An immunochromatographic test strip for detecting antibodies against the new coronavirus

Abstract

The present invention discloses an immunochromatographic test strip for detecting antibodies against novel coronavirus, and belongs to the technical field of biological diagnostic products. The immunochromatographic test strip consists of a sample pad, a coupling pad, a chromatographic membrane and an absorption pad, wherein SiO2 coupled to SARS-CoV-2S protein is dripped onto the coupling pad. 2 @Au@CDs, the T line and C line of the chromatographic membrane are coated with rabbit anti-mouse antibody and SARS-CoV-2 IgG mouse antibody respectively. The immunochromatographic test strip provided by the present invention: (1) can detect novel coronavirus antibodies within 15 minutes, with a relatively fast detection speed; (2) the detection limit of novel coronavirus antibodies is as low as 0.1 pg / mL, with extremely high detection sensitivity; (3) has colorimetric and fluorescent dual functions; (4) is environmentally friendly, low-cost, and has a simple preparation process.

Description

Technical Field

[0001] The present invention relates to an immunochromatographic test strip, and in particular to an immunochromatographic test strip for detecting antibodies against a novel coronavirus, and belongs to the technical field of biological diagnostic products. Background Art

[0002] The new coronavirus disease is caused by the new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which has spread through the community worldwide and has become an epidemic with a very high mortality rate. Vaccination with SARS-CoV-2 can trigger an immune response, thereby producing antibodies to the new coronavirus. A test strip with both qualitative and quantitative functions for antibodies to the new coronavirus has been developed, which can monitor the level of antibodies to the new coronavirus in the population and provide technical and theoretical support for controlling the time interval between vaccinations.

[0003] Fluorescence resonance energy transfer means that if the distance between the noble metal particles and the fluorescent substance is appropriate, the energy of the noble metal particles can be transferred to the fluorescent substance, so that the fluorescence signal of the fluorescent substance is amplified. Commonly used noble metal particles are mainly silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs). Due to the poor stability of AgNPs, it is usually necessary to add a protective agent or coat them with gold. AuNPs have strong plasma resonance coupling activity and are commonly used materials for synthesizing controllable nanostructures. They have simple synthesis methods and excellent stability. However, conventional colloidal gold test strips have poor sensitivity and are difficult to use for the detection of low-concentration samples. Summary of the invention

[0004] In order to address the deficiencies of the prior art, the object of the present invention is to provide an immunochromatographic test strip with extremely high detection sensitivity for novel coronavirus antibodies (detection limit as low as 0.1 pg / mL).

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] An immunochromatographic test strip for detecting antibodies against the novel coronavirus, comprising a sample pad, a coupling pad, a chromatographic membrane and an absorption pad, characterized in that:

[0007] The coupling pad is dripped with SiO2 coupled with SARS-CoV-2S protein. 2 @Au@CDs;

[0008] The T line and C line of the chromatographic membrane are coated with rabbit anti-mouse antibody and SARS-CoV-2 IgG mouse antibody respectively.

[0009] Preferably, the coupling pad is prepared by the following method:

[0010] Soak the glass cellulose membrane in the coupling pad treatment solution at 0-4°C overnight, dry it and set it aside;

[0011] The SiO2 coupled with SARS-CoV-2 S protein 2 @Au@CDs were evenly dropped onto the glass cellulose membrane treated with the coupling pad treatment solution, and the coupling pad was obtained after vacuum freeze-drying.

[0012] Preferably, the coupling pad treatment solution is prepared by the following method:

[0013] First, dissolve 0.001-0.01 g of polyvinyl alcohol, 0.001-0.01 g of Tween-20, 0.01-0.1 g of bovine serum albumin and 0.1-1.0 g of sucrose in a small amount of PBS buffer at pH 7-9, and then make up to 5 mL with PBS buffer.

[0014] Preferably, the SiO coupled to the SARS-CoV-2S protein 2 @Au@CDs was prepared by the following method:

[0015] (1) SiO 2 The nanoparticles were mixed with PEI solution, incubated with ultrasound, and then washed twice with deionized water by centrifugation to prepare SiO 2 @PEI, SiO 2 @PEI was redispersed in deionized water to obtain SiO 2 @PEI dispersion;

[0016] (2) To SiO 2 AuNPs were added to the PEI dispersion and ultrasonically mixed to prepare SiO 2 @Au, SiO collected by centrifugation 2 @Au, and disperse it in deionized water to obtain SiO 2 @Au dispersion;

[0017] (3) SiO 2 @Au dispersion was mixed with PEI aqueous solution and ultrasonicated to make PEI cover the SiO 2 The surface of SiO@Au was then washed twice by centrifugation with deionized water to prepare 2 @Au@PEI;

[0018] (4) To SiO 2 CDs were added to @Au@PEI and ultrasonically mixed to form SiO 2 @Au@CDs, and then centrifuged, and the precipitate was stored in ethanol to obtain the fluorescent composite material SiO 2 @Au@CDs;

[0019] (5) SiO2 stored in ethanol 2 @Au@CDs were centrifuged, resuspended in MES buffer, and then mixed with EDC and NHS and sonicated;

[0020] (6) centrifuging the solution, resuspending the precipitate in PBS buffer, then adding SARS-CoV-2S protein to the resuspension and incubating on a shaker at room temperature;

[0021] (7) Add BSA to the incubation solution to 2 The unreacted carboxyl groups on the surface of @Au@CDs were blocked to prepare SiO2 coupled to SARS-CoV-2 S protein. 2 @Au@CDs.

[0022] Preferably, the preparation of SiO 2 @SiO used in PEI 2 The nanoparticles have a core-shell structure and a particle size of 50 to 500 nm.

[0023] Preferably, the preparation of SiO 2 @AuNPs The AuNPs used are prepared by a reduction method based on sodium borohydride and sodium citrate, and the particle size of the AuNPs is 1 to 20 nm.

[0024] Preferably, the concentration of the rabbit anti-mouse antibody used for coating the T line of the chromatographic membrane is 0.5-2.1 mg / mL, and the concentration of the SARS-CoV-2 IgG mouse antibody used for coating the C line of the chromatographic membrane is 5-10 mg / mL.

[0025] Preferably, the sample pad is prepared by the following method:

[0026] Dissolve 0.01-0.1 g polyvinyl pyrrolidone, 0.01-0.1 g TritonX-100, 0.01-0.1 g bovine serum albumin and 0.1-1.0 g sucrose in a small amount of PBS buffer at pH 7-9, and then dilute to 5 mL with PBS buffer to prepare a sample pad treatment solution;

[0027] The polyester film is immersed in the sample pad treatment solution at 0-4°C overnight, and then dried to obtain the sample pad.

[0028] Preferably, pure cotton linter pulp filter paper without any treatment is used as the absorption pad.

[0029] The present invention is beneficial in that:

[0030] (1) The immunochromatographic test strip provided by the present invention can detect novel coronavirus antibodies within 15 minutes, and has a relatively fast detection speed;

[0031] (2) The immunochromatographic test strip provided by the present invention is based on the fluorescence resonance energy transfer between carbon quantum dots (CDs) and AuNPs, which effectively solves the problem of low sensitivity of traditional colloidal gold test strips. The detection limit of novel coronavirus antibodies is as low as 0.1 pg / mL, which has extremely high detection sensitivity.

[0032] (3) The immunochromatographic test strip provided by the present invention has dual functions of colorimetry and fluorescence. It can not only perform qualitative detection of novel coronavirus antibodies by colorimetry based on AuNPs, but also detect the fluorescence intensity on the T line of the test strip by a fluorescence detector, and determine the concentration of novel coronavirus antibodies in serum, thereby achieving quantitative detection of novel coronavirus antibodies in serum;

[0033] (4) The immunochromatographic test strip provided by the present invention does not contain heavy metal elements and is environmentally friendly;

[0034] (5) The immunochromatographic test strip provided by the present invention requires readily available raw materials, is low-cost, and does not require the use of large and expensive testing instruments;

[0035] (6) The immunochromatographic test strip provided by the present invention has a simple preparation process and only requires basic experimental steps such as ultrasound and centrifugation. It does not require complicated experimental operations and has low operating difficulty. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Is in SiO 2 Flow chart of the assembly of AuNPs and CDs on nanoparticle surfaces;

[0037] Figure 2 SiO 2 Flowchart of coupling of @Au@CDs with SARS-CoV-2S protein;

[0038] Figure 3 SiO 2 @Au@CDs test strips for detecting antibodies against the novel coronavirus;

[0039] Figure 4 SiO 2 @TEM image of AuNPs;

[0040] Figure 5 SiO 2 TEM images of @Au@CDs;

[0041] Figure 6 SiO 2 Comparison of the colorimetric detection capabilities of @Au@CDs test strips and traditional colloidal gold test strips;

[0042] Figure 7 SiO2 @Au@CDs test strip specificity test result diagram;

[0043] Figure 8 SiO 2 、SiO 2 @Au and SiO 2 Particle size distribution test results of @Au@CDs;

[0044] Fig. 9 SiO 2 、SiO 2 @PEI、SiO 2 @Au、SiO 2 @Au@PEI and SiO 2 Zeta potential test results of @Au@CDs;

[0045] Fig.10 The SiO 2 @Au@CDs test strip quantitative linear standard curve of novel coronavirus antibodies;

[0046] Fig.11 This is a graph of the fluorescence intensity detection results of different positive samples (P1, P2, P3, P4, P5) and different negative samples (N1, N2, N3, N4, N5). DETAILED DESCRIPTION

[0047] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0048] 1. Preparation based on SiO 2 @Au@CDs immunochromatographic test strips for detecting antibodies against novel coronavirus

[0049] 1. Preparation of AuNPs

[0050] AuNPs were prepared by a reduction method based on sodium borohydride and sodium citrate. The preparation process is as follows:

[0051] (1) Add 0.01 g of trisodium citrate and 0.15 g of tetrachloroauric acid to 98 mL of deionized water and stir vigorously to obtain a mixture;

[0052] (2) 1.5 mL of freshly prepared sodium borohydride solution with a concentration of 0.2 mol / L was quickly added to the above mixture and stirred continuously for 4 h to prepare AuNPs.

[0053] 2. Preparation of fluorescent composite material - SiO 2 @Au@CDs

[0054] According to the electrostatic adsorption strategy mediated by polyethyleneimine (PEI), the SiO 2 AuNPs and CDs were assembled on the nanoparticle surface.

[0055] Reference Figure 1 , in SiO 2 The process of assembling AuNPs and CDs on the surface of nanoparticles is as follows:

[0056] (1) SiO 2 Nanoparticles (0.25 mL, 1 mmol / L) were mixed with PEI solution (20 mL, 0.5 mg / mL) and incubated with ultrasound for 30 min. During this process, the positively charged PEI was coated on the SiO 2 After the incubation, the surface of the nanoparticles was washed twice with deionized water by centrifugation (6000 rpm, 6 min) to remove the unreacted PEI and prepare SiO 2 @PEI, the prepared SiO 2 @PEI was redispersed in deionized water (5 mL) to obtain SiO 2 @PEI dispersion;

[0057] (2) To SiO 2 4 nm AuNPs (25 mL) were added to the PEI dispersion (5 mL) and ultrasonically mixed for 30 min to prepare SiO 2 @AuNPs (also known as SiO 2 @Au), and then centrifuged (6000rpm, 6min) to collect SiO 2 @Au, and dispersed it in deionized water (2.5 mL) to obtain SiO 2 @Au dispersion;

[0058] (3) SiO 2 The Au dispersion (2.5 mL) was mixed with the PEI aqueous solution (25 mL, 0.5 mg / mL) and ultrasonicated for 60 min to cover the SiO 2 The surface of @Au was then washed twice with deionized water by centrifugation (6000 rpm, 6 min) to prepare SiO 2 @Au@PEI;

[0059] (4) To SiO 2 CDs (10 mL, 1 nmol / L) were added to @Au@PEI and ultrasonically mixed for 30 min to form SiO 2 @Au@CDs, and then centrifuged (6000rpm, 6min), and the precipitate was stored in ethanol (5mL) to obtain the fluorescent composite material SiO 2@Au@CDs.

[0060] The core of the fluorescent composite material is a core-shell structure SiO 2 Nanoparticles, core-shell structure SiO 2 Nanoparticles have a large specific surface area and can provide higher fluorescence intensity. In addition, their surface can be easily modified with active groups, and antigens can be labeled using chemical coupling methods to stably bind to new coronavirus antibodies in serum and plasma.

[0061] Polyethyleneimine can control the distance between AuNPs and CDs, effectively preventing CDs from being quenched, thus obtaining a stable and strong fluorescence signal. Studies have found that the optimal distance between AuNPs and CDs is 10-20nm. Compared with the distance of 5nm or 8nm, no obvious fluorescence quenching is observed, and the best fluorescence enhancement effect is obtained.

[0062] The SiO obtained in step (2) 2 @Au was characterized by transmission electron microscopy, and the TEM image obtained is shown in Figure 4 ,Depend on Figure 4 It can be seen that SiO 2 The particle size of the nanoparticles is about 200nm, and uniformly sized AuNPs are successfully assembled on their surface, with the particle size of the AuNPs ranging from 1 to 20nm.

[0063] The SiO obtained in step (3) 2 @Au@CDs were characterized by transmission electron microscopy, and the TEM images are shown in Figure 5 ,Depend on Figure 5 It can be seen that SiO 2 CDs were successfully assembled on the surface of @Au. The distance between AuNPs and CDs was 10-20nm. 2 The particle size of @Au@CDs is about 275nm.

[0064] 3. SiO 2 @Au@CDs coupled to SARS-CoV-2 S protein

[0065] SARS-CoV-2 S protein was reacted with SiO2 in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS). 2 @Au@CDs for coupling, refer to Figure 2 , the coupling process is as follows:

[0066] (1) SiO2 stored in ethanol 2@Au@CDs (1 mL) were centrifuged and then resuspended in MES buffer (1 mL, 0.1 mol / L, pH = 5.5), then mixed with EDC (10 μL, 0.1 mol / L) and NHS (20 μL, 0.1 mol / L) and sonicated for 15 min;

[0067] (2) The solution was centrifuged (6000 rpm, 6 min) to remove excess EDC and NHS, the precipitate was collected, and the precipitate was resuspended in PBS buffer (200 μL, 10 mM, pH = 7.4), and then SARS-CoV-2S protein (10 μg) was added to the resuspension and incubated on a shaker at room temperature for 2 h;

[0068] (3) Add 3% BSA (250 μL) to the incubation solution to 2 The unreacted carboxyl groups on the surface of @Au@CDs were blocked for 1 h to obtain SiO2@@Au@CDs coupled to SARS-CoV-2 S protein. 2 @Au@CDs, that is, S protein-coupled marker.

[0069] Per 1mL SiO 2 The amount of @Au@CDs that can be coupled to SARS-CoV-2S protein is 10μg.

[0070] 4. Prepare the sample pad

[0071] Prepare the sample pad treatment solution: first dissolve 0.01-0.1g polyvinyl pyrrolidone, 0.01-0.1g TritonX-100, 0.01-0.1g bovine serum albumin and 0.1-1.0g sucrose in a small amount of PBS buffer with a pH of 7-9, and then dilute to 5mL with PBS buffer to prepare the sample pad treatment solution.

[0072] In this specific embodiment, the sample pad treatment solution used has the following formula: 0.05 g polyvinyl pyrrolidone, 0.05 g Triton X-100, 0.05 g bovine serum albumin and 0.2 g sucrose, and the volume is fixed to 5 mL with PBS buffer (pH=7-9).

[0073] The polyester film was soaked in the sample pad treatment solution, placed in a refrigerator (0-4°C) overnight, and dried to obtain the sample pad.

[0074] 5. Preparation of coupling pad

[0075] Prepare the coupling pad treatment solution: first dissolve 0.001-0.01g polyvinyl alcohol, 0.001-0.01g Tween-20, 0.01-0.1g bovine serum albumin and 0.1-1.0g sucrose in a small amount of PBS buffer with pH=7-9, and then make up to 5mL with PBS buffer.

[0076] In this specific embodiment, the coupling pad treatment solution used has the following formula: 0.005 g polyvinyl alcohol, 0.005 g Tween-20, 0.05 g bovine serum albumin and 0.2 g sucrose, and the volume is fixed to 5 mL with PBS buffer (pH=7-9).

[0077] The glass cellulose membrane was soaked in the coupling pad treatment solution, placed in a refrigerator (0-4°C) overnight, and dried for later use.

[0078] The prepared SiO2 coupled with SARS-CoV-2S protein 2 @Au@CDs were evenly dropped onto the glass cellulose membrane treated with the coupling pad treatment solution, and the coupling pad was obtained after vacuum freeze-drying.

[0079] 6. Preparation of Chromatography Membrane

[0080] A rabbit anti-mouse antibody with a concentration of 0.5 to 2.1 mg / mL is coated on a nitrocellulose membrane (NC membrane) through a capillary to form a T line of the chromatographic membrane, and a SARS-CoV-2 IgG mouse antibody (new coronavirus antibody) with a concentration of 5 to 10 mg / mL is coated on the NC membrane through a capillary to form a C line of the chromatographic membrane. The interval between the C line and the T line is 4 to 6 mm. The NC membrane coated with the rabbit anti-mouse antibody and the SARS-CoV-2 IgG mouse antibody is dried to obtain a chromatographic membrane.

[0081] In this specific embodiment, the concentration of the rabbit anti-mouse antibody used to coat the T line is 1.05 mg / mL, the concentration of the SARS-CoV-2 IgG mouse antibody used to coat the C line is 7.4 mg / mL, and the interval between the C line and the T line is 5 mm.

[0082] 7. Prepare the absorbent pad

[0083] Use pure cotton lint filter paper without any treatment as the absorption pad.

[0084] 8. Assembly

[0085] Lay the chromatographic membrane (NC membrane coated with T line and C line) flat on a clean PVC plate (bottom plate), lay the coupling pad flat on the T line end of the chromatographic membrane, and lay the absorption pad flat on the C line end of the chromatographic membrane. The overlapping width of the coupling pad and the chromatographic membrane and the overlapping width of the absorption pad and the chromatographic membrane are both 2 mm. Lay the sample pad flat on the coupling pad. The overlapping width of the sample pad and the coupling pad is 2 mm. Place the pasted test paper between the two flat plates and press it tightly for 15 minutes. Then cut the test paper into strips with a width of 3 to 10 mm. Finally, seal and dry them to obtain immunochromatographic test strips for detecting antibodies to the new coronavirus and store them for future use.

[0086] The prepared immunochromatographic test strip is recorded as SiO 2 @Au@CDs test strips.

[0087] The SiO 2 @Au@CDs test strips for the principle of detecting antibodies against the new coronavirus Figure 3 .

[0088] As a zero-dimensional carbon-based material, CDs not only have good optical properties and photochemical stability, but also have many advantages such as good water solubility, low toxicity, environmental friendliness, low cost, good biocompatibility, etc. The present invention uses AuNPs and CDs as donors and acceptors in the fluorescence resonance energy transfer system to develop a new type of fluorescent composite material SiO 2 @Au@CDs, and combined with immunochromatography technology to 2 @Au@CDs was used as a marker to couple the SARS-CoV-2 S protein, and a colorimetric and fluorescent dual-function immunochromatographic test strip was developed. CDs were excited by ultraviolet light with a wavelength of 365nm, and 605nm fluorescence was emitted as the detection signal. Under the excitation of ultraviolet light with a wavelength of 365nm, AuNPs can effectively enhance the fluorescence intensity of CDs through fluorescence resonance energy transfer. By detecting the fluorescence intensity on the T line of the test strip through a fluorescence detector, the concentration of novel coronavirus antibodies in serum and plasma can be accurately and sensitively detected.

[0089] 2. Preparation based on SiO 2 @CDs immunochromatographic test strips for detecting antibodies to the novel coronavirus

[0090] Based on SiO 2 The preparation process of @CDs' immunochromatographic test strips for detecting novel coronavirus antibodies is as follows:

[0091] (1) SiO 2 Nanoparticles (0.25 mL, 1 mmol / L) were mixed with PEI solution (20 mL, 0.5 mg / mL) and incubated with ultrasound for 30 min. During this process, the positively charged PEI was coated on the SiO2 After the incubation, the surface of the nanoparticles was washed twice with deionized water by centrifugation (6000 rpm, 6 min) to remove the unreacted PEI and prepare SiO 2 @PEI, the prepared SiO 2 @PEI was redispersed in deionized water (5 mL) to obtain SiO 2 @PEI dispersion;

[0092] (2) To SiO 2 CDs (20 mL, 1 nmol / L) were added to the PEI dispersion (5 mL) and ultrasonically mixed for 30 min to form SiO 2 @CDs, and then centrifuged (6000rpm, 6min) to collect SiO 2 @CDs and stored it in ethanol (5 mL) to obtain SiO 2 @CDs dispersion;

[0093] (3) SiO2 stored in ethanol 2 @CDs (1 mL) were centrifuged and then resuspended in MES buffer (1 mL, 0.1 mol / L, pH = 5.5), then mixed with EDC (10 μL, 0.1 mol / L) and NHS (20 μL, 0.1 mol / L) and sonicated for 15 min;

[0094] (4) The solution was centrifuged (6000 rpm, 6 min) to remove excess EDC and NHS, the precipitate was collected, and the precipitate was resuspended in PBS buffer (200 μL, 10 mM, pH = 7.4), and then SARS-CoV-2S protein (10 μg) was added to the resuspension and incubated on a shaker at room temperature for 2 h;

[0095] (5) Add 3% BSA (250 μL) to the incubation solution to 2 The unreacted carboxyl groups on the surface of @CDs were blocked for 2 h to obtain SiO2 coupled to SARS-CoV-2 S protein. 2 @CDs;

[0096] (6) Use and preparation of SiO 2 @Au@CDs test strips are similar to the method used to couple SiO2 with SARS-CoV-2 S protein. 2 @CDs is an S protein-coupled marker for preparing immunochromatographic test strips.

[0097] The prepared immunochromatographic test strip is recorded as SiO 2 @CDs test strips.

[0098] 3. Comparison of SiO2 @Au@CDs test strips and SiO 2 @CDs test strips to detect the results of the new coronavirus antibodies

[0099] With SiO 2 @Au@CDs test strips and SiO 2 @CDs test strips detect antibodies to the new coronavirus. Each serum sample is tested three times to collect fluorescence data. The specific detection process is as follows:

[0100] (1) Add the sample (50 μL) to the SiO 2 @Au@CDs test strips and SiO 2 On the sample pad of the @CDs test strip, when the sample migrates from the sample pad to the absorption pad, the T line and C line continuously capture the S protein-coupled marker;

[0101] (2) After 15 min of reaction, the fluorescence intensity of the T line was measured using a fluorescence detector (excitation at 365 nm).

[0102] SiO 2 @Au@CDs test strips and SiO 2 @CDs test strips test results for novel coronavirus antibodies are shown in the table below:

[0103] Table 1 Detection results of novel coronavirus antibodies

[0104]

[0105] From Table 1, we can see that: 2 @Au@CDs test strips for detecting novel coronavirus antibodies and using SiO 2 @CDs test strips detect antibodies to the new coronavirus, and the two methods have a good correlation.

[0106] 4. Comparison of SiO 2 Colorimetric detection capabilities of @Au@CDs test strips and traditional colloidal gold test strips

[0107] The preparation process of traditional colloidal gold test strips is as follows:

[0108] (1) Use K at a concentration of 0.2 mol / L 2 CO 3 The solution was adjusted to pH 8-9 with 10 μg of SARS-CoV-2S protein, and then the SARS-CoV-2S protein was incubated with 1 mL of AuNPs for 15 min to obtain an incubation solution;

[0109] (2) adding 50 μL of 10% (W / V) bovine serum albumin solution to the incubation solution to block the unreacted sites of AuNPs for 1 h to obtain S protein-AuNPs;

[0110] (3) The prepared S protein-AuNPs were centrifuged, the precipitate was collected, and then resuspended with 200 μL of storage solution to obtain a resuspension, wherein the storage solution was prepared by mixing BSA, PVP, sucrose, Tween-20 and 10 mM PBS solution, and each 5 mL of PBS solution contained 0.01 g BSA, 0.01 g PVP, 1 g sucrose and 0.005 g Tween-20;

[0111] (4) dropping the resuspended solution onto the gold pad and drying it, leaving it to stand for 1 h, drying it at room temperature, and storing it at 4° C. to prepare a coupling pad;

[0112] (5) Assemble the coupling pad on the test paper and cut the test paper into strips with a width of 2 to 10 mm for subsequent use.

[0113] Compare SiO 2 The colorimetric detection capabilities of @Au@CDs test strips and traditional colloidal gold test strips are compared as follows:

[0114] (1) Add the sample (50 μL) to the SiO 2 On the sample pads of @Au@CDs test strips and traditional colloidal gold test strips, when the sample migrates from the sample pad to the absorption pad, the T line and C line continuously capture the S protein-coupled marker;

[0115] (2) After 15 minutes of reaction, observe SiO 2 Colorimetric signals on @Au@CDs test strips and traditional colloidal gold test strips.

[0116] The comparison results are shown in Figure 6 .

[0117] Depend on Figure 6 It can be seen that SiO 2 @Au@CDs test strips have the same colorimetric detection capabilities as traditional colloidal gold test strips.

[0118] 5. Detection of SiO 2 @Au@CDs test strip specificity

[0119] Detection of SiO 2 @Au@CDs test strips are specific. Each serum sample is tested three times to collect fluorescence data. The specific testing process is as follows:

[0120] (1) Add 50 μL of different antibody samples (N=4) of the same concentration to the SiO 2On the sample pad of the @Au@CDs test strip, when the sample migrates from the sample pad to the absorption pad, the T line and C line continuously capture the S protein-coupled marker;

[0121] (2) After 15 min of reaction, the fluorescence intensity was measured using a fluorescence detector (365 nm excitation, 605 nm emission).

[0122] Test results are shown in Figure 7 .

[0123] Depend on Figure 7 It can be seen that SiO 2 @Au@CDs test strips have almost no cross-reaction to MERS-CoV-2 antibodies, RSV antibodies, and ASF1B antibodies, and are highly specific to SARS-CoV-2 antibodies (new coronavirus antibodies).

[0124] 6. Detection and Preparation of SiO 2 @Au@CDs uses SiO 2 In the preparation of SiO 2 SiO obtained in the process of @Au@CDs 2 @AuNPs and the final SiO 2 Particle size distribution of @Au@CDs.

[0125] The particle size distribution test is helpful to detect whether the electrostatic adsorption of PEI can successfully adsorb AuNPs and CDs to SiO 2 On the surface of nanoparticles, if the particle size increases, it means that SiO 2 @AuNPs or SiO 2 @Au@CDs.

[0126] SiO 2 、SiO 2 @AuNPs、SiO 2 The particle size distribution of @Au@CDs is shown in Figure 8 .

[0127] Depend on Figure 8 It can be seen that SiO 2 The particle size of SiO is about 200nm. 2 The particle size of @AuNPs is about 240nm. 2 The particle size of @Au@CDs is about 275nm. 2 The particle size of @Au@CDs is larger than that of SiO 2 The particle size of AuNPs is 35nm larger than that of SiO 2 The particle size is as large as 75nm.

[0128] 7. Detection of SiO2 、SiO 2 @PEI、SiO 2 @AuNPs、SiO 2 @Au@PEI、SiO 2 Zeta potential (ζ-potential) of @Au@CDs

[0129] The regular changes in ζ-potential confirm that SiO 2 The self-assembly of @Au@CDs was driven by electrostatic adsorption. The zeta potential of the nanocomposite material increased significantly after PEI coating, and the zeta potential of AuNPs and CDs decreased significantly after electrostatic adsorption. These results indicate that SiO 2 @Au@CDs.

[0130] Detection and preparation of SiO 2 @Au@CDs uses SiO 2 In the preparation of SiO 2 SiO obtained in the process of @Au@CDs 2 @PEI、SiO 2 @AuNPs and SiO 2 @Au@PEI and the final SiO 2 Zeta potential of @Au@CDs.

[0131] SiO 2 、SiO 2 @PEI、SiO 2 @AuNPs、SiO 2 @Au@PEI and SiO 2 The results of the Zeta potential test of @Au@CDs are shown in Fig. 9 .

[0132] Depend on Fig. 9 It can be seen that the zeta potential of the nanocomposite material increased significantly after PEI coating, and the zeta potential of AuNPs and CDs decreased significantly after electrostatic adsorption. These results indicate that SiO 2 @Au@CDs.

[0133] 8. Drawing SiO 2 @Au@CDs test strip quantitative novel coronavirus antibody linear standard curve

[0134] The process of drawing the standard curve is as follows:

[0135] (1) Prepare a series of standard antibody solutions with different concentrations using PBS buffer;

[0136] (2) Add 50 μL of sample to the SiO 2@Au@CDs test strips were placed on the sample pad and SiO was detected by fluorescence detector after 15 min. 2 The fluorescence intensity of the T line of the @Au@CDs test strip;

[0137] (3) Using the concentration of the standard antibody solution as the horizontal axis and the fluorescence intensity of the T line as the vertical axis, the SiO 2 Standard linear curve of @Au@CDs test strips.

[0138] Draw the obtained SiO 2 @Au@CDs test strip quantitative novel coronavirus antibody linear standard curve see Fig.10 .

[0139] Depend on Fig.10 It can be seen that SiO 2 The detection limit of the @Au@CDs test strip is as low as 0.1pg / mL, which is 6 orders of magnitude lower than the antibody level in the human body (μg / mL). It can be used for low-concentration detection of new coronavirus antibodies in serum and plasma.

[0140] 9. Use SiO 2 @Au@CDs test strips for detecting novel coronavirus antibodies in actual serum samples

[0141] Sample processing: The blood samples were centrifuged at 600 rpm for 10 min to obtain serum samples. 0.74 μg of SARS-CoV-2 IgG mouse antibody was added to one part of the serum samples (used to simulate the actual positive samples) by the spike method, and SARS-CoV-2 IgG mouse antibody was not added to the other part of the serum samples (used to simulate the actual negative samples). Then, a series of dilutions were performed on the simulated positive and negative samples with PBS buffer (pH = 7-9).

[0142] Detection: Add 50 μL of sample to SiO 2 @Au@CDs test strips were placed on the sample pad and SiO was detected by fluorescence detector after 15 min. 2 The fluorescence intensity of the T line of the @Au@CDs test strip.

[0143] The SiO corresponding to each sample 2 The detection results of the fluorescence intensity of the T line of the @Au@CDs test strip are shown in Fig.11 .

[0144] Depend on Fig.11 It can be seen that the SiO 2 The @Au@CDs test strips had a detection rate of 100% for positive samples.

[0145] It should be noted that the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. An immunochromatographic test strip for detecting antibodies against the new coronavirus, comprising a sample pad, a coupling pad, a chromatographic membrane and an absorption pad, characterized in that: SiO2@Au@CDs coupled with SARS-CoV-2S protein are dripped onto the coupling pad; The T line and C line of the chromatographic membrane are coated with rabbit anti-mouse antibody and SARS-CoV-2 IgG mouse antibody respectively.

2. The immunochromatographic test strip for detecting novel coronavirus antibodies according to claim 1, characterized in that: The coupling pad is prepared by the following method: Soak the glass cellulose membrane in the coupling pad treatment solution at 0-4°C overnight, dry it and set it aside; SiO2@Au@CDs coupled with SARS-CoV-2S protein were evenly dropped onto the glass cellulose membrane treated with coupling pad treatment solution, and the coupling pad was obtained after vacuum freeze-drying.

3. The immunochromatographic test strip for detecting novel coronavirus antibodies according to claim 2, characterized in that: The coupling pad treatment solution is prepared by the following method: First, dissolve 0.001-0.01 g of polyvinyl alcohol, 0.001-0.01 g of Tween-20, 0.01-0.1 g of bovine serum albumin and 0.1-1.0 g of sucrose in a small amount of PBS buffer at pH 7-9, and then make up to 5 mL with PBS buffer.

4. The immunochromatographic test strip for detecting novel coronavirus antibodies according to claim 2, characterized in that: The SiO2@Au@CDs coupled with SARS-CoV-2S protein is prepared by the following method: (1) mixing SiO2 nanoparticles with PEI solution, incubating with ultrasound, and washing twice with deionized water by centrifugation to prepare SiO2@PEI, and redispersing SiO2@PEI in deionized water to obtain SiO2@PEI dispersion; (2) adding AuNPs to the SiO2@PEI dispersion, mixing by ultrasonication to prepare SiO2@Au, collecting SiO2@Au by centrifugation, and dispersing it in deionized water to obtain a SiO2@Au dispersion; (3) mixing the SiO2@Au dispersion with the PEI aqueous solution, ultrasonicating to cover the surface of SiO2@Au with PEI, and then washing twice with deionized water by centrifugation to prepare SiO2@Au@PEI; (4) adding CDs to SiO2@Au@PEI, ultrasonically mixing to form SiO2@Au@CDs, and then centrifuging and storing the precipitate in ethanol to obtain the fluorescent composite material SiO2@Au@CDs; (5) SiO2@Au@CDs stored in ethanol were centrifuged, resuspended in MES buffer, mixed with EDC and NHS, and sonicated; (6) centrifuging the solution, resuspending the precipitate in PBS buffer, then adding SARS-CoV-2S protein to the resuspension and incubating on a shaker at room temperature; (7) BSA was added to the incubation solution to block the unreacted carboxyl groups on the surface of SiO2@Au@CDs to obtain SiO2@Au@CDs coupled with SARS-CoV-2S protein.

5. The immunochromatographic test strip for detecting novel coronavirus antibodies according to claim 4, characterized in that: The SiO2 nanoparticles used to prepare SiO2@PEI are of core-shell structure and have a particle size of 50 to 500 nm.

6. The immunochromatographic test strip for detecting novel coronavirus antibodies according to claim 4, characterized in that: The AuNPs used to prepare SiO2@AuNPs are prepared by a reduction method based on sodium borohydride and sodium citrate, and the particle size of the AuNPs is 1 to 20 nm.

7. The immunochromatographic test strip for detecting novel coronavirus antibodies according to claim 1, characterized in that: The concentration of rabbit anti-mouse antibody used for coating the T line of the chromatographic membrane was 0.5-2.1 mg / mL.

8. The immunochromatographic test strip for detecting novel coronavirus antibodies according to claim 1, characterized in that: The concentration of SARS-CoV-2 IgG mouse antibody used to coat the C line of the chromatographic membrane is 5-10 mg / mL.

9. The immunochromatographic test strip for detecting novel coronavirus antibodies according to claim 1, characterized in that: The sample pad is prepared by the following method: Dissolve 0.01-0.1 g polyvinyl pyrrolidone, 0.01-0.1 g TritonX-100, 0.01-0.1 g bovine serum albumin and 0.1-1.0 g sucrose in a small amount of PBS buffer at pH 7-9, and then dilute to 5 mL with PBS buffer to prepare a sample pad treatment solution; The polyester film is immersed in the sample pad treatment solution at 0-4°C overnight, and then dried to obtain the sample pad.

10. The immunochromatographic test strip for detecting novel coronavirus antibodies according to claim 1, characterized in that: Use pure cotton lint filter paper without any treatment as the absorption pad.

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