CN119119388B - Colloidal gold-labeled hydrogel polymer nanoparticles and their application in SARS-CoV-2 virus detection - Google Patents
Colloidal gold-labeled hydrogel polymer nanoparticles and their application in SARS-CoV-2 virus detectionInfo
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Abstract
本发明公开了一种胶体金标记的水凝胶聚合物纳米颗粒，它是由非生物抗体外壳和示踪标记物内核形成的核壳结构，所述非生物抗体外壳是由N‑丙烯酰基‑L‑色氨酸、N‑烷基丙烯酰胺、N,N’‑亚甲基双丙烯酰胺进行自由基链式聚合反应形成的水凝胶共聚物，所述示踪标记物内核是金纳米颗粒，将胶体金标记的水凝胶聚合物纳米颗粒作为亲和探针，利用其能与SARS‑CoV‑2病毒S蛋白及其受体结合域RBD特异性结合形成稳定复合物的特性，实现对SARS‑CoV‑2病毒抗原的非生物抗体快速识别与检测。本发明克服了生物抗体开发难度大、成本高、易失活等限制，提供的侧向层析检测方法准确性好、灵敏度高，为SARS‑CoV‑2病毒的体外诊断与环境样本检测提供了新方法。
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
The invention belongs to the field of biotechnology, and relates to a colloidal gold-labeled hydrogel polymer nanoparticle, and a preparation method and application of the colloidal gold-labeled hydrogel polymer nanoparticle in SARS-CoV-2 virus detection.
Background
The novel coronavirus (SARS-CoV-2), abbreviated as "New coronavirus", is a third highly pathogenic beta-human coronavirus found after SARS-CoV and MERS-CoV and capable of causing severe acute respiratory diseases. As a serious epidemic disease, rapid diagnosis of a new coronavirus is important, and the detection method mainly comprises an etiology detection method, a molecular biology detection method, an immunological antibody detection method and an antigen detection method. Antigen detection is usually used for early detection of patients suspected to be infected with a new coronavirus, mainly detects a new coronavirus structural protein nucleocapsid protein (N) or spike protein (S), wherein both structural proteins are specific proteins of the new coronavirus, and the detection principle is based on antigen-antibody immune reaction or aptamer hybridization, and the detection principle is converted into detection signals by adopting colorimetric, fluorescent, electrochemical and other modes.
Lateral chromatography detection is a platform based on paper chromatography and is widely used for rapid and on-site detection, wherein the detection result can be obtained based on antigen-antibody immune reaction and can be called as flow-measuring immunochromatography detection. As a more common antigen detection method, the flow measurement immunochromatography detection combines the immunodetection and the chromatographic separation, and has the advantages of simple operation, low cost, easy carrying, instant diagnosis and the like. The colored reagents such as colloidal gold, latex, europium, chelate-loaded silicon dioxide and the like are color detection reagents commonly used in lateral chromatography technology, wherein the colloidal gold (gold nanoparticles, au) is the most commonly used, and the novel coronavirus antigen detection kit based on the colloidal gold method is a common means for performing self-diagnosis on the early stage of a novel coronavirus patient. The colloid Jin Kangyuan detection test strip comprises a carrier, a nitrocellulose membrane coated with a detection line (T line and containing monoclonal antibody) and a quality control line (C line and containing anti-antibody), a gold-labeled pad for carrying a gold-labeled antibody (colloidal gold-labeled monoclonal antibody), a sample pad for receiving a sample solution and absorbent paper for guiding the sample solution to sequentially pass through the sample pad, the gold-labeled pad and the nitrocellulose membrane. During detection, a sample to be detected is dripped into a sample hole, and is subjected to forward chromatography under the capillary action, when the chromatography reaches a T line, an antibody-antigen-gold-labeled antibody complex is formed when a target antigen exists, so that a red reaction line appears, namely positive, and no red reaction line appears, namely negative. A red reaction line appears on the C line no matter whether the target antigen exists or not, so that whether the chromatographic process is normal or not can be judged, and whether the detection is effective or not can be judged.
The hydrogel polymer nano-particles are high molecular polymers formed by linking C-C bonds, and are formed by free radical initiated polymerization of various functional monomers, wherein the main chain is a C-C long chain, the side chain is various functional monomers, and the functional monomers commonly related to the hydrogel polymer nano-particles include acrylamide and methacrylic acid derivatives, such as N-acryloyl-L-amino acid and 2-methacryloyl-L-amino acid-NH 2. Whether the hydrogel copolymer can realize a specific molecular recognition function is critical to whether the functional monomer introduced into the side chain of the hydrogel copolymer can provide multiple interactions such as hydrophobic, hydrogen bond, static electricity, pi-pi stacking effect and the like which are highly complementary with the target biomolecule binding site, and the interactions are closely related to the hydrophobic, hydrophilic and/or charged groups carried by the functional monomer. The variety and the proportion of each functional monomer are adjustable, so that the synthesized hydrogel polymer nanoparticles have wider chemical diversity, and by utilizing the characteristics, a polymer nanoparticle library with flexible and diverse structures and functions can be constructed. The hydrogel polymer nanoparticle has the advantages of simple preparation process, low cost, contribution to large-scale and rapid production, good stability and robustness, strong tolerance to external environments such as pH, temperature and the like, and is used for extracting, separating, purifying and removing target biomolecules in various complex systems, and is more expected to replace natural antibodies to play specific roles and functions in the field of biotechnology.
Functional monomers, particularly amino acid monomers, introduced into the side chains of the artificially synthesized polymer nanoparticles are similar to the structure and properties of amino acid side groups in the peptide chains of protein antibodies, and can specifically recognize target biological molecules to achieve the effect similar to that of biological antibodies/receptors, and are also called as "polymer bionic affinity ligands" or "non-biological antibodies" because the functional monomers can simulate the specific multiple complementary interactions (hydration complementarity, hydropathic complementarity) between natural biological macromolecules such as antigen-antibodies, so that the functional monomers have the function similar to that of antibody recognition antigens. Therefore, the invention utilizes structural proteins coded by SARS-CoV-2, such as S protein, to analyze the crystal structure information and molecular recognition mechanism of the S protein combined with host cell receptor, obtains amino acid functional monomers with higher affinity to the S protein receptor combined domain (receptor binding domain, RBD) through means of molecular docking and the like, regulates and controls the types and the proportion of candidate functional monomers, synthesizes a series of polymer nano particles with different structures and surface chemical properties to form a library, and screens out hydrogel polymer nano particles with stronger combining ability from the library through an affinity experiment to the S protein RBD. By utilizing the method based on the combination of rational design and directed chemical evolution, the hydrogel polymer bionic affinity ligand with high affinity and high selectivity for the novel coronavirus S protein is finally obtained. On the basis, the nanometer particle is polymerized with colloid Jin Yuanwei to obtain hydrogel polymer nanometer particles marked by colloidal gold with a core-shell structure, and a lateral chromatography visual detection platform which replaces biological antibodies with artificially synthesized non-biological antibodies is established based on antigen detection technology, thereby providing a new method for SARS-CoV-2 in-vitro diagnosis and environmental sample detection and providing more possibility for rapid response of epidemic situation of new emergent infectious diseases in the future.
Disclosure of Invention
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 uses the characteristic that the colloidal gold-labeled hydrogel polymer nanoparticle can specifically bind with spike protein (spike, S) of a novel coronavirus (SARS-CoV-2) and a receptor binding domain (receptor binding domain, RBD) thereof to form a stable complex, so as to realize rapid identification and detection of an abiotic antibody of a SARS-CoV-2 virus antigen.
The invention provides an affinity probe which is a core-shell structure formed by a non-biological antibody shell and a trace marker inner core, wherein the non-biological antibody shell is a hydrogel copolymer (ATrp-NPs) formed by free radical chain polymerization reaction of N-acryl-L-tryptophan (ATrp), N-alkyl acrylamide and N, N' -methylene bisacrylamide, and the trace marker inner core is gold nanoparticles.
Further, the molar ratio of the N-acryloyl-L-tryptophan, the N-alkyl acrylamide and the N, N' -methylene bisacrylamide is 20-80:15-80:1-5.
A method for preparing the colloidal gold-labeled hydrogel polymer nanoparticles (Au@ATrp-NPs) comprises the following steps of firstly carrying out 3-butenamine hydrochloride modification on gold nanoparticles, then adding N-acryl-L-tryptophan, N-alkylacrylamide and N, N' -methylenebisacrylamide, then adding an initiator, and carrying out free radical chain polymerization reaction on the surfaces of the gold nanoparticles under the protection of nitrogen to generate hydrogel copolymer in situ and polymerizing on the surfaces of the colloidal gold.
Preferably, the initiator is azobisisobutyronitrile.
The invention further provides application of the colloidal gold-labeled hydrogel polymer nanoparticle in preparing a SARS-CoV-2 virus detection test strip and the SARS-CoV-2 virus detection test strip.
Preferably, the test strip is a lateral chromatographic test strip of an abiotic antibody for detecting SARS-CoV-2 virus S protein, and the test strip contains the colloidal gold-labeled hydrogel polymer nanoparticles.
Further preferably, the test strip comprises a sample pad, a nitrocellulose membrane and a water absorption pad, wherein the nitrocellulose membrane is provided with a detection line and a quality control line, the hydrogel polymer nanoparticles marked by colloidal gold are dripped on 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.
Further preferably, the colloidal gold-labeled hydrogel polymer nanoparticles are used at a concentration of 0.01-0.2mg/mL and a drop volume of 20-100. Mu.L.
Further preferably, the hydrogel polymer nanoparticles with positive charges on the surface are formed by free radical chain polymerization of 2-methacryloyl-lysine-NH 2 (MLys), N-alkylacrylamide and N, N '-methylenebisacrylamide, wherein the N-alkylacrylamide consists of N-isopropylacrylamide and N-tert-butylacrylamide, and the molar ratio of the four 2-methacryloyl-lysine-NH 2, N-isopropylacrylamide, N-tert-butylacrylamide and N, N' -methylenebisacrylamide is 20-40:15-35:30-50:1-5.
Another object of the present invention is to provide a method for detecting SARS-CoV-2 virus non-diagnostic purpose, comprising the steps of:
S1, taking gold nanoparticles as the inner core of a tracer marker, carrying out 3-butenamine hydrochloride modification on the surface of the gold nanoparticles, then adding N-acryl-L-tryptophan, N-alkylacrylamide, N' -methylenebisacrylamide and an initiator into the gold nanoparticles, carrying out free radical chain polymerization under the protection of nitrogen, taking the colloidal gold-labeled hydrogel polymer nanoparticles obtained by the reaction as an affinity probe, and concentrating for later use;
s2, adding an initiator into 2-methacryloyl-lysine-NH 2, N-alkyl acrylamide and N, N' -methylene bisacrylamide, and carrying out free radical chain polymerization under the protection of nitrogen to synthesize hydrogel polymer nano particles with positive charges on the surfaces;
S3, assembling a sample pad, a nitrocellulose membrane and a water absorption pad on a supporting bottom plate, spotting a sample solution to be detected on a detection line of the nitrocellulose membrane, marking hydrogel polymer nano particles with positive charges on the surface prepared in the step S2 on a quality control line of the nitrocellulose membrane, and then drying;
S4, dripping the affinity probe prepared in the step S1 onto a sample pad for lateral chromatography detection, wherein if the detection line and the quality control line develop color simultaneously, the detection result is SARS-CoV-2 positive, and if the detection line does not develop color, the quality control line develops color, and the detection result is SARS-CoV-2 negative.
In order to realize the non-biological antibody detection of SARS-CoV-2 virus, two hydrogel polymer nano particles are prepared respectively, one is a hydrogel copolymer synthesized by taking N-acryl-L-tryptophan as a main functional monomer, and the hydrogel copolymer is taken as a non-biological antibody shell to wrap gold nano particles, so that a colloidal gold marked hydrogel polymer nano particle affinity probe (Au@ATrp-NPs) is formed, the colloidal gold marked hydrogel polymer nano particle affinity probe can specifically identify and combine with the RBD structural domain of SARS-CoV-2S protein, and the other is a hydrogel polymer nano particle (MLys-NPs) synthesized by taking 2-methacryl-lysine-NH 2 as the main functional monomer, and the hydrogel polymer nano particle is combined with the affinity probe based on electrostatic action, so that the application of a quality control line (C) of a detection platform is realized. Based on the two abiotic antibodies, a lateral chromatography detection platform is established, and a direct method is adopted to realize the rapid detection of the novel coronavirus.
The detection method constructed by the invention can be used for diagnosing SARS-CoV-2 virus diseases, and can also be used for screening and identifying SARS-CoV-2 virus laboratory and detecting environment for non-disease diagnosis.
The beneficial effects of the invention are as follows:
the invention uses hydrogel polymer nano particles as non-biological antibody to specifically bind SARS-CoV-2S protein, the non-biological antibody has excellent affinity, and compared with biological antibody, the invention has the advantages of mild synthesis condition, simple preparation process, low cost, good stability, and higher biocompatibility and plasticity.
The non-biological antibody lateral chromatography visual detection platform established by the invention takes a colloidal gold-labeled hydrogel polymer nanoparticle affinity probe (Au@ATrp-NPs) as a detection standard, takes a hydrogel polymer nanoparticle (MLys-NPs) as a quality control standard, has no biological antibody, is low in preparation cost, easy to store and good in stability, is used for replacing the original biological antibody, overcomes the limitations of high development difficulty, high cost, poor environmental tolerance, easy inactivation, severe storage and reaction conditions and the like of the biological antibody, simultaneously has the advantages of no need of a special interpretation instrument, short reaction time, capability of completing detection within 15-20 min, visual reading of results, no need of mastering complex expertise and certain application potential in the aspects of qualitative and semi-quantitative detection.
Drawings
FIG. 1 is a transmission electron microscope image of an affinity probe Au@ATrp-NPs.
FIG. 2 screening of hydrogel polymer nanoparticles at the quality control line (C).
FIG. 3 is a schematic diagram of the test strip construction and the detection steps thereof.
FIG. 4 is a schematic diagram of the test result of the test strip practical sample.
FIG. 5 shows the results of a specific assay for SARS-CoV-2S protein detection.
FIG. 6 shows the results of a sensitivity test for SARS-CoV-2S protein detection.
Detailed Description
The following description of the embodiments of the present invention will be made more apparent and fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.
The following examples are not to be construed as limiting the scope of the invention in any way, and all materials and reagents used may be obtained from the usual commercial sources, unless otherwise specified, as per the conditions under which they are conventionally used or as suggested by the manufacturer. The uncovered 96-well black flat-bottom polystyrene high-binding-force microwell plate is purchased from corning company, U.S., wild recombinant SARS-CoV-2S protein trimer, S protein RBD, S protein NTD, S2 protein from general biological (Wuhan) technology Co., ltd, and Fluorescein Isothiocyanate (FITC) labeling is performed by Beijing biological technology Co., ltd (Beijing).
EXAMPLE 1 preparation of hydrogel Polymer nanoparticles
Step one, synthesis of N-acryl-L-tryptophan monomer
30Mmol of L-tryptophan is dissolved in 15mL of water solution containing 60mmol of sodium hydroxide, external ice bath is kept at the temperature of 0-4 ℃, after the solid is completely dissolved, 30mmol of acryloyl chloride is slowly and dropwise added into the mixture by using a separating funnel for more than 30min, stirring reaction is continued for 1h under the ice bath condition after the dropwise addition, and then the temperature is raised to room temperature and stirring is further carried out for 2h. After the pH of the obtained product is regulated by 1M hydrochloric acid, solid is separated out and precipitated, the crude product is obtained by vacuum suction filtration, and the product is recrystallized in water to finally obtain the light yellow solid product N-acryloyl-L-tryptophan.
Step two, synthesis of hydrogel Polymer nanoparticles (ATrp-NPs)
The hydrogel polymer nanoparticles are synthesized by a free radical initiated polymerization method based on different functional monomers, wherein N-acryl-L-tryptophan is used as a main functional monomer, N-isopropyl acrylamide (NIPAm) is used as a coexisting monomer, N, N' -methylenebisacrylamide (BIS) is used as a cross-linking agent, sodium Dodecyl Sulfate (SDS) is used as a surfactant, and Azobisisobutyronitrile (AIBN) is used as an initiator to synthesize the hydrogel polymer nanoparticles with different surface properties.
The total concentration of monomers reacted was 65mM and the total volume of solution was 25mL. 0.975mmol (60% of the total monomer amount), 0.6175mmol (38% of the total monomer amount) of N-isopropyl acrylamide and 0.0325: 0.0325mmoL (2% of the total monomer amount) of N, N' -methylene bisacrylamide were taken respectively, wherein the amino acid functional monomer was dissolved in ethanol, the remaining monomer was dissolved in water, and then added to a 100mL branched reaction tube, 1mL of surfactant SDS (10 mg/mL) was added, and the total volume was made up to 25mL with ultrapure water. The rubber stopper was sealed, magnetically stirred, deoxygenated 3 times with nitrogen, preheated to 65 ℃, and 1mL of initiator AIBN (30 mg/mL) was injected from the rubber stopper sealed neck with a syringe. The reaction was continued with stirring at 65 ℃ under nitrogen protection for 3 hours, then after cooling to room temperature, the reacted liquid was transferred to a dialysis bag prepared in advance, and dialyzed in 3L of ultrapure water for 4 days to purify hydrogel polymer nanoparticles, water was changed once a day in the morning and evening, and after the dialysis was completed, the obtained hydrogel polymer nanoparticles were stored in a 4 ℃ refrigerator.
The hydrogel polymer nanoparticles (ATrp-NPs) synthesized in the second step are used for affinity adsorption of three domains of SARS-CoV-2S protein RBD, NTD and S2, and as a result, the high affinity of the hydrogel polymer nanoparticles to the S protein RBD is found, and the affinity effect to the NTD and the S2 is hardly observed, so that the RBD is the main area of the binding of the hydrogel polymer nanoparticles (ATrp-NPs) to the SARS-CoV-2S protein. In addition, the binding capacity of the hydrogel polymer nanoparticles to the S protein RBD was examined by referring to the method of CN 115141311A, and the result shows that the maximum binding capacity of ATrp-NPs to RBD-Fitc protein is 2620.6mg/g, and the binding capacity of ATrp-NPs to the S protein RBD is stronger than that of the polymer nanoparticles which are constructed before and take N-acryl-L-phenylalanine as a main functional monomer (the maximum binding capacity is 1035.7 mg/g).
Example 2 Synthesis of colloidal gold-labeled hydrogel Polymer nanoparticle affinity probes (Au@ATrp-NPs)
The hydrogel copolymer is used as a non-biological antibody shell, the gold nanoparticles are used as a tracing marker and are wrapped in the inner core by the hydrogel copolymer, and a colloidal gold-labeled hydrogel polymer nanoparticle affinity probe (Au@ATrp-NPs) is prepared so as to realize the lateral chromatography rapid detection of SARS-CoV-2 virus. Firstly, synthesizing gold nanoparticles by adopting a sodium citrate reduction method, then modifying 3-butenamine hydrochloride (BA) containing double bonds to the surfaces of the gold nanoparticles, and finally polymerizing functional monomers (ATrp) and coexisting monomers (NIPAm) to the surfaces of the gold nanoparticles in situ by a free radical polymerization reaction according to the method of example 1 to form the colloidal gold-labeled hydrogel polymer nanoparticle affinity probe with a core-shell structure. The method comprises the following specific steps:
step one, synthesis of gold nanoparticles
500ML of ultrapure water was added to the round-bottomed flask, and then 5mL of HAuCl 4 (25 mM) was taken in the round-bottomed flask, magnetically stirred, and heated in an oil bath. While the solution was boiling, 5mL of 3% (m/v) aqueous sodium citrate (0.15 g) was added to the solution at the same time, and the stirring and heating reaction was continued for 15min. The solution changed from transparent to grey and then from grey to reddish wine during the reaction. After the reaction is finished, the round-bottom flask is removed from the oil bath, and is continuously stirred and cooled to room temperature to obtain gold nanoparticles (Au NPs), and the gold nanoparticles are stored in a refrigerator at 4 ℃.
Step two, modification of gold nanoparticles
100ML of the gold nanoparticle solution is taken, 200 mu L of SDS solution (10 mg/mL) is added, the reaction is carried out for 20min at room temperature, and the magnetic stirring is carried out during the reaction process. After the completion of the reaction, 200. Mu.L of 3-butenamine hydrochloride (BA) solution (1 mg/mL) was added to the above solution, followed by stirring and reaction for 20 minutes. After the reaction was completed, the reaction solution was centrifuged to remove excess BA and SDS at 8000rpm for 1 hour. After centrifugation, the solution was concentrated to 20mL for use.
Step three, synthesis of gel gold marked hydrogel polymer nanoparticle affinity probe (Au@ATrp-NPs)
A certain amount of ultrapure water was added to the branched reaction tube, then 1mL of SDS solution (10 mg/mL) was added, and then 4mL of the BA-modified gold nanoparticle concentrated solution was added to the above solution. The reaction tube is placed in a water bath kettle, nitrogen and oxygen are continuously introduced while magnetic stirring is carried out, when the temperature of the water bath kettle is raised to 65 ℃, a small amount of solution is used for dissolving 0.24mmol (60%), 0.152mmol (38%) of N-acryl-L-tryptophan and 0.008mmoL (2%) of N, N' -methylenebisacrylamide monomers, the dissolved functional monomer mixed solution is slowly dripped into the reaction tube, the reaction tube is sealed, the magnetic stirring is carried out for 10min to ensure that the monomers are uniformly dispersed, then 1mL of AIBN initiator (3 mg/mL) is added into the reaction tube by a syringe, the magnetic stirring is carried out continuously, the reaction is carried out for 3h at 65 ℃, and the total reaction volume is 40mL. After the reaction is finished, the mixture is centrifuged and washed for 3 times to remove unreacted monomers and SDS, the centrifugation speed is 8000rpm, the centrifugation time is 1h, finally, the synthesized affinity probe Au@ATrp-NPs solution is concentrated to 20mL, the concentration is 0.05mg/mL (gold is taken as the standard, gold loss is ignored), and the purified affinity probe solution is placed in a refrigerator at 4 ℃ for standby.
The transmission electron microscope of the affinity probe Au@ATrp-NPs in FIG. 1 shows that a layer of hydrogel copolymer is obviously wrapped outside the gold nanoparticles, so that an obvious core-shell microsphere structure is formed, and the dispersion is uniform, thus showing that the probe is successfully synthesized.
Example 3 Synthesis of hydrogel Polymer nanoparticles at quality control line (C)
The red reaction presented by the quality control line (C) can judge whether the chromatography process of the affinity probe is normal or not, and whether the color development direct relation detection result is effective or not. The red color reaction at the quality control line (C) is realized, and the material at the C line and the affinity probe only need to act. The detection affinity probe is a hydrogel polymer nanoparticle affinity probe Au@ATrp-NPs marked by colloidal gold, and the surface of the detection affinity probe presents negative charges. Based on the principle of electrostatic interaction, hydrogel polymer nanoparticles with opposite charges are selected as the quality control line (C).
Step one, synthesis of positively charged hydrogel polymer nanoparticles
According to the method for synthesizing the hydrogel polymer nanoparticles in reference example 1, the mixture ratio of three positively charged amino acid monomers, namely 2-methacryloyl-arginine-NH 2 (MArg), 2-methacryloyl-lysine-NH 2 (MLys) and 2-methacryloyl-histidine-NH 2 (MHis), are used as main functional monomers, N-tertiary butyl acrylamide (TBAm) and N-isopropyl acrylamide (NIPAm) are used as coexisting monomers, N, N' -methylenebisacrylamide (BIS) is used as a cross-linking agent, cetyltrimethylammonium bromide (CTAB) is used as a surfactant, azobisisobutyronitrile (AIBN) is used as an initiator, and the hydrogel polymer nanoparticles with positive charges and different surface properties are synthesized by adopting a free radical initiated polymerization method.
Step two, screening non-biological antibody hydrogel polymer nano particles at the quality control line (C)
And (3) assembling the sample pad, the nitrocellulose membrane and the water absorption pad on a supporting bottom plate, directly scribing the hydrogel polymer nano particles with positive charges synthesized in the step (I) at the position of a C line, placing a scribed test strip with the scribing concentration of 1mg/mL in a 37 ℃ oven for drying, and cutting the test strip into thin strips with the thickness of about 4mm for later use. The affinity probe Au@ATrp-NPs synthesized in example 2 above was resuspended in 10mM PBST buffer pH 6.0 and was applied dropwise to the sample pad for lateral chromatography detection at a probe volume of 80. Mu.L. After waiting for 15min, comparing the color development condition of the C line of each group of test strips to evaluate the binding performance of the hydrogel polymer nanoparticles with different positive charges on the affinity probes, and screening out the hydrogel polymer nanoparticles meeting the requirements.
In fig. 2, the color development of the combination of the positively charged hydrogel polymer nanoparticles with different monomers and different ratios and the affinity probe au@atrp-NPs shows that the color development degree of the two hydrogel polymer nanoparticles of NP6 and NP9 is obvious, and the NP6 is more stable from the stability point of view, namely, the hydrogel polymer nanoparticles (MLys-NPs) prepared when MLys accounts for 30% of the total monomers, NIPAm accounts for 28% of the total monomers and TBAm accounts for 40% of the total monomers are used as the quality control standard at the quality control line (C) of the lateral chromatography visual detection platform.
Example 4 establishment of lateral chromatography visual detection platform
The method comprises the steps of adopting a lateral chromatography detection method to carry out measurement, assembling a sample pad, a nitrocellulose membrane and a water absorption pad on a supporting bottom plate, directly connecting a SARS-CoV-2S protein sample to a detection line (T) of the nitrocellulose membrane, directly connecting hydrogel polymer nano particles (MLys-NPs) synthesized in the embodiment 3 to a quality control line (C) of the nitrocellulose membrane, dripping an affinity probe Au@ATrp-NPs solution prepared in the embodiment 2 to the sample pad after drying, moving the affinity probe forward by capillary action to the T line, forming an Au@ATrp-NPs-antigen complex if SARS-CoV-2S protein exists in the sample, forming a red reaction line at the T line, continuously moving the unbound affinity probe forward to the C line, combining the positively charged hydrogel polymer nano particles (MLys-NPs) to form a red reaction line through electrostatic action, judging whether target antigen exists at the T line or not, and forming the red reaction line at the C line, so that the detection result is normal, and the detection process is effective. The T line and the C line develop color simultaneously, the detection result is SARS-CoV-2S protein positive, the T line does not develop color, the C line develops color, the detection result is SARS-CoV-2S protein negative, the C line does not develop color, and the detection result is invalid. Meanwhile, the SARS-CoV-2S protein can be semi-quantitatively detected according to the shade of the color on the T line.
Step one, synthesizing and scribing hydrogel polymer nano particles at the position of a quality control line (C)
The hydrogel polymer nanoparticles were synthesized as described in example 3, with a total monomer concentration of 16.25mM and a total volume of 25mL determined. The ratio of each monomer was 2-methacryloyl-lysine-NH 2 0.1219mmol (30% of the total monomer), N-isopropylacrylamide (NIPAm) 0.1138mmol (28% of the total monomer), N-t-butylacrylamide (TBAm) 0.1625mmol (40% of the total monomer), N' -methylenebisacrylamide (BIS) 0.008125mmol (2% of the total monomer). Cetyl trimethylammonium bromide (CTAB) is used as a surfactant and Azobisisobutyronitrile (AIBN) is used as an initiator. The resulting hydrogel polymer nanoparticles (MLys-NPs) were stored in a 4℃refrigerator and the concentration was determined to be 3.98mg/mL.
In order to optimize the scribing concentration of the hydrogel polymer nanoparticles (MLys-NPs) on the quality control line (C), the scribing concentrations of 0.1, 0.5, 1, 2 and 3.98mg/mL are selected, the scribing concentrations are respectively scribed at the quality control line (C), and then the scribing concentrations are dried in a 37 ℃ oven, so that the test strip is cut into thin strips with the thickness of about 4mm for standby. The synthesized Au@ATrp-NPs probe solution was centrifuged to remove the supernatant, then resuspended in 10mM PBST buffer, and lateral chromatography was performed by dropping onto the sample pad, and the color development of the C line was observed to give an optimal streaking concentration of 2mg/mL.
Construction of lateral chromatography visual detection platform
Unlike the common sandwich lateral chromatographic colloidal gold method, the detection method utilizes two non-biological antibody hydrogel polymer nanoparticles and adopts a direct method for detection. The sample pad, nitrocellulose membrane and absorbent pad were assembled on a support base plate, and a test sample containing SARS-CoV-2S protein and positively charged hydrogel polymer nanoparticles (MLys-NPs) were spotted on a detection line (T) and a quality control line (C) of the nitrocellulose membrane, respectively, and then dried in an oven at 37 ℃. Subsequently, an affinity probe Au@ATrp-NPs solution was dropped on the sample pad, and the affinity probe was moved forward by capillary action to bind to SARS-CoV-2S protein on the T line and MLys-NPs on the C line for color development. The structure of the test strip and its detection schematic diagram are shown in FIG. 3.
Step three, optimizing the concentration and the volume of the lateral chromatography detection affinity probe
In the lateral flow chromatography detection experiments, the amount of the added affinity probe affects the T-line color development result, so that the probe use concentration and volume are necessary to be optimized.
Positively charged hydrogel polymer nanoparticles (MLys-NPs) were directly streaked at the C-line position at a streaking concentration of 2mg/mL, and SARS-CoV-2S protein solution was directly spotted at the T-line position. Subsequently, the test strips were dried in an oven at 37℃and cut into thin strips of about 4mm for use, the synthesized Au@ATrp-NPs probe solution was centrifuged, the supernatant was removed, and then resuspended in 10mM PBST buffer pH 6.0 and added dropwise to the sample pad for lateral chromatography detection. When the probe use concentration is optimized, the immobilized probe use volume is 40 mu L, the probe use concentration is controlled to be in the range of 0.0125-0.2mg/mL (calculated by gold), chromatographic detection is carried out according to the steps, the color development condition of the T line is observed, the optimal probe use concentration is optimized, when the probe use volume is optimized, the immobilized probe use concentration is 0.1mg/mL (calculated by gold), the probe use volume is 20-100 mu L, chromatographic detection is carried out according to the steps, the color development condition of the T line is observed, and the optimal probe use volume is optimized.
And (3) analyzing the result that when the concentration of the affinity probe is too low, the color development is not obvious enough after chromatography, when the concentration is too high, the viscosity of the probe solution is increased, the probe runs slowly in the chromatography process, the optimized optimal probe concentration is 0.1mg/mL (calculated by gold), the color development degree of the lateral chromatography test strip is increased along with the increase of the probe volume, the stability is realized, and the optimized optimal probe volume is 80 mu L.
The lateral chromatography visual detection method is finally determined by combining the tests and is as follows:
S1, taking gold nanoparticles as a tracing marker kernel, carrying out 3-butenamine hydrochloride modification on the surface of the gold nanoparticles, then adding three monomers of N-acryl-L-tryptophan, N-isopropyl acrylamide and N, N' -methylene bisacrylamide and an initiator azodiisobutyronitrile into the gold nanoparticles, carrying out free radical chain polymerization under a nitrogen atmosphere, taking colloidal gold-labeled hydrogel polymer nanoparticles (Au@ATrp-NPs) obtained by the reaction as an affinity probe, and concentrating for standby, wherein the molar ratio of the three monomers is 60:38:2;
S2, adding an initiator azodiisobutyronitrile into four monomers of 2-methacryloyl-lysine-NH 2, N-isopropyl acrylamide, N-tert-butyl acrylamide and N, N' -methylene bisacrylamide, and carrying out free radical chain polymerization reaction in a nitrogen atmosphere to synthesize hydrogel polymer nano particles (MLys-NPs) with positive charges on the surfaces, wherein the molar ratio of the four monomers is 30:28:40:2;
S3, assembling a sample pad, a nitrocellulose membrane and a water absorption pad on a supporting base plate, spotting a sample solution to be detected on a detection line (T) of the nitrocellulose membrane, marking hydrogel polymer nano particles (MLys-NPs) with positive charges of 2mg/mL on a quality control line (C) of the nitrocellulose membrane, and drying in a 37 ℃ oven;
S4, dropwise adding an affinity probe (Au@ATrp-NPs) to the sample pad for lateral chromatography detection, wherein the concentration of the affinity probe is 0.1mg/mL (calculated by gold), the dropwise adding volume is 80 mu L, the detection result is SARS-CoV-2 positive if the detection line and the quality control line are both colored, and the detection result is SARS-CoV-2 negative if the detection line is not colored and the quality control line is colored.
The actual lateral chromatography assay was performed with 0.3mg/mL SARS-CoV-2S protein and the results are shown in FIG. 4.
Example 5 evaluation of lateral chromatography detection method
1. Selectivity evaluation
SARS-CoV-2S protein, normal proteins in blood and protease stock solution were diluted with 10mM PBST buffer solution containing 0.05% Tween-20 at pH 6 to give SARS-CoV-2S protein, lysozyme (Lysozyme), pepsin (Pepsion), trypsin (Trypsion), human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) at a concentration of 1mg/mL, and then each protein sample was directly connected to a detection line (T) of nitrocellulose membrane, followed by dropping 80. Mu.L of an affinity probe (Au@ATrp-NPs) solution having a gold content of 0.1mg/mL onto the sample pad. And judging the interference of different conventional proteins and proteases on an affinity probe (Au@ATrp-NPs) to detect SARS-CoV-2 by using the established lateral chromatography visual detection platform.
The detection result of the selectively combined lateral chromatographic test strip and the relative gray value of the corresponding T line are shown in FIG. 5. Experiments show that the color development of lysozyme, pepsin, trypsin, human serum albumin and bovine serum albumin is almost negligible compared with the detection of SARS-CoV-2S protein under the same concentration condition. The result shows that the gold-labeled hydrogel polymer nanoparticle affinity probe prepared by the invention can specifically bind SARS-CoV-2S protein.
2. Sensitivity evaluation
Directly spotting SARS-CoV-2S protein at the T line, wherein the concentrations are 1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015 and 0.0075mg/mL respectively, placing the spotted test strip in a 37 ℃ oven for drying, dripping 80 mu L of Au@ATrp-NPs affinity probe solution with the gold content of 0.1mg/mL on a sample pad, and performing lateral chromatography detection for about 15-20 min.
FIG. 6 shows the results of lateral chromatography detection of SARS-CoV-2S protein at different concentrations, and the results of lateral flow chromatography test strip development using imageJ software, i.e. T-line relative gray scale values, were data processed. As shown in the figure, the Au@ATrp-NPs affinity probe prepared by the invention can be used as a non-biological antibody to effectively bind SARS-CoV-2S protein, has good linear relation within the concentration range of 0.015-1 mg/mL, and has the lowest detection limit of 0.0075mg/mL.
The detection method based on the artificial synthetic non-biological antibody is low in cost, easy to store and good in stability, and overcomes the limitations of high development difficulty, high cost, poor environmental tolerance, easy inactivation, severe storage and reaction conditions and the like of the biological antibody. The invention can be used for rapid detection and screening of actual samples, and has certain application potential in qualitative and semi-quantitative detection. The established detection method provides a new method for in vitro diagnosis of SARS-CoV-2 and detection of environmental samples, and simultaneously provides more possibility for rapid response of epidemic situation of new outburst infectious disease in the future, and has important significance for prevention and control of the outburst infectious disease including SARS-CoV-2.
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