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

Abstract

The present invention provides a latex microsphere immunochromatographic test strip for detecting heavy metal mercury ions, which belongs to the field of rapid heavy metal detection. The latex microsphere 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 latex microsphere immune probe labeled with an anti-mercury ion monoclonal antibody; the anti-mercury ion monoclonal antibody is an anti-Hg 2+ ‑ITCBE hapten is a monoclonal antibody secreted by mouse hybridoma cell line 7A1, mouse hybridoma cell line 7A1, with the deposit number of CGMCC No. 23879. The latex microsphere immunochromatographic test strip has a detection threshold of 100 ng / mL and a detection limit of 0.78 ng / mL. It has strong specificity, high sensitivity, good repeatability, stable performance, and low operating requirements. It is very suitable for rapid detection in grains and is of great significance for monitoring the residue of heavy metal mercury.

Description

Technical Field

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

[0002] Mercury (Hg), also known as quicksilver, has a density of 13.49 g / cm 3 Under standard conditions, the melting point and boiling point of mercury are -38.87°C and 356.6°C respectively. Due to its low melting and boiling points, as well as its volatility, it has strong mobility and activity. The main sources of mercury pollution in the environment are: natural sources, atmospheric deposition, waste incineration, mining activities, etc. In addition, mercury-related products such as cement, pesticides, electric lights, thermometers, dental materials, etc. will produce mercury-containing waste during the production process. When they are discarded, their incineration or landfill will cause secondary pollution, and will inevitably increase the total amount of mercury in nature. In nature, mercury is usually distributed in the following three forms: elemental mercury in the gas phase (Hg0), particulate mercury (Hgp), and oxidized gas phase mercury (Hg 2+ ). Among them, combined methylmercury is the most toxic, with neurotoxicity, cardiovascular toxicity, renal toxicity, reproductive toxicity, immune system effects and carcinogenicity. Inorganic mercury is second, and free mercury rarely exists in nature. Although its toxicity is relatively low, long-term exposure can cause chronic poisoning. 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. At present, the commonly used analytical methods for mercury determination mainly include hydride generation atomic fluorescence spectrometry (HG-AFS), cold vapor atomic absorption spectrometry (CVAAS), inductively coupled plasma mass spectrometry (ICP-MS), immunochromatography and atomic absorption spectrometry (AFS). Although these traditional laboratory detection methods have high analytical sensitivity and technical maturity, they usually require complex sample pretreatment, which is time-consuming, labor-intensive and costly, and cannot meet the simple and rapid on-site requirements of mercury content in actual samples. Therefore, the on-site rapid detection technology of heavy metal mercury has been a hot topic in the relevant field in recent years. The present invention uses activated latex microsphere particles to couple antibodies to prepare immunoassay probes. Since the latex microspheres have a large particle size (200 nm), which is larger than other widely used nanoparticles on the market, they have a large surface area and can be coupled with more antibodies. By utilizing the inherent advantages of these particles and combining them with immunochromatographic test strip technology, latex microsphere immunochromatographic test strips have been developed. The immunochromatographic test strips are simple to assemble, highly specific, and only target Hg. 2+-ITCBE is competitive, has no cross-reaction to other metal ions, has 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 grains, which is of great practical significance for the safe monitoring of heavy metal residues in grains. Summary of the invention

[0003] The present invention aims at the above problems and provides a latex microsphere immunochromatographic test strip for detecting heavy metal mercury ions and its application. The latex microsphere immunochromatographic test strip prepared by the present invention has simple assembly, 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 solutions:

[0005] A latex microsphere immunochromatographic test strip for detecting heavy metal mercury ions, the latex microsphere immunochromatographic test strip comprising 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 latex microsphere immunoprobe 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 the 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 method for preparing the latex microsphere immunochromatographic test strip for detecting heavy metal mercury ions comprises the following steps:

[0007] (1) Preparation of latex microsphere immunoprobe labeled with anti-mercuric ion monoclonal antibody:

[0008] 1) Take 20 μL of latex microsphere suspension in a 1.5 mL EP tube, add 1 mL of MES buffer with a pH of 6.0, suspend the microspheres, and centrifuge at 12,000 rpm at 4 °C for 10 min;

[0009] 2) Discard the supernatant, resuspend the precipitate with 1 mL of MES buffer (pH 6.0), and then add 1 mg of EDC to the resuspended solution;

[0010] 3) Place the resuspended liquid in step 2) on a constant temperature shaker at 25°C and 160 rpm for 15 min. Make sure to mix well, otherwise there will be lumps, indicating that the experiment has failed.

[0011] 4) Take out the activated latex microsphere suspension in step 3), centrifuge at 12000 rpm at 4 °C for 10 min, and then discard the supernatant;

[0012] 5) Add 0.5 mL of 50 g / L glycine solution with a pH of 6.5 to suspend the precipitate, and then add 15 μL of anti-Hg 2+ -ITCBE monoclonal antibody, incubated at room temperature on a shaker at 160 rpm for 2 h;

[0013] 6) After incubation, centrifuge at 4°C 12,000 rpm for 10 min, discard the supernatant, and wash once under the same conditions;

[0014] 7) Add 0.5 mL of glycine and shake at 160 rpm for 30 min at room temperature to quench the microspheres;

[0015] 8) Take out and centrifuge at 12000 rpm for 10 min at 4℃, and wash again under the same conditions;

[0016] 9) The supernatant was discarded, and the precipitate was blown up with 50 μL of resuspension solution to obtain the latex microsphere immunoprobe labeled with anti-mercuric ion monoclonal antibody, which was placed at 4°C for use; the composition of the resuspension solution was: 0.5% BSA + 0.5% glycine + 5% trehalose + 0.5 vol% Tween-20 diluted with 1× PBS buffer;

[0017] (2) 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;

[0018] (3) NC membrane streaking: Take goat anti-mouse secondary antibody and draw C line on NC membrane. The final content of goat anti-mouse secondary antibody on C line is 0.4 ng / cm. After cutting, the content of goat anti-mouse secondary antibody on each 4 mm wide card strip is 0.16 ng. Take complete antigen Hg 2+ -ITCBE-BSA is drawn on the NC membrane with a T line, and finally the complete antigen Hg is drawn on the T line 2+ -ITCBE-BSA content is 0.025 ng / cm. After cutting, each 4 mm wide card strip is marked with complete antigen Hg 2+The content of -ITCBE-BS was 0.01 ng, and the distance between the C and T lines on the same NC membrane was 0.5 cm;

[0019] (4) Preparation of the immunoprobe binding pad: Add 2 μL of the latex microsphere immunoprobe labeled with the anti-mercuric ion monoclonal antibody prepared in step (1) onto the pretreated immunoprobe binding pad;

[0020] (5) Assembly of immunochromatographic test strips: Assemble the NC membrane marked in step (3), the immunoprobe binding pad prepared in step (4), the sample pad pretreated in step (2), and the absorbent pad in sequence on the bottom plate; 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.

[0021] The above-mentioned nanoflower immunochromatographic test strip for detecting heavy metal mercury ions is used in detecting heavy metal mercury ions.

[0022] 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 latex microsphere immunoprobe is first added to the immunoprobe binding pad, and then dried according to the schematic diagram ( Figure 2 ) 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 latex microsphere immunoprobe that has not bound to the antigen in the sample will completely bind to the antigen on the T line and will 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 latex microsphere immunoprobe that has 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.

[0023] Beneficial effects of the present invention:

[0024] (1) This invention proposes for the first time a method of using latex microspheres to couple anti-Hg 2+ -ITCBE monoclonal antibody latex microsphere immunochromatographic test strip technology for detecting heavy metal mercury ions;

[0025] (2) The latex microsphere immunochromatographic test strip can be used to detect heavy metal mercury ions in grain. Under visual conditions, the minimum detection limit is 0.78 ng / mL, the detection threshold (complete disappearance of the T line) is 100 ng / mL, and the detection time is only 10 min. It meets the requirements of national food safety standards and can be used for actual on-site testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Identification of the purification results of 7A1 monoclonal antibody by SDS-PAEG.

[0027] Figure 2 Schematic diagram of the structural model of latex microsphere immune layer test strip.

[0028] Figure 3 Determination of the optimal pH of latex microsphere activation buffer.

[0029] Figure 4 Determination of the optimal EDC amount for activated latex microsphere immunoprobe.

[0030] Figure 5 Determination of the optimal glycine buffer pH for latex microsphere immunoprobe.

[0031] Figure 6 Determination of the optimal amount of antibody coupled to latex microsphere immunoprobe.

[0032] Figure 7 Determination of the optimal resuspension of latex microsphere immunoprobe.

[0033] Figure 8 Selection of the optimal dilution of line C in latex microsphere immunolayer test strips.

[0034] Fig. 9 Selection of the optimal dilution of the T line of latex microsphere immunochromatographic test strips.

[0035] Fig.10 Determination of the optimal probe amount for latex microsphere immunolayer test strips.

[0036] Fig.11 Specificity determination of latex microsphere immunochromatographic test strips.

[0037] Fig.12 Sensitivity determination of latex microsphere immunochromatographic test strips.

[0038] Fig.13 Actual sample testing of latex microsphere immunolayer test strips. DETAILED DESCRIPTION

[0039] 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.

[0040] Example 1 Preparation of monoclonal antibodies against heavy metal mercury ions

[0041] Anti-Hg monoclonal antibodies are 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 the 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.

[0042] 1. Ascites Preparation

[0043] (1) Take a 1 mL sterile syringe and inject 500 μL of paraffin into the peritoneal cavity of a 12-week-old Balb / c mouse. Fully lubricate the mouse's peritoneal cavity. When injecting, insert the syringe vertically into the back of the mouse's peritoneal cavity. Disinfect with alcohol before and after the injection site to avoid bleeding.

[0044] (2) One week after sensitization, take the mouse hybridoma cell line 7A1 that has just grown to the logarithmic phase and has covered about 80% of the culture dish, discard the supernatant, use a pipette to take 1 mL of fresh culture medium to blow down the cells at the bottom of the culture dish, blow and wash evenly, use a 1 mL sterile syringe to draw 200 μL of the cell suspension, and then vertically inject it into the abdominal cavity of the sensitized mouse. Be careful to insert the needle into the abdominal cavity accurately and without obstruction, and do not inject it into the abdominal tissue to avoid tumor formation and waste of mice;

[0045] (3) About a week after the cells were injected into the mouse's peritoneal cavity, under normal circumstances, the mouse's abdomen was obviously bloated, and the mouse's growth was sluggish and it did not like to exercise. It was time to prepare for ascites extraction;

[0046] (4) Prepare a blood collection needle, a 1.5 mL EP tube, cotton and other items. Pick up the mouse with one hand and wipe the blood collection site with alcohol cotton. With the other hand, carefully insert the blood collection needle into the side of the abdominal cavity. Place the blood collection needle against the EP tube to collect ascites. You can see ascites flowing into the tube along the blood collection needle. The color is reddish. This may be due to excessive ascites production and hemolysis, so the color is reddish, but it does not affect the nature of the antibodies in the ascites. When collecting blood, the mouse must be fixed to prevent the mouse from struggling and causing ascites loss. Only about 1.5 mL of ascites can be collected from each mouse at a time. Put the mouse after the tail blood is collected in a mouse cage to continue to produce ascites. Ascites can be collected every other day until the mouse dies.

[0047] (5) After the mouse ascites is balanced, place it in a 4 ℃ centrifuge and centrifuge it at 12000 rpm for 20 min. It can be observed that the liquid in the centrifuge tube is divided into three layers, with a layer of fat floating on the top. Carefully remove the middle layer of ascites, place it in a new 1.5 mL EP tube, mark it, and store it in a -20 ℃ refrigerator.

[0048] 2. Ascites purification

[0049] The ascites was equilibrated with buffer (Na 2 HPO 4 The ascites was diluted 1:10 with 4.383 g / L NaCl (3.5814 g / L, pH=7.0) and filtered through a 0.45 μm filter and 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 (8.0 g NaCl; 0.2 g KCl; and 1.5 g NaCl). 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 13wt% SDS-PAGE gel electrophoresis. Figure 1 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 freeze-dried powder, the anti-divalent mercuric ion (Hg 2+ -ITCBE) monoclonal antibody and store it in a -20 ℃ refrigerator for later use.

[0050] Example 2 Preparation of latex microsphere immunoprobe

[0051] 1. Determination of the optimal pH value of MES buffer system for activated latex microspheres

[0052] First, take 20 μL of latex microsphere suspension (purchased from Bang Laboratories USA, the particle size is about 200 nm under electron microscope) into four 1.5 mL EP tubes, then add 1 mL of MES buffer with pH values ​​of 5.5, 6.0, 6.5, and 7.0 respectively, and mark the corresponding pH values. Blow up the washed latex microspheres, centrifuge at 12000 rpm at 4 ℃ for 10 min, and discard the supernatant. Next, resuspend the precipitate with 1 mL of MES buffer of the corresponding pH value, then add 1 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) to the resuspended solution, and place it in a constant temperature shaker at 25 °C and 160 rpm for 15 min for activation. Note that it must be mixed well, otherwise there will be lumps, indicating that the experiment has failed; then take out the activated latex microsphere suspension and centrifuge it at 12000 rpm and 4 °C for 10 min, discard the supernatant and add 0.5 mL of glycine buffer solution (50 g / L) with a pH of 6.5 to suspend the precipitate, and finally add 15 μL of 1.25 mg / mL anti-Hg 2+ -ITCBE monoclonal antibody, incubated at room temperature on a shaker at 160 rpm for 2 h; finally, the coupled latex microsphere suspension was taken out and centrifuged at 4 ℃ 12000 rpm for 10 min, the supernatant was discarded, and washed once under the same conditions; 0.5 mL of glycine buffer was added and shaken at room temperature at 160 rpm for 30 min to quench the latex microspheres; taken out and centrifuged at 4 ℃ 12000 rpm for 10 min, and washed again under the same conditions; the supernatant was discarded, and the precipitate was suspended with 50 μL of resuspension solution (0.5% BSA + 0.5% glycine + 5% trehalose + 0.5% Tween-20 (1×PBS)), and finally anti-Hg 2+ -ITCBE latex microsphere immunoprobe, stored at 4 ℃ for future use.

[0053] The components of the latex microsphere immunochromatographic test strip include a plastic shell, a sample pad, an immunoprobe binding pad, a nitrocellulose membrane and a water-absorbing pad. The plastic shell contains an immunochromatographic test strip, which is assembled on a bottom plate in sequence, with the immunoprobe binding pad and the sample pad overlapping and staggered, with the same ends separated by 2 mm, and the water-absorbing pad and the nitrocellulose membrane overlapping by 2 mm from end to end. The plastic shell cover is covered, dried and packaged, and stored at 4°C. The immunoprobe is located on the probe binding pad ( Figure 2 schematic diagram).

[0054] The test strips showed that the pH of the MES buffer (2-morpholineethanesulfonic acid) activation buffer had a great influence on the activated carboxyl groups on the latex microspheres. When the pH was 5.5 and 7.0, the test strips had almost no bands or the bands were very shallow. When the pH was 6.0, the bands were most obvious, indicating that the optimal pH environment for the activation of latex microspheres was 6.0 ( Figure 3 ).

[0055] 2. Determination of EDC content of the optimal activated latex microsphere immunoprobe

[0056] First, take 20 μL of latex microsphere suspension into 4 1.5 mL EP tubes, add 1 mL of MES buffer with the optimal pH (pH 6.0) determined in 1 above, blow up the washed latex microspheres, centrifuge at 4°C 12000 rpm for 10 min, and discard the supernatant. Next, the precipitate was resuspended with 1 mL of MES buffer at pH 6.0, and then 0.6, 0.8, 1.0, and 1.2 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) were added to the resuspended solution, respectively, and the added EDC mass value was used as a mark. The solution was placed in a constant temperature shaker at 25°C and 160 rpm for 15 min for activation. Note that the solution must be mixed well, otherwise there will be lumps, indicating that the experiment has failed. The activated latex microsphere suspension was then taken out and centrifuged at 12000 rpm and 4°C for 10 min. The supernatant was discarded and 0.5 mL of glycine coupling buffer solution (50 g / L) at pH 6.5 was added to suspend the precipitate. Finally, 15 μL of 1.25 mg / mL anti-Hg was added. 2+ -ITCBE monoclonal antibody, incubated at room temperature on a shaker at 160 rpm for 2 h; finally, the coupled latex microsphere suspension was taken out and centrifuged at 4 ℃ 12000 rpm for 10 min, the supernatant was discarded, and washed once under the same conditions; 0.5 mL of glycine buffer was added and shaken at room temperature at 160 rpm for 30 min to quench the latex microspheres; taken out and centrifuged at 4 ℃ 12000 rpm for 10 min, and washed again under the same conditions; the supernatant was discarded, and the precipitate was suspended with 50 μL of resuspension solution (0.5% BSA + 0.5% glycine + 5% trehalose + 0.5% Tween-20 (1 * PBS)), and finally anti-Hg 2+-ITCBE latex microsphere immunoprobe, stored at 4 ℃ for future use. According to the test strip display results: when the amount of latex microsphere mother solution added is 20 μL, 1mL of MES buffer with a pH of 6.0 is added, the amount of EDC required for carboxyl activation is 1.0 mg, and under the same conditions, the C and T lines are most obvious. When the amount of EDC added is 1.2 mg, precipitation and aggregation can be clearly seen in the suspension after activation, indicating that too much is added, resulting in aggregation and precipitation of latex microspheres, and finally reducing the proportion of successfully prepared probes, or even failure in preparation ( Figure 4 ).

[0057] 3. Determination of the optimal glycine buffer pH for latex microsphere immunoprobe

[0058] First, take 20 μL of latex microsphere suspension into three 1.5 mL EP tubes, add 1 mL of MES buffer with a pH of 6.0, blow up the latex microspheres to wash them, centrifuge at 12000 rpm at 4 °C for 10 min, and discard the supernatant. Next, resuspend the precipitate with 1 mL of MES buffer with a pH of 6.0, and then add 1.0 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) to the resuspended solution. Next, prepare three glycine buffers with different pH values ​​of 5.5, 6.5, and 7.5 (weigh 50 g of glycine and dilute to 1 L with double distilled water as the mother solution, then divide the 50 mL volumetric flask into three bottles with 40 mL / bottle, adjust the three bottles of glycine solution to 5.5, 6.5, and 7.5 with 1 M NaOH, and mark them), add glycine coupling buffers with pH values ​​of 5.5, 6.5, and 7.5 to the above three tubes, mark them according to the corresponding pH values, resuspend the precipitate, and add 15 μL of 1.25 mg / mL anti-Hg 2+ -ITCBE monoclonal antibody, incubated at room temperature on a shaker at 160 rpm for 2 h; finally, the coupled latex microsphere suspension was taken out and centrifuged at 4 ℃ 12000 rpm for 10 min, the supernatant was discarded, and washed once under the same conditions; then 0.5 mL of glycine coupling buffer of the corresponding pH was added and shaken at room temperature at 160 rpm for 30 min to quench the latex microspheres; taken out and centrifuged at 4 ℃ 12000 rpm for 10 min, and washed again under the same conditions; the supernatant was discarded, and the precipitate was suspended with 50 μL of resuspension solution (0.5% BSA + 0.5% glycine + 5% trehalose + 0.5% Tween-20 (1×PBS)), and finally anti-Hg 2+-ITCBE latex microsphere immunoprobe, stored at 4 ℃ for future use. According to the test strip display results: only when the pH value of the glycine coupling buffer is 6.5, the card strip C and T lines will be colored, and the color is obvious, while when the pH value is 5.5 and 7.5, the card strip C and T lines do not show color, and after adding the coupling buffer of these two pH values, precipitates appear in the EP tube, and after resuspension, small pieces still exist. The suspension cannot flow through the chromatographic membrane, so there is no color ( Figure 5 ).

[0059] 4. Determination of the optimal antibody amount and resuspension for latex microsphere immunoprobe

[0060] First, take 20 μL of latex microsphere suspension into 4 1.5 mL EP tubes, add 1 mL of MES buffer with pH 6.0, blow up and wash the latex microspheres, centrifuge at 4 ℃ 12000 rpm for 10 min, and discard the supernatant. Next, resuspend the precipitate with 1 mL of MES buffer with pH 6.0, and then weigh 1.0 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) in the resuspension. The resuspension is placed on a constant temperature shaker at 25 ℃ 160 rpm for 15 min. Be careful to mix it, otherwise there will be lumps, indicating that the experiment has failed; take out the activated latex microspheres mentioned above and centrifuge them at 12000 rpm 4 ℃ for 10 min, then discard the supernatant; add 0.5 mL of 50 g / L glycine solution with pH 6.5, suspend the precipitate, and then add 5, 10, 15, and 20 μL of four different volumes of anti-Hg 2+ -ITCBE monoclonal antibody (1.25 mg / mL), marked in turn according to the volume of antibody added. Incubate at room temperature on a 160 rpm shaker for 2 h; finally, take out the coupled latex microsphere suspension and centrifuge at 4 ℃ 12000 rpm for 10 min, discard the supernatant, and wash once under the same conditions; add 0.5 mL of glycine (pH 6.5 50 g / L) and shake at room temperature 160 rpm for 30 min to quench the latex microspheres; take out and centrifuge at 4 ℃ 12000 rpm for 10 min, and wash again under the same conditions; discard the supernatant, and suspend the precipitate with 50 μL of resuspension solution (0.5% BSA + 0.5% glycine + 5% trehalose + 0.5% Tween-20 (1×PBS)), and finally obtain anti-Hg 2+ -ITCBE latex microsphere immunoprobe, stored at 4 ℃ for future use. The final results showed that ( Figure 6), when the volume of latex microsphere mother solution added is 20 μL, the optimal amount of adding the above antibody is 15 μL. At this time, the color of C and T lines is close to saturation, which is similar to adding 20 μL of anti-Hg 2+ -The colors of the C and T lines of the ITCBE monoclonal antibody strip are consistent, and the colors of the C and T lines of the immune layer test strips added with 5 μL and 10 μL of monoclonal antibody are significantly darker, indicating that the amount of coupled antibody is insufficient.

[0061] When resuspending, first take 20 μL of latex microsphere suspension into three 1.5 mL EP tubes, add 1 mL of MES buffer with a pH of 6.0, blow up the washed latex microspheres, centrifuge at 4°C 12000 rpm for 10 min, and discard the supernatant. Next, resuspend the precipitate with 1 mL of MES buffer with a pH of 6.0, and then weigh 1.0 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) into the resuspension. The resuspension was placed on a constant temperature shaker at 25°C and 160 rpm for 15 min. Be careful to mix it evenly, otherwise there will be lumps, which means the experiment has failed. Take out the activated latex microspheres and centrifuge them at 12000 rpm and 4°C for 10 min, then discard the supernatant; add 0.5 mL of 50 g / L glycine solution with a pH of 6.5 to suspend the precipitate, and then add four different volumes of anti-Hg2+-ITCBE monoclonal antibodies (1.25 mg / mL) of 5, 10, 15, and 20 μL, respectively, and mark them according to the volume of the antibody added. Incubate at room temperature in a 160 rpm shaker for 2 h; finally, take out the coupled latex microsphere suspension and centrifuge at 4 ℃ 12000 rpm for 10 min, discard the supernatant, and wash once under the same conditions; add 0.5 mL glycine (pH 6.5 50 g / L) and shake at room temperature 160 rpm for 30 min to quench the latex microspheres; take out and centrifuge at 4 ℃ 12000 rpm for 10 min, discard the supernatant, keep the precipitate, and prepare three different resuspensions 0.5% BSA (ddH 2 O), 0.5% BSA + 0.5% glycine + 5% trehalose + 0.5% Tween-20 (1×PBS) and glycine buffer (50 g / L) with a pH of 6.5. After quenching and centrifugation, the supernatant was discarded and 50 μL of the above three resuspensions were taken to suspend the precipitate. Finally, according to the spotting of the immune layer test strip, it was found that compared with the two resuspensions of 0.5% BSA and glycine buffer (50 g / L) with a pH of 6.5, when the resuspension formula was 0.5% BSA + 0.5% glycine + 5% trehalose + 0.5% Tween-20 (1×PBS), the immune probe was the most stable, and the C and T lines of the test strip were stably colored ( Figure 7 ).

[0062] Example 3 Performance verification of latex microsphere immunochromatographic test strips

[0063] 1. Pretreatment of probe binding pad and sample pad

[0064] Cut the untreated probe binding pad and sample pad into 1.3 cm wide strips with scissors, place them in a large culture dish, submerge them with the pre-prepared blocking solution (5wt% BSA + 1vol% Tween-20), and then transfer them to a 37 ℃ constant temperature incubator for blocking for 2 h. Remove them from the incubator and filter out the blocking solution and continue to dry them in the incubator. Store them at 4 ℃ after drying. Cut them into 1.3 cm long and 4 mm wide for subsequent use.

[0065] 2. Selection of the optimal dilution of the C line of latex microsphere immunolayer test strips

[0066] 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, 0.67, 0.5, and 0.4 mg / mL. 10 μL of each concentration was streaked on the NC membrane; at a streaking speed of 0.2 μL / cm, the final contents of goat anti-mouse secondary antibody streaked on line C were 1, 0.67, 0.5, and 0.4 ng / cm, respectively. After cutting, the amount of goat anti-mouse secondary antibody streaked on each 4 mm wide card strip was 0.4, 0.268, 0.2, and 0.16 ng. The NC membrane with the markings was placed in a 37 ℃ constant temperature incubator to dry for 20 min and then cut into strips with a width of 4 mm. Next, 3 μL of gold-labeled probe was dripped onto the four assembled latex microsphere 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 0.4 ng / cm (i.e., the content of the goat anti-mouse secondary antibody on each 4 mm wide card strip after cutting was 0.16 ng) was selected as the optimal C line marking concentration ( Figure 8 ).

[0067] 3. Selection of the optimal dilution of the T line of latex microsphere immunochromatographic test strips

[0068] Complete antigen Hg 2+ -ITCBE-BSA was diluted with 0.01 M PBS according to the final concentration of the T line. The final dilution was 0.1, 0.05, 0.033, and 0.025 mg / mL. 10 μL of each concentration was streaked on the NC membrane. When using the gold spray instrument to streak, the streaking speed was 0.2 μL / cm, and the complete antigen Hg was finally streaked on the T line. 2+-ITCBE-BSA contents were 0.1, 0.05, 0.033, and 0.025 ng / cm , respectively. After cutting, each 4 mm wide card strip was marked with complete antigen Hg 2+ The content of -ITCBE-BSA was 0.04, 0.02, 0.0132, and 0.01 ng. The NC membrane with the lines was placed in a 37 ℃ constant temperature incubator to dry for 20 minutes and then cut into strips with a width of 4 mm. Next, 3 μL of gold-labeled probe was dripped onto the 4 assembled immune layer test strips, and 100 μL of 0.01 M PBS was slowly dripped onto the sample pad. The reaction was placed at room temperature for 10 minutes, and the color depth of the T line of the test strip was observed. Finally, the concentration of 0.025 ng / cm (i.e., the complete antigen Hg on each 4 mm wide card strip after cutting) was selected through observation. 2+ -ITCBE-BSA content is 0.01 ng) is the optimal T-line concentration ( Fig. 9 ).

[0069] 4. Determination of the optimal probe amount for latex microsphere immune layer test strips

[0070] According to the optimized dilution ratio of the best C line (0.4 ng / cm) and the best T line (0.025 ng / cm), the NC membrane with the lines was placed in a 37 °C constant temperature incubator to dry for 20 min and then cut into strips with a width of 4 mm. Next, 1, 2, 3, and 4 μL of latex microsphere immune probes were dripped onto the probe binding pads of the four assembled immune layer test strips, respectively. 100 μL of 0.01 M PBS was slowly dripped onto the sample pad and reacted at room temperature for 10 min. The color depth of the C and T lines of the test strips was observed. Finally, 2 μL of the concentration was selected as the optimal latex microsphere probe spotting amount ( Fig.10 ).

[0071] 5. Latex microsphere immunochromatographic test strip specificity determination

[0072] According to the optimized dilution ratio of the best C line (0.4 ng / cm) and the best T line (0.025 ng / cm), 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 the optimal 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, Zn2+ -ITCBE and Pb 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.11 ).

[0073] 6. Sensitivity determination of latex microsphere immunochromatographic test strips

[0074] According to the optimized dilution ratio of the best C line (0.4 ng / cm) and the best T line (0.025 ng / cm), 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 the 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 200 ng / mL, 100 ng / mL, 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, and 0 ng / mL. Then 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 obtain the detection range of the test strip. The results showed that when Hg 2+ When the amount of -ITCBE is 100 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 decrease of antigen concentration, it can be observed that the color of T line gradually darkens. Under visual conditions, when the concentration is 0.78 ng / mL, there is a significant change in the color of T line and control card strip. Therefore, the detection limit of this immune layer test strip item is 0.78 ng / mL ( Fig.12 ).

[0075] Example 4 Actual sample testing

[0076] 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 10 mL centrifuge tubes, and 2 mL of deionized water was added to them. After fully uniform, centrifuged at 12000 rpm for 10 min, 1 mL of supernatant was taken, and marked. 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 four sample extracts and the control solution, respectively, and shaken at 37 ℃ for 3 h. Then centrifuge at 10,000 rpm for 20 minutes, collect the supernatant, filter through a 0.22 μm filter membrane, and take the filtrate for use. Then take 100 μL of rice, wheat, sweet potato, and corn filtrates and drop them on the sample pad of the test strip, place them at room temperature for 10 minutes, and observe the disappearance of the T line of the test strip. The results showed that compared with the control group (PBS buffer solution), the T line color of the four samples of rice, wheat, sweet potato, and corn did not change significantly, and was not detected, indicating that there was no mercury ion residue in the sample; at the same time, in the sample with a spiked amount of 200 ng of mercury ions, the T line of the test strip disappeared significantly compared with the control group ( Fig.13 ), 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.

[0077] Table 1 Actual sample testing

[0078]

[0079] Note: The minimum detection limit of atomic fluorescence spectrometry is 0.003 μg / mL

[0080] 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. A latex microsphere immunochromatographic test strip for detecting heavy metal mercury ions, characterized in that: The latex microsphere 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 latex microsphere immunoprobe labeled with an anti-mercuric ion monoclonal antibody; the anti-mercuric 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 the China Microbiological Culture Collection Administration, and the deposit address is: 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; the preparation method of the latex microsphere immunochromatographic test strip comprises the following steps: (1) Preparation of latex microsphere immunoprobe labeled with anti-mercuric ion monoclonal antibody: 1) Take 20 μL of latex microsphere suspension in a 1.5 mL EP tube, add 1 mL of MES buffer with a pH of 6.0, suspend the microspheres, and centrifuge at 4°C and 12,000 rpm for 10 min; 2) Discard the supernatant, resuspend the precipitate with 1 mL of MES buffer (pH 6.0), and then add 1 mg of EDC to the resuspended solution; 3) Place the resuspended solution in step 2) on a constant temperature shaker at 25°C and 160 rpm for 15 min. Make sure to mix well, otherwise there will be lumps, indicating that the experiment has failed. 4) Take out the activated latex microsphere suspension in step 3), centrifuge at 12000 rpm and 4°C for 10 min, and then discard the supernatant; 5) Add 0.5 mL of 50 g / L glycine solution with a pH of 6.5 to suspend the precipitate, then add 15 μL of anti-Hg 2+ -ITCBE monoclonal antibody, incubated at room temperature on a shaker at 160 rpm for 2 h; 6) After incubation, centrifuge at 4°C and 12,000 rpm for 10 min, discard the supernatant, and wash once under the same conditions; 7) Add 0.5 mL of glycine and shake at 160 rpm for 30 min at room temperature to quench the microspheres; 8) Take out and centrifuge at 4°C and 12000 rpm for 10 min, and wash again under the same conditions; 9) The supernatant was discarded, and the precipitate was blown up with 50 μL of resuspension solution to prepare the latex microsphere immunoprobe labeled with anti-mercuric ion monoclonal antibody, which was placed at 4°C for use; the composition of the resuspension solution was: 0.5% BSA + 0.5% glycine + 5% trehalose + 0.5 vol% Tween-20 diluted with 1× PBS buffer; (2) 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. (3) NC membrane streaking: Take goat anti-mouse secondary antibody and draw C line on NC membrane. The final content of goat anti-mouse secondary antibody on C line is 0.4ng / cm. After cutting, the content of goat anti-mouse secondary antibody on each 4mm wide card strip is 0.16ng. Take complete antigen Hg 2+ -ITCBE-BSA is streaked on the NC membrane with a T line, and finally the complete antigen Hg is streaked on the T line 2+ -ITCBE-BSA content is 0.025ng / cm. After cutting, each 4mm wide card strip is marked with complete antigen Hg 2+ -The content of ITCBE-BSA is 0.01 ng, and the distance between the C and T lines on the same NC membrane is 0.5 cm; (4) Preparation of the immunoprobe binding pad: Add 2 μL of the latex microsphere immunoprobe labeled with the anti-mercuric ion monoclonal antibody prepared in step (1) onto the pretreated immunoprobe binding pad; (5) Assembly of immunochromatographic test strips: Assemble the NC membrane marked in step (3), the immunoprobe binding pad prepared in step (4), the sample pad pretreated in step (2), 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.

2. Use of the latex microsphere immunochromatographic test strip according to claim 1 in detecting heavy metal mercury ions.

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