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

Abstract

The present invention discloses colloidal gold-labeled hydrogel polymer nanoparticles, which have a core-shell structure formed by a non-biological antibody shell and a tracer marker core. The non-biological antibody shell is a hydrogel copolymer formed by a free radical chain polymerization reaction of N-acryloyl-L-tryptophan, N-alkyl acrylamide, and N,N'-methylenebisacrylamide. The tracer marker core is a gold nanoparticle. The colloidal gold-labeled hydrogel polymer nanoparticles are used as affinity probes, utilizing their ability to specifically bind to the SARS-CoV-2 virus S protein and its receptor binding domain (RBD) to form a stable complex, thereby enabling rapid identification and detection of non-biological antibodies against SARS-CoV-2 viral antigens. The present invention overcomes the limitations of biological antibody development, such as the difficulty, high cost, and easy inactivation. The provided lateral flow detection method has good accuracy and high sensitivity, providing a new method for in vitro diagnosis of SARS-CoV-2 virus and environmental sample detection.

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 novel coronavirus (SARS-CoV-2), also known as the "new coronavirus," is the third highly pathogenic human beta coronavirus discovered after SARS-CoV and MERS-CoV that can cause severe acute respiratory illness. As a serious epidemic disease, rapid diagnosis of the novel 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 novel coronavirus. It mainly detects the structural proteins of the novel coronavirus, the nucleocapsid protein (N) or the spike protein (S). Both structural proteins are specific proteins of the novel coronavirus. The detection principle is based on antigen-antibody immune reaction or aptamer hybridization, and uses colorimetry, fluorescence, electrochemistry, and other methods to convert them into detection signals.

[0003] Lateral flow testing (LFTA) is a paper-based chromatography platform widely used for rapid and on-site testing. Flow-through immunochromatographic testing (FLIT) relies on antigen-antibody immune reactions to generate test results. As a common antigen detection method, FLIT combines immunoassays with chromatographic separations, offering advantages such as ease of use, low cost, portability, and immediate diagnosis. Colored reagents such as colloidal gold, latex, europium, and chelated silica are commonly used in LFTA. Colloidal gold (gold nanoparticles, Au) is the most commonly used. SARS-CoV-2 antigen detection kits based on FLIT are a common tool for early self-diagnosis of COVID-19 patients. The FLIT strip consists of a carrier, a nitrocellulose membrane coated with a test line (T line, containing a monoclonal antibody) and a control line (C line, containing an anti-antibody), a gold label pad for carrying the gold-labeled antibody (colloidal gold-labeled monoclonal antibody), a sample pad for receiving the sample solution, and absorbent paper for guiding the sample solution through the sample pad, gold label pad, and nitrocellulose membrane. During the test, the sample is added to the sample well and chromatographed forward under capillary action. When the target antigen is present at the T line, a red reaction line appears, indicating a positive result. No red reaction line appears, indicating a negative result. Regardless of the presence of the target antigen, a red reaction line appears at the C line, confirming the normal chromatography process and the validity of the test.

[0004] Hydrogel polymer nanoparticles are high-molecular-weight polymers linked by C-C bonds. They are formed through free radical-initiated polymerization of various functional monomers. The backbone consists of a long C-C chain, while the side chains are composed of 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 hydrogel copolymers can achieve specific molecular recognition depends on whether the functional monomers introduced into the side chains can provide multiple interactions with the target biomolecule binding sites, including hydrophobic, hydrogen bonding, electrostatic, and π-π stacking interactions. These interactions are closely related to the hydrophobic, hydrophilic, and / or charged groups carried by the functional monomers. The adjustable type and ratio of the functional monomers allows for a broad chemical diversity in the synthesized hydrogel polymer nanoparticles. This characteristic can be exploited to construct a library of polymer nanoparticles with flexible structures and diverse functions. The preparation process of hydrogel polymer nanoparticles is simple and low-cost. The one-step synthesis method facilitates their scalability 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] Functional monomers, particularly amino acid monomers, introduced into the side chains of synthetic polymer nanoparticles have similar structures and properties to the amino acid side groups in protein and antibody peptide chains. Their ability to specifically recognize target biomolecules and achieve efficacy similar to that of biological antibodies / receptors stems from their ability to mimic the specific multiple complementary interactions (hydropathic complementarity) between natural biomacromolecules, such as antigen-antibody interactions, giving them a function similar to antibody-antigen recognition. Consequently, they are also referred to as "polymer biomimetic affinity ligands" or "non-biological antibodies." Therefore, the present invention utilizes structural proteins encoded by SARS-CoV-2, such as the S protein, to analyze the crystal structure and molecular recognition mechanism of S protein binding to its host cell receptors. Through molecular docking and other methods, amino acid functional monomers with high affinity for the S protein receptor-binding domain (RBD) are obtained. The types and ratios of candidate functional monomers are then manipulated to synthesize a library of polymer nanoparticles with varying structures and surface chemical properties. Affinity experiments with the S protein RBD are then performed to screen for hydrogel polymer nanoparticles with enhanced binding capacity from the library. Using this approach, which combines rational design with directed chemical evolution, they ultimately obtained a hydrogel polymer biomimetic affinity ligand with high affinity and selectivity for the SARS-CoV-2 S protein. Furthermore, they then polymerized it with colloidal gold in situ to produce colloidal gold-labeled hydrogel polymer nanoparticles with a core-shell structure. Based on antigen detection technology, they established a lateral flow visualization detection platform that replaces biological antibodies with synthetic non-biological antibodies. This platform provides a new approach for in vitro diagnosis of SARS-CoV-2 and detection of environmental samples, and opens up more possibilities for rapid response to emerging infectious disease outbreaks in the future. Summary of the Invention

[0006] The first purpose 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 (S) and its receptor binding domain (RBD) of the new coronavirus (SARS-CoV-2) to form a stable complex, thereby realizing the 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. 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 gold nanoparticles.

[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 gold nanoparticles with 3-butenamine 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 colloidal gold surface.

[0010] Preferably, the initiator is azobisisobutyronitrile.

[0011] The present invention further provides the use of the colloidal gold-labeled hydrogel polymer nanoparticles in preparing 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-absorbing pad, wherein the nitrocellulose membrane is provided with a detection line and a quality control line, the colloidal gold-labeled hydrogel polymer nanoparticles are dropped onto the sample pad; the quality control line contains hydrogel polymer nanoparticles with a positive surface charge; 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, wherein the N-alkyl acrylamide is composed of N-isopropyl acrylamide and N-tert-butyl acrylamide, and the molar ratio of the 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 the SARS-CoV-2 virus for non-diagnostic purposes, comprising the following steps:

[0017] S1. Using gold nanoparticles as the core of the tracer marker, the surface of the gold nanoparticles is modified with 3-butenamine hydrochloride. Then, N-acryloyl-L-tryptophan, N-alkyl acrylamide, N,N'-methylenebisacrylamide, and an initiator are added to the gold nanoparticles. A free radical chain polymerization reaction is carried out under nitrogen protection. The resulting colloidal gold-labeled hydrogel polymer nanoparticles are used as affinity probes and concentrated 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 surface charges;

[0019] S3, assembling the sample pad, nitrocellulose membrane, and absorbent pad on a supporting base, spotting the sample solution on the detection line of the nitrocellulose membrane, and streaking 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 and the quality control line is colored, the test result is SARS-CoV-2 negative.

[0021] In order to achieve non-biological antibody detection of SARS-CoV-2 virus, the present invention prepared two types of hydrogel polymer nanoparticles. One is a hydrogel copolymer synthesized with N-acryloyl-L-tryptophan as the main functional monomer, and it is used 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. It binds to the above affinity probe based on electrostatic interaction to achieve the application of the detection platform quality control line (C). Based on the above two non-biological antibodies, a lateral flow detection platform was established, and a direct method was used to achieve 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, as well as 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 the SARS-CoV-2 S 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 flow visualization detection platform established by the present invention uses a colloidal gold-labeled hydrogel polymer nanoparticle affinity probe (Au@ATrp-NPs) as the detection standard and a hydrogel polymer nanoparticle (MLys-NPs) as the 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, overcoming the limitations of biological antibody development, such as high difficulty, 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, and no complex professional knowledge is required. 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-2 S protein detection.

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

[0032] The technical solution of the present invention will be clearly and completely described below with reference to 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 specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. All raw materials and reagents used were obtained from common commercial sources unless otherwise specified. Uncapped 96-well black flat-bottom polystyrene high-binding microplates were purchased from Corning Incorporated. 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 30 mmol of L-tryptophan in 15 mL of aqueous solution containing 60 mmol of sodium hydroxide. Maintain an external ice bath at 0-4°C. Once the solid has completely dissolved, slowly add 30 mmol of acryloyl chloride dropwise to the mixture using a separatory funnel. Add over 30 minutes. Continue stirring in an ice bath for 1 hour, then warm to room temperature and stir for an additional 2 hours. After adjusting the pH of the resulting product with 1 M hydrochloric acid, a solid precipitates. Vacuum filtration yields the crude product, which is then recrystallized from water to obtain N-acryloyl-L-tryptophan as a pale yellow solid.

[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 for the reaction was 65 mM, and the total solution volume was 25 mL. 0.975 mmol of N-acryloyl-L-tryptophan (60% of the total monomer content), 0.6175 mmol of N-isopropylacrylamide (38% of the total monomer content), and 0.0325 mmol of N,N'-methylenebisacrylamide (2% of the total monomer content) were dissolved in ethanol, while the remaining monomers were dissolved in water. These were then added to a 100 mL branched reaction tube. 1 mL of the surfactant SDS (10 mg / mL) was added, and the total volume was brought to 25 mL with ultrapure water. The tube was sealed with a rubber stopper, magnetically stirred, and deoxygenated three times with nitrogen. The tube was preheated to 65°C, and 1 mL of the initiator AIBN (30 mg / mL) was injected via syringe through the rubber stopper. 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 structural domains of the SARS-CoV-2 S protein RBD, NTD, and S2. The results showed that they had a high affinity for the S protein RBD, but had almost no affinity effect on the NTD and S2, indicating that the 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 for the S protein RBD was tested according to the method of CN 115141311A. The results showed that the maximum binding capacity of ATrp-NPs for 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 have a stronger binding ability to the S protein RBD.

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

[0042] The hydrogel copolymer is used as a non-biological antibody shell, and gold nanoparticles are wrapped in the core of 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 3-buteneamine hydrochloride (BA) containing a double bond 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 a 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] To a round-bottom flask, add 500 mL of ultrapure water, then add 5 mL of 25 mM HAuCl₄. The mixture is magnetically stirred and heated in an oil bath. When the solution reaches boiling, add 5 mL of a 3% (m / v) aqueous sodium citrate solution (0.15 g). Stirring and heating are continued for 15 minutes. During the reaction, the solution changes from transparent to gray and then to wine red. After the reaction is complete, remove the round-bottom flask from the oil bath, continue stirring, and cool to room temperature to obtain gold nanoparticles (Au NPs). The mixture is then stored in a refrigerator at 4°C.

[0045] Step 2: Modification of gold nanoparticles

[0046] To 100 mL of the gold nanoparticle solution, add 200 μL of 10 mg / mL SDS solution. Incubate at room temperature for 20 minutes with magnetic stirring. After the reaction, add 200 μL of 1 mg / mL 3-butenylamine hydrochloride (BA) solution to the solution and continue stirring for 20 minutes. After the reaction, centrifuge the solution at 8000 rpm for 1 hour to remove excess BA and SDS. After centrifugation, concentrate the solution to 20 mL for later use.

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

[0048] A certain amount of ultrapure water was added to a branched reaction tube, followed by 1 mL of SDS solution (10 mg / mL). 4 mL of concentrated solution of BA-modified gold nanoparticles was then added to the solution. The reaction tube was placed in a water bath with magnetic stirring and nitrogen flow for deoxygenation. When the water bath temperature reached 65°C, 0.24 mmol (60%) of N-acryloyl-L-tryptophan, 0.152 mmol (38%) of N-isopropylacrylamide, and 0.008 mmol (2%) of N,N'-methylenebisacrylamide were dissolved in a small amount of solvent. The dissolved functional monomer mixture was slowly added dropwise to the reaction tube. The reaction tube was sealed and magnetically stirred for 10 minutes to ensure uniform dispersion of the monomers. Then, 1 mL of AIBN initiator (3 mg / mL) was added to the reaction tube via syringe. Magnetic stirring was continued and the reaction was carried out at 65°C for 3 hours, resulting in a total reaction volume of 40 mL. After the reaction, the solution was centrifuged and washed three times with water 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 refrigerator at 4 °C for future use.

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

[0050] Example 3: Synthesis of hydrogel polymer nanoparticles at the 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 develops color directly affects whether the test results are valid. To achieve the red color reaction at the quality control line (C), it is only necessary to allow the material at line C to interact 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 surface charge. Based on the principle of electrostatic interaction, hydrogel polymer nanoparticles with opposite charges were 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), were used as main functional monomers, N-tert-butylacrylamide (TBAm) and N-isopropylacrylamide (NIPAm) were used as co-monomers, N,N'-methylenebisacrylamide (BIS) was used as a cross-linker, cetyltrimethylammonium bromide (CTAB) was used as a surfactant, and azobisisobutyronitrile (AIBN) was used as an initiator. The ratio of each monomer was changed, and hydrogel polymer nanoparticles with positive charge and different surface properties were synthesized by free radical polymerization.

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

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

[0056] Figure 2 The color development of positively charged hydrogel polymer nanoparticles with different monomers and different ratios combined with the affinity probe Au@ATrp-NPs shows 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 was used for determination. The sample pad, nitrocellulose membrane and absorbent pad were assembled on the supporting base. The SARS-CoV-2S protein sample was directly spotted on the detection line (T) of the nitrocellulose membrane, and the hydrogel polymer nanoparticles (MLys-NPs) synthesized in Example 3 were directly spotted on the quality control line (C) of the nitrocellulose membrane. After drying, the affinity probe Au@ATrp-NPs solution prepared in Example 2 was dropped onto the sample pad. The affinity probe moved forward to the T line by capillary action. If SARS-CoV-2S protein was present in the sample, an "Au@ATrp-NPs-antigen" complex was formed, forming a red reaction line at the T line. The unbound affinity probe continued to move forward to the C line and combined with the positively charged hydrogel polymer nanoparticles (MLys-NPs) through electrostatic interaction to form a red reaction line. Regardless of whether the target antigen was present at the T line, a red reaction line would appear at the C line to determine that the chromatography process was normal and the test result was valid. If both the T and C lines develop color simultaneously, the test result is positive for SARS-CoV-2 S protein. If the T line does not develop color but the C line does, the test result is negative for SARS-CoV-2 S protein. If the C line does not develop color, the test result is invalid. Semi-quantitative detection of SARS-CoV-2 S protein can also be performed based on the depth of color on the T line.

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

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

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

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

[0063] Unlike the more common sandwich lateral chromatography 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 detection 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, and combines 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 are shown in the figure. Figure 3 shown.

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

[0065] In lateral flow chromatography (LFC) assays, 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] Positively charged hydrogel polymer nanoparticles (MLys-NPs) were directly streaked onto the C line at a concentration of 2 mg / mL, and a SARS-CoV-2 S protein solution was directly spotted onto the T line. The test strips were then dried in a 37°C oven and cut into approximately 4 mm strips for later use. The synthesized Au@ATrp-NPs probe solution was centrifuged, the supernatant removed, and then resuspended in 10 mM PBST buffer, pH 6.0, and applied dropwise to the sample pad for lateral flow chromatography. When optimizing the probe concentration, 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), and the chromatography test is performed according to the above steps. The T-line color development is observed to optimize the optimal probe concentration. When optimizing the probe volume, the fixed probe concentration is 0.1 mg / mL (in terms of gold), the probe volume is 20-100 μL, and the chromatography test is performed according to the above steps. The T-line color development is observed to optimize the optimal probe volume.

[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 chromatography. The optimal probe concentration after optimization is 0.1 mg / mL (in terms of gold). The color development degree 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, the surface of the gold nanoparticles was modified with 3-butenylamine hydrochloride. Then, three monomers, N-acryloyl-L-tryptophan, N-isopropylacrylamide, and N,N'-methylenebisacrylamide, and the initiator azobisisobutyronitrile were added to the gold nanoparticles. A free radical chain polymerization reaction was carried out under a nitrogen atmosphere. The resulting colloidal gold-labeled hydrogel polymer nanoparticles (Au@ATrp-NPs) were used as affinity probes and concentrated for later use. The molar ratio of the three monomers was 60:38:2.

[0070] S2. Adding an initiator azobisisobutyronitrile to four monomers, namely 2-methacryloyl-lysine-NH2, N-isopropylacrylamide, N-tert-butylacrylamide, and N,N'-methylenebisacrylamide, to carry out a free radical chain polymerization reaction under a nitrogen atmosphere to synthesize hydrogel polymer nanoparticles (MLys-NPs) with positive surface charges, 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 a support base. Spot the sample solution on the detection line (T) of the nitrocellulose membrane. Streak 2 mg / mL positively charged hydrogel polymer nanoparticles (MLys-NPs) on the control line (C) of the nitrocellulose membrane, and then dry in a 37°C oven.

[0072] S4. Add the affinity probe (Au@ATrp-NPs) dropwise onto the sample pad for lateral flow 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 and the quality control line is colored, the test result is SARS-CoV-2 negative.

[0073] The actual sample was tested by lateral flow chromatography with 0.3 mg / mL of 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-2 S protein, conventional blood proteins, and protease stock solutions were diluted with 10mM PBST buffer (pH 6, containing 0.05% Tween-20) to obtain 1mg / mL concentrations of SARS-CoV-2 S protein, lysozyme, pepsin, trypsin, human serum albumin (HSA), and bovine serum albumin (BSA). Each protein sample was then spotted directly onto the detection line (T) of a nitrocellulose membrane. Subsequently, 80μL of an affinity probe (Au@ATrp-NPs) solution containing 0.1mg / mL gold was dripped onto the sample pad. Using the established lateral flow visualization detection platform, the interference of various conventional proteins and proteases with the affinity probe (Au@ATrp-NPs) detection of SARS-CoV-2 was determined.

[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. Experiments show that the colorimetric activity of lysozyme, pepsin, trypsin, human serum albumin, and bovine serum albumin is negligible compared to the detection of SARS-CoV-2 S protein at the same concentration. These results indicate that the gold-labeled hydrogel polymer nanoparticle affinity probe prepared by the present invention can specifically bind to the SARS-CoV-2 S protein.

[0078] 2. Sensitivity evaluation

[0079] SARS-CoV-2S protein was directly spotted at the T line at 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 detection. The detection time was about 15 to 20 minutes.

[0080] Figure 6 The following figure shows the lateral flow chromatography test results for different concentrations of SARS-CoV-2 S protein. ImageJ software was used to process the lateral flow chromatography test strip color development results, namely the relative grayscale value of the T line. As can be seen from the figure, the Au@ATrp-NPs affinity probe prepared in this invention can effectively bind to the SARS-CoV-2 S protein as a non-biological antibody, with good linearity in the concentration range of 0.015 to 1 mg / mL, and 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, such as 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. It will also provide more possibilities for rapid response to new and emerging infectious disease outbreaks in the future, and is of great significance to the prevention and control of emerging infectious diseases including SARS-CoV-2.

Claims

1. A colloidal gold-labeled hydrogel polymer nanoparticle having 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, wherein: 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-butenamine hydrochloride, and then N-acryloyl-L-tryptophan, N-alkyl acrylamide, and N,N'-methylenebisacrylamide were added. Then, 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 that was in situ polymerized on the colloidal gold surface.

4. The preparation method according to claim 3, wherein: 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 SARS-CoV-2 virus S protein, 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 surface charges; 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 dropwise addition volume is 20-100 μL.

9. The SARS-CoV-2 virus detection test strip according to claim 7, wherein: The hydrogel polymer nanoparticles with positive surface charges 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 components 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, the surface of the gold nanoparticles is modified with 3-butenamine hydrochloride. Then, N-acryloyl-L-tryptophan, N-alkyl acrylamide, N,N'-methylenebisacrylamide, and an initiator are added to the gold nanoparticles. A free radical chain polymerization reaction is carried out under nitrogen protection. The resulting colloidal gold-labeled hydrogel polymer nanoparticles are used as affinity probes and concentrated 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 surface charges; S3, assembling the sample pad, nitrocellulose membrane, and absorbent pad on a supporting base, spotting the sample solution on the detection line of the nitrocellulose membrane, and streaking 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 and the quality control line is colored, the test result is SARS-CoV-2 negative.

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