Immunochromatography test strip for enhancing near-infrared two-region fluorescence through supramolecular host-guest interaction and preparation method of immunochromatography test strip

Abstract

The invention relates to the technical field of clinical medical examination, in particular to an immunochromatography test strip for enhancing near-infrared two-region fluorescence through supramolecular host-guest interaction and a preparation method of the immunochromatography test strip. The invention provides a preparation method of an immunochromatographic test strip. The preparation method comprises the following steps: preparing a rare earth nanoparticle (RENPs) labeling solution; preparing a microsphere combination pad; preparing a sample pad; coating an antibody with a nitrocellulose membrane; and assembling and manufacturing the card. The aggregation phenomenon and partial fluorescence quenching effect caused in the surface modification process of the rare earth microspheres are reduced by utilizing an efficient surface modification strategy of a supramolecular host compound, the fluorescence luminescence property of the rare earth microspheres is effectively improved, and meanwhile, the antibody labeling efficiency of the rare earth microspheres is further improved by utilizing a supramolecular self-assembly strategy; the prepared test strip is obviously improved in analysis sensitivity, has the advantages of simplicity in operation, low cost and short detection time, and is suitable for rapid and sensitive detection of low-concentration markers in complex biological samples.

Description

Technical Field

[0001] The invention relates to the technical field of clinical medical testing, and in particular to an immunochromatographic test strip for enhancing near-infrared second-zone fluorescence by supramolecular host-guest interaction and a preparation method thereof. Background Art

[0002] Near-infrared II (NIR-II) fluorescence has significant advantages such as long emission wavelength (1000-1700nm), strong tissue penetration ability, and low light scattering and autofluorescence interference, which can effectively avoid the problem of spontaneous fluorescence interference of biological samples (blood, etc.). Choosing to use near-infrared II fluorescence as a detection signal in the fluorescent immunochromatography technology platform can reduce background interference and signal attenuation, thereby further improving the analytical performance of the platform. Therefore, the development of efficient near-infrared II fluorescent probes with high fluorescence quantum efficiency, good photostability and easy surface modification has become the key to improving the analytical performance of the fluorescent immunochromatography technology platform.

[0003] Among the numerous NIR-II molecular probes, rare earth nanoparticles (RENPs) have the characteristics of large Stokes shift (greater than 200 nm), long fluorescence decay lifetime, high quantum yield and easy surface modification, which have great application advantages and development potential in the fluorescence immunochromatography technology platform. The surface hydrophilization modification process of RENPs is an important link affecting its luminescence performance. There are two main surface modification methods currently used: (1) The ligand direct exchange method uses the different binding forces between different ligands and the surface of RENPs to remove surface ligands. For example, the binding force between borate (NaBF4) and RENPs is much higher than that between carboxylates. Then, a hydrophilic polymer (polyacrylic acid PAA, etc.) is added, and the surface carboxyl-modified hydrophilic RENPs-PAA is obtained by heating reaction. However, this method is difficult to control that all binding groups from each polymer molecule can be attached to RENPs in an orderly and selective manner. At the same time, after the surface of RENPs is carboxylated, the -COOH and -OH groups on its surface will cause the non-radiative relaxation enhancement effect of lanthanide elements (doping elements and sensitizers), resulting in partial fluorescence quenching. (2) The acid washing method is to first use a dilute hydrochloric acid solution and a mixture of RENPs (RENPs-OA) with hydrophobic oleic acid ligands on the surface to remove the surface oleic acid ligands and then add a hydrophilic polymer (PAA, etc.), and then heat the reaction to obtain surface carboxylated RENPs. However, after the surface ligands are removed, the RENPs are not stable and are prone to hydrolysis, corrosion, and agglomeration. Similarly, the partial fluorescence quenching phenomenon caused by the surface modification of hydrophilic polymers on RENPs is also unavoidable.

[0004] Therefore, how to develop new and efficient RENPs surface modification technology to avoid or minimize the particle aggregation phenomenon and the partial fluorescence quenching effect caused during the surface modification process has become an urgent problem to be solved, and it is also the key to improving the analytical performance of fluorescent immunochromatography reagents. Summary of the invention

[0005] In order to solve the above technical problems existing in the prior art, the present invention provides an immunochromatographic test strip for enhancing near-infrared second-zone fluorescence by supramolecular host-guest interaction and a preparation method thereof.

[0006] The first aspect of the present invention is to provide a method for preparing an immunochromatographic test strip for enhancing near-infrared second-region fluorescence by supramolecular host-guest interaction, comprising the following steps:

[0007] (1) Preparing a rare earth nanoparticle (RENPs) labeling solution, comprising the steps of:

[0008] S1, supramolecular host compound modified rare earth nanoparticles;

[0009] S2, 1-adamantane carboxylate sodium (ADCNa) pre-activation;

[0010] S3, forming a rare earth nanosphere labeling solution by combining RENPs modified with the supramolecular main compound prepared in S1, the activated ADCNa prepared in S2 and the antibody;

[0011] (2) preparing a microsphere binding pad;

[0012] (3) preparing a sample pad;

[0013] (4) Nitrocellulose membrane coated with antibodies;

[0014] (5) Assembling the card.

[0015] Among them, the microspheres are rare earth-doped near-infrared second-zone fluorescent nano-microspheres.

[0016] The invention provides a method for preparing an immunochromatographic test strip with supramolecular host-guest action enhanced near-infrared zone II fluorescence. The method uses supramolecular host compound-modified enhanced near-infrared zone II fluorescence rare earth nano-microspheres as fluorescent probes. On the one hand, the supramolecular host compound surface modification strategy is used to enhance the luminescence effect of rare earth nano-microspheres (RENPs) to reduce particle aggregation and fluorescence quenching phenomena. On the other hand, the supramolecular host compound-modified rare earth nano-microspheres (RENPs) can increase the antibody binding amount on the microsphere surface by means of host-guest self-assembly, thereby effectively improving the antibody labeling efficiency. On this basis, an immunochromatographic method is established and a fluorescent immunochromatographic reagent strip is prepared, which can more sensitively and accurately measure the concentration of trace markers in biological samples.

[0017] Further, the steps of S3 are as follows:

[0018] The self-assembled complex was prepared by self-assembly of RENPs modified with supramolecular host compounds and activated ADCNa;

[0019] The self-assembled complex is coupled with the antibody to form a rare earth nanosphere labeling solution.

[0020] Further, the steps of S3 are as follows:

[0021] ADCNa is coupled to an antibody to form an antibody complex (ADC-Ab);

[0022] The RENPs modified by the supramolecular host compound and the antibody complex self-assemble to form a rare earth nanosphere labeling liquid.

[0023] Furthermore, in step (1), the specific preparation steps of the rare earth nanosphere marking liquid are as follows:

[0024] The RENPs modified with oleic acid ligand (RENPs-OA) were dissolved in chloroform, and the supramolecular host compound aqueous solution was added. After stirring at room temperature overnight, the solution was centrifuged at high speed and washed with pure water, and finally resuspended with pure water by ultrasonication to obtain the RENPs solution modified with the supramolecular host compound.

[0025] 1-adamantanecarboxylic acid (ADC) and NaOH were dissolved in ethanol, stirred at room temperature for overnight reaction and then vacuum dried to obtain 1-adamantanecarboxylic acid sodium (ADCNa), a certain amount of ADCNa was dissolved in MES buffer (2-(N-morpholino)ethanesulfonic acid), freshly prepared EDC solution (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and Sulfo-NHS solution (N-hydroxysulfosuccinimide) were added, and activation reaction was carried out at room temperature in the dark to obtain an activation solution;

[0026] Add the RENPs solution modified by the supramolecular host compound to the activation solution, continue stirring at room temperature to perform a self-assembly reaction, centrifuge at high speed, discard the supernatant to obtain a white precipitate, wash the white precipitate with BBS (borate buffer, pH=8.0), and then add BBS for ultrasonic re-suspending to obtain a self-assembly complex solution;

[0027] The self-assembled complex solution was added to an antibody solution of a certain concentration, and the reaction was oscillated at room temperature for 2-3 hours. The blocking solution was added, and the solution was washed with a microsphere washing solution after high-speed centrifugation. The microsphere protection solution was added and ultrasonically resuspended to obtain a rare earth nano-microsphere labeling solution, which was stored at 4° C. for future use.

[0028] Furthermore, in step (1), the specific preparation steps of the rare earth nanosphere marking liquid are as follows:

[0029] The RENPs modified with oleic acid ligands were dissolved in chloroform, and the supramolecular host compound aqueous solution was added. After stirring at room temperature for overnight reaction, the mixture was centrifuged at high speed and washed with pure water. Finally, the RENPs solution modified with supramolecular host compound was obtained by ultrasonic resuspending with pure water.

[0030] 1-adamantanecarboxylic acid (ADC) and NaOH were dissolved in ethanol, stirred at room temperature for overnight reaction, and then vacuum dried to obtain 1-adamantanecarboxylic acid sodium (ADCNa) white powder. A certain amount of ADCNa was dissolved in MES buffer, and freshly prepared EDC solution and Sulfo-NHS solution were added, and activation reaction was carried out at room temperature in the dark to obtain an activation solution;

[0031] The activation solution and antibody were dissolved in BBS buffer, and freshly prepared EDC solution and Sulfo-NHS solution were added. The mixture was stirred at 4°C for overnight reaction, and the antibody complex (ADC-Ab) was concentrated and separated by centrifugation. The concentration of ADC-Ab was determined by UV-visible spectrophotometry (UV-vis).

[0032] The RENPs solution modified by the supramolecular host compound and the ADC-Ab solution were mixed, stirred and reacted at 4°C overnight, blocked by adding blocking solution, washed with microsphere washing solution by high-speed centrifugation, and ultrasonically resuspended by adding microsphere protection solution to obtain rare earth nanosphere labeling solution, which was stored at 4°C for future use.

[0033] Furthermore, the molar ratios of the ADC to EDC, and the molar ratios of EDC to Sulfo-NHS are both between 1-10.

[0034] Furthermore, the molar ratio of ADC, EDC and Sulfo-NHS is 1:5:10.

[0035] Furthermore, the antibody addition amount is 50-200 μg; the blocking solution is 40 mM ethanolamine solution or 10% BSA solution (bovine serum albumin); the microsphere washing solution is 20 mM Tris buffer containing 0.5% BSA and 0.1% Tween-20; the microsphere protection solution is 20 mM Tris buffer containing 0.5% BSA, 5% trehalose and 0.1% Tween-20.

[0036] Furthermore, the activation time of the ADC is 20-40 minutes; the self-assembly reaction time of the RENPs modified by the supramolecular host compound and the ADC is 10-30 minutes.

[0037] Furthermore, the preparation steps of the microsphere binding pad are as follows: adding microsphere diluent to the rare earth nano-microsphere labeling liquid, and then evenly spraying it onto the pretreated glass fiber or polyester fiber, and drying it in an oven; the preparation steps of the sample pad are as follows: selecting glass fiber or polyester fiber, immersing it in the sample pad treatment liquid, oscillating it at room temperature for 2-3 hours, and drying it in an oven; the preparation steps of the nitrocellulose membrane-coated antibody are as follows: using borate (BBS) buffer to prepare capture antibody and quality control antibody solutions respectively, using a three-dimensional film drawing instrument to spray the detection line and quality control line on the nitrocellulose membrane in parallel, and drying it in an oven; the steps of assembling the card are as follows: sticking the prepared sample pad, microsphere binding pad, nitrocellulose membrane and absorbent paper on the bottom plate in turn and overlapping each other by 2 mm, cutting them into test strips with an automatic chopper, loading them into a card shell, making them into a detection reagent card, and storing them in low-temperature drying.

[0038] Furthermore, the sample pad treatment solution is a 20 mM Tris buffer containing 0.5% Tetronic 1307 (S9), 0.05% bovine serum albumin (BSA), 0.05% Tween-20, 0.3% polyvinylpyrrolidone-K30 (PVP-K30), and 0.05% Proclin-300.

[0039] Furthermore, the film-stripping concentration of the test line and the quality control line is 0.5-2.0 mg / mL; the film-stripping spray volume of the test line and the quality control line is 0.5-2 μL / cm.

[0040] The second aspect of the present invention is to provide an immunochromatographic test strip for enhancing near-infrared second-zone fluorescence by supramolecular host-guest interaction, which is prepared by the above-mentioned preparation method. The fluorescent immunochromatographic test strip comprises a PVC base plate, a sample pad, a microsphere binding pad, a nitrocellulose membrane and absorbent paper; the sample pad, the microsphere binding pad, the nitrocellulose membrane and the absorbent paper are sequentially overlapped 2mm in the horizontal direction and pasted on the base plate; the microsphere binding pad is fixed with a RENPs-coupled antibody complex modified by a supramolecular host compound; the nitrocellulose membrane is respectively coated with an antibody detection line and a quality control line.

[0041] Furthermore, the immunochromatographic test strip uses rare earth nanoparticles with near-infrared second-zone fluorescence enhanced by supramolecular main compounds as fluorescent probes to label antibodies, and the particle size of the rare earth nanoparticles is 100-500 nm.

[0042] Furthermore, the excitation wavelength of the rare earth nanospheres is 700-900 nm; and the fluorescence emission wavelength range is 900-1700 nm.

[0043] Furthermore, the rare earth ion is Nd 3+ , Yb 3+ , Er 3+ 、Ho3+ or Ce 3+ Any one or more of .

[0044] Furthermore, the rare earth nanospheres are composed of a core and a shell structure.

[0045] Furthermore, the inner core is an inorganic matrix doped with ions, including NaLnF4, LiLnF4 or LnF3, wherein Ln=Y, Yb, Gd or Lu; the shell is one or more layers of inorganic matrix, coated outside the inner core, including NaLnF4, LiLnF4 or CaF2, wherein Ln=Y, Yb, Gd or Lu.

[0046] Furthermore, the supramolecular host compound is any one or more of cyclodextrin (CD), cucurbituril (CB), crown ether (CE), pillar aromatics (PA), and calixarene (CA), and the microspheres are rare earth nano-microspheres whose surfaces are modified by one or more of cyclodextrin (CD), cucurbituril (CB), crown ether (CE), pillar aromatics (PA), and calixarene (CA).

[0047] Furthermore, the cyclodextrin includes α-CD, β-CD or γ-CD.

[0048] Furthermore, the cucurbituril comprises CB[6], CB[7], CB[8] or CB

[10] .

[0049] Furthermore, the crown ethers include 15-crown-5 and 18-crown-6.

[0050] Furthermore, the pillar aromatics include PA[6], PA[7], PA[9] or PA

[10] .

[0051] Furthermore, the calixarene includes CA[4], CA[6] or CA[8].

[0052] Compared with the prior art, the present invention has the following technical effects:

[0053] The test strip of the present invention adopts an efficient surface modification strategy of supramolecular host compounds, which can effectively reduce the microsphere aggregation phenomenon and the partial fluorescence quenching phenomenon caused by the RENPs surface modification process, and has the advantages of better luminescence stability, stronger fluorescence, higher antibody labeling efficiency and higher analysis sensitivity. At the same time, the prepared test strip also has the characteristics of low cost, simple operation and fast analysis speed. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 A schematic diagram of the structure of an immunochromatographic test strip for enhancing near-infrared second-zone fluorescence using a supramolecular host compound (CB[7]) prepared in the present invention;

[0055] Figure 2 Transmission electron microscopy image of RENPs-CB[7] prepared in an embodiment of the present invention;

[0056] Figure 3 The particle size distribution diagram of RENPs-CB[7] prepared in the embodiment of the present invention;

[0057] Figure 4 The fluorescence emission spectra of RENPs-CB[7] prepared in the embodiment of the present invention and the rare earth nanoparticles in the prior art;

[0058] Figure 5 The luminescence stability spectra of RENPs-CB[7] prepared in the embodiment of the present invention and RENPs-PAA in the prior art;

[0059] Figure 6 Schematic diagram of the self-assembly of RENPs-CB[7] and ADC-Ab prepared in an embodiment of the present invention;

[0060] Figure 7 The conjugation amount spectrum of RENPs-CB[7] prepared in the embodiment of the present invention and RENPs-PAA antibody in the prior art;

[0061] Figure 8 A standard curve diagram of PSA concentration detected by a fluorescent immunochromatographic test strip prepared in an embodiment of the present invention;

[0062] Fig. 9 It is a comparison chart of the detection results of the fluorescent immunochromatography method of the present invention and the Roche chemiluminescence method;

[0063] Figure numerals: 1, base plate; 2, sample pad; 3, microsphere binding pad; 4, nitrocellulose membrane; 5, absorbent paper; 6, detection line; 7, quality control line; 8, RENPs-CB[7]-ADC complex. DETAILED DESCRIPTION

[0064] The following specific embodiments illustrate the implementation of the present invention, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Although the description of the present invention will be introduced in conjunction with the preferred embodiment, this does not mean that the features of this invention are limited to this implementation. On the contrary, the purpose of introducing the invention in conjunction with the implementation is to cover other options or modifications that may extend based on the claims of the present invention. In order to provide a deep understanding of the present invention, the following description will include many specific details. The present invention can also be implemented without using these details. In addition, in order to avoid confusion or blurring the focus of the present invention, some specific details will be omitted in the description. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0065] like Figure 1 As shown, a supramolecular host-guest interaction enhanced near-infrared second-zone fluorescence immunochromatographic test strip comprises a base plate 1, a sample pad 2, a microsphere binding pad 3, a nitrocellulose membrane 4 and a blotting paper 5. The sample pad 2, the microsphere binding pad 3, the nitrocellulose membrane 4 and the blotting paper 5 are sequentially overlapped and pasted on the base plate 1 in the horizontal direction, and the adjacent parts overlap each other by 2 mm on the base plate 1; the microsphere binding pad 3 is fixed with a RENPs-CB[7] coupled antibody complex; the nitrocellulose membrane 4 is coated with a detection line 6 of a prostate-specific antigen (PSA) capture antibody and a quality control line 7 of goat anti-mouse IgG.

[0066] The microspheres are rare earth nano-microspheres (NaYF4: X% Nd@NaYF4, X=1-100) having a fluorescence emission peak in the second near-infrared region;

[0067] The particle size of the rare earth nano-microspheres is 100-500nm.

[0068] Example

[0069] (1) Preparation of rare earth nanosphere labeling solution

[0070] ① Preparation of RENPs-CB[7]: 1 mL of oleic acid ligand-modified RENPs (RENPs-OA) was dissolved in 3 mL of chloroform, and then 5 mL of CB[7] aqueous solution (2 mg / mL) was added. After magnetic stirring at room temperature overnight, the mixture was centrifuged at high speed and washed with pure water. Finally, 1 mL of pure water was used for ultrasonic resuspending to obtain RENPs-CB[7];

[0071] ②ADC pre-activation: First, prepare sodium 1-adamantanecarboxylate (ADCNa), that is, dissolve 200 mg 1-adamantanecarboxylic acid (ADC) and 50 mg NaOH in 10 mL ethanol, stir at room temperature for overnight reaction, and then vacuum dry to obtain a white powder (ADCNa); then, dissolve 3 mg ADCNa in 3 mL 50 mM MES buffer, add freshly prepared 10 mg / mL EDC solution and 10 mg / mL Sulfo-NHS solution, each in a volume of 200 μL, activate the reaction at room temperature in the dark for 30 min, centrifuge at high speed for 15 min, discard the supernatant, wash the precipitate twice with 50 mM BBS solution, add 0.1 mL BBS solution and sonicate until fully resuspended;

[0072] ③ADC-coupled PSA detection antibody: Add the above 0.1 mL of ADCNa activation solution to 1 mL of PSA antibody solution (0.10 mg / mL), shake and react at room temperature for 2 hours. After the reaction is completed, collect and concentrate the ADC-Ab coupling liquid with a centrifugal filter, make the volume to 1 mL, and determine its concentration by UV-vis spectroscopy.

[0073] ④Self-assembly of RENPs-CB[7] and ADC-Ab: Add 0.1 mL of RENPs-CB[7] solution to the above ADC-Ab coupling solution, stir at room temperature for 15 min to fully carry out the host-guest self-assembly reaction, add 40 mM ethanolamine solution to block for 40 min, centrifuge at high speed and wash twice with microsphere washing solution, add microsphere protection solution and ultrasonically resuspend to obtain rare earth nanosphere labeling solution (RENPs-CB-ADC-Ab), and store at 4°C for later use.

[0074] (2) Preparation of microsphere binding pad

[0075] Add microsphere diluent (10 mM sodium citrate solution containing 1% sucrose) to the rare earth nanosphere labeling solution prepared in step (1) to dilute it to 10% of the original concentration, then spread it evenly on glass fiber or polyester fiber at 2 μL / cm and dry it in a 45°C oven for 12 hours.

[0076] (3) Sample pad preparation

[0077] The sample pad is made of glass fiber or polyester fiber, which is soaked in the sample pad treatment solution, shaken at room temperature for 2 hours, and dried in a 45°C oven for 12 hours.

[0078] (4) Coating treatment of nitrocellulose membrane

[0079] The preparation of the test line 6 is to prepare 2 mg / mL of PSA capture antibody with 50 mM BBS buffer (pH=8.0), spray it in parallel on the nitrocellulose membrane 4 at a volume of 1.2 μL / cm using a three-dimensional film scribing instrument, the scribing speed is 50 mm / s, and dry it in a 45°C oven for 12 h;

[0080] The quality control line 7 was prepared by preparing a sheep anti-mouse IgG solution with 50 mM BBS buffer (pH = 8.0), and the quality control line 7 was sprayed in parallel on the nitrocellulose membrane 4 at a volume of 1.2 μL / cm using a three-dimensional film scribing instrument. The interval between the quality control line 7 and the detection line 6 was 5 mm, the scribing speed was 50 mm / s, and the membrane was dried in a 45°C oven for 12 h.

[0081] (5) Preparation of absorbent paper

[0082] Cut the absorbent paper into strips of 30*2.5cm each.

[0083] (6) Card assembly

[0084] The prepared sample pad, microsphere binding pad, nitrocellulose membrane and absorbent paper were sequentially attached to the PVC base plate and overlapped with each other by 2 mm. The test strips were cut into 4.0 mm wide strips using an automatic cutter and loaded into a card shell to make a test reagent card. The test strips were dried and stored at low temperature for later use.

[0085] See also Figure 2 The RENPs-CB[7] microspheres prepared in this example have regular shapes and good dispersibility and can be used for subsequent antibody coupling. Figure 3 The RENPs-CB[7] microspheres prepared in this example have a moderate particle size (216.6 nm) and a low dispersion index (PDI = 0.252), indicating that the particle size is relatively uniform. Figure 4 The fluorescence intensity of RENPs-CB[7] microspheres prepared in this example is close to that of RENPs-OA and better than that of RENPs-PAA, indicating that cucurbit[7]uril (CB[7]) modified RENPs can effectively avoid particle aggregation and fluorescence quenching. Figure 5 , 808nm laser continuously excited RENPs-CB[7] microspheres for 24h, and its fluorescence intensity hardly changed, indicating good photostability. Figure 6 The RENPs-CB[7] microspheres prepared in this example and ADC formed a supramolecular self-assembled complex (RENPs-CB[7]-ADC), indicating that the host-guest interaction occurred between the two. Figure 7 , the amount of bound antibody of RENPs-CB[7] microspheres prepared in this example is higher than that of RENPs-PAA, indicating that the former has a higher antibody labeling efficiency.

[0086] Application Examples

[0087] Detection of prostate cancer specific antigen (PSA) using the reagent card of the embodiment

[0088] The PSA serum quality control product and the reagent card prepared in the embodiment were restored to room temperature, and 40 μL of PSA serum quality control products with concentrations of 0, 3.0, 16.0, 45.0, 90.0 and 200.0 ng / mL were added to the reagent card sample wells, and then 40 μL of sample diluent was added, respectively, and the reaction was carried out for 15 minutes. Then, the reagent card was placed in the NIR-II fluorescence immunoassay analyzer for testing, and the fluorescence intensity of the T line and C line was recorded, and the T / C ratio was calculated. The standard curve of concentration and fluorescence ratio was established (see Figure 8 Each concentration point was tested in parallel 3 times.

[0089] 55 clinical samples were taken and tested by the reagent card of the present invention. The T / C ratio was calculated and substituted into the standard curve equation to obtain the sample concentration value and compare the correlation of the detection results of the two methods. The experimental results show that the two detection methods have good consistency (see Fig. 9 ), indicating that the test strip of the present invention has high accuracy and reliability in detecting clinical samples.

[0090] The present invention uses rare earth nano-microspheres with near-infrared second-zone fluorescence enhanced by supramolecular main compounds as fluorescent probes to label antibodies, and prepares test strips for immunochromatographic detection; the efficient surface modification strategy of the supramolecular main compounds can significantly reduce the aggregation phenomenon and partial fluorescence quenching effect caused in the surface modification process of the rare earth microspheres, and can effectively improve the fluorescence luminescence performance of the rare earth microspheres. At the same time, the supramolecular self-assembly strategy can further improve the antibody labeling efficiency of the rare earth microspheres, so that the analytical sensitivity of the prepared test strip is significantly improved. At the same time, it also has the advantages of simple operation, low cost, short detection time, etc., and is more suitable for rapid and sensitive detection of low-concentration markers in complex biological samples.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an immunochromatographic test strip for enhancing near-infrared second-region fluorescence by supramolecular host-guest interaction, characterized in that: The following steps are involved: (1) Preparing a rare earth nanoparticle (RENPs) labeling solution, comprising the steps of: S1, supramolecular host compound modified rare earth nanoparticles; S2, 1-adamantane carboxylate sodium (ADCNa) pre-activation; S3, forming a rare earth nanosphere labeling solution by combining RENPs modified with the supramolecular main compound prepared in S1, the activated ADCNa prepared in S2 and the antibody; (2) preparing a microsphere binding pad; (3) preparing a sample pad; (4) Nitrocellulose membrane coated with antibodies; (5) Assembling the card.

2. The method for preparing the immunochromatographic test strip for enhancing near-infrared second-region fluorescence by supramolecular host-guest interaction according to claim 1, characterized in that: The steps for S3 are as follows: The self-assembled complex was prepared by self-assembly of RENPs modified with supramolecular host compounds and activated ADCNa; The self-assembled complex is coupled with the antibody to form a rare earth nanosphere labeling solution.

3. The method for preparing the immunochromatographic test strip for enhancing near-infrared second-region fluorescence by supramolecular host-guest interaction according to claim 1, characterized in that: The steps for S3 are as follows: ADCNa is coupled to an antibody to form an antibody complex (ADC-Ab); The RENPs modified by the supramolecular host compound and the antibody complex self-assemble to form a rare earth nanosphere labeling liquid.

4. The method for preparing the immunochromatographic test strip for enhancing near-infrared second-region fluorescence by supramolecular host-guest interaction according to claim 2, characterized in that: In step (1), the specific preparation steps of the rare earth nanosphere labeling liquid are as follows: The RENPs modified with oleic acid ligand (RENPs-OA) were dissolved in chloroform, and the supramolecular host compound aqueous solution was added, stirred at room temperature, centrifuged and washed, and finally resuspended with pure water by ultrasonication to obtain the RENPs solution modified with the supramolecular host compound; 1-adamantanecarboxylic acid (ADC) and NaOH were dissolved in ethanol, stirred at room temperature for reaction, and then vacuum dried to obtain 1-adamantanecarboxylic acid sodium (ADCNa). A certain amount of ADCNa was dissolved in MES buffer, and EDC solution and Sulfo-NHS solution were added to perform activation reaction at room temperature in the dark to obtain an activation solution; Adding the RENPs solution modified by the supramolecular host compound to the activation solution, stirring at room temperature for self-assembly reaction, centrifuging, discarding the supernatant to obtain a white precipitate, washing the white precipitate with BBS, and then adding BBS for ultrasonic re-suspending to obtain a self-assembly complex solution; The self-assembled complex solution was added to an antibody solution of a certain concentration, and the reaction was carried out under shaking at room temperature for 2-3 hours. A blocking solution was added, and the solution was washed with a microsphere washing solution after centrifugation. The microsphere protection solution was added and ultrasonically resuspended to obtain a rare earth nano-microsphere labeling solution, which was stored at 4° C. for future use.

5. The method for preparing the immunochromatographic test strip for enhancing near-infrared second-region fluorescence by supramolecular host-guest interaction according to claim 3, characterized in that: In step (1), the specific preparation steps of the rare earth nanosphere labeling liquid are as follows: The RENPs modified with oleic acid ligands were dissolved in chloroform, and the supramolecular host compound aqueous solution was added, stirred at room temperature, centrifuged and washed with pure water, and finally resuspended with pure water by ultrasonication to obtain the RENPs solution modified with the supramolecular host compound; 1-adamantanecarboxylic acid (ADC) and NaOH were dissolved in ethanol, stirred at room temperature for reaction, and then vacuum dried to obtain 1-adamantanecarboxylic acid sodium (ADCNa). A certain amount of ADCNa was dissolved in MES buffer, and EDC solution and Sulfo-NHS solution were added to perform activation reaction at room temperature in the dark to obtain an activation solution; The activation solution and antibody were dissolved in BBS buffer, EDC solution and Sulfo-NHS solution were added, the reaction was stirred at 4°C, the antibody complex (ADC-Ab) was concentrated and separated by centrifugation, and the concentration of ADC-Ab was determined by UV-visible spectrophotometry; The RENPs solution modified by the supramolecular host compound and the ADC-Ab solution were mixed, stirred at 4°C for reaction, a blocking solution was added, the mixture was centrifuged and washed with a microsphere washing solution, and a microsphere protection solution was added for ultrasonic re-suspending to obtain a rare earth nano-microsphere labeling solution, which was stored at 4°C for later use.

6. The method for preparing the immunochromatographic test strip for enhancing near-infrared second-region fluorescence by supramolecular host-guest interaction according to claim 4 or 5, characterized in that: The molar ratios of ADC to EDC and EDC to Sulfo-NHS are both between 1-10.

7. The method for preparing the immunochromatographic test strip for enhancing near-infrared second-region fluorescence by supramolecular host-guest interaction according to claim 4 or 5, characterized in that: The antibody addition amount is 50-200 μg, the blocking solution is ethanolamine solution or BSA solution, the microsphere washing solution is Tris buffer containing BSA and Tween-20, and the microsphere protection solution is Tris buffer containing BSA, trehalose and Tween-20.

8. An immunochromatographic test strip for enhancing near-infrared second-zone fluorescence by supramolecular host-guest interaction, characterized in that: It is prepared by the preparation method described in any one of claims 1 to 5.

9. The immunochromatographic test strip for enhancing near-infrared second-region fluorescence by supramolecular host-guest interaction according to claim 8, characterized in that: The immunochromatographic test strip uses rare earth nano-microspheres with supramolecular main compound-enhanced near-infrared second-zone fluorescence as fluorescent probes to mark antibodies, and the particle size of the rare earth nano-microspheres is 100-500nm.

10. The immunochromatographic test strip for enhancing near-infrared second-region fluorescence by supramolecular host-guest interaction according to claim 9, characterized in that: The supramolecular main compound is any one or more of cyclodextrin (CD), cucurbituril (CB), crown ether (CE), pillar aromatics (PA) and calix aromatics (CA).

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