A multi-target universal detection probe, immunochromatographic test strip, and preparation and detection method thereof

Abstract

The present invention provides a universal multi-target detection probe, an immunochromatographic test strip, and methods for preparing and detecting the same. The probe comprises a magnetic organic metal framework (MOF) hybrid material probe and a test strip body. The test strip body comprises a sample pad, a nitrocellulose membrane, and a water-absorbing pad. The surface of the MOF hybrid material probe is coated with noble metal nanoparticles, and the MOF hybrid material probe contains a magnetic material. The sensitivity of colorimetric chromatographic detection is enhanced by exploring and optimizing a novel MOF hybrid material probe with the "three-in-one" capabilities of high colorimetric performance, magnetic separation, and multi-site catalysis, and utilizing this probe to trigger a gold deposition catalytic amplification strategy.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunochromatographic detection, and in particular relates to a multi-target universal detection probe, an immunochromatographic test strip, and a preparation and detection method thereof. Background Art

[0002] Low-density immunoassay (LFIA) offers advantages such as ease of use, visual results, and universal accessibility, meeting the urgent need for point-of-care (POCT) testing. However, traditional colloidal gold low-density immunoassay (Au-LFIA) primarily uses colloidal gold as its output signal. Due to the inherent optical density deficiency of the colloidal gold probe, the detection sensitivity is low. This makes it difficult to detect low-abundance targets in early-stage disease specimens, hindering early screening and diagnosis.

[0003] Currently, a variety of amplification methods have been developed to improve the sensitivity of chromatographic detection, but most of them are only targeted at nucleic acid targets. Among them, the gold deposition catalytic amplification strategy involves secondary amplification of the band signal after the chromatographic reaction. This strategy has the advantage of universal applicability to analytes and is a commonly used method to improve the sensitivity of Au-LFIA. However, the gold nanoparticles used in traditional colloidal gold have limited effect on gold deposition catalytic amplification, and false positives caused by self-nucleation during the growth process can seriously affect the accuracy and reproducibility of detection. Therefore, there is an urgent need to find a new probe that is sensitive to the gold deposition catalytic reaction and has excellent amplification effect to replace traditional colloidal gold probes, minimize the false positive interference caused by self-nucleation, achieve visual detection, and expand the scope of application. Summary of the Invention

[0004] In order to solve the problems in the prior art, the present invention provides a multi-target universal detection probe, an immunochromatographic test strip, and a preparation and detection method thereof by combining a constructed new multifunctional nanohybrid material with an efficient gold deposition signal amplification strategy.

[0005] The present invention solves the technical problem by adopting the following technical solutions:

[0006] The first purpose of the present invention is to provide a universal detection probe for multiple targets, characterized in that the detection probe is a magnetic organic metal framework hybrid material probe, which includes a metal-organic framework material, a magnetic material and noble metal nanoparticles, and the metal-organic framework material is arranged between the magnetic material core and the noble metal nanoparticle adsorption outer layer, and the surface of the noble metal nanoparticles is enriched with nucleic acid aptamer sequences / detection antibodies.

[0007] Furthermore, the preparation method of the magnetic organic metal framework hybrid material probe is as follows: noble metal nanoparticles treated with anionic / cationic surfactants are mixed with a metal organic framework material loaded with a magnetic material to obtain a magnetic organic metal framework hybrid material; the intermediate product of the reaction of the magnetic organic metal framework hybrid material with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide sulfonic acid sodium salt in a buffer solution is coupled with a nucleic acid aptamer sequence / detection antibody to obtain the magnetic organic metal framework hybrid material probe. The noble metal nanoparticles have different groups (including carboxyl, amino, thiol, etc.) on their surface. After being treated with anionic / cationic surfactants, they are easily adsorbed and enriched on the organic metal framework shell. The anionic / cationic surfactants selected are PVP, PEI, etc.

[0008] Furthermore, the preparation method of the magnetic organic metal framework hybrid material probe is:

[0009] A magnetic material is added to an ethanol solution of ferric chloride, followed by ultrasonic dispersion, and an ethanol solution of trimesic acid is added under stirring, followed by mixing and reaction to obtain a metal organic framework material loaded with a magnetic material;

[0010] Then, the surfactant and the noble metal nanoparticles are mixed and centrifuged, and the surfactant is added again after washing with an organic solvent, and the metal organic framework material loaded with the magnetic material is added and washed to obtain a magnetic organic metal framework hybrid material;

[0011] The magnetic organic metal framework hybrid material is dispersed in a buffer solution, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide sulfonic acid sodium salt are added to the solution. The intermediate product is mixed and reacted at room temperature to obtain an intermediate product. The intermediate product is dissolved in a buffer solution, and a nucleic acid adapter sequence / detection antibody coupling is added to obtain the magnetic organic metal framework hybrid material probe.

[0012] Furthermore, the magnetic material is Fe3O4 magnetic beads.

[0013] Furthermore, the preparation method of the Fe3O4 magnetic beads is: mixing ferric chloride and trisodium citrate and dissolving them in ethylene glycol, and adding sodium acetate under stirring to obtain the Fe3O4 magnetic beads.

[0014] Furthermore, the molar ratio of ferric chloride, trisodium citrate, and sodium acetate is 1 to 10:1:1000. Preferably, the molar ratio of ferric chloride, trisodium citrate, and sodium acetate is 6:1:1000.

[0015] Furthermore, the Fe3O4 magnetic beads dispersed in ethanol were mixed with ferric chloride and trimesic acid to obtain a metal organic framework material loaded with Fe3O4 magnetic beads.

[0016] Furthermore, the molar ratio of the ferric chloride to trimesic acid is 0.5 to 3:1.

[0017] Furthermore, the mass of the added nucleic acid aptamer sequence / detection antibody is 1% to 10% of the total mass of the magnetic organic metal framework hybrid material probe.

[0018] Furthermore, the magnetic organic metal framework hybrid material probe is prepared and then blocked by adding bovine serum albumin or 6-mercaptohexanol.

[0019] The second object of the present invention is to provide a high-performance immunochromatographic test strip that is universal for multiple targets, characterized in that it includes a magnetic organic metal framework hybrid material probe and a test strip body, the test strip body includes a sample pad, a nitrocellulose membrane and a water-absorbent pad, the surface of the magnetic organic metal framework hybrid material probe is coated with precious metal nanoparticles, and the magnetic organic metal framework hybrid material probe is loaded with magnetic material; the test strip also includes gold nanoparticles coupled to a chicken IgY marker.

[0020] Furthermore, the test strip also includes a detection auxiliary chain with a modified group at the end.

[0021] Furthermore, the detection auxiliary chain is a FAM-modified nucleic acid adapter sequence detection auxiliary chain.

[0022] Furthermore, the detection auxiliary chain can specifically bind to the nucleic acid target to be detected, and the magnetic organic metal framework hybrid material probe can specifically bind to the target to be detected through the nucleic acid adapter sequence / detection antibody.

[0023] Furthermore, the magnetic material is Fe3O4 magnetic beads.

[0024] Furthermore, the high-performance immunochromatographic test strip further includes a conjugate pad, which is staggered below the sample pad.

[0025] Furthermore, the high-performance immunochromatographic test strip further comprises a bottom plate, on which the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are all laid. Preferably, the bottom plate is a PVC plate.

[0026] Furthermore, a quality control line and a detection line are drawn on the nitrocellulose membrane.

[0027] Furthermore, the organic metal framework in the magnetic organic metal framework hybrid material probe is a metal-organic framework material, which is disposed between the magnetic material and the noble metal nanoparticles. The metal-organic framework separates the magnetic material from the noble metal nanoparticles, preventing magnetism loss while increasing the loading capacity of the noble metal nanoparticles.

[0028] Furthermore, the metal organic framework material is any one of COF, ZIF, UiO, PCN, and MIL. Preferably, the metal organic framework material is MIL.

[0029] Furthermore, the noble metal nanoparticles are selected from gold, silver, and platinum noble metal nanoparticles.

[0030] Furthermore, the particle size of the noble metal nanoparticles is 2 to 100 nm.

[0031] Furthermore, the diameter of the magnetic material is 20 to 500 nm.

[0032] Furthermore, the metal organic framework material has a thickness of 10 to 200 nm.

[0033] Furthermore, the particle size of the magnetic organic metal framework hybrid material probe is 50 to 800 nm, and the coefficient of variation is less than 5%. Preferably, the particle size of the magnetic organic metal framework hybrid material probe is 310 nm.

[0034] The third object of the present invention is to provide a method for preparing a high-performance immunochromatographic test strip that is universal for multiple targets, characterized in that it comprises the following steps:

[0035] Preparation of magnetic organic metal framework hybrid material probes;

[0036] Preparation of chicken IgY-labeled gold nanoparticles;

[0037] The magnetic organic metal framework hybrid material probe, the chicken IgY labeled coupled gold nanoparticles, the test solution, and the sedimentation mixture were vacuum-stored separately for later use;

[0038] Preparation of the test strip body: A quality control line and a test line are drawn on a nitrocellulose membrane and dried, and a sample pad and a water-absorbing pad are placed at both ends of the nitrocellulose membrane to obtain the test strip body; the quality control line is sprayed with an activity verification substance, and the test line is sprayed with a second biomarker that can specifically bind to the target to be detected or the terminal group of the detection auxiliary chain.

[0039] Furthermore, the test solution includes NaCl solution, B66 solution, and PBS solution; and the deposition mixture includes tetrachloroauric acid solution, hydroxylamine hydrochloride solution, and deionized water.

[0040] Furthermore, the concentration of NaCl in the test solution is 2%, the concentration of B66 is 0.1%, and the concentration of PBS is 0.01M; the pH value of the hydroxylamine hydrochloride solution is 1-2; and the molar ratio of the tetrachloroauric acid solution to the hydroxylamine hydrochloride solution is 1-10:10-20.

[0041] Furthermore, the content of the second biomarker is 2 mg / mL to 0.5 mg / mL, and the content of the active verification substance is 2 mg / mL to 0.5 mg / mL.

[0042] Furthermore, the activity verification substance is anti-chicken IgY sheep anti-chicken IgY, and the second biomarker is anti-FAMIgG antibody or detection antibody.

[0043] Furthermore, the particle size of the magnetic organic metal framework hybrid material is 310 nm, and the coefficient of variation is less than 5%.

[0044] A fourth object of the present invention is to provide a multi-target universal high-performance immunochromatographic test strip detection method, characterized in that a target analyte of known concentration is diluted to different concentration gradients, and then mixed with a magnetic organic metal framework hybrid material probe, gold nanoparticles coupled to a chicken IgY marker, and a test solution to form a test sample, the test sample is loaded onto a sample pad of a high-performance immunochromatographic test strip, and after 5 to 15 minutes, the mixed solution is loaded again and deposited, and after 3 to 20 minutes, the colorimetric intensity of the test line and the quality control line is measured, and the intensity ratio is calculated to establish a standard curve of the intensity ratio and the target analyte concentration;

[0045] The collected sample is mixed with a magnetic organic metal framework hybrid material probe, chicken IgY-labeled gold nanoparticles, and a test solution, and then loaded onto the sample pad of a high-performance immunochromatographic test strip. After 5 to 15 minutes, the mixed solution is loaded and deposited again. After 3 to 20 minutes, the colorimetric intensity of the test line and the quality control line is measured, the intensity ratio is calculated, and compared with the standard curve to obtain the concentration of the target detection object in the collected sample.

[0046] By secondary amplifying the deposition contrast signal after chromatography, the gold deposition reaction is inhibited by the reducing property of hydroxylamine hydrochloride in a low pH liquid environment, and the reducing property of hydroxylamine hydrochloride is re-triggered when precious metal nanoparticles are present, thereby avoiding false positives caused by self-nucleation during the signal amplification process.

[0047] A fifth object of the present invention is to provide a high-performance, multi-target immunochromatographic test strip for use in nucleic acid and protein marker detection. Preferably, nucleic acid and protein marker detection refers to miRNA-21, novel coronavirus, or cTnT detection.

[0048] Compared with the prior art, the beneficial technical effects of the present invention are:

[0049] The present invention realizes the "three-in-one" function of high colorimetry, magnetic separation, and multi-site catalysis through a magnetic organic metal framework hybrid material probe, and utilizes a magnetic organic metal framework hybrid material probe to replace the traditional colloidal gold probe with low optical density, thereby realizing gold deposition catalytic amplification triggered by a noble metal seed, and improving the sensitivity of detection. The gold deposition catalytic amplification of the present invention is a secondary amplification of the colorimetric signal performed after the chromatography reaction is completed. It utilizes the characteristics that the reducing property of hydroxylamine hydrochloride is suppressed in a low pH liquid environment, and when the noble metal nanoparticle seeds are present, the reducing property of hydroxylamine hydrochloride is re-triggered, thereby avoiding the occurrence of false positive results caused by self-nucleation during the signal amplification process. The present invention achieves a breakthrough in the sensitivity bottleneck of the traditional visual lateral flow chromatography detection method, obtains visual detection of a variety of trace targets, and the detection is fast and accurate, low cost, and greatly improves the convenience of detection.

[0050] 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 and other purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are specifically listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a TEM image of magnetic materials with different particle sizes in a high-performance immunochromatographic test strip universal for multiple targets and its preparation and detection method of the present invention.

[0052] Figure 2 TEM images of magnetic materials coated with organic metal framework materials of different thicknesses in a high-performance immunochromatographic test strip universal for multiple targets and its preparation and detection method of the present invention.

[0053] Figure 3 This is a TEM image of a magnetic organic metal framework hybrid material adsorbed with different noble metal nanoparticles in a multi-target universal high-performance immunochromatographic test strip and its preparation and detection method of the present invention.

[0054] Figure 4 This is a structural diagram of a multi-target universal high-performance immunochromatographic test strip and a chromatography test strip in a preparation and detection method thereof of the present invention.

[0055] Figure 5 This is a mobile phone photo and standard curve diagram of the detection of miRNA in serum samples in Example 2 of the present invention's high-performance universal multi-target immunochromatographic test strip and its preparation and detection method.

[0056] Figure 6 This is a mobile phone photo and standard curve diagram of Example 3 of the present invention, a high-performance immunochromatographic test strip for multiple targets and its preparation and detection method, for detecting novel coronavirus nucleic acid in serum samples.

[0057] Figure 7 This is a mobile phone photo and standard curve diagram of the detection of cTnT in serum samples in Example 4 of the present invention's high-performance, universal multi-target immunochromatographic test strip and its preparation and detection method. DETAILED DESCRIPTION

[0058] The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings and 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 intended by the present invention.

[0059] In addition, unless otherwise specified, the various raw materials, reagents, instruments, and equipment used in the present invention can be purchased from the market or prepared by existing methods. The anti-FAM IgG antibody and goat anti-chicken IgY antibody in the present invention were purchased from Jingda Bio. The MIL metal-organic framework material in the present invention was from Materials of Institut Lavoisier, the UiO metal-organic framework material was from University of Oslo, the ZIF metal-organic framework material was from Zeolitic imidazolate framework, the COF metal-organic framework material was from Covalent organic framework, and the PCN metal-organic framework material was from Porous Coordination Network.

[0060] Example 1: Preparation of magnetic organic metal framework hybrid materials

[0061] A high-performance, multi-target immunochromatographic test strip comprises a sample pad, a nitrocellulose membrane, and an absorbent pad. The sample pad is coated with a magnetic organic metal framework hybrid material probe, which is coated with precious metal nanoparticles. The magnetic material loaded on the magnetic organic metal framework hybrid probe is Fe3O4 magnetic beads. The sample pad is also coated with gold nanoparticles, which are coupled to a chicken IgY marker and a detection auxiliary chain with a terminal modification group.

[0062] The metal organic framework (MOF) in the magnetic metal organic framework hybrid material probe is a metal organic framework (MOF) material, which is interposed between the magnetic material and noble metal nanoparticles. The metal organic framework (MOF) material can be any of COF, ZIF, UiO, PCN, and MIL. The noble metal nanoparticles are selected from gold, silver, and platinum noble metal nanoparticles.

[0063] The preparation method of the magnetic organic metal framework hybrid material is as follows:

[0064] 1. Take 0.3g, 0.65g, and 1.08g of ferric chloride and 0.2g of trisodium citrate respectively, and dissolve them in 20mL of ethylene glycol;

[0065] 2. Add 1.2 g of sodium acetate while stirring and continue stirring vigorously for 30 minutes;

[0066] 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 ethanol and deionized water to obtain magnetic beads (Fe3O4) with different particle sizes. The transmission electron microscopy images are shown in FIG. Figure 1 .

[0067] 4. Add 50 mg of Fe3O4 magnetic beads to 2 mL, 4 mL, and 8 mL of 10 mM ferric chloride ethanol solution, respectively, and disperse by ultrasonication;

[0068] 5. Add 4 mL of 10 nM trimesic acid ethanol solution under continuous stirring for 10 min and stir at 70 °C for 2 h;

[0069] 6. After magnetic separation of the product in step 5, it was washed with ethanol three times, and then the product was repeated in steps 4-6 for a total of 5 times to obtain a metal organic framework material (Fe3O4@MIL) with Fe3O4 magnetic beads of different thicknesses. The transmission electron microscopy images are shown in Figure 2 .

[0070] 7. Dissolve 20 mg of PVP K30 in 5 mL of gold, silver, and platinum nanoparticle solutions respectively and stir for 15 hours;

[0071] 8. Centrifuge at 14000 rpm for 10 min, wash with methanol three times, and reconstitute into 2 mL solution;

[0072] 9. Add 4 mg of Fe3O4@MIL, stir for 1 hour, and wash with ethanol after magnetic separation to obtain magnetic organic metal framework hybrid materials (Fe3O4@MIL-Au, Ag or Pt) adsorbed by different noble metal nanoparticles. The transmission electron microscopy images are shown in Figure 3 .

[0073] See attached Figure 1-3It can be seen that the ratio of ferric chloride to trisodium citrate has a significant impact on the particle size of the magnetic core material. The addition ratio can be adjusted according to the desired particle size of the magnetic material, with the diameter of the magnetic material ranging from 20 to 500 nm. The amount of ferric chloride ethanol solution added also has a significant impact on the thickness of the metal-organic framework material. The greater the amount of ferric chloride ethanol solution added, the thicker the metal-organic framework material, with a thickness ranging from 10 to 200 nm. By varying the type of noble metal seed, magnetic metal-organic framework hybrid materials (Fe3O4@MIL-Au, Ag, or Pt) adsorbed with different noble metal nanoparticles can be produced. The morphology and size of the magnetic metal-organic framework hybrid materials are uniform, and the magnetic metal-organic framework hybrid materials have a stronger adsorption capacity for gold nanoparticles.

[0074] Example 2: Preparation and detection of miRNA-21 immunochromatographic test strips

[0075] Preparation method of miRNA-21 immunochromatographic test strips:

[0076] 1. Preparation of magnetic organic metal framework hybrid material probes:

[0077] 1) 5 mg of the gold-based magnetic metal organic framework hybrid material (Fe3O4@MIL-Au) prepared in Example 1 was redissolved in 1 mL of deionized water, vortexed, magnetically separated and washed, and then dispersed in a MES (morpholineethanesulfonic acid) coupling buffer at pH 6.0;

[0078] 2) Add 5 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 5 mg of NHSS (N-hydroxysuccinimide sulfonic acid sodium salt to assist amide bond) to the solution, vortex to mix, and react at room temperature for 30 min;

[0079] 3) Wash by centrifugation, then redissolve in 1 mL of TBS storage buffer (pH 8.0), add 50 μL of 100 μM amino-modified miRNA-21 aptamer sequence (complementary to half of the miRNA-21 sequence), and incubate at room temperature for 4 h to obtain a magnetic organic metal framework hybrid material probe;

[0080] 4) Wash by centrifugation, then redissolve in 1 mL of TBS storage buffer (pH 8.0), add 10 μL of 1 mM MCH (6-mercaptohexanol), and block at room temperature for 15 minutes;

[0081] 5) Wash by centrifugation, then redissolve in 1 mL of TBS storage buffer (pH 8.0) and store at 4°C until use.

[0082] 2. Preparation of nitrocellulose membrane:

[0083] Anti-FAM IgG antibody (Jingda Biotechnology) and goat anti-chicken IgY antibody (Jingda Biotechnology) were streaked onto nitrocellulose membranes at a concentration of 1 mg / mL using a PBS buffer (4% sucrose, 4% NaCl, and 0.01 M PBS) with a streak instrument as the test line and quality control line, respectively, with an interval of 8 mm, and then dried at 37°C overnight.

[0084] 3. Assembly of miRNA-21 immunochromatographic test strips:

[0085] See attached Figure 4 Glass fiber was used as the sample pad. On a white PVC base, the sample pad, along with a NC membrane marked with a T line (test line) for anti-FAM IgG antibodies and a C line (control line) for goat anti-chicken LgY antibodies, were placed alternately 3 mm apart. Finally, an absorbent pad was applied. The assembled chromatography plate was then cut into 4 mm wide test strips using a high-speed chopper. The test strips were then secured with two matching upper and lower plastic cartridges to create the immunochromatographic test strips. The plastic cartridges of the immunochromatographic test strips are equipped with a sample loading port and an observation port. The sample loading port is located above the sample pad for easy sample loading, while the observation port is located at the T and C lines of the NC membrane.

[0086] Detection method of miRNA-21 in serum using miRNA-21 immunochromatographic test strips:

[0087] 1. Dilute the synthesized miRNA-21 with TE buffer to different concentrations, namely 1nM, 0.1nM, 10pM, 1pM, 0.1pM, 10fM, 1fM, 0.1fM, 10aM, 1aM, 0.

[0088] 2. Take 10 μL of miRNA-21 dilution of different concentrations and add it to 50 μL of chromatography buffer to obtain a mixed solution. The chromatography buffer includes a magnetic organic metal framework hybrid material probe, a FAM-modified miRNA-21 detection auxiliary chain (complementary to the other half of the miRNA-21 sequence), chicken IgY-labeled gold nanoparticles, and a test solution, and mix them thoroughly for reaction.

[0089] 3. Add 60 μL of the mixed solution to the sample well of the immunochromatographic test strip (the location corresponding to the sample loading area on the plastic cartridge). The mixture will pass through the sample zone, detection zone, and water absorption zone sequentially through capillary action. When the test sample contains the target analyte (miRNA-21), miRNA-21 first binds to the magnetic organic metal framework hybrid material probe and FAM-modified miRNA-21 detection auxiliary chain in the sample zone. It then migrates with the liquid to the T line, where it binds to the anti-FAM IgG antibody to form a sandwich immune complex. Simultaneously, the chicken IgY-labeled gold nanoparticles in the sample zone also migrate with the liquid to the C line, where they bind to the goat anti-chicken IgY antibody. If the test sample does not contain miRNA-21, the probe migrates directly to the water absorption zone.

[0090] 4. After 10 minutes of reaction, 60 μL of fresh deposition reaction solution (22.5 μL of 1% tetrachloroauric acid solution + 22.5 μL of 160 mM hydroxylamine hydrochloride solution + 15 μL of deionized water) was added dropwise to the sample wells of the immunochromatographic test strip. In the presence of the target analyte (miRNA-21), the novel high-performance signal probe retained on the T-line triggers gold deposition via the gold nanoparticles adsorbed on its surface, causing the strip color to darken from brown to purple-black. In the absence of miRNA-21, the reducing properties of hydroxylamine hydrochloride are suppressed by the low pH environment, preventing nonspecific gold deposition amplification on the T-line and avoiding false positives.

[0091] 5. After the deposition reaction has been carried out for 10 minutes, use a mobile phone to take a picture of the immunochromatographic test strip result image, and use PS software to process and analyze the grayscale image. Use the logarithmic relationship between the grayscale value ratio of the T line and the C line and the concentration of miRNA-21 to establish a standard curve, such as Figure 5 As shown, the lowest detection limit was 0.1 fM.

[0092] 6. Collect a patient serum sample and add it to 50 μL of chromatography buffer to prepare a mixture. The chromatography buffer contains a magnetic organic metal framework hybrid material probe, a FAM-modified miRNA-21 detection auxiliary chain (complementary to the other half of the miRNA-21 sequence), chicken IgY-labeled gold nanoparticles, and a test solution. The mixture is thoroughly mixed and reacted. 60 μL of the mixed solution is added to the sample well of the immunochromatographic test strip (the corresponding sample loading area on the plastic cartridge). After the reaction is allowed to proceed for 10 minutes, 60 μL of fresh deposition reaction solution (22.5 μL of 1% tetrachloroauric acid solution + 22.5 μL of 160 mM hydroxylamine hydrochloride solution + 15 μL of deionized water) is again added dropwise to the sample well of the immunochromatographic test strip. After the deposition reaction proceeds for 10 minutes, the color changes of the T and C lines are observed to determine the presence of miRNA-21. The grayscale value ratio of the T and C lines is compared with the standard curve in step 5 to determine the concentration of the target analyte in the collected sample.

[0093] Example 3: Preparation and detection of novel coronavirus nucleic acid immunochromatographic test strips

[0094] Preparation method of novel coronavirus nucleic acid immunochromatographic test strips:

[0095] 1. Preparation of magnetic organic metal framework hybrid material probes:

[0096] 1) 5 mg of the gold-based magnetic metal organic framework hybrid material (Fe3O4@MIL-Au) prepared in Example 1 was redissolved in 1 mL of deionized water, vortexed, magnetically separated and washed, and then dispersed in a MES (morpholineethanesulfonic acid) coupling buffer at pH 6.0;

[0097] 2) Add 5 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 5 mg of NHSS (N-hydroxysuccinimide sulfonic acid sodium salt to assist amide bond) to the solution, vortex to mix, and react at room temperature for 30 min;

[0098] 3) Wash by centrifugation, then redissolve in 1 mL of TBS storage buffer at pH 8.0, add 70 μL of 100 μM amino-modified SARS-CoV-2 nucleic acid fragment adapter sequence (complementary to half of the SARS-CoV-2 nucleic acid fragment sequence), and incubate at room temperature for 4 hours to obtain a magnetic organic metal framework hybrid material probe;

[0099] 4) Wash by centrifugation, then redissolve in 1 mL of TBS storage buffer (pH 8.0), add 10 μL of 1 mM MCH (6-mercaptohexanol), and block at room temperature for 15 minutes;

[0100] 5) Wash by centrifugation, then redissolve in 1 mL of TBS storage buffer (pH 8.0) and store at 4°C until use.

[0101] 2. Preparation of nitrocellulose membrane:

[0102] Anti-FAM IgG antibody (Jingda Biotechnology) and goat anti-chicken IgY antibody (Jingda Biotechnology) were streaked onto nitrocellulose membranes at a concentration of 2 mg / mL using a PBS buffer (4% sucrose, 4% NaCl, and 0.01 M PBS) with a streak instrument as the test line and quality control line, respectively, with an interval of 8 mm, and then dried at 37°C overnight.

[0103] 3. Assembly of the novel coronavirus nucleic acid immunochromatographic test strips:

[0104] A glass fiber sample pad is used. On a white PVC base, the sample pad, a NC membrane marked with a T line (test line) for anti-FAM IgG antibodies, and a C line (control line) for goat anti-chicken LgY antibodies are placed, staggered 3 mm apart. A glass fiber binding pad is placed between the NC membrane and the sample pad, staggered 3 mm between the sample pad, binding pad, and NC membrane. Finally, an absorbent pad is applied. The assembled chromatography plate is then cut into 4 mm wide test strips using a high-speed chopper. The test strips are then secured with two matching upper and lower plastic holders to create the immunochromatographic test strips. The plastic holders of the immunochromatographic test strips are equipped with a sample loading port and an observation port. The sample loading port is located above the sample pad for easy sample loading, while the observation port is located at the T and C lines of the NC membrane.

[0105] Detection method of SARS-CoV-2 nucleic acid immunochromatographic test strips for detecting SARS-CoV-2 nucleic acid in serum:

[0106] 1. Use TE buffer to dilute the synthesized new coronavirus nucleic acid fragments to different concentrations, namely 1nM, 0.1nM, 10pM, 1pM, 0.1pM, 10fM, 1fM, 0.1fM, 10aM, 1aM, 0.

[0107] 2. Take 10 μL of different concentrations of SARS-CoV-2 nucleic acid fragment dilutions and add them to 50 μL of chromatography buffer to obtain a mixed solution. The chromatography buffer includes a magnetic organic metal framework hybrid material probe, a FAM-modified SARS-CoV-2 nucleic acid fragment detection auxiliary chain (complementary to the other half of the sequence of the SARS-CoV-2 nucleic acid fragment), chicken IgY-labeled gold nanoparticles, and a test solution, and mix them thoroughly for reaction.

[0108] 3. Add 60 μL of the evenly mixed solution to the sample well of the immunochromatographic test strip (the position corresponding to the sample loading area on the plastic cartridge). The mixed solution will pass through the sample area, detection area, and water absorption area in sequence through capillary action. When the test sample contains the target analyte (COVID-19 nucleic acid), the COVID-19 nucleic acid fragment first binds to the magnetic organic metal framework hybrid material probe and FAM-modified COVID-19 nucleic acid fragment detection auxiliary chain in the sample area, then swims with the liquid to the T line, where it binds to the anti-FAM IgG antibody to form a sandwich immune complex. At the same time, the chicken IgY-labeled gold nanoparticles in the sample area also swim with the liquid to the C line and bind to the goat anti-chicken IgY antibody. When the test sample does not contain COVID-19 nucleic acid, the probe swims directly to the water absorption area.

[0109] 4. After 10 minutes of reaction, add 60 μL of fresh deposition reaction solution (22.5 μL of 1% tetrachloroauric acid solution + 22.5 μL of 160 mM hydroxylamine hydrochloride solution + 15 μL of deionized water) to the sample well of the immunochromatographic test strip. When the target analyte (COVID-19 nucleic acid) is present, the new high-performance signal probe trapped on the T line triggers gold deposition through the gold nanoparticles adsorbed on its surface, causing the strip color to deepen from brown to purple-black. When the COVID-19 nucleic acid is absent, the reducing properties of hydroxylamine hydrochloride are suppressed by the low pH environment, and nonspecific gold deposition amplification will not occur on the T line, avoiding false positives.

[0110] 5. After the deposition reaction has been carried out for 10 minutes, use a mobile phone to take a picture of the immunochromatographic test strip result, process and analyze the grayscale image using PS software, and use the logarithmic relationship between the grayscale value ratio of the T line and the C line and the concentration of the new coronavirus nucleic acid to establish a standard curve, such as Figure 6 As shown, the lowest detection limit was 0.1 fM.

[0111] 6. Collect the patient's serum sample and add the sample to 50 μL of chromatography buffer to obtain a mixture. The chromatography buffer includes a magnetic organic metal framework hybrid material probe, a FAM-modified novel coronavirus nucleic acid fragment detection auxiliary chain, chicken IgY-labeled gold nanoparticles, and a test solution. Mix the mixture thoroughly and add 60 μL of the mixed solution to the sample well of the immunochromatographic test strip (the position corresponding to the sample loading area on the plastic card). After the reaction is carried out for 10 minutes, 60 μL of fresh deposition reaction solution (22.5 μL 1% tetrachloroauric acid solution + 22.5 μL 160mM hydroxylamine hydrochloride solution + 15 μL deionized water) is added dropwise to the sample well of the immunochromatographic test strip. After the deposition reaction is carried out for 10 minutes, observe the color changes of the T line and C line to determine whether the novel coronavirus nucleic acid is present. At the same time, compare the gray value ratio of the T line and the C line with the standard curve in step 5 to determine the concentration of the target test substance in the collected sample.

[0112] Example 4: Preparation and detection of cTnT immunochromatographic test strips

[0113] Preparation method of cTnT immunochromatographic test strips:

[0114] 1. Preparation of magnetic organic metal framework hybrid material probes:

[0115] 1) 5 mg of the gold-based magnetic metal organic framework hybrid material (Fe3O4@MIL-Au) prepared in Example 1 was redissolved in 1 mL of deionized water, vortexed, magnetically separated and washed, and then dispersed in a MES (morpholineethanesulfonic acid) coupling buffer at pH 6.0;

[0116] 2) Add 5 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 5 mg of NHSS (N-hydroxysuccinimide sulfonic acid sodium salt to assist amide bond) to the solution, vortex to mix, and react at room temperature for 30 min;

[0117] 3) washing by centrifugation, then redissolving in 1 mL of TBS storage buffer (pH 8.0), adding 1 mg of cTnT monoclonal antibody, and incubating at room temperature for 4 h to obtain a magnetic organic metal framework hybrid material probe;

[0118] 4) Wash by centrifugation, then redissolve in 1 mL of TBS storage buffer (pH 8.0), add 1 mL of 1% BSA, and block at room temperature for 15 minutes;

[0119] 5) Wash by centrifugation, then redissolve in 1 mL of TBS storage buffer (pH 8.0) and store at 4°C until use.

[0120] 2. Preparation of nitrocellulose membrane:

[0121] Using PBS buffer (4% sucrose, 4% NaCl and 0.01 M PBS), cTnT monoclonal antibody (Jingda Biotechnology) and goat anti-chicken IgY antibody (Jingda Biotechnology) were streaked on nitrocellulose membranes at a concentration of 1 mg / mL using a streaking instrument as the test line and quality control line, respectively, with an interval of 8 mm, and then dried at 37°C overnight.

[0122] 3. Assembly of cTnT immunochromatographic test strips:

[0123] On a white PVC base, a sample pad, a NC membrane marked with a T line (test line) for cTnT monoclonal antibody, and a C line (control line) for goat anti-chicken LgY antibody are placed, staggered 3 mm apart. A fiberglass binding pad is placed between the NC membrane and the sample pad, staggered 3 mm between the sample pad, binding pad, and NC membrane. Finally, an absorbent pad is applied. The assembled chromatography plate is then cut into 4 mm wide test strips using a high-speed chopper. The strips are then secured with two matching upper and lower plastic holders to create the immunochromatographic test strips. The plastic holders of the immunochromatographic test strips are equipped with a sample loading port and an observation port. The sample loading port is located above the sample pad for easy sample loading, while the observation port is located at the T and C lines of the NC membrane.

[0124] Detection method of cTnT in serum using cTnT immunochromatographic test strips:

[0125] 1. Dilute the concentrated cTnT stock solution with PBS buffer to different concentrations, namely 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1 ng / mL, 0.5 ng / mL, 250 pg / mL, 100 pg / mL, 50 pg / mL, 25 pg / mL, 10 pg / mL, 5 pg / mL, 2.5 pg / mL, and 0 pg / mL.

[0126] 2. Take 10 μL of cTnT dilutions of different concentrations and add them to 50 μL of chromatography buffer to obtain a mixed solution. The chromatography buffer contains magnetic organic metal framework hybrid material probe, chicken IgY-labeled gold nanoparticles, and test solution, and mix them thoroughly for reaction.

[0127] 3. Add 60 μL of the mixed solution to the sample well of the immunochromatographic test strip (the location corresponding to the sample loading area on the plastic cartridge). The mixture will pass through the sample area, detection area, and water absorption area in sequence through capillary action. When the test sample contains the target analyte (cTnT), the cTnT first binds to the magnetic organic metal framework hybrid material probe in the sample area. It then migrates with the liquid to the T line, where it binds to the cTnT monoclonal antibody to form a sandwich immune complex. Simultaneously, the chicken IgY-labeled gold nanoparticles in the sample area also migrate with the liquid to the C line and bind to the goat anti-chicken IgY antibody. If the test sample does not contain cTnT, the probe migrates directly to the water absorption area.

[0128] 4. After 10 minutes of reaction, add 60 μL of fresh deposition reaction solution (22.5 μL of 1% tetrachloroauric acid solution + 22.5 μL of 160 mM hydroxylamine hydrochloride solution + 15 μL of deionized water) to the sample wells of the immunochromatographic test strip. When the target analyte (cTnT) is present, the novel high-performance signal probe retained on the T line triggers gold deposition via the gold nanoparticles adsorbed on its surface, causing the strip color to darken from brown to purple-black. In the absence of cTnT, the reducing properties of hydroxylamine hydrochloride are suppressed by the low pH environment, preventing nonspecific gold deposition and amplification on the T line, thus avoiding false positives.

[0129] 5. After the deposition reaction has been carried out for 10 minutes, use a mobile phone to take a picture of the immunochromatographic test strip result image, and use PS software to process and analyze the grayscale image. Use the logarithmic relationship between the grayscale value ratio of the T line and the C line and the cTnT concentration to establish a standard curve, such as Figure 7 As shown, the lowest detection limit was 0.1 fM.

[0130] 6. Collect a patient serum sample and add it to 50 μL of chromatography buffer to obtain a mixture. The chromatography buffer contains a magnetic organic metal framework hybrid material probe, chicken IgY-labeled gold nanoparticles, and a test solution. Mix thoroughly and react. Add 60 μL of the mixed solution to the sample well of the immunochromatographic test strip (the corresponding sample loading area on the plastic cartridge). After the reaction is allowed to proceed for 10 minutes, add 60 μL of fresh deposition reaction solution (22.5 μL of 1% tetrachloroauric acid solution + 22.5 μL of 160 mM hydroxylamine hydrochloride solution + 15 μL of deionized water) dropwise to the sample well of the immunochromatographic test strip. After the deposition reaction is allowed to proceed for 10 minutes, observe the color changes of the T and C lines to determine the presence of cTnT. At the same time, compare the grayscale value ratio of the T and C lines with the standard curve in step 5 to determine the concentration of the target analyte in the collected sample.

[0131] 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.

[0132] 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 high-performance immunochromatographic test strip with universal detection probes for multiple targets, characterized by: Including magnetic organic metal framework hybrid material probe and test strip body, The magnetic organic metal framework hybrid material probe comprises a metal organic framework material, a magnetic material and noble metal nanoparticles, wherein the metal organic framework material is arranged between the magnetic material core and the noble metal nanoparticle adsorption outer layer, the surface of the noble metal nanoparticles is enriched with nucleic acid aptamer sequences, and the noble metal nanoparticles are selected from gold nanoparticles; The preparation method of the magnetic organic metal framework hybrid material probe comprises: mixing noble metal nanoparticles treated with anionic / cationic surfactants with a metal organic framework material loaded with a magnetic material to obtain a magnetic organic metal framework hybrid material; coupling the intermediate product of the reaction of the magnetic organic metal framework hybrid material with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide sulfonic acid sodium salt in a buffer solution with a nucleic acid aptamer sequence to obtain the magnetic organic metal framework hybrid material probe; The test strip body includes a sample pad, a nitrocellulose membrane and a water-absorbing pad; the surface of the magnetic organic metal framework hybrid material probe is coated with precious metal nanoparticles, and the magnetic organic metal framework hybrid material probe contains magnetic material; the test strip also includes gold nanoparticles coupled with chicken IgY marker; the test strip also includes a detection auxiliary chain with a group modified at the end, and the detection auxiliary chain is a FAM-modified nucleic acid adapter sequence detection auxiliary chain.

2. The high-performance immunochromatographic test strip of a multi-target universal detection probe according to claim 1, characterized in that: The preparation method of the magnetic organic metal framework hybrid material probe is: A magnetic material is added to an ethanol solution of ferric chloride, followed by ultrasonic dispersion, and an ethanol solution of trimesic acid is added under stirring, followed by mixing and reaction to obtain a metal organic framework material loaded with a magnetic material; Then, the surfactant and the noble metal nanoparticles are mixed and centrifuged, and the surfactant is added again after washing with an organic solvent, and the metal organic framework material loaded with the magnetic material is added and washed to obtain a magnetic organic metal framework hybrid material; The magnetic organic metal framework hybrid material is dispersed in a buffer solution, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide sulfonic acid sodium salt are added to the solution. The intermediate product is mixed and reacted at room temperature to obtain an intermediate product. The intermediate product is dissolved in a buffer solution, and a nucleic acid adapter sequence is added for coupling to obtain the magnetic organic metal framework hybrid material probe.

3. A method for preparing a high-performance immunochromatographic test strip with a universal multi-target detection probe according to any one of claims 1 to 2, characterized in that: The following steps are involved: Preparation of magnetic organic metal framework hybrid material probes; Preparation of chicken IgY-labeled gold nanoparticles; The magnetic organic metal framework hybrid material probe, the chicken IgY-labeled gold nanoparticles, the test solution, and the deposition mixture are respectively vacuum-stored for standby use, wherein the test solution includes a NaCl solution, a B66 solution, and a PBS solution; the deposition mixture includes a tetrachloroauric acid solution, a hydroxylamine hydrochloride solution, and deionized water, wherein the pH value of the hydroxylamine hydrochloride solution is 1-2; and the molar ratio of the tetrachloroauric acid solution to the hydroxylamine hydrochloride solution is 1-10:10-20; Preparation of the test strip body: A quality control line and a test line are drawn on a nitrocellulose membrane and dried, and a sample pad and a water-absorbing pad are placed at both ends of the nitrocellulose membrane to obtain the test strip body; the quality control line is sprayed with an activity verification substance, and the test line is sprayed with a second biomarker that can specifically bind to the target object to be detected or the terminal group of the detection auxiliary chain. The activity verification substance is anti-chicken IgY sheep anti-chicken IgY, and the second biomarker is an anti-FAM IgG antibody.

4. A method for detecting a multi-target universal detection probe using a high-performance immunochromatographic test strip obtained according to the preparation method of claim 3, characterized in that: After the target analyte of known concentration is diluted to different concentration gradients, it is mixed with the magnetic organic metal framework hybrid material probe, gold nanoparticles conjugated with chicken IgY marker, and test solution to form a test sample. The test sample is loaded onto the sample pad of a high-performance immunochromatographic test strip. After 5-15 minutes, the mixed solution is loaded again and deposited. After 3-20 minutes, the colorimetric intensity of the test line and the quality control line is measured, and the intensity ratio is calculated. A standard curve of the intensity ratio and the target analyte concentration is established; The collected sample is mixed with a magnetic organic metal framework hybrid material probe, chicken IgY-labeled gold nanoparticles, and a test solution, and then loaded onto the sample pad of a high-performance immunochromatographic test strip. After 5 to 15 minutes, the mixed solution is loaded again and deposited. After 3 to 20 minutes, the colorimetric intensity of the test line and the quality control line is measured, the intensity ratio is calculated, and compared with the standard curve to obtain the concentration of the target analyte in the collected sample.

5. An application of a high-performance immunochromatographic test strip comprising the multi-target universal detection probe prepared according to the preparation method of claim 3 in nucleic acid detection, wherein the application is for non-disease diagnosis or treatment purposes.

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