A nanoflower immunochromatographic test strip for detecting heavy metal mercury ions and its application

Abstract

The present invention provides a nanoflower immunochromatographic test strip for detecting heavy metal mercury ions, which belongs to the technical field of rapid heavy metal detection. The nanoflower immunochromatographic test strip comprises the following components: a plastic shell, a sample pad, an immune probe binding pad, a nitrocellulose membrane and a water-absorbing pad; the immune probe binding pad is dripped with a gold nanoimmunoprobe labeled with an anti-mercury ion monoclonal antibody; the anti-mercury ion monoclonal antibody is an anti-Hg 2+ ‑ITCBE hapten monoclonal antibody secreted by mouse hybridoma cell line 7A1; the deposit number of mouse hybridoma cell line 7A1 is CGMCC No.23879. The nanoflower immunochromatographic test strip detects heavy metal hapten Hg 2+ ‑ITCBE, the detection threshold is 50 ng / mL, and the detection limit is 0.39 ng / mL; it has strong specificity, high sensitivity, good repeatability, and rapid detection, and is of great significance for monitoring the residues of heavy metal mercury.

Description

Technical Field

[0001] The invention belongs to the technical field of rapid detection of heavy metal pollution, and specifically relates to a nanoflower immunochromatographic test strip for detecting heavy metal mercury ions and an application thereof. Background Art

[0002] With the continuous improvement of living standards in today's society, a large amount of industrial pollution and agricultural activities that have not been effectively treated have led to excessive discharge of heavy metals, which in turn directly or indirectly harm the environment and human health through atmospheric deposition, agricultural fertilization, human ingestion, etc. In the process of heavy metals being discharged into the environment, they will quickly settle into the sediment, which can easily cause secondary release, continue to pollute the water body, and bring great harm to aquatic organisms such as fish; if contaminated sewage containing heavy metals is used to irrigate farmland, it will cause serious pollution to the soil and crops. These heavy metal pollutions will be enriched hundreds of times under the biomagnification of the food chain, and eventually enter the human organs and accumulate, posing a huge threat to human health. Plants have the ability to absorb and accumulate mercury, and absorb and accumulate more mercury as the mercury content in water and soil increases. The mercury in plants can be used as animal feed and circulate into the animal body. Traditional detection methods for mercury and other heavy metals, such as atomic absorption spectrometry, inductively coupled plasma mass spectrometry and other analytical methods, can meet the needs of trace detection, but these methods are expensive, time-consuming and labor-intensive, and costly, and are not suitable for the needs of rapid on-site detection. The immunoassay method is fast, sensitive, and suitable for on-site detection, providing a new option for the detection of mercury and other heavy metals. The present invention uses gold nanoflower particles to label antibodies to prepare immunoassay probes, and combines immunochromatographic test strip technology to develop nanoflower immunochromatographic test strips. The immunochromatographic test strips are simple to assemble, highly specific, and only target Hg 2+ It has no competition, no cross-reaction to other metal ions, good stability, high sensitivity, excellent repeatability, and a detection limit lower than the national food safety standard. It can effectively and accurately detect heavy metal mercury ions residual in grain, which is of great practical significance for the safe monitoring of heavy metal residues in grain. Summary of the invention

[0003] In view of the above problems, the present invention provides a nanoflower immunochromatographic test strip for detecting heavy metal mercury ions and its application. The nanoflower immunochromatographic test strip of the present invention has strong specificity, high sensitivity and good stability, and can be used for rapid detection of mercury ions in food.

[0004] To achieve the above object, the present invention adopts the following technical solution:

[0005] A nanoflower immunochromatographic test strip for detecting heavy metal mercury ions, the nanoflower immunochromatographic test strip comprising the following components: a plastic shell, a sample pad, an immune probe binding pad, a nitrocellulose membrane and a water-absorbing pad; the immune probe binding pad is dripped with a gold nanoimmunoprobe labeled with an anti-mercury ion monoclonal antibody; the anti-mercury ion monoclonal antibody is an anti-Hg 2+ -ITCBE hapten The monoclonal antibody secreted by the mouse hybridoma cell line 7A1, the mouse hybridoma cell line 7A1, has been deposited in the General Microbiology Center of China Culture Collection Administration, the deposit address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the deposit date is November 23, 2021, and the deposit number is CGMCC No.23879.

[0006] The preparation method of the above-mentioned nanoflower immunochromatographic test strip for detecting heavy metal mercury ions comprises the following steps:

[0007] (1) Preparation of gold nanoflower solution: In a 250 mL conical flask that has been soaked in aqua regia and washed and sterilized at high temperature, add 100 mL of double distilled water and adjust the pH to 7.5 with 1 M NaOH; under stirring, add 750 μL of 1 wt% chloroauric acid, 500 μL of colloidal gold as seeds, 300 μL of 1 wt% trisodium citrate and 1 mL of newly prepared 0.03 M hydroquinone solution in sequence, and continue stirring until a stable blue color is obtained to obtain a gold nanoflower solution;

[0008] (2) Preparation of immune probe: Take 10 mL of gold nanoflower solution and place it in a sterilized bottle. Add 60 μL of 0.1 MK 2 CO 3 , stirred evenly, then added 60 μL of 1.25 mg / mL anti-mercury ion monoclonal antibody dropwise, continued stirring for 1 h, then added BSA according to 1% of the total volume, continued stirring for 30 min under ice bath, then added PEG 20000 according to 0.5% of the total volume, continued stirring for 30 min under ice bath, finally sealed the labeled solution and placed in a 4°C refrigerator for equilibrium overnight to obtain anti-mercury ion monoclonal antibody labeled gold nanoimmunoprobe (AuNF-mAb); the preparation method of the anti-mercury ion monoclonal antibody is: the mouse hybridoma cell line 7A1 in the logarithmic growth phase is cultured at 1×10 6 cell / was injected into the abdominal cavity of 9-week-old Balb / c female mice sensitized with paraffin. When the abdomen of the mice was swollen and tense, ascites was extracted and anti-Hg was obtained by separation and purification. 2+ -ITCBE monoclonal antibody;

[0009] (3) Pretreatment of the immunoprobe binding pad and sample pad: Cut the untreated immunoprobe binding pad and sample pad into strips with a width of 1.3 cm with scissors, place them in a large culture dish, submerge them with the blocking solution prepared in advance, and then transfer them to a 37°C constant temperature incubator for blocking for 2 h. After taking them out of the incubator, filter out the blocking solution and continue to place them in the incubator for drying. After drying, store them at 4°C; for subsequent use, cut them into strips with a length of 1.3 cm and a width of 4 mm; the composition of the blocking solution is: 5wt% BSA + 1vol% Tween-20;

[0010] (4) NC membrane streaking: Take goat anti-mouse secondary antibody and streak it on the NC membrane. The final concentration on line C is 0.53 ng / cm. After cutting, the content of goat anti-mouse secondary antibody streaked on each 4 mm wide card strip is 0.212 ng. Take complete antigen Hg 2+ -ITCBE-BSA was streaked on the NC membrane, and the final concentration on the T line was 0.043 ng / cm. After cutting, each 4 mm wide card strip contained complete antigen Hg 2+ -ITCBE-BSA content is 0.0172 ng, which is the T line; on the same NC membrane, the distance between the C and T lines is 0.5 cm;

[0011] (5) Preparation of the immunoprobe binding pad: Add 3 μL of the gold nanoimmunoprobe labeled with the anti-mercuric ion monoclonal antibody prepared in step (2) onto the pretreated immunoprobe binding pad;

[0012] (6) Assembly of immunochromatographic test strips: Assemble the NC membrane marked in step (4), the immunoprobe binding pad prepared in step (5), the sample pad pretreated in step (3), and the absorbent pad in sequence on the bottom plate; wherein the immunoprobe binding pad and the sample pad are overlapped and pasted alternately, with the same ends separated by 2 mm, and the absorbent pad overlaps the NC membrane by 2 mm from beginning to end. Cover with a plastic shell lid, dry and seal, and store at 4°C.

[0013] The above-mentioned nanoflower immunochromatographic test strip for detecting heavy metal mercury ions is used in detecting heavy metal mercury ions.

[0014] The detection principle of a nanoflower immunochromatographic test strip for detecting heavy metal mercury ions: When the test strip is used for detection, the prepared gold-labeled probe is first dropped onto the gold-labeled pad, and then the probe is dried and then placed according to the schematic diagram ( Figure 1) and drop the sample on the sample pad. The liquid flows to one end of the absorbent paper through chromatography. When passing through the T line, the gold-labeled probe that has not bound to the antigen in the sample will completely bind to the T line and stay on the T line. As a result, the T line will be colored. The liquid will continue to flow forward. When passing through the C line, the gold-labeled probe that is bound to the antigen in the sample or not will bind to the C line until saturation. The bound gold-labeled probe will stay on the C line, and the C line will be colored. The entire color reaction takes about 10 minutes.

[0015] Beneficial effects of the present invention:

[0016] (1) The present invention develops a nanoflower immunochromatographic test strip for detecting heavy metal mercury ions. The nanoflower immunochromatographic test strip can be used to detect the content of heavy metal mercury ions in food. The detection threshold is 50 ng / mL, and the minimum detection limit is 1.56 ng / mL under visual conditions; the minimum detection limit is 0.39 ng / mL under card reader detection conditions, and the detection time only takes 5 min.

[0017] (2) The present invention provides a cell line of monoclonal antibodies against heavy metal mercury ions, and determines the affinity constant Kaff =7.2×10 9 L / mol, with high affinity, it can better recognize mercury ion hapten, and the 50% inhibition rate of mercury ion IC50=37.426 ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Structural model of nanoflower immune layer test strip.

[0019] Figure 2 Agarose gel analysis of antigen conjugates. Lane 1: KLH Lane 2: Hg 2+ -ITCBE-KLH Lane 3: Hg 2+ -ITCBE-BSA Lane 4: BSA.

[0020] Figure 3 Mouse tail blood titer and icELISA determination; A Mouse tail blood titer; B Mouse tail blood competition.

[0021] Figure 4 Identification of 7A1 monoclonal antibody isotypes.

[0022] Figure 5 Identification of the purification results of 7A1 monoclonal antibody by SDS-PAEG.

[0023] Figure 6 Graph of purified antibody and ascites titer.

[0024] Figure 7 Monoclonal antibody affinity determination.

[0025] Figure 8 Antibody cross-reactivity analysis.

[0026] Fig. 9 Anti-Hg 2+ -Plotting of competition curve of ITCBE monoclonal antibody.

[0027] Fig.10 UV full wavelength scanning image and TEM image of gold nanoflower solution. A: Full wavelength scanning image; B: TEM image.

[0028] Fig.11 Determination of the optimal amount of antibody and pH for gold nanoflower labeling. The optimal amount of A antibody labeled gold nanoflower is 4.98 μg / mL; the optimal pH of B antibody labeled gold nanoflower system is 6.5.

[0029] Fig.12 Selection of the best C and T lines for gold nanoflower-labeled antibodies. A The best C line concentration is 0.53 μg / cm; B The best T line concentration is 0.043 μg / cm.

[0030] Fig.13 Determination of specificity of the Nanoflower Immunolayer Test Strip. A. Determination of specificity of the Nanoflower Immunolayer Test Strip; B. Determination of T-line value by the immunoreader.

[0031] Fig.14 Nanoflower immunolayer test strip sensitivity determination. A. Determination of the sensitivity of the immunolayer test strip; B. Determination of the sensitivity value using an immunoreader.

[0032] Fig.15 Actual sample testing of Nanoflower Immunolayer Test Strips. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention but are not intended to limit the present invention.

[0034] Example 1 Screening of mercury ion-resistant monoclonal cell line 7A1 and antibody preparation

[0035] 1. Preparation of artificial complete antigen

[0036] 10 mg of hemocyanin KLH was dissolved in 1 mL of HBS buffer and adjusted to pH 8 with 1 mol / mL NaOH. Then, 10 mg of 1-(4-isothiocyanatobenzyl)ethylenediamine-N,N,N,N-tetraacetic acid (ITCBE) was added and mixed evenly. The mixture was cross-linked at room temperature at 180 r / min in the dark overnight to obtain the hapten. Then, 130 μL of mercury ion standard solution (1 mg / mL) was slowly added dropwise. The reaction was cross-linked at 180 r / min in the dark for 6 h. The mixture was washed with 0.01 M PBS (NaCl 18.0 g; KCl 0.2 g; Na 2 HPO 4 1.44 g; KH 2 PO 4 0.24 g; add distilled water to 1000 ml, adjust pH to 7.4) and dialyze for two days, changing the dialysate every 4 h. After dialysis, concentrate with PEG 20000 to obtain the immune antigen Hg 2+ -ITCBE-KLH. Replace hemocyanin KLH with bovine serum albumin (BSA) to prepare the detection antigen Hg 2+ -ITCBE-BSA, prepared as above ( Figure 2 ).

[0037] 2. Animal immunization

[0038] Artificial complete antigen Hg 2+ -ITCBE-KLH was used as the immune antigen to immunize 6-week-old Balb / c mice. 2+ -ITCBE-KLH was mixed with an equal volume of Freund's complete adjuvant, and after complete emulsification by vortex oscillator and syringe suction, 100 μg / 200 μL per mouse was injected subcutaneously at multiple points on the back of the mouse's neck. For subsequent immunizations, the immunizing antigen was mixed with Freund's incomplete adjuvant in a 1:1 ratio and emulsified completely, and 50 μg / 200 μL per mouse was injected subcutaneously at multiple points, with an interval of 14 days between each immunization. Within 5-7 days after each immunization starting from the fourth time, blood was collected from the tail vein, serum was separated, and the mouse serum titer was detected by indirect ELISA, and the mouse serum IC50 ( Figure 3 ). Select mice with relatively good titer and IC50, and use 2 times the previous immunization dose of immune antigen Hg 2+ -ITCBE-KLH was diluted to 500 μL with normal saline and injected intraperitoneally for the last booster immunization. The above indirect ELISA and indirect competitive ELISA were both conventional ELISA operations.

[0039] 3. Cell fusion

[0040] 3-4 days after the final booster immunization, spleen cells of mice were obtained, 50% polyethylene glycol 1450 (PEG 1450) was used as a fusion agent, spleen cells and SP2 / 0 myeloma cells were mixed in 20 mL of fresh RPMI 1640 incomplete culture medium at a cell number ratio of 4:1, and after gentle mixing, the mixture was centrifuged at 1100 rpm for 7 min, the supernatant was discarded, and the centrifuge tube was flicked to loosen the cells at the bottom. Then, the centrifuge tube was placed in a 37°C water bath, and 1 mL of 50% PEG1450 preheated at 37°C was added slowly and then quickly within 1 min, while shaking the centrifuge tube to promote cell contact, and the mixture was allowed to stand for 0.5 min. Then, 1 mL of RPMI 1640 incomplete culture medium was slowly added within the first min, and RPMI 1640 incomplete culture medium was added slowly and then quickly within 2 min to terminate the effect of PEG; centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and 5 mL of fresh culture medium was gently mixed, and the cell suspension was transferred to 95 mL of complete culture medium containing 2% HAT (RPMI 1640 culture medium containing 20% ​​FBS), mixed, and then plated to a 96-well plate pre-plated with feeder cells (about 2000 feeder cells per well in RPMI 1640 culture medium containing 10% FBS), 100 μL per well; after 15 days of fusion, the complete culture medium with 2% HAT was replaced. When the cells grew to 5-7 days after fusion, 100 μL of supernatant was aspirated from each well, and 120 μL of complete culture medium containing 2% HAT (RPMI 1640 culture medium containing 20% ​​FBS) was added. On the 10th-12th day of fusion, the ELISA method was used for screening in two steps. The first step was to use indirect ELISA to screen anti-Hg 2+ -ITCBE but not resistant to carrier protein; in the second step, the positive wells screened in the first step were screened by indirect competitive ELISA with Hg 2+ -ITCBE was used as a competitive antigen for indirect competitive ELISA; wells with higher absorbance and sensitivity were selected (the absorbance here refers to the first step test result of the same cell well; the sensitivity here refers to the concentration of the competitive antigen when the inhibition rate is 50%, that is, the second step test result of the same cell well), and subcloning was performed by limiting dilution method. 7-8 days after the first subcloning, the same two-step screening was used for detection, and wells with higher absorbance and sensitivity were selected. 10-12 days after the remaining subclones, only indirect ELISA method was used for detection, and wells with higher absorbance were selected. After repeating subcloning 2-3 times, until the positive rate reached 100%, the mouse hybridoma cell line 7A1 was obtained.

[0041] The mouse hybridoma cell line 7A1 has been deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is November 23, 2021, and the deposit number is CGMCC No. 23879.

[0042] 4. Antibody subtype identification

[0043] The anti-Hg secreted by hybridoma cell line 7A1 was identified using a commercially available isoform determination kit (manufacturer SIGMA, catalog number ISO2-1KT). 2+ -Monoclonal antibodies of ITCBE, identification results as Figure 4 As shown, the cell line is of IgG1 subtype.

[0044] Example 2 Anti-Hg 2+ - Preparation and characterization of ITCBE monoclonal antibodies

[0045] 1. Preparation and purification of ascites

[0046] 1) Preparation of ascites

[0047] The mouse hybridoma cell line 7A1 in the logarithmic growth phase was cultured at a rate of approximately 1×10 6 cell / mouse was injected into the abdominal cavity of 9-week-old Balb / c female mice sensitized with paraffin (500μL / mouse). The mice were observed one week later. When the abdomen of the mice was swollen and tense, the ascites was extracted and centrifuged at 4℃ and 13000 r / min for 20 min. After centrifugation, it was divided into three layers (from bottom to top: aggregates, middle layer containing a large amount of antibodies, lipid layer), and the middle layer was taken.

[0048] 2) Antibody purification

[0049] The ascites was equilibrated with buffer (Na 2 HPO 4 The ascites was diluted 1:10 with 4.383 g / L NaCl (pH 7.0) and filtered through a 0.45 μm filter and then passed through a Protein G affinity chromatography column. The commercial Protein G affinity chromatography instructions were followed and the purified antibodies were dialysis-converted in a dialysis bag with PBS (NaCl 8.0 g; KCl 0.2 g; Na 2 HPO 4 1.44 g; KH 2 PO 4 0.24 g; add distilled water to 1000 ml, adjust pH to 7.4) and dialyze for 3 days, then dialyze for 1 day with ultrapure water, and finally concentrate with PEG 20000 to obtain anti-Hg 2+-ITCBE monoclonal antibody, store the monoclonal antibody in a -20℃ refrigerator for future use. Verify the purity of the antibody by 13% SDS-PAGE gel electrophoresis. Figure 5 As shown in the figure, the purified antibody has bands at 25 kDa and 50 kDa, corresponding to the light chain and heavy chain of IgG antibody, respectively, and there are almost no mixed bands, indicating that the purification effect is good. After complete dialysis with ultrapure water, freeze-drying, and collecting the lyophilized powder, the anti-divalent mercuric ion monoclonal antibody 7A1 is obtained, and the antibody is placed in a -20 ℃ refrigerator for use.

[0050] 3) Titer determination: The titer of ascites and purified antibodies was determined by conventional non-competitive enzyme-linked immunosorbent assay (ELISA). Figure 6 As shown, the titer of ascites exceeded 1.28×10 5 After purification, the antibody titer exceeded 0.64×10 5 , indicating that the antibody still maintains high activity after purification.

[0051] 2. Characterization and identification of monoclonal antibodies

[0052] 1) Affinity determination

[0053] Monoclonal antibody affinity constants by indirect ELISA according to the Beatty method Kaff Hg was measured by ELISA coating buffer. 2+ -ITCBE-BSA was diluted to 10, 5, 2.5, and 1.25 μg / mL and coated on the ELISA plate. The remaining steps were performed according to the conventional indirect ELISA procedure. Origin 8.0 was used for analysis. Figure 7 As shown, the affinity constant of the antibody is calculated according to the following formula, and the result is 7.3×10 9 L / mol, which is a high affinity antibody.

[0054]

[0055] Where: [Ab] represents the antibody concentration at IC50 when the antigen concentration is [Ag];

[0056] [Ab]t represents the antibody concentration at IC50 when the antigen concentration is [Ag]t;

[0057] n=[Ag] / [Ag]t

[0058] 2) Antibody cross-reactivity analysis

[0059] Detection of antigen Hg 2+ -ITCBE-BSA was coated on the ELISA plate at 5 μg / mL, Hg 2+ , Zn2+ ,Mg 2+ , Cu 2+ , Ca 2+ , Mn 2+ , Fe 2+ , Cd 2+ ,Cr 3+ Nine metal ion standards were chelated with ITCBE, and ITCBE was used as a competitive antigen. Ten competitive antigens were diluted to 200 ng / mL, and antibody 7A1 was diluted to 1:64k. The specificity was identified by conventional indirect competitive ELISA. The results are as follows: Figure 8 shown.

[0060] 4) Antibody sensitivity determination

[0061] The conventional indirect competitive ELISA method was used to identify its activity against Hg 2+ -ITCBE sensitivity, draw a standard competition curve, the result is as follows Fig. 9 As shown in A, the curve equation is y=0.0366+{0.8753 / [1+(x / 37.426) 0.9067 ]},R 2 =0.99017, where IC50=37.426 ng / mL, showing a good linear relationship between IC10 and IC90, and the linear equation of the linear part is y =0.89963-0.26524x, R 2 =0.97494, the result is Fig. 9 As shown in B, the linear range is 0.00099 ~ 1.024 μg / mL.

[0062] Example 3 Preparation of Nanoflower Immunochromatographic Test Strip Card

[0063] (1) Preparation of gold nanoflower working solution

[0064] Prepare a 250 mL conical flask that has been soaked in anhydride and washed and sterilized at high temperature, add 100 mL of double distilled water, and adjust to about pH 7.5 with 1M NaOH. Under stirring conditions, add 750 μL of 1wt% chloroauric acid, 500 μL of colloidal gold as seeds, 300 μL of 1wt% trisodium citrate, and 1 mL of newly prepared 0.03 M hydroquinone solution in sequence, and stir until dark blue to prepare a gold nanoflower solution. Next, use an ELISA reader to scan the prepared gold nanoflower solution in the range of 400-900nm to observe its maximum absorption peak wavelength (such as Fig.10 A). The prepared gold nanoflower solution needs to be subjected to scanning electron microscopy (TEM) to observe the size of the gold nanoflower particles and calculate the average diameter (e.g. Fig.10 B). Then seal the prepared solution and store it in a refrigerator at 4°C.

[0065] (2) Labeling of gold nanoflower immunoprobes

[0066] 1) Determination of the optimal amount of antibody labeled with gold nanoflowers

[0067] First, 200 μL of gold nanoflower working solution was added to 8 ELISA wells respectively. The first well was used as a blank control, and 1 μL of 1.25 mg / mL anti-mercury ion (Hg 2+ -ITCBE) monoclonal antibody, then add 2μL, 3μL, 4μL, 5μL, 6μL and 7μL of anti-mercury ion monoclonal antibody to the remaining 6 wells in turn, mix with a pipette, and place in a 37℃ constant temperature incubator for 30 min. Then add 20μL of 10wt% NaCl solution to each well, mix well and continue to stand in a 37℃ constant temperature incubator for 5min. After taking out, observe the color change of the gold nanoflower working solution, and detect the absorbance of each well at 590 nm by an enzyme marker (such as Fig.11 A). The results show that when 1 μL of anti-mercuric ion monoclonal antibody was added, the 590 nm, the absorbance can reach the maximum value, and the solution color of this well has basically no obvious change compared with the control group, indicating that adding 1 μL1.25 mg / mL monoclonal antibody to the gold nanoflower working solution system is the most stable.

[0068] 2) Determination of the optimal pH for gold nanoflower labeling

[0069] After optimizing the amount of labeled antibody, the pH of the working solution for labeling antibodies needs to be optimized. The steps are as follows: First, take 8 enzyme-labeled wells and add 200 μL of gold nanoflower working solution to each well. Add 1 μL of the previously determined optimal antibody (1.25 mg / mL) to the gold nanoflower working solution and mix gently with a pipette. The first well is used as a blank control, and 1 μL of 0.1 MK is added to the second well. 2 CO 3 Then, add 2 μL, 3 μL, 4 μL, 5 μL, 6 μL and 7 μL of 0.1 MK to the remaining 6 wells. 2 CO 3 The solution was mixed and placed in a 37 ℃ constant temperature incubator for 30 min. Then 20 μL of 10% NaCl solution was added to each well, mixed and placed at 37 ℃ for 5 min, and the color change of the gold nanoflower working solution was observed. The OD of each well was detected by an enzyme marker. 590 The absorbance at nm (e.g. Fig.11 B). The results show that: adding 1 μL 0.1 MK 2 CO 3 When OD 590nm, the absorbance can reach the maximum value, and the solution color of this well has basically no significant change compared with the control group, indicating that 1 μL of 0.1 MK 2 CO 3 Most stable.

[0070] (3) Gold nanoflower labeled anti-mercury ion monoclonal antibody

[0071] Take 10 mL of gold nanoflower working solution and place it in a 20 mL narrow-necked bottle that has been sterilized at high temperature. Add 60 μL of the optimal K 2 CO 3 (0.1 M) (considering the loss of antibodies during the addition or labeling process, the optimal antibody amount is added at 1.2 times the volume), stir evenly, then add 60 μL of the optimal antibody labeling amount dropwise, continue stirring for 1 h, then add 0.1012 g of BSA protein at 1% of the total volume, continue stirring for 30 min in an ice bath, then add 0.0506 g of PEG20000 at 0.5% of the total volume, continue stirring for 30 min in an ice bath, finally seal the labeled solution and place it in a 4°C refrigerator for equilibrium overnight.

[0072] (4) Selection of optimal conditions for rapid detection of gold nanoflower test strips

[0073] 1) Treatment of probe binding pad and sample pad

[0074] Cut the untreated gold label pad and sample pad into 1.3 cm wide strips with scissors, place them in a large culture dish, submerge them with the prepared blocking solution (5% BSA + 1% Tween-20), and then transfer them to a 37°C constant temperature incubator for blocking for 2 hours. Remove the blocking solution from the incubator and continue to dry in the incubator. Store at 4°C after drying. Cut them into 1.3 cm long and 4 mm wide for subsequent use.

[0075] 2) Selection of the optimal dilution of the C line of the test strip

[0076] 2 mg / mL goat anti-mouse secondary antibody was diluted with 0.01 M PBS according to the final concentration of line C, and the final dilution was 1.33, 0.89, 0.67, and 0.53 μg / mL. 10 μL of each concentration was streaked on the NC membrane. When using a gold spray instrument to streak, the streaking speed was 0.2 μL / cm, and repeated 5 times. The final concentrations on line C were 1.33, 0.89, 0.67, and 0.53 ng / cm, respectively. The streaked NC membrane was placed in a 37°C constant temperature incubator to dry for 15 min and then cut into strips with a width of 4 mm. After cutting, the contents of goat anti-mouse secondary antibody on each 4 mm wide card strip were 0.532, 0.356, 0.268, and 0.212 ng, respectively. Next, 3 μL of gold-labeled probe was dripped onto the four assembled immune layer test strips, and 100 μL of 0.01 M PBS was slowly dripped onto the sample pad. The strips were allowed to react at room temperature for 10 min, and the color depth of the C line of the test strips was observed. Finally, through observation, a concentration of 0.53 ng / cm (i.e., the content of goat anti-mouse secondary antibody on each 4 mm wide card strip after cutting was 0.212 ng) was selected as the optimal C line concentration ( Fig.12 A).

[0077] 3) Selection of the optimal dilution of the test strip T line

[0078] Complete antigen Hg 2+ -ITCBE-BSA was diluted with 0.01 M PBS according to the final concentration of the T line, and the final dilution was 0.21, 0.11, 0.071, 0.053, and 0.043 μg / mL. 10 μL of each concentration was streaked on the NC membrane. When using a gold spray instrument to streak, the streaking was repeated five times at a streaking speed of 0.2 μL / cm. The final concentrations streaked on the T line were 0.21, 0.11, 0.071, 0.053, and 0.043 ng / cm, respectively. The streaked NC membrane was placed in a 37 ℃ constant temperature incubator to dry for 15 min and then cut into strips with a width of 4 mm. After cutting, the amount of antigen on each 4 mm wide card strip was 0.084, 0.044, 0.0284, 0.0212, and 0.0172 ng, respectively. Next, 3 μL of gold-labeled probe was dripped onto each of the five assembled immune layer test strips, and 100 μL of 0.01 M PBS was slowly dripped onto the sample pad. The mixture was allowed to react at room temperature for 10 min, and the color depth of the T line of the test strip was observed. Finally, the concentration of 0.043 ng / cm (i.e., the concentration of complete antigen Hg on each 4 mm wide card after cutting) was selected by observation. 2+ -ITCBE-BSA content is 0.0172 ng) is the optimal T-line concentration ( Fig.12 B).

[0079] (5) Gold nanoflower rapid test strip test

[0080] 1) Specificity determination of gold nanoflower chromatography test strips

[0081] According to the optimized optimal C line and optimal T line dilution ratio, the NC membrane was streaked at the same time. The streaked NC membrane was placed in a 37°C constant temperature incubator to dry for 15 min and then cut into strips. Next, 3 μL of immune probe was added to the probe binding pad and the test strip was assembled. Hg was diluted with 0.01 M PBS. 2+ -ITCBE, Ca 2+ -ITCBE, Mg 2+ -ITCBE、Mn 2+ -ITCBE, Cu 2+ -ITCBE, Fe 2+ -ITCBE and Zn 2+ -ITCBE was diluted to a final concentration of 200 ng / mL. 100 μL of each was added to the sample pad of the test strip and allowed to react at room temperature for 10 min. The disappearance of the T line of the test strip was observed to determine the specificity of the test strip. The results showed that the test strip was only sensitive to the added Hg 2+ -ITCBE complex has a competitive reaction, the T line disappears, and there is no obvious cross-reactivity to other ion chelates, indicating that the test strip has good specificity ( Fig.13 ).

[0082] 2) Gold nanoflower chromatography test strip sensitivity determination

[0083] According to the optimized optimal C line and optimal T line dilution ratio, the NC membrane was streaked at the same time, and the streaked NC membrane was placed in a 37 ℃ constant temperature incubator to dry for 15 min before cutting the strips; then 3 μL of immune probe was added to the probe binding pad and the test strip was assembled. Hg was diluted with 0.01 M PBS. 2+ -ITCBE was diluted to a final concentration of 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, 1.56 ng / mL, 0.78 ng / mL, 0.39 ng / mL and 0 ng / mL. Then 100 μL of each was added to the sample pad of the test strip, and the mixture was allowed to react at room temperature for 10 min. The disappearance of the T line of the test strip was observed, and the detection range of the test strip was obtained. The results showed that when Hg 2+ When the amount of -ITCBE is 50 ng / mL, the T line disappears significantly, indicating that the epitope of the gold-labeled antibody has been completely covered by the hapten Hg in the sample. 2+-ITCBE combined, with the concentration of the double dilution, it can be observed that the color of the T line gradually darkens. Under visual conditions, when the concentration is 0.78 ng / mL, the color of the T line and the control card strip has obvious changes. When the concentration is 0.39 ng / mL, the inhibition rate can reach 72% compared with the T line of the control group. Therefore, the detection limit of the immunolayer test strip is 1.56 ng / mL, and the detection limit of the immunolayer reader is 0.39 ng / mL ( Fig.14 ).

[0084] (6) Actual sample testing

[0085] According to the above optimized optimal C line and optimal T line dilution concentration, the NC membrane was streaked at the same time, and the streaked NC membrane was placed in a 37 ℃ constant temperature incubator to dry for 15 min before cutting. 3 μL of immune probe was added to the probe binding pad and the test strip was assembled. Four major food crops were randomly purchased from the supermarket as the test objects, ground into powder with a mortar, passed through a 0.5 mm experimental sample sieve, and the sieved powder was collected. 1 mg of sieved rice, wheat, sweet potato, and corn were taken respectively, placed in 4 2 mL centrifuge tubes, and 1 mL of deionized water was added to each of the 4 centrifuge tubes. In addition, 1 mL of 200 ng / mL divalent mercuric ions was taken as a control, and an excess (400 ng) of ITCBE was added to the five solutions respectively, and shaken on a shaker at 37 ℃ for 3 h. Then centrifuged at 1000 r / min for 20 min, the supernatant was collected respectively, and the filtrate was filtered through a 0.22 μm filter membrane and the filtrate was taken for use. Then, 100 μL of rice, wheat, sweet potato, and corn filtrate were dripped onto the sample pad of the test strip, placed at room temperature for 10 min, and the T line of the test strip was observed to disappear. The results showed that compared with the control group (spotted with PBS buffer), the T line color of the four samples of rice, wheat, sweet potato, and corn did not change significantly, indicating that the samples did not contain mercury ion residues; at the same time, in the sample spiked with 200 ng of mercury ions, the T line of the test strip disappeared significantly compared with the control group ( Fig.15 ), which is consistent with the results of atomic fluorescence spectrometry (Table 1), indicating that the test strip has good stability and can be used for on-site detection of actual samples. It has practical significance for detecting the residues of heavy metal mercury ions in grain.

[0086] Table 1 Actual sample testing

[0087]

[0088] Note: The minimum detection limit of atomic fluorescence spectrometry is 0.003 μg / mL

[0089] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Application of mouse hybridoma cell line 7A1 in the preparation of nanoflower immunochromatographic test strips for detecting heavy metal mercury ions, characterized in that: The nanoflower immunochromatographic test strip comprises the following components: a plastic shell, a sample pad, an immunoprobe binding pad, a nitrocellulose membrane and a water-absorbing pad; the immunoprobe binding pad is dripped with a gold nanoimmunoprobe labeled with an anti-mercury ion monoclonal antibody; the anti-mercury ion monoclonal antibody is an anti-Hg 2+ -ITCBE hapten The monoclonal antibody secreted by the mouse hybridoma cell line 7A1, the mouse hybridoma cell line 7A1, has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the deposit date is November 23, 2021, and the deposit number is CGMCC No.23879; The preparation method of the nanoflower immunochromatographic test strip comprises the following steps: (1) Preparation of gold nanoflower solution: Add 100 mL of double distilled water to a 250 mL conical flask that has been soaked in aqua regia and sterilized at high temperature, and adjust the pH to 7.5 with 1 M NaOH; under stirring, add 750 μL of 1 wt% chloroauric acid, 500 μL of colloidal gold as seeds, 300 μL of 1 wt% trisodium citrate and 1 mL of newly prepared 0.03 M hydroquinone solution in sequence, and continue stirring until a stable blue color is obtained to obtain a gold nanoflower solution; (2) Preparation of immune probe: Take 10mL of gold nanoflower solution and place it in a narrow-necked bottle that has been sterilized at high temperature. Add 60μL of 0.1M K2CO3 under ice bath and stir evenly. Then add 60μL of 1.25mg / mL anti-mercury ion monoclonal antibody dropwise and continue stirring for 1h. Then add BSA according to 1% of the total volume and continue stirring for 30min under ice bath. Next, add PEG20000 according to 0.5% of the total volume and continue stirring for 30min under ice bath. Finally, seal the labeled solution and place it in a 4℃ refrigerator for equilibrium overnight to obtain the gold nanoimmunoprobe labeled with anti-mercury ion monoclonal antibody. (3) Pretreatment of the immunoprobe binding pad and the sample pad: Cut the untreated immunoprobe binding pad and the sample pad into strips with a width of 1.3 cm with scissors, place them in a large culture dish, submerge them with the blocking solution prepared in advance, and then transfer them to a 37°C constant temperature incubator for blocking for 2 h. After taking them out of the incubator, filter out the blocking solution and continue to place them in the incubator for drying. After drying, store them at 4°C. For subsequent use, cut them into strips with a length of 1.3 cm and a width of 4 mm. The composition of the blocking solution is: 5wt% BSA + 1vol% Tween-20. (4) NC membrane streaking: Take goat anti-mouse secondary antibody and streak it on the NC membrane. The final concentration on line C is 0.53 ng / cm. After cutting, the content of goat anti-mouse secondary antibody streaked on each 4 mm wide card strip is 0.212 ng. Take complete antigen Hg 2+ -ITCBE-BSA was streaked on the NC membrane, and the final concentration on the T line was 0.043ng / cm. After cutting, each 4mm wide card strip contained complete antigen Hg 2+ -ITCBE-BSA content is 0.0172ng, which is the T line; on the same NC membrane, the distance between the C and T lines is 0.5cm; (5) Preparation of the immunoprobe binding pad: Add 3 μL of the gold nanoimmunoprobe labeled with the anti-mercuric ion monoclonal antibody prepared in step (2) onto the pretreated immunoprobe binding pad; (6) Assembly of the immunochromatographic test strip: assemble the NC membrane after the marking in step (4), the immunoprobe binding pad prepared in step (5), the sample pad pretreated in step (3), and the absorbent pad in sequence on the bottom plate; wherein the immunoprobe binding pad and the sample pad are overlapped and pasted alternately, with the same ends separated by 2 mm, and the absorbent pad overlaps the NC membrane by 2 mm from beginning to end, cover with a plastic shell cover, dry and seal, and store at 4°C; The method for preparing the anti-mercuric ion monoclonal antibody in step (2) is as follows: 1×10 6 cell / was injected into the abdominal cavity of 9-week-old Balb / c female mice sensitized with paraffin. When the abdomen of the mice was swollen and tense, ascites was extracted and anti-Hg was obtained by separation and purification. 2+ -ITCBE monoclonal antibody.

2. Application of the nanoflower immunochromatographic test strip described in claim 1 in detecting heavy metal mercury ions.

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