A novel coronavirus test strip and its preparation method and use method

Abstract

The present invention discloses a novel coronavirus detection test strip and a preparation method and a use method thereof, including a liner, a sample pad, a gold labeling pad, a detection pad and a water absorbing plate are sequentially arranged from one end to the other end of the liner, 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 fixed substance, and the sialic acid ligand fixed substance includes a sialic acid ligand molecule with high affinity; the quality control C line is coated with a control substance, and the control substance is a novel coronavirus S protein; the gold labeling pad is sprayed with a colloidal gold marker, and the colloidal gold marker is a gold nanoparticle with a sialic acid ligand molecule with medium affinity fixed on the surface. The present invention provides a test strip that can directly detect novel coronavirus particles, which can quickly and conveniently determine whether the sample contains virus particles, and then quickly determine whether the subject is infectious.

Description

Technical Field

[0001] The present invention belongs to the field of biochemical technology and relates to a novel coronavirus detection test strip and a preparation method and a use method thereof. Background Art

[0002] A key to preventing and controlling COVID-19 is to develop effective medical diagnosis and treatment methods for the virus, among which the ability to detect the virus in a timely and accurate manner is crucial to preventing and controlling the epidemic.

[0003] 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 produced by the human immune system after the new coronavirus infection. Among them, the detection of the new coronavirus RNA mainly includes the real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR) method and the CRISPR (clustered regularly interspaced short palindromic repeats) method. The former uses the PCR reaction to amplify the RNA of the test sample to identify whether it is the new coronavirus RNA, and the latter uses gene editing tools that can specifically identify the RNA sequence of the new coronavirus to detect and identify whether the test sample contains the new coronavirus RNA. The specific IgM and IgG antibodies produced by the immune system after the human body is infected with the new coronavirus are used to indirectly detect whether there is a new coronavirus infection, among which the colloidal gold method is particularly common. The colloidal gold method is also called immunochromatography. Its main principle is to use the sample solution dripped on one end of the membrane to move to the other end under the capillary action of the membrane (lateral flow based on chromatography). During the movement, the analyte binds to the receptor fixed on a certain area of ​​the membrane and is solid-phased, and the solid-phased result is determined by the color development of the marker, usually with colloidal gold as the color development marker. The IgG / IgM test card currently on the market for the new coronavirus also uses immunochromatography to detect whether there is new coronavirus IgG / IgM antibody in human blood. It mainly labels anti-IgG / anti-IgM antibodies on colloidal gold. After immunological binding with IgG or IgM in the blood, it then binds to the receptor fixed on the membrane and solidifies, which can quickly determine the result.

[0004] The current mainstream method for detecting the RNA of the new coronavirus is the RT-PCR method. Although it can directly detect the virus, it requires the manual extraction of RNA viruses from the sample, which is affected by multiple factors such as the sampling method, sample transportation and storage, and manual operation. According to frontline medical staff, this method is often accompanied by serious false negative interference, which poses a challenge to the test results; in addition, a small number of people with re-positive RNA test results did not show infectiousness, so there is also false positive interference in the test results. Their re-positive test results may contain remnants of the new coronavirus nucleic acid in the test sample, and there are no infectious new coronavirus particles. The method of detecting IgG / IgM antibodies produced by the immune system after human infection with the new coronavirus is an indirect detection method. Since the human immune system needs to produce an immune response to produce IgG and IgM after a certain period of infection, this type of method can only reflect whether the subject has been infected with the new coronavirus, and cannot immediately indicate whether there are new coronavirus particles in the human body. It is also not suitable for the detection of patients in the early or latent period of infection, and thus cannot make a direct judgment on the infectiousness of the test subject.

[0005] In summary, there is currently no method to directly detect new coronavirus particles, so the development of new coronavirus particle detection methods is of great significance in the prevention and control of new coronavirus pneumonia. Summary of the invention

[0006] 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 a preparation method and use method thereof.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] On the one hand, the present invention provides a novel coronavirus detection test strip, comprising a liner, on which a sample pad, a gold labeling pad, a detection pad and a water absorption plate are arranged in sequence from one end to the other end of the liner, 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 labeling pad, and the quality control C line is close to the water absorption plate;

[0009] The two ends of the detection pad are respectively overlapped and connected with the gold marking pad and the water absorption plate, and the sample pad is pressed on the gold marking pad;

[0010] 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. The general structural formula of the sialic acid ligand molecule with high affinity is shown in Formula II: wherein R1 is selected from methoxy or substituted methoxy; R2 is selected from substituted acetylamino, benzylamino, substituted benzylamino, alkoxycarbonylamide, triazolyl or substituted triazolyl; R3 is selected from hydroxyl, methoxy, substituted methoxy, acetylamino, substituted acetylamino, sulfonamide or phosphoramide; 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, and R4 is selected from amino or thiol;

[0011] The quality control C line is coated with a control substance, and the control substance is the S protein of the new coronavirus;

[0012] The gold label pad is sprayed with colloidal gold markers, which are gold nanoparticles with medium-affinity sialic acid ligand molecules fixed on the surface; the general structural formula of the medium-affinity sialic acid ligand molecules is shown in Formula I: wherein R5 is selected from hydroxy, methoxy, substituted methoxy, acetylamino, substituted acetylamino, sulfonamide or phosphoramide; 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.

[0013] Furthermore, a sample adding hole is provided on the sample pad.

[0014] Further, the substituted methoxy group is X1-CH2O-, wherein X1 is selected from phenyl or vinyl;

[0015] The substituted acetylamino group is X2-CH2CONH-, wherein X2 is selected from methyl, ethyl, n-propyl, isopropyl, hydroxymethyl or hydroxyethyl;

[0016] The substituted benzylamino group is X3-BzNH-, wherein the number of X3 is at least 1 and is located at any position of the benzene ring, and X3 is selected from at least one of a halogen atom, a methyl group, a methoxy group, and a nitro group;

[0017] The alkoxycarbonylamide group is X4-OC(O)NH-, wherein X4 is selected from benzyl, allyl, tert-butyl or trichloroethyl;

[0018] The substituted triazole group is wherein X5 is selected from substituted phenylalkyl (X6-Ph-(CH2) n6 -) or substituted carbonyl (X7-C(O)-), wherein the number of X6 is at least 1 and is located at any position of the benzene ring, X6 is selected from at least one of a halogen atom, a methyl group, a methoxy group, and a nitro group, and n6 is selected from 0, 1, and 2; X7 is selected from an alkoxy group CH3-(CH2) n7 -O- or alkylamino CH3-(CH2)n7 -NH-, n7 is selected from 0, 1, 2, 3, 4, 5.

[0019] Furthermore, the sialic acid ligand immobilizer comprises an immobilized protein, a sialic acid ligand molecule with high affinity and a connecting molecule, wherein the immobilized protein is a protein macromolecule used for blocking and immobilization;

[0020] Preferably, the immobilized protein is selected from bovine serum albumin BSA, skimmed milk powder or casein and other protein macromolecules commonly used for blocking and immobilization;

[0021] Preferably, the general structural formula of the linker molecule is R6-linker-R7, wherein R6 is a group that can react and connect with R4, and more preferably, the R6 is selected from a carboxyl group, an ester group, a sulfonic acid group, a phosphate group or a maleimide group; R7 is a group that can react with an active reactive group (such as an amino group, a carboxyl group, a thiol group) present on the surface of the fixed protein, and more preferably, the R7 is selected from a carboxyl group, an ester group, an amino group or a maleimide group; the linker is selected from an alkyl group, a cycloalkyl group, a polyethylene glycol chain or an aromatic group; more preferably, the linker molecule is selected from a bifunctional compound such as diethyl squarate, diglycolic acid, maleimide-polyethylene glycol carboxylic acid, 1,4-cyclohexanedicarboxylic acid, etc.

[0022] Furthermore, the colloidal gold label comprises gold nanoparticles, sialic acid ligand molecules with medium affinity and blocking molecules;

[0023] Preferably, the particle size of the gold nanoparticles is 30-50 nm;

[0024] Preferably, the blocking molecule is a small molecule thiol polyethylene glycol, and more preferably, the general structural formula of the blocking molecule is R8-(C2H4O) n3 -C2H4-SH, n3 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and R8 is selected from hydroxy, methoxy or carboxyl.

[0025] In another aspect, the present invention provides a method for preparing any of the above-mentioned novel coronavirus detection test strips, comprising the following steps:

[0026] preparing a colloidal gold label and spraying the colloidal gold label on a gold label pad;

[0027] preparing a sialic acid ligand immobilized substance, and coating the sialic acid ligand immobilized substance on a detection T line;

[0028] Coat the SARS-CoV-2 S protein on the quality control C line;

[0029] Glue the sample pad, gold label pad, detection pad and absorbent plate to the backing plate to obtain the novel coronavirus detection test strip;

[0030] Preferably, the colloidal gold marker uses gold nanoparticles as the labeling core, fixes sialic acid ligand molecules with medium affinity on the surface of the gold nanoparticles through S-Au bonds, and uses small molecule polyethylene glycol as a blocking molecule;

[0031] Preferably, the sialic acid ligand immobilizer is a complex in which an immobilized protein is connected to a sialic acid ligand molecule with high affinity via a linker molecule, and the immobilized protein is a protein macromolecule used for closed immobilization.

[0032] Furthermore, the preparation method of the colloidal gold marker is as follows: a sialic acid ligand molecule with medium affinity is configured into an aqueous solution, mixed with a gold nanoparticle solution, stirred at 20 to 30° C. for 12 to 24 hours, fixed on the surface of the gold nanoparticles by forming S-Au bonds on the surface of the gold nanoparticles, then an excess of small molecule thiol polyethylene glycol is added, and stirring is continued at 20 to 30° C. for 12 to 24 hours to block the remaining reaction sites on the surface of the gold nanoparticles, and the sialic acid-gold nanoparticle marker complex is obtained by centrifugal purification;

[0033] Preferably, the molar ratio of the sialic acid ligand molecules with medium affinity to the gold nanoparticles is 1×10 4 :1~2×10 4 :1;

[0034] Preferably, the preparation method of the sialic acid ligand molecule with medium affinity is: As the starting material, the R9 protecting group is introduced into the carboxyl group to obtain In the next step, the fully acetylated chlorosugar is generated by the action of chlorination and acetylation reagents. Then, the functional side chain precursor is derived at the 2-position of the sugar ring through glycosylation reaction. After deacetylation, several hydroxyl groups on the sugar ring are exposed to obtain Then, the R5 group was introduced by utilizing the difference in reactivity 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 medium affinity is synthesized through group conversion and deprotection reaction; wherein R9 is selected from methyl or benzyl, and A2 is or -(CH2)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 The groups on the molecule do not need to be introduced into the relevant reaction process of the R5 group;

[0035] Preferably, the preparation method of the gold nanoparticle solution is: heating the chloroauric acid aqueous solution to boiling, adding sodium citrate, and keeping boiling for 15 to 30 minutes, and cooling to obtain a gold nanoparticle solution with a particle size of 30 to 50 nm and a surface covered with sodium citrate, wherein the concentration of the chloroauric acid aqueous solution is 1 mM, and the molar ratio of chloroauric acid to sodium citrate is 1:2-4.

[0036] Furthermore, the preparation method of the sialic acid ligand immobilized substance is as follows: mixing a sialic acid ligand molecule with high affinity with a linker molecule in water, stirring at 20-30° C. for 12-24 hours, wherein the sialic acid ligand molecule with high affinity reacts with the R6 group of the linker molecule through the active reaction group R4 on the functional side chain A to covalently link to obtain a sialic acid-linker molecule complex; then adding an immobilized protein, standing at 20-25° C. for 15-30 hours, and forming a covalent bond between the R7 group on the sialic acid-linker molecule complex and the active reaction group on the surface of the immobilized protein to obtain a target sialic acid ligand immobilized substance;

[0037] Preferably, the molar ratio of the high-affinity sialic acid ligand molecule, the linker molecule, and the fixed protein is 5-10:12-15:1;

[0038] Preferably, the method for preparing the high-affinity sialic acid ligand molecule is: As the starting material, R was introduced into the carboxyl group after carboxyl protection. 11 Protecting group obtained In the next step, the fully acetylated chlorosugar is generated by the action of chlorination and acetylation reagents. Then, the functional side chain precursor is derived at the 2nd position of the sugar ring through glycosylation reaction. After deacetylation and carboxyl protection, the intermediate with both amino and hydroxyl groups exposed on the sugar ring was obtained. The groups were introduced in the order of R2→R1→R3 to obtain Alternatively, the groups are introduced in the order of R2 → R3 → R1 to obtain 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. This ligand molecule can be further matched to the sugar binding domain pocket of the SARS-CoV-2 S protein by the derivatization of R1, R2 and R3 groups, and has high specificity and binding force to the SARS-CoV-2 S protein; among them, R 11 is selected from methyl or benzyl, A3 is or -(CH2)m4-R 12 , R 12is 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 R3 is hydroxyl, it is sialic acid The groups on the molecule do not need to be introduced into the relevant reaction process of the R3 group.

[0039] In another aspect, the present invention provides a method for using any of the above-mentioned novel coronavirus detection test strips, comprising the following steps:

[0040] 1) Place the COVID-19 test strip horizontally, drop the test sample onto the sample pad, and let it stand for 10-15 minutes;

[0041] 2) Result determination:

[0042] Positive result: the test T line appears red, and the quality control C line appears red;

[0043] Negative result: the test T line does not show color, and the quality control C line shows red;

[0044] Invalid result: Regardless of whether the test T line is colored or not, if the quality control C line does not appear red, another COVID-19 test strip should be taken for retesting.

[0045] Furthermore, the test samples include nasopharyngeal swabs, saliva, blood, and excrement.

[0046] The present invention provides a novel coronavirus detection test strip, which can be used to directly detect novel coronavirus particles. Compared with the existing novel coronavirus and related detection methods, it has the following beneficial effects:

[0047] 1) The novel coronavirus test strip provided by the present invention directly detects virus particles based on the recognition and binding of sialic acid ligands and S proteins on the surface of novel coronavirus particles;

[0048] 2) The present invention provides a test strip that can directly detect novel coronavirus particles, which can quickly and conveniently determine whether a sample contains virus particles;

[0049] 3) The novel coronavirus test strip provided by the present invention can directly detect novel coronavirus particles in a sample, and then quickly determine whether the subject is infectious;

[0050] 4) Compared with the nucleic acid detection method, the novel coronavirus detection test strip provided by the present invention does not involve multiple procedures such as sample RNA extraction and PCR. It can be directly added after sampling, which simplifies the detection process and avoids 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 test results.

[0051] 5) Compared with the IgG / IgM detection method for the detection of the new coronavirus, the new coronavirus detection test strip provided by the present invention is a direct detection method that directly detects the new coronavirus particles and can instantly characterize whether there are new coronavirus particles in the sample;

[0052] 6) The novel coronavirus detection test strip provided by the present invention detects by binding and recognizing sialic acid small molecules with the novel coronavirus surface S protein. Compared with the traditional colloidal gold immunochromatography method using biological macromolecules such as antigens and antibodies, the small molecule properties of sialic acid detection molecules have obvious advantages such as good economy, high stability, and large-scale preparation;

[0053] 7) The novel coronavirus test strip provided by the present invention directly detects virus particles, and has rich sample selection, including nasopharyngeal swabs, saliva, blood, excrement and other samples that may contain virus particles commonly used in nucleic acid testing;

[0054] 8) The present invention provides a method for preparing a small molecule-colloidal gold labeled complex (colloidal gold marker) that can be used for colloidal gold method detection. In addition to having advantages in economy and availability, the small molecule-colloidal gold labeled complex also expands the types and selectable range of gold markers in the colloidal gold method;

[0055] 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, and expands the types and selectable range of molecules that can be detected by the colloidal gold method. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 1 is a schematic diagram of the structure of the novel coronavirus detection test strip of the present invention, wherein (a) is a top view and (b) is a side view;

[0057] Figure 2 It is a schematic diagram of the structure of the colloidal gold label of the present invention;

[0058] Figure 3 is a flow chart for preparing the sialic acid ligand molecule with medium affinity of the present invention;

[0059] Figure 4 It is a preparation flow chart of the colloidal gold label of the present invention;

[0060] Figure 5 is a schematic structural diagram of the sialic acid ligand immobilized substance of the present invention;

[0061] Figure 6 is a flow chart for preparing the high-affinity sialic acid ligand molecule of the present invention;

[0062] Figure 7 is a flow chart for preparing the sialic acid ligand immobilized substance of the present invention;

[0063] Figure 8 Schematic diagram of the principle of directly detecting novel coronavirus particles in the present invention;

[0064] Fig. 9 It is a schematic diagram of the positive, negative, and invalid detection principles of the present invention;

[0065] Among them, 1. backing plate, 2. sample pad, 3. gold label pad, 4. detection pad, 5. water absorption plate, 6. detection T line, 7. quality control C line, 8. sample addition hole, Colloidal gold marker, is a sialic acid ligand immobilizer, For control objects. DETAILED DESCRIPTION

[0066] The novel coronavirus is a type of β-coronavirus. 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. S1 mainly contains a C-terminus domain (S1-CTD) and an N-terminus domain (S1-NTD), which together form a receptor binding domain (RBD). Generally, S1-NTD recognizes and binds to sialic acid residues on the surface of host cells, while S1-CTD recognizes and binds to peptide molecules on the surface of host cells. The results of the structural analysis of the novel coronavirus S protein show that the S1-NTD part of the novel coronavirus has a carbohydrate recognition domain (CRD) pocket structure, which can recognize and bind to sialic acid ligand molecules. Therefore, this recognition and binding effect can be used to design corresponding sialic acid ligand molecules as detection molecules to directly detect novel coronavirus particles, and to design a point-of-care (POC) detection device for novel coronavirus particles.

[0067] The present invention provides a test strip for directly detecting novel coronavirus particles in a sample to be tested, and detects the novel coronavirus particles that are the source of pathogenicity and infectivity of COVID-19 by using sialic acid ligands to recognize and bind to the S protein molecules on the surface of the novel coronavirus, and can immediately and quickly determine whether novel coronavirus particles exist in the sample of the subject, thereby making a rapid judgment on the infectivity of the subject. The structure of the test strip is as follows Figure 1As shown, Figure (a) is a top view: from left to right, it is sample pad 2 → gold label pad 3 → detection pad 4 → water absorption plate 5, wherein, colloidal gold label is sprayed on gold label pad 3, detection pad 4 has detection T line 6 and quality control C line 7, which are formed by fixing sialic acid ligand fixation and control material on detection pad 4 NC membrane (nitrocellulose membrane), and sample pad 2 is provided with sample loading hole 8; Figure (b) is a side view: it is mainly composed of 5 parts: bottom liner 1 (such as polyvinyl chloride PVC), sample pad 2, gold label pad 3, detection pad 4, water absorption plate 5, detection pad 4 has detection T line 6 and quality control C line 7. The structural schematic diagram of colloidal gold label is shown in Figure 2 As shown: Gold nanoparticles are used as the labeling core, and sialic acid ligand molecules with medium affinity are fixed on the surface of the nanogold through S-Au bonds, and small molecule polyethylene glycol is used as a blocking molecule. The preparation flow chart of the sialic acid ligand molecules with medium affinity is shown in Figure 3 As shown in the flow chart of the preparation of colloidal gold markers Figure 4 As shown in the figure; the virus particles recognize and bind to the colloidal gold marker on the gold label pad to form a virus-gold label complex. The structural diagram of the sialic acid ligand immobilizer is shown in the figure. Figure 5 As shown: a complex formed by a high-affinity sialic acid ligand molecule and a fixed protein through a linker molecule is fixed to a detection pad through a fixed protein unit, wherein the preparation flow chart of a high-affinity sialic acid ligand molecule is as shown Figure 6 As shown in FIG. 1 , the preparation flow chart of the sialic acid ligand immobilized substance is as follows Figure 7 As shown; the virus-gold labeling complex is fixed and colored by binding the remaining S protein on the virus-gold labeling complex through the sialic acid ligand unit, or by exchanging a part of the colloidal gold labeling fixed on the virus surface through the ligand exchange reaction. Control: The new coronavirus S protein, which can be obtained through commercial channels and fixed in the C line area of ​​the detection pad, is fixed and colored by identifying the sialic acid ligand bound to the surface of the free colloidal gold labeling.

[0068] The principle of directly detecting new coronavirus particles in the present invention is as follows: the present invention provides a colloidal gold method similar to the double antibody sandwich method for detecting new coronavirus particles, in which sialic acid ligands and nanogold particles are prepared into colloidal gold labels, and the new coronavirus and the colloidal gold labels are recognized and bound by the surface protein-sialic acid ligands to form a virus-gold label complex, and the virus-gold label complex is fixed by recognizing and binding with the remaining S protein on the surface of the virus particles under the action of the sialic acid ligand fixator, or exchanging part of the colloidal gold labels on the virus-gold label complex, and the detection result is determined by color change, as shown in Figure 8.

[0069] Specifically, the positive detection process, principle and results are as follows: when the new coronavirus in the sample to be tested flows through the gold-labeled pad, the new coronavirus recognizes and binds to the sialic acid ligand on the colloidal gold-labeled surface through the surface S protein to form a labeled complex. When the complex unfolds on the detection pad and flows through the test line (Test line, T line), the remaining S protein on the surface of the new coronavirus recognizes and binds to the fixed sialic acid ligand, or through a ligand exchange reaction, it exchanges with the gold label partially fixed on the surface of the virus particles. The virus-gold label complex is fixed, thereby showing red on the T line. When the excess colloidal gold label continues to unfold and flow through the quality control line (Control line, C line), it is bound and fixed by the controlled substance, thereby showing red on the C line;

[0070] Negative test process, principle and results: The sample to be tested does not contain the new coronavirus. When the sample flows through the gold label pad, it cannot form a labeling complex. When it continues to flow through the T line, the fixed sialic acid ligand cannot fix the colloidal gold label, so the T line does not show red. When the sample continues to flow through the C line, the colloidal gold label is bound and fixed by the control substance, so it shows red on the C line;

[0071] Invalid test result: If the C line does not show color, regardless of whether the T line shows red, it is an invalid result and another test strip should be taken for retesting.

[0072] In order to better understand the content of the present invention, the content of the present invention is further described below in conjunction with specific implementation methods, but the protection content of the present invention is not limited to the following embodiments.

[0073] Example 1: Synthesis of sialic acid ligand molecule 1 with medium affinity

[0074]

[0075] 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 stirred at 50 ° C for 2 hours. The reaction was complete by TLC detection, and the reaction liquid was dried by rotary evaporation. The residue was evaporated three times by adding toluene and dried to obtain a crude intermediate of formula 1-1, which was directly used in the next step;

[0076] 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 a crude product of formula 1-2, which was directly used in the next step;

[0077] The crude product of formula 1-2 obtained in the previous step was mixed with HOPEG4OBn (13.79 g, 48.5 mmol) in dry dichloromethane (60 mL), and silver carbonate (17.83 g, 64.7 mmol) was added under ice bath, and the reaction was carried out for 10 h. The reaction was completed by TLC detection, and the insoluble matter was filtered out. The residue was concentrated and separated by 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;

[0078] 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, and stirred for 30 min. The reaction was completed by TLC detection, and the reaction solution was neutralized to neutrality. The solvent was evaporated, and toluene was added to the residue and evaporated three times to obtain compound 1-4 (15.2 g, 98.7%). ESI-MS m / z calcd for [C 33 H 48 NO 13 ] + (M+H) + :666.73,found: 666.72;

[0079] 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, and p-toluenesulfonyl chloride (6.52 g, 34.25 mmol) was added in batches, and stirred overnight. The reaction was complete by TLC detection. Methanol was added and stirred for 10 minutes. The solvent was evaporated and the residue was dissolved in dichloromethane. The organic phase was washed with brine and dried over anhydrous sodium sulfate. The solvent was evaporated and the residue was dissolved in N,N-dimethylformamide (20 mL). Sodium azide (3.70 g, 57.08 mmol) was added and stirred in an oil bath at 50 ° C for 12 hours. The reaction was complete by TLC detection. The solvent was evaporated and the residue was dissolved in tetrahydrofuran (40 mL). Triphenylphosphine (10.48 g, 39.95mmol), 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 (20mL), acetic anhydride (3.03g, 29.68mmol) was added dropwise, and the solvent was evaporated after stirring for 12 hours. The residue was directly mixed with a sample column for separation to obtain compound 1-5 (11.6g, 71.9%), ESI-MS m / z calcd for [C 35 H 51 N2O 13] + (M+H) + :707.79,found:707.80;

[0080] The compound of formula 1-5 (11.6 g, 16.41 mmol) was dissolved in methanol (40 mL), and Pd / C catalyst (0.1 g) was added. The mixture was stirred for 6 hours in a 4 atm H2 atmosphere. The reaction was complete when detected by TLC. The Pd / C powder was filtered off and the filtrate was concentrated to obtain the compound of formula 1-6 (8.4 g, 97.2%). ESI-MS m / z calcd for [C 21 H 39 N2O 13 ] + (M+H) + :527.54,found:527.54;

[0081] 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, and p-toluenesulfonyl chloride (4.56 g, 23.93 mmol) was added in batches, and stirred overnight. The reaction was complete by TLC detection. Methanol was added and stirred for 10 minutes. The solvent was evaporated and the residue was dissolved in dichloromethane. The organic phase was washed with brine and dried over anhydrous sodium sulfate. After concentration, the residue was dissolved in N,N-dimethylformamide (50 mL), potassium thioacetate (3.54 g, 30.97 mmol) was added, and stirred in an oil bath at 50°C for 12 hours. The reaction was complete by TLC detection. The solvent was evaporated and the residue was separated and purified by column chromatography to obtain the compound of formula 1-7 (7.6 g, 81.5%), ESI-MS m / z calcd for [C 23 H 41 N2O 13 S] + (M+H) + :585.63,found:585.62;

[0082] 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 the mixture was stirred at room temperature for 15 minutes. After TLC detection, the reaction was complete, and 732-type H + After stirring for 5 minutes, the resin was filtered off and the filtrate was concentrated to obtain the compound of formula 1 (6.9 g, 97.8%). ESI-MS m / z calculated for [C 21 H 39 N2O 12 S] + (M+H) +: 542.60,found:542.59.

[0083] Example 2: Synthesis of sialic acid ligand molecule 5 with medium affinity:

[0084]

[0085] The preparation process of the compound of formula 5-1 is the same as that of the compound of formula 1-4 in the embodiment, wherein only the HOPEG4OBn reagent is replaced with HOPEG3ONap;

[0086] The compound of formula 5-1 (5 g, 7.44 mmol) was dissolved in methanol (50 mL), Bu2SnO (2.22 g, 8.93 mmol) was added, and the mixture was refluxed at 80 ° C for 3 hours until the reaction solution was clear. The solvent was evaporated, and the residue was rotary evaporated three times with toluene, dissolved in dry DMF (40 mL), and benzyl bromide (1.91 g, 11.17 mmol) and CsF (1.36 g, 8.93 mmol) were added. The mixture was reacted in a N2 atmosphere for 12 hours. The reaction was complete when detected by TLC. 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 / zcalcd for [C 42 H 52 NO 12 ] + (M+H) + :762.87,found:762.88;

[0087] The compound of formula 5-2 (5.2 g, 6.83 mmol) was dissolved in a DCM / MeOH mixed solvent (v / v=2:1, 100 mL), 2,3-dichloro-5,6-dicyano-p-benzoquinone DDQ (2.32 g, 10.24 mmol) was added, and stirred for 30 minutes under light protection. The reaction was complete when detected by TLC. The reaction solution was concentrated to obtain the intermediate of formula 5-3, which was directly used in the next step;

[0088] The intermediate of formula 5-3 obtained in the previous step was dissolved in a 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) was added, and the reaction was stirred at room temperature for 6 hours. After TLC detection, the reaction was complete, methanol (1 mL) was added to quench the reaction, and the solvent was evaporated. The residue was dissolved in acetonitrile (40 mL), KSAc (1.17 g, 10.24 mmol) was added, and stirred at room temperature for 3 hours. After TLC detection, the reaction was complete, the solvent was evaporated, and the residue was dissolved in dichloromethane and successively reacted with sat.NaHCO 3a.q.The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain the compound of formula 5-4 (3.5 g, 75.4%). ESI-MS m / z calcd for [C 33 H 46 NO 12 S] + (M+H) + :680.78,found:680.77;

[0089] The compound of formula 5-4 (3.5 g, 5.15 mmol) was dissolved in methanol (40 mL), and a NaOH aqueous solution (1.5 M, 20 mL) was added dropwise, and stirred until the reaction was complete as detected by TLC. The reaction solution was neutralized, the solvent was evaporated, and the residue was separated by a rapid column to obtain the compound of formula 5 (2.7 g, 95.8%). ESI-MS m / z calcd for [C 24 H 38 NO 11 S] + (M+H) + :548.62,found:548.62;

[0090] Example 3: Synthesis of sialic acid ligand molecule 6 with medium affinity

[0091]

[0092] The preparation process of the compound of formula 6 is the same as that of the compound of formula 1, wherein only the HOPEG4OBn reagent is replaced by HOC5H 10 OBn, and the Ac2O reagent in the last step of the preparation of the compound of formula 1-4 can be replaced with TsCl reagent, ESI-MS m / z calcd for [C 23 H 37 N2O 10 S2] + (M+H) + :565.18,found:565.17.

[0093] Example 4: Synthesis of high-affinity sialic acid ligand molecule 2

[0094]

[0095] Concentrated hydrochloric acid (1 mL) was added dropwise to methanol (100 mL), and natural sialic acid Neu5Ac (10 g, 32.3 mmol) was added in batches. The reaction mixture was stirred until the solution was clear, and the reaction solution was neutralized. The solvent was evaporated, and the residue was evaporated three times with toluene and dried to obtain a crude intermediate of formula 2-1, which was directly used in the next step.

[0096] 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 a crude product of formula 2-2, which was directly used in the next step;

[0097] The crude product of formula 2-2 obtained in the previous step was mixed with HOPEG4OBn (13.8 g, 48.5 mmol) in dry dichloromethane (60 mL), and silver carbonate (17.83 g, 64.7 mmol) was added under ice bath. The reaction was carried out for 12 h. The reaction was completed by TLC detection. The insoluble matter was filtered out, and the residue was concentrated and separated by 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.

[0098] The compound of formula 2-3 (19.3 g, 25.47 mmol) was dissolved in methanol (40 mL), and a NaOH aqueous solution (1.5 M, 20 mL) was added dropwise, and the mixture was stirred until the reaction was complete as detected by TLC. The reaction solution was neutralized, and the solvent was evaporated. The residue was dissolved in a 2M NaOH aqueous solution (40 mL), and refluxed in an oil bath at 95°C for 10 hours. The reaction solution was neutralized, and the solvent was evaporated. The residue was azeotropically evaporated with toluene three times and then dissolved in hydrochloric acid / methanol (v / v=1:99, 50 mL). The mixture was stirred until the reaction was complete as detected by TLC. The reaction solution was neutralized, and the solvent was evaporated to obtain a crude compound of formula 2-4, which was directly used in the next step. ESI-MS m / z calcd for [C 25 H 42 NO 12 ] + (M+H) + :548.60,found:548.59;

[0099] Sodium azide NaN3 (1.99 g, 30.56 mmol) was dissolved in water (30 mL), and trifluoromethanesulfonic anhydride (8.62 g, 30.56 mmol) was slowly added dropwise, and stirred for 15 minutes. The aqueous phase was extracted with toluene, and the organic phase was dried over anhydrous sodium sulfate to obtain trifluoromethanesulfonyl azide TfN3. The crude compound of formula 2-4 obtained in the previous step was dissolved in ethanol, and the prepared TfN3 and copper sulfate CuSO4 (0.812 g, 5.09 mmol) were added. Stir for 6 hours, and the solvent was evaporated. The residue was dissolved in pyridine (50 mL), and acetic anhydride (26 g, 254.7 mmol) was added dropwise under ice bath conditions. Stir at room temperature for 12 hours, and methanol was added dropwise under ice bath to quench the reaction. The solvent was evaporated, and the residue was dissolved in dichloromethane and successively treated with 1N HCl a.q. 、sat.NaHCO3a.q. The mixture was washed with saturated brine, 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 N3O 16 ] + (M+H) + :742.74,found: 742.73;

[0100] 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 tetrahydrofuran (THF) (60 mL). N, N-diisopropylethylamine DIPEA (4.73 gm36.6 mmol) was added dropwise and stirred at room temperature for 1 hour. The reaction solvent was evaporated and the residue was dissolved in dichloromethane and successively treated with 1M ammonia water and 1N HCl. a.q. 、sat. NaHCO 3a.q. The product was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain the compound of formula 2-6 (19.6 g, 97.3%). ESI-MS m / z calculated for [C 37 H 52 N3O 18 ] + (M+H) + :826.82,found:826.81;

[0101] 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, and the mixture was stirred at room temperature for 30 minutes. Hydrochloric acid was added dropwise to neutralize the reaction solution, and the solvent was evaporated to obtain a crude product of formula 2-7, which was directly used in the next step;

[0102] 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. The mixture was stirred at room temperature for 6 hours, and methanol was added to quench the reaction. The solvent was evaporated, and the residue was dissolved in dichloromethane and washed with brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated. The residue was dissolved in N, N-dimethylformamide (50 mL), and NaN3 (4.63 g, 71.20 mmol) was added. The mixture was stirred in an oil bath at 50°C for 12 hours. The reaction was complete by TLC detection, and the solvent was evaporated. The residue was dissolved in dichloromethane, washed with brine, dried over anhydrous sodium sulfate, concentrated, and then column chromatography was performed to obtain the compound of formula 2-8 (14.3 g, 88.3%). ESI-MS m / z calcd for [C 29 H 43 N6O 13 ] + (M+H) + :683.68,found:683.69;

[0103] 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, and the mixture was stirred until no bubbles were generated. Stirring was continued for 12 hours, and the solvent was evaporated. The residue was dissolved in ethanol, and benzoyl chloride (3.53 g, 25.14 mmol) was slowly added dropwise. The mixture was stirred at room temperature for 1 hour, and the solvent was evaporated. 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 N4O 14 ] + (M+H) + :761.79,found:761.78;

[0104] The compound of formula 2-9 (13.1 g, 17.22 mmol) was dissolved in acetonitrile (50 mL), acetone dimethyl acetal (5.38 g, 51.66 mmol) and a catalytic amount of p-toluenesulfonic acid (0.3 g, 1.72 mmol) were added, and refluxed in an oil bath at 80°C for 12 hours. The reaction was complete when detected by TLC. 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 N4O 13 ] + (M+H) + :801.86,found:801.86;

[0105] 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), iodomethane (3.40 g, 23.97 mmol) was added, and stirred at room temperature for 4 hours. The reaction was complete after TLC detection. The insoluble matter was filtered off, the solvent was evaporated, and 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 N4O 14 ] + (M+H) + :815.89,found:815.88;

[0106] The compound of formula 2-11 (11.6 g, 14.24 mmol) was dissolved in methanol, p-toluenesulfonic acid (0.25 g, 1.42 mmol) was added, and the mixture was refluxed in an oil bath at 80°C for 6 hours. The reaction was complete when detected by TLC. Triethylamine was added to neutralize the reaction solution, and the solvent was evaporated. The residue was dissolved in dichloromethane, washed with brine, dried over anhydrous sodium sulfate, concentrated, and dissolved in methanol. Pd / C powder (0.1 g) was added, and the mixture was reacted in a H2 atmosphere at 4 atm pressure for 2 hours. The Pd / C powder was filtered off, and the filtrate was concentrated to obtain a crude compound of formula 2-12, which was directly used in the next step. ESI-MS m / z calcd for [C 30 H 45 N4O 14 ] + (M+H) + :685.70,found:685.69;

[0107] 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 p-toluenesulfonyl chloride (4.07 g, 21.35 mmol) was added in batches after stirring for 5 minutes. The mixture was stirred at room temperature for 6 hours, and methanol was added to quench the reaction. The solvent was evaporated, and the residue was dissolved in dichloromethane and washed with brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated. The residue was dissolved in N, N-dimethylformamide (40 mL), and NaN3 (2.78 g, 42.71 mmol) was added. The mixture was stirred in an oil bath at 50°C for 12 hours. The reaction was complete when detected by TLC. The solvent was evaporated, and the residue was dissolved in dichloromethane, washed with brine, dried over anhydrous sodium sulfate, concentrated, and then column chromatography was performed to obtain the compound of formula 2-13 (8.2 g, 81.2%). ESI-MS m / z calcd for [C 30 H 43 N7O13 ] + (M+H) + :710.71,found: 710.70;

[0108] The compound of formula 2-13 (8.2 g, 11.55 mmol) was dissolved in methanol (30 mL), and an aqueous solution of NaOH (1.5 M, 10 mL) was added. The mixture was stirred at room temperature for 30 minutes, and 732 H + The reaction solution was neutralized with cationic resin, the resin was filtered off, Pd / C powder (0.1 g) was added to the filtrate, and the mixture was reacted for 2 hours in a 4 atm H2 atmosphere, 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 N5O 13 ] + (M+H) + :670.69,found:670.68.

[0109] Example 5: Synthesis of high affinity sialic acid ligand molecule 7

[0110]

[0111] 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 HOPEG4OBn reagent is replaced with HOPEG3OBn;

[0112] The compound of formula 7-1 was dissolved in ethanol, benzoyl chloride was added dropwise under ice bath, and the reaction was continued with stirring for 1 hour. The solvent was evaporated to obtain a crude compound of formula 7-2, which was directly used in the next step reaction;

[0113] 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 Ac2O reagent is replaced by TsCl;

[0114] The process of preparing the compound of formula 7 from the compound of formula 7-3 is the same as the process of preparing the compound of formula 2 from the compound of formula 2-9.

[0115] Example 6: Synthesis of high affinity sialic acid ligand molecule 8

[0116]

[0117] 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 HOPEG4OBn reagent is replaced by HOC5H 10 OAll is enough;

[0118] 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 with benzyloxycarbonyl chloride CbzCl;

[0119] 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;

[0120] 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;

[0121] 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, and the mixture was stirred at room temperature for 12 hours. The reaction was complete when detected by TLC. Methanol was added dropwise to quench the reaction. The reaction solution was diluted with dichloromethane (300 mL), washed three times with saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography 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;

[0122] 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, 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, 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, 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;

[0123] 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;

[0124] The compound of formula 8-7 (4 g, 5.66 mmol) was dissolved in methanol (40 mL), and a NaOH aqueous solution (1 M, 20 mL) was added dropwise, and stirred until the reaction was complete as detected by TLC. The reaction solution was neutralized, and the solvent was evaporated. The residue was dissolved in tetrahydrofuran (40 mL), and Ph3P (4.45 g, 16.98 mmol) was added. The mixture was stirred at room temperature for 12 hours. The reaction was complete as detected by TLC, and the solvent was evaporated. The residue was directly purified by column chromatography to obtain the compound of formula 8 (3.2 g, 84.8%). ESI-MS m / z calcd for [C 36 H 47 N2O 10 ] + (M+H) + :667.77,found:667.76;

[0125] Example 7: Preparation of sialic acid-gold nanoparticle labeled complex 3

[0126]

[0127] The preparation method of the gold nanoparticle solution stabilized by sodium citrate and having a particle size of 30 nm is as follows: take 100 mL of 1 mM HAuCl4 aqueous solution into a two-necked bottle, heat it to 100°C, add sodium citrate (dihydrate) (118 mg, 0.3 mmol), stir for 15 minutes, take out and slowly cool to room temperature to obtain the solution.

[0128] The sialic acid molecule 1 obtained in Example 1 was dissolved in water to prepare an aqueous solution (10 mM), 100 μL was taken and added to a sodium citrate-stabilized gold nanoparticle solution with a particle size of 30 nm (OD=15, 5 mL), and after standing for 15 hours, an excess of thiotetraglycol (PEG4-SH) blocking solution (0.1 M, 100 uL) was added, and after shaking for 12 hours, the solution was centrifuged at 15000 rpm for 15 minutes twice, and the aggregates in the centrifuge tube substrate were redispersed in deionized water (5 mL) to obtain the sialic acid-gold nanoparticle labeled complex 3.

[0129] Example 8: Preparation of Sialic Acid Ligand Immobilized Material 4

[0130]

[0131] The sialic acid ligand molecule 2 (1 g, 1.46 mmol) obtained in Example 2 was dissolved in methanol (20 mL), diethyl squarate (2.48 g, 14.58 mmol) was added, and the mixture was stirred at room temperature for 24 h. The reaction solution was directly concentrated and subjected to column chromatography to obtain the intermediate compound of formula 4-1 (1.1 g, 93.1%). ESI-MS m / z calculated for [C 35 H 47 N5O 17 ]+ (M+H) + :810.78,found:810.77, recycle excess diethyl squarate;

[0132] The obtained compound of formula 4-1 (1.1 g, 1.36 mmol) was mixed with bovine serum albumin BSA (66.43 kDa, 3 g, 45 umol) in water and allowed to stand for 24 hours. The reaction solution was purified by gel column to obtain a complex of formula 4 (3.8 g), which was dissolved in ultrapure water to prepare a 1 nM solution.

[0133] Example 9: Preparation of a novel coronavirus test strip

[0134] The sample pad, gold label pad, detection pad, and absorbent pad were cut to have length × width specifications of 2 cm × 2 cm, 1 cm × 2 cm, 6 cm × 2 cm, and 1 cm × 2 cm, respectively. The sialic acid-gold nanoparticle labeling complex 3 solution (10 μL) prepared in Example 7 was sprayed on the gold label pad, and the sialic acid ligand fixative 4 solution (5 μL) prepared in Example 8 was sprayed on the T line (0.1 × 2 cm) area of ​​the detection pad. A commercial SARS-CoV-2 S protein solution (1 nM, 5 μL) was used as a control substance and sprayed on the C line (0.1 × 2 cm) area of ​​the detection pad. The sample pad, gold label pad, detection pad, and absorbent pad were placed in the same manner as above. Figure 1 The layers and positions shown in the structure of the detection device are affixed to the PVC liner, with each layer overlapping by 0.5 cm to prepare a COVID-19 test strip.

[0135] Example 10: Detection Experiment

[0136] The novel coronavirus is highly pathogenic and contagious. The detection experiment was conducted using novel coronavirus simulated particles. The commercial novel coronavirus S protein was fixed on the surface of SiO2 nanospheres with a diameter of about 100 nm to construct novel coronavirus simulated particles. One drop of novel coronavirus simulated particle aqueous solution (10 -6 / mL, about 50μL), add it dropwise to the sample well of the new coronavirus test strip, let it stand horizontally for 15 minutes, and the T line of the kit will turn red and the C line will turn red.

[0137] Example 11: Parallel detection experiment

[0138] Take two new coronavirus test strips, numbered as test strip A and test strip B respectively, take saliva from a healthy person, divide it into two parts and mark them as saliva A and saliva B respectively, add the new coronavirus simulated particle solution prepared in Example 10 to saliva A, add deionized water to saliva B, take 1 drop (about 50 μL) of each sample, drop them into the sample holes of test strip A and test strip B respectively, and let them stand horizontally for 15 minutes. The T line of test strip A shows red and the C line shows color. The T line of test strip B does not show color, but the C line shows color.

[0139] In summary, the embodiments of the present invention provide a test strip that can directly detect novel coronavirus particles, which can very quickly and conveniently determine whether a sample contains virus particles, and then quickly determine whether the subject is contagious.

[0140] The above description is only a specific implementation mode of the present invention, not all implementation modes. Any equivalent changes made to the technical solution of the present invention by ordinary technicians in this field after reading the specification of the present invention are covered by the claims of the present invention.

Claims

1. A novel coronavirus test strip, characterized in that: The invention comprises a backing plate (1), wherein a sample pad (2), a gold labeling pad (3), a detection pad (4) and a water absorption plate (5) are sequentially arranged from one end to the other end of the backing 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 labeling 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 to the gold label pad (3) and the water absorption plate (5) in an overlapping manner, and the sample pad (2) is pressed on the gold label pad (3); 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. The general structural formula of the sialic acid ligand molecule with high affinity is shown in Formula II: wherein R1 is selected from methoxy or substituted methoxy; R2 is selected from substituted acetylamino, benzylamino, substituted benzylamino, alkoxycarbonylamide, triazolyl or substituted triazolyl; R3 is selected from hydroxyl, methoxy, substituted methoxy, acetylamino, substituted acetylamino, sulfonamide or phosphoramide; 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, and R4 is selected from amino or thiol; The quality control C line (7) is coated with a control substance, and the control substance is the S protein of the new coronavirus; The gold label pad (3) is sprayed with colloidal gold labeling material, which is a gold nanoparticle with a sialic acid ligand molecule with medium affinity fixed on the surface; the general structural formula of the sialic acid ligand molecule with medium affinity is shown in Formula I: wherein R5 is selected from hydroxy, methoxy, substituted methoxy, acetylamino, substituted acetylamino, sulfonamide or phosphoramide; 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.

2. The novel coronavirus test strip according to claim 1, characterized in that: The sample pad (2) is provided with a sample addition hole (8).

3. The novel coronavirus test strip according to claim 1, characterized in that: The substituted methoxy group is X1-CH2O-, wherein X1 is selected from phenyl or vinyl; The substituted acetylamino group is X2-CH2CONH-, wherein X2 is selected from methyl, ethyl, n-propyl, isopropyl, hydroxymethyl or hydroxyethyl; The substituted benzylamino group is X3-BzNH-, wherein the number of X3 is at least 1 and is located at any position of the benzene ring, and X3 is selected from at least one of a halogen atom, a methyl group, a methoxy group, and a nitro group; The alkoxycarbonylamide group is X4-OC(O)NH-, wherein X4 is selected from benzyl, allyl, tert-butyl or trichloroethyl; The substituted triazole group is Wherein, X5 is selected from substituted phenylalkyl X6-Ph-(CH2) n6 - or substituted carbonyl X7-C(O)-, wherein the number of X6 is at least 1 and is located at any position of the benzene ring, X6 is selected from at least one of a halogen atom, a methyl group, a methoxy group, and a nitro group, and n6 is selected from 0, 1, and 2; X7 is selected from an alkoxy group CH3-(CH2) n7 -O- or alkylamino CH3-(CH2) n7 -NH-, n7 is selected from 0, 1, 2, 3, 4, 5.

4. The novel coronavirus test strip according to claim 1, characterized in that: The sialic acid ligand immobilizer comprises an immobilized protein, a sialic acid ligand molecule with high affinity and a connecting molecule, wherein the immobilized protein is a protein macromolecule used for blocking and immobilization.

5. The novel coronavirus test strip according to claim 4, characterized in that: The fixed protein is selected from bovine serum albumin BSA, skimmed milk powder or casein.

6. The novel coronavirus test strip according to claim 4, characterized in that: The general structural formula of the linker molecule is R6-linker-R7, wherein R6 is a group that can react and connect with R4.

7. The novel coronavirus test strip according to claim 6, characterized in that: The R6 is selected from a carboxyl group, an ester group, a sulfonic acid group, a phosphoric acid group or a maleimide group; and R7 is a group that can react with an active reactive group on the surface of the fixed protein.

8. The novel coronavirus test strip according to claim 6, characterized in that: The R7 is selected from a carboxyl group, an ester group, an amino group or a maleimide group; the linker is selected from an alkyl group, a cycloalkyl group, a polyethylene glycol chain or an aromatic group.

9. The novel coronavirus test strip according to claim 6, characterized in that: The linking molecule is selected from diethyl squarate, diglycolic acid, maleimide-polyethylene glycol carboxylic acid, and 1,4-cyclohexanedicarboxylic acid.

10. The novel coronavirus test strip according to claim 1, characterized in that: The colloidal gold label comprises gold nanoparticles, sialic acid ligand molecules with medium affinity and blocking molecules.

11. The novel coronavirus test strip according to claim 10, characterized in that: The particle size of the gold nanoparticles is 30-50 nm.

12. The novel coronavirus test strip according to claim 10, characterized in that: The blocking molecule is a small molecule thiol polyethylene glycol.

13. The novel coronavirus test strip according to claim 12, characterized in that: The structural formula of the blocking molecule is R8-(C2H4O) n3 -C2H4-SH, n3 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and R8 is selected from hydroxy, methoxy or carboxyl.

14. The method for preparing the novel coronavirus detection test strip according to any one of claims 1 to 13, characterized in that: The following steps are involved: preparing a colloidal gold label and spraying the colloidal gold label onto a gold label pad (3); preparing a sialic acid ligand immobilized substance, and coating the sialic acid ligand immobilized substance on a detection T line (6); The SARS-CoV-2 S protein was coated on the quality control C line (7); The sample pad (2), the gold labeling pad (3), the detection pad (4) and the absorbent plate (5) are adhered to the backing plate (1) to obtain the novel coronavirus detection test strip.

15. The preparation method according to claim 14, characterized in that: The colloidal gold marker uses gold nanoparticles as the marking core, fixes sialic acid ligand molecules with medium affinity on the surface of the gold nanoparticles through S-Au bonds, and uses small molecule polyethylene glycol as a blocking molecule.

16. The preparation method according to claim 14, characterized in that: The sialic acid ligand immobilizer is a complex in which an immobilized protein is connected to a sialic acid ligand molecule with high affinity via a connecting molecule, and the immobilized protein is a protein macromolecule used for closed immobilization.

17. The preparation method according to claim 14, characterized in that: The preparation method of the colloidal gold marker is as follows: sialic acid ligand molecules with medium affinity are configured into an aqueous solution, mixed with a gold nanoparticle solution, stirred at 20 to 30° C. for 12 to 24 hours, fixed on the surface of the gold nanoparticles by forming S-Au bonds on the surface of the gold nanoparticles, then an excess of small molecule thiol polyethylene glycol is added, and stirring is continued at 20 to 30° C. for 12 to 24 hours to block the remaining reaction sites on the surface of the gold nanoparticles, and the sialic acid-gold nanoparticle marker complex is obtained by centrifugal purification.

18. The preparation method according to claim 17, characterized in that: The molar ratio of the sialic acid ligand molecule with medium affinity to the gold nanoparticles is 1×10 4 :1~2×10 4 :

1.

19. The preparation method according to claim 17, characterized in that: The preparation method of the sialic acid ligand molecule with medium affinity is as follows: As the starting material, the R9 protecting group is introduced into the carboxyl group to obtain In the next step, the fully acetylated chlorosugar is generated by the action of chlorination and acetylation reagents. Then, the functional side chain precursor is derived at the 2-position of the sugar ring through glycosylation reaction. After deacetylation, several hydroxyl groups on the sugar ring are exposed to obtain Then, the R5 group was introduced by utilizing the difference in reactivity 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 medium affinity is synthesized through group conversion and deprotection reaction; wherein R9 is selected from methyl or benzyl, and A2 is or -(CH2)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 is selected from 2, 3, 4, 5, 6, 7, 8, 9; when R5 is hydroxyl, it is sialic acid The groups on the molecule do not need to be introduced into the relevant reaction process of the R5 group.

20. The preparation method according to claim 17, characterized in that: The preparation method of the gold nanoparticle solution is as follows: heating a chloroauric acid aqueous solution to boiling, adding sodium citrate, and keeping boiling for 15 to 30 minutes, and cooling to obtain a gold nanoparticle solution with a particle size of 30 to 50 nm and a surface covered with sodium citrate, wherein the concentration of the chloroauric acid aqueous solution is 1 mM, and the molar ratio of chloroauric acid to sodium citrate is 1:2-4.

21. The preparation method according to claim 14, characterized in that: The preparation method of the sialic acid ligand immobilized substance is as follows: The high-affinity sialic acid ligand molecule and the linker molecule are mixed in water and stirred at 20-30°C for 12-24 hours. The high-affinity sialic acid ligand molecule reacts with the R6 group of the linker molecule through the active reaction group R4 on the functional side chain A to covalently link to obtain a sialic acid-linker molecule complex. Then, an immobilized protein is added and allowed to stand at 20-25°C for 15-30 hours. A covalent bond is formed between the R7 group on the sialic acid-linker molecule complex and the active reaction group on the surface of the immobilized protein to obtain a target sialic acid ligand immobilized substance.

22. The preparation method according to claim 21, characterized in that: The molar ratio of the high-affinity sialic acid ligand molecule, the linking molecule and the fixed protein is 5-10:12-15:

1.

23. The preparation method according to claim 21, characterized in that: The method for preparing the high-affinity sialic acid ligand molecule is as follows: As the starting material, R was introduced into the carboxyl group after carboxyl protection. 11 Protecting group obtained In the next step, the fully acetylated chlorosugar is generated by the action of chlorination and acetylation reagents. Then, the functional side chain precursor is derived at the 2nd position of the sugar ring through glycosylation reaction. After deacetylation and carboxyl protection, the intermediate with both amino and hydroxyl groups exposed on the sugar ring was obtained. The groups were introduced in the order of R2→R1→R3 to obtain Alternatively, the groups are introduced in the order of R2 → R3 → R1 to obtain 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; wherein R11 is selected from methyl or benzyl, and A3 is or -(CH2)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 R3 is hydroxyl, it is sialic acid The groups on the molecule do not need to be introduced into the relevant reaction process of the R3 group.

24. A method for using the COVID-19 test strip according to any one of claims 1 to 13, characterized in that: The following steps are involved: 1) Place the COVID-19 test strip horizontally, drop the test sample onto the sample pad (2), and let it stand for 10-15 minutes; 2) Result determination: Positive result: the test T line (6) appears red, and the quality control C line (7) appears red; Negative result: the test T line (6) does not show color, and the quality control C line (7) shows red; Invalid result: Regardless of whether the test T line (6) is colored or not, the quality control C line (7) does not appear red, and another COVID-19 test strip should be taken for retesting.

25. The method of use according to claim 24, characterized in that: The test samples include nasopharyngeal swabs, saliva, blood, and excrement.

Images