CN114441753A

CN114441753A - New coronavirus detection test strip and preparation method and application method thereof

Info

Publication number: CN114441753A
Application number: CN202011230093.9A
Authority: CN
Inventors: 李伟; 王怀雨
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2020-11-06
Filing date: 2020-11-06
Publication date: 2022-05-06

Abstract

The invention discloses a neocoronaviruse detection test strip as well as a preparation method and a use method thereof, wherein the neocoronaviruse detection test strip comprises a lining plate, a sample pad, a gold mark pad, a detection pad and a water absorption plate are sequentially arranged from one end to the other end of the lining plate, a nitrocellulose membrane is arranged on the surface of the detection pad, and a T line and a C line are arranged on the nitrocellulose membrane; the detection T line is coated with a sialic acid ligand fixture, and the sialic acid ligand fixture comprises sialic acid ligand molecules with high affinity; the quality control C line is coated with a controller, and the controller is a new coronavirus S protein; and a colloidal gold marker is sprayed on the gold labeling pad, and the colloidal gold marker is gold nanoparticles of which the surfaces are fixed with sialic acid ligand molecules with medium affinity. The invention provides a test strip capable of directly detecting new coronavirus particles, which can quickly and conveniently judge whether a sample contains the virus particles or not, and further quickly judge whether a subject is infectious or not.

Description

New coronavirus detection test strip and preparation method and application method thereof

Technical Field

The invention belongs to the technical field of biochemistry, and relates to a novel coronavirus detection test strip, and a preparation method and a use method thereof.

Background

One key point of preventing and controlling the new coronary pneumonia is to develop an effective medical diagnosis and treatment method aiming at the new coronary viruses, wherein the method is crucial to timely and accurately detecting the new coronary viruses for preventing and controlling epidemic situations.

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 IgM and IgG antibodies generated by a human immune system after the new coronavirus is infected. The detection aiming at the new coronavirus RNA mainly comprises a real-time fluorescence Reverse transcription-polymerase chain reaction (RT-PCR) method and a CRISPR (clustered regulated amplified polymorphic short palindromic repeats) method, wherein the former amplifies the RNA of a detection sample through PCR reaction to identify whether the RNA is the new coronavirus RNA, and the latter detects and identifies whether the RNA of the detection sample contains the new coronavirus RNA by using a gene editing tool capable of specifically identifying the RNA sequence of the new coronavirus. The immune system can generate specific IgM and IgG antibodies after human is infected with new coronavirus to indirectly detect whether the new coronavirus is infected, wherein the colloidal gold method is common. The colloidal gold method is also called immunochromatography, and its main principle is that a sample solution dropped on one end of a membrane moves to the other end by capillary action (lateral flow) of the membrane, an analyte binds to a receptor immobilized on a certain region of the membrane during the movement and becomes solid-phase, and the result of the solid-phase is judged by color development of a label, and the label is usually developed at a colloidal gold position. The IgG/IgM detection card for the new coronavirus in the market at present also detects whether the new coronavirus IgG/IgM antibody exists in human blood by an immunochromatography method, mainly labels an anti-IgG/anti-IgM antibody on colloidal gold, after the anti-IgG/anti-IgM antibody is combined with IgG or IgM in the blood in an immune manner, the anti-IgG/anti-IgM antibody is combined with a receptor fixed on a membrane to be immobilized, and the result can be rapidly judged.

The currently mainstream RNA detection method for the new coronavirus is an RT-PCR method, which can directly detect the virus, but needs to manually extract the RNA virus in a sample, has the influence of multiple factors such as a sample sampling method, sample transportation and storage, manual operation and the like, and has the problem that the method is often accompanied by more serious false negative interference according to the reflection of first-line medical personnel, so that the detection result is challenged; in addition, a few of the positive-recovering RNA test results do not show infectivity, so that false positive interference exists in the test results, and the positive-recovering test results can indicate that the test sample contains new coronavirus nucleic acid residues and does not have new coronavirus particles causing infection. The method for detecting IgG/IgM antibody produced by immune system reaction after human body is infected with new coronavirus belongs to an indirect detection method, because the human immune system can produce immune response to produce IgG and IgM after being infected for a certain time, the method can only reflect whether the subject is infected with new coronavirus, can not instantly indicate whether new coronavirus particles exist in the human body, is not suitable for detection of patients in early stage or latent stage of infection, and can not directly judge the infectivity of the detected object.

In conclusion, at present, no method for directly detecting the new coronavirus particles exists, so that the development of the new coronavirus particle detection method has important significance in prevention and control of the new coronary pneumonia.

Disclosure of Invention

In order to solve the problems in the background art, the present invention provides a novel coronavirus detection test strip, and a preparation method and a use method thereof.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

on one hand, the invention provides a test strip for detecting neocoronavirus, which comprises a lining plate, wherein a sample pad, a gold mark pad, a detection pad and a water absorption plate are sequentially arranged from one end to the other end of the lining plate, a nitrocellulose membrane is arranged on the surface of the detection pad, a detection T line and a quality control C line are arranged on the nitrocellulose membrane, the detection T line is close to the gold mark pad, and the quality control C line is close to the water absorption plate;

the two ends of the detection pad are respectively connected with the gold mark pad and the water absorption plate in an overlapping manner, and the sample pad is pressed and stuck on the gold mark pad;

the detection T line is coated with a sialic acid ligand fixture, the sialic acid ligand fixture comprises sialic acid ligand molecules with high affinity, and the structural general formula of the sialic acid ligand molecules with high affinity is shown as formula II:

wherein R is₁Selected from methoxy or substituted methoxy; r₂Selected from substituted acetamido, benzoylamino, substituted benzoylamino, alkoxycarbonylamino, trisAn azolyl group or a substituted triazolyl group; r₃Selected from hydroxy, methoxy, substituted methoxy, acetamido, substituted acetamido, sulfonamido 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, R₄Selected from amino or mercapto;

the quality control C line is coated with a controller, and the controller is a new coronavirus S protein;

a colloidal gold marker is sprayed on the gold labeling pad, and the colloidal gold marker is gold nanoparticles of which the surfaces are fixed with sialic acid ligand molecules with medium affinity; the sialic acid ligand molecule with medium affinity has a structural general formula shown in a formula I:

wherein R is₅Selected from hydroxy, methoxy, substituted methoxy, acetamido, substituted acetamido, sulfonamido or phosphoramido; a1 is

n2 is selected from 0,1,2,3,4,5,6,7, m2 is selected from 2,3,4,5,6,7,8, 9.

Furthermore, a sample adding hole is formed in the sample pad.

Further, the substituted methoxy group is X₁-CH₂O-, in which X₁Selected from phenyl or vinyl;

the substituted acetamido group is X₂-CH₂CONH-, wherein X₂Selected from methyl, ethyl, n-propyl, isopropyl, hydroxymethyl or hydroxyethyl;

the substituted benzoylamino group is X₃-BzNH-, wherein X₃Is at least 1 and is located at any position of the benzene ring, X₃At least one selected from halogen atoms, methyl, methoxy and nitro;

the alkoxy carbonyl amido is X₄-OC (O) NH-, in which X₄Selected from benzylAlkyl, allyl, tert-butyl or trichloroethyl;

the substituted triazolyl is

Wherein, X₅Selected from substituted phenylalkyl (X)₆-Ph-(CH₂)_n6-) or a substituted carbonyl group (X₇-C (O) -, wherein X₆Is at least 1 and is located at any position of the benzene ring, X₆At least one selected from halogen atoms, methyl, methoxy and nitro, wherein n6 is selected from 0,1, 2; x₇Is selected from alkoxy CH₃-(CH₂)_n7-O-or alkylamino CH₃-(CH₂)_n7-NH-, n7 is selected from 0,1,2,3,4, 5.

Further, the sialic acid ligand fixture comprises an immobilized protein, a sialic acid ligand molecule with high affinity and a linking molecule, wherein the immobilized protein is a protein macromolecule used for closed immobilization;

preferably, the immobilized protein is selected from protein macromolecules such as bovine serum albumin BSA, skimmed milk powder or casein which are commonly used as blocking immobilization;

preferably, the structural general formula of the connecting molecule is R₆-linker-R₇Wherein R is₆Can be identical with R₄A reactive linking group, more preferably, said R₆Selected from carboxyl, ester group, sulfonic group, phosphoric group or maleimide group; r is₇Is a group capable of reacting with a reactive group (e.g., amino group, carboxyl group, mercapto group) present on the surface of the immobilized protein, more preferably, the R group₇Selected from carboxyl, ester, amino or maleimide; the linker is selected from alkyl, cycloalkyl, polyethylene glycol chain or aryl; more preferably, the linker molecule is selected from difunctional compounds such as diethyl squarate, diglycolic acid, maleimide-polyethylene glycol carboxylic acid, 1, 4-cyclohexanedicarboxylic acid, and the like.

Further, the colloidal gold label comprises gold nanoparticles, sialic acid ligand molecules with medium affinity and blocking molecules;

preferably, the particle size of the gold nanoparticles is 30-50 nm;

preferably, the blocking molecule is a small molecule sulfhydryl polyethylene glycol, and more preferably, the structural general formula of the blocking molecule is R₈-(C₂H₄O)_n3-C₂H₄-SH, n3 is selected from 1,2,3,4,5,6,7,8,9, R₈Selected from hydroxyl, methoxy or carboxyl.

On the other hand, the invention provides a preparation method of the novel coronavirus detection test strip, which comprises the following steps:

preparing a colloidal gold marker, and spraying the colloidal gold marker on a gold marking pad;

preparing a sialic acid ligand fixture, and coating the sialic acid ligand fixture on a detection T line;

coating the new coronavirus S protein on the quality control C line;

sticking a sample pad, a gold mark pad, a detection pad and a water absorption plate on a lining plate to obtain the new coronavirus detection test strip;

preferably, the colloidal gold marker takes gold nanoparticles as a marked inner core, sialic acid ligand molecules with medium affinity are fixed on the surfaces of the gold nanoparticles through S-Au bonds, and micromolecular polyethylene glycol is taken as a closed molecule;

preferably, the sialic acid ligand anchor is a complex of an immobilised protein, which is a protein macromolecule used as a closed immobilisation, linked to sialic acid ligand molecules with high affinity via a linking molecule.

Further, the preparation method of the colloidal gold marker comprises the following steps: preparing sialic acid ligand molecules with medium affinity into an aqueous solution, mixing the aqueous solution with a gold nanoparticle solution, stirring for 12-24 hours at 20-30 ℃, fixing the aqueous solution on the surface of the gold nanoparticle by forming an S-Au bond on the surface of the gold nanoparticle, adding excessive micromolecule sulfhydryl polyethylene glycol, continuously stirring for 12-24 hours at 20-30 ℃ to seal residual reaction sites on the surface of the gold nanoparticle, and obtaining a sialic acid-gold nanoparticle labeled compound through centrifugal purification;

preferably, the sialic acid ligand molecule with moderate affinity is bound to gold nanoparticlesThe molar ratio of the particles is 1X 10⁴:1～2×10⁴:1；

Preferably, the sialic acid ligand molecule with medium affinity is prepared by: with sialic acid

Introducing R into carboxyl through carboxyl protection as a starting material₉Protection group is obtained

The fully acetylated chloro sugar is generated in the next step under the action of chloro and acetylating reagents

Then a functional side chain precursor is derived at the 2-position of the sugar ring through glycosylation reaction

Exposing several hydroxyls on the sugar ring after deacetylation reaction to obtain

Then introducing an R5 group by utilizing the reactivity difference of the primary hydroxyl of the sugar ring 9-OH and other secondary hydroxyl

Finally, sialic acid ligand molecules with medium affinity are synthesized through group conversion and deprotection reaction; wherein R is₉Selected from methyl or benzyl, A₂Is composed of

Or- (CH)₂)m3-R₁₀，R₁₀Is 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 R is₅When it is a hydroxyl group, it is a sialic acid

Groups on the molecule, not requiring the introduction of R₅Related reaction process of radical；

Preferably, the preparation method of the gold nanoparticle solution comprises the following steps: heating the chloroauric acid aqueous solution to boiling, adding sodium citrate, keeping boiling for 15-30 minutes, and cooling to obtain the gold nanoparticle solution with the particle size of 30-50nm and the surface covered with the sodium citrate, wherein the concentration of the chloroauric acid aqueous solution is 1mM, and the molar ratio of the chloroauric acid to the sodium citrate is 1: 2-4.

Further, the preparation method of the sialic acid ligand fixture comprises the following steps: mixing sialic acid ligand molecules with high affinity and connecting molecules in water, stirring for 12-24 hours at 20-30 ℃, and allowing the sialic acid ligand molecules with high affinity to pass through active reaction groups R on functional side chains A₄R to a linker molecule₆Reacting groups to covalently link to obtain a sialic acid-linked molecule complex; then adding immobilized protein, standing for 15-30 hours at 20-25 ℃, and connecting R on the molecular complex through sialic acid₇Forming covalent bonds between the groups and surface active reaction groups of the immobilized protein to obtain a target sialic acid ligand fixture;

preferably, the mole ratio of the sialic acid ligand molecules with high affinity, the connecting molecules and the immobilized protein is 5-10: 12-15: 1;

preferably, the sialic acid ligand molecule with high affinity is prepared by: with sialic acid

Introducing R into carboxyl through carboxyl protection as a starting material₁₁Protection group is obtained

Followed by deacetylation and carboxyl protectionThen obtaining an intermediate with all naked amino and hydroxyl on the sugar ring

According to R₂→R₁→R₃Sequentially introducing groups to respectively obtain

Or, according to R₂→R₃→R₁By introduction of radicals in the order of (1) to give

Finally, through side chain group conversion and sugar ring protecting group removing reaction, sialic acid ligand molecule with high affinity is synthesized, and the ligand molecule can pass through R₁，R₂And R₃The derivation of the group further matches and occupies a carbohydrate binding domain pocket of the new coronavirus S protein, and has high specificity and binding force on the new coronavirus S protein; wherein R is₁₁Selected from methyl or benzyl, A₃Is composed of

Or- (CH)₂)m4-R₁₂，R₁₂Selected 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 an abbreviation for protecting group; when R is₃When it is a hydroxyl group, it is a sialic acid

Groups on the molecule, not requiring the introduction of R₃The relevant reaction process of the group.

In another aspect, the present invention provides a method for using the novel coronavirus detection test strip, which comprises the following steps:

1) horizontally placing the new coronavirus detection test strip, dripping a detection sample into a sample pad, and standing for 10-15 minutes;

2) and (4) judging a result:

positive results: detecting that the T line shows red, and controlling the quality to show red by the C line;

negative results: detecting that the T line does not develop color, and controlling the quality of the C line to develop red;

invalid result: no matter whether the detection T line is colored or not, the quality control C line is not colored in red, and a new coronavirus detection test strip is taken for retesting.

Further, the detection sample comprises nasopharyngeal swabs, saliva, blood and excrement.

The invention provides a test strip for detecting new coronavirus, which can be used for directly detecting new coronavirus particles and has the following beneficial effects compared with the existing new coronavirus and related detection methods:

1) the test strip for detecting the new coronavirus directly detects the virus particles on the basis of the recognition and combination of a sialic acid ligand and S protein on the surface of the new coronavirus particles;

2) the invention provides a test strip capable of directly detecting new coronavirus particles, which can rapidly and conveniently judge whether virus particles are contained in a sample or not;

3) the test strip for detecting the new coronavirus can directly detect the new coronavirus particles in a sample, and further quickly judges whether a subject is infectious;

4) compared with the nucleic acid detection method, the test strip for detecting the new coronavirus does not relate to multiple procedures such as sample RNA extraction and PCR (polymerase chain reaction), and can be directly sampled and then dripped, so that the detection process is simplified, and the interference caused by manual operation errors is avoided; on the other hand, the detection can be completed only by taking about 15 minutes, and the detection result can be conveniently judged through color reaction;

5) compared with a new coronavirus IgG/IgM detection method, the new coronavirus detection test strip provided by the invention is used for detecting new coronaviruses, belongs to a direct detection method, directly detects new coronaviruses particles, and can immediately represent whether the new coronaviruses particles exist in a sample;

6) compared with the traditional colloidal gold immunochromatography method using biological macromolecules such as antigen antibodies, the sialic acid detection molecule has the obvious advantages of good economy, high stability, large-scale preparation and the like;

7) the novel coronavirus detection test strip provided by the invention can be used for directly detecting virus particles, has richness in sample selection, and can be used for detecting common nasopharynx swabs, saliva, blood, excrement and other samples possibly containing virus particles for nucleic acid detection;

8) the invention provides a preparation method of a micromolecule-colloidal gold labeled compound (colloidal gold labeled compound) for colloidal gold detection, wherein the micromolecule-colloidal gold labeled compound has the advantages of economy and availability, and the types and the selectable range of the colloidal gold labeled compound in the colloidal gold method are expanded;

9) the invention provides a small molecule-immobilized protein complex (sialic acid ligand fixture) for colloidal gold detection, which has obvious economic and availability advantages and expands the types and selectable range of colloidal gold detection molecules.

Drawings

FIG. 1 is a schematic structural diagram of the novel coronavirus detection test strip of the present invention, wherein FIG. (a) is a top view, and FIG. (b) is a side view;

FIG. 2 is a schematic diagram of the structure of the colloidal gold-labeled compound of the present invention;

FIG. 3 is a flow chart of the preparation of sialic acid ligand molecules of the present invention with moderate avidity;

FIG. 4 is a flow chart of the preparation of the colloidal gold conjugate of the present invention;

FIG. 5 is a schematic diagram of the structure of the sialic acid ligand immobilizates of the present invention;

FIG. 6 is a flow chart of the preparation of a high affinity sialic acid ligand molecule of the present invention;

FIG. 7 is a flow chart of the preparation of sialic acid ligand immobilizates of the present invention;

FIG. 8 is a schematic diagram of the principle of the present invention for direct detection of new coronavirus particles;

FIG. 9 is a schematic diagram of the positive, negative, and negative detection principles of the present invention;

wherein, 1, a lining board, 2, a sample pad, 3, a gold mark pad, 4, a detection pad, 5, a water absorption plate, 6, a detection T line, 7, a quality control C line, 8, a sample adding hole,

is a colloidal gold label, and is characterized in that,

is a sialic acid ligand fixture, and is used as a medicine,

is a control substance.

Detailed Description

One of the new coronaviruses beta-coronavirus, the surface Spike protein (S protein) is a trimer composed of three monomers, each monomer is composed of S1 unit and conserved S2 unit, wherein S1 mainly comprises C-terminal binding domain (S1-CTD) and N-terminal binding domain (N-terminal domain, S1-NTD), which together form Receptor Binding Domain (RBD), generally, S1-NTD recognizes sialic acid residue bound to the surface of host cell, and S1-CTD recognizes peptide molecule bound to the surface of host cell. The research result of the structure analysis of the S protein of the new coronavirus shows that a Carbohydrate recognition threshold (CRD) pocket structure exists in the S1-NTD part of the new coronavirus, and the CRD pocket structure can be recognized and combined with sialic acid ligand molecules, so that the recognition and combination effect can be utilized to design corresponding sialic acid ligand molecules as detection molecules, the new coronavirus particles can be directly detected, and a point-of-care (POC) detection device for detecting the new coronavirus particles is designed and provided.

The invention provides a test strip for directly detecting new corona virus particles in a sample to be detected, which utilizes sialic acid ligand to identify and combine S protein molecules on the surface of new corona virus for detection, directly detects new corona virus particles serving as pathogenicity and infectivity of new corona pneumonia, can immediately and quickly judge whether new corona virus particles exist in a sample of a subject, and thus quickly judges the infectivity of the subject, wherein the structure of the test strip is shown in figure 1, and the figure (a) is a top view: the detection device comprises a sample pad 2 → a gold mark pad 3 → a detection pad 4 → a water absorption plate 5 from left to right, wherein a colloidal gold mark is sprayed on the gold mark pad 3, a detection T line 6 and a quality control C line 7 are arranged on the detection pad 4, the detection pad 4 is formed by fixing a sialic acid ligand fixture and a control object on a detection pad 4NC membrane (nitrocellulose membrane), and a sample adding hole 8 is arranged on the sample pad 2; fig. (b) is a side view: it is mainly composed of 5 parts: the device comprises a bottom lining plate 1 (such as polyvinyl chloride PVC), a sample pad 2, a gold mark pad 3, a detection pad 4 and a water absorption plate 5, wherein a detection T line 6 and a quality control C line 7 are arranged on the detection pad 4. The structure diagram of the colloidal gold label is shown in figure 2: the preparation method comprises the following steps of (1) fixing sialic acid ligand molecules with medium affinity on the surface of nano gold by taking gold nanoparticles as a labeling inner core through an S-Au bond, and taking micromolecular polyethylene glycol as a closed molecule, wherein the preparation flow chart of the sialic acid ligand molecules with medium affinity is shown in figure 3, and the preparation flow chart of a colloidal gold label is shown in figure 4; recognition and combination of the virus particles and the colloidal gold label are carried out on the gold-labeled pad to form a virus-gold labeled complex. A schematic diagram of the structure of the sialic acid ligand immobilizate is shown in FIG. 5: the sialic acid ligand molecule with high affinity is fixed on the detection pad through a fixing protein unit by a compound formed by a connecting molecule and a fixing protein, wherein the preparation flow chart of the sialic acid ligand molecule with high affinity is shown in figure 6, and the preparation flow chart of the sialic acid ligand fixture is shown in figure 7; the S protein remained on the virus-gold labeled complex is combined through a sialic acid ligand unit, or a part of the colloidal gold label fixed on the virus surface is exchanged through a ligand exchange reaction, so that the fixed virus-gold labeled complex is developed. The control substances are: is a new coronavirus S protein which can be obtained commercially and fixed in the C line area of the detection pad, and the colloidal gold marker is fixed for color development by recognizing sialic acid ligand bound on the surface of the free colloidal gold marker.

The principle of the invention for directly detecting the new coronavirus particles is as follows: the invention provides a colloidal gold method for detecting new corona virus particles similar to a double antibody sandwich method, sialic acid ligands and nano gold particles are prepared into colloidal gold labels, the new corona virus and the colloidal gold labels are identified and combined through protein-sialic acid ligands on the surfaces to form virus-gold label complexes, and the virus-gold label complexes are identified and combined with residual S protein on the surfaces of the virus particles under the action of sialic acid ligand fixtures or exchange part of the colloidal gold labels on the virus-gold label complexes, so that the virus-gold label complexes are fixed, and the detection result is judged through color change, as shown in figure 8.

Specifically, the positive detection process, principle and result are as follows: when new coronavirus of a sample to be detected flows through the gold-labeled pad, the new coronavirus is identified and combined with sialic acid ligands on the surface of the colloidal gold-labeled pad through surface S protein to form a labeled compound, when the compound is expanded on the detection pad and flows through a detection line (T line), the residual S protein on the surface of the new coronavirus is identified and combined with the fixed sialic acid ligands, or is exchanged with part of gold labels fixed on the surfaces of virus particles through ligand exchange reaction, the virus-gold label compound is fixed, so that red is displayed on the T line, and when the redundant colloidal gold label is continuously expanded and flows through a quality Control line (C line), the residual S protein is combined and fixed by a controller, so that red is displayed on the C line;

negative detection process, principle and results: the sample to be detected does not contain new coronavirus, a labeled complex cannot be formed when the sample flows through the gold labeled pad, the fixed sialic acid ligand cannot fix the colloidal gold label when the sample continues to expand and flow through the T line, so that red is not displayed on the T line, and the colloidal gold label is combined and fixed by the control object when the sample continues to expand and flow through the C line, so that red is displayed on the C line;

and (4) invalid detection results: the line C is not colored, and no matter whether the line T shows red, the line C is an invalid result, and the test paper strip is taken to detect again.

For a better understanding of the present invention, the following examples are given to illustrate the present invention, but the present invention is not limited to the following examples.

Example 1: synthesis of sialic acid ligand molecule 1 with intermediate avidity

Dissolving natural sialic acid Neu5Ac (10g,32.3mmol) in ethanol (100mL), adding potassium carbonate (5.36g,38.8mmol), stirring for 15 minutes, dropwise adding benzyl bromide (6.08g,35.6mmol), stirring at 50 ℃ for 2 hours, detecting by TLC to complete the reaction, spin-drying the reaction solution, adding toluene to the residue for spin-steaming three times, and pumping to obtain a crude intermediate product of the formula 1-1, wherein the crude intermediate product is directly used for the next reaction;

dissolving the crude product of the formula 1-1 obtained in the previous step in chloroacetyl (20mL), stirring at room temperature until the reaction is complete, and evaporating to remove the solvent to obtain a crude product of the formula 1-2, which is directly used for the next step;

the crude product of the formula 1-2 obtained in the previous step and HOPEG₄OBn (13.79g,48.5mmol) was mixed with dry dichloromethane (60mL), silver carbonate (17.83g,64.7mmol) was added under ice bath, reacted for 10h, checked by TLC for completion, insoluble material was filtered off, residue was concentrated and separated on column to give a compound of formula 1-3 (19.3g, 71.6% in 3steps), ESI-MS m/z calcd for [ C₄₁H₅₆NO₁₇]⁺(M+H)⁺: 834.88,found:834.88；

Dissolving a compound (19.3g,23.14mmol) shown in a formula 1-3 in methanol (60mL), adding sodium methoxide (0.1M) to adjust the pH of a reaction solution to 8-10, stirring for 30min, detecting by TLC to complete the reaction, neutralizing the reaction solution to be neutral, evaporating the solvent, adding toluene into a residue, and carrying out rotary evaporation for three times to obtain a compound 1-4(15.2g, 98.7%), and ESI-MS M/z calcd for [ C ]₃₃H₄₈NO₁₃]⁺(M+H)⁺:666.73,found: 666.72；

Dissolving the compound of formula 1-4(15.2g, 22.83mmol) in dichloromethane (60mL), adding triethylamine (4.61g,45.66mmol), adding p-toluenesulfonyl chloride (6.52g,34.25mmol) in portions, stirring overnight, detecting reaction completion by TLC, adding methanol, stirring for 10 minutes, evaporating the solvent, dissolving the residue in dichloromethane, washing the organic phase with brine, drying over anhydrous sodium sulfate, evaporating the solvent, dissolving the residue in N, N-dimethylformamide (20mL), adding sodium azide (3.70g,57.08mmol), stirring in an oil bath at 50 ℃ for 12 hours, detecting reaction completion by TLC, evaporating the solvent, dissolving the residue in tetrahydrofuran (40mL), adding triphenylphosphine (10.48g, 39.95mmol), stirring for 15 minutes after no bubble is generatedThe reaction solution was directly distilled off the solvent, the residue was dissolved in ethanol (20mL), acetic anhydride (3.03g,29.68mmol) was added dropwise, the mixture was stirred for 12 hours, the solvent was distilled off, and the residue was directly subjected to column separation to give compound 1-5(11.6g, 71.9%), ESI-MS m/z calcd for [ C ]₃₅H₅₁N₂O₁₃]⁺(M+H)⁺:707.79,found:707.80；

The compound of formula 1-5(11.6g, 16.41mmol) was dissolved in methanol (40mL), Pd/C catalyst (0.1g) was added, and H was pressurized at 4atm₂Stirring for 6 hr under atmosphere, detecting by TLC to remove Pd/C powder, filtering to remove Pd/C powder, concentrating the filtrate to obtain compound of formula 1-6 (8.4g, 97.2%), ESI-MS m/z calcd for [ C ]₂₁H₃₉N₂O₁₃]⁺(M+H)⁺:527.54,found:527.54；

Dissolving the compound of formula 1-6 (8.4g,16.41mmol) in dichloromethane (40mL), adding triethylamine (3.23g,31.91mmol), adding p-toluenesulfonyl chloride (4.56g,23.93mmol) in portions, stirring overnight, detecting reaction by TLC, adding methanol, stirring for 10 minutes, evaporating off the solvent, dissolving the residue in dichloromethane, washing the organic phase with brine, drying over anhydrous sodium sulfate, concentrating, dissolving the residue in N, N-dimethylformamide (50mL), adding potassium thioacetate (3.54g,30.97mmol), stirring in 50 deg.C oil bath for 12 hours, detecting reaction by TLC completely, evaporating off the solvent, and purifying the residue by column chromatography to obtain the compound of formula 1-7 (7.6g, 81.5%), ESI-MS m/z calcd for [ C ] (7.6g, 81.5%)₂₃H₄₁N₂O₁₃S]⁺(M+H)⁺:585.63,found:585.62；

Dissolving the compound (7.6g,13.00mmol) of the formula 1-7 in methanol (50mL), adding sodium methoxide (0.1M in MeOH), adjusting the pH of the reaction solution to 8-10, stirring at room temperature for 15 minutes, detecting the completion of the reaction by TLC, and adding 732H⁺Cation resin, stirring for 5 minutes, filtering resin, concentrating filtrate to obtain formula 1 compound (6.9g, 97.8%), ESI-MS m/z calcd for [ C ]₂₁H₃₉N₂O₁₂S]⁺(M+H)⁺: 542.60,found:542.59。

Example 2: synthesis of sialic acid ligand molecule with moderate avidity 5:

the preparation of the compound of formula 5-1 was the same as the preparation of the compound of formula 1-4 in the examples, wherein only HOPEG was added₄Replacement of OBn reagent with HOPEG₃ONap;

the compound of formula 5-1 (5g,7.44mmol) was dissolved in methanol (50mL) and Bu was added₂SnO (2.22g,8.93mmol), refluxing at 80 ℃ for 3 hours until the reaction solution is clear, evaporating the solvent, rotary evaporating the residue with toluene three times, dissolving in dry DMF (40mL), adding benzyl bromide (1.91g,11.17mmol), CsF (1.36g,8.93mmol), and adding N₂Reacting for 12 hr in gas atmosphere, detecting by TLC, evaporating to remove solvent, performing column chromatography to the residue to obtain compound 5-2(5.2g, 91.7%), ESI-MS m/z calcd for [ C ]₄₂H₅₂NO₁₂]⁺(M+H)⁺:762.87,found:762.88；

Dissolving a compound shown as a formula 5-2(5.2g, 6.83mmol) in a DCM/MeOH mixed solvent (v/v ═ 2:1,100mL), adding 2, 3-dichloro-5, 6-dicyan p-benzoquinone DDQ (2.32g,10.24mmol), stirring for 30 minutes in the dark, completely detecting by TLC, concentrating the reaction solution to obtain an intermediate shown as a formula 5-3, and directly using the intermediate in the next reaction;

dissolving the intermediate of formula 5-3 obtained in the above step in dichloromethane/DMF mixed solvent (v/v is 3:1,40mL), adding triethylamine (2.07g, 20.48mmol), stirring for 5 min, adding TsCl (2.6g,13.65mmol), stirring at room temperature for 6 h, detecting by TLC to complete the reaction, adding methanol (1mL) to quench the reaction, evaporating off the solvent, dissolving the residue in acetonitrile (40mL), adding KSAc (1.17g, 10.24mmol), stirring at room temperature for 3 h, detecting by TLC to complete the reaction, evaporating off the solvent, dissolving the residue in dichloromethane, then sat_3a.q.Washing with saturated saline, drying over anhydrous sodium sulfate, concentrating, and purifying by column chromatography to obtain compound of formula 5-4 (3.5g, 75.4%), ESI-MS m/z calcd for [ C ]₃₃H₄₆NO₁₂S]⁺(M+H)⁺:680.78,found:680.77；

The compound of formula 5-4 (3.5g,5.15mmol) was dissolved in methanol (40mL), and aqueous NaOH (1.5M, 20mL) was added dropwise, followed by stirring until completion of the TLC detection reaction,the reaction solution was neutralized, the solvent was distilled off, and the residue was subjected to flash column separation to give the compound of formula 5 (2.7g, 95.8%), ESI-MS m/z calcd for [ C ]₂₄H₃₈NO₁₁S]⁺(M+H)⁺:548.62,found:548.62；

Example 3: synthesis of sialic acid ligand molecule 6 with moderate avidity

The process for preparing the compound of formula 6 is the same as the process for preparing the compound of formula 1, wherein only HOPEG is added₄Replacement of OBn reagent by HOC₅H₁₀OBn, and Ac, the last step in the preparation of the compounds of formulae 1-4₂Replacing the O reagent with TsCl reagent, ESI-MS m/z calcd for [ C ]₂₃H₃₇N₂O₁₀S₂]⁺(M+H)⁺:565.18,found:565.17。

Example 4: synthesis of sialic acid ligand molecule 2 with high affinity

Dripping concentrated hydrochloric acid (1mL) into methanol (100mL), adding natural sialic acid Neu5Ac (10g,32.3mmol) in batches, stirring to react until the solution is clear, neutralizing the reaction solution, evaporating to remove the solvent, adding toluene to the residue, performing rotary evaporation for three times, and draining to obtain an intermediate crude product of the formula 2-1, wherein the intermediate crude product is directly used for the next reaction;

dissolving the crude product of the formula 2-1 obtained in the previous step in chloroacetyl (40mL), stirring at room temperature until the reaction is complete, and evaporating to remove the solvent to obtain the crude product of the formula 2-2, which is directly used for the next step;

the crude product of the formula 2-2 obtained in the previous step and HOPEG₄OBn (13.8g,48.5mmol) was mixed in dry dichloromethane (60mL), silver carbonate (17.83g,64.7mmol) was added under ice bath, reaction was carried out for 12h, TLC detection of completion of reaction, insoluble material was filtered off, and the residue was concentrated and separated on a column to give a compound of formula 2-3 (19.3g, 78.8% in 3steps), ESI-MS m/z calcd for [ C₃₅H₅₂NO₁₇]⁺(M+H)⁺: 758.78,found:758.77.

Dissolving a compound (19.3g,25.47mmol) shown in a formula 2-3 in methanol (40mL), dropwise adding NaOH aqueous solution (1.5M, 20mL), stirring until TLC detection reaction is completed, neutralizing the reaction solution, evaporating the solvent, dissolving the residue in 2M NaOH aqueous solution (40mL), refluxing in an oil bath at 95 ℃ for 10 hours, neutralizing the reaction solution, evaporating the solvent, performing azeotropic rotary evaporation on the residue with toluene for three times, dissolving the residue in hydrochloric acid/methanol (v/v ═ 1:99,50mL), stirring until TLC detection reaction is completed, neutralizing the reaction solution, evaporating the solvent to obtain a crude compound shown in a formula 2-4, directly using the crude compound in the next step, and ESI-MS M/z calcd for [ C ] in₂₅H₄₂NO₁₂]⁺(M+H)⁺:548.60,found:548.59；

Sodium azide NaN₃(1.99g,30.56mmol) was dissolved in water (30mL), trifluoromethanesulfonic anhydride (8.62g, 30.56mmol) was slowly added dropwise, stirring was carried out for 15 minutes, the aqueous phase was extracted with toluene, and the organic phase was dried over anhydrous sodium sulfate to give trifluoromethanesulfonyl azide TfN₃Dissolving the crude compound of formula 2-4 in ethanol, and adding the TfN₃And copper sulfate CuSO4(0.812g, 5.09mmol), stirring for 6 hours, evaporating off the solvent, dissolving the residue in pyridine (50mL), adding acetic anhydride (26g, 254.7mmol) dropwise under ice bath conditions, stirring at room temperature for 12 hours, adding methanol dropwise under ice bath to quench the reaction, evaporating off the solvent, dissolving the residue in dichloromethane, then adding 1N HCl_a.q.、sat.NaHCO_3a.q.Washing with saturated saline, drying over anhydrous sodium sulfate, concentrating, and purifying by column chromatography to obtain compound of formula 2-5 (18.1g, 95.8%), ESI-MS m/z calcd for [ C ]₃₃H₄₇N₃O₁₆]⁺(M+H)⁺:742.74,found: 742.73；

The compound of formula 2-5 (18.1g,24.4mmol), methyl acrylate (2.46g,29.28mmol) and cuprous iodide (0.93g, 4.88mmol) were mixed in THF (60mL), N-diisopropylethylamine DIPEA (4.73gm 36.6mmol) was added dropwise, the reaction solvent was evaporated off, the residue was dissolved in dichloromethane, then 1M aqueous ammonia, 1N HCl and water were added_a.q.、sat. NaHCO_3a.q.Washing with saturated saline solution, drying with anhydrous sodium sulfate, concentrating, and purifying by column chromatography to obtain compound of formula 2-6Substance (19.6g, 97.3%), ESI-MS m/z calcd for [ C ]₃₇H₅₂N₃O₁₈]⁺(M+H)⁺:826.82,found:826.81；

Dissolving a compound (19.6g,23.73mmol) shown in a formula 2-6 in methanol (100mL), adding sodium methoxide (0.1M in MeOH), adjusting the pH value of the solution to 8-10, stirring at room temperature for 30 minutes, dropwise adding hydrochloric acid to neutralize the reaction solution, and evaporating to remove the solvent to obtain a crude product shown in a formula 2-7, wherein the crude product is directly used for the next reaction;

the crude product of formula 2-7 obtained in the above step was dissolved in dichloromethane (100mL), triethylamine (4.8g,47.47mmol) was added, p-toluenesulfonyl chloride (6.79g, 35.60mmol) was added in portions after stirring for 5 minutes, stirring was carried out at room temperature for 6 hours, the reaction was quenched by adding methanol, the solvent was evaporated off, the residue was dissolved in dichloro, washed with brine, the organic phase was dried over anhydrous sodium sulfate, the solvent was evaporated off, the residue was dissolved in N, N-dimethylformamide (50mL), NaN was added₃(4.63g,71.20mmol), stirring in 50 deg.C oil bath for 12 hours, TLC to detect completion of the reaction, distilling off the solvent, dissolving the residue in dichloromethane, washing with brine, drying over anhydrous sodium sulfate, concentrating and column chromatography to obtain the compound of formula 2-8 (14.3g, 88.3%), ESI-MS m/z calcd for [ C ]₂₉H₄₃N₆O₁₃]⁺(M+H)⁺:683.68,found:683.69；

Dissolving the compound of formula 2-8 (14.3g, 20.95mmol) in tetrahydrofuran (100mL), adding triphenylphosphine (16.48g, 62.84mmol) in portions, stirring until no bubble is generated, continuing to stir for 12 hours, evaporating off the solvent, dissolving the residue in ethanol, slowly dropping benzoyl chloride (3.53g,25.14mmol), stirring at room temperature for 1 hour, evaporating off the solvent, and purifying the residue by column chromatography to obtain the compound of formula 2-9 (13.1g, 82.2%), ESI-MS m/z calcd for [ C ] -MS m/z calcd₃₆H₄₉N₄O₁₄]⁺(M+H)⁺:761.79,found:761.78；

Dissolving the compound (13.1g,17.22mmol) of the formula 2-9 in acetonitrile (50mL), adding acetonide dimethyl acetal (5.38g,51.66mmol) and catalytic amount of p-toluenesulfonic acid (0.3g,1.72mmol), refluxing in an oil bath at 80 ℃ for 12 hours, detecting by TLC to complete the reaction, adding triethylamine to neutralize the reaction solution, evaporating to remove the solvent, and directly purifying the residue by column chromatography to obtain the compound (12.8g, 92.8%) of the formula 2-10 (ESI-MS)m/z calcd for[C₃₉H₅₃N₄O₁₃]⁺(M+H)⁺:801.86,found:801.86；

The compound of formula 2-10 (12.8g,15.98mmol) and freshly prepared silver oxide (7.41g,31.97mmol) were mixed in N, N-dimethylformamide (50mL), iodomethane (3.40g,23.97mmol) was added, stirring was carried out at room temperature for 4 hours, TLC detection was complete, insoluble material was filtered off, solvent was evaporated, the residue was dissolved in dichloromethane, washed with brine, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the compound of formula 2-11 (11.6g, 89.1%), ESI-MS m/z calcd for [ C ] C₄₀H₅₅N₄O₁₄]⁺(M+H)⁺:815.89,found:815.88；

Dissolving the compound of formula 2-11 (11.6g,14.24mmol) in methanol, adding p-toluenesulfonic acid (0.25g,1.42mmol), refluxing in 80 deg.C oil bath for 6 hr, detecting by TLC to complete reaction, neutralizing the reaction solution with triethylamine, evaporating off the solvent, dissolving the residue in dichloromethane, washing with brine, drying with anhydrous sodium sulfate, concentrating, dissolving in methanol, adding Pd/C powder (0.1g), and reacting under 4atm pressure H₂Reacting for 2 hours in atmosphere, filtering Pd/C powder, concentrating the filtrate to obtain crude compound of formula 2-12, directly using in next step, ESI-MS m/z calcd for [ C ]₃₀H₄₅N₄O₁₄]⁺(M+H)⁺:685.70,found:685.69；

Dissolving the crude compound of formula 2-12 obtained in the above step in dichloromethane (60mL), adding triethylamine (2.88g,28.47mmol), stirring for 5 minutes, adding p-toluenesulfonyl chloride (4.07g, 21.35mmol) in portions, stirring at room temperature for 6 hours, adding methanol to quench the reaction, evaporating off the solvent, dissolving the residue in dichloro, washing with brine, drying the organic phase over anhydrous sodium sulfate, evaporating off the solvent, dissolving the residue in N, N-dimethylformamide (40mL), adding NaN₃(2.78g,42.71mmol), stirring in 50 deg.C oil bath for 12 hours, TLC to detect completion of the reaction, distilling off the solvent, dissolving the residue in dichloromethane, washing with brine, drying over anhydrous sodium sulfate, concentrating and column chromatography to obtain the compound of formula 2-13 (8.2g, 81.2%), ESI-MS m/z calcd for [ C ]₃₀H₄₃N₇O₁₃]⁺(M+H)⁺:710.71,found: 710.70；

The compound of formula 2-13 (8.2g,11.55mmol) was dissolved in methanol (30mL), aqueous NaOH (1.5M,10mL) was added, stirring was carried out at room temperature for 30 minutes, and form 732H was added⁺The reaction solution was neutralized with cationic resin, the resin was filtered off, and Pd/C powder (0.1g) was added to the filtrate under 4atm pressure H₂Reacting for 2 hours in atmosphere, filtering Pd/C powder, concentrating the filtrate to obtain the compound of formula 2 (7.1g, 90.0%), ESI-MS m/z calcd for [ C ]₂₉H₄₄N₅O₁₃]⁺(M+H)⁺:670.69,found:670.68。

Example 5: synthesis of sialic acid ligand molecule 7 with high affinity

The compound of formula 7-1 is prepared by the same process as the compound of formula 2-4, except that HOPEG is added₄Replacement of OBn reagent with HOPEG₃OBn is just needed;

dissolving the compound shown in the formula 7-1 in ethanol, dripping benzoyl chloride in ice bath, continuously stirring for reacting for 1 hour, and evaporating to remove the solvent to obtain a crude compound shown in the formula 7-2, wherein the crude compound is directly used for the next reaction;

the process for preparing the compound of formula 7-3 from the compound of formula 7-2 is the same as the process for preparing the compound of formula 1-5 from the compound of formula 1-4, wherein Ac is only added₂Replacing the O reagent with TsCl;

the process for preparing the compound of formula 7 starting from the compound of formula 7-3 is the same as the process for preparing the compound of formula 2 starting from the compound of formula 2-9.

Example 6: synthesis of sialic acid ligand molecule 8 with high affinity

The compound of formula 8-1 is prepared by the same procedure as the compound of formula 2-4, except that HOPEG is added₄OBn reagent replacement with HOC₅H₁₀OAll is carried out;

the process for preparing the compound of formula 8-2 from the compound of formula 8-1 is the same as the process for preparing the compound of formula 7-2 from the compound of formula 7-1, except that the BzCl reagent is replaced by benzyloxycarbonylchloride CbzCl;

the process for preparing the compound of formula 8-3 from the compound of formula 8-2 is the same as the process for preparing the compound of formula 5-2 from the compound of formula 5-1;

the process for preparing the compound of formula 8-4 from the compound of formula 8-3 is the same as the process for preparing the compound of formula 7-4 from the compound of formula 7-3;

dissolving a compound (10g,14.89mmol) of a formula 8-4 in DMF (100mL), dropwise adding benzyl bromide (3.82g,22.83mmol), adding barium hydroxide (5.1g,29.77mmol), stirring at room temperature for 12 hours, detecting complete reaction by TLC, dropwise adding methanol to quench reaction, adding dichloromethane (300mL) to dilute reaction liquid, washing with saturated saline solution for three times, drying with anhydrous sodium sulfate, concentrating, and performing column chromatography to separate to obtain a compound (10.1g, 89%) of a formula 8-5 (ESI-MS m/z calcd for [ C ])₄₃H₅₆NO₁₁]⁺(M+H)⁺:762.91,found:762.90；

Dissolving a compound (10.1g,13.26mmol) of a formula 8-5 in methanol (100mL), adding TsOH (114mg,0.66mmol), placing the reaction solution in an oil bath at 80 ℃ for reflux reaction for 12 hours, detecting complete reaction by TLC, cooling the reaction solution to room temperature, adding triethylamine to neutralize the reaction solution to neutrality, adding palladium chloride (4.7g,26.51mmol), reacting at room temperature for 1 hour, detecting complete reaction by TLC, evaporating the solvent, dissolving the residue in dichloromethane, washing with saturated saline, drying with anhydrous sodium sulfate, concentrating, and performing column chromatography to obtain a compound (7.4g, 81.9%) of a formula 8-6, ESI-MS m/z calc for [ C ] lc₃₇H₄₈NO₁₁]⁺(M+H)⁺:682.78,found:682.77；

The process for preparing the compound of formula 8-7 from the compound of formula 8-6 is the same as the process for preparing the compound of formula 7-7 from the compound of formula 7-6;

dissolving the compound of formula 8-7 (4g,5.66mmol) in methanol (40mL), adding dropwise NaOH aqueous solution (1M,20mL), stirring until TLC detection reaction is complete, neutralizing the reaction solution, evaporating solvent, dissolving the residue in tetrahydrofuran (40mL), adding Ph3P (4.45g, 16.98mmol), stirring at room temperature for 12 hr, TLC detection reaction is complete, evaporating solvent, and purifying the residue by direct column chromatography to obtain the compound of formula 8 (3.2g, 84.8%), ESI-MS M/z calcd for [ C ] M/z₃₆H₄₇N₂O₁₀]⁺(M+H)⁺:667.77,found:667.76；

Example 7: preparation of sialic acid-gold nanoparticle labeled Complex 3

The preparation method of the gold nanoparticle solution with stable sodium citrate and 30nm of particle size comprises the following steps: 100mL of 1mM HAuCl was taken₄Putting the aqueous solution into a two-mouth bottle, heating to 100 ℃, adding sodium citrate (dihydrate) (118mg,0.3mmol), stirring for 15 minutes, taking out, and slowly cooling to room temperature to obtain the sodium citrate dihydrate.

Sialic acid molecule 1 obtained in example 1 was dissolved in water to prepare an aqueous solution (10mM), 100 μ L of the solution was added to a gold nanoparticle solution having a particle size of 30nm and stabilized with sodium citrate (OD 15, 5mL), and after standing for 15 hours, an excess amount of mercaptotetraethyleneglycol (PEG) was added₄-SH) blocking solution (0.1M, 100uL), after shaking for 12 hours, centrifuging for 2 times at 15000rpm for 15 minutes, and redispersing the centrifuge tube substrate aggregate in deionized water (5mL) to obtain the sialic acid-gold nanoparticle labeled complex 3.

Example 8: preparation of sialic acid ligand anchor 4

Sialic acid ligand molecule 2(1g,1.46mmol) obtained in example 2 was dissolved in methanol (20mL), diethyl squarate (2.48g, 14.58mmol) was added thereto, the mixture was stirred at room temperature for 24 hours, the reaction mixture was directly concentrated by column chromatography to isolate an intermediate (1.1g, 93.1%) of the compound of formula 4-1, ESI-MS m/z calcd for [ C₃₅H₄₇N₅O₁₇]⁺(M+H)⁺810.78, found 810.77, and recovering redundant diethyl squarate;

the compound of formula 4-1 (1.1g,1.36mmol) and bovine serum albumin BSA (66.43kDa,3g,45umol) were mixed in water, allowed to stand for 24 hours, and the reaction solution was purified by a gel column to give a complex of formula 4 (3.8g), which was dissolved in ultrapure water to give a 1nM solution.

Example 9: preparing a test strip for detecting the new coronavirus

Cutting a sample pad, a gold marking pad, a detection pad and a water absorption pad, wherein the length and width specifications are respectively 2cm multiplied by 2cm, 1cm multiplied by 2cm, 6cm multiplied by 2cm and 1cm multiplied by 2cm, spraying the sialic acid-gold nanoparticle marking compound 3 solution (10 mu L) prepared in the example 7 on the gold marking pad, spraying the sialic acid ligand fixture 4 solution (5 mu L) prepared in the example 8 on a T line (0.1 multiplied by 2cm) area of the detection pad, spraying the commercialized new coronavirus S protein solution (1nM, 5 mu L) as a control substance on a C line (0.1 multiplied by 2cm) area of the detection pad, and using water absorption paper as the water absorption pad, attaching the sample pad, the gold marking pad, the detection pad and the water absorption pad on a PVC lining board according to the levels and positions shown in the detection device structure of the figure 1, and overlapping each layer by 0.5cm to prepare the new coronavirus detection test strip.

Example 10: detection experiment

The new coronavirus has strong pathogenicity and infectivity, the detection experiment is carried out by the new coronavirus mimic particle, and the commercial new coronavirus S protein is fixed on SiO with the diameter of about 100nm₂Constructing new coronavirus simulated particles on the surfaces of the nanospheres, and taking 1 drop (10 drops) of aqueous solution of the new coronavirus simulated particles^-6one/mL, about 50 mu L) is added into a sample hole of a new coronavirus detection test strip, the test strip is horizontally placed for 15 minutes, and a T line and a C line of the kit are respectively red.

Example 11: parallel detection experiment

Taking two new coronavirus detection test strips, wherein the test strips A and B are numbered respectively, taking healthy human saliva, dividing the saliva into two parts and respectively marking the two parts as saliva A and B, adding the saliva A into the new coronavirus simulation particle solution prepared in the embodiment 10, adding deionized water into the saliva B, respectively taking 1 drop (about 50 mu L) of two samples, respectively dropping sample holes of the test strips A and B, horizontally standing for 15 minutes, wherein the T line of the test strip A shows red color, the C line shows color, the T line of the test strip B does not show color, and the C line shows color.

In summary, the embodiment of the present invention provides a test strip capable of directly detecting new coronavirus particles, which can rapidly and conveniently determine whether a sample contains virus particles, and further rapidly determine whether a subject is infectious.

The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent modifications of the technical solutions of the present invention, which are made by those skilled in the art through reading the present specification, are covered by the claims of the present invention.

Claims

1. The test strip for detecting the neocoronaviruses is characterized by comprising a lining plate (1), wherein a sample pad (2), a gold mark pad (3), a detection pad (4) and a water absorption plate (5) are sequentially arranged from one end to the other end of the lining plate (1), a nitrocellulose membrane is arranged on the surface of the detection pad (4), a detection T line (6) and a quality control C line (7) are arranged on the nitrocellulose membrane, the detection T line (6) is close to the gold mark pad (3), and the quality control C line (7) is close to the water absorption plate (5);

the two ends of the detection pad (4) are respectively connected with the gold mark pad (3) and the water absorption plate (5) in an overlapping manner, and the sample pad (2) is pressed on the gold mark pad (3);

the detection T line (6) is coated with a sialic acid ligand fixture, the sialic acid ligand fixture comprises sialic acid ligand molecules with high affinity, and the structural general formula of the sialic acid ligand molecules with high affinity is shown as a formula II:

wherein R is₁Selected from methoxy or substituted methoxy; r₂Selected from substituted acetamido, benzoylamino, substituted benzoylamino, alkoxycarbonylamino, triazolyl or substituted triazolyl; r₃Selected from hydroxy, methoxy, substituted methoxy, acetamido, substituted acetamido, sulfonamido or phosphoramido; a is

the quality control C line (7) is coated with a controller, and the controller is a new coronavirus S protein;

the above-mentionedA colloidal gold marker is sprayed on the gold labeling pad (3), and the colloidal gold marker is gold nanoparticles with sialic acid ligand molecules with medium affinity fixed on the surface; the sialic acid ligand molecule with medium affinity has a structural general formula shown in a formula I:

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. The test strip for detecting new coronavirus according to claim 1, wherein the sample pad (2) is provided with a sample adding hole (8).

3. The test strip for detecting a novel coronavirus according to claim 1,

the substituted methoxy group is X₁-CH₂O-, in which X₁Selected from phenyl or vinyl;

the substituted benzoylamino group is X₃-BzNH-, wherein X₃Is at least 1 and is positioned at any position of a benzene ring, and X3 is selected from at least one of halogen atoms, methyl, methoxy and nitro;

the alkoxy carbonyl amido is X₄-OC (O) NH-, in which X₄Selected from benzyl, allyl, tert-butyl or trichloroethyl;

the substituted triazolyl is

Wherein, X₅Selected from substituted phenylalkyl X₆-Ph-(CH₂)_n6-or substituted carbonyl X₇-C (O) -, wherein X₆Is at least 1 and is located at any position of the benzene ring, X₆At least one selected from halogen atoms, methyl, methoxy and nitro, wherein n6 is selected from 0,1, 2; x₇Is selected from alkoxy CH₃-(CH₂)_n7-O-or alkylamino CH₃-(CH₂)_n7-NH-, n7 is selected from 0,1,2,3,4, 5.

4. The test strip of claim 1, wherein the sialic acid ligand conjugate comprises an immobilized protein, a sialic acid ligand molecule with high affinity, and a linker molecule, wherein the immobilized protein is a protein macromolecule used as a blocking immobilization;

preferably, the immobilized protein is selected from bovine serum albumin BSA, skim milk powder or casein;

preferably, the structural general formula of the connecting molecule is R₆-linker-R₇Wherein R is₆Can be identical with R₄A reactive linking group, more preferably, said R₆Selected from carboxyl, ester group, sulfonic group, phosphoric group or maleimide group; r₇Is a group capable of reacting with a reactive group present on the surface of the immobilized protein, more preferably, the R is₇Selected from carboxyl, ester, amino or maleimide; the linker is selected from alkyl, cycloalkyl, polyethylene glycol chain or aryl; more preferably, the linker molecule is selected from the group consisting of diethyl squarate, diglycolic acid, maleimide-polyethylene glycol carboxylic acid, 1, 4-cyclohexanedicarboxylic acid.

5. The test strip for detecting neocoronavirus according to claim 1, wherein the colloidal gold marker comprises gold nanoparticles, sialic acid ligand molecules with medium affinity and blocking molecules;

preferably, the particle size of the gold nanoparticles is 30-50 nm;

preferably, the blocking molecule is a small molecule of thiol-polyethylene glycol, more preferably, the structure of the blocking moleculeHas the general formula R₈-(C₂H₄O)_n3-C₂H₄-SH, n3 is selected from 1,2,3,4,5,6,7,8,9, R₈Selected from hydroxyl, methoxy or carboxyl.

6. The method for preparing the test paper strip for detecting the new coronavirus of any one of claims 1-5, which is characterized by comprising the following steps of:

preparing a colloidal gold marker, and spraying the colloidal gold marker on the gold-labeled pad (3);

preparing a sialic acid ligand fixture, and coating the sialic acid ligand fixture on a detection T line (6);

coating the new coronavirus S protein on a quality control C line (7);

sticking a sample pad (2), a gold mark pad (3), a detection pad (4) and a water absorption plate (5) on a lining plate (1) to obtain the new coronavirus detection test strip;

7. The method according to claim 6, wherein the colloidal gold-labeled compound is prepared by: preparing sialic acid ligand molecules with medium affinity into an aqueous solution, mixing the aqueous solution with a gold nanoparticle solution, stirring for 12-24 hours at 20-30 ℃, fixing the aqueous solution on the surface of the gold nanoparticle by forming an S-Au bond on the surface of the gold nanoparticle, adding excessive micromolecule sulfhydryl polyethylene glycol, continuously stirring for 12-24 hours at 20-30 ℃ to seal residual reaction sites on the surface of the gold nanoparticle, and obtaining a sialic acid-gold nanoparticle labeled compound through centrifugal purification;

preferably, the sialic acid ligand molecule with medium affinity is used with gold nanoparticlesThe molar ratio is 1X 10⁴:1～2×10⁴:1；

Or- (CH)₂)m3-R₁₀R10 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 R is₅When it is a hydroxyl group, it is a sialic acid

Groups on the molecule, not requiring the introduction of R₅The relevant reaction process of the group;

8. The method of claim 6, wherein the sialic acid ligand conjugate immobilizate is prepared by:

mixing sialic acid ligand molecules with high affinity and connecting molecules in water, stirring for 12-24 hours at 20-30 ℃, and allowing the sialic acid ligand molecules with high affinity to pass through active reaction groups R on functional side chains A₄R to a linker molecule₆Reacting groups to covalently link to obtain a sialic acid-linked molecule complex; then adding immobilized protein, standing for 15-30 hours at 20-25 ℃, and connecting R on the molecular complex through sialic acid₇Forming covalent bonds between the groups and surface active reaction groups of the immobilized protein to obtain a target sialic acid ligand fixture;

preferably, the sialic acid ligand molecule with high affinity is prepared by the following method: with sialic acid

Then obtaining an intermediate with all naked amino and hydroxyl on a sugar ring after deacetylation and carboxyl protection

Finally, synthesizing sialic acid ligand molecules with high affinity through side chain group conversion and sugar ring protecting group removing reaction; wherein R11 is selected from methyl or benzyl, A₃Is composed of

9. The method of using the test strip for detecting a novel coronavirus according to any one of claims 1 to 5, wherein the method comprises the following steps:

1) horizontally placing the new coronavirus detection test strip, dripping a detection sample into the sample pad (2), and standing for 10-15 minutes;

2) and (4) judging a result:

positive results: detecting that the T line (6) is red, and the quality control C line (7) is red;

negative results: detecting that the T line (6) is not developed, and the quality control C line (7) is developed red;

invalid result: no matter whether the detection T line (6) is colored or not, the quality control C line (7) is not colored in red, and a new coronavirus detection test strip is taken for retesting.

10. The use of claim 9, wherein the test sample comprises a nasopharyngeal swab, saliva, blood, or feces.