A detection probe based on polydopamine nanoparticles and a detection test strip and novel coronavirus antibody detection application based on the same

Abstract

The present invention discloses a detection probe based on polydopamine nanoparticles and a detection test paper and a novel coronavirus antibody detection application based thereon. Polydopamine nanoparticles are applied to lateral flow immunochromatographic test paper, and the novel coronavirus RBD antigen is specifically combined with novel coronavirus-specific immunoglobulins (IgG, IgM) to achieve the purpose of detecting whether the sample is infected with the novel coronavirus. Since the immunochromatographic test paper has the advantages of simple operation, convenient use, low cost, rapid detection, and applicability to a variety of detection environments, it is widely used and can be used as an important detection method.

Description

Technical Field

[0001] The present invention belongs to the technical field of test strips, and specifically relates to a detection probe based on polydopamine nanoparticles and a test strip and novel coronavirus antibody detection application based thereon. Background Art

[0002] Dopamine is a biological neurotransmitter that can form polydopamine (PDA) nanoparticles through self-assembly. It has good biocompatibility and no biotoxicity. The preparation process of PDA is simple, environmentally friendly and low-cost. PDA is a black nanoparticle with a wide absorption range in the spectrum. There are a large number of active groups on its surface, which can be functionalized with a variety of substances.

[0003] Lateral flow immunochromatography assay (LFIA) is a rapid detection technology based on the binding of antigens and antibodies through immune reactions. Lateral flow immunochromatography test strips are composed of a sample pad, a binding pad, an absorbent pad, a nitrocellulose membrane, and a glass fiber membrane. Under the action of the absorbent pad, the test liquid flows through the test line and the quality control line, and the test substance bound to the labeled antibody is captured by the capture antibody of the test line. The signal intensity is read by the aggregation of the marker to achieve qualitative or quantitative detection. Immunochromatography test strips are widely used because of their simple operation and rapid detection. Since PDA has a wide wavelength absorption range in the spectrum, the color change of the signal can be read by the naked eye for rapid and sensitive detection.

[0004] At present, commercial nanogold test strips used for novel coronavirus detection still have some problems: low detection sensitivity, high false negative rate, etc. Other high-sensitivity markers used for novel coronavirus detection include fluorescent markers, nanozymes, and magnetic nanoparticles, but these markers must rely on specific detection equipment to read fluorescent signals, enzyme signals, and magnetic signals. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the object of the present invention is to provide a detection probe based on polydopamine nanoparticles and a detection test strip and new coronavirus antibody detection application based thereon.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention discloses a dopamine nanoprobe, which has a core-shell structure. The core is a polydopamine nanoparticle, and the surface of the core-shell is modified with a polymer layer, a SiO2 layer, a PEG layer and a new coronavirus antibody detection layer.

[0008] Preferably, the diameter of the polydopamine nanoparticles is 200-400 nm.

[0009] Preferably, the chemical formula of the polydopamine monomer is C8H 11 NO2.

[0010] The present invention also discloses a method for preparing the above dopamine nanoprobe, comprising: preparing polydopamine nanoparticles, and sequentially modifying a polymer layer, a silicon dioxide layer and a PEG layer on the surface of the polydopamine nanoparticles to prepare the dopamine nanoprobe.

[0011] The present invention also discloses the use of the dopamine nanoprobe in preparing products for antigen-antibody detection, wherein the products include detection test strips, detection reagents or detection kits.

[0012] The present invention also discloses the use of the above-mentioned dopamine nanoprobe in the preparation of a probe for detecting antibodies against COVID-19.

[0013] The present invention discloses a dopamine nanoprobe for detecting novel coronavirus antibodies, which is prepared by combining the novel coronavirus antibodies with the above-mentioned dopamine nanoprobe.

[0014] Furthermore, the novel coronavirus antibodies select IgG and lgM.

[0015] The present invention also discloses the use of the above-mentioned dopamine nanoprobe for detecting new coronavirus antibodies in the preparation of products for detecting new coronavirus antibodies, and the products include detection test strips, detection reagents or detection kits.

[0016] The present invention discloses a polydopamine nanoparticle test paper for detecting novel coronavirus-specific immunoglobulin, which is prepared by combining the above-mentioned dopamine nanoparticle probe for detecting novel coronavirus antibodies with a binding pad of a lateral flow immunochromatography test paper.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The dopamine nanoprobe for detecting new coronavirus antibodies disclosed in the present invention uses polydopamine nanoparticles instead of gold nanoparticles as a colorimetric label. Dopamine is a biological neurotransmitter that can form polydopamine nanoparticles (PDA) through self-assembly. Therefore, polydopamine nanoparticles have good biocompatibility and are non-biotoxic. The preparation process of polydopamine nanoparticles is simple and the preparation cost is low. Polydopamine nanoparticles are black nanoparticles with a wide absorption range in the spectrum. There are a large number of active groups on the surface of polypamine, which can be functionalized with a variety of substances. After connecting with different detection probes, they can be used for detection of various purposes.

[0019] Dopamine nanoprobes are further applied to lateral flow immunochromatography test strips. Compared with the most widely used commercial nanogold test strips, polydopamine nanoparticle test strips have the advantages of high biosafety and environmental friendliness. Aggregation on the test strip test line can form a more obvious color reaction. Compared with the red detection line presented by the nanogold test strip, the polydopamine nanoparticle test strip is more conducive to naked eye observation and significantly improves the sensitivity of new coronavirus detection. Therefore, the present invention can be applied to food safety, environmental monitoring, and detection of various diseases, providing a powerful detection method.

[0020] Furthermore, the present invention is a polydopamine nanoparticle test paper for detecting novel coronavirus-specific immunoglobulins (IgG, lgM). The method applies polydopamine nanoparticles to lateral flow immunochromatographic test paper, and uses the specific binding properties of the novel coronavirus RBD antigen and the novel coronavirus-specific immunoglobulin (IgG, lgM) to achieve the purpose of detecting whether the sample is infected with the novel coronavirus. Immunochromatographic test paper is widely used because it has the advantages of simple operation, easy use, low cost, rapid detection, and applicability to a variety of detection environments. The commercial nanogold test paper that is currently most widely used for novel coronavirus detection still has some problems: low detection sensitivity, high false negative rate, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a photo of the result of the lateral flow test strip of Example 1;

[0022] Figure 2 This is a photo of the results of the lateral flow test strip of Example 2;

[0023] Figure 3 This is a photo of the result of the lateral flow test strip of Example 3;

[0024] Figure 4a This is a photo of the PDA@PEG probe test results;

[0025] Figure 4b This is a photo of the PDA@SiO2@PEG probe test results. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0027] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0028] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0029] Composition of dopamine nanoprobe: The nanoprobe has a core-shell structure, the core of which is polydopamine nanoparticles with a diameter of about 200-400 nanometers. Its monomer chemical formula is C8H 11 NO2. The surface is modified with a polymer layer, a SiO2 layer, a PEG layer and a detection antibody layer.

[0030] Test strip composition: The structure includes a sample pad, a conjugate pad, an NC membrane, a water absorbent pad and a base plate.

[0031] The specific preparation process is as follows:

[0032] 1. Preparation of PDA@PSS@PAH@PVP@SiO2@PEG

[0033] (1) Synthesis of polydopamine (PDA)

[0034] Place 22.5 ml of ultrapure water in a 100 ml round-bottom flask and heat in a water bath while stirring. Add 10 ml of anhydrous ethanol and 0.75 ml of ammonia water and stir for a while. Quickly add dopamine solution and stir overnight. Then drop acetone into the reaction solution and let it stand for sedimentation. Centrifuge to remove the supernatant and resuspend in ultrapure water to obtain PDA solution.

[0035] (2) Surface modified polyelectrolytes

[0036] 1. Add 5 ml of PDA solution dropwise to 5 mL of 2 g / L poly(styrene sulfonic acid) sodium salt (PSS) aqueous solution containing 6 mM sodium chloride under stirring for 3 hours to obtain a PDA@PSS solution.

[0037] 2. The PDA@PSS solution was centrifuged twice to remove excess electrolytes, and the precipitate was resuspended in 5 mL of ultrapure water to obtain a PDA@PSS aqueous solution.

[0038] 3. Add 5 ml of PDA@PSS aqueous solution dropwise to 5 mL of 2 g / L poly(propyleneamine hydrochloride (PAH) aqueous solution containing 6 mM sodium chloride under stirring for 3 hours to obtain a PDA@PSS@PAH solution.

[0039] 4. The PDA@PSS@PAH solution was centrifuged twice to remove excess electrolytes, and the precipitate was resuspended in 5 mL of ultrapure water to obtain a PDA@PSS@PAH aqueous solution.

[0040] 5. Add 5 ml of PDA@PSS@PAH aqueous solution dropwise into 5 mL of 4 g / L polyvinylpyrrolidone (PVP) aqueous solution containing 6 mM sodium chloride under stirring and stir for 3 hours to obtain a PDA@PSS@PAH@PVP solution.

[0041] 6. The PDA@PSS@PAH@PVP solution was centrifuged to remove excess electrolytes, and the precipitate was resuspended in 0.2 mL of ultrapure water to obtain a PDA@PSS@PAH@PVP aqueous solution.

[0042] 7. Add 1 mL of isopropanol to the PDA@PSS@PAH@PVP aqueous solution.

[0043] (3) Surface modified silicon layer

[0044] 1. Add 1.2 mL of PDA@PSS@PAH@PVP isopropanol solution into water under stirring.

[0045] 2. Add 1.43 mL of ammonia-isopropanol solution to the PDA@PSS@PAH@PVP isopropanol solution, and then add 0.4 mL of TEOS-isopropanol solution and stir for two hours to obtain the PDA@PSS@PAH@PVP@SiO2 solution.

[0046] 3. Centrifuge the PDA@PSS@PAH@PVP@SiO2 solution three times to remove excess impurities, and then resuspend it in methanol to obtain a PDA@PSS@PAH@PVP@SiO2 methanol solution.

[0047] (4) Surface modification with PEG

[0048] 1. Dissolve COOH-PEG-Silane in methanol and add it to the PDA@PSS@PAH@PVP@SiO2 methanol solution. Stir for 2 hours to obtain a PDA@PSS@PAH@PVP@SiO2@PEG solution.

[0049] 2. The PDA@PSS@PAH@PVP@SiO2@PEG solution was centrifuged and washed twice to remove excess impurities, and finally the PDA@PSS@PAH@PVP@SiO2@PEG was resuspended in ultrapure water.

[0050] @PEG aqueous solution.

[0051] 2. Preparation of PDA probes for detecting COVID-19 antibodies

[0052] 1. Take the PDA@PSS@PAH@PVP@SiO2@PEG solution, centrifuge and discard the supernatant.

[0053] 2. Wash the PDA@PSS@PAH@PVP@SiO2@PEG solution with DMSO-MES buffer, centrifuge and discard the supernatant, and resuspend it in DMSO-MES buffer.

[0054] 3. Add EDC and NHS to the PDA@PSS@PAH@PVP@SiO2@PEG solution at room temperature and activate it under ultrasonic conditions for 30 minutes to obtain the activated PDA@PSS@PAH@PVP@SiO2@PEG solution.

[0055] 4. Centrifuge the activated PDA@PSS@PAH@PVP@SiO2@PEG solution, discard the supernatant, and wash once with DMSO-MES buffer.

[0056] 5. Resuspend the activated PDA@PSS@PAH@PVP@SiO2@PEG in DMSO-MES buffer, and then add 20mM MES buffer, RBD antibody and mouse IgG solution.

[0057] 6. Incubate the reaction solution at 37°C on a shaker for 2 hours to obtain a PDA@PSS@PAH@PVP@SiO2@PEG-antibody solution.

[0058] 7. Add Glycine stop buffer to the PDA@PSS@PAH@PVP@SiO2@PEG-antibody solution, vortex, sonicate for 30 seconds and stop for 15 seconds, repeat 3 times, centrifuge and discard the supernatant.

[0059] 8. Resuspend the PDA@PSS@PAH@PVP@SiO2@PEG-antibody solution in ultrapure water and place in a 4°C refrigerator overnight.

[0060] 3. Preparation of test strips

[0061] (1) Use plastic adhesive to mount the binding pad (1.9 cm × 30 cm), nitrocellulose membrane (2.0 cm × 30 cm) and absorption pad (2.5 cm × 30 cm) on a support pad (6.0 cm × 30 cm) with their long sides overlapping by 2 mm. Cut the assembled pad along the long side to obtain 120 test strips (6 cm × 0.25 cm).

[0062] (2) Add 0.5 μl of anti-human IgG antibody and 0.5 μl of anti-human IgM antibody to the two test lines and 0.5 μl of goat anti-mouse IgG to the quality control line; place the test strip with the antibody and secondary antibody in an oven at 37°C for 2 hours to dry, and then store at 25°C for further use.

[0063] Example 1 Carcinoembryonic antigen (CEA) detection

[0064] 0.5 μL of 1 mg / mL CEA antigen and 0.5 μL of 0.2 mg / mL goat anti-mouse IgG were added to the lateral flow test strip (6 cm×0.25 cm) to produce the test area and the control area. The obtained nitrocellulose membrane was dried in an electric drying oven at 37°C for 6 hours. The PDA probe solution included 10 μL of PDA-antigen complex and 60 μL of HSLF buffer (270 mM NaCl, 100 mM HEPES buffer, 0.5% w / v tween 20, 1% w / v BSA). After mixing 30 μL of anti-CEA antibody solution of different concentrations with the PDA probe solution, the resulting solution was added to the sample pad of the lateral flow test strip. The sample addition concentrations of lanes 1 to 8 on the test strip are shown in Table 1 below. After 20 minutes, the results of the lateral flow test strip were obtained by taking pictures with a Nikon D90 camera as shown in the following table. Figure 1 As shown, from Figure 1 It can be seen that the test paper can be applied to the CEA antibody detection of cancer, and can specifically distinguish between negative and positive samples. As the antibody concentration increases, the color reaction of the test line becomes more obvious, and it can be used for qualitative and quantitative detection.

[0065] Table 1

[0066] sample 1 2 3 4 5 6 7 8 ng / mL blank 0 0.1 1 10 100 1000 10000

[0067] Example 2 Detection of COVID-19 IgG and IgM

[0068] 0.5 μL of 1 mg / mL SARS-CoV-2 RBD antigen and 0.5 μL of 0.2 mg / mL goat anti-mouse IgG were added to the lateral flow test strip (6 cm×0.25 cm) to produce the test area and the control area. The obtained nitrocellulose membrane was dried in an electric drying oven at 37°C for 6 hours. The PDA probe solution includes 10 μL PDA-antigen complex and 60 μL HSLF buffer (270 mM NaCl, 100 mM HEPES buffer, 0.5% w / v tween 20, 1% w / v BSA). After mixing 30 μL of different concentrations of new crown IgG and lgM antibody solutions with the PDA probe solution, the resulting solution was added to the lateral flow test strip sample pad. The sample addition concentrations for lanes 1 to 8 on the test strip are shown in Table 2 below. After 20 minutes, the results of the lateral flow test strip were obtained by taking pictures with a Nikon D90 camera, as shown in Figure 2 As shown, from Figure 2 It can be seen that the test paper can be applied to the IgG-lgM combined antibody detection of COVID-19, can specifically distinguish between negative and positive samples, and can specifically bind to lgG and lgM with high sensitivity.

[0069] Table 2

[0070] sample 1 2 3 4 IgG (ng / mL) 0 10 50 100 sample 5 6 7 8 lgM(ng / mL) 0 10 50 100

[0071] Example 3 Neutralizing Antibody Detection

[0072] 0.5 μL of 1 mg / mL SARS-CoV-2 ACE2 antigen and 0.5 μL of 0.2 mg / mL goat anti-mouse IgG were added to the lateral flow test strip (6 cm × 0.25 cm) to produce the test area and the control area. The obtained nitrocellulose membrane was dried at 37 ° C in an electric drying oven for 6 hours. The PDA probe solution includes 10 μL of PDA-antigen complex and 60 μL of HSLF buffer (270 mM NaCl, 100 mM HEPES buffer, 0.5% w / v tween 20, 1% w / v BSA). After mixing 30 μL of standard neutralizing antibody solution of different concentrations with the PDA probe solution, the resulting solution was added to the lateral flow test strip sample pad, and the sample addition concentrations of lanes 1 to 12 on the test paper are shown in Table 3 below. After 20 minutes, the results of the lateral flow test strip were obtained by taking pictures with a Nikon D90 camera.

[0073] Table 3

[0074] sample 1 2 3 4 5 6 ng / mL 10000 5000 2500 1250 625 312 sample 7 8 9 10 11 12 ng / mL 156 78 39 19.5 9.75 0

[0075] Example 4 Comparative Experimental Example

[0076] PDA has strong adhesion, which makes them easy to attach to lateral flow test strips. Figure 4a and Figure 4b Therefore, if the antibody is directly modified on the PDA@PEG surface, the resulting PDA probe will be mostly nonspecifically adsorbed on the test line, resulting in false negative results ( Figure 4a To solve this problem, a dense silicon layer was formed on PDA to prevent the nonspecific adsorption of PDA. The resulting PDA@SiO2@PEG probe can effectively reduce the nonspecific adsorption problem on the positive group test line ( Figure 4b (shown)

[0077] Table 4

[0078]

[0079] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A dopamine nanoprobe, characterized in that: The dopamine nanoprobe has a core-shell structure, the core is a polydopamine nanoparticle, and the core-shell surface is modified with a polymer layer, a SiO2 layer, a PEG layer, and a COVID-19 antibody detection layer; the dopamine nanoprobe is prepared according to the following method: Prepare polydopamine nanoparticles, and sequentially modify the surface of the polydopamine nanoparticles with a polymer layer, a silica layer, a PEG layer, and a COVID-19 antibody detection layer to prepare a dopamine nanoprobe; The components contained in the polymer layer include: polydopamine PDA, poly(styrene sulfonic acid) sodium salt PSS, polyacrylamine hydrochloride PAH and polyvinylpyrrolidone PVP.

2. The dopamine nanoprobe according to claim 1, characterized in that: The diameter of the polydopamine nanoparticles is 200-400 nm.

3. The dopamine nanoprobe according to claim 1, characterized in that: The chemical formula of polydopamine monomer is C8H 11 NO2.

4. The dopamine nanoprobe according to claim 1, characterized in that: The novel coronavirus antibodies selected are IgG and lgM.

5. The method for preparing the dopamine nanoprobe according to any one of claims 1 to 4, characterized in that: include: Prepare polydopamine nanoparticles, and sequentially modify the surface of the polydopamine nanoparticles with a polymer layer, a silica layer, a PEG layer, and a COVID-19 antibody detection layer to prepare a dopamine nanoprobe; The components contained in the polymer layer include: polydopamine PDA, poly(styrene sulfonic acid) sodium salt PSS, polyacrylamine hydrochloride PAH and polyvinylpyrrolidone PVP.

6. Use of the dopamine nanoprobe according to any one of claims 1 to 4 in the preparation of a product for antibody detection, characterized in that: The product includes a test strip or a test kit.

7. Use of the dopamine nanoprobe described in any one of claims 1 to 4 in the preparation of a probe for detecting antibodies against COVID-19.

8. A polydopamine nanoparticle test strip for detecting novel coronavirus-specific immunoglobulin, characterized in that: The dopamine nanoprobe according to any one of claims 1 to 4 is combined with a conjugate pad of a lateral flow immunochromatography test paper to obtain the nanoprobe.

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