CN110646606A - Fluorescent quantitative rapid detection test strip for heavy metal mercury ions and preparation method and application thereof - Google Patents
Fluorescent quantitative rapid detection test strip for heavy metal mercury ions and preparation method and application thereof Download PDFInfo
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Abstract
A fluorescent quantitative rapid detection test strip for heavy metal mercury ions and 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 10 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.
Description
Technical Field
The invention relates to a fluorescent quantitative rapid detection test strip for heavy metal mercury ions, a preparation method and application thereof, and belongs to the technical field of food detection.
Background
Mercury is a common chemical substance, is one of heavy metal ions with strong toxicity, and has great harm to aquatic organisms and human health. Mercury poisoning mainly damages the nervous system of humans and animals, damages brain tissue, has a great influence on the digestive system, hearing, skin and the like of humans, and may adversely affect the developing brain and nervous system of infants.
There are many methods for detecting heavy metal ions, and the commonly used methods include inductively coupled plasma emission method, atomic fluorescence spectrometry, atomic absorption spectrometry, hydride generation atomic fluorescence spectrometry, and the like. In recent years, many new methods for detecting mercury ions have appeared, such as the detection of mercury ions by various biochemical substances: fluorescent substances, gold nanoparticles, organic molecules, DNase, electrochemistry and the like are used as basic sensor methods, the sensitivity of the methods is high, but some methods have the defects of complicated sample pretreatment, expensive instruments, time consumption and the like. The fluorescent quantitative rapid detection test strip has the advantages of convenience, rapidness, low cost, simple operation and the like, and can be used for on-site temporary detection. The research establishes a fluorescent quantitative detection test strip method for rapidly and directly detecting mercury ions by using the mercury ion monoclonal antibody, the method is rapid and accurate, a complex sample pretreatment process is not needed, and the rapid detection requirements of different samples can be met.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides the fluorescent quantitative detection test strip for heavy metal mercury ions, and the preparation method and the application thereof, and can be used for rapidly, conveniently and accurately detecting residual heavy metal mercury in grains and processed products thereof on a large scale.
According to the technical scheme, the fluorescent quantitative rapid detection test strip for heavy metal mercury 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 ABA highly secreting heavy metal mercury ion specific antibodies has been deposited in China general microbiological culture Collection center (CGMCC), and is classified and named as a monoclonal cell strain with the preservation number of CGMCC No.17388 and the preservation date of 2019, 3 months and 7 days.
The preparation method of the fluorescent quantitative rapid detection test strip for heavy metal mercury ions comprises the following specific steps:
(1) preparation of mercury 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 34. mu.L of ITCBE solution, stirring at room temperature for reacting for 8 hours, then dropwise adding 187. mu.L (1 mg/mL) of mercury 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-mercury ion monoclonal antibody: after the bifunctional chelating agent ITCBE is coupled with KLH, heavy metal mercury ions are added for chelation, and the heavy metal mercury ions are used as complete immunogen. After the complete immunogen was mixed and emulsified with an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous multipoint injection at the back of the neck (except for the booster immunization). The first immunization adopts the complete antigen of heavy metal mercury 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 mercury ions to be mixed with incomplete Freund's adjuvant, and the dosage is 50 mug/mouse; and finally, mixing heavy metal mercury 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 a monoclonal hybridoma cell strain ABA of the heavy metal mercury ion high-secretion specific antibody.
(4) Purifying heavy metal mercury ion antibodies: carrying out amplification culture on the high-secretion specific antibody monoclonal hybridoma cell strain ABA, and injecting the cell into a mouse body to induce ascites; adding 1mL of ascites into 1mL of sodium acetate buffer solution with the volume equivalent to that of the 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 1mL of saturated ammonium sulfate, and standing for 1-2 h; centrifuging at 8000rpm for 5min, discarding supernatant, dissolving the precipitate in PBS solution, dialyzing for 3 days to obtain anti-mercury ion monoclonal antibody;
(5) the preparation method of the fluorescent quantitative rapid detection test strip for heavy metal mercury ions comprises the following steps:
a. preparing a fluorescent microsphere stock solution: taking 400 mu L of 0.05M boric acid buffer solution with pH of 8 into a 2mL centrifuge tube, adding 100 mu L-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 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 mercury 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 supernatant by using 0.5mL of boric acid buffer solution, redissolving the supernatant by using the boric acid buffer solution after washing and centrifuging, performing ultrasonic dispersion to obtain a fluorescence-labeled heavy metal mercury ion antibody, and storing the antibody at 4 ℃;
c. preparing a fluorescent microsphere marking pad: uniformly spraying the fluorescence-labeled heavy metal mercury 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;
d. preparation of a nitrocellulose membrane detection layer: uniformly spraying the diluted 0.2mg/mL heavy metal mercury 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 and the water absorption pad to obtain the fluorescent quantitative rapid detection test strip for the heavy metal mercury ions of the product.
The method is applied to rapid detection of heavy metal mercury ions in food, is suitable for customs, enterprises, inspection and quarantine units and the like, and can realize rapid detection of heavy metal mercury 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 line of the rice sample is 10 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 ABA capable of highly secreting heavy metal mercury ion specific antibodies is preserved in China general microbiological culture Collection center, and the preservation addresses are as follows: the classification name of No. 3 Xilu-1 of Beijing, Chaoyang, China academy of sciences microbial research is monoclonal cell strain with the collection number of CGMCC No.17388 and the collection date of 2019, 3 months and 7 days.
Drawings
FIG. 1 is a schematic structural diagram of the test strip of the present invention.
FIG. 2 is a standard curve for mercury 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
Example 1
As shown in fig. 1, a fluorescent quantitative rapid detection test strip for heavy metal mercury ions 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 base plate 7;
the two ends of the PVC bottom plate 7 are respectively provided with a sample pad 1 and a water absorption pad 5; 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 mark pad 2 is mutually overlapped with the sample pad 1, and the other end 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.
The preparation method of the fluorescent quantitative rapid detection test strip for heavy metal mercury ions comprises the following specific steps:
(1) preparation of mercury 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 34. mu.L of ITCBE solution, stirring at room temperature for reacting for 8 hours, then dropwise adding 187. mu.L (1 mg/mL) of mercury 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-mercury ion monoclonal antibody: after the bifunctional chelating agent ITCBE is coupled with KLH, heavy metal mercury ions are added for chelation, the heavy metal mercury ions are used as a complete immunogen to be mixed and emulsified with an equal amount of Freund adjuvant, and then the BALB/c mouse is subjected to neck-back subcutaneous multipoint injection immunization (except for sprint immunization). The first immunization adopts the complete antigen of heavy metal mercury 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 mercury ions to be mixed with incomplete Freund's adjuvant, and the dosage is 50 mug/mouse; and finally, mixing heavy metal mercury 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 a monoclonal hybridoma cell strain ABA of the heavy metal mercury ion high-secretion specific antibody.
(4) Purifying heavy metal mercury ion antibodies: carrying out amplification culture on the high-secretion specific antibody monoclonal hybridoma cell strain ABA, and injecting the cell into a mouse body to induce ascites; adding 1mL of ascites into 1mL of sodium acetate buffer solution with the volume equivalent to that of the 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 1mL of saturated ammonium sulfate, and standing for 1-2 h; centrifuging at 8000rpm for 5min, discarding supernatant, dissolving the precipitate in PBS solution, dialyzing for 3 days to obtain anti-mercury ion monoclonal antibody;
(5) the preparation method of the fluorescent quantitative rapid detection test strip for heavy metal mercury ions comprises the following steps:
a. preparing a fluorescent microsphere stock solution: taking 400 mu L of 0.05M boric acid buffer solution with pH of 8 into a 2mL centrifuge tube, adding 100 mu L of 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 20000rpm at 10 ℃ for 10min, discarding the supernatant, redissolving with 0.5mL of 0.05M boric acid buffer solution with pH =8, and ultrasonically dispersing; adding heavy metal mercury ion antibody to make the final concentration of protein 30 μ g/mL, and placing on a 250r shaking bed for 2 h; adding a blocking solution, namely BSA with the final concentration of 1%, placing the mixture in a shaking table for blocking for 2h, centrifuging at 2000rpm for 10min, discarding the supernatant, redissolving the supernatant by using 0.5mL of boric acid buffer solution, washing and centrifuging the supernatant, redissolving the supernatant by using the boric acid buffer solution, performing ultrasonic dispersion to obtain a fluorescence-labeled heavy metal mercury ion antibody, and storing the antibody at 4 ℃;
c. preparing a fluorescent microsphere marking pad: uniformly spraying the fluorescence-labeled heavy metal mercury ion antibody prepared in the step b with the concentration of 0.2mg/mL on a glass fiber membrane detection layer, drying at 37 ℃ overnight, and sealing for later use;
d. preparation of a nitrocellulose membrane detection layer: uniformly spraying the diluted 0.2mg/mL heavy metal mercury 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 (3) 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 mercury ions of the product.
Example 2
The sample is processed according to the following steps during detection: accurately weighing 3g of rice sample crushed to a proper particle size in a 50mL centrifuge tube, adding 9mL of 0.5M dilute hydrochloric acid solution and 6mL of dichloromethane, shaking for 3min on a shaker, centrifuging for 3min at 8000rpm, taking 500 μ L of supernatant, adding 1mL of n-hexane, taking 200 μ L of lower layer solution, and adding 1M NaHCO3Diluting 4 times for standby, and simultaneously making reagent blank.
Dripping 20 mu L of diluted sample solution on a sample pad, and after 5min, putting the test strip into a fluorescent quantitative detector to detect the concentration of heavy metal mercury ions, wherein FIG. 2 is a standard curve for detecting the mercury ions in the rice sample; if the test strip quality control line 4 does not develop color, the test strip quality is problematic.
Claims (6)
1. The utility model provides a fluorescence quantitative short-term test paper strip of heavy metal mercury 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 mercury ions in 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 mercury ion antibody to make the final concentration of protein be 30-50 μ g/mL, placing on 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 supernatant by using 0.5mL of boric acid buffer solution, washing and centrifuging the supernatant, redissolving the supernatant by using the boric acid buffer solution, performing ultrasonic dispersion to obtain a fluorescence-labeled heavy metal mercury ion antibody, and storing the antibody at 4 ℃;
(3) preparing a fluorescent microsphere marking pad: uniformly spraying the fluorescence-labeled heavy metal mercury 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 mercury 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 the bag 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 mercury ions of the product.
3. The preparation method of the fluorescent quantitative rapid detection test strip for heavy metal mercury ions according to claim 2, which is characterized in that: the heavy metal mercury ion antibody is obtained by secreting a monoclonal hybridoma cell strain ABA which highly secretes an anti-mercury ion specific antibody.
4. A monoclonal hybridoma cell strain ABA highly secreting heavy metal mercury ion specific antibodies has been deposited in China general microbiological culture Collection center (CGMCC), and is classified and named as a monoclonal cell strain with the preservation number of CGMCC No.17388 and the preservation date of 2019, 3 months and 7 days.
5. The monoclonal antibody for resisting mercury ions is characterized in that: using the monoclonal hybridoma cell strain ABA of claim 4, carrying out amplification culture, and injecting cells into a mouse body to induce ascites; adding 1mL of ascites into 1mL of sodium acetate buffer solution with the volume equivalent to that of the 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 1mL of saturated ammonium sulfate, and standing for 1-2 h; centrifuging at 8000rpm for 5min, discarding supernatant, dissolving the precipitate in PBS solution, and dialyzing for 3 days to obtain the anti-mercury ion monoclonal antibody.
6. The application of the fluorescent quantitative rapid detection test strip for heavy metal mercury ions in claim 1 is characterized in that: the method is applied to the rapid detection of heavy metal mercury ions in food.
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