A dynamic dual-mode, ultrasensitive immunoassay test strip and its preparation method

Abstract

This invention relates to the field of immunoassay technology, specifically disclosing a dynamic dual-mode, ultrasensitive immunoassay test strip and its preparation method. The method includes the following steps: MAF nanoparticles are surface-modified chemically, then mixed and incubated with AuNPs to self-assemble into MAF@AuNPs composite nanoparticles. These are further mixed and incubated with Ab1, followed by the addition of a blocking agent and continued incubation. After centrifugation, the precipitate is reconstituted with a reconstituted solvent to obtain Ab1-modified MAF@AuNPs composite nanoparticles. These nanoparticles are then dropped onto a labeling pad and dried. Anti-Ab1 is streaked onto a chromatography membrane and labeled as the C line; Ab2 is streaked onto a chromatography membrane and labeled as the T line, and then dried. The sample pad is treated with borate buffer to obtain the immunoassay test strip. This invention achieves dynamic complementarity of fluorescence-colorimetric dual-mode signals, and the sensitivity of the fluorescence mode is more than 100 times higher than that of the colorimetric mode.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, specifically to a dynamic dual-mode, ultrasensitive immunoassay test strip and its preparation method. Background Technology

[0002] Immunoassay test strips are one of the most widely used point-of-care in vitro diagnostic technologies, offering advantages such as speed, convenience, low cost, and naked-eye visualization. Their practical applications have expanded from high-concentration target detection (ng / mL to ug / mL) to low-concentration target detection (pg / mL to ng / mL). Most existing immunoassay test strips are based on AuNPs or fluorescent microspheres / quantum dots, employing single-modal colorimetry or fluorescence signal detection. The key challenge lies in the following: AuNP immunoassay test strips are easily identifiable and quantified by the naked eye when detecting high-concentration targets, but their sensitivity is insufficient for effectively detecting low-concentration targets; fluorescent microsphere / quantum dot immunoassay test strips offer significantly improved sensitivity, effectively detecting low-concentration targets, but their fluorescence signal tends to saturate when detecting high-concentration targets, making effective naked-eye identification or quantification difficult.

[0003] Currently, a few studies have reported on fluorescence-colorimetric dual-modal immunoassay strips based on fluorescent quantum dot @AuNPs composite materials. Although these strips achieve dual-modal detection signal readout, the sensitivity of the fluorescence signal mode is not significantly improved compared to the colorimetric signal mode, remaining within the same order of magnitude. On the one hand, they fail to achieve dynamic naked-eye recognition covering high and low concentrations and simultaneous ultrasensitive quantitative analysis. On the other hand, the fluorescent quantum dot @AuNPs composite materials reported in these studies are based on a stepwise synthesis method with layer-by-layer coating, which is relatively complex to prepare. Furthermore, subsequent antibody and protein labeling still rely on traditional chemical covalent modification processes, increasing the technical difficulty and labor and material costs, and reducing the low-cost economics of immunoassay strip technology.

[0004] To meet the application needs of different detection scenarios and solve the problem of complex preparation processes, it is essential to develop a dynamic dual-mode, ultrasensitive immunoassay test strip and its preparation method. This strip should be able to achieve ultrasensitive detection using the fluorescence signal mode at low target concentrations, while simultaneously enabling effective naked-eye recognition and quantitative analysis using the colorimetric signal mode at high target concentrations. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a dynamic dual-mode, ultrasensitive immunoassay test strip and its preparation method, which can achieve ultrasensitive detection using the fluorescence signal mode when the target concentration is low, while simultaneously achieving effective naked-eye recognition and quantitative analysis using the colorimetric signal mode when the target concentration is high.

[0006] The first aspect of the present invention provides a method for preparing a dynamic dual-mode, ultrasensitive immunoassay test strip.

[0007] Specifically, it includes the following steps:

[0008] (1) Fix the base plate and the absorbent pad, chromatography membrane, labeling pad and sample pad on the base plate in sequence;

[0009] (2) MAF nanoparticles were synthesized using tetra(4-carboxyphenyl)ethylene as an organic ligand and zirconium chloride as a metal ion salt.

[0010] (3) AuNPs were prepared by coating gold nanoparticles with sodium citrate;

[0011] (4) After surface chemical modification, MAF nanoparticles are mixed with AuNPs and incubated for 0.5 to 2 hours to form MAF@AuNPs composite nanoparticles by self-assembly.

[0012] (5) Mix MAF@AuNPs composite nanoparticles with primary antibody protein (Ab1) and incubate for 1-3 h, then add blocking agent and continue incubation for 0.1-1 h. After centrifugation, take the precipitate and reconstitute it with a reconstituted solvent to obtain Ab1 modified MAF@AuNPs composite nanoparticles.

[0013] (6) Add Ab1-modified MAF@AuNPs composite nanoparticles to a labeling pad and dry;

[0014] (7) Streak the primary anti-protein antibody (Anti-Ab1) on the chromatography membrane and mark it as the C line; streak the secondary anti-protein antibody (Ab2) on the chromatography membrane and mark it as the T line, then dry.

[0015] (8) The sample pad was treated with borate buffer to prepare the immunoassay strip.

[0016] Preferably, in step (2), the particle size of the MAF nanoparticles is 150-250 nm.

[0017] More preferably, the MAF nanoparticles have a particle size of 200 nm.

[0018] Preferably, in step (5), the MAF@AuNPs composite nanoparticles are 1 to 3 parts by volume, the primary antibody protein is 0.02 to 0.05 parts, the blocking agent is 0.1 to 0.4 parts, and the resolvent is 0.2 to 0.5 parts.

[0019] Preferably, in step (7), the primary antibody and the secondary antibody are 2 to 5 parts by weight.

[0020] Preferably, in step (3), the particle size of the AuNPs is 10-30 nm.

[0021] More preferably, the AuNPs have a particle size of 15 nm.

[0022] Preferably, in step (4), the surface chemical modification step includes: mixing MAF nanoparticles with positively charged polymer molecules and incubating for 0.1 to 1 h, centrifuging, and then re-dissolving the precipitate.

[0023] More preferably, the surface chemical modification step includes: mixing negatively charged MAF nanoparticles with positively charged polymer molecules and incubating for 0.5 h at room temperature and with stirring; centrifuging the mixture and removing the supernatant; and redissolving the precipitate in an equal volume of deionized water.

[0024] Preferably, in step (4), after surface chemical modification of MAF nanoparticles, they are mixed with AuNPs and incubated for 1 hour.

[0025] In a further preferred embodiment, in step (4), after surface chemical modification of MAF nanoparticles under room temperature and stirring conditions, they are mixed with negatively charged AuNPs and incubated for 1 hour. The mixture is centrifuged to remove the supernatant, and the precipitate is redissolved in an equal volume of deionized water to obtain self-assembled MAF@AuNPs composite nanoparticles.

[0026] AuNPs adsorb onto the surface of fluorescent MAF nanoparticles via electrostatic interactions, avoiding the traditional complex layer-by-layer encapsulation synthesis process and effectively simplifying the preparation process. At the same time, AuNPs exposed to solvents can be used to directly adsorb labeled antibody proteins, avoiding traditional carboxyl / amino functional modifications and covalent cross-linking, further simplifying the preparation process and improving economic efficiency.

[0027] Preferably, the positively charged polymer molecule is at least one selected from polylysine, ε-polylysine, polyethyleneamine, polyacrylamide, polyamide, and polyethyleneimine.

[0028] More preferably, the positively charged polymer molecule is polyethyleneimine.

[0029] Preferably, the molecular weight of the polyethyleneimine is 600 to 20,000.

[0030] More preferably, the molecular weight of the polyethyleneimine is 600, 2000, 5000, 10000, or 20000.

[0031] More preferably, the molecular weight of the polyethyleneimine is 10,000.

[0032] Preferably, the mass concentration of the polyethyleneimine is 0.01% to 1%.

[0033] More preferably, the mass concentration of the polyethyleneimine is 0.1%.

[0034] When MAF nanoparticles are surface chemically modified with 0.1% polyethyleneimine-10000 and then mixed and incubated with AuNPs, under the same conditions, the amount of AuNPs added determines the number of AuNPs adsorbed on the surface of a single MAF nanoparticle, wherein the amount of AuNPs added is 1 to 5 parts by volume.

[0035] Preferably, the amount of AuNPs added is 2.5 parts by volume.

[0036] Preferably, in step (5), the MAF@AuNPs composite nanoparticles are mixed with primary antibody protein (Ab1) and incubated for 2 hours, then a blocking agent is added and incubated for another 0.5 hours. After centrifugation, the precipitate is reconstituted with a reconstituted solvent to obtain Ab1-modified MAF@AuNPs composite nanoparticles. When incubating the MAF@AuNPs composite nanoparticles with Ab1, pH adjustment is not required.

[0037] Preferably, in step (5), the blocking agent and the resolvent are phosphate buffer solutions containing any one of bovine serum albumin, polyethylene glycol, gelatin, and Tween.

[0038] More preferably, the blocking agent and the resolvent are phosphate buffer containing bovine serum albumin.

[0039] Preferably, in step (6), 0.001 to 0.003 parts by volume of Ab1-modified MAF@AuNPs composite nanoparticles are dropped onto the labeling pad and dried.

[0040] In a further preferred embodiment, in step (6), 0.002.5 parts by volume of Ab1-modified MAF@AuNPs composite nanoparticles are dropped onto the labeling pad and then dried.

[0041] A second aspect of the present invention provides a dynamic dual-mode, ultrasensitive immunoassay test strip.

[0042] Specifically, the immunoassay test strip includes a base plate and an absorbent pad, a chromatography membrane, a labeling pad, and a sample pad located on the base plate; the length of the immunoassay test strip is 40-100 mm and the width is 2-10 mm.

[0043] Preferably, the base plate is made of polyvinyl chloride.

[0044] Preferably, the absorbent pad is filter paper.

[0045] Preferably, the chromatography membrane is a nitrocellulose membrane; the chromatography membrane is marked with C-lines and T-lines at intervals of 5-10 mm.

[0046] More preferably, the width of the scribe lines, namely the C-line and the T-line, is 0.3 to 1.0 mm.

[0047] More preferably, the width of the scribing marks, namely the C-line and the T-line, is 0.35 mm.

[0048] Preferably, the pore size of the chromatography membrane is 6–10 μm.

[0049] More preferably, the pore size of the chromatography membrane is 8 μm.

[0050] Preferably, the marking pad is made of glass fiber.

[0051] Preferably, the sample pad is glass fiber treated with the treatment solution.

[0052] More preferably, the treatment solution is a borate buffer solution.

[0053] A third aspect of the present invention provides a detection method for a dynamic dual-mode, ultrasensitive immunoassay test strip.

[0054] Specifically, the detection method includes the following steps:

[0055] Add the sample solution to be tested to the sample pad of the immunoassay strip, then add the working buffer to the sample pad. After chromatography is complete, observe the colorimetric signal of the T / C line when the sample solution is at a high concentration, and observe the fluorescence signal of the T / C line under ultraviolet light when the sample solution is at a low concentration.

[0056] Preferably, the chromatography time is 10 to 15 minutes.

[0057] More preferably, the chromatography time is 15 minutes.

[0058] Preferably, the working buffer is a deionized aqueous solution containing Triton X-100, and the amount of working buffer added is 0.01 to 0.05 parts by volume. The working buffer is used to support the chromatographic flow.

[0059] Preferably, the wavelength of the ultraviolet light is 302 nm.

[0060] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0061] The immunoassay test strip prepared in this invention achieves dynamic complementarity of fluorescence and colorimetric dual-mode signals. It enables ultrasensitive detection using the fluorescence signal mode at low target concentrations, while simultaneously achieving effective naked-eye recognition and quantitative analysis using the colorimetric signal mode at high target concentrations. Furthermore, the sensitivity of the fluorescence mode is more than 100 times higher than that of the colorimetric mode. The immunoassay test strip of this invention has a wide range of applications, covering both conventional and ultrasensitive analytes. The preparation method of this invention has a simpler overall process, saving manpower and material costs. The AuNPs treated in this invention can directly adsorb labeled antibody proteins, avoiding chemical modification and covalent cross-linking. Attached Figure Description

[0062] Figure 1 The results of the dynamic dual-mode, ultrasensitive immunoassay strip detection model for PCT are shown in the figure.

[0063] Figure 2 The results of the dynamic dual-mode, ultrasensitive immunoassay strip detection model of D-dimer are shown in the figure.

[0064] Figure 3 The results of the dynamic dual-mode, ultrasensitive immunoassay strip detection model for NT-proBNP are shown in the figure.

[0065] Figure 4 The image shows the results of a dynamic dual-mode, ultrasensitive immunoassay strip detection model for cTnI. Detailed Implementation

[0066] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0067] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0068] The inventive concept of this invention aims to meet the application needs of different detection scenarios and solve the problem of complex preparation processes. It achieves a dynamic, ultrasensitive immunoassay strip with both fluorescence and colorimetric modalities. This invention utilizes surface chemical design to enable the self-assembly of fluorescent MAF nanoparticles and AuNPs to form a MAF@AuNPs nanocomposite, which possesses both fluorescence and colorimetric modal signals. In the self-assembled MAF@AuNPs nanocomposite, AuNPs are adsorbed on the surface of the MAF particles, avoiding complex synthesis processes. Furthermore, the AuNPs exposed in solution can be used for subsequent direct adsorption of labeled antibody proteins, further simplifying the overall process. The immunoassay strip based on the MAF@AuNPs nanocomposite achieves ultra-high sensitivity for low-concentration target detection using the fluorescence modal signal of MAF, and enables naked-eye recognition and efficient quantitative analysis for high-concentration targets using the colorimetric modal signal of AuNPs. The sensitivity of the fluorescence modal signal is more than 100 times higher than that of the colorimetric modal signal, forming a dual-modal dynamic complementary real-time detection system that addresses various potential high and low concentration target detection needs.

[0069] Example 1

[0070] A dynamic dual-mode, ultrasensitive immunoassay test strip and its preparation method.

[0071] Example 1 uses procalcitonin (PCT) detection as an example.

[0072] The materials / reagents used in this embodiment include, by volume, the following:

[0073] 2 parts MAF@AuNPs composite nanoparticles, 0.03 parts primary antibody protein PCT-Ab1, 0.2 parts blocking agent, 0.3 parts reconstitution solvent, and 0.04 parts working buffer.

[0074] Included in parts by weight:

[0075] 2.5 parts of primary antibody Anti-PCT-Ab1 and 2.5 parts of secondary antibody PCT-Ab2.

[0076] The preparation method includes the following steps:

[0077] (1) Fix the base plate and the absorbent pad, chromatography membrane, labeling pad and sample pad on the base plate in sequence;

[0078] (2) MAF nanoparticles were synthesized using tetra(4-carboxyphenyl)ethylene as an organic ligand and zirconium chloride as a metal ion salt.

[0079] (3) AuNPs were prepared by coating gold nanoparticles with sodium citrate;

[0080] (4) At room temperature and with stirring, negatively charged MAF nanoparticles were mixed with 0.1% polyethyleneimine-10000 and incubated for 0.5 h. After centrifugation, the supernatant was removed, and the precipitate was redissolved in an equal volume of deionized water to obtain surface chemically modified MAF nanoparticles. Then, they were mixed with 2.5 parts of negatively charged AuNPs and incubated for 1 h. After centrifugation, the supernatant was removed, and the precipitate was redissolved in an equal volume of deionized water to obtain self-assembled MAF@AuNPs composite nanoparticles.

[0081] (5) Mix MAF@AuNPs composite nanoparticles with PCT-Ab1 and incubate in a shaker for 2 hours. Then add a blocking agent and continue incubation for 0.5 hours. After centrifugation at 8000 rpm for 10 minutes, remove the supernatant and take the precipitate to redissolve with a reconstituted solvent to obtain PCT-Ab1 modified MAF@AuNPs composite nanoparticles.

[0082] (6) 0.0025 parts by volume of PCT-Ab1 modified MAF@AuNPs composite nanoparticles were dropped onto a labeling pad and dried;

[0083] (7) Streak Anti-PCT-Ab1 on the chromatography membrane and mark it as line C; streak PCT-Ab2 on the chromatography membrane and mark it as line T, then dry.

[0084] (8) The sample pad was treated with borate buffer to prepare the immunoassay strip.

[0085] The detection method includes the following steps:

[0086] Add a standard solution containing the target to be detected to the sample pad, and then add the corresponding working buffer to the sample pad. After chromatography is completed (15 min), record the colorimetric and fluorescence signals of the T line and C line respectively.

[0087] Test results as follows Figure 1 As shown, the sensitivity of the colorimetric modality is 5 ng / mL, and the sensitivity of the fluorescence modality is 10 pg / mL (clinical threshold 50 pg / mL). The sensitivity of the fluorescence modality is about 500 times higher than that of the colorimetric modality.

[0088] Example 2

[0089] A dynamic dual-mode, ultrasensitive immunoassay test strip and its preparation method.

[0090] Example 2 uses the detection of D-dimer as an example.

[0091] The materials / reagents used in this embodiment include, by volume, the following:

[0092] 2 parts MAF@AuNPs composite nanoparticles, 0.03 parts primary antibody protein D-dimer-Ab1, 0.2 parts blocking agent, 0.3 parts reconstitution solvent, and 0.05 parts working buffer.

[0093] Included in parts by weight:

[0094] 2.5 parts of primary antibody Anti-PCT-Ab1 and 2.5 parts of secondary antibody PCT-Ab2.

[0095] The preparation method includes the following steps:

[0096] (1) Fix the base plate and the absorbent pad, chromatography membrane, labeling pad and sample pad on the base plate in sequence;

[0097] (2) MAF nanoparticles were synthesized using tetra(4-carboxyphenyl)ethylene as an organic ligand and zirconium chloride as a metal ion salt.

[0098] (3) AuNPs were prepared by coating gold nanoparticles with sodium citrate;

[0099] (4) At room temperature and with stirring, negatively charged MAF nanoparticles were mixed with 0.1% polyethyleneimine-10000 and incubated for 0.5 h. After centrifugation, the supernatant was removed, and the precipitate was redissolved in an equal volume of deionized water to obtain surface chemically modified MAF nanoparticles. Then, they were mixed with 2.5 parts of negatively charged AuNPs and incubated for 1 h. After centrifugation, the supernatant was removed, and the precipitate was redissolved in an equal volume of deionized water to form MAF@AuNPs composite nanoparticles.

[0100] (5) Mix MAF@AuNPs composite nanoparticles with D-dimer-Ab1 and incubate in a shaker for 2 hours. Then add a blocking agent and continue incubation for 0.5 hours. After centrifugation at 8000 rpm for 10 minutes, remove the supernatant and take the precipitate to redissolve with a reconstituted solvent to obtain D-dimer-Ab1 modified MAF@AuNPs composite nanoparticles.

[0101] (6) 0.0025 parts by volume of D-dimer-Ab1 modified MAF@AuNPs composite nanoparticles were dropped onto a labeling pad and dried;

[0102] (7) Streak Anti-D-dimer-Ab1 on the chromatography membrane and mark it as line C; streak D-dimer-Ab2 on the chromatography membrane and mark it as line T, then dry.

[0103] (8) The sample pad was treated with borate buffer to prepare the immunoassay strip.

[0104] The detection method includes the following steps:

[0105] Add a standard solution containing the target to be detected to the sample pad, and then add the corresponding working buffer to the sample pad. After chromatography is completed (15 min), record the colorimetric and fluorescence signals of the T line and C line respectively.

[0106] Test results as follows Figure 2 As shown, the sensitivity of the colorimetric modality is 20 ng / mL, and the sensitivity of the fluorescence modality is 100 pg / mL (clinical threshold 500 ng / mL). The sensitivity of the fluorescence modality is about 200 times higher than that of the colorimetric modality.

[0107] Example 3

[0108] A dynamic dual-mode, ultrasensitive immunoassay test strip and its preparation method.

[0109] Example 3 uses NT-proBNP (N-terminal pro-B-type natriuretic peptide) detection as an example.

[0110] The materials / reagents used in this embodiment include, by volume, the following:

[0111] 2 parts MAF@AuNPs composite nanoparticles, 0.03 parts primary antibody protein NT-proBNP-Ab1, 0.2 parts blocking agent, 0.03 parts reconstitution solvent, and 0.05 parts working buffer.

[0112] Included in parts by weight:

[0113] 2.5 parts of primary antibody Anti-PCT-Ab1 and 2.5 parts of secondary antibody PCT-Ab2.

[0114] The preparation method includes the following steps:

[0115] (1) Fix the base plate and the absorbent pad, chromatography membrane, labeling pad and sample pad on the base plate in sequence;

[0116] (2) MAF nanoparticles were synthesized using tetra(4-carboxyphenyl)ethylene as an organic ligand and zirconium chloride as a metal ion salt.

[0117] (3) AuNPs were prepared by coating gold nanoparticles with sodium citrate;

[0118] (4) At room temperature and with stirring, negatively charged MAF nanoparticles were mixed with 0.1% polyethyleneimine-10000 and incubated for 0.5 h. After centrifugation, the supernatant was removed, and the precipitate was redissolved in an equal volume of deionized water to obtain surface chemically modified MAF nanoparticles. Then, they were mixed with 2.5 parts of negatively charged AuNPs and incubated for 1 h. After centrifugation, the supernatant was removed, and the precipitate was redissolved in an equal volume of deionized water to form MAF@AuNPs composite nanoparticles.

[0119] (5) Mix MAF@AuNPs composite nanoparticles with NT-proBNP-Ab1 and incubate in a shaker for 2 hours. Then add a blocking agent and continue incubation for 0.5 hours. After centrifugation at 8000 rpm for 10 minutes, remove the supernatant and take the precipitate to redissolve with a reconstituted solvent to obtain NT-proBNP-Ab1 modified MAF@AuNPs composite nanoparticles.

[0120] (6) 0.0025 parts by volume of NT-proBNP-Ab1 modified MAF@AuNPs composite nanoparticles were dropped onto a labeling pad and dried;

[0121] (7) Streak Anti-NT-proBNP-Ab1 on the chromatography membrane and mark it as line C; streak NT-proBNP-Ab2 on the chromatography membrane and mark it as line T, then dry.

[0122] (8) The sample pad was treated with borate buffer to prepare the immunoassay strip.

[0123] The detection method includes the following steps:

[0124] Add a standard solution containing the target to be detected to the sample pad, and then add the corresponding working buffer to the sample pad. After chromatography is completed (15 min), record the colorimetric and fluorescence signals of the T line and C line respectively.

[0125] Test results as follows Figure 3 As shown, the sensitivity of the colorimetric modality is 0.5 ng / mL, and the sensitivity of the fluorescence modality is 2 pg / mL (clinical threshold 300 pg / mL). The sensitivity of the fluorescence modality is about 250 times higher than that of the colorimetric modality.

[0126] Example 4

[0127] A dynamic dual-mode, ultrasensitive immunoassay test strip and its preparation method.

[0128] Example 4 uses cTnI (cardiac troponin) detection as an example.

[0129] The materials / reagents used in this embodiment include, by volume, the following:

[0130] 2 parts MAF@AuNPs composite nanoparticles, 0.03 parts primary antibody protein cTnI-Ab1, 0.2 parts blocking agent, 0.3 parts reconstitution solvent, and 0.05 parts working buffer.

[0131] Included in parts by weight:

[0132] 2.5 parts of primary antibody Anti-PCT-Ab1 and 2.5 parts of secondary antibody PCT-Ab2.

[0133] The preparation method includes the following steps:

[0134] (1) Fix the base plate and the absorbent pad, chromatography membrane, labeling pad and sample pad on the base plate in sequence;

[0135] (2) MAF nanoparticles were synthesized using tetra(4-carboxyphenyl)ethylene as an organic ligand and zirconium chloride as a metal ion salt.

[0136] (3) AuNPs were prepared by coating gold nanoparticles with sodium citrate;

[0137] (4) At room temperature and with stirring, negatively charged MAF nanoparticles were mixed with 0.1% polyethyleneimine-10000 and incubated for 0.5 h. After centrifugation, the supernatant was removed, and the precipitate was redissolved in an equal volume of deionized water to obtain surface chemically modified MAF nanoparticles. Then, they were mixed with 2.5 parts of negatively charged AuNPs and incubated for 1 h. After centrifugation, the supernatant was removed, and the precipitate was redissolved in an equal volume of deionized water to form MAF@AuNPs composite nanoparticles.

[0138] (5) Mix MAF@AuNPs composite nanoparticles with cTnI-Ab1 and incubate in a shaker for 2 hours. Then add a blocking agent and continue incubation for 0.5 hours. After centrifugation at 8000 rpm for 10 minutes, remove the supernatant and take the precipitate to redissolve with a reconstituted solvent to obtain cTnI-Ab1 modified MAF@AuNPs composite nanoparticles.

[0139] (6) 0.0025 parts by volume of cTnI-Ab1 modified MAF@AuNPs composite nanoparticles were dropped onto a labeling pad and dried;

[0140] (7) Streak Anti-cTnI-Ab1 on the chromatography membrane and mark it as the C line; streak cTnI-Ab2 on the chromatography membrane and mark it as the T line, then dry.

[0141] (8) The sample pad was treated with borate buffer to prepare the immunoassay strip.

[0142] The detection method includes the following steps:

[0143] Add a standard solution containing the target to be detected to the sample pad, and then add the corresponding working buffer to the sample pad. After chromatography is completed (15 min), record the colorimetric and fluorescence signals of the T line and C line respectively.

[0144] Test results as follows Figure 4As shown, the sensitivity of the colorimetric modality is 20 ng / mL, and the sensitivity of the fluorescence modality is 10 pg / mL (clinical threshold 12 pg / mL). The sensitivity of the fluorescence modality is about 2000 times higher than that of the colorimetric modality.

[0145] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, any technical solutions obtained by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concept of the present invention and on the existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for preparing a dynamic dual-mode immunoassay test strip, characterized in that, Includes the following steps: (1) Fix the base plate and the absorbent pad, chromatography membrane, labeling pad and sample pad on the base plate in sequence; (2) MAF nanoparticles were synthesized using tetra(4-carboxyphenyl)ethylene as an organic ligand and zirconium chloride as a metal ion salt; (3) AuNPs were prepared by coating gold nanoparticles with sodium citrate; (4) Mix MAF nanoparticles with polyethyleneimine molecules at a mass concentration of 0.01~1% and incubate for 0.1~1h. After centrifugation, take the precipitate and re-dissolve. Mix the modified MAF nanoparticles with AuNPs and incubate for 0.5~2h to self-assemble into MAF@AuNPs composite nanoparticles. (5) Mix MAF@AuNPs composite nanoparticles with primary antibody protein and incubate for 1-3 h, then add blocking agent and continue incubation for 0.1-1 h. After centrifugation, take the precipitate and reconstitute it with a reconstituted solvent to obtain Ab1 modified MAF@AuNPs composite nanoparticles. (6) Add Ab1-modified MAF@AuNPs composite nanoparticles to the labeling pad and dry; (7) Streak the primary anti-protein antibody on the chromatographic membrane and mark it as the C line; streak the secondary anti-protein antibody on the chromatographic membrane and mark it as the T line, then dry it; (8) The sample pad was treated with borate buffer to prepare the immunoassay strip.

2. The preparation method according to claim 1, characterized in that, In step (5), the MAF@AuNPs composite nanoparticles are 1 to 3 parts by volume, the primary antibody protein is 0.02 to 0.05 parts, the blocking agent is 0.1 to 0.4 parts, and the resolvent is 0.2 to 0.5 parts.

3. The preparation method according to claim 1, characterized in that, In step (7), the primary antibody and the secondary antibody are 2 to 5 parts by weight.

4. The preparation method according to claim 1, characterized in that, The molecular weight of the polyethyleneimine is 600 to 20,000.

5. The preparation method according to claim 1, characterized in that, In step (5), the blocking agent and the resolvent are phosphate buffer solutions containing any one of bovine serum albumin, polyethylene glycol, gelatin, and Tween.

6. The immunoassay test strip prepared by the preparation method according to any one of claims 1 to 5, characterized in that, The immunoassay test strip includes a base plate and an absorbent pad, a chromatography membrane, a labeling pad, and a sample pad located on the base plate; the length of the immunoassay test strip is 40~100mm and the width is 2~10mm.

7. The detection method of the immunoassay test strip according to claim 6, characterized in that, The detection method includes the following steps: Add the sample solution to be tested to the sample pad of the immunoassay strip, then add the working buffer to the sample pad. After chromatography is complete, observe the colorimetric signal of the T / C line when the sample solution is at a high concentration, and observe the fluorescence signal of the T / C line under ultraviolet light when the sample solution is at a low concentration.

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