阅读说明：本技术 一种重金属铅离子的荧光定量快速检测试纸条及其制备方法和应用 (Fluorescent quantitative rapid detection test strip for heavy metal lead ions, and preparation method and application thereof ) 是由 胥传来 李少珍 匡华 徐丽广 马伟 朱建平 刘丽强 吴晓玲 宋珊珊 胡拥明 于 2019-10-12 设计创作，主要内容包括：一种重金属铅离子的荧光定量快速检测试纸条及其制备方法和应用,属于食品检测技术领域。本发明PVC底板两端分别设有样品垫和吸水垫；在PVC底板中部设有硝酸纤维素膜检测层,在硝酸纤维素膜检测层与样品垫之间设有荧光微球标记垫；所述荧光微球标记垫一端与样品垫互相叠加,另一端与硝酸纤维素膜检测层互相叠加；所述硝酸纤维素膜检测层上依次设有质控线和检测线。本发明检测速度快,全过程只需要5min即可,可以实施大批量样品的快速检测；本发明灵敏度高,大米样本检测限为5ng/mL；本发明过程简单,直接上样检测,样品前处理简单,无需经过专业培训,易于推广,不需要仪器,适合现场检测。(A fluorescent quantitative rapid detection test strip for heavy metal lead ions, a preparation method and application thereof belong to the technical field of food detection. The two ends of the PVC base plate are respectively provided with a sample pad and a water absorption pad; a nitrocellulose membrane detection layer is arranged in the middle of the PVC base plate, and a fluorescent microsphere marking pad is arranged between the nitrocellulose membrane detection layer and the sample pad; one end of the fluorescent microsphere marking pad is mutually overlapped with the sample pad, and the other end of the fluorescent microsphere marking pad is mutually overlapped with the nitrocellulose membrane detection layer; and the nitrocellulose membrane detection layer is sequentially provided with a quality control line and a detection line. The invention has fast detection speed, the whole process only needs 5min, and the rapid detection of a large batch of samples can be implemented; the invention has high sensitivity, and the detection limit of the rice sample is 5 ng/mL; the method has the advantages of simple process, direct sample loading detection, simple sample pretreatment, no need of professional training, easy popularization, no need of instruments and suitability for field detection.)

1. The utility model provides a fluorescence quantitative short-term test paper strip of heavy metal lead ion which characterized in that: comprises a sample pad (1), a fluorescent microsphere marking pad (2), a detection line (3), a quality control line (4), a water absorption pad (5), a nitrocellulose membrane detection layer (6) and a PVC bottom plate (7);

a sample pad (1) and a water absorption pad (5) are respectively arranged at two ends of the PVC bottom plate (7); a nitrocellulose membrane detection layer (6) is arranged in the middle of the PVC bottom plate (7), and a fluorescent microsphere marking pad (2) is arranged between the nitrocellulose membrane detection layer (6) and the sample pad (1); one end of the fluorescent microsphere marking pad (2) is mutually overlapped with the sample pad (1), and the other end of the fluorescent microsphere marking pad is mutually overlapped with the nitrocellulose membrane detection layer (6); and the nitrocellulose membrane detection layer (6) is sequentially provided with a quality control line (4) and a detection line (3).

2. The preparation method of the fluorescent quantitative rapid detection test strip for heavy metal lead ions of claim 1 is characterized by comprising the following steps:

(1) preparing a fluorescent microsphere stock solution: taking 400 mu L of 0.05M boric acid buffer solution with the pH value of 8 into a 2mL centrifuge tube, adding 100-120 mu L fluorescent microspheres, carrying out vortex oscillation, and uniformly mixing for later use;

(2) preparing a fluorescent microsphere labeled antibody: taking 20 mu L of 10mg/mL EDC solution, shaking and activating at room temperature for 15min, centrifuging at 10 ℃ and 2000rpm for 10min, discarding supernatant, redissolving with 0.5mL of 0.05M boric acid buffer solution with pH =8, and ultrasonically dispersing; adding heavy metal lead ion antibody to make the final concentration of protein be 30-50 mug/mL, and placing on a 250r shaking bed for 2 h; adding a blocking solution, namely BSA with the final concentration of 1-2%, placing the mixture in a shaking table for blocking for 1-2h, centrifuging the mixture at 2000rpm for 10min, discarding the supernatant, redissolving the mixture by using 0.5mL of boric acid buffer solution, washing and centrifuging the mixture, redissolving the mixture by using the boric acid buffer solution, performing ultrasonic dispersion to obtain a fluorescence-labeled heavy metal lead ion antibody, and storing the fluorescence-labeled heavy metal lead ion antibody at 4 ℃;

(3) preparing a fluorescent microsphere marking pad: uniformly spraying the fluorescence-labeled heavy metal lead ion antibody prepared in the step (2) of 0.2mg/mL on a glass fiber membrane, wherein the spraying amount is 0.5-1 mu L/cm, drying at 37 ℃ overnight, and sealing for later use;

(4) preparation of a nitrocellulose membrane detection layer: uniformly spraying the diluted 0.2mg/mL heavy metal lead ion antigen on a nitrocellulose membrane detection layer (6) to obtain a detection line (3); uniformly spraying the diluted goat anti-mouse IgG on a nitrocellulose membrane detection layer (6) to obtain a quality control line (4), drying at 37 ℃ overnight, and sealing for later use;

(5) combining: and (2) combining the PVC base plate (7), the sample pad (1), the fluorescent microsphere marking pad (2), the nitrocellulose membrane detection layer (6) coated with the detection line (3) and the quality control line (4) and the water absorption pad (5) to obtain the fluorescent quantitative rapid detection test strip for the heavy metal lead ions.

3. The preparation method of the fluorescent quantitative rapid detection test strip for heavy metal lead ions according to claim 2, which is characterized in that: the heavy metal lead ion antibody is obtained by secreting a monoclonal hybridoma cell strain Rob with high-secretion anti-lead ion specific antibody.

4. A monoclonal hybridoma cell strain Rob with high secretion of a lead ion-resistant specific antibody is preserved in the China general microbiological culture Collection center, is named as a monoclonal cell strain in a classification way, and has a preservation number of CGMCC No.17387 and a preservation date of 2019, 3 months and 7 days.

5. The monoclonal antibody against lead ions is characterized in that: carrying out amplification culture on the monoclonal hybridoma cell strain Rob of claim 4, and injecting the cells into a mouse body to induce ascites; adding 1mL of sodium acetate buffer solution with the same volume to 1mL of ascites, dropwise adding 33.3 mu L of octanoic acid under stirring at room temperature, shaking for 30min, and centrifuging at 8000rpm for 5min to obtain supernatant; adding 1.5-2mL of saturated ammonium sulfate, and standing for 1-2h at 4 ℃; centrifuging at 8000rpm for 5min, discarding supernatant, dissolving the precipitate in PBS solution, dialyzing for 2-3 days to obtain the anti-lead ion monoclonal antibody.

6. The application of the fluorescent quantitative rapid detection test strip for heavy metal lead ions in claim 1 is characterized in that: the method is applied to the rapid detection of heavy metal lead ions in food.

Technical Field

The invention relates to a fluorescent quantitative rapid detection test strip for heavy metal lead ions, and a preparation method and application thereof, and belongs to the technical field of food detection.

Background

In recent years, with the development of science and technology, the quality of life of people is better, but the people's life is also puzzled by the accompanying environmental problems. Industrial pollution and the use of a large amount of chemical fertilizers cause the content of heavy metals in grains to exceed the standard. Lead, as a heavy metal element difficult to be eliminated by human bodies, is difficult to be completely eliminated after entering human bodies, can injure brain cells of human bodies and influence normal activities of human brains, and lead metal seriously influences the nervous systems of fetuses and causes the problems of low intelligence and the like.

It is imperative to establish a rapid and accurate detection method for lead in food samples. At present, the traditional heavy metal detection method mainly comprises the following steps: atomic Absorption Spectrometry (AAS), Atomic Emission Spectrometry (AES), inductively coupled plasma mass spectrometry (ICP-MS), Atomic Fluorescence Spectrometry (AFS), ultraviolet-visible spectrophotometry (UV-VIS), Anodic Stripping Voltammetry (ASV), and the like. However, these methods have high professional requirements, long analysis time and high analysis price, and are not beneficial to popularization and field detection. Compared with the analysis method, the immunoassay method has the advantages of strong specificity, simple and convenient operation, low instrumentization degree and analysis cost, and the fluorescent quantitative detection test strip can carry out quantitative or semi-quantitative determination, has quite high sensitivity and can meet the rapid detection requirements of different samples.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides the fluorescent quantitative detection test strip for heavy metal lead ions, and the preparation method and the application thereof, and can be used for rapidly, conveniently and accurately detecting the residual heavy metal lead in grains and processed products thereof on a large scale.

According to the technical scheme, the fluorescent quantitative rapid detection test strip for the heavy metal lead ions comprises a sample pad, a fluorescent microsphere marking pad, a detection line, a quality control line, a water absorption pad, a nitrocellulose membrane detection layer and a PVC bottom plate;

a sample pad and a water absorption pad are respectively arranged at two ends of the PVC bottom plate; a nitrocellulose membrane detection layer is arranged in the middle of the PVC base plate, and a fluorescent microsphere marking pad is arranged between the nitrocellulose membrane detection layer and the sample pad; one end of the fluorescent microsphere marking pad is mutually overlapped with the sample pad, and the other end of the fluorescent microsphere marking pad is mutually overlapped with the nitrocellulose membrane detection layer; and the nitrocellulose membrane detection layer is sequentially provided with a quality control line and a detection line.

A monoclonal hybridoma cell strain Rob with high secretion of a lead ion-resistant specific antibody is stored in the China general microbiological culture Collection center (CGMCC), is named as a monoclonal cell strain in a classification way, and has a storage number of CGMCC No.17387 and a storage date of 2019, 3 months and 7 days.

The preparation method of the fluorescent quantitative rapid detection test strip for heavy metal lead ions comprises the following specific steps:

(1) preparation of lead antigen: first, 10mg of ITCBE was dissolved in 1mL of anhydrous DMSO, mixed by shaking, and stored at-20 ℃. Weighing 20mg of BSA, adding a 5mLHBS (0.01M PH = 9) solution for dissolving, dropwise adding 66. mu.L of ITCBE solution, stirring at room temperature for reacting for 8 hours, then dropwise adding 596. mu.L (1 mg/mL) of lead standard solution, adjusting the pH with 0.5M NaOH to maintain the pH of the solution at 8-9, and reacting for 1 hour. After the reaction is finished, centrifuging 6500rpm by using an Amicon Ultra-4 Ultra cel-3K ultrafiltration centrifugal tube with the cutoff of 3000 for 20min, and resuspending by using 3mL of HBS solution after each ultrafiltration is finished. The ultrafiltration was repeated three times, and 10mL of HBS solution was added to give a final concentration of 2mg/mL of protein, and the resulting mixture was frozen and stored at-20 ℃.

(2) Preparing the anti-lead ion monoclonal antibody: after coupling the bifunctional chelating agent ITCBE and KLH, adding heavy metal lead ions for chelation to serve as a complete immunogen, mixing and emulsifying the complete immunogen and an equivalent amount of Freund's adjuvant, and then carrying out neck and back subcutaneous multipoint injection immunization (except for puncture immunization) on BALB/c mice. The first immunization adopts the complete antigen of heavy metal lead ions and the complete Freund adjuvant to be mixed, and the dosage is 100 mu g/mouse; multiple booster immunizations adopt complete antigen of heavy metal lead ions and incomplete Freund's adjuvant mixed, the dosage is 50 mu; and finally, mixing heavy metal lead ion complete antigen and normal saline for spurting immunization. Intraperitoneal injection is adopted, and the dosage is 25 mu g per mouse. The interval between the first immunization and the second boosting immunization is one month, the interval between the multiple boosting immunizations is 21 days, and the interval between the sprint immunization and the last boosting immunization is 18-21 days. The titer and inhibition of the mouse serum are detected by observing the immune effect of the mouse by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).

(3) Cell fusion and cell line establishment: fusing mouse spleen cells and mouse myeloma cells by a polyethylene glycol (PEG 4000) method, screening hybridoma cells by using a selective medium (HAT medium), and performing cell culture by using an HT medium. And detecting the positive cell holes by using an ic-ELISA method after one week of fusion, further determining the inhibition effect of the positive cell holes by using the ic-ELISA method, performing subcloning on the positive cell holes with better inhibition by using a limiting dilution method, and detecting, selecting and subcloning again after one week. And carrying out subcloning for three times according to the method to obtain the heavy metal lead ion monoclonal hybridoma cell strain Rob with high secretion of the anti-lead ion specific antibody.

(4) Purifying heavy metal lead ion antibodies: carrying out amplification culture on a monoclonal hybridoma cell strain Rob which highly secretes an anti-lead ion specific antibody, and injecting hybridoma cells into a mouse body to induce ascites; adding 1mL of sodium acetate buffer solution with the same volume to 1mL of ascites, dropwise adding 33.3 mu L of octanoic acid under stirring at room temperature, shaking for 30min, and centrifuging at 8000rpm for 5min to obtain supernatant; adding 1.5-2mL of saturated ammonium sulfate, and standing for 1-2h at 4 ℃; centrifuging at 8000rpm for 5min, discarding supernatant, dissolving the precipitate in PBS solution, dialyzing for 2-3 days to obtain the anti-lead ion monoclonal antibody.

(5) The preparation of the fluorescent quantitative rapid detection test strip for heavy metal lead ions comprises the following steps:

a. preparing a fluorescent microsphere stock solution: taking 400 mu L of 0.05M boric acid buffer solution with the pH value of 8 into a 2mL centrifuge tube, adding 100-120 mu L fluorescent microspheres, carrying out vortex oscillation, and uniformly mixing for later use;

b. preparing a fluorescent microsphere labeled antibody: taking 20 mu L of 10mg/mL EDC solution, shaking and activating at room temperature for 15min, centrifuging at 2000rpm at 10 ℃ for 10min, discarding supernatant, redissolving with 0.5mL of 0.05M boric acid buffer solution with pH =8, and ultrasonically dispersing; adding heavy metal lead ion antibody to make the final concentration of protein be 30-50 mug/mL, and placing on a 250r shaking bed for 2 h; adding a blocking solution, namely BSA (bovine serum albumin) with the final concentration of 1-2%, placing the mixture in a shaking table for blocking for 1-2h, centrifuging at 2000rpm for 10min, discarding the supernatant, redissolving the mixture by using 0.5mL of boric acid buffer solution, washing and centrifuging the mixture, redissolving the mixture by using a coupling storage buffer solution, performing ultrasonic dispersion to obtain a fluorescence-labeled heavy metal lead ion antibody, and storing the fluorescence-labeled heavy metal lead ion antibody at 4 ℃;

c. preparing a fluorescent microsphere marking pad: uniformly spraying the fluorescence-labeled heavy metal lead ion antibody prepared in the step b with the concentration of 0.2mg/mL on a glass fiber membrane, drying at 37 ℃ overnight, and sealing for later use;

uniformly spraying the d0.2mg/mL heavy metal lead ion antigen on a nitrocellulose membrane detection layer to obtain a detection line; uniformly spraying the diluted goat anti-mouse IgG on a nitrocellulose membrane to obtain a quality control line, drying at 37 ℃ overnight, and sealing for later use;

e. combining: and combining the PVC base plate, the sample pad, the fluorescent microsphere marking pad, the nitrocellulose membrane detection layer coated with the detection line and the quality control line, the water absorption pad and the combination to obtain the fluorescent quantitative rapid detection test strip for the heavy metal lead ions of the product.

The fluorescent quantitative rapid detection test strip for the heavy metal lead ions is applied to rapid detection of the heavy metal lead ions in food, is suitable for customs, enterprises, inspection and quarantine units and the like, and can realize rapid detection of the heavy metal lead ions in rice samples.

The invention has the beneficial effects that: the invention has fast detection speed, the whole process only needs 5min, and the rapid detection of a large batch of samples can be implemented; the invention has high sensitivity, and the detection limit of the rice sample is 5 ng/mL;

the method has the advantages of simple process, direct sample loading detection, simple sample pretreatment, no need of professional training, easy popularization, no need of instruments and suitability for field detection.

Biological material sample preservation: a monoclonal hybridoma cell strain Rob with high secretion of a specific anti-lead ion antibody is preserved in the China general microbiological culture Collection center, and the preservation addresses are as follows: the collection number of the microbial research institute of Chinese academy of sciences is CGMCC No.17387, the collection date is 3 months and 7 days in 2019.

Drawings

FIG. 1 is a schematic structural diagram of the test strip of the present invention.

FIG. 2 is a standard curve of lead ion detection of rice samples.

Description of reference numerals: 1. a sample pad; 2. a fluorescent microsphere label pad; 3. detecting lines; 4. a quality control line; 5. a water absorbent pad; 6. a nitrocellulose membrane detection layer; 7. PVC bottom plate.

Detailed Description