Colloidal gold labeled hydrogel polymer nanoparticles and application thereof in SARS-CoV-2 virus detection

Abstract

The invention discloses a colloidal gold labeled hydrogel polymer nanoparticle which is of a core-shell structure formed by a non-biological antibody shell and a tracing marker core, and the non-biological antibody shell is formed by N-acryloyl-L-tryptophan, N-alkyl acrylamide, N, N-dimethyl formamide, N, N-dimethyl formamide, N, N-dimethyl formamide, N, N-dimethyl formamide, N, N-dimethyl formamide, N, N-dimethyl formamide, N, N-dimethyl formamide, N, N-dimethyl formamide and N, N-dimethyl formamide. The method comprises the following steps: carrying out free radical chain polymerization reaction on colloidal gold and N, N '-methylene bisacrylamide to form a hydrogel copolymer, taking colloidal gold-labeled hydrogel polymer nanoparticles as an affinity probe, and taking the colloidal gold-labeled hydrogel polymer nanoparticles as a cross-linking agent to carry out cross-linking reaction on the cross-linking agent and N, N'-methylene bisacrylamide to form the cross-linking agent. By utilizing the characteristic that the antibody can be specifically combined with SARS-CoV-2 virus S protein and a receptor binding domain RBD of the SARS-CoV-2 virus S protein to form a stable compound, rapid recognition and detection of the abiotic antibody of the SARS-CoV-2 virus antigen are realized. According to the invention, the limitations of large development difficulty, high cost, easy inactivation and the like of biological antibodies are overcome, the provided lateral chromatography detection method is good in accuracy and high in sensitivity, and a new method is provided for in-vitro diagnosis and environmental sample detection of the SARS-CoV-2 virus.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to colloidal gold-labeled hydrogel polymer nanoparticles. The present invention also relates to a preparation method of the colloidal gold-labeled hydrogel polymer nanoparticles and an application thereof in SARS-CoV-2 virus detection. Background Art

[0002] The new coronavirus (SARS-CoV-2), referred to as "new coronavirus", is the third highly pathogenic human beta coronavirus that can cause severe acute respiratory diseases after SARS-CoV and MERS-CoV. As a serious epidemic disease, rapid diagnosis of the new coronavirus is crucial. Its detection methods mainly include pathogen detection, molecular biology detection, and immunological antibody and antigen detection. Antigen detection is usually used for early detection of patients suspected of being infected with the new coronavirus. It mainly detects the structural protein nucleocapsid protein (nucleocapsid, N) or spike protein (spike, S) of the new coronavirus. Both structural proteins are specific proteins of the new coronavirus. The detection principle is based on antigen-antibody immune reaction or aptamer hybridization, and is converted into detection signals by colorimetry, fluorescence, electrochemistry, etc.

[0003] Lateral flow detection is a paper chromatography-based platform that is widely used for rapid and on-site detection. Among them, the detection results based on antigen-antibody immune reaction can be called lateral flow immunochromatographic detection. As a more common antigen detection method, lateral flow immunochromatographic detection combines immunoassay and chromatographic separation, and has the advantages of simple operation, low cost, easy to carry, and instant diagnosis. Colloidal gold, latex, europium, chelated silica and other colored reagents are commonly used color detection reagents for lateral flow chromatography technology, among which colloidal gold (gold nanoparticles, Au) is the most commonly used. The new coronavirus antigen detection kit based on the colloidal gold method is a common means for self-diagnosis of new coronavirus patients in the early stage. The colloidal gold antigen detection test strip consists of a carrier, a nitrocellulose membrane coated with a detection line (T line, containing monoclonal antibodies) and a quality control line (C line, containing anti-antibodies), a gold label pad for carrying gold-labeled antibodies (colloidal gold-labeled monoclonal antibodies), a sample pad for receiving sample solutions, and absorbent paper for guiding the sample solution to pass through the sample pad, gold label pad and nitrocellulose membrane in sequence. During the test, the sample to be tested is dripped into the sample hole and chromatographed forward under the capillary action. When the chromatography reaches the T line, if the target antigen exists, a complex of "antibody-antigen-gold-labeled antibody" is formed, and a red reaction line appears, which is positive; if no red reaction line appears, it is negative. Regardless of whether there is a target antigen, a red reaction line will appear on the C line to determine whether the chromatography process is normal and whether the test is effective.

[0004] Hydrogel polymer nanoparticles are high molecular polymers linked by C—C bonds. They are formed by various functional monomers through free radical-induced polymerization reactions. The main chain is a long C—C chain, and the side chains are various functional monomers. Commonly used functional monomers include acrylamide and methacrylic acid derivatives, such as N-acryloyl-L-amino acid and 2-methacryloyl-L-amino acid-NH2. Whether the hydrogel copolymer can achieve specific molecular recognition depends on whether the functional monomers introduced in its side chain can provide multiple interactions such as hydrophobicity, hydrogen bonding, electrostatics, and π-π stacking that are highly complementary to the binding sites of target biomolecules. These interactions are closely related to the hydrophobicity, hydrophilicity, and (or) charged groups of the functional monomers. Since the types and ratios of the functional monomers are adjustable, the synthesized hydrogel polymer nanoparticles have a wider range of chemical diversity. By utilizing this feature, a library of polymer nanoparticles with flexible structures and functions can be constructed. The preparation process of hydrogel polymer nanoparticles is simple and low-cost. The one-step synthesis method facilitates their scale-up and rapid production. They have good stability and robustness, and have strong tolerance to external environments such as pH and temperature. They have been used for the extraction, separation, purification and removal of target biological molecules in various complex systems, and are expected to replace natural antibodies to play specific roles and functions in the field of biotechnology.

[0005] The functional monomers introduced into the side chains of artificially synthesized polymer nanoparticles, especially amino acid monomers, are similar in structure and properties to the amino acid side groups in the peptide chains of protein antibodies. The reason why they can specifically recognize target biological molecules and achieve similar effects to biological antibodies / receptors is that they can simulate natural biological macromolecules, such as the specific multiple complementary interactions between antigens and antibodies (hydration complementarity, hydropathic complementarity), so that they have functions similar to antibody recognition of antigens, and are therefore also called "polymer biomimetic affinity ligands" or "non-biological antibodies". Therefore, the present invention utilizes structural proteins such as S protein encoded by SARS-CoV-2, analyzes the crystal structure information and molecular recognition mechanism of S protein binding to its host cell receptor, obtains amino acid functional monomers with high affinity for the receptor binding domain (RBD) of S protein by means of molecular docking, and regulates the types and ratios of candidate functional monomers to synthesize a series of polymer nanoparticles with different structures and surface chemical properties to form a library, and screens hydrogel polymer nanoparticles with stronger binding ability from the library through affinity experiments on S protein RBD. By using this method based on the combination of rational design and directed chemical evolution, we finally obtained a hydrogel polymer biomimetic affinity ligand with high affinity and high selectivity for the S protein of the new coronavirus. On this basis, we polymerized it with colloidal gold in situ to obtain colloidal gold-labeled hydrogel polymer nanoparticles with a core-shell structure, and based on antigen detection technology, established a lateral chromatography visualization detection platform that uses artificially synthesized non-biological antibodies instead of biological antibodies, providing new methods for in vitro diagnosis of SARS-CoV-2 and environmental sample detection, and providing more possibilities for rapid response to new and emerging infectious diseases in the future. Summary of the invention

[0006] The first object of the present invention is to provide a colloidal gold-labeled hydrogel polymer nanoparticle, which is used as an affinity probe, and utilizes its ability to specifically bind to the spike protein (spike, S) of the new coronavirus (SARS-CoV-2) and its receptor binding domain (receptor binding domain, RBD) to form a stable complex, so as to achieve rapid recognition and detection of non-biological antibodies to SARS-CoV-2 viral antigens.

[0007] The affinity probe provided by the present invention has a core-shell structure formed by a non-biological antibody shell and a tracer marker core, wherein the non-biological antibody shell is a hydrogel copolymer (ATrp-NPs) formed by a free radical chain polymerization reaction of N-acryloyl-L-tryptophan (ATrp), N-alkyl acrylamide, and N,N'-methylenebisacrylamide, and the tracer marker core is a gold nanoparticle.

[0008] Furthermore, the molar ratio of the N-acryloyl-L-tryptophan, N-alkyl acrylamide and N,N'-methylenebisacrylamide is 20-80:15-80:1-5.

[0009] A method for preparing the colloidal gold-labeled hydrogel polymer nanoparticles (Au@ATrp-NPs) comprises the following steps: firstly, modifying the gold nanoparticles with 3-butenylamine hydrochloride, then adding N-acryloyl-L-tryptophan, N-alkyl acrylamide, and N,N'-methylenebisacrylamide, and then adding an initiator and performing a free radical chain polymerization reaction on the surface of the gold nanoparticles under nitrogen protection to generate a hydrogel copolymer in situ and polymerize it on the surface of the colloidal gold.

[0010] Preferably, the initiator is azobisisobutyronitrile.

[0011] The present invention further provides the use of the colloidal gold-labeled hydrogel polymer nanoparticles in the preparation of a SARS-CoV-2 virus detection test strip and a SARS-CoV-2 virus detection test strip.

[0012] Preferably, the test strip is a non-biological antibody lateral flow test strip for detecting the S protein of the SARS-CoV-2 virus, and the test strip contains the colloidal gold-labeled hydrogel polymer nanoparticles.

[0013] Further preferably, the test strip comprises a sample pad, a nitrocellulose membrane and a water absorbent pad, the nitrocellulose membrane is provided with a detection line and a quality control line, the colloidal gold-labeled hydrogel polymer nanoparticles are dropped on the sample pad; the quality control line contains hydrogel polymer nanoparticles with positive charge on the surface; and the detection line contains a sample solution to be tested.

[0014] Further preferably, the colloidal gold-labeled hydrogel polymer nanoparticles are used at a concentration of 0.01-0.2 mg / mL, and the dropwise addition volume is 20-100 μL.

[0015] Further preferably, the hydrogel polymer nanoparticles with positive surface charge are generated by free radical chain polymerization of 2-methacryloyl-lysine-NH2 (MLys), N-alkyl acrylamide and N,N'-methylenebisacrylamide, the N-alkyl acrylamide is composed of N-isopropyl acrylamide and N-tert-butyl acrylamide, and the molar ratio of 2-methacryloyl-lysine-NH2, N-isopropyl acrylamide, N-tert-butyl acrylamide and N,N'-methylenebisacrylamide is 20-40:15-35:30-50:1-5.

[0016] Another object of the present invention is to provide a method for detecting SARS-CoV-2 virus for non-diagnostic purposes, comprising the following steps:

[0017] S1. Using gold nanoparticles as the core of the tracer marker, modifying the surface of the gold nanoparticles with 3-buteneamine hydrochloride, then adding N-acryloyl-L-tryptophan, N-alkyl acrylamide, N,N'-methylenebisacrylamide and an initiator to the gold nanoparticles, performing a free radical chain polymerization reaction under nitrogen protection, and using the colloidal gold-labeled hydrogel polymer nanoparticles obtained by the reaction as affinity probes and concentrating them for later use;

[0018] S2, adding an initiator to 2-methacryloyl-lysine-NH2, N-alkyl acrylamide, and N,N'-methylenebisacrylamide, and performing a free radical chain polymerization reaction under nitrogen protection to synthesize hydrogel polymer nanoparticles with positive charges on the surface;

[0019] S3, assembling the sample pad, the nitrocellulose membrane and the absorbent pad on the supporting bottom plate, applying the sample solution to be tested on the detection line of the nitrocellulose membrane, and drawing the hydrogel polymer nanoparticles with positive surface charge prepared in step S2 on the quality control line of the nitrocellulose membrane, and then drying;

[0020] S4. Add the affinity probe prepared in step S1 to the sample pad for lateral flow chromatography detection. If the test line and the quality control line are colored at the same time, the test result is SARS-CoV-2 positive; if the test line does not color, but the quality control line is colored, the test result is SARS-CoV-2 negative.

[0021] In order to realize the non-biological antibody detection of SARS-CoV-2 virus, the present invention prepares two kinds of hydrogel polymer nanoparticles respectively, one is a hydrogel copolymer synthesized with N-acryloyl-L-tryptophan as the main functional monomer, and uses it as a non-biological antibody shell to wrap gold nanoparticles to form a colloidal gold-labeled hydrogel polymer nanoparticle affinity probe (Au@ATrp-NPs), which can specifically recognize and bind to the RBD domain of SARS-CoV-2S protein; the other is a hydrogel polymer nanoparticle (MLys-NPs) synthesized with 2-methacryloyl-lysine-NH2 as the main functional monomer, which combines with the above affinity probe based on electrostatic interaction to realize the application of the quality control line (C) of the detection platform. Based on the above two non-biological antibodies, a lateral flow detection platform is established, and the direct method is adopted to realize the rapid detection of the new coronavirus.

[0022] The detection method constructed by the present invention can be used for disease diagnosis of SARS-CoV-2 virus, and can also be used for laboratory screening and identification of SARS-CoV-2 virus for non-disease diagnosis purposes, environmental testing, etc.

[0023] The beneficial effects of the present invention are:

[0024] The present invention uses hydrogel polymer nanoparticles as non-biological antibodies to specifically bind to SARS-CoV-2S protein. The non-biological antibody has excellent affinity. Compared with biological antibodies, it has mild synthesis conditions, simple preparation process, low cost, good stability, and high biocompatibility and plasticity.

[0025] The non-biological antibody lateral chromatography visualization detection platform established by the present invention uses a colloidal gold-labeled hydrogel polymer nanoparticle affinity probe (Au@ATrp-NPs) as a detection standard and a hydrogel polymer nanoparticle (MLys-NPs) as a quality control standard. The entire detection platform does not contain any biological antibodies, has low preparation cost, is easy to store, and has good stability. It is used to replace the original biological antibodies, and overcomes the limitations of biological antibody development, high cost, poor environmental tolerance, easy inactivation, and harsh storage and reaction conditions. At the same time, the detection platform does not require special interpretation instruments, has a short reaction time, and can complete the detection in only 15 to 20 minutes. The results are intuitively read without the need to master complex professional knowledge. It also has certain application potential in qualitative and semi-quantitative detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Transmission electron microscopy image of affinity probe Au@ATrp-NPs.

[0027] Figure 2 : Screening of hydrogel polymer nanoparticles at the quality control line (C).

[0028] Figure 3 : Schematic diagram of the test strip structure and its detection steps.

[0029] Figure 4 : Schematic diagram of the actual sample test results of the test strip.

[0030] Figure 5 : Specificity test results for SARS-CoV-2S protein detection.

[0031] Figure 6 : Sensitivity test results for SARS-CoV-2S protein detection. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments.

[0033] In the following examples, if the specific conditions are not specified, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials and reagents used can be derived from ordinary commercial channels. The uncapped 96-well black flat-bottom polystyrene high-binding microplate was purchased from Corning, USA, and the wild-type recombinant SARS-CoV-2 S protein trimer, S protein RBD, S protein NTD, and S2 protein were purchased from Pujian Biotechnology (Wuhan) Technology Co., Ltd. and labeled with fluorescein isothiocyanate (FITC) by Beijing Biosynthetic Biotechnology Co., Ltd. (Beijing).

[0034] Example 1: Preparation of hydrogel polymer nanoparticles

[0035] Step 1: Synthesis of N-acryloyl-L-tryptophan monomer

[0036] Dissolve 30mmol L-tryptophan in 15mL aqueous solution containing 60mmol sodium hydroxide, keep an external ice bath, and keep the temperature at 0-4°C. After the solid is completely dissolved, add 30mmol acryloyl chloride slowly dropwise to the above mixture using a separatory funnel. The dropwise addition time is longer than 30min. After the dropwise addition is completed, continue to stir the reaction under ice bath conditions for 1h, then warm to room temperature and stir for another 2h. After the obtained product is adjusted to pH with 1M hydrochloric acid, solids precipitate and precipitate. Vacuum filtration is performed to obtain a crude product, which is recrystallized in water to finally obtain a light yellow solid product N-acryloyl-L-tryptophan.

[0037] Step 2: Synthesis of hydrogel polymer nanoparticles (ATrp-NPs)

[0038] Hydrogel polymer nanoparticles are synthesized by free radical polymerization of different functional monomers, in which N-acryloyl-L-tryptophan is used as the main functional monomer, N-isopropylacrylamide (NIPAm) is used as a co-monomer, N,N'-methylenebisacrylamide (BIS) is used as a cross-linker, sodium dodecyl sulfate (SDS) is used as a surfactant, and azobisisobutyronitrile (AIBN) is used as an initiator to synthesize hydrogel polymer nanoparticles with different surface properties.

[0039] The total monomer concentration of the reaction is 65mM, and the total volume of the solution is 25mL. Take 0.975mmol of N-acryloyl-L-tryptophan (accounting for 60% of the total monomer), 0.6175mmol of N-isopropylacrylamide (accounting for 38% of the total monomer), and 0.0325mmoL of N,N'-methylenebisacrylamide (accounting for 2% of the total monomer), wherein the amino acid functional monomer is dissolved in ethanol, and the remaining monomers are dissolved in water, and then added to a 100mL branched reaction tube, 1mL of surfactant SDS (10mg / mL) is added, and the total volume is supplemented to 25mL with ultrapure water. Seal with a rubber stopper, stir magnetically, pass nitrogen to deoxygenate 3 times, preheat to 65℃, and inject 1mL of initiator AIBN (30mg / mL) from the bottle mouth sealed with a rubber stopper with a syringe. Under nitrogen protection, the reaction was continued with stirring at 65°C for 3 hours. After cooling to room temperature, the reaction liquid was transferred to a dialysis bag prepared in advance and dialyzed in 3L ultrapure water for 4 days to purify the hydrogel polymer nanoparticles. The water was changed once in the morning and evening every day. After the dialysis, the obtained hydrogel polymer nanoparticles were stored in a refrigerator at 4°C.

[0040] The hydrogel polymer nanoparticles (ATrp-NPs) synthesized in step 2 were used for affinity adsorption of the three domains of SARS-CoV-2 S protein RBD, NTD and S2. The results showed that it had a high affinity for the S protein RBD, but had almost no affinity effect on NTD and S2, indicating that RBD is the main region for the hydrogel polymer nanoparticles (ATrp-NPs) to bind to the SARS-CoV-2 S protein. In addition, the binding capacity of the hydrogel polymer nanoparticles to the S protein RBD was detected by referring to the method of CN 115141311A. The results showed that the maximum binding capacity of ATrp-NPs to RBD-Fitc protein was 2620.6 mg / g. Compared with the previously constructed polymer nanoparticles with N-acryloyl-L-phenylalanine as the main functional monomer (maximum binding capacity of 1035.7 mg / g), ATrp-NPs has a stronger binding ability to the S protein RBD.

[0041] Example 2: Synthesis of colloidal gold labeled hydrogel polymer nanoparticle affinity probe (Au@ATrp-NPs)

[0042] The hydrogel copolymer is used as a non-biological antibody shell, and gold nanoparticles are wrapped in the core by the hydrogel copolymer as a tracer marker to prepare a colloidal gold-labeled hydrogel polymer nanoparticle affinity probe (Au@ATrp-NPs) to achieve rapid lateral chromatography detection of SARS-CoV-2 virus. First, the gold nanoparticles are synthesized by sodium citrate reduction method, and then the double-bonded 3-buteneamine hydrochloride (BA) is modified to the surface of the gold nanoparticles. Finally, according to the method of Example 1, the functional monomer (ATrp) and the coexisting monomer (NIPAm) are in situ polymerized to the surface of the gold nanoparticles through free radical polymerization reaction to form a colloidal gold-labeled hydrogel polymer nanoparticle affinity probe with a core-shell structure. The specific steps are as follows:

[0043] Step 1: Synthesis of gold nanoparticles

[0044] Add 500mL of ultrapure water to the round-bottom flask, then take 5mL of HAuCl4 (25mM) into the round-bottom flask, stir magnetically, and heat in an oil bath. When the solution boils, add 5mL of 3% (m / v) sodium citrate (0.15g) aqueous solution to the solution, and continue to stir and heat the reaction for 15min. During the reaction, the solution changes from transparent to gray, and then from gray to wine red. After the reaction is completed, remove the round-bottom flask from the oil bath, continue to stir and cool to room temperature, and obtain gold nanoparticles (Au NPs), which are stored in a 4°C refrigerator.

[0045] Step 2: Modification of gold nanoparticles

[0046] Take 100mL of the gold nanoparticle solution, add 200μL SDS solution (10mg / mL), react at room temperature for 20min, and stir magnetically during the reaction. After the reaction is completed, add 200μL 3-buteneamine hydrochloride (BA) solution (1mg / mL) to the above solution, continue stirring, and react for 20min. After the reaction is completed, centrifuge the reaction solution to remove excess BA and SDS, the centrifugal speed is 8000rpm, and the centrifugal time is 1h. After the centrifugation is completed, concentrate the solution to 20mL for use.

[0047] Step 3: Synthesis of colloidal gold labeled hydrogel polymer nanoparticle affinity probes (Au@ATrp-NPs)

[0048] Add a certain amount of ultrapure water to a branched reaction tube, then add 1mL SDS solution (10mg / mL), and then add 4mL of concentrated solution of gold nanoparticles modified with BA to the above solution. Place the reaction tube in a water bath, stir magnetically, and continue to pass nitrogen to deoxygenate. When the temperature of the water bath rises to 65℃, use a small amount of solution to dissolve 0.24mmol (60%) of N-acryloyl-L-tryptophan, 0.152mmol (38%) of N-isopropylacrylamide, and 0.008mmoL (2%) of N,N'-methylenebisacrylamide. Slowly add the dissolved functional monomer mixture to the reaction tube, seal the reaction tube, stir magnetically for 10min to make the monomers evenly dispersed, then add 1mL of AIBN initiator (3mg / mL) to the reaction tube with a syringe, continue magnetic stirring, react at 65℃ for 3h, and the total reaction volume is 40mL. After the reaction, the solution was centrifuged and washed with water three times to remove unreacted monomers and SDS. The centrifugal speed was 8000 rpm and the centrifugal time was 1 h. Finally, the synthesized affinity probe Au@ATrp-NPs solution was concentrated to 20 mL with a concentration of 0.05 mg / mL (based on gold, ignoring gold loss). The purified affinity probe solution was stored in a 4 °C refrigerator for future use.

[0049] Figure 1 The transmission electron microscopy image of the affinity probe Au@ATrp-NPs showed that the gold nanoparticles were obviously wrapped with a layer of hydrogel copolymer, forming an obvious core-shell microsphere structure with uniform dispersion, indicating that the probe was successfully synthesized.

[0050] Example 3: Synthesis of hydrogel polymer nanoparticles at the quality control line (C)

[0051] The red reaction presented by the quality control line (C) can determine whether the affinity probe chromatography process is normal, and whether it is colored is directly related to whether the test result is valid. To achieve the red color development reaction at the quality control line (C), it is only necessary to make the material at the C line react with the affinity probe. The detection affinity probe of the present invention is a colloidal gold-labeled hydrogel polymer nanoparticle affinity probe Au@ATrp-NPs, which has a negative charge on its surface. Based on the principle of electrostatic interaction, hydrogel polymer nanoparticles with opposite charges are selected as the quality control line (C).

[0052] Step 1: Synthesis of positively charged hydrogel polymer nanoparticles

[0053] Referring to the synthesis method of hydrogel polymer nanoparticles in Example 1, three positively charged amino acid monomers, 2-methacryloyl-arginine-NH2 (MArg), 2-methacryloyl-lysine-NH2 (MLys), and 2-methacryloyl-histidine-NH2 (MHis), are used as main functional monomers, N-tert-butylacrylamide (TBAm) and N-isopropylacrylamide (NIPAm) are used as co-existing monomers, N,N'-methylenebisacrylamide (BIS) is used as a cross-linking agent, cetyltrimethylammonium bromide (CTAB) is used as a surfactant, and azobisisobutyronitrile (AIBN) is used as an initiator. The ratio of each monomer is changed, and hydrogel polymer nanoparticles with positive charge and different surface properties are synthesized by free radical initiated polymerization.

[0054] Step 2: Screening of non-bioantibody hydrogel polymer nanoparticles at the control line (C)

[0055] Assemble the sample pad, nitrocellulose membrane and absorbent pad on the supporting base plate, and directly draw the positively charged hydrogel polymer nanoparticles synthesized in the above step 1 at the C line position, the drawing concentration is: 1 mg / mL, the drawn test strips are placed in a 37°C oven to dry, and the test strips are cut into thin strips of about 4 mm for standby use. The affinity probe Au@ATrp-NPs synthesized in the above Example 2 is resuspended with pH 6.0, 10mM PBST buffer, and dripped onto the sample pad for lateral chromatography detection, and the probe volume is 80μL. After waiting for 15 minutes, compare the color development at the C line of each group of test strips to evaluate the binding performance of different positively charged hydrogel polymer nanoparticles to the affinity probe, and screen out the hydrogel polymer nanoparticles that meet the requirements.

[0056] Figure 2 From the color development of positively charged hydrogel polymer nanoparticles with different monomers and different ratios combined with the affinity probe Au@ATrp-NPs, it can be clearly seen that the color development of the two hydrogel polymer nanoparticles NP6 and NP9 to the affinity probe Au@ATrp-NPs is more obvious, and from the perspective of stability, NP6 is more stable, that is, the hydrogel polymer nanoparticles (MLys-NPs) prepared when MLys accounts for 30% of the total monomer amount, NIPAm accounts for 28% of the total monomer amount, and TBAm accounts for 40% of the total monomer amount are used as the quality control standard at the quality control line (C) of the lateral flow visualization detection platform.

[0057] Example 4: Establishment of a Lateral Flow Visualization Detection Platform

[0058] The lateral flow detection method is used for determination. The sample pad, the nitrocellulose membrane and the absorbent pad are assembled on the supporting bottom plate. The SARS-CoV-2S protein sample is directly dotted on the detection line (T) of the nitrocellulose membrane. The hydrogel polymer nanoparticles (MLys-NPs) synthesized in Example 3 are directly dotted on the quality control line (C) of the nitrocellulose membrane. After drying, the affinity probe Au@ATrp-NPs solution prepared in Example 2 is dripped on the sample pad. The affinity probe moves forward to the T line through capillary action. If SARS-CoV-2S protein is present in the sample, an "Au@ATrp-NPs-antigen" complex is formed, and a red reaction line is formed at the T line. The unbound affinity probe continues to move forward to the C line and combines with the positively charged hydrogel polymer nanoparticles (MLys-NPs) through electrostatic action to form a red reaction line. Regardless of whether the target antigen is present at the T line, a red reaction line will appear at the C line to determine that the chromatography process is normal and the detection result is valid. If the T line and C line are colored at the same time, the test result is SARS-CoV-2S protein positive; if the T line is not colored, the C line is colored, and the test result is SARS-CoV-2S protein negative; if the C line is not colored, the test result is invalid. At the same time, the SARS-CoV-2S protein can also be semi-quantitatively detected according to the depth of the color on the T line.

[0059] Step 1: Synthesis and streaking of hydrogel polymer nanoparticles at the control line (C)

[0060] According to the method of hydrogel polymer nanoparticles in Example 3, the total monomer concentration of the reaction system was determined to be 16.25mM and the total volume was 25mL. The proportions of each monomer are as follows: 2-methacryloyl-lysine-NH2 0.1219mmol (30% of the total monomer amount), N-isopropylacrylamide (NIPAm) 0.1138mmol (28% of the total monomer amount), N-tert-butylacrylamide (TBAm) 0.1625mmol (40% of the total monomer amount), N,N'-methylenebisacrylamide (BIS) 0.008125mmol (2% of the total monomer amount). Hexadecyltrimethylammonium bromide (CTAB) was used as a surfactant and azobisisobutyronitrile (AIBN) was used as an initiator. The obtained hydrogel polymer nanoparticles (MLys-NPs) were stored in a refrigerator at 4°C and the concentration was determined to be 3.98mg / mL.

[0061] In order to optimize the streaking concentration of hydrogel polymer nanoparticles (MLys-NPs) on the quality control line (C), streaking concentrations of 0.1, 0.5, 1, 2, and 3.98 mg / mL were selected and streaked on the quality control line (C), and then dried in an oven at 37°C. The test strips were cut into thin strips of about 4 mm for later use. The synthesized Au@ATrp-NPs probe solution was centrifuged, the supernatant was removed, and then resuspended with pH 6.0, 10mM PBST buffer, and dropped onto the sample pad for lateral chromatography. The color development of the C line was observed, and the optimal streaking concentration was 2 mg / mL.

[0062] Step 2: Construction of lateral flow visualization detection platform

[0063] Unlike the more common sandwich lateral flow colloidal gold method, this detection method utilizes two non-biological antibody hydrogel polymer nanoparticles and adopts a direct method for determination. The sample pad, nitrocellulose membrane and absorbent pad are assembled on the supporting base plate, and the test sample containing SARS-CoV-2S protein and positively charged hydrogel polymer nanoparticles (MLys-NPs) are respectively spotted on the test line (T) and quality control line (C) of the nitrocellulose membrane, and then dried in an oven at 37°C. Subsequently, the affinity probe Au@ATrp-NPs solution is dropped onto the sample pad, and the affinity probe moves forward chromatography by capillary action, combining with the SARS-CoV-2S protein on the T line and the MLys-NPs on the C line to develop color. The structure of the test strip and its detection schematic diagram are shown in the figure. Figure 3 shown.

[0064] Step 3: Optimization of lateral flow assay affinity probe concentration and volume

[0065] In lateral flow chromatography detection experiments, the amount of affinity probe added will affect the T-line color development results, so it is necessary to optimize the probe concentration and volume.

[0066] The positively charged hydrogel polymer nanoparticles (MLys-NPs) were directly streaked at the C line position with a streaking concentration of 2 mg / mL, and the SARS-CoV-2S protein solution was directly spotted at the T line position. Subsequently, the test strip was placed in a 37°C oven to dry and cut into thin strips of about 4 mm for later use. The synthesized Au@ATrp-NPs probe solution was centrifuged, the supernatant was removed, and then resuspended with pH 6.0, 10 mM PBST buffer and dropped onto the sample pad for lateral flow detection. When the probe concentration is optimized, the fixed probe volume is 40 μL, the probe concentration is controlled within the range of 0.0125-0.2 mg / mL (in terms of gold), the chromatography test is performed according to the above steps, the T line color development is observed, and the optimal probe concentration is optimized; when the probe volume is optimized, the fixed probe concentration is 0.1 mg / mL (in terms of gold), the probe volume is 20-100 μL, the chromatography test is performed according to the above steps, the T line color development is observed, and the optimal probe volume is optimized.

[0067] Analysis of results: When the affinity probe concentration is too low, the color development after chromatography is not obvious enough. When the concentration is too high, the viscosity of the probe solution increases and the probe runs very slowly during the chromatography process. The optimal probe concentration after optimization is 0.1 mg / mL (in terms of gold). The color development of the lateral flow test strip increases with the increase of the probe volume and then tends to stabilize. The optimal volume of the optimized probe is 80 μL.

[0068] Based on the above experiments, the lateral flow visualization detection method was finally determined as follows:

[0069] S1. Using gold nanoparticles as the core of the tracer marker, modifying the surface of the gold nanoparticles with 3-buteneamine hydrochloride, then adding three monomers, N-acryloyl-L-tryptophan, N-isopropylacrylamide and N,N'-methylenebisacrylamide, and an initiator, azobisisobutyronitrile, to the gold nanoparticles, performing a free radical chain polymerization reaction under a nitrogen atmosphere, and using the colloidal gold-labeled hydrogel polymer nanoparticles (Au@ATrp-NPs) obtained by the reaction as affinity probes and concentrating them for later use, wherein the molar ratio of the three monomers is 60:38:2;

[0070] S2, adding an initiator azobisisobutyronitrile to four monomers of 2-methacryloyl-lysine-NH2, N-isopropylacrylamide, N-tert-butylacrylamide, and N,N'-methylenebisacrylamide, and performing a free radical chain polymerization reaction under a nitrogen atmosphere to synthesize hydrogel polymer nanoparticles (MLys-NPs) with positive charges on the surface, wherein the molar ratio of the four monomers is 30:28:40:2;

[0071] S3, assemble the sample pad, nitrocellulose membrane and absorbent pad on the support base, spot the sample solution on the detection line (T) of the nitrocellulose membrane, draw 2 mg / mL positively charged hydrogel polymer nanoparticles (MLys-NPs) on the quality control line (C) of the nitrocellulose membrane, and then dry in an oven at 37°C;

[0072] S4. Add the affinity probe (Au@ATrp-NPs) to the sample pad for lateral flow chromatography detection. The affinity probe is used at a concentration of 0.1 mg / mL (in terms of gold) and the addition volume is 80 μL. If both the test line and the quality control line are colored, the test result is SARS-CoV-2 positive; if the test line does not color, the quality control line is colored, and the test result is SARS-CoV-2 negative.

[0073] The actual sample was tested by lateral flow chromatography with 0.3 mg / mL SARS-CoV-2 S protein. The results are as follows Figure 4 shown.

[0074] Example 5: Evaluation of Lateral Flow Detection Method

[0075] 1. Selective evaluation

[0076] SARS-CoV-2S protein, conventional proteins in blood and protease stock solution were diluted with 10mM PBST buffer solution at pH 6 containing 0.05% Tween-20 to obtain SARS-CoV-2S protein, lysozyme, pepsin, trypsin, human serum albumin (HSA), and bovine serum albumin (BSA) at a concentration of 1mg / mL. Then, each protein sample was directly spotted on the detection line (T) of the nitrocellulose membrane, and then 80μL of affinity probe (Au@ATrp-NPs) solution with a gold content of 0.1mg / mL was dripped on the sample pad. The above-established lateral flow visualization detection platform was used to determine the interference of different conventional proteins and proteases on the affinity probe (Au@ATrp-NPs) detection of SARS-CoV-2.

[0077] The test results of the selectively bound lateral flow test strip and the corresponding T-line relative gray value are as follows Figure 5 As shown. The experiment shows that the colorimetric of lysozyme, pepsin, trypsin, human serum albumin, and bovine serum albumin is almost negligible compared to the detection of SARS-CoV-2S protein under the same concentration conditions. The above results show that the gold-labeled hydrogel polymer nanoparticle affinity probe prepared by the present invention can specifically bind to SARS-CoV-2S protein.

[0078] 2. Sensitivity evaluation

[0079] SARS-CoV-2S protein was directly spotted at the T line with concentrations of 1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015, and 0.0075 mg / mL, respectively. The spotted test strips were placed in a 37°C oven to dry, and 80 μL of Au@ATrp-NPs affinity probe solution with a gold content of 0.1 mg / mL was dropped onto the sample pad for lateral flow chromatography detection, with a detection time of about 15 to 20 min.

[0080] Figure 6 The lateral flow chromatography test results of SARS-CoV-2S protein at different concentrations are shown in Figure 1. The lateral flow chromatography test strip color development results, i.e., the relative gray value of the T line, are processed using ImageJ software. As can be seen from the figure, the Au@ATrp-NPs affinity probe prepared by the present invention can effectively bind to the SARS-CoV-2S protein as a non-biological antibody, and has a good linear relationship in the concentration range of 0.015 to 1 mg / mL, with a minimum detection limit of 0.0075 mg / mL.

[0081] No biological antibodies were used in this study. This detection method based on artificially synthesized non-biological antibodies is low-cost, easy to store, and has good stability. It overcomes the limitations of biological antibody development, high cost, poor environmental tolerance, easy inactivation, and harsh storage and reaction conditions. The present invention can be used for rapid detection and screening of actual samples, and it also has certain application potential in qualitative and semi-quantitative detection. The established detection method will provide a new method for in vitro diagnosis of SARS-CoV-2 and detection of environmental samples, and also provide more possibilities for rapid response to new and emerging infectious diseases in the future, which is of great significance for the prevention and control of emerging infectious diseases including SARS-CoV-2.

Claims

1. A colloidal gold-labeled hydrogel polymer nanoparticle, which has a core-shell structure formed by a non-biological antibody shell and a tracer marker core, wherein the non-biological antibody shell is a hydrogel copolymer formed by free radical chain polymerization of N-acryloyl-L-tryptophan, N-alkyl acrylamide, and N,N'-methylenebisacrylamide, and the tracer marker core is a gold nanoparticle.

2. The colloidal gold-labeled hydrogel polymer nanoparticles according to claim 1, characterized in that: The molar ratio of the N-acryloyl-L-tryptophan, N-alkyl acrylamide and N,N'-methylenebisacrylamide is 20-80:15-80:1-5.

3. A method for preparing the colloidal gold-labeled hydrogel polymer nanoparticles according to claim 1, characterized in that: First, gold nanoparticles were modified with 3-buteneamine hydrochloride, and then N-acryloyl-L-tryptophan, N-alkyl acrylamide, and N,N'-methylenebisacrylamide were added. Subsequently, an initiator was added and a free radical chain polymerization reaction was carried out on the surface of the gold nanoparticles under nitrogen protection to generate a hydrogel copolymer which was in situ polymerized on the colloidal gold surface.

4. The preparation method according to claim 3, characterized in that: The initiator is azobisisobutyronitrile.

5. Use of the colloidal gold-labeled hydrogel polymer nanoparticles according to claim 1 in preparing a SARS-CoV-2 virus detection test strip.

6. A SARS-CoV-2 virus detection test strip, which is a non-biological antibody lateral flow test strip for detecting the S protein of the SARS-CoV-2 virus, and the test strip contains the colloidal gold-labeled hydrogel polymer nanoparticles according to claim 1.

7. The SARS-CoV-2 virus detection test strip according to claim 6, characterized in that: The test strip comprises a sample pad, a nitrocellulose membrane and a water-absorbing pad. The nitrocellulose membrane is provided with a detection line and a quality control line. The colloidal gold-labeled hydrogel polymer nanoparticles are dripped onto the sample pad; the quality control line contains hydrogel polymer nanoparticles with positive charges on the surface; and the detection line contains a sample solution to be tested.

8. The SARS-CoV-2 virus detection test strip according to claim 7, characterized in that: The colloidal gold-labeled hydrogel polymer nanoparticles are used at a concentration of 0.01-0.2 mg / mL, and the drop volume is 20-100 μL.

9. The SARS-CoV-2 virus detection test strip according to claim 7, characterized in that: The hydrogel polymer nanoparticles with positive charges on the surface are generated by free radical chain polymerization of 2-methacryloyl-lysine-NH2, N-alkyl acrylamide and N,N'-methylenebisacrylamide, wherein the N-alkyl acrylamide is composed of N-isopropyl acrylamide and N-tert-butyl acrylamide, and the molar ratio of the four of 2-methacryloyl-lysine-NH2, N-isopropyl acrylamide, N-tert-butyl acrylamide and N,N'-methylenebisacrylamide is 20-40:15-35:30-50:1-5.

10. A method for detecting SARS-CoV-2 virus for non-diagnostic purposes, characterized in that The following steps are involved: S1. Using gold nanoparticles as the core of the tracer marker, modifying the surface of the gold nanoparticles with 3-buteneamine hydrochloride, then adding N-acryloyl-L-tryptophan, N-alkyl acrylamide, N,N'-methylenebisacrylamide and an initiator to the gold nanoparticles, performing a free radical chain polymerization reaction under nitrogen protection, and using the colloidal gold-labeled hydrogel polymer nanoparticles obtained by the reaction as affinity probes and concentrating them for later use; S2, adding an initiator to 2-methacryloyl-lysine-NH2, N-alkyl acrylamide, and N,N'-methylenebisacrylamide, and performing a free radical chain polymerization reaction under nitrogen protection to synthesize hydrogel polymer nanoparticles with positive charges on the surface; S3, assembling the sample pad, the nitrocellulose membrane and the absorbent pad on the supporting bottom plate, applying the sample solution to be tested on the detection line of the nitrocellulose membrane, and drawing the hydrogel polymer nanoparticles with positive surface charge prepared in step S2 on the quality control line of the nitrocellulose membrane, and then drying; S4. Add the affinity probe prepared in step S1 to the sample pad for lateral flow chromatography detection. If the test line and the quality control line are colored at the same time, the test result is SARS-CoV-2 positive; if the test line does not color, but the quality control line is colored, the test result is SARS-CoV-2 negative.

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