 μl, mix well and react in the dark at room temperature for 2 hours; add 100 μl of BSA (bovine serum albumin) with a mass volume ratio of 10% , react at room temperature for 2 hours after mixing; centrifuge at 10000×g for two minutes, collect the precipitate after centrifugation and use 200 μl reconstitution solution (pH value 7.2, 0.01M PBS solution containing 20% trehalose by mass, 20% by mass % sucrose, 1% by volume Triton X-100 and 2% by mass PEG (polyethylene glycol) 4000) were dissolved to obtain fluorescently labeled complete antigen, which was stored at 4°C in the dark.

(2) Preparation of fluorescently labeled BSA: Add 100μl fluorescent latex microspheres to a 1.5ml EP tube, then add 400μl triple distilled water, mix well, and then ultrasonicate (36000Hz) for 3 minutes; then add NHS with a concentration of 100mg/ml (N-hydroxysuccinimide) 40μl and EDC (carbodiimide) 60μl with a concentration of 100mg/ml, mix well and react at room temperature for 30 minutes; centrifuge at 10000×g for two minutes, collect the precipitate after centrifugation and distill with 500μl Disperse with water and mix well; add 100 μl of BSA (bovine serum albumin) with a mass volume ratio of 10%, and react for 2 hours at room temperature in the dark after mixing; centrifuge at 10,000×g for two minutes, collect the precipitate after centrifugation and dissolve it with 200 μl reconstitution solution Dissolve to obtain fluorescently labeled BSA, and store in the dark at 4°C.

(3) Preparation of colloidal gold-labeled anti-EDTA-Cr antibody:

A, preparation of colloidal gold:

100ml of three-distilled water is installed in a 250ml Erlenmeyer flask, which is placed on a magnetic heating stirrer and heated to boiling; after accurately adding 2ml of 1% by mass percentage of chloroauric acid, 4ml of 1% by mass of lemon dihydrate trisodium acid, heated and stirred for 10 minutes, then stopped heating and continued to stir for 10 minutes; the prepared colloidal gold solution was cooled and stored at 4°C for later use.

B. Preparation of colloidal gold-labeled antibody:

Take 5 mL of the colloidal gold solution prepared in step A, and adjust the pH value of the colloidal gold solution to 8.0 with 0.25 M potassium carbonate. Add 100 μl 0.04mg/ml anti-Cr-EDTA antibody (Colloidal gold nanoparticle probe-based immunochromatographic assay for the rapid detection of chromium ions in water and serum samples. Analytica Chimica Acta.2012,745:99-105) while stirring, and stir for 30 minutes; add 550ml of 10% BSA by mass, stir for 30 minutes; centrifuge at 12000r/min for 15 minutes, discard the supernatant, and redissolve the precipitate with 200ml redissolving solution (pH value 7.2, 0.01M PBS solution containing 20% by mass trehalose, 20% by mass sucrose, 1% by volume Triton X-100 and 2% by mass PEG (polyethylene glycol) 4000), and stored at 4°C for later use.

(4) Assembly of immune FERT test strips

As shown in FIG. 1 , the immune FERT test strip includes a substrate 11 , and a sample absorbent pad 12 , a binding pad 13 , a chromatographic membrane 14 and a water absorbent pad 15 attached to the substrate and closely connected in sequence.

Spray the colloidal gold-labeled antibody onto the polyester membrane binding pad at an amount of 2.5 mg/cm with a gold sprayer, as shown in Figure 1.18; follow the direction from the binding pad to the water-absorbing pad, and dilute the antibody by volume sequentially. 300-fold fluorescence-labeled complete antigen and fluorescent-labeled BSA diluted 300 times by volume were coated on the chromatographic membrane (nitrocellulose membrane) at an amount of 1 mg/cm with a gold-spraying device, and were used as the detection line 16 and the calibration line, respectively. Line 17, and bake in a 37°C oven for two days. Lay the sample absorbent pad, conjugation pad, chromatographic membrane and water absorbent pad sequentially on the backing, and cut the obtained test paper board into 4mm test paper strips as required. Then put it into the plastic cartridge (the plastic cartridge has a sample hole and a detection hole, the position of the sample hole is located at the position of the sample absorption pad; the position of the detection hole is located in the area where the detection line and the calibration line are located) to form a complete immune FERT test strip.

(5) Sample pretreatment

Take 100ml of the water sample from Liede Bridge on the Pearl River, filter it with filter paper to remove impurities, take 50ml of it, adjust the pH value to 7.0~7.2 with 10M NaOH and 10M hydrochloric acid, then add 0.5μM chelating agent EDTA-Na ₂ 0.5ml, react for 10 ~15min.

(6) Use of test strips

Add chromium trichloride to the EDTA-Na ₂ aqueous solution with a concentration of 50nM, and prepare standard solutions with the following concentrations respectively: the concentration of trivalent chromium ions is 800ng/ml, 400ng/ml, 200ng/ml, 100ng/ml, 50ng/ml , 25ng/ml, 12.5ng/ml, 6.25ng/ml, 3.125ng/ml, 1.5625ng/ml, 0ng/ml. Add 60 μl of the standard solutions of 11 concentrations prepared above to the sample wells of the immunoFERT test strips prepared in step (4), and calculate the ratio of the fluorescence intensity on the detection line to the fluorescence intensity on the calibration line after 15 minutes. (After taking pictures with the fluorescent test strip camera shown in Figure 2, use Image J software to analyze the fluorescence intensity F _T of the T line and the fluorescence intensity F _C of the C line, and calculate F _T /F _C ), and draw a standard curve. Add the pretreated water sample into the sample hole of the test paper card during sample detection, calculate the ratio of the fluorescence intensity between the detection line and the calibration line, and then calculate the concentration of trivalent chromium ions in the water sample through the standard curve . The sensitivity of this experiment is 6.25ng/ml, and the linearity is y=0.1192x-0.0432, R ² =0.9934 in the detection range from 6.25ng/ml to 800ng/ml.

The structure of the fluorescent test strip camera shown in Figure 2 is as follows: the shell 1 is a box structure enclosed by 6 PVC boards, and the camera 3, two LED light sources 4, and two LED lights are installed inside to dissipate heat 2, two filters 5 for filtering the luminous wavelength of the LED light source (filtering peak is 450nm), one filter 6 for filtering the luminous wavelength of fluorescent substances (filtering peak is 508nm), and the sampling tank 7; among them, The camera 3, the two LED light sources 4 and the two LED light radiators 2 are located on the same side of the sampling slot 7; the two LED light sources 4 are connected in parallel, and each LED light source 4 is connected to an LED light transformer; The LED light source is a blue LED light source with a peak wavelength of 450-455nm; an LED light radiator 2 is arranged next to each LED light source 4, and there are several holes on the side wall of the shell near the LED light radiator; A filter 5 with a filtering peak value of 450nm is arranged between each LED light source 4 and the sampling tank 7; a camera 3 is arranged between the two LED light sources 4, and the lens of the camera 3 faces the center of the sampling tank. A filter 6 with a filtering peak of 508nm is installed in front of the lens of the camera 3; the sampling slot 7 is in a sliding connection with the bottom plate of the housing 1, and the side of the sampling slot 7 in contact with the fluorescent test strip is black.

(7) Observation of results: A positive result means that the test line and the calibration line show fluorescence, and a negative result means that only the calibration line shows fluorescence; if the calibration line has no fluorescence, the test strip is invalid.

The sensitivity of the test strip is Cr ³⁺ 6.25ng/ml, and the sensitivity of the colloidal gold test strip is 5ng/ml.

Example 2

The preparation and application of a fluorescent test strip based on energy resonance transfer for the rapid detection of furaltadone metabolites (AMOZ) in meat foods includes the following steps:

(1) Preparation of fluorescently labeled complete antigen CP-AMOZ-BSA: Add 100 μl fluorescent latex microspheres (PS-G0200-010, Suzhou Zhaokang Biotechnology Co., Ltd.) into a 1.5ml EP tube, then add 400 μl triple distilled water , after mixing, ultrasonic (36000 Hz) treatment for 3 minutes; then add 40 μl of NHS (N-hydroxysuccinimide) with a concentration of 100 mg/ml and 60 μl of EDC (carbodiimide) with a concentration of 100 mg/ml, and mix well Then react at room temperature for 30 minutes; centrifuge at 10,000×g for two minutes, collect the precipitate after centrifugation and disperse with 500 μl triple distilled water and mix well; add complete antigen CP-AMOZ-BSA (Yanlu. Preparation of ketone metabolite AMOZ monoclonal antibody and research on immunological detection method [D] Doctoral thesis. Guangzhou: Department of Bioengineering, Jinan University, 2011) 10 μl, mixed and reacted at room temperature for 2 hours in the dark; adding 10% by mass BSA (Bovine Serum Albumin) 100μl, mix well and react at room temperature for 2 hours; centrifuge at 10000×g for two minutes, collect the precipitate after centrifugation and use 200μl redissolving solution (pH value 7.2, 0.01M PBS solution containing 20 % trehalose, 20% by mass of sucrose, 1% by volume of Triton X-100 and 2% by mass of PEG (polyethylene glycol) 4000 were dissolved to obtain fluorescently labeled complete antigen, which was stored at 4°C in the dark.

(2) Preparation of fluorescently labeled BSA: Add 100μl fluorescent latex microspheres into a 1.5ml EP tube, then add 400μl triple distilled water, mix well, and ultrasonicate (36000 Hz) for 3 minutes; then add 100mg/ml BSA 40 μl of NHS (N-hydroxysuccinimide) and 60 μl of EDC (carbodiimide) at a concentration of 100 mg/ml, mix well and react at room temperature for 30 minutes; centrifuge at 10,000×g for two minutes, and collect the precipitate with 500 μl of three Disperse with distilled water and mix well; add 100 μl of 10% BSA (bovine serum albumin) by mass, mix well and react in the dark at room temperature for 2 hours; centrifuge at 10000×g for two minutes, collect the precipitate after centrifugation and use 200 μl reconstitution solution ( pH value 7.2, 0.01M PBS solution containing 20% by mass of trehalose, 20% by mass of sucrose, 1% by volume of Triton X-100 and 2% by mass of PEG (polyethylene glycol) 4000 Dissolve to obtain fluorescently labeled BSA, and store in the dark at 4°C.

(3) Preparation of colloidal gold-labeled anti-furaltadone metabolite antibody:

A, preparation of colloidal gold:

100ml of three-distilled water is installed in a 250ml Erlenmeyer flask, which is placed on a magnetic heating stirrer and heated to boiling; after accurately adding 2ml of 1% by mass percentage of chloroauric acid, 3ml of 1% by mass of lemon dihydrate trisodium acid, heated and stirred for 10 minutes, then stopped heating and continued to stir for 10 minutes; the prepared colloidal gold solution was cooled and stored at 4°C for later use.

B. Preparation of colloidal gold-labeled antibody:

Take 5 mL of the colloidal gold solution prepared in step A, and adjust the pH value of the colloidal gold solution to 8.0 with 0.25 M potassium carbonate. While stirring, add 100μl 0.04mg/ml anti-furaltadone metabolite antibody (Yan Lu. Preparation of furaltadone metabolite AMOZ monoclonal antibody and research on immunological detection method[D]. Guangzhou: Bioengineering of Jinan University Department of Science, 2011), stirring for 30 minutes; adding 550ml of 10% BSA in mass percentage, stirring for 30 minutes; PBS solution containing 20% by mass of trehalose, 20% by mass of sucrose, 1% by volume of Triton X-100 and 2% by mass of PEG (polyethylene glycol) 4000) was dissolved and stored at 4°C for later use .

(4) Assembly of immune FERT test strips

As shown in FIG. 1 , the immune FERT test strip includes a substrate 11 , and a sample absorbent pad 12 , a binding pad 13 , a chromatographic membrane 14 and a water absorbent pad 15 attached to the substrate and closely connected in sequence.

Spray the colloidal gold-labeled antibody onto the polyester binding pad at an amount of 2.5 mg/cm with a gold sprayer, as shown in Figure 1. 18; follow the direction from the binding pad to the water-absorbing pad, sequentially dilute 300 by volume times the fluorescently labeled complete antigen and the fluorescently labeled BSA diluted 300 times by volume were coated on the chromatographic membrane (nitrocellulose membrane) at 1 mg/ml with a gold spraying device, and were used as the detection line 16 and the calibration line, respectively. 17, and bake in a 37°C oven for two days. Lay the sample absorbent pad, conjugation pad, chromatographic membrane and water absorbent pad sequentially on the bottom liner, and cut the obtained test paper board into 4mm test paper strips as required. Then put it into the plastic cartridge (the plastic cartridge has a sample hole and a detection hole, the position of the sample hole is located at the position of the sample absorption pad; the position of the detection hole is located in the area where the detection line and the calibration line are located) to form a complete immune FERT test strip.

(5) Sample pretreatment: Weigh 1.0 g of homogenized chicken tissue sample, add it to 4 mL of deionized water, 0.5 mL of 1mol/L hydrochloric acid solution and 100 μL of 50 mmol/L 4-CBA (p-aldehyde benzoic acid), and use The oscillator fully oscillates for 2min. Incubate overnight at 37°C for 16h. Then add 5mL 0.1mol/L dipotassium hydrogen phosphate solution, 0.4ml 1mol/L sodium hydroxide solution and 5mL ethyl acetate, vibrate vigorously with an oscillator for 30s, and centrifuge at 4000r/min for 10min. Take 2.5mL of ethyl acetate phase into a 10mL dry glass test tube, and blow dry under nitrogen flow at 50-60°C. Add 1mL of n-hexane, vortex for 30s with a vortex, then add 1mL of 0.01M, pH 7.2 PBS, and vortex for 1min to fully mix. Centrifuge at 4000r/min for 10min. The upper organic phase was removed, and 50 μL of the lower aqueous phase was taken for analysis.

(6) Standard 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml, 0.625ng/ml, 0.3125ng/ml, 0.15625ng/ml CP-AMOZ standard (use 0.01M, PH7 .2 in PBS) was added dropwise into the sample well, and the standard curve was drawn by calculating the ratio of the fluorescence intensity on the detection line to the fluorescence intensity on the calibration line. When the sample is detected, the concentration of the detected substance can be calculated through the standard curve by calculating the ratio of the fluorescence intensity of the test strip detection line and the calibration line. The sensitivity of this experiment is 0.3125ng/ml.

(7) Observation of results: A positive result means that the test line and the calibration line show fluorescence, and a negative result means that only the calibration line shows fluorescence; if the calibration line has no fluorescence, the test strip is invalid. The sensitivity of the test strip is Cr ³⁺ 0.3ng/ml, and the sensitivity of the colloidal gold test strip is 3ng/ml.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (9)

1. A fluorescent test strip based on energy resonance transfer, comprising a substrate, a sample absorption pad, a binding pad, a chromatographic membrane and a water absorption pad; the sample absorption pad, the binding pad, the chromatography membrane and the water absorption pad are closely connected in sequence, and It is attached to the substrate; it is characterized in that: a fluorescent receptor-labeled antibody is attached to the binding pad; a detection line and a calibration line are set on the chromatographic membrane, the detection line is close to the binding pad, and the calibration line is close to the water-absorbing pad; fluorescent substances are attached to the detection line Labeled antigen, fluorescent substance-labeled protein attached to the calibration line; fluorescent substance-labeled antigen and fluorescent receptor-labeled antibody can specifically bind through antigen-antibody reaction, fluorescent substance-labeled protein does not react with fluorescent receptor-labeled antibody ; The fluorescent substances are fluorescent dyes, fluorescent latex microspheres, lanthanides, quantum dots, fluorescent polymers or fluorescent proteins; The fluorescent acceptor is colloidal gold nanoparticles, silver nanoparticles or carbon nanoparticles; The protein is bovine serum albumin or ovalbumin. 2. The fluorescent test strip based on energy resonance transfer according to claim 1, characterized in that: the described fluorescent test strip based on energy resonance transfer also includes a plastic shell, and the plastic shell wraps the bottom liner and is attached to the bottom liner The sample absorption pad, binding pad, chromatographic membrane and water absorption pad are tightly connected in sequence; among them, the plastic shell is provided with a sample injection hole and an observation hole, the sample injection hole is located at the position of the sample absorption pad, and the observation hole is located at the detection line and the calibration line. line area. 3. The fluorescent test strip based on energy resonance transfer according to claim 1, characterized in that: the bottom liner is an impermeable substance; the material of the sample absorption pad is glass fiber; the bonding pad The material of the chromatographic membrane is polyester film or glass cellulose membrane; the material of the chromatographic membrane is nitrocellulose membrane; the material of the absorbent pad is absorbent paper. 4. the fluorescent test strip based on energy resonance transfer according to claim 1, is characterized in that: The fluorescent receptor-labeled antibody is prepared by the following method: mixing the fluorescent receptor and the antibody, stirring evenly; adding bovine serum albumin to block; The fluorescent substance-labeled antigen is prepared by the following method: through the action of N-hydroxysuccinimide and carbodiimide, the antigen is coupled to the fluorescent substance; The protein labeled with the fluorescent substance is prepared by the following method: through the action of N-hydroxysuccinimide and carbodiimide, bovine serum albumin or ovalbumin is coupled with the fluorescent substance. 5. the fluorescent test strip based on energy resonance transfer according to claim 4, is characterized in that: The fluorescent receptor-labeled antibody is prepared by the following method: ① Adjust the pH of the fluorescent receptor substance to 7.0-8.2, add the antibody while stirring, and stir to react; ② Add bovine serum albumin and stir for reaction; centrifuge, and the precipitate is fluorescent receptor-labeled antibody; The antigen labeled with fluorescent substance is prepared by the following method: (1) Mix the fluorescent substance, N-hydroxysuccinimide and carbodiimide and react; centrifuge to collect the precipitate, disperse it with triple distilled water and mix it; (2) Then add the antigen, mix well and react in the dark; (3) Add bovine serum albumin and react after mixing; collect the precipitate after centrifugation to obtain the antigen labeled with fluorescent substance; wherein, the fluorescent substance is fluorescent latex microspheres, fluorescent latex microspheres, N-hydroxysuccinimide , carbodiimide, antigen and bovine serum albumin in a mass ratio of 90-100:4:6:0.3:10; The fluorescent substance-labeled protein is prepared by the following method: (1) react after mixing the fluorescent substance, N-hydroxysuccinimide and carbodiimide; centrifuge to collect the precipitate, disperse and mix with triple distilled water; (II) Then add protein, mix and react; collect the precipitate after centrifugation to obtain fluorescent substance-labeled protein; wherein, the fluorescent substance is fluorescent latex microspheres, fluorescent latex microspheres, N-hydroxysuccinimide, carbon dioxide The ratio of imine and protein is 90-100:4:6:10 by mass. 6. the fluorescent test strip based on energy resonance transfer according to claim 5, is characterized in that: The antibody described in step ① is an anti-Cr-EDTA antibody or an anti-furaltadone metabolite antibody; The time of the stirring reaction described in step 1. and step 2. is 25～35min; The fluorescent substance described in the step (1) is a fluorescent latex microsphere; The condition of the reaction described in step (1) is 20～30 ℃ of reaction 30min; The condition of the light-shielding reaction described in step (2) is 20～30 ℃ of reaction 120min; The antigen described in step (2) is the complete antigen Cr-iEDTA-BSA or the complete antigen CP-AMOZ-BSA; The reaction condition described in step (3) is 20～30 ℃ of reaction 120min; Fluorescent substance described in step (1) is fluorescent latex microsphere; The condition of the reaction described in step (1) is 20～30 ℃ of reaction 30min; The protein described in step (II) is bovine serum albumin or ovalbumin; The condition of the reaction described in the step (II) is 20-30° C. for 120 minutes. 7. the preparation method of the fluorescent test strip based on energy resonance transfer according to claim 1, is characterized in that comprising the following steps: (1) Disperse the fluorescent receptor-labeled antibody on the binding pad; (2) Attach the fluorescent substance-labeled antigen to the chromatographic membrane in a linear manner to form a detection line; attach the fluorescent substance-labeled protein to the chromatographic membrane in a linear manner to form a calibration line; the detection line and the calibration line are parallel relation; (3) Lap the sample absorbent pad, the binding pad obtained in step (1), the chromatographic membrane obtained in step (2) and the water-absorbing pad successively on the substrate, wherein the detection line on the chromatographic membrane is close to the binding pad, The calibration line is away from the binding pad and close to the absorbent pad; resulting in a fluorescent strip based on energy resonance transfer. 8. the preparation method of the fluorescent test paper strip based on energy resonance transfer according to claim 7, is characterized in that: step (1) is: spray the antibody of fluorescent acceptor mark on the binding pad with gold-spraying film-drawing instrument; The fluorescent substance-labeled antigen described in step (2) is wrapped onto the chromatographic membrane by a gold-spraying film marking device; The fluorescent substance-labeled protein described in step (2) is wrapped onto the chromatographic membrane by a gold-spraying film-drawing device. 9. Application of the fluorescent test strip based on energy resonance transfer according to any one of claims 1 to 6 in the detection field.