Colorimetric immunochromatographic detection probe, test strip, detection method and application

Abstract

The present invention belongs to the field of immunochromatographic detection technology, and specifically relates to a colorimetric immunochromatographic detection probe, a test strip, a detection method and an application. The detection probe is a magnetic mesoporous metal nanomaterial probe, which includes a magnetic material, a mesoporous silicon material, a dopamine hydrochloride flexible support material and precious metal nanoparticles. The mesoporous silicon material is located between the core of the magnetic material and the dopamine hydrochloride flexible support material, and the precious metal nanoparticles are adsorbed on the surface and pores of the mesoporous silicon material. The surface of the dopamine hydrochloride flexible support and the precious metal nanoparticles is enriched with detection antibodies. The precious metal nanoparticles coated on the colorimetric immunochromatographic detection probe of the present invention can quickly trigger the gold plasma surface resonance effect, so that Au 3+ The reduced gold particles are deposited on the chromatographic test strip, thereby enhancing the colorimetric intensity. Notably, adjusting the pH of the hydroxylamine solution effectively reduces false-positive interference caused by self-nucleation. Therefore, this immunoassay method enables rapid, ultrasensitive, and visual detection of biomarkers.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunochromatographic detection, and in particular relates to a colorimetric immunochromatographic detection probe, a test strip, a detection method and an application. Background Art

[0002] Colorimetric lateral flow immunochromatographic assays (LFIAs) have attracted significant interest in point-of-care (POCT) due to their rapidity, ease of use, and low cost. To date, gold nanoparticle (Au NPs)-labeled LFIAs have been the preferred method for large-scale, early-stage biomarker screening. However, the limited size of Au NPs and the suboptimal brightness of the colorimetric signal produced by these assays have resulted in conventional Au NPs-labeled LFIAs being insufficiently sensitive for detecting low-concentration targets.

[0003] Currently, some research teams are leveraging the catalytic activity of nanozymes to catalyze the color change of TMB and AEC colorimetric solutions, as well as plasmon surface resonance (SPR) methods to enhance colorimetric intensity. SPR has attracted considerable attention due to its rapid catalytic activity and large deposition particles. For example, one research team has exploited the SPR properties of Au NPs to trigger gold deposition, increasing intrinsic colorimetric brightness and thus improving detection sensitivity. However, the small specific surface area of ​​conventional Au NPs limits their effectiveness in catalyzing gold deposition, and the self-nucleation of gold during in-situ growth can cause false positives, thus affecting detection results. Summary of the Invention

[0004] In order to solve the problems in the prior art, the present invention provides a colorimetric immunochromatographic detection probe, a test strip, a detection method and an application. The designed probe has high colorimetric intensity, super strong catalytic activity and the like, which can quickly trigger Au 3+ The reduction to gold particles enhances the colorimetric intensity of the test strip's detection area, enabling rapid, ultrasensitive, and visual detection of trace analytes. Simultaneously, by adjusting the pH of hydroxylamine, false positives caused by self-nucleation of gold atoms can be effectively reduced, making test results more accurate.

[0005] The present invention provides a method for preparing and detecting a universal multi-target detection probe by combining the constructed magnetic mesoporous metal nanomaterial with an efficient gold plasma signal amplification strategy.

[0006] The present invention solves the technical problem by adopting the following technical solutions:

[0007] A colorimetric immunochromatographic detection probe is a magnetic mesoporous metal nanomaterial probe, which includes a magnetic material, a mesoporous silicon material, a dopamine hydrochloride flexible support material and precious metal nanoparticles. The mesoporous silicon material is located between the magnetic material core and the dopamine hydrochloride flexible support, the precious metal nanoparticles are adsorbed on the surface and pores of the mesoporous silicon material, and detection antibodies are enriched on the surface of the dopamine hydrochloride flexible support and the precious metal nanoparticles.

[0008] Furthermore, the preparation method of the probe includes: first mixing dopamine hydrochloride with a mesoporous silicon material loaded with a magnetic material, stirring the reaction and then washing; then mixing the product with a noble metal nanoparticle material to obtain a magnetic mesoporous metal nanomaterial; redissolving the magnetic mesoporous metal nanomaterial in a buffer solution, and then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and a detection antibody, and coupling the magnetic mesoporous metal nanomaterial and the detection antibody through a carboxyl and amino bond energy reaction, and coupling the magnetic mesoporous metal nanomaterial and the detection antibody through an amino and carboxyl bond energy reaction to obtain a magnetic mesoporous metal nanomaterial probe, and adding bovine serum albumin to block the magnetic mesoporous metal nanomaterial probe after preparation.

[0009] Dopamine hydrochloride is added to a mesoporous silica material loaded with a magnetic material, and after mixing and stirring, a mesoporous silica material loaded with a magnetic material coated with a dopamine flexible scaffold is obtained. The dopamine flexible scaffold facilitates the physical adsorption of a large number of precious metal particles and enhances the intrinsic colorimetric intensity. The magnetic mesoporous metal nanomaterial probe contains a mesoporous silica material, which is disposed between the magnetic material and the precious metal nanoparticles. The mesoporous silica material separates the magnetic material from the precious metal nanoparticles, preventing the reduction of magnetism while increasing the loading capacity of the precious metal nanoparticles. The precious metal nanoparticles are adsorbed on the surface and interior of the mesoporous silica material loaded with the dopamine flexible scaffold, providing a large number of catalytic sites for triggering the gold deposition reaction.

[0010] Furthermore, surfactant PVP is added during the synthesis of the precious metal nanoparticles, which is conducive to the adsorption of the precious metal particles on the surface and inside of the mesoporous silicon. The precious metal nanoparticles are platinum precious metal nanoparticles, and the particle size is 3 to 5 nm.

[0011] Furthermore, the preparation method of mesoporous silicon material loaded with magnetic material includes: adding magnetic material to a hexadecyltrimethylammonium bromide aqueous solution and then ultrasonically dispersing it, controlling the temperature at 65°C, and adding triethanolamine, cyclohexane and tetraethoxysilane solutions in a volume ratio of 1 to 10: 100 to 1000: 1 to 10 in sequence under stirring, the volume ratio of triethanolamine, cyclohexane and tetraethoxysilane solution is preferably 5:500:2, and after mixing reaction, a mesoporous silicon material loaded with magnetic material is obtained, and the thickness of the mesoporous silicon material is 40 to 60 nm.

[0012] Furthermore, the magnetic material is Fe3O4 nanoparticles with a diameter of 100 to 300 nm, preferably 200 nm. The preparation method is: ferric chloride hexahydrate and trisodium citrate dihydrate are mixed and dissolved in ethylene glycol, and then anhydrous sodium acetate is added to obtain Fe3O4 nanoparticles. The mass ratio of ferric chloride hexahydrate, trisodium citrate dihydrate, and anhydrous sodium acetate is 30 to 80:20 to 40:50 to 200. Preferably, the mass ratio of ferric chloride hexahydrate, trisodium citrate dihydrate, and anhydrous sodium acetate is 65:20:120.

[0013] A colorimetric immunochromatographic test strip comprises a base plate, a sample pad, a nitrocellulose membrane and a water-absorbing pad. The sample pad contains the colorimetric immunochromatographic detection probe of the present invention. The nitrocellulose membrane is provided with a detection line and a quality control line. The detection line is sprayed with mouse anti-human IgG antibody; the quality control line is sprayed with goat anti-mouse polyclonal IgG antibody.

[0014] Furthermore, the mass ratio of the magnetic mesoporous metal nanomaterial to the detection antibody in the colorimetric immunochromatographic detection probe is 0.1 to 50:1, preferably 10:1.

[0015] A colorimetric immunochromatographic detection method comprises adding a sample liquid into a chromatography buffer to obtain a mixed solution, wherein the mixed solution contains the colorimetric immunochromatographic detection probe of the present invention; then adding the mixed solution to the sample addition wells of the colorimetric immunochromatographic detection test strip of the present invention; then adding a prepared deposition mixed solution to the sample addition wells; and triggering gold deposition after the chromatography reaction is completed, wherein the deposition mixed solution comprises a tetrachloroauric acid solution and a hydroxylamine hydrochloride solution.

[0016] Furthermore, the mass concentration of the tetrachloroauric acid solution is 0.1 to 10 wt%, preferably 1 wt%, 2 wt% or 4 wt%, the molar concentration of the hydroxylamine hydrochloride solution is 100 to 800 mM, preferably 160 mM, 320 mM or 640 mM, and the pH value of the hydroxylamine hydrochloride solution is 0.5 to 9.5, preferably 1 to 2.

[0017] The pH value of the hydroxylamine hydrochloride solution is 1-2, which can inhibit its reducing properties. In the presence of magnetic mesoporous metal nanomaterials, the reducing properties of hydroxylamine hydrochloride are restored, thereby reducing tetrachloroauric acid to gold particles, which effectively prevents tetrachloroauric acid from self-nucleating.

[0018] The deposition mixture is available in multiple combinations. As a preferred technical solution, the optimal combination is a 1wt% mass concentration of tetrachloroauric acid solution and a 640mM molar concentration of hydroxylamine hydrochloride solution. This optimal combination maximizes colorimetric brightness and improves detection sensitivity.

[0019] A colorimetric immunochromatographic detection probe, a test strip, and a detection method are used in protein and nucleic acid detection, mainly involving the detection of protein markers. The protein markers include cardiac troponin I (cTnI) and alpha-fetoprotein (AFP). Magnetic mesoporous metal nanomaterials are combined with detection antibodies, which are human cardiac troponin I antibodies and human alpha-fetoprotein antibodies. The magnetic mesoporous metal nanomaterial probe can specifically bind to the target to be detected through the detection of polyclonal antibodies.

[0020] Compared with the prior art, the beneficial technical effects of the present invention are:

[0021] 1. The present invention realizes magnetic separation, high load, and super catalytic function through magnetic mesoporous metal nanomaterial probes. The magnetic mesoporous metal nanomaterial probes are used to replace traditional colloidal gold probes to realize precious metal seed-triggered gold deposition, further enhancing the colorimetric brightness of the magnetic mesoporous metal nanomaterial probes, thereby improving the detection sensitivity.

[0022] 2. The magnetic mesoporous silicon material of the present invention has the characteristics of high specific surface area and adjustable pore size, which can adsorb a large amount of precious metal particles and promote full contact between precious metal nanoparticles and gold deposition mixture, making up for the defect of small specific surface area of ​​traditional colloidal gold.

[0023] 3. Gold deposition catalytic amplification is a signal amplification strategy performed after the chromatographic reaction. It utilizes a strong acidic environment to inhibit the reducing properties of hydroxylamine hydrochloride. Hydroxylamine hydrochloride triggers the reduction reaction of tetrachloroauric acid only in the presence of a magnetic mesoporous metal nanomaterial probe. This effectively avoids false positives caused by self-nucleation of gold particles. This invention enables rapid, visual, and ultrasensitive detection of multiple target markers using a lateral flow chromatography platform.

[0024] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are specifically listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 These are TEM images of the magnetic material, mesoporous silicon material-coated magnetic material, magnetic mesoporous silicon material-coated dopamine hydrochloride, and magnetic mesoporous metal nanomaterial adsorbed by noble metal nanoparticles in the present invention.

[0026] Figure 2 These are mobile phone photos and a standard curve diagram of the detection of cTnI in serum samples in Example 4 of the present invention.

[0027] Figure 3 These are mobile phone photos and a standard curve diagram of the detection of AFP in serum samples in Example 7 of the present invention. DETAILED DESCRIPTION

[0028] The technical solutions of the present invention are further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0029] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0030] A colorimetric immunochromatographic detection probe is a magnetic mesoporous metal nanomaterial probe, which includes a magnetic material, a mesoporous silicon material, a dopamine hydrochloride flexible support material and precious metal nanoparticles. The mesoporous silicon material is located between the magnetic material core and the dopamine hydrochloride flexible support, the precious metal nanoparticles are adsorbed on the surface and pores of the mesoporous silicon material, and detection antibodies are enriched on the surface of the dopamine hydrochloride flexible support and the precious metal nanoparticles.

[0031] A universal colorimetric immunochromatographic test strip for multiple targets comprises a base plate, a sample pad, a nitrocellulose membrane and a water-absorbing pad. The sample pad is sprayed with a magnetic mesoporous metal nanomaterial probe coated with noble metal nanoparticles.

[0032] Example 1

[0033] The preparation method of magnetic mesoporous metal nanomaterials is as follows:

[0034] 1. Mix 0.65g of ferric chloride hexahydrate and 0.2g of trisodium citrate dihydrate and dissolve them in 20mL of ethylene glycol;

[0035] 2. Add 1.2g of anhydrous sodium acetate and stir until there are no particles;

[0036] 3. The mixture obtained in step 2 was transferred to a reactor and reacted at 200°C for 10 h. The product was washed three times with anhydrous ethanol and deionized water respectively. The obtained Fe3O4 nanoparticles of about 200 nm were dispersed in deionized water and stored at 4°C.

[0037] 4. Add 50 mg of Fe3O4 nanoparticles to 190 mL of aqueous solution containing 3.2 g of hexadecyltrimethylammonium bromide, keep stirring vigorously, and control the temperature at 65 °C;

[0038] 5. Add 500 μL of triethanolamine, 50 mL of cyclohexane, and 200 μL of tetraethoxysilane solution in sequence under stirring, and mix and react for 11 hours to obtain a mesoporous silica material loaded with magnetic material;

[0039] 6. The product of step 5 was washed with anhydrous ethanol and deionized water three times respectively, and then dispersed in anhydrous ethanol for later use to obtain a mesoporous silicon material loaded with magnetic material;

[0040] 7. Add 8 mL of Tris-HCl buffer to 8 mL (4 mg / mL) of the product of step 6, add 32 mg of dopamine hydrochloride dropwise under stirring, and react for 5 hours. Wash with anhydrous ethanol and deionized water three times respectively, and then disperse in deionized water for use to obtain a mesoporous silica material coated with a dopamine flexible scaffold loaded with magnetic material;

[0041] 8. Dissolve 3.185 mL (100 mM) chloroplatinic acid and 0.225 g PVP in 10 mL ethylene glycol, respectively, and mix and stir for 3 h. Control the temperature at 110 ° C. Wash once with 3 volumes of acetone by centrifugation, and then disperse in 20 mL deionized water to obtain 3-5 nm platinum nanoparticles for use.

[0042] 9. Take 8 mL (2 mg / mL) of the product of step 7 and 5 mL of the product of step 8, mix and stir for 10 hours to obtain magnetic mesoporous metal nanomaterials. Wash the magnetic mesoporous metal nanomaterials with anhydrous ethanol and deionized water three times respectively, and then disperse them in deionized water for use.

[0043] Figure 1 (a) is the TEM image of Fe3O4 nanoparticles prepared in step 3. Figure 1 (b) is a TEM image of the mesoporous silicon material loaded with magnetic material obtained in step 6. Figure 1 (c) is a TEM image of the mesoporous silicon material loaded with magnetic material coated with the dopamine flexible scaffold prepared in step 7. Figure 1 (d) is the TEM image of the magnetic mesoporous metal nanomaterial prepared in step 9.

[0044] Example 2

[0045] Preparation of magnetic mesoporous metal nanomaterial probes:

[0046] 1) 2 mg of the magnetic mesoporous metal nanomaterial (Fe3O4@MSN@PDA@Pt) prepared in Example 1 was redissolved in 2.5 mL of buffer and vortexed;

[0047] 2) Add 0.8 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) to the solution and vortex to mix:

[0048] 3) Add 0.2 mg of cTnI detection antibody to the solution and incubate at room temperature for 2 h;

[0049] 4) Finally, 291 μL (10 wt%) bovine serum albumin solution was added to the solution and blocked for 1 h to obtain a magnetic mesoporous metal nanomaterial probe;

[0050] 5) After magnetic separation, the solution was washed three times and then redissolved in 2 mL of a buffer containing 0.2 wt% bovine serum albumin and stored for later use.

[0051] Example 3

[0052] Preparation method of cTnI immunochromatographic test strips:

[0053] 1. Preparation of nitrocellulose membrane:

[0054] cTnI monoclonal antibody (Phipeng Bio) and goat anti-mouse antibody (Phipeng Bio) were added to a buffer solution (4% sucrose, 4% sodium chloride, and 0.01 M phosphate buffer) to a final concentration of 1 mg / mL. A nitrocellulose membrane was attached to a substrate. Using a streaking instrument, cTnI monoclonal antibody and goat anti-mouse antibody were sprayed onto the test and control lines of the nitrocellulose membrane, respectively, with a 5 mm interval. The membrane was then dried overnight at 37°C.

[0055] 2. Assembly of cTnI immunochromatographic test strips:

[0056] A sample pad (17.5 mm) and a water-absorbing pad (22.5 mm) are sequentially attached to the base plate, with the excess 3 mm of the sample pad and water-absorbing pad covering the nitrocellulose membrane. The assembled chromatography plate is then cut into 4 mm wide test strips using a high-speed chopper. The test strips are then affixed to the matching plastic card holder, completing the assembly of the immunochromatographic test strip. The plastic card holder is equipped with a sample loading port and a viewing window. The sample loading port is located above the sample pad for easy sample loading; the viewing window is located above the detection line and quality control line of the nitrocellulose membrane.

[0057] Example 4

[0058] Method for detecting cTnI in serum:

[0059] 1) A series of serial dilutions of cTnI stock solution with known concentrations were performed using buffer, with the concentrations being 53.042 ng / mL, 35.361 ng / mL, 23.574 ng / mL, 15.716 ng / mL, 3.143 ng / mL, 629 pg / mL, 126 pg / mL, 25.1 pg / mL, 5 pg / mL, 1 pg / mL, and 0 pg / mL, respectively;

[0060] 2) Add 20 μL of each cTnI dilution at different concentrations to 40 μL of chromatography buffer to create a mixture. The chromatography buffer also contains 2.5 μL of magnetic mesoporous metal nanomaterial probes. Vortex the mixture and wait for 1 minute. Use an external magnetic field to magnetically attract the magnetic mesoporous metal nanomaterial probes and then redissolve them in 60 μL of chromatography buffer for later use.

[0061] 3) Add the diluted mixed solution containing different concentrations of cTnI to the sample well of the immunochromatographic test strip, and the mixed solution passes through the sample pad, detection area, and absorbent pad in sequence by capillary action. The cTnI in the mixed solution combines with the magnetic mesoporous metal nanomaterial probe, and then flows to the detection line with capillary action, and combines with the cTnI monoclonal antibody fixed on the detection line to form a sandwich immune complex; at the same time, the remaining magnetic mesoporous metal nanomaterial probe continues to flow with the liquid to the quality control line, combines with the goat anti-mouse antibody, and two strips appear in the window. When there is no cTnI in the test sample, the magnetic mesoporous metal nanomaterial probe flows directly to the quality control line and combines with the goat anti-mouse antibody, and one strip appears in the window;

[0062] 4) After 10 minutes of reaction, add 50 μL of freshly prepared deposition solution (25 μL of 1 wt% tetrachloroauric acid solution + 25 μL of 640 mM hydroxylamine hydrochloride solution) dropwise to the sample well of the immunochromatographic test strip. When cTnI is present, the magnetic mesoporous metal nanomaterial probe on the test line triggers gold deposition through the platinum nanoparticles adsorbed on its surface and internally, causing the strip color to change from brownish-yellow to grayish-black. When cTnI is absent, the reducing properties of hydroxylamine hydrochloride are suppressed by the strong acid environment, and nonspecific gold deposition amplification will not occur on the test line, avoiding false positives.

[0063] 5) After the deposition reaction has been carried out for 5 minutes, use a mobile phone to take a picture of the immunochromatographic test strip result, and use a colloidal gold test strip reader to output the optical density value of the test line. Use the relationship between the optical density value of the test line and the cTnI concentration to establish a standard curve, such as Figure 2 As shown, the minimum detection limit was calculated to be 16.0 pg / mL (the average optical density of the blank sample plus 3 times the standard deviation);

[0064] 6) Collect a serum sample from the patient, take 20 μL of the sample solution and add it to 40 μL of chromatography buffer to obtain a mixed solution, which also contains 2.5 μL of magnetic mesoporous metal nanomaterial probe. After vortexing the mixed solution and waiting for 1 minute, use an external magnetic field to magnetically attract the magnetic mesoporous metal nanomaterial probe and then redissolve it in 60 μL of chromatography buffer. Then, add the evenly mixed 60 μL of the mixed solution to the sample well of the immunochromatographic test strip. After the reaction is carried out for 10 minutes, add 50 μL of freshly prepared deposition solution (25 μL 1wt% tetrachloroauric acid solution + 25 μL 640mM hydroxylamine hydrochloride solution) dropwise to the sample well of the immunochromatographic test strip. After the deposition reaction is carried out for 5 minutes, the presence of cTnI is determined by observing the color changes of the T line and C line. At the same time, the optical density value obtained by the T line is compared with the standard curve in step 5 to determine the concentration of the target analyte in the collected sample.

[0065] Example 5

[0066] Preparation of magnetic mesoporous metal nanomaterial probes:

[0067] 1) 2 mg of the magnetic mesoporous metal nanomaterial (Fe3O4@MSN@PDA@Pt) prepared in Example 1 was redissolved in 2.5 mL of buffer and vortexed;

[0068] 2) Add 0.8 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) to the solution and vortex to mix:

[0069] 3) Add 0.2 mg of AFP detection antibody to the solution and incubate at room temperature for 2 h;

[0070] 4) Finally, 291 μL (10 wt%) bovine serum albumin solution was added to the solution and blocked for 1 h to obtain a magnetic mesoporous metal nanomaterial probe;

[0071] 5) After magnetic separation, the solution was washed three times and then redissolved in 2 mL of a buffer solution containing 0.2 wt% bovine serum albumin and stored for later use.

[0072] Example 6

[0073] Preparation method of AFP immunochromatographic test strips:

[0074] 1. Preparation of nitrocellulose membrane:

[0075] AFP monoclonal antibody (Phipeng Bio) and goat anti-mouse antibody (Phipeng Bio) were added to a buffer solution (4% sucrose, 4% sodium chloride, and 0.01 M phosphate buffer) to a final concentration of 1 mg / mL. A nitrocellulose membrane was attached to a substrate. Using a membrane streaking instrument, AFP antibody and goat anti-mouse antibody were sprayed onto the test and control lines of the nitrocellulose membrane, respectively, with a 5 mm interval. The membrane was then dried overnight at 37°C.

[0076] 2. Assembly of AFP immunochromatographic test strips:

[0077] A sample pad (17.5 mm) and a water-absorbing pad (22.5 mm) are sequentially attached to the base plate, with the excess 3 mm of the sample pad and water-absorbing pad covering the nitrocellulose membrane. The assembled chromatography plate is then cut into 4 mm wide test strips using a high-speed chopper. The test strips are then affixed to the matching plastic card holder, completing the assembly of the immunochromatographic test strip. The plastic card holder is equipped with a sample loading port and a viewing window. The sample loading port is located above the sample pad for easy sample loading; the viewing window is located above the detection line and quality control line of the nitrocellulose membrane.

[0078] Example 7

[0079] Method for detecting AFP in serum:

[0080] 1) A series of gradient dilutions of AFP stock solution samples with known concentrations were performed with buffer, and the concentrations were 2965.9 ng / mL, 1482.95 ng / mL, 741.475 ng / mL, 370.7375 ng / mL, 185.3688 ng / mL, 370.7375 ng / mL, 185.3688 ng / mL, 92.6844 ng / mL, 46.3422 ng / mL, 23.17 11ng / mL, 11.5855ng / mL, 5.7928ng / mL, 2.8964ng / mL, 1.4482ng / mL, 0.7241ng / mL, 0.3620ng / mL , 0.1810ng / mL, 0.0901ng / mL, 0.0452ng / mL, 0.0226ng / mL, 0.0113ng / mL, 0.0057ng / mL, 0ng / mL;

[0081] 2) Add 20 μL of each AFP dilution at different concentrations to 40 μL of chromatography buffer to create a mixture. The chromatography buffer also contains 2.5 μL of magnetic mesoporous metal nanomaterial probe. Vortex the mixture and wait for 1 minute. Use an external magnetic field to magnetically attract the magnetic mesoporous metal nanomaterial probe and then redissolve it in 60 μL of chromatography buffer for later use.

[0082] 3) Add the diluted mixed solution containing different concentrations of AFP to the sample loading hole of the immunochromatographic test strip, and the mixed solution passes through the sample pad, detection area, and absorbent pad in sequence through capillary action. The AFP in the mixed solution combines with the magnetic mesoporous metal nanomaterial probe, and then flows to the detection line with capillary action, and combines with the AFP antibody fixed on the detection line to form a sandwich-type immune complex; at the same time, the remaining magnetic mesoporous metal nanomaterial probe continues to flow with the liquid to the quality control line, combines with the goat anti-mouse antibody, and two strips appear in the window. When there is no AFP in the test sample, the magnetic mesoporous metal nanomaterial probe flows directly to the quality control line and combines with the goat anti-mouse antibody, and one strip appears in the window;

[0083] 4) After 10 minutes of reaction, add 50 μL of freshly prepared deposition solution (25 μL of 1wt% tetrachloroauric acid solution + 25 μL of 640mM hydroxylamine hydrochloride solution) dropwise to the sample well of the immunochromatographic test strip. When AFP is present, the magnetic mesoporous metal nanomaterial probe on the detection line triggers gold deposition through the platinum nanoparticles adsorbed on its surface and inside, causing the strip color to change from brown-yellow to gray-black; when AFP is not present, the reducing property of hydroxylamine hydrochloride is suppressed by the strong acid environment, and nonspecific gold deposition amplification will not appear on the detection line, avoiding false positives;

[0084] 5) After the deposition reaction has been carried out for 5 minutes, use a mobile phone to take a picture of the immunochromatographic test strip result, and use a colloidal gold test strip reader to output the optical density value of the test line. Use the relationship between the optical density value of the test line and the AFP concentration to establish a standard curve, such as Figure 3 As shown, the minimum detection limit was calculated to be 11.7 pg / mL (the average optical density of the blank sample plus 3 times the standard deviation);

[0085] 6) Collect a serum sample from the patient, take 20 μL of the sample solution and add it to 40 μL of chromatography buffer to obtain a mixed solution, which also contains 2.5 μL of magnetic mesoporous metal nanomaterial probe. After vortexing the mixed solution and waiting for 1 minute, use an external magnetic field to magnetically absorb the magnetic mesoporous metal nanomaterial probe and then redissolve it in 60 μL of chromatography buffer. Then, add the evenly mixed 60 μL of the mixed solution to the sample well of the immunochromatographic test strip. After the reaction is carried out for 10 minutes, add 50 μL of freshly prepared deposition solution (25 μL 1wt% tetrachloroauric acid solution + 25 μL 640mM hydroxylamine hydrochloride solution) dropwise to the sample well of the immunochromatographic test strip. After the deposition reaction is carried out for 5 minutes, the presence of AFP is determined by observing the color changes of the T line and the C line. At the same time, the optical density value obtained by the T line is compared with the standard curve in step 5 to determine the concentration of the target detection substance in the collected sample.

[0086] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0087] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. A colorimetric immunochromatographic detection probe, characterized in that: The detection probe is a magnetic mesoporous metal nanomaterial probe, which includes a magnetic material, a mesoporous silicon material, a dopamine hydrochloride flexible scaffold material, and noble metal nanoparticles. The mesoporous silicon material is located between the core of the magnetic material and the dopamine hydrochloride flexible scaffold, and the noble metal nanoparticles are adsorbed on the surface and pores of the mesoporous silicon material. Detection antibodies are enriched on the surface of the dopamine hydrochloride flexible scaffold and the noble metal nanoparticles. The preparation method of the probe includes: first mixing dopamine hydrochloride with a mesoporous silicon material loaded with a magnetic material; then mixing the product with a noble metal nanoparticle material to obtain a magnetic mesoporous metal nanomaterial; redissolving the magnetic mesoporous metal nanomaterial in a buffer solution, and adding a detection antibody to obtain a magnetic mesoporous metal nanomaterial probe; The preparation method of a mesoporous silicon material loaded with a magnetic material comprises: adding a magnetic material to a hexadecyltrimethylammonium bromide aqueous solution, ultrasonically dispersing the magnetic material, sequentially adding a triethanolamine, cyclohexane, and tetraethoxysilane solution in a volume ratio of 1-10:100-1000:1-10 under stirring, and mixing and reacting to obtain a mesoporous silicon material loaded with a magnetic material; The magnetic material is Fe3O4 nanoparticles with a diameter of 100 to 300 nm. The preparation method is as follows: ferric chloride hexahydrate and trisodium citrate dihydrate are mixed and dissolved in ethylene glycol, and then anhydrous sodium acetate is added to obtain Fe3O4 nanoparticles. The mass ratio of ferric chloride hexahydrate, trisodium citrate dihydrate, and anhydrous sodium acetate is 30-80:20-40:50-200. The noble metal nanoparticles are platinum noble metal nanoparticles. The preparation method of the platinum noble metal nanoparticles comprises: dissolving chloroplatinic acid and PVP in ethylene glycol respectively, and then mixing and stirring to obtain the platinum noble metal nanoparticles.

2. A colorimetric immunochromatographic detection probe according to claim 1, characterized in that: The particle size of the platinum noble metal nanoparticles is 1 to 20 nm.

3. A colorimetric immunochromatographic test strip, characterized in that: The invention comprises a bottom plate, a sample pad, a nitrocellulose membrane and a water-absorbing pad. The sample pad contains the colorimetric immunochromatographic detection probe as claimed in claim 1 or 2. The nitrocellulose membrane is provided with a detection line and a quality control line. The detection line is sprayed with an antibody; the quality control line is sprayed with a secondary antibody. The test strip is fixed on a matching plastic card shell, and the plastic card shell is provided with a sample addition hole.

4. A colorimetric immunochromatographic test strip as claimed in claim 3, characterized in that: The mass ratio of the magnetic mesoporous metal nanomaterial to the detection antibody in the colorimetric immunochromatographic detection probe is 0.1~50:

1.

5. A colorimetric immunochromatographic detection method, characterized in that: The detection method includes adding a sample liquid to a chromatography buffer to obtain a mixed solution, wherein the mixed solution contains the colorimetric immunochromatographic detection probe as described in claim 1 or 2, and then adding the mixed solution to the sample addition well of the colorimetric immunochromatographic detection test strip as described in claim 3 or 4, and then adding the prepared deposition mixed solution to the sample addition well, wherein the deposition mixed solution includes tetrachloroauric acid solution and hydroxylamine hydrochloride solution, and triggering gold deposition after the chromatography reaction is completed.

6. A colorimetric immunochromatographic detection method according to claim 5, characterized in that: The mass concentration of the tetrachloroauric acid solution is 0.1-10 wt %, the molar concentration of the hydroxylamine hydrochloride solution is 100-800 mM, and the pH value of the hydroxylamine hydrochloride solution is 0.5-9.

5.

7. Use of a colorimetric immunochromatographic test strip as claimed in claim 3 or 4 in protein and nucleic acid detection.

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