WO2022094932A1 - Novel coronavirus detection test strip, and preparation method and use method therefor - Google Patents

Novel coronavirus detection test strip, and preparation method and use method therefor Download PDF

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WO2022094932A1
WO2022094932A1 PCT/CN2020/127148 CN2020127148W WO2022094932A1 WO 2022094932 A1 WO2022094932 A1 WO 2022094932A1 CN 2020127148 W CN2020127148 W CN 2020127148W WO 2022094932 A1 WO2022094932 A1 WO 2022094932A1
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sialic acid
group
molecule
acid ligand
detection
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PCT/CN2020/127148
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French (fr)
Chinese (zh)
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李伟
王怀雨
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深圳先进技术研究院
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Priority to PCT/CN2020/127148 priority Critical patent/WO2022094932A1/en
Publication of WO2022094932A1 publication Critical patent/WO2022094932A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the technical field of biochemistry, and relates to a new coronavirus detection test strip and a preparation method and use method thereof.
  • the novel coronavirus pneumonia (“COVID-19” for short) is a major public health emergency that has infected tens of millions of people and killed hundreds of thousands of people worldwide.
  • the pathogen causing new coronary pneumonia is a new type of coronavirus (referred to as "new coronavirus”), which was named SARS-Cov-2 (Severe acute respiratory syndromes coronavirus 2) by the International Committee on Taxonomy of Viruses.
  • a key to the prevention and control of new coronary pneumonia is to develop effective medical diagnosis and treatment methods for the new coronavirus, of which timely and accurate detection of the new coronavirus is crucial to the prevention and control of the epidemic.
  • the detection technology of the new coronavirus mainly focuses on the detection of the RNA sequence of the new coronavirus, and the detection of the IgM and IgG antibodies produced by the human immune system after the infection of the new coronavirus.
  • the detection of new coronavirus RNA mainly includes real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR) method and CRISPR (clustered regularly interspaced short palindromic repeats) method.
  • RT-PCR real-time fluorescent reverse transcription-polymerase chain reaction
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the human immune system will produce specific IgM and IgG antibodies after infection with the new coronavirus to indirectly detect whether there is a new coronavirus infection, especially the colloidal gold method.
  • the colloidal gold method is also known as immunochromatography. Its main principle is to use the sample solution dropped on one end of the membrane to move to the other end by the capillary action of the membrane (lateral flow based on chromatography), and the analyte moves during the movement. It binds to a receptor immobilized on a certain area of the membrane and becomes immobilized, and the immobilization result is judged by developing the color of a marker, which is usually colored by colloidal gold.
  • the IgG/IgM test cards for 2019-nCoV on the market also use immunochromatography to detect the presence of 2019-nCoV IgG/IgM antibodies in human blood, mainly by labeling anti-IgG/anti-IgM antibodies on colloidal gold. After the IgG or IgM in the membrane is immunocombined, it binds to the receptor immobilized on the membrane and becomes immobilized, and the result can be quickly determined.
  • the current mainstream new coronavirus RNA detection method is RT-PCR. Although it can directly detect the virus, it needs to manually extract the RNA virus in the sample, which is affected by multiple factors such as sample sampling method, sample transportation and preservation, and manual operation. Staff reported that this method was often accompanied by serious false-negative interference, which challenged the test results; in addition, a small number of RNA test results with positive results did not show contagiousness, so there was also false-positive interference in test results, and their positive results
  • the test result may be that the test sample contains nucleic acid residues of the new coronavirus, and there are no infectious new coronavirus particles.
  • the method of detecting IgG/IgM antibodies produced by the immune system response after human infection with the new coronavirus belongs to an indirect detection method. Since the human immune system needs a certain period of time after infection to produce an immune response to appear IgG and IgM, this type of method can only reflect Whether the subject has been infected with the new coronavirus does not immediately indicate whether there are new coronavirus particles in the human body, nor is it suitable for the detection of patients in the early stage of infection or the incubation period, so it is impossible to directly judge the infectivity of the test object.
  • the purpose of the present invention is to provide a new coronavirus detection test strip and its preparation method and use method.
  • the present invention provides a new coronavirus detection test strip, including a liner, one end of the liner is provided with a sample pad, a gold marker pad, a detection pad and a water absorption plate in sequence, the detection pad is The surface of the nitrocellulose membrane is provided with a nitrocellulose membrane, and the nitrocellulose membrane is provided with a detection T line and a quality control C line, the detection T line is close to the gold marker pad, and the quality control C line is close to the water absorption plate;
  • the two ends of the detection pad are respectively overlapped with the gold marking pad and the water absorbing plate, and the gold marking pad is pressed with a sample pad;
  • the detection T line is coated with a sialic acid ligand immobilizer, and the sialic acid ligand immobilizer includes a sialic acid ligand molecule with high affinity, and the structure of the sialic acid ligand molecule with high affinity
  • the general formula is shown in formula II: Wherein, R 1 is selected from methoxy or substituted methoxy; R 2 is selected from substituted acetamido, benzamido, substituted benzamido, alkoxycarbonamido, triazolyl or substituted Triazolyl; R is selected from hydroxyl, methoxy, substituted methoxy, acetamido, substituted acetamido, sulfonamide or phosphoramido; A is wherein n1 is selected from 0,1,2,3,4,5,6,7, m1 is selected from 2,3,4,5,6,7,8,9, R4 is selected from amino or mercapto ;
  • the quality control line C is coated with a control substance, and the control substance is the new coronavirus S protein;
  • the gold labeling pad is sprayed with colloidal gold labels, and the colloidal gold labels are gold nanoparticles with sialic acid ligand molecules with medium affinity immobilized on the surface;
  • the general structural formula is shown in formula I: Wherein, R 5 is selected from hydroxyl, methoxy, substituted methoxy, acetamido, substituted acetamido, sulfonamide or phosphoramido; A1 is n2 is selected from 0,1,2,3,4,5,6,7, and m2 is selected from 2,3,4,5,6,7,8,9.
  • sample pad is provided with a sample injection hole.
  • substituted methoxy group is X 1 -CH 2 O-, wherein X 1 is selected from phenyl or vinyl;
  • the substituted acetamido is X 2 -CH 2 CONH-, wherein X 2 is selected from methyl, ethyl, n-propyl, isopropyl, hydroxymethyl or hydroxyethyl;
  • the substituted benzamido is X 3 -BzNH-, wherein the number of X 3 is at least 1 and is located at any position of the benzene ring, and X 3 is selected from halogen atoms, methyl groups, methoxyl groups, nitro groups. at least one;
  • the alkoxycarbonamide group is X 4 -OC(O)NH-, wherein X 4 is selected from benzyl, allyl, tert-butyl or trichloroethyl;
  • the substituted triazolyl is wherein X 5 is selected from substituted phenylalkyl (X 6 -Ph-(CH 2 ) n6 -) or substituted carbonyl (X 7 -C(O)-), wherein X 6 is at least 1 in number and located in Any position on the benzene ring, X 6 is selected from at least one of halogen, methyl, methoxy, and nitro, n6 is selected from 0, 1, 2; X 7 is selected from alkoxy CH 3 -(CH 2 ) n7 -O- or alkylamino CH 3 -(CH 2 ) n7 -NH-, n7 is selected from 0,1,2,3,4,5.
  • the sialic acid ligand immobilizer includes an immobilized protein, a sialic acid ligand molecule with high affinity and a linking molecule, and the immobilized protein is a protein macromolecule used for sealing and immobilization;
  • the immobilized protein is selected from protein macromolecules commonly used for sealing and immobilization, such as bovine serum albumin BSA, skimmed milk powder or casein;
  • the general structural formula of the linker molecule is R 6 -linker-R 7 , wherein R 6 is a group that can react and link with R 4 , more preferably, the R 6 is selected from a carboxyl group, an ester group, sulfonic acid group, phosphoric acid group or maleimide group; R 7 is a group that can react with active reactive groups (such as amino group, carboxyl group, sulfhydryl group) existing on the surface of the immobilized protein, more preferably, the R 7 is selected from from carboxyl group, ester group, amino group or maleimide group; the linker is selected from alkyl, cycloalkyl, polyethylene glycol chain or aryl; more preferably, the linker is selected from diethyl squaraine Esters, diglycolic acid, maleimide-polyethylene glycol carboxylic acid, 1,4-cyclohexanedicarboxylic acid and other bifunctional compounds.
  • R 6 is a group that can react
  • colloidal gold label includes gold nanoparticles, sialic acid ligand molecules with moderate affinity and blocking molecules;
  • the particle size of the gold nanoparticles is 30-50 nm;
  • the blocking molecule is a small molecule thiol polyethylene glycol, more preferably, the general structural formula of the blocking molecule is R 8 -(C 2 H 4 O) n3 -C 2 H 4 -SH, and n3 is selected from From 1, 2, 3, 4, 5, 6, 7, 8, 9, R 8 is selected from hydroxy, methoxy or carboxyl.
  • the present invention provides a preparation method of any of the above-mentioned new coronavirus detection test strips, comprising the following steps:
  • the colloidal gold marker uses gold nanoparticles as the inner core of the marker, immobilizes sialic acid ligand molecules with medium affinity on the surface of the gold nanoparticles through S-Au bonds, and uses small molecular polyethylene glycol as the blocking molecule;
  • the sialic acid ligand immobilizer is a complex in which an immobilized protein is linked with a sialic acid ligand molecule with high affinity through a linking molecule, and the immobilized protein is a protein macromolecule used for blocking and immobilization.
  • the preparation method of the colloidal gold label is as follows: disposing a sialic acid ligand molecule with a moderate affinity into an aqueous solution, mixing with the gold nanoparticle solution, stirring at 20-30° C. for 12-24 hours, and passing through the solution. S-Au bonds were formed on the surface of gold nanoparticles and fixed on the surface of gold nanoparticles, then an excess of small molecular mercapto polyethylene glycol was added, and the remaining reaction sites on the surface of gold nanoparticles were further stirred at 20-30 °C for 12-24 hours.
  • the sialic acid-gold nanoparticle-labeled complex was obtained by centrifugation and purification;
  • the molar ratio of the medium affinity sialic acid ligand molecule to the gold nanoparticle is 1 ⁇ 10 4 :1 ⁇ 2 ⁇ 10 4 :1;
  • the preparation method of the sialic acid ligand molecule with medium affinity is: using sialic acid As the starting material, through the protection of the carboxyl group, the R 9 protecting group is introduced into the carboxyl group to obtain Generation of peracetylated chlorosaccharides in the next step of chlorination and acetylation reagents Then, a functional side chain precursor is derived from the 2-position of the sugar ring through a glycosylation reaction After deacetylation, several hydroxyl groups on the sugar ring are exposed to obtain Then, the R5 group was introduced by using the reactivity difference between the primary hydroxyl group of the sugar ring 9-OH and other secondary hydroxyl groups to obtain Finally, a sialic acid ligand molecule with moderate affinity is synthesized through group conversion and deprotection reaction; wherein R 9 is selected from methyl or benzyl, and A 2 is or -(CH 2 )m3-R 10 , R 10 is selected from benzyloxy,
  • the preparation method of the gold nanoparticle solution is as follows: heating the aqueous solution of chloroauric acid to boiling, adding sodium citrate, keeping boiling for 15-30 minutes, and cooling to obtain a particle size of 30-50 nm and a surface covered with citric acid
  • the sodium gold nanoparticle solution wherein the concentration of the chloroauric acid aqueous solution is 1 mM, and the molar ratio of chloroauric acid and sodium citrate is 1:2-4.
  • the preparation method of the sialic acid ligand immobilizer is as follows: mixing the sialic acid ligand molecule with high affinity and the linking molecule in water, and stirring at 20-30° C. for 12-24 hours, which has high affinity
  • the sialic acid ligand molecule is covalently linked by the reactive reactive group R 4 on the functional side chain A and the R 6 group of the linking molecule to obtain a sialic acid-linking molecule complex;
  • Standing at °C for 15-30 hours, the R 7 group on the sialic acid-linking molecule complex forms a covalent bond with the surface active reactive group of the immobilized protein to obtain the target sialic acid ligand immobilizer;
  • the molar ratio of the high-affinity sialic acid ligand molecule, linker molecule, and immobilized protein is 5-10:12-15:1;
  • the preparation method of the sialic acid ligand molecule with high affinity is: using sialic acid As the starting material, through the protection of the carboxyl group, the R 11 protecting group is introduced into the carboxyl group to obtain Generation of peracetylated chlorosaccharides in the next step of chlorination and acetylation reagents Then the functional side chain precursor is derived from the glycosylation reaction at the 2-position of the sugar ring After deacetylation and carboxyl protection, an intermediate with all the exposed amino and hydroxyl groups on the sugar ring is obtained Introduce groups in the order of R 2 ⁇ R 1 ⁇ R 3 to obtain Alternatively, the introduction of groups in the order of R 2 ⁇ R 3 ⁇ R 1 can be obtained, respectively.
  • the sugar-binding domain pocket of the new coronavirus S protein has high specificity and binding force to the new coronavirus S protein; wherein R 11 is selected from methyl or benzyl, and A 3 is or -(CH 2 )m4-R 12 , R 12 is selected from benzyloxy, 2-naphthylmethoxy or allyloxy, n5 is selected from 0,1,2,3,4,5,6,7, m4 is selected from 2, 3, 4, 5, 6, 7, 8, 9, PG is the abbreviation of protection group; when R 3 is hydroxyl, it is sialic acid The group on the molecule does not need to introduce the relevant reaction process of the R3 group.
  • the present invention provides a method for using any of the above-mentioned new coronavirus detection test strips, comprising the following steps:
  • the detection T line is red
  • the quality control C line is red
  • the detection samples include nasopharyngeal swabs, saliva, blood, and excrement.
  • the present invention provides a new coronavirus detection test strip, which can be used to directly detect new coronavirus particles, and has the following beneficial effects compared with the existing new coronavirus and related detection methods:
  • the new coronavirus detection test strip provided by the present invention directly detects virus particles based on the recognition and combination of sialic acid ligands and the surface S protein of new coronavirus particles;
  • the present invention provides a detection test strip that can directly detect new coronavirus particles, which can quickly and conveniently determine whether a sample contains virus particles;
  • the new coronavirus detection test strip provided by the present invention can directly detect the new coronavirus particles in the sample, and then quickly determine whether the subject is infectious;
  • the new coronavirus detection test strip provided by the present invention does not involve multiple procedures such as sample RNA extraction and PCR, and can be directly sampled and added dropwise, which simplifies the detection process. It avoids the interference caused by manual operation errors; on the other hand, it only takes about 15 minutes to complete the detection, and the color reaction is used to facilitate the judgment of the detection results;
  • the new coronavirus detection test strip is a direct detection method, which can directly detect the new coronavirus particles, and can immediately characterize whether there are new coronavirus particles in the sample;
  • the new coronavirus detection test strip provided by the present invention is detected by the recognition and combination of sialic acid small molecules and the surface S protein of the new coronavirus.
  • the small molecule properties of the molecule have obvious advantages such as good economy, high stability, and mass preparation;
  • the new coronavirus detection test strip provided by the present invention can directly detect virus particles, and is rich in sample selection, including nasopharyngeal swabs commonly used in nucleic acid detection, saliva, blood, excrement and other samples that may contain virus particles. ;
  • the present invention provides a preparation method of a small molecule-colloidal gold-labeled complex (colloidal gold label) that can be used for colloidal gold detection, and the small-molecule-colloidal gold-labeled complex has advantages in economy and availability. , and also expanded the types and selectable range of gold markers in the colloidal gold method;
  • the present invention provides a small molecule-immobilized protein complex (sialic acid ligand immobilized substance) that can be used for colloidal gold detection.
  • the small molecule-immobilized protein complex has obvious advantages in economy and availability. The types and selectable ranges of molecules detected by colloidal gold method are described.
  • Fig. 1 is the structural representation of the new coronavirus detection test strip of the present invention, Fig. (a) is a top view, and Fig. (b) is a side view;
  • Fig. 2 is the structural representation of the colloidal gold marker of the present invention
  • Fig. 3 is the preparation flow chart of the sialic acid ligand molecule with medium affinity of the present invention
  • Fig. 4 is the preparation flow chart of the colloidal gold marker of the present invention.
  • Fig. 5 is the structural representation of sialic acid ligand immobilizer of the present invention.
  • Fig. 6 is the preparation flow chart of the high-affinity sialic acid ligand molecule of the present invention.
  • Fig. 7 is the preparation flow chart of sialic acid ligand immobilized matter of the present invention.
  • Figure 8 is a schematic diagram of the principle of the present invention for directly detecting new coronavirus particles
  • Sample addition hole is a colloidal gold marker, is a sialic acid ligand immobilizer, for control.
  • the new coronavirus is one of the beta-coronaviruses, and its surface Spike protein (S protein) is a trimer composed of three monomers, each of which is composed of an S1 unit and a conserved S2 unit, of which S1 mainly contains C-terminal binding domain (C-terminus domain, S1-CTD) and N-terminal binding domain (N-terminus domain, S1-NTD), the two together constitute the receptor binding domain (Receptor binding domain, RBD), generally, S1-NTD recognizes sialic acid residues that bind to the surface of host cells, while S1-CTD recognizes peptide molecules that bind to the surface of host cells.
  • S protein surface Spike protein
  • S protein surface Spike protein
  • S protein is a trimer composed of three monomers, each of which is composed of an S1 unit and a conserved S2 unit, of which S1 mainly contains C-terminal binding domain (C-terminus domain, S1-CTD) and N-terminal binding domain (
  • the invention provides a test strip for directly detecting new coronavirus particles in a sample to be tested, using sialic acid ligands to detect the identification and binding of S protein molecules on the surface of the new coronavirus, and directly detecting it as a source of pathogenicity and infectivity of new coronary pneumonia It can instantly and quickly determine whether there are new coronavirus particles in the sample of the subject, so as to make a rapid judgment on the infectivity of the subject.
  • the structure of the test strip is shown in Figure 1, and Figure (a) is a top view.
  • sample pad 2 ⁇ gold marking pad 3 ⁇ testing pad 4 ⁇ water absorbing plate 5 are sample pad 2 ⁇ gold marking pad 3 ⁇ testing pad 4 ⁇ water absorbing plate 5, among which, gold marking pad 3 is sprayed with colloidal gold marker, and detection pad 4 has detection T line 6 and quality control
  • the C line 7 is formed by the sialic acid ligand immobilizer and the control substance immobilized on the detection pad 4NC membrane (nitrocellulose membrane) respectively, and the sample pad 2 is provided with a sample addition hole 8;
  • Figure (b) is a side view: the main It consists of 5 parts: bottom liner 1 (such as polyvinyl chloride PVC), sample pad 2, gold marking pad 3, detection pad 4, water absorption plate 5, detection pad 4 has detection T line 6 and quality control C line 7 .
  • bottom liner 1 such as polyvinyl chloride PVC
  • the schematic diagram of the structure of colloidal gold label is shown in Figure 2: Gold nanoparticles are used as the core of the label, and sialic acid ligand molecules with medium affinity are immobilized on the surface of gold nanoparticles through S-Au bonds, and small molecules of polyethylene glycol are used.
  • a blocking molecule As a blocking molecule, the flow chart of preparation of sialic acid ligand molecules with moderate affinity is shown in Figure 3, and the preparation flow chart of colloidal gold label is shown in Figure 4; virus particles and colloidal gold labeling occur on the gold labeling pad The recognition and binding of the compound forms a virus-gold-labeled complex.
  • the schematic diagram of the structure of the sialic acid ligand immobilizer is shown in Figure 5: it is a complex formed by a high-affinity sialic acid ligand molecule and an immobilized protein through a linker molecule, and is immobilized on the detection pad through the immobilized protein unit.
  • the flow chart of the preparation of ligand molecules is shown in Figure 6, and the flow chart of the preparation of sialic acid ligand immobilized substances is shown in Figure 7;
  • the ligand exchange reaction exchanges part of the colloidal gold label immobilized on the surface of the virus, so that the immobilized virus-gold label complex develops color.
  • Control substance the new coronavirus S protein, which can be obtained through commercialization, and is immobilized on the C-line area of the detection pad, and the colloidal gold label is immobilized by recognizing the sialic acid ligand bound to the surface of the free colloidal gold label to develop color.
  • the present invention provides a colloidal gold method similar to the double-antibody sandwich method to detect the new coronavirus particles.
  • the gold label is recognized and bound by the protein-sialic acid ligand on the surface to form a virus-gold label complex.
  • the virus-gold label complex is recognized and bound to the remaining S protein on the surface of the virus particle under the action of the sialic acid ligand immobilizer. , or exchange part of the colloidal gold label on the virus-gold-labeled complex, so as to fix the virus-gold-labeled complex, and determine the detection result by color change, as shown in Figure 8.
  • the positive detection process when the new coronavirus of the sample to be tested flows through the gold-labeled pad, the new coronavirus recognizes and binds to the sialic acid ligand on the surface of the colloidal gold-labeled surface through the surface S protein to form a labeling complex.
  • T line Test line
  • the remaining S protein on the surface of the new coronavirus recognizes and binds to the immobilized sialic acid ligand, or through a ligand exchange reaction, it is partially immobilized on the surface of the virus particle.
  • the gold label is exchanged, and the virus-gold label complex is immobilized, thereby showing red on the T line.
  • Control line, C line Control line, C line
  • the controlled substance is bound and immobilized, so that the Red on line C;
  • the sample to be tested does not contain the new coronavirus, and the labeled complex cannot be formed when the sample flows through the gold labeling pad.
  • the immobilized sialic acid ligand cannot immobilize the colloidal gold label, so No red is displayed on the T line, and when the sample continues to flow through the C line, the colloidal gold label is bound and fixed by the control substance, thus showing red on the C line;
  • Invalid test result The C line does not show color, and whether the T line shows red or not, it is an invalid result, and another test strip should be taken to test again.
  • the compound of formula 1-4 (15.2 g, 22.83 mmol) was dissolved in dichloromethane (60 mL), triethylamine (4.61 g, 45.66 mmol) was added, p-toluenesulfonyl chloride (6.52 g, 34.25 mmol) was added in batches, and the mixture was stirred overnight , TLC detected that the reaction was complete, methanol was added, and after stirring for 10 minutes, the solvent was evaporated, the residue was dissolved in dichloromethane, the organic phase was washed with brine, dried over anhydrous sodium sulfate, evaporated to remove the solvent, and the residue was dissolved in N,N- Dimethylformamide (20 mL) was added with sodium azide (3.70 g, 57.08 mmol), and the mixture was stirred in an oil bath at 50° C.
  • the compound of formula 1-6 (8.4 g, 16.41 mmol) was dissolved in dichloromethane (40 mL), triethylamine (3.23 g, 31.91 mmol) was added, p-toluenesulfonyl chloride (4.56 g, 23.93 mmol) was added in batches, and the mixture was stirred overnight , TLC detected that the reaction was complete, methanol was added, and after stirring for 10 minutes, the solvent was evaporated, the residue was dissolved in dichloromethane, the organic phase was washed with brine, dried over anhydrous sodium sulfate, and after concentration, the residue was dissolved in N,N-dimethylmethane Formamide (50 mL), potassium thioacetate (3.54 g, 30.97 mmol) was added, and the mixture was stirred in an oil bath at 50° C.
  • the compound of formula 1-7 (7.6 g, 13.00 mmol) was dissolved in methanol (50 mL), sodium methoxide (0.1 M in MeOH) was added, the pH of the reaction solution was adjusted to 8-10, and stirred at room temperature for 15 minutes. TLC detected that the reaction was complete, and added 732 Form H + cation resin, after stirring for 5 minutes, filter off the resin, and concentrate the filtrate to obtain the compound of formula 1 (6.9 g, 97.8%), ESI-MS m/z calcd for [C 21 H 39 N 2 O 12 S] + (M+H) + : 542.60, found: 542.59.
  • the preparation process of the compound of formula 5-1 is the same as the compound of formula 1-4 in the embodiment, wherein only the HOPEG 4 OBn reagent is replaced by HOPEG 3 ONap;
  • the compound of formula 5-4 (3.5 g, 5.15 mmol) was dissolved in methanol (40 mL), NaOH aqueous solution (1.5 M, 20 mL) was added dropwise, stirred until the reaction was completed by TLC, the reaction solution was neutralized, and the solvent was evaporated. Column separation to obtain the compound of formula 5 (2.7g, 95.8%), ESI-MS m/z calcd for [C 24 H 38 NO 11 S] + (M+H) + : 548.62, found: 548.62;
  • the preparation process of the compound of formula 6 is the same as the preparation process of the compound of formula 1, wherein only the HOPEG 4 OBn reagent is replaced by HOC 5 H 10 OBn, and the Ac 2 O reagent in the last step of the step of preparing the compound of formula 1-4 is replaced by a TsCl reagent That is, ESI-MS m/z calcd for [C 23 H 37 N 2 O 10 S 2 ] + (M+H) + : 565.18, found: 565.17.
  • the compound of formula 2-3 (19.3 g, 25.47 mmol) was dissolved in methanol (40 mL), NaOH aqueous solution (1.5 M, 20 mL) was added dropwise, stirred until the reaction was complete by TLC detection, the reaction solution was neutralized, the solvent was evaporated, and the residue was dissolved in 2M NaOH aqueous solution (40 mL) was refluxed in an oil bath at 95°C for 10 hours, the reaction solution was neutralized, and the solvent was evaporated.
  • the compound of formula 2-5 (18.1 g, 24.4 mmol) was mixed with methyl acrylate (2.46 g, 29.28 mmol) and cuprous iodide (0.93 g, 4.88 mmol) and dissolved in THF (60 mL), and N,N was added dropwise.
  • -Diisopropylethylamine DIPEA (4.73gm 36.6mmol) stirred at room temperature for 1 hour, evaporated the reaction solvent, the residue was dissolved in dichloromethane, successively passed through 1M ammonia water, 1N HCl aq , sat.NaHCO 3a.q.
  • the compound of formula 2-6 (19.6 g, 23.73 mmol) was dissolved in methanol (100 mL), sodium methoxide (0.1 M in MeOH) was added, the pH of the solution was adjusted to 8-10, stirred at room temperature for 30 minutes, and hydrochloric acid was added dropwise to neutralize the reaction solution, The solvent is evaporated to obtain the crude product of formula 2-7, which is directly used in the next step reaction;
  • the compound of formula 2-9 (13.1 g, 17.22 mmol) was dissolved in acetonitrile (50 mL), acetone acetal (5.38 g, 51.66 mmol) and a catalytic amount of p-toluenesulfonic acid (0.3 g, 1.72 mmol) were added, and the mixture was heated at 80° C.
  • the crude compound of formula 2-12 obtained in the previous step was dissolved in dichloromethane (60 mL), triethylamine (2.88 g, 28.47 mmol) was added, and after stirring for 5 minutes, p-toluenesulfonyl chloride (4.07 g, 21.35 mmol) was added in batches at room temperature.
  • the compound of formula 2-13 (8.2g, 11.55mmol) was dissolved in methanol (30mL), NaOH aqueous solution (1.5M, 10mL) was added, stirred at room temperature for 30 minutes, 732 type H + cation resin was added to neutralize the reaction solution, and the resin was filtered off , Pd/C powder (0.1 g) was added to the filtrate, and the reaction was carried out in an atmosphere of 4 atm H2 for 2 hours, the Pd/C powder was filtered off, and the filtrate was concentrated to obtain the compound of formula 2 (7.1 g, 90.0%), ESI-MS m /z calcd for [C 29 H 44 N 5 O 13 ] + (M+H) + : 670.69, found: 670.68.
  • the preparation process of the compound of formula 7-1 is the same as that of the compound of formula 2-4, except that the HOPEG 4 OBn reagent is replaced by HOPEG 3 OBn;
  • the process of preparing the compound of formula 7-3 from the compound of formula 7-2 is the same as the process of preparing the compound of formula 1-5 from the compound of formula 1-4, wherein only the Ac 2 O reagent is replaced by TsCl;
  • the procedure for preparing the compound of formula 7 from the compound of formula 7-3 is the same as the procedure for preparing the compound of formula 2 from the compound of formula 2-9.
  • the preparation process of the compound of formula 8-1 is the same as that of the compound of formula 2-4, except that the HOPEG 4 OBn reagent is replaced with HOC 5 H 10 OAll;
  • the process of preparing the compound of formula 8-2 from the compound of formula 8-1 is the same as the process of preparing the compound of formula 7-2 from the compound of formula 7-1, except that the BzCl reagent is replaced by benzyloxycarbonyl chloride CbzCl;
  • the process of preparing the compound of formula 8-3 from the compound of formula 8-2 is the same as the process of preparing the compound of formula 5-2 from the compound of formula 5-1;
  • the process of preparing the compound of formula 8-4 from the compound of formula 8-3 is the same as the process of preparing the compound of formula 7-4 from the compound of formula 7-3;
  • the compound of formula 8-5 (10.1 g, 13.26 mmol) was dissolved in methanol (100 mL), TsOH (114 mg, 0.66 mmol) was added, and the reaction solution was placed in an oil bath at 80 °C for reflux reaction for 12 hours. TLC detected that the reaction was complete, and the reaction solution was cooled. To room temperature, triethylamine was added to neutralize the reaction solution to neutrality, palladium chloride (4.7 g, 26.51 mmol) was added, and the reaction was carried out at room temperature for 1 hour. TLC detected that the reaction was complete. The solvent was evaporated and the residue was dissolved in dichloromethane.
  • the process of preparing the compound of formula 8-7 from the compound of formula 8-6 is the same as the process of preparing the compound of formula 7-7 from the compound of formula 7-6;
  • the compound of formula 8-7 (4 g, 5.66 mmol) was dissolved in methanol (40 mL), NaOH aqueous solution (1 M, 20 mL) was added dropwise, stirred until the reaction was completed by TLC, the reaction solution was neutralized, the solvent was evaporated, and the residue was dissolved in tetrahydrofuran ( 40mL), Ph3P (4.45g, 16.98mmol) was added, stirred at room temperature for 12 hours, TLC detected that the reaction was complete, the solvent was evaporated, and the residue was directly purified by column chromatography to obtain the compound of formula 8 (3.2g, 84.8%), ESI- MS m/z calcd for [C 36 H 47 N 2 O 10 ] + (M+H) + : 667.77, found: 667.76;
  • the preparation method of gold nanoparticle solution with stable sodium citrate and particle size of 30nm is as follows: take 100mL of 1mM HAuCl4 aqueous solution into a two - necked flask, heat it to 100°C, and add sodium citrate (dihydrate) (118mg, 0.3mmol) , After stirring for 15 minutes, take it out and slowly lower it to room temperature.
  • PEG 4 -SH mercaptotetraethylene glycol
  • the obtained compound of formula 4-1 (1.1g, 1.36mmol) was mixed with bovine serum albumin BSA (66.43kDa, 3g, 45umol) in water, and allowed to stand for 24h.
  • the reaction solution was purified by gel column to obtain the compound of formula 4 (3.8 g), dissolved in ultrapure water to make a 1nM solution.
  • the length x width are respectively 2cm x 2cm, 1cm x 2cm, 6cm x 2cm, and 1cm x 2cm.
  • the sialic acid-gold nanoparticle-labeled complex prepared in Example 7 The 3 solution (10 ⁇ L) was sprayed on the gold-labeled pad, and the sialic acid ligand immobilizer 4 solution (5 ⁇ L) prepared in Example 8 was sprayed on the T line (0.1 ⁇ 2 cm) area of the detection pad to commercialize the new coronavirus S protein.
  • the solution (1nM, 5 ⁇ L) was sprayed on the C-line (0.1 ⁇ 2cm) area of the detection pad as the control substance, and the absorbent paper was used as the absorbent pad.
  • the layers and positions shown are pasted on the PVC liner, and each layer is overlapped by 0.5cm to prepare a new coronavirus detection test strip.
  • the 2019-nCoV is highly pathogenic and infectious.
  • the detection experiment was carried out by using 2019-nCoV simulated particles.
  • the commercialized 2019-nCoV S protein was immobilized on the surface of SiO 2 nanospheres with a diameter of about 100nm to construct 2019-nCoV simulative particles.
  • the T line of the kit is red
  • the C line is red.
  • test strip A and test strip B Take two new coronavirus detection test strips, numbered test strip A and test strip B, respectively, take the saliva of healthy people, and mark them as saliva A and saliva B respectively, and add saliva A to the new crown prepared in Example 10.
  • Virus simulation particle solution add deionized water to saliva B, take 1 drop (about 50 ⁇ L) of the two samples respectively, drop them into the sample holes of test strip A and test strip B respectively, and let stand horizontally for 15 minutes.
  • the T line is red, the C line is colored, the T line of the test strip B is not colored, and the C line is colored.
  • the embodiment of the present invention provides a test strip that can directly detect new coronavirus particles, which can quickly and conveniently determine whether the sample contains virus particles, and then test whether the subject is infectious. judge quickly.

Abstract

A novel coronavirus detection test strip, and a preparation method and a use method therefor. The test strip comprises a lining plate (1), and a sample pad (2), a gold labeling pad (3), a detection pad (4) and a water absorption plate (5), which are sequentially arranged on the lining plate (1) from one end to the other end. A nitrocellulose membrane is arranged on the surface of the detection pad (4), and a detection T line and a quality control C line are disposed on the nitrocellulose membrane, wherein the detection T line is coated with a sialic acid ligand immobilization substance, which comprises a sialic acid ligand molecule with high affinity; and the quality control C line is coated with a control substance, which is SARS-CoV-2 S protein. A colloidal gold label is sprayed on the gold labeling pad (3), and the colloidal gold label is a gold nanoparticle having a sialic acid ligand molecule with moderate affinity immobilized on the surface. The test strip can be used to quickly and conveniently determine whether a sample contains SARS-CoV-2 particles, thereby quickly determining whether a subject is contagious.

Description

一种新冠病毒检测试纸条及其制备方法和使用方法A new coronavirus detection test strip and its preparation method and use method 技术领域technical field
本发明属于生物化学技术领域，涉及一种新冠病毒检测试纸条及其制备方法和使用方法。The invention belongs to the technical field of biochemistry, and relates to a new coronavirus detection test strip and a preparation method and use method thereof.
背景技术Background technique
新型冠状病毒肺炎(简称“新冠肺炎”)属重大突发公共卫生事件，目前全球范围内已造成上千万人感染，数十万人死亡。引起新冠肺炎的病原体是新型冠状病毒(简称“新冠病毒”)，国际病毒分类委员会将其命名为SARS-Cov-2(Severe acute respiratory syndromes coronavirus 2)。The novel coronavirus pneumonia ("COVID-19" for short) is a major public health emergency that has infected tens of millions of people and killed hundreds of thousands of people worldwide. The pathogen causing new coronary pneumonia is a new type of coronavirus (referred to as "new coronavirus"), which was named SARS-Cov-2 (Severe acute respiratory syndromes coronavirus 2) by the International Committee on Taxonomy of Viruses.
防控新冠肺炎的一个关键是针对新冠病毒发展有效的医疗诊治方法，其中能够及时、准确的检测新冠病毒对疫情的防控至关重要。A key to the prevention and control of new coronary pneumonia is to develop effective medical diagnosis and treatment methods for the new coronavirus, of which timely and accurate detection of the new coronavirus is crucial to the prevention and control of the epidemic.
目前新冠病毒的检测技术主要集中在对新冠病毒RNA序列的检测，以及对新冠病毒感染后人体免疫系统产生的IgM和IgG抗体的检测。其中，针对新冠病毒RNA的检测主要包括实时荧光逆转录聚合酶链反应(Reverse transcription-polymerase chain reaction,RT-PCR)法和CRISPR(clustered regularly interspaced short palindromic repeats)法，前者通过PCR反应对检测样本RNA进行扩增来鉴定其是否为新冠病毒RNA，后者利用能特异性识别新冠病毒RNA序列的基因编辑工具来对检测样本中是否含新冠病毒RNA进行检测鉴定。利用人感染新冠病毒后免疫系统会产生特异的IgM和IgG抗体来间接检测是否有新冠病毒感染，其中尤以胶体金法常见。胶体金法也称免疫层析法，其主要原理是利用滴在膜一端的样品溶液受膜的毛细作用(基于层析作用的横流(lateral flow))向另一端移动，移动过程中被分析物与固定在膜上某一区域的受体结合而被固相化，并通过标记物显色来判定该固相化结果，通常以胶体金位显色标记物。目前市面上用于新冠病毒的IgG/IgM检测卡同样通过免疫层析法来检测人体血液中是否存在新冠病毒IgG/IgM抗体，主要通过在胶体金上标记抗IgG/抗IgM抗体，其与血液中的IgG或IgM免疫结合后，继而与被固定于膜上的受体结合而固相化，能快速判定结果。At present, the detection technology of the new coronavirus mainly focuses on the detection of the RNA sequence of the new coronavirus, and the detection of the IgM and IgG antibodies produced by the human immune system after the infection of the new coronavirus. Among them, the detection of new coronavirus RNA mainly includes real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR) method and CRISPR (clustered regularly interspaced short palindromic repeats) method. The former uses PCR reaction to detect samples. The RNA is amplified to identify whether it is the new coronavirus RNA, and the latter uses a gene editing tool that can specifically recognize the new coronavirus RNA sequence to detect and identify whether the test sample contains the new coronavirus RNA. The human immune system will produce specific IgM and IgG antibodies after infection with the new coronavirus to indirectly detect whether there is a new coronavirus infection, especially the colloidal gold method. The colloidal gold method is also known as immunochromatography. Its main principle is to use the sample solution dropped on one end of the membrane to move to the other end by the capillary action of the membrane (lateral flow based on chromatography), and the analyte moves during the movement. It binds to a receptor immobilized on a certain area of the membrane and becomes immobilized, and the immobilization result is judged by developing the color of a marker, which is usually colored by colloidal gold. At present, the IgG/IgM test cards for 2019-nCoV on the market also use immunochromatography to detect the presence of 2019-nCoV IgG/IgM antibodies in human blood, mainly by labeling anti-IgG/anti-IgM antibodies on colloidal gold. After the IgG or IgM in the membrane is immunocombined, it binds to the receptor immobilized on the membrane and becomes immobilized, and the result can be quickly determined.
目前主流的新冠病毒RNA检测方法是RT-PCR法，其虽然能直接检测病毒，但是需要人工提取样本中RNA病毒，存在样本取样方法、样本运输保存、人工操作等多重因素的影响，据一线医务人员反映该方法常伴有较为严重的假阴性干扰，对检测结果产生了挑战；此外，有少数RNA检测结果复阳者并未表现出传染性，因此也存在检测结果假阳性干扰，其复阳检测结果可能是检测样本中含有新冠病毒核酸残余物，并没有传染致病的新冠病毒颗粒。检测人感染新冠病毒后免疫系统反应产生的IgG/IgM抗体的方法属于一种间接检测方法，由于人体免疫系统需要在感染一定时间后才能产生免疫应答出现IgG和IgM，因此该类方法仅能反映受试 者是否感染过新冠病毒，并不能即时指征人体内是否存在新冠病毒颗粒，也不适用于感染早期或潜伏期患者的检测，从而无法对检测对象的传染性做出直接判断。The current mainstream new coronavirus RNA detection method is RT-PCR. Although it can directly detect the virus, it needs to manually extract the RNA virus in the sample, which is affected by multiple factors such as sample sampling method, sample transportation and preservation, and manual operation. Staff reported that this method was often accompanied by serious false-negative interference, which challenged the test results; in addition, a small number of RNA test results with positive results did not show contagiousness, so there was also false-positive interference in test results, and their positive results The test result may be that the test sample contains nucleic acid residues of the new coronavirus, and there are no infectious new coronavirus particles. The method of detecting IgG/IgM antibodies produced by the immune system response after human infection with the new coronavirus belongs to an indirect detection method. Since the human immune system needs a certain period of time after infection to produce an immune response to appear IgG and IgM, this type of method can only reflect Whether the subject has been infected with the new coronavirus does not immediately indicate whether there are new coronavirus particles in the human body, nor is it suitable for the detection of patients in the early stage of infection or the incubation period, so it is impossible to directly judge the infectivity of the test object.
综上，目前尚无直接检测新冠病毒颗粒的方法，因此发展新冠病毒颗粒检测方法在新冠肺炎防控中具有重要意义。To sum up, there is no direct detection method for 2019-nCoV particles at present, so the development of detection methods for 2019-nCoV particles is of great significance in the prevention and control of 2019-nCoV.
发明内容SUMMARY OF THE INVENTION
为了解决上述背景技术中所提出的问题，本发明的目的在于提供一种新冠病毒检测试纸条及其制备方法和使用方法。In order to solve the problems raised in the above background technology, the purpose of the present invention is to provide a new coronavirus detection test strip and its preparation method and use method.
为达到上述目的，本发明所采用的技术方案为：In order to achieve the above object, the technical scheme adopted in the present invention is:
一方面，本发明提供了一种新冠病毒检测试纸条，包括衬板，所述衬板上的一端至另一端依次设有样品垫、金标记垫、检测垫和吸水板，所述检测垫的表面设有硝酸纤维素膜，所述硝酸纤维素膜上设有检测T线和质控C线，所述检测T线靠近金标记垫，所述质控C线靠近吸水板；In one aspect, the present invention provides a new coronavirus detection test strip, including a liner, one end of the liner is provided with a sample pad, a gold marker pad, a detection pad and a water absorption plate in sequence, the detection pad is The surface of the nitrocellulose membrane is provided with a nitrocellulose membrane, and the nitrocellulose membrane is provided with a detection T line and a quality control C line, the detection T line is close to the gold marker pad, and the quality control C line is close to the water absorption plate;
所述检测垫的两端分别与金标记垫和吸水板相互交叠连接，所述金标记垫上压贴有样品垫；The two ends of the detection pad are respectively overlapped with the gold marking pad and the water absorbing plate, and the gold marking pad is pressed with a sample pad;
所述检测T线上包被有唾液酸配体固定物，所述唾液酸配体固定物包括具高亲合力的唾液酸配体分子，所述具高亲合力的唾液酸配体分子的结构通式如式II所示：
Figure PCTCN2020127148-appb-000001
其中，R 1选自甲氧基或取代的甲氧基；R 2选自取代的乙酰氨基、苯甲酰氨基、取代的苯甲酰氨基、烷氧羰酰胺基、三氮唑基或取代的三氮唑基；R 3选自羟基、甲氧基、取代的甲氧基、乙酰氨基、取代的乙酰氨基、磺酰胺基或磷酰胺基；A为
Figure PCTCN2020127148-appb-000002
其中n1选自0,1,2,3,4,5,6,7，m1选自2,3,4,5,6,7,8,9，R 4选自氨基或巯基； The detection T line is coated with a sialic acid ligand immobilizer, and the sialic acid ligand immobilizer includes a sialic acid ligand molecule with high affinity, and the structure of the sialic acid ligand molecule with high affinity The general formula is shown in formula II:
Figure PCTCN2020127148-appb-000001
Wherein, R 1 is selected from methoxy or substituted methoxy; R 2 is selected from substituted acetamido, benzamido, substituted benzamido, alkoxycarbonamido, triazolyl or substituted Triazolyl; R is selected from hydroxyl, methoxy, substituted methoxy, acetamido, substituted acetamido, sulfonamide or phosphoramido; A is
Figure PCTCN2020127148-appb-000002
wherein n1 is selected from 0,1,2,3,4,5,6,7, m1 is selected from 2,3,4,5,6,7,8,9, R4 is selected from amino or mercapto ;
所述质控C线上包被有控制物，所述控制物为新冠病毒S蛋白；The quality control line C is coated with a control substance, and the control substance is the new coronavirus S protein;
所述金标记垫上喷有胶体金标记物，所述胶体金标记物为表面固定了具中等亲合力的唾液酸配体分子的金纳米粒；所述具中等亲合力的唾液酸配体分子的结构通式如式I所示：
Figure PCTCN2020127148-appb-000003
其中，R 5选自羟基、甲氧基、取代的甲氧基、乙酰氨基、取代的乙酰氨基、磺酰胺基或磷酰胺基；A1为
Figure PCTCN2020127148-appb-000004
n2选自0,1,2,3,4,5,6,7，m2选自2,3,4,5,6,7,8,9。 The gold labeling pad is sprayed with colloidal gold labels, and the colloidal gold labels are gold nanoparticles with sialic acid ligand molecules with medium affinity immobilized on the surface; The general structural formula is shown in formula I:
Figure PCTCN2020127148-appb-000003
Wherein, R 5 is selected from hydroxyl, methoxy, substituted methoxy, acetamido, substituted acetamido, sulfonamide or phosphoramido; A1 is
Figure PCTCN2020127148-appb-000004
n2 is selected from 0,1,2,3,4,5,6,7, and m2 is selected from 2,3,4,5,6,7,8,9.
进一步地，所述样品垫上设有加样孔。Further, the sample pad is provided with a sample injection hole.
进一步地，所述取代的甲氧基为X 1-CH 2O-，其中，X 1选自苯基或乙烯基； Further, the substituted methoxy group is X 1 -CH 2 O-, wherein X 1 is selected from phenyl or vinyl;
所述取代的乙酰氨基为X 2-CH 2CONH-，其中，X 2选自甲基、乙基、正丙基、异丙基、羟甲基或羟乙基； The substituted acetamido is X 2 -CH 2 CONH-, wherein X 2 is selected from methyl, ethyl, n-propyl, isopropyl, hydroxymethyl or hydroxyethyl;
所述取代的苯甲酰氨基为X 3-BzNH-，其中，X 3的数量至少为1且位于苯环的任意位置，X 3选自卤原子、甲基、甲氧基、硝基中的至少一种； The substituted benzamido is X 3 -BzNH-, wherein the number of X 3 is at least 1 and is located at any position of the benzene ring, and X 3 is selected from halogen atoms, methyl groups, methoxyl groups, nitro groups. at least one;
所述烷氧羰酰胺基为X 4-OC(O)NH-，其中，X 4选自苄基、烯丙基、叔丁基或三氯乙基； The alkoxycarbonamide group is X 4 -OC(O)NH-, wherein X 4 is selected from benzyl, allyl, tert-butyl or trichloroethyl;
所述取代的三氮唑基为
Figure PCTCN2020127148-appb-000005
其中，X 5选自取代的苯烷基(X 6-Ph-(CH 2) n6-)或取代的羰基(X 7-C(O)-)，其中，X 6的数量至少为1且位于苯环的任意位置，X 6选自卤原子、甲基、甲氧基、硝基中的至少一种，n6选自0,1,2；X 7选自烷氧基CH 3-(CH 2) n7-O-或烷胺基CH 3-(CH 2) n7-NH-，n7选自0,1,2,3,4,5。 The substituted triazolyl is
Figure PCTCN2020127148-appb-000005
wherein X 5 is selected from substituted phenylalkyl (X 6 -Ph-(CH 2 ) n6 -) or substituted carbonyl (X 7 -C(O)-), wherein X 6 is at least 1 in number and located in Any position on the benzene ring, X 6 is selected from at least one of halogen, methyl, methoxy, and nitro, n6 is selected from 0, 1, 2; X 7 is selected from alkoxy CH 3 -(CH 2 ) n7 -O- or alkylamino CH 3 -(CH 2 ) n7 -NH-, n7 is selected from 0,1,2,3,4,5.
进一步地，所述唾液酸配体固定物包括固定蛋白、具高亲合力的唾液酸配体分子和连接分子，所述固定蛋白为用作封闭固定的蛋白大分子；Further, the sialic acid ligand immobilizer includes an immobilized protein, a sialic acid ligand molecule with high affinity and a linking molecule, and the immobilized protein is a protein macromolecule used for sealing and immobilization;
优选地，所述固定蛋白选自牛血清白蛋白BSA、脱脂奶粉或酪蛋白等常用作封闭固定的蛋白大分子；Preferably, the immobilized protein is selected from protein macromolecules commonly used for sealing and immobilization, such as bovine serum albumin BSA, skimmed milk powder or casein;
优选地，所述连接分子的结构通式为R 6-linker-R 7，其中，R 6为能同R 4反应连接的基团，更优选地，所述R 6选自羧基、酯基、磺酸基、磷酸基或马来酰亚胺基；R 7为能与固定蛋白表面存在的活性反应基团(比如氨基、羧基、巯基)反应的基团，更优选地，所述R 7选自羧基、酯基、氨基或马来酰亚胺基；所述linker选自烷基、环烷基、聚乙二醇链或芳基；更优选的，所述连接分子选自方酸二乙酯、二甘醇酸，马来酰亚胺-聚乙二醇羧酸、1,4-环己烷二羧酸等双官能团化合物。 Preferably, the general structural formula of the linker molecule is R 6 -linker-R 7 , wherein R 6 is a group that can react and link with R 4 , more preferably, the R 6 is selected from a carboxyl group, an ester group, sulfonic acid group, phosphoric acid group or maleimide group; R 7 is a group that can react with active reactive groups (such as amino group, carboxyl group, sulfhydryl group) existing on the surface of the immobilized protein, more preferably, the R 7 is selected from from carboxyl group, ester group, amino group or maleimide group; the linker is selected from alkyl, cycloalkyl, polyethylene glycol chain or aryl; more preferably, the linker is selected from diethyl squaraine Esters, diglycolic acid, maleimide-polyethylene glycol carboxylic acid, 1,4-cyclohexanedicarboxylic acid and other bifunctional compounds.
进一步地，所述胶体金标记物包括金纳米粒、具中等亲合力的唾液酸配体分子和封闭分子；Further, the colloidal gold label includes gold nanoparticles, sialic acid ligand molecules with moderate affinity and blocking molecules;
优选地，所述金纳米粒的粒径为30-50nm；Preferably, the particle size of the gold nanoparticles is 30-50 nm;
优选地，所述封闭分子为小分子巯基聚乙二醇，更优选地，所述封闭分子的结构通式为R 8-(C 2H 4O) n3-C 2H 4-SH，n3选自1,2,3,4,5,6,7,8,9，R 8选自羟基、甲氧基或羧基。 Preferably, the blocking molecule is a small molecule thiol polyethylene glycol, more preferably, the general structural formula of the blocking molecule is R 8 -(C 2 H 4 O) n3 -C 2 H 4 -SH, and n3 is selected from From 1, 2, 3, 4, 5, 6, 7, 8, 9, R 8 is selected from hydroxy, methoxy or carboxyl.
另一方面，本发明提供了一种上述任一所述的新冠病毒检测试纸条的制备方法，包括以下步骤：On the other hand, the present invention provides a preparation method of any of the above-mentioned new coronavirus detection test strips, comprising the following steps:
制备胶体金标记物，将胶体金标记物喷在金标记垫上；Prepare colloidal gold marker, spray the colloidal gold marker on the gold marker pad;
制备唾液酸配体固定物，将唾液酸配体固定物包被在检测T线上；Prepare a sialic acid ligand immobilized material, and coat the sialic acid ligand immobilized material on the detection T line;
将新冠病毒S蛋白包被在质控C线上；Coat the new coronavirus S protein on the quality control line C;
将样品垫、金标记垫、检测垫和吸水板黏贴到衬板上，获得所述新冠病毒检测试纸条；Paste the sample pad, the gold marker pad, the detection pad and the absorbent plate to the liner to obtain the new coronavirus detection test strip;
优选地，所述胶体金标记物以金纳米粒为标记内核，在金纳米粒表面通过S-Au键固定具中等亲合力的唾液酸配体分子，以小分子聚乙二醇作为封闭分子；Preferably, the colloidal gold marker uses gold nanoparticles as the inner core of the marker, immobilizes sialic acid ligand molecules with medium affinity on the surface of the gold nanoparticles through S-Au bonds, and uses small molecular polyethylene glycol as the blocking molecule;
优选地，所述唾液酸配体固定物为通过连接分子将固定蛋白与具高亲合力的唾液酸配体分子连接的复合物，所述固定蛋白为用作封闭固定的蛋白大分子。Preferably, the sialic acid ligand immobilizer is a complex in which an immobilized protein is linked with a sialic acid ligand molecule with high affinity through a linking molecule, and the immobilized protein is a protein macromolecule used for blocking and immobilization.
进一步地，所述胶体金标记物的制备方法为：将具中等亲合力的唾液酸配体分子配置成水溶液，同金纳米粒溶液混合后，在20～30℃下搅拌12～24小时，通过在金纳米粒表面形成S-Au键而固定于金纳米粒表面，继而加入过量的小分子巯基聚乙二醇，在20～30℃下继续搅拌12～24小时对金纳米粒表面剩余反应位点进行封闭，通过离心纯化获得唾液酸-金纳米粒标记复合物；Further, the preparation method of the colloidal gold label is as follows: disposing a sialic acid ligand molecule with a moderate affinity into an aqueous solution, mixing with the gold nanoparticle solution, stirring at 20-30° C. for 12-24 hours, and passing through the solution. S-Au bonds were formed on the surface of gold nanoparticles and fixed on the surface of gold nanoparticles, then an excess of small molecular mercapto polyethylene glycol was added, and the remaining reaction sites on the surface of gold nanoparticles were further stirred at 20-30 °C for 12-24 hours. The sialic acid-gold nanoparticle-labeled complex was obtained by centrifugation and purification;
优选地，所述具中等亲合力的唾液酸配体分子与金纳米粒的摩尔比为1×10 4:1～2×10 4:1； Preferably, the molar ratio of the medium affinity sialic acid ligand molecule to the gold nanoparticle is 1×10 4 :1˜2×10 4 :1;
优选地，所述具中等亲合力的唾液酸配体分子的制备方法为：以唾液酸
Figure PCTCN2020127148-appb-000006
为起始原料，经羧基保护在羧基引入R 9保护基得到
Figure PCTCN2020127148-appb-000007
在氯代和乙酰化试剂作用下一步生成全乙酰化氯代糖
Figure PCTCN2020127148-appb-000008
继而经糖基化反应在糖环2-位衍生出功能侧链前体
Figure PCTCN2020127148-appb-000009
经脱乙酰化反应后裸露出糖环上的数个羟基得到
Figure PCTCN2020127148-appb-000010
继而利用糖环9-OH的伯羟基与其他仲羟基的反应活性差异引入R5基团得到
Figure PCTCN2020127148-appb-000011
最后经基团转换、脱保护反应合成具中等亲合力的唾液酸配体分子；其中R 9选自甲基或苄基，A 2为
Figure PCTCN2020127148-appb-000012
或-(CH 2)m3-R 10，R 10选自苄氧基、2-萘甲氧基或烯丙氧基，n4选自0,1,2,3,4,5,6,7，m3选自2,3,4,5,6,7,8,9；当R 5为羟基时，其为唾液酸
Figure PCTCN2020127148-appb-000013
分子上的基团，不需要引入R 5基团的相关反应过程； Preferably, the preparation method of the sialic acid ligand molecule with medium affinity is: using sialic acid
Figure PCTCN2020127148-appb-000006
As the starting material, through the protection of the carboxyl group, the R 9 protecting group is introduced into the carboxyl group to obtain
Figure PCTCN2020127148-appb-000007
Generation of peracetylated chlorosaccharides in the next step of chlorination and acetylation reagents
Figure PCTCN2020127148-appb-000008
Then, a functional side chain precursor is derived from the 2-position of the sugar ring through a glycosylation reaction
Figure PCTCN2020127148-appb-000009
After deacetylation, several hydroxyl groups on the sugar ring are exposed to obtain
Figure PCTCN2020127148-appb-000010
Then, the R5 group was introduced by using the reactivity difference between the primary hydroxyl group of the sugar ring 9-OH and other secondary hydroxyl groups to obtain
Figure PCTCN2020127148-appb-000011
Finally, a sialic acid ligand molecule with moderate affinity is synthesized through group conversion and deprotection reaction; wherein R 9 is selected from methyl or benzyl, and A 2 is
Figure PCTCN2020127148-appb-000012
or -(CH 2 )m3-R 10 , R 10 is selected from benzyloxy, 2-naphthylmethoxy or allyloxy, n4 is selected from 0,1,2,3,4,5,6,7, m3 is selected from 2,3,4,5,6,7,8,9 ; when R5 is hydroxyl, it is sialic acid
Figure PCTCN2020127148-appb-000013
The group on the molecule does not need to introduce the relevant reaction process of the R 5 group;
优选地，所述金纳米粒溶液的制备方法为：将氯金酸水溶液加热至沸腾，加入柠檬酸钠，保持沸腾15～30分钟，冷却后即得粒径为30～50nm、表面覆盖柠檬酸钠的金纳米粒溶液，其中，氯金酸水溶液的浓度为1mM，氯金酸、柠檬酸钠的摩尔比为1:2-4。Preferably, the preparation method of the gold nanoparticle solution is as follows: heating the aqueous solution of chloroauric acid to boiling, adding sodium citrate, keeping boiling for 15-30 minutes, and cooling to obtain a particle size of 30-50 nm and a surface covered with citric acid The sodium gold nanoparticle solution, wherein the concentration of the chloroauric acid aqueous solution is 1 mM, and the molar ratio of chloroauric acid and sodium citrate is 1:2-4.
进一步地，所述唾液酸配体固定物的制备方法为：将具高亲合力的唾液酸配体分子同连接分子混合在水中，在20～30℃下搅拌12～24小时，具高亲合力的唾液酸配体分子通过功能侧链A上的活性反应基团R 4与连接分子的R 6基团反应共价连接，获得唾液酸-连接分子复合物；然后加入固定蛋白，在20～25℃下静置15～30小时，通过唾液酸-连接分子复合物上R 7基团与固定蛋白表面活性反应基团形成共价键，获得目标唾液酸配体固定物； Further, the preparation method of the sialic acid ligand immobilizer is as follows: mixing the sialic acid ligand molecule with high affinity and the linking molecule in water, and stirring at 20-30° C. for 12-24 hours, which has high affinity The sialic acid ligand molecule is covalently linked by the reactive reactive group R 4 on the functional side chain A and the R 6 group of the linking molecule to obtain a sialic acid-linking molecule complex; Standing at ℃ for 15-30 hours, the R 7 group on the sialic acid-linking molecule complex forms a covalent bond with the surface active reactive group of the immobilized protein to obtain the target sialic acid ligand immobilizer;
优选地，所述具高亲合力的唾液酸配体分子、连接分子、固定蛋白的摩尔比为5～10:12～15:1；Preferably, the molar ratio of the high-affinity sialic acid ligand molecule, linker molecule, and immobilized protein is 5-10:12-15:1;
优选地，所述具高亲合力的唾液酸配体分子的制备方法为：以唾液酸
Figure PCTCN2020127148-appb-000014
为起始原料，经羧基保护在羧基引入R 11保护基得到
Figure PCTCN2020127148-appb-000015
在氯代和乙酰化试剂作用下一步生成全乙酰化氯代糖
Figure PCTCN2020127148-appb-000016
继而经糖基化反应在糖环2位衍生出功能 侧链前体
Figure PCTCN2020127148-appb-000017
随后经脱乙酰基、羧基保护后得到糖环上氨基和羟基全部裸露的中间体
Figure PCTCN2020127148-appb-000018
按照R 2→R 1→R 3顺序进行基团的引入分别得到
Figure PCTCN2020127148-appb-000019
或者，按照R 2→R 3→R 1的顺序进行基团的引入分别得到
Figure PCTCN2020127148-appb-000020
Figure PCTCN2020127148-appb-000021
最后通过侧链基团转换和糖环保护基脱除反应，合成具高亲合力的唾液酸配体分子，该配体分子可通过R 1，R 2和R 3基团的衍生，进一步匹配占据新冠病毒S蛋白的糖结合域口袋，对新冠病毒S蛋白具有很高的特异性和结合力；其中R 11选自甲基或苄基，A 3为
Figure PCTCN2020127148-appb-000022
或-(CH 2)m4-R 12，R 12选自苄氧基、2-萘甲氧基或烯丙氧基，n5选自0,1,2,3,4,5,6,7，m4选自2,3,4,5,6,7,8,9，PG是保护基protection group的缩写；当R 3为羟基时，其为唾液酸
Figure PCTCN2020127148-appb-000023
分子上的基团，不需要引入R 3基团的相关反应过程。 Preferably, the preparation method of the sialic acid ligand molecule with high affinity is: using sialic acid
Figure PCTCN2020127148-appb-000014
As the starting material, through the protection of the carboxyl group, the R 11 protecting group is introduced into the carboxyl group to obtain
Figure PCTCN2020127148-appb-000015
Generation of peracetylated chlorosaccharides in the next step of chlorination and acetylation reagents
Figure PCTCN2020127148-appb-000016
Then the functional side chain precursor is derived from the glycosylation reaction at the 2-position of the sugar ring
Figure PCTCN2020127148-appb-000017
After deacetylation and carboxyl protection, an intermediate with all the exposed amino and hydroxyl groups on the sugar ring is obtained
Figure PCTCN2020127148-appb-000018
Introduce groups in the order of R 2 →R 1 →R 3 to obtain
Figure PCTCN2020127148-appb-000019
Alternatively, the introduction of groups in the order of R 2 →R 3 →R 1 can be obtained, respectively.
Figure PCTCN2020127148-appb-000020
Figure PCTCN2020127148-appb-000021
Finally, through the conversion of side chain groups and the removal of sugar ring protecting groups, a sialic acid ligand molecule with high affinity is synthesized, which can be further matched and occupied by derivatization of R 1 , R 2 and R 3 groups. The sugar-binding domain pocket of the new coronavirus S protein has high specificity and binding force to the new coronavirus S protein; wherein R 11 is selected from methyl or benzyl, and A 3 is
Figure PCTCN2020127148-appb-000022
or -(CH 2 )m4-R 12 , R 12 is selected from benzyloxy, 2-naphthylmethoxy or allyloxy, n5 is selected from 0,1,2,3,4,5,6,7, m4 is selected from 2, 3, 4, 5, 6, 7, 8, 9, PG is the abbreviation of protection group; when R 3 is hydroxyl, it is sialic acid
Figure PCTCN2020127148-appb-000023
The group on the molecule does not need to introduce the relevant reaction process of the R3 group.
再一方面，本发明提供了一种上述任一所述的新冠病毒检测试纸条的使用方法，包括以下步骤：In another aspect, the present invention provides a method for using any of the above-mentioned new coronavirus detection test strips, comprising the following steps:
1)将新冠病毒检测试纸条水平放置，取检测样本滴入样品垫，静置10-15分钟；1) Place the new coronavirus detection test strip horizontally, take the test sample and drop it into the sample pad, and let it stand for 10-15 minutes;
2)结果判定：2) Result judgment:
阳性结果：检测T线显红色，质控C线显红色；Positive results: the detection T line is red, and the quality control C line is red;
阴性结果：检测T线不显色，质控C线显红色；Negative result: the T line of the test is not colored, and the quality control C line is red;
无效结果：无论检测T线显色与否，质控C线不显红色，应另取新冠病毒检测试纸条重新检测。Invalid result: No matter whether the detection T line is colored or not, the quality control C line does not show red, and another new coronavirus detection test strip should be taken for re-testing.
进一步地，所述检测样本包括鼻咽拭子、唾液、血液、排泄物。Further, the detection samples include nasopharyngeal swabs, saliva, blood, and excrement.
本发明提供了一种新冠病毒检测试纸条，该试纸条可以用于直接检测新冠病毒颗粒，与现有的新冠病毒及相关检测方法相比，具有以下有益效果：The present invention provides a new coronavirus detection test strip, which can be used to directly detect new coronavirus particles, and has the following beneficial effects compared with the existing new coronavirus and related detection methods:
1)本发明提供的新冠病毒检测试纸条基于唾液酸配体和新冠病毒颗粒表面S蛋白识别结合来直接检测病毒颗粒；1) The new coronavirus detection test strip provided by the present invention directly detects virus particles based on the recognition and combination of sialic acid ligands and the surface S protein of new coronavirus particles;
2)本发明提供了一种能直接检测新冠病毒颗粒的检测试纸条，能非常快速、便捷的对样本中是否含病毒颗粒进行快速判定；2) The present invention provides a detection test strip that can directly detect new coronavirus particles, which can quickly and conveniently determine whether a sample contains virus particles;
3)本发明提供的新冠病毒检测试纸条能直接检测样本中的新冠病毒颗粒，进而对受试者是否具有传染性进行迅速判定；3) The new coronavirus detection test strip provided by the present invention can directly detect the new coronavirus particles in the sample, and then quickly determine whether the subject is infectious;
4)本发明提供的新冠病毒检测试纸条用于新冠病毒检测与核酸检测方法相比，一方面不涉及样本RNA提取、PCR等多重程序，直接取样后滴加即可，简化了检测过程，避免了人工操作误差引起的干扰；另一方面仅需耗时约15分钟即可完成检测，并通过颜色反应来方便判断检测结果；4) Compared with the nucleic acid detection method, the new coronavirus detection test strip provided by the present invention does not involve multiple procedures such as sample RNA extraction and PCR, and can be directly sampled and added dropwise, which simplifies the detection process. It avoids the interference caused by manual operation errors; on the other hand, it only takes about 15 minutes to complete the detection, and the color reaction is used to facilitate the judgment of the detection results;
5)本发明提供的新冠病毒检测试纸条用于新冠病毒检测与新冠病毒IgG/IgM检测方法相比，属直接检测方法，直接检测新冠病毒颗粒，可即时表征样本中是否存在新冠病毒颗粒；5) Compared with the new coronavirus IgG/IgM detection method, the new coronavirus detection test strip provided by the present invention is a direct detection method, which can directly detect the new coronavirus particles, and can immediately characterize whether there are new coronavirus particles in the sample;
6)本发明提供的新冠病毒检测试纸条通过唾液酸小分子与新冠病毒表面S蛋白识别结合来进行检测，与传统胶体金免疫层析法使用抗原抗体等生物大分子相比，唾液酸检测分子的小分子属性具有经济性好、稳定性高、可大量制备等明显优点；6) The new coronavirus detection test strip provided by the present invention is detected by the recognition and combination of sialic acid small molecules and the surface S protein of the new coronavirus. The small molecule properties of the molecule have obvious advantages such as good economy, high stability, and mass preparation;
7)本发明提供的新冠病毒检测试纸条直接检测病毒颗粒，在样本选择上具有丰富性，包括核酸检测常用的鼻咽拭子、唾液、血液、排泄物等可能含病毒颗粒的样本均可；7) The new coronavirus detection test strip provided by the present invention can directly detect virus particles, and is rich in sample selection, including nasopharyngeal swabs commonly used in nucleic acid detection, saliva, blood, excrement and other samples that may contain virus particles. ;
8)本发明提供了一种可用于胶体金法检测的小分子-胶体金标记复合物(胶体金标记物)制备方法，小分子-胶体金标记复合物在经济性和可获得性具有优势以外，还扩展了胶体金法中金标记物类型和可选择范围；8) The present invention provides a preparation method of a small molecule-colloidal gold-labeled complex (colloidal gold label) that can be used for colloidal gold detection, and the small-molecule-colloidal gold-labeled complex has advantages in economy and availability. , and also expanded the types and selectable range of gold markers in the colloidal gold method;
9)本发明提供了一种可用于胶体金法检测的小分子-固定蛋白复合物(唾液酸配体固定物)，小分子-固定蛋白复合物具有明显的经济性和可获得性优势，扩展了胶体金法检测分子的类型和可选择范围。9) The present invention provides a small molecule-immobilized protein complex (sialic acid ligand immobilized substance) that can be used for colloidal gold detection. The small molecule-immobilized protein complex has obvious advantages in economy and availability. The types and selectable ranges of molecules detected by colloidal gold method are described.
附图说明Description of drawings
图1是本发明新冠病毒检测试纸条的结构示意图，图(a)为俯视图，图(b)为侧视图；Fig. 1 is the structural representation of the new coronavirus detection test strip of the present invention, Fig. (a) is a top view, and Fig. (b) is a side view;
图2是本发明胶体金标记物的结构示意图；Fig. 2 is the structural representation of the colloidal gold marker of the present invention;
图3是本发明具中等亲合力的唾液酸配体分子的制备流程图；Fig. 3 is the preparation flow chart of the sialic acid ligand molecule with medium affinity of the present invention;
图4是本发明胶体金标记物的制备流程图；Fig. 4 is the preparation flow chart of the colloidal gold marker of the present invention;
图5是本发明唾液酸配体固定物的结构示意图；Fig. 5 is the structural representation of sialic acid ligand immobilizer of the present invention;
图6是本发明具高亲和力唾液酸配体分子的制备流程图；Fig. 6 is the preparation flow chart of the high-affinity sialic acid ligand molecule of the present invention;
图7是本发明唾液酸配体固定物的制备流程图；Fig. 7 is the preparation flow chart of sialic acid ligand immobilized matter of the present invention;
图8本发明直接检测新冠病毒颗粒的原理示意图；Figure 8 is a schematic diagram of the principle of the present invention for directly detecting new coronavirus particles;
图9为本发明阳性、阴性、以及无效检测原理示意图；9 is a schematic diagram of the positive, negative, and invalid detection principles of the present invention;
其中，1.衬板，2.样品垫，3.金标记垫，4.检测垫，5.吸水板，6.检测T线，7.质控C线，8.加样孔，
Figure PCTCN2020127148-appb-000024
为胶体金标记物，
Figure PCTCN2020127148-appb-000025
为唾液酸配体固定物，
Figure PCTCN2020127148-appb-000026
为控制物。 Among them, 1. Backing plate, 2. Sample pad, 3. Gold marker pad, 4. Detection pad, 5. Water absorption plate, 6. Detection T line, 7. Quality control C line, 8. Sample addition hole,
Figure PCTCN2020127148-appb-000024
is a colloidal gold marker,
Figure PCTCN2020127148-appb-000025
is a sialic acid ligand immobilizer,
Figure PCTCN2020127148-appb-000026
for control.
具体实施方式Detailed ways
新冠病毒属β-冠状病毒中的一种，其表面Spike蛋白(S蛋白)是由三个单体组成的三聚体，每个单体由S1单元和保守的S2单元组成，其中S1主要包含C端结合域(C-terminus domain,S1-CTD)和N端结合域(N-terminus domain，S1-NTD)，二者共同组成了受体结合域(Receptor binding domain,RBD)，一般的，S1-NTD识别结合宿主细胞表面的唾液酸残基，而S1-CTD则识别结合宿主细胞表面的肽类分子。新冠病毒S蛋白结构解析研究结果表面新冠病毒S1-NTD部分存在糖识别阈(Carbohydrate recognition domain,CRD)口袋结构，其能与唾液酸配体分子识别结合，因此可利用这种识别结合作用来设计相应的唾液酸配体分子作为检测分子，直接对新冠病毒颗粒进行检测，设计提供一种point-of-care(POC)检测新冠病毒颗粒的检测装置。The new coronavirus is one of the beta-coronaviruses, and its surface Spike protein (S protein) is a trimer composed of three monomers, each of which is composed of an S1 unit and a conserved S2 unit, of which S1 mainly contains C-terminal binding domain (C-terminus domain, S1-CTD) and N-terminal binding domain (N-terminus domain, S1-NTD), the two together constitute the receptor binding domain (Receptor binding domain, RBD), generally, S1-NTD recognizes sialic acid residues that bind to the surface of host cells, while S1-CTD recognizes peptide molecules that bind to the surface of host cells. The research results of the structural analysis of the S protein of the new coronavirus show that there is a carbohydrate recognition threshold (CRD) pocket structure in the S1-NTD part of the new coronavirus, which can recognize and bind to the sialic acid ligand molecule, so this recognition and binding effect can be used to design Corresponding sialic acid ligand molecules are used as detection molecules to directly detect new coronavirus particles, and a detection device for point-of-care (POC) detection of new coronavirus particles is designed and provided.
本发明提供了一种直接检测待测样本中新冠病毒颗粒的试纸条，利用唾液酸配体对新冠病毒表面S蛋白分子的识别结合进行检测，直接检测作为新冠肺炎致病性、传染性来源的新冠病毒颗粒，能即时、快速判断受试者样本中是否存在新冠病毒颗粒，从而对受试者传染性做出迅速判断，试纸条的结构如图1所示，图(a)为俯视图：从左往右依次是样品垫2→金标记垫3→检测垫4→吸水板5，其中，金标记垫3上喷有胶体金标记物，检测垫4上有检测T线6和质控C线7，分别由唾液酸配体固定物和控制物固定于检测垫4NC膜(硝酸纤维素膜)上形成，样品垫2上设有加样孔8；图(b)为侧视图：主要由5个部分组成：底层衬板1(如聚氯乙烯PVC)，样品垫2，金标记垫3，检测垫4，吸水板5，检测垫4上有检测T线6和质控C线7。胶体金标记 物的结构示意图如图2所示：以金纳米粒为标记内核，在纳米金表面通过S-Au键固定了具中等亲合力的唾液酸配体分子，以小分子聚乙二醇作为封闭分子，其中具中等亲合力的唾液酸配体分子的制备流程图如图3所示，胶体金标记物的制备流程图如图4所示；在金标记垫发生病毒颗粒同胶体金标记物的识别结合形成病毒-金标记复合物。唾液酸配体固定物的结构示意图如图5所示：为具高亲和力唾液酸配体分子通过连接分子同固定蛋白形成的复合物，通过固定蛋白单元固定于检测垫，其中具高亲和力唾液酸配体分子的制备流程图如图6所示，唾液酸配体固定物的制备流程图如图7所示；通过唾液酸配体单元结合病毒-金标记复合物上剩余的S蛋白，或通过配体交换反应交换一部分固定于病毒表面的胶体金标记物，从而固定病毒-金标记复合物显色。控制物：为新冠病毒S蛋白，其可通过商业化途径获得，并固定于检测垫C线区域，通过识别结合游离胶体金标记物表面的唾液酸配体来固定胶体金标记物显色。The invention provides a test strip for directly detecting new coronavirus particles in a sample to be tested, using sialic acid ligands to detect the identification and binding of S protein molecules on the surface of the new coronavirus, and directly detecting it as a source of pathogenicity and infectivity of new coronary pneumonia It can instantly and quickly determine whether there are new coronavirus particles in the sample of the subject, so as to make a rapid judgment on the infectivity of the subject. The structure of the test strip is shown in Figure 1, and Figure (a) is a top view. : From left to right are sample pad 2→gold marking pad 3→testing pad 4→water absorbing plate 5, among which, gold marking pad 3 is sprayed with colloidal gold marker, and detection pad 4 has detection T line 6 and quality control The C line 7 is formed by the sialic acid ligand immobilizer and the control substance immobilized on the detection pad 4NC membrane (nitrocellulose membrane) respectively, and the sample pad 2 is provided with a sample addition hole 8; Figure (b) is a side view: the main It consists of 5 parts: bottom liner 1 (such as polyvinyl chloride PVC), sample pad 2, gold marking pad 3, detection pad 4, water absorption plate 5, detection pad 4 has detection T line 6 and quality control C line 7 . The schematic diagram of the structure of colloidal gold label is shown in Figure 2: Gold nanoparticles are used as the core of the label, and sialic acid ligand molecules with medium affinity are immobilized on the surface of gold nanoparticles through S-Au bonds, and small molecules of polyethylene glycol are used. As a blocking molecule, the flow chart of preparation of sialic acid ligand molecules with moderate affinity is shown in Figure 3, and the preparation flow chart of colloidal gold label is shown in Figure 4; virus particles and colloidal gold labeling occur on the gold labeling pad The recognition and binding of the compound forms a virus-gold-labeled complex. The schematic diagram of the structure of the sialic acid ligand immobilizer is shown in Figure 5: it is a complex formed by a high-affinity sialic acid ligand molecule and an immobilized protein through a linker molecule, and is immobilized on the detection pad through the immobilized protein unit. The flow chart of the preparation of ligand molecules is shown in Figure 6, and the flow chart of the preparation of sialic acid ligand immobilized substances is shown in Figure 7; The ligand exchange reaction exchanges part of the colloidal gold label immobilized on the surface of the virus, so that the immobilized virus-gold label complex develops color. Control substance: the new coronavirus S protein, which can be obtained through commercialization, and is immobilized on the C-line area of the detection pad, and the colloidal gold label is immobilized by recognizing the sialic acid ligand bound to the surface of the free colloidal gold label to develop color.
本发明直接检测新冠病毒颗粒的原理为：本发明提供了一种类似双抗夹心法的胶体金法检测新冠病毒颗粒，将唾液酸配体同纳米金颗粒制备成胶体金标记，新冠病毒同胶体金标记通过表面的蛋白-唾液酸配体识别结合形成病毒-金标复合物，该病毒-金标复合物通过在唾液酸配体固定物的作用下，同病毒颗粒表面剩余的S蛋白识别结合，或者交换病毒-金标复合物上的部分胶体金标记，从而将病毒-金标复合物固定，通过颜色变化进行检测结果判定，如图8所示。The principle of the present invention for directly detecting the new coronavirus particles is as follows: the present invention provides a colloidal gold method similar to the double-antibody sandwich method to detect the new coronavirus particles. The gold label is recognized and bound by the protein-sialic acid ligand on the surface to form a virus-gold label complex. The virus-gold label complex is recognized and bound to the remaining S protein on the surface of the virus particle under the action of the sialic acid ligand immobilizer. , or exchange part of the colloidal gold label on the virus-gold-labeled complex, so as to fix the virus-gold-labeled complex, and determine the detection result by color change, as shown in Figure 8.
具体地，阳性检测过程、原理及结果：待测样品的新冠病毒流经金标记垫时，新冠病毒通过表面S蛋白同胶体金标记表面的唾液酸配体识别结合形成标记复合物，该复合物在检测垫上展开流经检测线(Test line,T线)时，新冠病毒表面剩余的S蛋白同被固定的唾液酸配体识别结合，或者通过配体交换反应，同部分固定于病毒颗粒表面的金标交换，病毒-金标复合物被固定，从而在T线上显示红色，多余的胶体金标记继续展开流经质控线(Control line,C线)时，被控制物结合固定，从而在C线上显示红色；Specifically, the positive detection process, principle and results: when the new coronavirus of the sample to be tested flows through the gold-labeled pad, the new coronavirus recognizes and binds to the sialic acid ligand on the surface of the colloidal gold-labeled surface through the surface S protein to form a labeling complex. When the test line (Test line, T line) is unfolded on the detection pad, the remaining S protein on the surface of the new coronavirus recognizes and binds to the immobilized sialic acid ligand, or through a ligand exchange reaction, it is partially immobilized on the surface of the virus particle. The gold label is exchanged, and the virus-gold label complex is immobilized, thereby showing red on the T line. When the excess colloidal gold label continues to flow through the quality control line (Control line, C line), the controlled substance is bound and immobilized, so that the Red on line C;
阴性检测过程、原理及结果：待测样品不含新冠病毒，样品流经金标记垫时无法形成标记复合物，继续展开流经T线时，固定的唾液酸配体无法固定胶体金标记，从而在T线上不显示红色，样品继续展开流经C线时，胶体金标记被控制物结合固定，从而在C线上显示红色；Negative detection process, principle and results: the sample to be tested does not contain the new coronavirus, and the labeled complex cannot be formed when the sample flows through the gold labeling pad. When the sample continues to flow through the T line, the immobilized sialic acid ligand cannot immobilize the colloidal gold label, so No red is displayed on the T line, and when the sample continues to flow through the C line, the colloidal gold label is bound and fixed by the control substance, thus showing red on the C line;
无效检测结果：C线不显色，无论T线是否显示红色，均为无效结果，应另取试纸条重新检测。Invalid test result: The C line does not show color, and whether the T line shows red or not, it is an invalid result, and another test strip should be taken to test again.
为了更好地理解本发明的内容，下面结合具体实施方法对本发明内容作进一步说明，但本发明的保护内容不局限以下实施例。In order to better understand the content of the present invention, the content of the present invention will be further described below in conjunction with specific implementation methods, but the protection content of the present invention is not limited to the following examples.
实施例1：具中等亲合力的唾液酸配体分子1的合成Example 1: Synthesis of sialic acid ligand molecule 1 with moderate affinity
Figure PCTCN2020127148-appb-000027
Figure PCTCN2020127148-appb-000027
天然唾液酸Neu5Ac(10g,32.3mmol)溶于乙醇(100mL)，加入碳酸钾(5.36g,38.8mmol)，搅拌15分钟后，滴入溴苄(6.08g,35.6mmol)，于50℃中搅拌2小时，TLC检测反应完全，旋干反应液，残余物经加甲苯旋蒸三次，抽干，得式1-1中间体粗品，直接用于下步反应；Natural sialic acid Neu5Ac (10 g, 32.3 mmol) was dissolved in ethanol (100 mL), potassium carbonate (5.36 g, 38.8 mmol) was added, and after stirring for 15 minutes, benzyl bromide (6.08 g, 35.6 mmol) was added dropwise, and the mixture was stirred at 50 °C After 2 hours, TLC detected that the reaction was complete, the reaction solution was spin-dried, the residue was spin-evaporated three times with toluene, and drained to obtain the crude intermediate of formula 1-1, which was directly used in the next step reaction;
上步所得式1-1粗品溶于氯乙酰(20mL)，室温下搅拌至反应完全，蒸除溶剂，得式1-2粗品，直接用于下步反应；The crude product of formula 1-1 obtained in the previous step was dissolved in chloroacetyl (20 mL), stirred at room temperature until the reaction was complete, and the solvent was evaporated to obtain the crude product of formula 1-2, which was directly used in the next step reaction;
上步所得式1-2粗品与HOPEG 4OBn(13.79g,48.5mmol)混于干燥二氯甲烷(60mL)，冰浴下加入碳酸银(17.83g,64.7mmol)，反应10h，经TLC检测反应完全，滤除不溶物，残余物浓缩拌样柱分离，得式1-3化合物(19.3g,71.6％in 3 steps),ESI-MS m/z calcd for[C 41H 56NO 17] +(M+H) +:834.88,found:834.88； The crude product of formula 1-2 obtained in the previous step was mixed with HOPEG 4 OBn (13.79 g, 48.5 mmol) in dry dichloromethane (60 mL), and silver carbonate (17.83 g, 64.7 mmol) was added under an ice bath to react for 10 h, and the reaction was detected by TLC. Complete, insoluble matter was filtered off, and the residue was concentrated and separated with a sample column to obtain the compound of formula 1-3 (19.3 g, 71.6% in 3 steps), ESI-MS m/z calcd for [C 41 H 56 NO 17 ] + ( M+H) + :834.88,found:834.88;
式1-3化合物(19.3g,23.14mmol)溶于甲醇(60mL)，加入甲醇钠(0.1M)调节反应液pH 8～10，搅拌30min，TLC检测反应完全，中和反应液至中性，蒸除溶剂，残余物加入甲苯旋蒸三次后，即得化合物1-4(15.2g,98.7％),ESI-MS m/z calcd for[C 33H 48NO 13] +(M+H) +:666.73,found:666.72； The compound of formula 1-3 (19.3 g, 23.14 mmol) was dissolved in methanol (60 mL), sodium methoxide (0.1 M) was added to adjust the pH of the reaction solution to 8-10, stirred for 30 min, TLC detected that the reaction was complete, and the reaction solution was neutralized to neutrality, The solvent was evaporated, and the residue was added to toluene for rotary evaporation three times to obtain compound 1-4 (15.2g, 98.7%), ESI-MS m/z calcd for [C 33 H 48 NO 13 ] + (M+H) + :666.73,found:666.72;
式1-4化合物(15.2g,22.83mmol)溶于二氯甲烷(60mL)，加入三乙胺(4.61g,45.66mmol)，分批加入对甲苯磺酰氯(6.52g,34.25mmol)，搅拌过夜，TLC检测反应完全，加入甲醇，搅拌10分钟后，蒸除溶剂，残余物溶于二氯甲烷，有机相经盐水洗涤，无水硫酸钠干燥，蒸除溶剂，残余物溶于N,N-二甲基甲酰胺(20mL)，加入叠氮化钠(3.70g,57.08mmol)，于50℃油浴中搅拌12小时，TLC检测反应完全，蒸除溶剂，残余物溶于四氢呋喃(40mL)，加入三苯基膦(10.48g,39.95mmol)，至无气泡产生后继续搅拌15分钟，反应液直接蒸除溶剂，残余物溶于乙醇(20mL)， 滴入乙酸酐(3.03g,29.68mmol)，搅拌12小时后，蒸除溶剂，残余物直接拌样柱分离，得化合物1-5(11.6g,71.9％),ESI-MS m/z calcd for[C 35H 51N 2O 13] +(M+H) +:707.79,found:707.80； The compound of formula 1-4 (15.2 g, 22.83 mmol) was dissolved in dichloromethane (60 mL), triethylamine (4.61 g, 45.66 mmol) was added, p-toluenesulfonyl chloride (6.52 g, 34.25 mmol) was added in batches, and the mixture was stirred overnight , TLC detected that the reaction was complete, methanol was added, and after stirring for 10 minutes, the solvent was evaporated, the residue was dissolved in dichloromethane, the organic phase was washed with brine, dried over anhydrous sodium sulfate, evaporated to remove the solvent, and the residue was dissolved in N,N- Dimethylformamide (20 mL) was added with sodium azide (3.70 g, 57.08 mmol), and the mixture was stirred in an oil bath at 50° C. for 12 hours. TLC detected that the reaction was complete. The solvent was evaporated and the residue was dissolved in tetrahydrofuran (40 mL). Triphenylphosphine (10.48 g, 39.95 mmol) was added, and stirring was continued for 15 minutes until no bubbles were generated. The reaction solution was directly evaporated to remove the solvent, the residue was dissolved in ethanol (20 mL), and acetic anhydride (3.03 g, 29.68 mmol) was added dropwise. , after stirring for 12 hours, the solvent was evaporated, and the residue was directly separated with a sample column to obtain compound 1-5 (11.6 g, 71.9%), ESI-MS m/z calcd for [C 35 H 51 N 2 O 13 ] + (M+H) + :707.79,found:707.80;
式1-5化合物(11.6g,16.41mmol)溶于甲醇(40mL)，加入Pd/C催化剂(0.1g)，在4atm压力H 2氛围中搅拌6小时，TLC检测反应完全，滤除Pd/C粉末，滤液浓缩，得式1-6化合物(8.4g,97.2％),ESI-MS m/z calcd for[C 21H 39N 2O 13] +(M+H) +:527.54,found:527.54； The compound of formula 1-5 (11.6 g, 16.41 mmol) was dissolved in methanol (40 mL), Pd/C catalyst (0.1 g) was added, and the mixture was stirred at 4 atm pressure H atmosphere for 6 hours. TLC detected that the reaction was complete, and the Pd/C was filtered off. Powder, the filtrate was concentrated to obtain the compound of formula 1-6 (8.4g, 97.2%), ESI-MS m/z calcd for [C 21 H 39 N 2 O 13 ] + (M+H) + : 527.54, found: 527.54 ;
式1-6化合物(8.4g,16.41mmol)溶于二氯甲烷(40mL)，加入三乙胺(3.23g,31.91mmol)，分批加入对甲苯磺酰氯(4.56g,23.93mmol)，搅拌过夜，TLC检测反应完全，加入甲醇，搅拌10分钟后，蒸除溶剂，残余物溶于二氯甲烷，有机相经盐水洗涤，无水硫酸钠干燥，浓缩后残余物溶于N,N-二甲基甲酰胺(50mL)，加入硫代乙酸钾(3.54g,30.97mmol)，于50℃油浴中搅拌12小时，TLC检测反应完全，蒸除溶剂，残余物经柱层析分离纯化，得式1-7化合物(7.6g,81.5％),ESI-MS m/z calcd for[C 23H 41N 2O 13S] +(M+H) +:585.63,found:585.62； The compound of formula 1-6 (8.4 g, 16.41 mmol) was dissolved in dichloromethane (40 mL), triethylamine (3.23 g, 31.91 mmol) was added, p-toluenesulfonyl chloride (4.56 g, 23.93 mmol) was added in batches, and the mixture was stirred overnight , TLC detected that the reaction was complete, methanol was added, and after stirring for 10 minutes, the solvent was evaporated, the residue was dissolved in dichloromethane, the organic phase was washed with brine, dried over anhydrous sodium sulfate, and after concentration, the residue was dissolved in N,N-dimethylmethane Formamide (50 mL), potassium thioacetate (3.54 g, 30.97 mmol) was added, and the mixture was stirred in an oil bath at 50° C. for 12 hours. TLC detected that the reaction was complete, the solvent was evaporated, and the residue was separated and purified by column chromatography to obtain the formula Compound 1-7 (7.6 g, 81.5%), ESI-MS m/z calcd for [C 23 H 41 N 2 O 13 S] + (M+H) + : 585.63, found: 585.62;
式1-7化合物(7.6g,13.00mmol)溶于甲醇(50mL)，加入甲醇钠(0.1M in MeOH)，调节反应液pH 8～10，室温下搅拌15分钟，TLC检测反应完全，加入732型H +阳离子树脂，搅拌5分钟后，滤除树脂，浓缩滤液，得式1化合物(6.9g，97.8％)，ESI-MS m/z calcd for[C 21H 39N 2O 12S] +(M+H) +:542.60,found:542.59。 The compound of formula 1-7 (7.6 g, 13.00 mmol) was dissolved in methanol (50 mL), sodium methoxide (0.1 M in MeOH) was added, the pH of the reaction solution was adjusted to 8-10, and stirred at room temperature for 15 minutes. TLC detected that the reaction was complete, and added 732 Form H + cation resin, after stirring for 5 minutes, filter off the resin, and concentrate the filtrate to obtain the compound of formula 1 (6.9 g, 97.8%), ESI-MS m/z calcd for [C 21 H 39 N 2 O 12 S] + (M+H) + : 542.60, found: 542.59.
实施例2：具中等亲合力的唾液酸配体分子5的合成：Example 2: Synthesis of medium affinity sialic acid ligand molecule 5:
Figure PCTCN2020127148-appb-000028
Figure PCTCN2020127148-appb-000028
式5-1化合物的制备过程同实施例中式1-4化合物，其中仅将HOPEG 4OBn试剂替换为HOPEG 3ONap即可； The preparation process of the compound of formula 5-1 is the same as the compound of formula 1-4 in the embodiment, wherein only the HOPEG 4 OBn reagent is replaced by HOPEG 3 ONap;
式5-1化合物(5g,7.44mmol)溶于甲醇(50mL)，加入Bu 2SnO(2.22g,8.93mmol)，置80℃下回流3小时至反应液澄清，蒸除溶剂，残余物经甲苯旋蒸旋蒸三次后，溶于干燥DMF(40mL)，加入溴苄(1.91g,11.17mmol)、CsF(1.36g,8.93mmol)，在N 2气氛围中反应12小时，TLC检测反应完全，蒸除溶剂，残余物直接柱层析，得化合物5-2(5.2g,91.7％)，ESI-MS m/z calcd for[C 42H 52NO 12] +(M+H) +:762.87,found:762.88； The compound of formula 5-1 (5 g, 7.44 mmol) was dissolved in methanol (50 mL), Bu 2 SnO (2.22 g, 8.93 mmol) was added, and refluxed at 80° C. for 3 hours until the reaction solution was clear, the solvent was evaporated, and the residue was washed with toluene After rotary evaporation three times, dissolve in dry DMF (40 mL), add benzyl bromide (1.91 g, 11.17 mmol), CsF (1.36 g, 8.93 mmol), react in N atmosphere for 12 hours, TLC detects that the reaction is complete, The solvent was evaporated, and the residue was directly subjected to column chromatography to obtain compound 5-2 (5.2 g, 91.7%), ESI-MS m/z calcd for [C 42 H 52 NO 12 ] + (M+H) + : 762.87, found:762.88;
式5-2化合物(5.2g,6.83mmol)溶于DCM/MeOH混合溶剂(v/v＝2:1,100mL)，加入2,3-二氯-5,6-二氰对苯醌DDQ(2.32g,10.24mmol)，避光下搅拌30分钟，TLC检测反应完全，反应液浓缩得式5-3中间体，直接用于下步反应；The compound of formula 5-2 (5.2 g, 6.83 mmol) was dissolved in DCM/MeOH mixed solvent (v/v=2:1, 100 mL), and 2,3-dichloro-5,6-dicyano-p-benzoquinone DDQ (2.32 g, 10.24mmol), stirred for 30 minutes in the dark, TLC detected that the reaction was complete, and the reaction solution was concentrated to obtain the intermediate of formula 5-3, which was directly used in the next step reaction;
上步所得式5-3中间体溶于二氯甲烷/DMF混合溶剂(v/v＝3:1,40mL)，加入三乙胺(2.07g,20.48mmol)，搅拌5分钟后，加入TsCl(2.6g,13.65mmol)，室温下搅拌反应6小时，TLC检测反应完全，加入甲醇(1mL)淬灭反应，蒸除溶剂，残余物溶于乙腈(40mL)，加入KSAc(1.17g,10.24mmol)，室温下搅拌3小时，TLC检测反应完全，蒸除溶剂，残余物溶于二氯甲烷，先后经sat.NaHCO 3a.q.和饱和食盐水洗涤，无水硫酸钠干燥，浓缩后柱层析纯化，得式5-4化合物(3.5g,75.4％),ESI-MS m/z calcd for[C 33H 46NO 12S] +(M+H) +:680.78,found:680.77； The intermediate of formula 5-3 obtained in the previous step was dissolved in dichloromethane/DMF mixed solvent (v/v=3:1, 40 mL), triethylamine (2.07 g, 20.48 mmol) was added, and after stirring for 5 minutes, TsCl ( 2.6 g, 13.65 mmol), the reaction was stirred at room temperature for 6 hours, TLC detected that the reaction was complete, methanol (1 mL) was added to quench the reaction, the solvent was evaporated, the residue was dissolved in acetonitrile (40 mL), and KSAc (1.17 g, 10.24 mmol) was added. , stirred at room temperature for 3 hours, TLC detected that the reaction was complete, the solvent was evaporated, the residue was dissolved in dichloromethane, washed with sat.NaHCO 3a.q. and saturated brine successively, dried over anhydrous sodium sulfate, and concentrated after column chromatography Purified to obtain the compound of formula 5-4 (3.5g, 75.4%), ESI-MS m/z calcd for [C 33 H 46 NO 12 S] + (M+H) + : 680.78, found: 680.77;
式5-4化合物(3.5g,5.15mmol)溶于甲醇(40mL)，滴入NaOH水溶液(1.5M，20mL)，搅拌至TLC检测反应完全，中和反应液，蒸除溶剂，残余物经快速柱分离，得式5化合物(2.7g,95.8％),ESI-MS m/z calcd for[C 24H 38NO 11S] +(M+H) +:548.62,found:548.62； The compound of formula 5-4 (3.5 g, 5.15 mmol) was dissolved in methanol (40 mL), NaOH aqueous solution (1.5 M, 20 mL) was added dropwise, stirred until the reaction was completed by TLC, the reaction solution was neutralized, and the solvent was evaporated. Column separation to obtain the compound of formula 5 (2.7g, 95.8%), ESI-MS m/z calcd for [C 24 H 38 NO 11 S] + (M+H) + : 548.62, found: 548.62;
实施例3：具中等亲合力的唾液酸配体分子6的合成Example 3: Synthesis of medium affinity sialic acid ligand molecule 6
Figure PCTCN2020127148-appb-000029
Figure PCTCN2020127148-appb-000029
式6化合物的制备过程同式1化合物的制备过程，其中，仅将HOPEG 4OBn试剂替换为HOC 5H 10OBn，以及制备式1-4化合物步骤中最后一步反应Ac 2O试剂替换为TsCl试剂即可，ESI-MS m/z calcd for[C 23H 37N 2O 10S 2] +(M+H) +:565.18,found:565.17。 The preparation process of the compound of formula 6 is the same as the preparation process of the compound of formula 1, wherein only the HOPEG 4 OBn reagent is replaced by HOC 5 H 10 OBn, and the Ac 2 O reagent in the last step of the step of preparing the compound of formula 1-4 is replaced by a TsCl reagent That is, ESI-MS m/z calcd for [C 23 H 37 N 2 O 10 S 2 ] + (M+H) + : 565.18, found: 565.17.
实施例4：具高亲合力的唾液酸配体分子2的合成Example 4: Synthesis of high affinity sialic acid ligand molecule 2
Figure PCTCN2020127148-appb-000030
Figure PCTCN2020127148-appb-000030
浓盐酸(1mL)滴入甲醇(100mL)中，分批加入天然唾液酸Neu5Ac(10g,32.3mmol)，搅拌反应至溶液澄清，中和反应液，蒸除溶剂，残余物经加甲苯旋蒸三次，抽干，得式2-1中间体粗品，直接用于下步反应；Concentrated hydrochloric acid (1 mL) was dropped into methanol (100 mL), natural sialic acid Neu5Ac (10 g, 32.3 mmol) was added in batches, the reaction was stirred until the solution was clear, the reaction solution was neutralized, the solvent was evaporated, and the residue was rotary evaporated three times by adding toluene , drained to obtain the crude intermediate of formula 2-1, which was directly used in the next step reaction;
上步所得式2-1粗品溶于氯乙酰(40mL)，室温下搅拌至反应完全，蒸除溶剂，得式2-2粗品，直接用于下步反应；The crude product of formula 2-1 obtained in the previous step was dissolved in chloroacetyl (40 mL), stirred at room temperature until the reaction was complete, and the solvent was evaporated to obtain the crude product of formula 2-2, which was directly used in the next step reaction;
上步所得式2-2粗品与HOPEG 4OBn(13.8g,48.5mmol)混于干燥二氯甲烷(60mL)，冰浴下加入碳酸银(17.83g,64.7mmol)，反应12h，经TLC检测反应完全，滤除不溶物，残余物浓缩拌样柱分离，得式2-3化合物(19.3g,78.8％in 3 steps),ESI-MS m/z calcd for[C 35H 52NO 17] +(M+H) +:758.78,found:758.77. The crude product of formula 2-2 obtained in the previous step was mixed with HOPEG 4 OBn (13.8 g, 48.5 mmol) in dry dichloromethane (60 mL), and silver carbonate (17.83 g, 64.7 mmol) was added under an ice bath to react for 12 h, and the reaction was detected by TLC. Complete, insoluble matter was filtered off, and the residue was concentrated and separated with a sample column to obtain the compound of formula 2-3 (19.3 g, 78.8% in 3 steps), ESI-MS m/z calcd for [C 35 H 52 NO 17 ] + ( M+H) + :758.78,found:758.77.
式2-3化合物(19.3g,25.47mmol)溶于甲醇(40mL)，滴入NaOH水溶液(1.5M，20mL)，搅拌至TLC检测反应完全，中和反应液，蒸除溶剂，残余物溶于2M NaOH水溶液(40mL)，于95℃油浴中回流10小时，中和反应液，蒸除溶剂，残余物经甲苯共沸旋蒸三次后溶于盐酸/甲醇(v/v＝1:99,50mL)，搅拌至TLC检测反应完全，中和反应液，蒸除溶剂，得式2-4化合物粗品，直接用于下步反应，ESI-MS m/z calcd for[C 25H 42NO 12] +(M+H) +:548.60,found:548.59； The compound of formula 2-3 (19.3 g, 25.47 mmol) was dissolved in methanol (40 mL), NaOH aqueous solution (1.5 M, 20 mL) was added dropwise, stirred until the reaction was complete by TLC detection, the reaction solution was neutralized, the solvent was evaporated, and the residue was dissolved in 2M NaOH aqueous solution (40 mL) was refluxed in an oil bath at 95°C for 10 hours, the reaction solution was neutralized, and the solvent was evaporated. 50mL), stir until TLC detects that the reaction is complete, neutralize the reaction solution, evaporate the solvent to obtain the crude product of the compound of formula 2-4, which is directly used in the next step reaction, ESI-MS m/z calcd for [C 25 H 42 NO 12 ] + (M+H) + :548.60,found:548.59;
叠氮化钠NaN 3(1.99g,30.56mmol)溶于水(30mL)，缓慢滴入三氟甲磺酸酐(8.62g,30.56mmol)，搅拌15分钟，用甲苯萃取水相，有机相经无水硫酸钠干燥后制得三氟甲磺酰叠 氮TfN 3，将上步所得式2-4化合物粗品溶于乙醇，加入所制TfN 3和硫酸铜CuSO4(0.812g,5.09mmol)，搅拌6小时，蒸除溶剂，所得残余物溶于吡啶(50mL)，冰浴条件下滴入乙酸酐(26g,254.7mmol)，室温下搅拌12小时，冰浴下滴入甲醇淬灭反应，蒸除溶剂，残余物溶于二氯甲烷，先后经1N HCl a.q.、sat.NaHCO 3a.q.和饱和食盐水洗涤，无水硫酸钠干燥，浓缩后柱层析纯化，得式2-5化合物(18.1g,95.8％),ESI-MS m/z calcd for[C 33H 47N 3O 16] +(M+H) +:742.74,found:742.73； Sodium azide NaN 3 (1.99g, 30.56mmol) was dissolved in water (30mL), trifluoromethanesulfonic anhydride (8.62g, 30.56mmol) was slowly added dropwise, stirred for 15 minutes, the aqueous phase was extracted with toluene, and the organic phase was washed without After drying with sodium sulfate, trifluoromethanesulfonyl azide TfN 3 was obtained, the crude compound of formula 2-4 obtained in the previous step was dissolved in ethanol, the prepared TfN 3 and copper sulfate CuSO 4 (0.812g, 5.09mmol) were added, and stirred for 6 After 1 hour, the solvent was evaporated, the obtained residue was dissolved in pyridine (50 mL), acetic anhydride (26 g, 254.7 mmol) was added dropwise under an ice bath, stirred at room temperature for 12 hours, methanol was added dropwise under an ice bath to quench the reaction, and the solvent was evaporated. , the residue was dissolved in dichloromethane, washed with 1N HCl aq , sat.NaHCO 3a.q. and saturated brine successively, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain the compound of formula 2-5 (18.1 g , 95.8%), ESI-MS m/z calcd for [C 33 H 47 N 3 O 16 ] + (M+H) + : 742.74, found: 742.73;
式2-5化合物(18.1g,24.4mmol)与丙烯酸甲酯(2.46g,29.28mmol)、碘化亚铜(0.93g,4.88mmol)混于溶于四氢呋喃THF(60mL)，滴入N,N-二异丙基乙胺DIPEA(4.73gm 36.6mmol)，室温下搅拌1小时，蒸除反应溶剂，残余物溶于二氯甲烷，先后经1M氨水、1N HCl a.q.、sat.NaHCO 3a.q.和饱和食盐水洗涤，无水硫酸钠干燥，浓缩后柱层析纯化，得式2-6化合物(19.6g,97.3％),ESI-MS m/z calcd for[C 37H 52N 3O 18] +(M+H) +:826.82,found:826.81； The compound of formula 2-5 (18.1 g, 24.4 mmol) was mixed with methyl acrylate (2.46 g, 29.28 mmol) and cuprous iodide (0.93 g, 4.88 mmol) and dissolved in THF (60 mL), and N,N was added dropwise. -Diisopropylethylamine DIPEA (4.73gm 36.6mmol), stirred at room temperature for 1 hour, evaporated the reaction solvent, the residue was dissolved in dichloromethane, successively passed through 1M ammonia water, 1N HCl aq , sat.NaHCO 3a.q. Washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain the compound of formula 2-6 (19.6g, 97.3%), ESI-MS m/z calcd for [C 37 H 52 N 3 O 18 ] + (M+H) + :826.82,found:826.81;
式2-6化合物(19.6g,23.73mmol)溶于甲醇(100mL)，加入甲醇钠(0.1M in MeOH)，调节溶液pH 8～10，室温下搅拌30分钟，滴入盐酸中和反应液，蒸除溶剂，得式2-7粗品，直接用于下步反应；The compound of formula 2-6 (19.6 g, 23.73 mmol) was dissolved in methanol (100 mL), sodium methoxide (0.1 M in MeOH) was added, the pH of the solution was adjusted to 8-10, stirred at room temperature for 30 minutes, and hydrochloric acid was added dropwise to neutralize the reaction solution, The solvent is evaporated to obtain the crude product of formula 2-7, which is directly used in the next step reaction;
上步所得式2-7粗品溶于二氯甲烷(100mL)，加入三乙胺(4.8g,47.47mmol)，搅拌5分钟后分批加入对甲苯磺酰氯(6.79g，35.60mmol)，室温下搅拌6小时，加入甲醇淬灭反应，蒸除溶剂，残余物溶于二氯，盐水洗涤，有机相经无水硫酸钠干燥，蒸除溶剂，残余物溶于N，N-二甲基甲酰胺(50mL)，加入NaN 3(4.63g,71.20mmol)，于50℃油浴中搅拌12小时，TLC检测反应完全，蒸除溶剂，残余物溶于二氯甲烷，盐水洗涤，无水硫酸钠干燥，浓缩后柱层析得式2-8化合物(14.3g,88.3％)，ESI-MS m/z calcd for[C 29H 43N 6O 13] +(M+H) +:683.68,found:683.69； The crude product of formula 2-7 obtained in the previous step was dissolved in dichloromethane (100 mL), triethylamine (4.8 g, 47.47 mmol) was added, and p-toluenesulfonyl chloride (6.79 g, 35.60 mmol) was added in batches after stirring for 5 minutes. After stirring for 6 hours, methanol was added to quench the reaction, the solvent was evaporated, the residue was dissolved in dichloride, washed with brine, the organic phase was dried over anhydrous sodium sulfate, the solvent was evaporated, and the residue was dissolved in N,N-dimethylformamide (50 mL), NaN 3 (4.63 g, 71.20 mmol) was added, and the mixture was stirred in an oil bath at 50° C. for 12 hours. TLC detected that the reaction was complete, and the solvent was evaporated. The residue was dissolved in dichloromethane, washed with brine, and dried over anhydrous sodium sulfate. , the compound of formula 2-8 (14.3g, 88.3%) was obtained by column chromatography after concentration, ESI-MS m/z calcd for [C 29 H 43 N 6 O 13 ] + (M+H) + : 683.68, found: 683.69;
式2-8化合物(14.3g，20.95mmol)溶于四氢呋喃(100mL)，分批加入三苯基膦(16.48g,62.84mmol)，搅拌至无气泡产生后，继续搅拌12小时，蒸除溶剂，残余物溶于乙醇，缓慢滴入苯甲酰氯(3.53g,25.14mmol)，室温下搅拌1小时，蒸除溶剂，残余物直接柱层析纯化得式2-9化合物(13.1g,82.2％)，ESI-MS m/z calcd for[C 36H 49N 4O 14] +(M+H) +:761.79,found:761.78； The compound of formula 2-8 (14.3 g, 20.95 mmol) was dissolved in tetrahydrofuran (100 mL), triphenylphosphine (16.48 g, 62.84 mmol) was added in batches, stirred until no bubbles were generated, continued stirring for 12 hours, and the solvent was evaporated. The residue was dissolved in ethanol, slowly added dropwise with benzoyl chloride (3.53 g, 25.14 mmol), stirred at room temperature for 1 hour, evaporated to remove the solvent, and the residue was directly purified by column chromatography to obtain the compound of formula 2-9 (13.1 g, 82.2%) , ESI-MS m/z calcd for [C 36 H 49 N 4 O 14 ] + (M+H) + : 761.79, found: 761.78;
式2-9化合物(13.1g,17.22mmol)溶于乙腈(50mL)，加入丙酮缩二甲醇(5.38g,51.66mmol)和催化量的对甲苯磺酸(0.3g,1.72mmol)，于80℃油浴中回流12小时，TLC检测反应完全，加入三乙胺中和反应液，蒸除溶剂，残余物直接柱层析纯化得式2-10化合物(12.8g,92.8％),ESI-MS m/z calcd for[C 39H 53N 4O 13] +(M+H) +:801.86,found:801.86； The compound of formula 2-9 (13.1 g, 17.22 mmol) was dissolved in acetonitrile (50 mL), acetone acetal (5.38 g, 51.66 mmol) and a catalytic amount of p-toluenesulfonic acid (0.3 g, 1.72 mmol) were added, and the mixture was heated at 80° C. Refluxed in an oil bath for 12 hours, TLC detected that the reaction was complete, triethylamine was added to neutralize the reaction solution, the solvent was evaporated, and the residue was directly purified by column chromatography to obtain the compound of formula 2-10 (12.8 g, 92.8%), ESI-MS m /z calcd for[C 39 H 53 N 4 O 13 ] + (M+H) + :801.86,found:801.86;
式2-10化合物(12.8g,15.98mmol)和新制氧化银(7.41g,31.97mmol)混于N,N-二甲基甲酰胺(50mL)，加入碘甲烷(3.40g,23.97mmol)，室温下搅拌4小时，TLC检测反应完全，滤除不溶物， 蒸除溶剂，残余物溶于二氯甲烷，盐水洗涤，无水硫酸钠干燥，浓缩后柱层析纯化得式2-11化合物(11.6g,89.1％)，ESI-MS m/z calcd for[C 40H 55N 4O 14] +(M+H) +:815.89,found:815.88； The compound of formula 2-10 (12.8 g, 15.98 mmol) and freshly prepared silver oxide (7.41 g, 31.97 mmol) were mixed in N,N-dimethylformamide (50 mL), methyl iodide (3.40 g, 23.97 mmol) was added, room temperature After stirring for 4 hours, TLC detected that the reaction was complete, insoluble matter was filtered off, the solvent was evaporated, the residue was dissolved in dichloromethane, washed with brine, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain the compound of formula 2-11 (11.6 g, 89.1%), ESI-MS m/z calcd for [C 40 H 55 N 4 O 14 ] + (M+H) + : 815.89, found: 815.88;
式2-11化合物(11.6g,14.24mmol)溶于甲醇，加入对甲苯磺酸(0.25g,1.42mmol)，置于80℃油浴中回流6小时，TLC检测反应完全，加入三乙胺中和反应液，蒸除溶剂，残余物溶于二氯甲烷，盐水洗涤，无水硫酸钠干燥，浓缩后溶于甲醇，加入Pd/C粉末(0.1g)，于4atm压力H 2氛围中反应2小时，滤除Pd/C粉末，滤液浓缩后得式2-12化合物粗品，直接用于下步反应，ESI-MS m/z calcd for[C 30H 45N 4O 14] +(M+H) +:685.70,found:685.69； The compound of formula 2-11 (11.6 g, 14.24 mmol) was dissolved in methanol, added p-toluenesulfonic acid (0.25 g, 1.42 mmol), placed in an oil bath at 80°C and refluxed for 6 hours, TLC detected that the reaction was complete, added to triethylamine and the reaction solution, evaporated to remove the solvent, the residue was dissolved in dichloromethane, washed with brine, dried over anhydrous sodium sulfate, concentrated and dissolved in methanol, added Pd/C powder (0.1 g), and reacted under 4 atm pressure H atmosphere for 2 hours, the Pd/C powder was filtered off, and the filtrate was concentrated to obtain the crude compound of formula 2-12, which was directly used in the next step reaction, ESI-MS m/z calcd for [C 30 H 45 N 4 O 14 ] + (M+H ) + :685.70,found:685.69;
上步所得式2-12化合物粗品溶于二氯甲烷(60mL)，加入三乙胺(2.88g,28.47mmol)，搅拌5分钟后分批加入对甲苯磺酰氯(4.07g，21.35mmol)，室温下搅拌6小时，加入甲醇淬灭反应，蒸除溶剂，残余物溶于二氯，盐水洗涤，有机相经无水硫酸钠干燥，蒸除溶剂，残余物溶于N，N-二甲基甲酰胺(40mL)，加入NaN 3(2.78g,42.71mmol)，于50℃油浴中搅拌12小时，TLC检测反应完全，蒸除溶剂，残余物溶于二氯甲烷，盐水洗涤，无水硫酸钠干燥，浓缩后柱层析得式2-13化合物(8.2g,81.2％)，ESI-MS m/z calcd for[C 30H 43N 7O 13] +(M+H) +:710.71,found:710.70； The crude compound of formula 2-12 obtained in the previous step was dissolved in dichloromethane (60 mL), triethylamine (2.88 g, 28.47 mmol) was added, and after stirring for 5 minutes, p-toluenesulfonyl chloride (4.07 g, 21.35 mmol) was added in batches at room temperature. After stirring for 6 hours, methanol was added to quench the reaction, the solvent was evaporated, the residue was dissolved in dichloride, washed with brine, the organic phase was dried over anhydrous sodium sulfate, the solvent was evaporated, and the residue was dissolved in N,N-dimethylmethane Amide (40 mL), NaN 3 (2.78 g, 42.71 mmol) was added, and the mixture was stirred in an oil bath at 50° C. for 12 hours. The reaction was completed by TLC, and the solvent was evaporated. The residue was dissolved in dichloromethane, washed with brine, and anhydrous sodium sulfate. After drying and concentration, the compound of formula 2-13 (8.2g, 81.2%) was obtained by column chromatography, ESI-MS m/z calcd for [C 30 H 43 N 7 O 13 ] + (M+H) + : 710.71, found :710.70;
式2-13化合物(8.2g,11.55mmol)溶于甲醇(30mL)，加入NaOH水溶液(1.5M,10mL)，室温下搅拌30分钟，加入732型H +阳离子树脂中和反应液，滤除树脂，滤液中加入Pd/C粉末(0.1g)，于4atm压力H 2氛围中反应2小时，滤除Pd/C粉末，滤液浓缩后得式2化合物(7.1g，90.0％)，ESI-MS m/z calcd for[C 29H 44N 5O 13] +(M+H) +:670.69,found:670.68。 The compound of formula 2-13 (8.2g, 11.55mmol) was dissolved in methanol (30mL), NaOH aqueous solution (1.5M, 10mL) was added, stirred at room temperature for 30 minutes, 732 type H + cation resin was added to neutralize the reaction solution, and the resin was filtered off , Pd/C powder (0.1 g) was added to the filtrate, and the reaction was carried out in an atmosphere of 4 atm H2 for 2 hours, the Pd/C powder was filtered off, and the filtrate was concentrated to obtain the compound of formula 2 (7.1 g, 90.0%), ESI-MS m /z calcd for [C 29 H 44 N 5 O 13 ] + (M+H) + : 670.69, found: 670.68.
实施例5：具高亲合力的唾液酸配体分子7的合成Example 5: Synthesis of high affinity sialic acid ligand molecule 7
Figure PCTCN2020127148-appb-000031
Figure PCTCN2020127148-appb-000031
式7-1化合物制备过程同式2-4化合物，仅将其中HOPEG 4OBn试剂替换为HOPEG 3OBn即可； The preparation process of the compound of formula 7-1 is the same as that of the compound of formula 2-4, except that the HOPEG 4 OBn reagent is replaced by HOPEG 3 OBn;
式7-1化合物溶于乙醇，冰浴下滴入苯甲酰氯，继续搅拌反应1小时，蒸除溶剂，得式7-2化合物粗品，直接用于下步反应；The compound of formula 7-1 is dissolved in ethanol, benzoyl chloride is added dropwise under an ice bath, the reaction is continued to stir for 1 hour, and the solvent is evaporated to obtain a crude product of the compound of formula 7-2, which is directly used in the next step reaction;
由式7-2化合物制备式7-3化合物的过程同式1-4化合物制备式1-5化合物的过程，其中仅将Ac 2O试剂替换为TsCl即可； The process of preparing the compound of formula 7-3 from the compound of formula 7-2 is the same as the process of preparing the compound of formula 1-5 from the compound of formula 1-4, wherein only the Ac 2 O reagent is replaced by TsCl;
从式7-3化合物出发制备式7化合物的过程同从式2-9化合物制备式2化合物的过程。The procedure for preparing the compound of formula 7 from the compound of formula 7-3 is the same as the procedure for preparing the compound of formula 2 from the compound of formula 2-9.
实施例6：具高亲合力的唾液酸配体分子8的合成Example 6: Synthesis of high affinity sialic acid ligand molecule 8
Figure PCTCN2020127148-appb-000032
Figure PCTCN2020127148-appb-000032
式8-1化合物的制备过程同式2-4化合物，仅将其中HOPEG 4OBn试剂替换为HOC 5H 10OAll即可； The preparation process of the compound of formula 8-1 is the same as that of the compound of formula 2-4, except that the HOPEG 4 OBn reagent is replaced with HOC 5 H 10 OAll;
式8-1化合物制备式8-2化合物的过程同式7-1化合物制备式7-2化合物过程，仅将BzCl试剂替换为苄氧羰酰氯CbzCl即可；The process of preparing the compound of formula 8-2 from the compound of formula 8-1 is the same as the process of preparing the compound of formula 7-2 from the compound of formula 7-1, except that the BzCl reagent is replaced by benzyloxycarbonyl chloride CbzCl;
式8-2化合物制备式8-3化合物的过程同式5-1化合物制备式5-2化合物的过程；The process of preparing the compound of formula 8-3 from the compound of formula 8-2 is the same as the process of preparing the compound of formula 5-2 from the compound of formula 5-1;
式8-3化合物制备式8-4化合物的过程同式7-3化合物制备式7-4化合物的过程；The process of preparing the compound of formula 8-4 from the compound of formula 8-3 is the same as the process of preparing the compound of formula 7-4 from the compound of formula 7-3;
式8-4化合物(10g,14.89mmol)溶于DMF(100mL)，滴入溴苄(3.82g,22.83mmol)，加入氢氧化钡(5.1g,29.77mmol)，室温下搅拌12小时，TLC检测反应完全，滴入甲醇淬灭反应，反应液加入二氯甲烷(300mL)稀释，饱和食盐水洗涤三次，无水硫酸钠干燥，浓缩后柱层析分离，得式8-5化合物(10.1g,89％),ESI-MS m/z calcd for[C 43H 56NO 11] +(M+H) +:762.91,found:762.90； The compound of formula 8-4 (10 g, 14.89 mmol) was dissolved in DMF (100 mL), benzyl bromide (3.82 g, 22.83 mmol) was added dropwise, barium hydroxide (5.1 g, 29.77 mmol) was added, stirred at room temperature for 12 hours, and detected by TLC The reaction was completed, methanol was added dropwise to quench the reaction, dichloromethane (300 mL) was added to the reaction solution to dilute, washed with saturated brine three times, dried over anhydrous sodium sulfate, and separated by column chromatography after concentration to obtain the compound of formula 8-5 (10.1 g, 89%), ESI-MS m/z calcd for [C 43 H 56 NO 11 ] + (M+H) + : 762.91, found: 762.90;
式8-5化合物(10.1g,13.26mmol)溶于甲醇(100mL)，加入TsOH(114mg,0.66mmol)，反应液置于80℃油浴中回流反应12小时，TLC检测反应完全，反应液冷却至室温，加入三乙胺中和反应液至中性，加入氯化钯(4.7g,26.51mmol)，室温下反应1小时，TLC检测反应完全，蒸除 溶剂，残余物溶于二氯甲烷，饱和食盐水洗涤，无水硫酸钠干燥，浓缩后柱层析分离，得式8-6化合物(7.4g,81.9％),ESI-MS m/z calcd for[C 37H 48NO 11] +(M+H) +:682.78,found:682.77； The compound of formula 8-5 (10.1 g, 13.26 mmol) was dissolved in methanol (100 mL), TsOH (114 mg, 0.66 mmol) was added, and the reaction solution was placed in an oil bath at 80 °C for reflux reaction for 12 hours. TLC detected that the reaction was complete, and the reaction solution was cooled. To room temperature, triethylamine was added to neutralize the reaction solution to neutrality, palladium chloride (4.7 g, 26.51 mmol) was added, and the reaction was carried out at room temperature for 1 hour. TLC detected that the reaction was complete. The solvent was evaporated and the residue was dissolved in dichloromethane. Washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and separated by column chromatography to obtain the compound of formula 8-6 (7.4 g, 81.9%), ESI-MS m/z calcd for [C 37 H 48 NO 11 ] + ( M+H) + :682.78,found:682.77;
式8-6化合物制备式8-7化合物的过程同式7-6化合物制备式7-7化合物的过程；The process of preparing the compound of formula 8-7 from the compound of formula 8-6 is the same as the process of preparing the compound of formula 7-7 from the compound of formula 7-6;
式8-7化合物(4g,5.66mmol)溶于甲醇(40mL)，滴入NaOH水溶液(1M,20mL)，搅拌至TLC检测反应完全，中和反应液，蒸除溶剂，残余物溶于四氢呋喃(40mL)，加入Ph3P(4.45g,16.98mmol)，室温下搅拌12小时，TLC检测反应完全，蒸除溶剂，残物语直接柱层析纯化，得式8化合物(3.2g,84.8％),ESI-MS m/z calcd for[C 36H 47N 2O 10] +(M+H) +:667.77,found:667.76； The compound of formula 8-7 (4 g, 5.66 mmol) was dissolved in methanol (40 mL), NaOH aqueous solution (1 M, 20 mL) was added dropwise, stirred until the reaction was completed by TLC, the reaction solution was neutralized, the solvent was evaporated, and the residue was dissolved in tetrahydrofuran ( 40mL), Ph3P (4.45g, 16.98mmol) was added, stirred at room temperature for 12 hours, TLC detected that the reaction was complete, the solvent was evaporated, and the residue was directly purified by column chromatography to obtain the compound of formula 8 (3.2g, 84.8%), ESI- MS m/z calcd for [C 36 H 47 N 2 O 10 ] + (M+H) + : 667.77, found: 667.76;
实施例7：唾液酸-金纳米粒标记复合物3的制备Example 7: Preparation of sialic acid-gold nanoparticle labeling complex 3
Figure PCTCN2020127148-appb-000033
Figure PCTCN2020127148-appb-000033
柠檬酸钠稳定、粒径30nm的金纳米粒溶液制备方法为：取100mL 1mM的HAuCl 4水溶液至两口瓶中，加热至100℃后，加入柠檬酸钠(二水合物)(118mg,0.3mmol)，搅拌15分钟后，取出缓慢降至室温，即得。 The preparation method of gold nanoparticle solution with stable sodium citrate and particle size of 30nm is as follows: take 100mL of 1mM HAuCl4 aqueous solution into a two - necked flask, heat it to 100°C, and add sodium citrate (dihydrate) (118mg, 0.3mmol) , After stirring for 15 minutes, take it out and slowly lower it to room temperature.
将实施例1中所得唾液酸分子1溶于水制成水溶液(10mM)，取100μL加至柠檬酸钠稳定、粒径30nm的金纳米粒溶液中(OD＝15，5mL)，静置15小时后，加入过量巯基四甘醇(PEG 4-SH)封闭溶液(0.1M，100uL)，震荡12小时后，经15000rpm离心15分钟2次，离心管底物聚集物重新分散于去离子水(5mL)，得唾液酸-金纳米粒标记复合物3。 The sialic acid molecule 1 obtained in Example 1 was dissolved in water to prepare an aqueous solution (10 mM), and 100 μL was added to the gold nanoparticle solution (OD=15, 5 mL) with a stable sodium citrate and a particle size of 30 nm (OD=15, 5 mL), and allowed to stand for 15 hours Then, excess mercaptotetraethylene glycol (PEG 4 -SH) blocking solution (0.1M, 100uL) was added, and after shaking for 12 hours, centrifuged at 15,000 rpm for 15 minutes twice, and the centrifuge tube substrate aggregates were redispersed in deionized water (5 mL). ) to obtain sialic acid-gold nanoparticle-labeled complex 3.
实施例8：唾液酸配体固定物4的制备Example 8: Preparation of Sialic Acid Ligand Immobilizer 4
Figure PCTCN2020127148-appb-000034
Figure PCTCN2020127148-appb-000034
实施例2所得唾液酸配体分子2(1g,1.46mmol)溶于甲醇(20mL)，加入方酸二乙酯(2.48g,14.58mmol)，室温下搅拌24h，反应液直接浓缩柱层析，分离得式4-1化合物中间体(1.1g,93.1％)，ESI-MS m/z calcd for[C 35H 47N 5O 17] +(M+H) +:810.78,found:810.77，回收多余的方酸二乙酯； Sialic acid ligand molecule 2 (1 g, 1.46 mmol) obtained in Example 2 was dissolved in methanol (20 mL), diethyl squaraine (2.48 g, 14.58 mmol) was added, stirred at room temperature for 24 h, and the reaction solution was directly concentrated by column chromatography, The intermediate compound of formula 4-1 was isolated (1.1 g, 93.1%), ESI-MS m/z calcd for [C 35 H 47 N 5 O 17 ] + (M+H) + : 810.78, found: 810.77, recovered excess diethyl squaraine;
所得式4-1化合物(1.1g,1.36mmol)同牛血清白蛋白BSA(66.43kDa,3g,45umol)混于水中，静置24h，反应液经凝胶柱纯化，得式4复合物(3.8g)，溶于超纯水，制成1nM溶液。The obtained compound of formula 4-1 (1.1g, 1.36mmol) was mixed with bovine serum albumin BSA (66.43kDa, 3g, 45umol) in water, and allowed to stand for 24h. The reaction solution was purified by gel column to obtain the compound of formula 4 (3.8 g), dissolved in ultrapure water to make a 1nM solution.
实施例9：制备新冠病毒检测试纸条Example 9: Preparation of new coronavirus detection test strips
裁剪样品垫，金标记垫，检测垫，吸水垫，长×宽规格分别为2cm×2cm，1cm×2cm，6cm×2cm，1cm×2cm将由实施例7制备的唾液酸-金纳米粒标记复合物3溶液(10μL)喷洒于金标垫上，将由实施例8制备的唾液酸配体固定物4溶液(5μL)喷于检测垫的T线(0.1×2cm)区域，以商业化的新冠病毒S蛋白溶液(1nM，5μL)作为控制物喷于检测垫的C线(0.1×2cm)区域，以吸水纸为吸水垫，将样品垫、金标记垫、检测垫、吸水垫按照图1检测装置结构所示的层次和位置贴于PVC衬板上，各层重叠0.5cm，制备获得新冠病毒检测试纸条。Cut out the sample pad, gold marker pad, detection pad, and water-absorbing pad, the length x width are respectively 2cm x 2cm, 1cm x 2cm, 6cm x 2cm, and 1cm x 2cm. The sialic acid-gold nanoparticle-labeled complex prepared in Example 7 The 3 solution (10 μL) was sprayed on the gold-labeled pad, and the sialic acid ligand immobilizer 4 solution (5 μL) prepared in Example 8 was sprayed on the T line (0.1×2 cm) area of the detection pad to commercialize the new coronavirus S protein. The solution (1nM, 5μL) was sprayed on the C-line (0.1×2cm) area of the detection pad as the control substance, and the absorbent paper was used as the absorbent pad. The layers and positions shown are pasted on the PVC liner, and each layer is overlapped by 0.5cm to prepare a new coronavirus detection test strip.
实施例10：检测实验Example 10: Detection experiment
新冠病毒具有很强的致病性和传染性，检测实验通过新冠病毒模拟颗粒来进行，将商业化新冠病毒S蛋白固定于约100nm直径的SiO 2纳米球表面构建新冠病毒模拟颗粒，取新冠病毒模拟颗粒水溶液1滴(10 -6个/mL，约50μL)，滴加至新冠病毒检测试纸条样品孔，水平静置15分钟，试剂盒的T线显红色，C线显红色。 The 2019-nCoV is highly pathogenic and infectious. The detection experiment was carried out by using 2019-nCoV simulated particles. The commercialized 2019-nCoV S protein was immobilized on the surface of SiO 2 nanospheres with a diameter of about 100nm to construct 2019-nCoV simulative particles. Add 1 drop ( 10-6 /mL, about 50 μL) of simulated particle aqueous solution to the sample well of the new coronavirus detection test strip, and let it stand for 15 minutes horizontally. The T line of the kit is red, and the C line is red.
实施例11：平行检测实验Example 11: Parallel detection experiment
取两个新冠病毒检测试纸条，编号分别为试纸条A和试纸条B，取健康人唾液，分两份分别标记为唾液A和唾液B，唾液A加入实施例10中所制新冠病毒模拟颗粒溶液，唾液B加入去离子水，分别取两种样本1滴(约50μL)，分别滴入试纸条A和试纸条B样品孔，水平静置15分钟，试纸条A的T线显红色，C线显色，试纸条B的T线不显色，C线显色。Take two new coronavirus detection test strips, numbered test strip A and test strip B, respectively, take the saliva of healthy people, and mark them as saliva A and saliva B respectively, and add saliva A to the new crown prepared in Example 10. Virus simulation particle solution, add deionized water to saliva B, take 1 drop (about 50 μL) of the two samples respectively, drop them into the sample holes of test strip A and test strip B respectively, and let stand horizontally for 15 minutes. The T line is red, the C line is colored, the T line of the test strip B is not colored, and the C line is colored.
综上，本发明实施例提供了一种能直接检测新冠病毒颗粒的检测试纸条，能非常快速、便捷的对样本中是否含病毒颗粒进行快速判定，进而对受试者是否具有传染性进行迅速判定。In summary, the embodiment of the present invention provides a test strip that can directly detect new coronavirus particles, which can quickly and conveniently determine whether the sample contains virus particles, and then test whether the subject is infectious. judge quickly.
以上所述仅为本发明的具体实施方式，不是全部的实施方式，本领域普通技术人员通过阅读本发明说明书而对本发明技术方案采取的任何等效的变换，均为本发明的权利要求所涵盖。The above descriptions are only specific implementations of the present invention, not all implementations. Any equivalent transformations taken by those of ordinary skill in the art to the technical solutions of the present invention by reading the specification of the present invention are covered by the claims of the present invention. .

Claims (10)

  1. 一种新冠病毒检测试纸条，其特征在于，包括衬板(1)，所述衬板(1)上的一端至另一端依次设有样品垫(2)、金标记垫(3)、检测垫(4)和吸水板(5)，所述检测垫(4)的表面设有硝酸纤维素膜，所述硝酸纤维素膜上设有检测T线(6)和质控C线(7)，所述检测T线(6)靠近金标记垫(3)，所述质控C线(7)靠近吸水板(5)；A new coronavirus detection test strip, characterized in that it comprises a backing plate (1), and one end of the backing plate (1) is provided with a sample pad (2), a gold marking pad (3), a detection A pad (4) and a water-absorbing plate (5), the surface of the detection pad (4) is provided with a nitrocellulose membrane, and the nitrocellulose membrane is provided with a detection T line (6) and a quality control C line (7) , the detection T line (6) is close to the gold marking pad (3), and the quality control C line (7) is close to the water absorption plate (5);
    所述检测垫(4)的两端分别与金标记垫(3)和吸水板(5)相互交叠连接，所述金标记垫(3)上压贴有样品垫(2)；The two ends of the detection pad (4) are respectively overlapped and connected with the gold marking pad (3) and the water absorbing plate (5), and the gold marking pad (3) is pressed with a sample pad (2);
    所述检测T线(6)上包被有唾液酸配体固定物，所述唾液酸配体固定物包括具高亲合力的唾液酸配体分子，所述具高亲合力的唾液酸配体分子的结构通式如式II所示：
    Figure PCTCN2020127148-appb-100001
    其中，R 1选自甲氧基或取代的甲氧基；R 2选自取代的乙酰氨基、苯甲酰氨基、取代的苯甲酰氨基、烷氧羰酰胺基、三氮唑基或取代的三氮唑基；R 3选自羟基、甲氧基、取代的甲氧基、乙酰氨基、取代的乙酰氨基、磺酰胺基或磷酰胺基；A为
    Figure PCTCN2020127148-appb-100002
    其中n1选自0,1,2,3,4,5,6,7，m1选自2,3,4,5,6,7,8,9，R 4选自氨基或巯基；     The detection T line (6) is coated with a sialic acid ligand immobilizer, and the sialic acid ligand immobilizer includes a sialic acid ligand molecule with high affinity, and the sialic acid ligand with high affinity The general structural formula of the molecule is shown in formula II:
    Figure PCTCN2020127148-appb-100001
    Wherein, R 1 is selected from methoxy or substituted methoxy; R 2 is selected from substituted acetamido, benzamido, substituted benzamido, alkoxycarbonamido, triazolyl or substituted Triazolyl; R is selected from hydroxyl, methoxy, substituted methoxy, acetamido, substituted acetamido, sulfonamide or phosphoramido; A is
    Figure PCTCN2020127148-appb-100002
    wherein n1 is selected from 0,1,2,3,4,5,6,7, m1 is selected from 2,3,4,5,6,7,8,9, R4 is selected from amino or mercapto ;
    所述质控C线(7)上包被有控制物，所述控制物为新冠病毒S蛋白；The quality control C line (7) is coated with a control substance, and the control substance is the new coronavirus S protein;
    所述金标记垫(3)上喷有胶体金标记物，所述胶体金标记物为表面固定了具中等亲合力的唾液酸配体分子的金纳米粒；所述具中等亲合力的唾液酸配体分子的结构通式如式I所示：
    Figure PCTCN2020127148-appb-100003
    其中，R 5选自羟基、甲氧基、取代的甲氧基、乙酰氨基、取代的乙酰氨基、磺酰胺基或磷酰胺基；A1为
    Figure PCTCN2020127148-appb-100004
    n2选自0,1,2,3,4,5,6,7，m2选自2,3,4,5,6,7,8,9。     The gold labeling pad (3) is sprayed with a colloidal gold label, and the colloidal gold label is a gold nanoparticle with a sialic acid ligand molecule with a medium affinity immobilized on the surface; the sialic acid with a medium affinity is The general structural formula of the ligand molecule is shown in formula I:
    Figure PCTCN2020127148-appb-100003
    Wherein, R 5 is selected from hydroxyl, methoxy, substituted methoxy, acetamido, substituted acetamido, sulfonamide or phosphoramido; A1 is
    Figure PCTCN2020127148-appb-100004
    n2 is selected from 0,1,2,3,4,5,6,7, m2 is selected from 2,3,4,5,6,7,8,9.
  2. 根据权利要求1所述的新冠病毒检测试纸条，其特征在于，所述样品垫(2)上设有加样孔(8)。The new coronavirus detection test strip according to claim 1, characterized in that, the sample pad (2) is provided with a sample addition hole (8).
  3. 根据权利要求1所述的新冠病毒检测试纸条，其特征在于，The new coronavirus detection test strip according to claim 1, characterized in that,
    所述取代的甲氧基为X 1-CH 2O-，其中，X 1选自苯基或乙烯基； The substituted methoxy group is X 1 -CH 2 O-, wherein X 1 is selected from phenyl or vinyl;
    所述取代的乙酰氨基为X 2-CH 2CONH-，其中，X 2选自甲基、乙基、正丙基、异丙基、羟甲基或羟乙基； The substituted acetamido is X 2 -CH 2 CONH-, wherein X 2 is selected from methyl, ethyl, n-propyl, isopropyl, hydroxymethyl or hydroxyethyl;
    所述取代的苯甲酰氨基为X 3-BzNH-，其中，X 3的数量至少为1且位于苯环的任意位置，X3选自卤原子、甲基、甲氧基、硝基中的至少一种； The substituted benzamido is X 3 -BzNH-, wherein the number of X 3 is at least 1 and is located at any position of the benzene ring, and X 3 is selected from at least one of halogen atoms, methyl groups, methoxy groups, and nitro groups. A sort of;
    所述烷氧羰酰胺基为X 4-OC(O)NH-，其中，X 4选自苄基、烯丙基、叔丁基或三氯乙基； The alkoxycarbonamide group is X 4 -OC(O)NH-, wherein X 4 is selected from benzyl, allyl, tert-butyl or trichloroethyl;
    所述取代的三氮唑基为
    Figure PCTCN2020127148-appb-100005
    其中，X 5选自取代的苯烷基X 6-Ph-(CH 2) n6-或取代的羰基X 7-C(O)-，其中，X 6的数量至少为1且位于苯环的任意位置，X 6选自卤原子、甲基、甲氧基、硝基中的至少一种，n6选自0,1,2；X 7选自烷氧基CH 3-(CH 2) n7-O-或烷胺基CH 3-(CH 2) n7-NH-，n7选自0,1,2,3,4,5。     The substituted triazolyl is
    Figure PCTCN2020127148-appb-100005
    Wherein, X 5 is selected from substituted phenylalkyl X 6 -Ph-(CH 2 ) n6 - or substituted carbonyl X 7 -C(O)-, wherein X 6 is at least 1 in number and located at any position in the benzene ring Position, X 6 is selected from at least one of halogen atom, methyl group, methoxy group and nitro group, n6 is selected from 0, 1, 2; X 7 is selected from alkoxy group CH 3 -(CH 2 ) n7 -O -or alkylamino CH3- ( CH2 ) n7 -NH-, n7 is selected from 0,1,2,3,4,5.
  4. 根据权利要求1所述的新冠病毒检测试纸条，其特征在于，所述唾液酸配体固定物包括固定蛋白、具高亲合力的唾液酸配体分子和连接分子，所述固定蛋白为用作封闭固定的蛋白大分子；The new coronavirus detection test strip according to claim 1, wherein the sialic acid ligand immobilizer comprises an immobilized protein, a sialic acid ligand molecule with high affinity and a linking molecule, and the immobilized protein is a For sealing and immobilizing protein macromolecules;
    优选地，所述固定蛋白选自牛血清白蛋白BSA、脱脂奶粉或酪蛋白；Preferably, the immobilized protein is selected from bovine serum albumin BSA, skimmed milk powder or casein;
    优选地，所述连接分子的结构通式为R 6-linker-R 7，其中，R 6为能同R 4反应连接的基团，更优选地，所述R 6选自羧基、酯基、磺酸基、磷酸基或马来酰亚胺基；R 7为能与固定蛋白表面存在的活性反应基团反应的基团，更优选地，所述R 7选自羧基、酯基、氨基或马来酰亚胺基；所述linker选自烷基、环烷基、聚乙二醇链或芳基；更优选的，所述连接分子选自方酸二乙酯、二甘醇酸、马来酰亚胺-聚乙二醇羧酸、1,4-环己烷二羧酸。 Preferably, the general structural formula of the linker molecule is R 6 -linker-R 7 , wherein R 6 is a group that can react and link with R 4 , more preferably, the R 6 is selected from a carboxyl group, an ester group, Sulfonic acid group, phosphoric acid group or maleimide group; R 7 is a group capable of reacting with active reactive groups existing on the surface of the immobilized protein, more preferably, said R 7 is selected from carboxyl group, ester group, amino group or Maleimide group; the linker is selected from alkyl, cycloalkyl, polyethylene glycol chain or aryl; more preferably, the linker is selected from diethyl squaraine, diglycolic acid, Leimide-polyethylene glycol carboxylic acid, 1,4-cyclohexanedicarboxylic acid.
  5. 根据权利要求1所述的新冠病毒检测试纸条，其特征在于，所述胶体金标记物包括金纳米粒、具中等亲合力的唾液酸配体分子和封闭分子；The new coronavirus detection test strip according to claim 1, wherein the colloidal gold marker comprises gold nanoparticles, sialic acid ligand molecules with medium affinity and blocking molecules;
    优选地，所述金纳米粒的粒径为30-50nm；Preferably, the particle size of the gold nanoparticles is 30-50 nm;
    优选地，所述封闭分子为小分子巯基聚乙二醇，更优选地，所述封闭分子的结构通式为R 8-(C 2H 4O) n3-C 2H 4-SH，n3选自1,2,3,4,5,6,7,8,9，R 8选自羟基、甲氧基或羧基。 Preferably, the blocking molecule is a small molecule thiol polyethylene glycol, more preferably, the general structural formula of the blocking molecule is R 8 -(C 2 H 4 O) n3 -C 2 H 4 -SH, and n3 is selected from From 1, 2, 3, 4, 5, 6, 7, 8, 9, R 8 is selected from hydroxy, methoxy or carboxyl.
  6. 权利要求1-5任一所述的新冠病毒检测试纸条的制备方法，其特征在于，包括以下步骤：The preparation method of the new coronavirus detection test strip described in any one of claims 1-5, is characterized in that, comprises the following steps:
    制备胶体金标记物，将胶体金标记物喷在金标记垫(3)上；Prepare a colloidal gold marker, and spray the colloidal gold marker on the gold marker pad (3);
    制备唾液酸配体固定物，将唾液酸配体固定物包被在检测T线(6)上；Prepare a sialic acid ligand immobilized substance, and coat the sialic acid ligand immobilized substance on the detection T line (6);
    将新冠病毒S蛋白包被在质控C线(7)上；Coat the new coronavirus S protein on the quality control line C (7);
    将样品垫(2)、金标记垫(3)、检测垫(4)和吸水板(5)黏贴到衬板(1)上，获得所述新冠病毒检测试纸条；Paste the sample pad (2), the gold marking pad (3), the detection pad (4) and the water absorption plate (5) on the liner (1) to obtain the new coronavirus detection test strip;
    优选地，所述胶体金标记物以金纳米粒为标记内核，在金纳米粒表面通过S-Au键固定具中等亲合力的唾液酸配体分子，以小分子聚乙二醇作为封闭分子；Preferably, the colloidal gold marker uses gold nanoparticles as the inner core of the marker, immobilizes sialic acid ligand molecules with medium affinity on the surface of the gold nanoparticles through S-Au bonds, and uses small molecular polyethylene glycol as the blocking molecule;
    优选地，所述唾液酸配体固定物为通过连接分子将固定蛋白与具高亲合力的唾液酸配体分子连接的复合物，所述固定蛋白为用作封闭固定的蛋白大分子。Preferably, the sialic acid ligand immobilizer is a complex in which an immobilized protein is linked with a sialic acid ligand molecule with high affinity through a linking molecule, and the immobilized protein is a protein macromolecule used for blocking and immobilization.
  7. 根据权利要求6所述的制备方法，其特征在于，所述胶体金标记物的制备方法为：将具中等亲合力的唾液酸配体分子配置成水溶液，同金纳米粒溶液混合后，在20～30℃下搅拌12～24小时，通过在金纳米粒表面形成S-Au键而固定于金纳米粒表面，继而加入过量的小分子巯基聚乙二醇，在20～30℃下继续搅拌12～24小时对金纳米粒表面剩余反应位点进行封闭，通过离心纯化获得唾液酸-金纳米粒标记复合物；The preparation method according to claim 6, characterized in that, the preparation method of the colloidal gold label is as follows: disposing the sialic acid ligand molecule with medium affinity into an aqueous solution, mixing with the gold nanoparticle solution, and then at 20 Stir for 12 to 24 hours at ~30 °C, and fix it on the surface of gold nanoparticles by forming S-Au bonds on the surface of gold nanoparticles, then add excess small molecular mercapto polyethylene glycol, and continue stirring at 20 ~ 30 °C for 12 The remaining reaction sites on the surface of gold nanoparticles are blocked for ~24 hours, and the sialic acid-gold nanoparticles labeling complex is obtained by centrifugal purification;
    优选地，所述具中等亲合力的唾液酸配体分子与金纳米粒的摩尔比为1×10 4:1～2×10 4:1； Preferably, the molar ratio of the medium affinity sialic acid ligand molecule to the gold nanoparticle is 1×10 4 :1˜2×10 4 :1;
    优选地，所述具中等亲合力的唾液酸配体分子的制备方法为：以唾液酸
    Figure PCTCN2020127148-appb-100006
    为起始原料，经羧基保护在羧基引入R 9保护基得到
    Figure PCTCN2020127148-appb-100007
    在氯代和乙酰化试剂作用下一步生成全乙酰化氯代糖
    Figure PCTCN2020127148-appb-100008
    继而经糖基化反应在糖环2-位衍生出功能侧链前体
    Figure PCTCN2020127148-appb-100009
    经脱乙酰化反应后裸露出糖环上的数个羟基得到
    Figure PCTCN2020127148-appb-100010
    继而利用糖环9-OH的伯羟基与其他仲羟基的反应活性差异引入R5基团得到
    Figure PCTCN2020127148-appb-100011
    最后经基团转换、脱保护反应合成具中等亲合力的唾液酸配体分子；其中R 9选自甲基或苄基，A 2为
    Figure PCTCN2020127148-appb-100012
    或-(CH 2)m3-R 10，R10选自苄氧基、 2-萘甲氧基或烯丙氧基，n4选自0,1,2,3,4,5,6,7，m3选自2,3,4,5,6,7,8,9；当R 5为羟基时，其为唾液酸
    Figure PCTCN2020127148-appb-100013
    分子上的基团，不需要引入R 5基团的相关反应过程；     Preferably, the preparation method of the sialic acid ligand molecule with medium affinity is: using sialic acid
    Figure PCTCN2020127148-appb-100006
    As the starting material, through the protection of the carboxyl group, the R 9 protecting group is introduced into the carboxyl group to obtain
    Figure PCTCN2020127148-appb-100007
    Generation of peracetylated chlorosaccharides in the next step of chlorination and acetylation reagents
    Figure PCTCN2020127148-appb-100008
    Then, a functional side chain precursor is derived from the 2-position of the sugar ring through a glycosylation reaction
    Figure PCTCN2020127148-appb-100009
    After deacetylation, several hydroxyl groups on the sugar ring are exposed to obtain
    Figure PCTCN2020127148-appb-100010
    Then, the R5 group was introduced by using the reactivity difference between the primary hydroxyl group of the sugar ring 9-OH and other secondary hydroxyl groups to obtain
    Figure PCTCN2020127148-appb-100011
    Finally, a sialic acid ligand molecule with moderate affinity is synthesized through group conversion and deprotection reaction; wherein R 9 is selected from methyl or benzyl, and A 2 is
    Figure PCTCN2020127148-appb-100012
    Or -(CH 2 )m3-R 10 , R10 is selected from benzyloxy, 2-naphthylmethoxy or allyloxy, n4 is selected from 0,1,2,3,4,5,6,7, m3 Selected from 2,3,4,5,6,7,8,9; when R5 is hydroxyl, it is sialic acid
    Figure PCTCN2020127148-appb-100013
    The group on the molecule does not need to introduce the relevant reaction process of the R 5 group;
    优选地，所述金纳米粒溶液的制备方法为：将氯金酸水溶液加热至沸腾，加入柠檬酸钠，保持沸腾15～30分钟，冷却后即得粒径为30～50nm、表面覆盖柠檬酸钠的金纳米粒溶液，其中，氯金酸水溶液的浓度为1mM，氯金酸、柠檬酸钠的摩尔比为1:2-4。Preferably, the preparation method of the gold nanoparticle solution is as follows: heating the aqueous solution of chloroauric acid to boiling, adding sodium citrate, keeping boiling for 15-30 minutes, and cooling to obtain a particle size of 30-50 nm and a surface covered with citric acid The sodium gold nanoparticle solution, wherein the concentration of chloroauric acid aqueous solution is 1 mM, and the molar ratio of chloroauric acid and sodium citrate is 1:2-4.
  8. 根据权利要求6所述的制备方法，其特征在于，所述唾液酸配体固定物的制备方法为：The preparation method according to claim 6, wherein the preparation method of the sialic acid ligand immobilizer is:
    将具高亲合力的唾液酸配体分子同连接分子混合在水中，在20～30℃下搅拌12～24小时，具高亲合力的唾液酸配体分子通过功能侧链A上的活性反应基团R 4与连接分子的R 6基团反应共价连接，获得唾液酸-连接分子复合物；然后加入固定蛋白，在20～25℃下静置15～30小时，通过唾液酸-连接分子复合物上R 7基团与固定蛋白表面活性反应基团形成共价键，获得目标唾液酸配体固定物； Mix the sialic acid ligand molecule with high affinity and the linking molecule in water, and stir at 20-30 °C for 12-24 hours, the sialic acid ligand molecule with high affinity passes through the active reactive group on the functional side chain A The group R 4 reacts with the R 6 group of the linking molecule and is covalently linked to obtain a sialic acid-linking molecule complex; then add immobilized protein, stand at 20 ~ 25 ℃ for 15 ~ 30 hours, through the sialic acid-linking molecule complex The R 7 group on the substance forms a covalent bond with the surface active reactive group of the immobilized protein to obtain the target sialic acid ligand immobilizer;
    优选地，所述具高亲合力的唾液酸配体分子、连接分子、固定蛋白的摩尔比为5～10:12～15:1；Preferably, the molar ratio of the high-affinity sialic acid ligand molecule, linker molecule, and immobilized protein is 5-10:12-15:1;
    优选地，所述具高亲合力的唾液酸配体分子的制备方法为：以唾液酸
    Figure PCTCN2020127148-appb-100014
    为起始原料，经羧基保护在羧基引入R 11保护基得到
    Figure PCTCN2020127148-appb-100015
    在氯代和乙酰化试剂作用下一步生成全乙酰化氯代糖
    Figure PCTCN2020127148-appb-100016
    继而经糖基化反应在糖环2位衍生出功能侧链前体
    Figure PCTCN2020127148-appb-100017
    随后经脱乙酰基、羧基保护后得到糖环上氨基和羟基全部裸露的中间体
    Figure PCTCN2020127148-appb-100018
    按照R 2→R 1→R 3顺序进行基团的引入分别得到
    Figure PCTCN2020127148-appb-100019
    或者，按照R 2→R 3→R 1的顺序进行基团的引入分别得到
    Figure PCTCN2020127148-appb-100020
    Figure PCTCN2020127148-appb-100021
    最后通过侧链基团转换和糖环保护基脱除反应，合成具高亲合力的唾液酸配体分子；其中R11选自甲基或苄基，A 3为
    Figure PCTCN2020127148-appb-100022
    或-(CH 2)m4-R 12，R 12选自苄氧基、2-萘甲氧基或烯丙氧基，n5选自0,1,2,3,4,5,6,7，m4选自2,3,4,5,6,7,8,9，PG是保护基protection group的缩写；当R 3为羟基时，其为唾液酸
    Figure PCTCN2020127148-appb-100023
    分子上的基团，不需要引入R 3基团的相关反应过程。     Preferably, the preparation method of the sialic acid ligand molecule with high affinity is: using sialic acid
    Figure PCTCN2020127148-appb-100014
    As the starting material, through the protection of the carboxyl group, the R 11 protecting group is introduced into the carboxyl group to obtain
    Figure PCTCN2020127148-appb-100015
    Generation of peracetylated chlorosaccharides in the next step of chlorination and acetylation reagents
    Figure PCTCN2020127148-appb-100016
    Then the functional side chain precursor is derived from the glycosylation reaction at the 2-position of the sugar ring
    Figure PCTCN2020127148-appb-100017
    After deacetylation and carboxyl protection, an intermediate with all the exposed amino and hydroxyl groups on the sugar ring is obtained
    Figure PCTCN2020127148-appb-100018
    Introduce groups in the order of R 2 →R 1 →R 3 to obtain
    Figure PCTCN2020127148-appb-100019
    Alternatively, the introduction of groups in the order of R 2 →R 3 →R 1 can be obtained, respectively.
    Figure PCTCN2020127148-appb-100020
    Figure PCTCN2020127148-appb-100021
    Finally, a sialic acid ligand molecule with high affinity is synthesized through the conversion of the side chain group and the removal of the sugar ring protecting group; wherein R11 is selected from methyl or benzyl, and A 3 is
    Figure PCTCN2020127148-appb-100022
    or -(CH 2 )m4-R 12 , R 12 is selected from benzyloxy, 2-naphthylmethoxy or allyloxy, n5 is selected from 0,1,2,3,4,5,6,7, m4 is selected from 2, 3, 4, 5, 6, 7, 8, 9, PG is the abbreviation of protection group; when R 3 is hydroxyl, it is sialic acid
    Figure PCTCN2020127148-appb-100023
    The group on the molecule does not need to introduce the relevant reaction process of the R3 group.
  9. 权利要求1-5任一所述的新冠病毒检测试纸条的使用方法，其特征在于，包括以下步骤：The use method of the new coronavirus detection test strip described in any one of claims 1-5, is characterized in that, comprises the following steps:
    1)将新冠病毒检测试纸条水平放置，取检测样本滴入样品垫(2)，静置10-15分钟；1) Place the new coronavirus detection test strip horizontally, take the test sample and drop it into the sample pad (2), and let it stand for 10-15 minutes;
    2)结果判定：2) Result judgment:
    阳性结果：检测T线(6)显红色，质控C线(7)显红色；Positive results: the detection T line (6) is red, and the quality control C line (7) is red;
    阴性结果：检测T线(6)不显色，质控C线(7)显红色；Negative result: the detection T line (6) is not colored, and the quality control C line (7) is red;
    无效结果：无论检测T线(6)显色与否，质控C线(7)不显红色，应另取新冠病毒检测试纸条重新检测。Invalid result: Regardless of whether the detection T line (6) is colored or not, the quality control C line (7) does not show red, and another new coronavirus detection test strip should be taken for re-testing.
  10. 根据权利要求9所述的使用方法，其特征在于，所述检测样本包括鼻咽拭子、唾液、血液、排泄物。The method of use according to claim 9, wherein the detection samples include nasopharyngeal swabs, saliva, blood, and excrement.
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