WO2022094932A1 - Bandelette réactive de détection de nouveau coronavirus, et son procédé de préparation et d'utilisation - Google Patents
Bandelette réactive de détection de nouveau coronavirus, et son procédé de préparation et d'utilisation Download PDFInfo
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- WO2022094932A1 WO2022094932A1 PCT/CN2020/127148 CN2020127148W WO2022094932A1 WO 2022094932 A1 WO2022094932 A1 WO 2022094932A1 CN 2020127148 W CN2020127148 W CN 2020127148W WO 2022094932 A1 WO2022094932 A1 WO 2022094932A1
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- WIPO (PCT)
- Prior art keywords
- sialic acid
- group
- molecule
- acid ligand
- detection
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- 238000001514 detection method Methods 0.000 title claims abstract description 105
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 85
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- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 claims abstract description 89
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- PIBWKRNGBLPSSY-UHFFFAOYSA-L palladium(II) chloride Chemical compound Cl[Pd]Cl PIBWKRNGBLPSSY-UHFFFAOYSA-L 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910001923 silver oxide Inorganic materials 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- NQPHMXWPDCSHTE-UHFFFAOYSA-N trifluoromethanesulfonyl azide Chemical compound FC(F)(F)S(=O)(=O)N=[N+]=[N-] NQPHMXWPDCSHTE-UHFFFAOYSA-N 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Definitions
- the invention belongs to the technical field of biochemistry, and relates to a new coronavirus detection test strip and a preparation method and use method thereof.
- the novel coronavirus pneumonia (“COVID-19” for short) is a major public health emergency that has infected tens of millions of people and killed hundreds of thousands of people worldwide.
- the pathogen causing new coronary pneumonia is a new type of coronavirus (referred to as "new coronavirus”), which was named SARS-Cov-2 (Severe acute respiratory syndromes coronavirus 2) by the International Committee on Taxonomy of Viruses.
- a key to the prevention and control of new coronary pneumonia is to develop effective medical diagnosis and treatment methods for the new coronavirus, of which timely and accurate detection of the new coronavirus is crucial to the prevention and control of the epidemic.
- the detection technology of the new coronavirus mainly focuses on the detection of the RNA sequence of the new coronavirus, and the detection of the IgM and IgG antibodies produced by the human immune system after the infection of the new coronavirus.
- the detection of new coronavirus RNA mainly includes real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR) method and CRISPR (clustered regularly interspaced short palindromic repeats) method.
- RT-PCR real-time fluorescent reverse transcription-polymerase chain reaction
- CRISPR clustered regularly interspaced short palindromic repeats
- the human immune system will produce specific IgM and IgG antibodies after infection with the new coronavirus to indirectly detect whether there is a new coronavirus infection, especially the colloidal gold method.
- the colloidal gold method is also known as immunochromatography. Its main principle is to use the sample solution dropped on one end of the membrane to move to the other end by the capillary action of the membrane (lateral flow based on chromatography), and the analyte moves during the movement. It binds to a receptor immobilized on a certain area of the membrane and becomes immobilized, and the immobilization result is judged by developing the color of a marker, which is usually colored by colloidal gold.
- the IgG/IgM test cards for 2019-nCoV on the market also use immunochromatography to detect the presence of 2019-nCoV IgG/IgM antibodies in human blood, mainly by labeling anti-IgG/anti-IgM antibodies on colloidal gold. After the IgG or IgM in the membrane is immunocombined, it binds to the receptor immobilized on the membrane and becomes immobilized, and the result can be quickly determined.
- the current mainstream new coronavirus RNA detection method is RT-PCR. Although it can directly detect the virus, it needs to manually extract the RNA virus in the sample, which is affected by multiple factors such as sample sampling method, sample transportation and preservation, and manual operation. Staff reported that this method was often accompanied by serious false-negative interference, which challenged the test results; in addition, a small number of RNA test results with positive results did not show contagiousness, so there was also false-positive interference in test results, and their positive results
- the test result may be that the test sample contains nucleic acid residues of the new coronavirus, and there are no infectious new coronavirus particles.
- the method of detecting IgG/IgM antibodies produced by the immune system response after human infection with the new coronavirus belongs to an indirect detection method. Since the human immune system needs a certain period of time after infection to produce an immune response to appear IgG and IgM, this type of method can only reflect Whether the subject has been infected with the new coronavirus does not immediately indicate whether there are new coronavirus particles in the human body, nor is it suitable for the detection of patients in the early stage of infection or the incubation period, so it is impossible to directly judge the infectivity of the test object.
- the purpose of the present invention is to provide a new coronavirus detection test strip and its preparation method and use method.
- the present invention provides a new coronavirus detection test strip, including a liner, one end of the liner is provided with a sample pad, a gold marker pad, a detection pad and a water absorption plate in sequence, the detection pad is The surface of the nitrocellulose membrane is provided with a nitrocellulose membrane, and the nitrocellulose membrane is provided with a detection T line and a quality control C line, the detection T line is close to the gold marker pad, and the quality control C line is close to the water absorption plate;
- the two ends of the detection pad are respectively overlapped with the gold marking pad and the water absorbing plate, and the gold marking pad is pressed with a sample pad;
- the detection T line is coated with a sialic acid ligand immobilizer, and the sialic acid ligand immobilizer includes a sialic acid ligand molecule with high affinity, and the structure of the sialic acid ligand molecule with high affinity
- the general formula is shown in formula II: Wherein, R 1 is selected from methoxy or substituted methoxy; R 2 is selected from substituted acetamido, benzamido, substituted benzamido, alkoxycarbonamido, triazolyl or substituted Triazolyl; R is selected from hydroxyl, methoxy, substituted methoxy, acetamido, substituted acetamido, sulfonamide or phosphoramido; A is wherein n1 is selected from 0,1,2,3,4,5,6,7, m1 is selected from 2,3,4,5,6,7,8,9, R4 is selected from amino or mercapto ;
- the quality control line C is coated with a control substance, and the control substance is the new coronavirus S protein;
- the gold labeling pad is sprayed with colloidal gold labels, and the colloidal gold labels are gold nanoparticles with sialic acid ligand molecules with medium affinity immobilized on the surface;
- the general structural formula is shown in formula I: Wherein, R 5 is selected from hydroxyl, methoxy, substituted methoxy, acetamido, substituted acetamido, sulfonamide or phosphoramido; A1 is n2 is selected from 0,1,2,3,4,5,6,7, and m2 is selected from 2,3,4,5,6,7,8,9.
- sample pad is provided with a sample injection hole.
- substituted methoxy group is X 1 -CH 2 O-, wherein X 1 is selected from phenyl or vinyl;
- the substituted acetamido is X 2 -CH 2 CONH-, wherein X 2 is selected from methyl, ethyl, n-propyl, isopropyl, hydroxymethyl or hydroxyethyl;
- the substituted benzamido is X 3 -BzNH-, wherein the number of X 3 is at least 1 and is located at any position of the benzene ring, and X 3 is selected from halogen atoms, methyl groups, methoxyl groups, nitro groups. at least one;
- the alkoxycarbonamide group is X 4 -OC(O)NH-, wherein X 4 is selected from benzyl, allyl, tert-butyl or trichloroethyl;
- the substituted triazolyl is wherein X 5 is selected from substituted phenylalkyl (X 6 -Ph-(CH 2 ) n6 -) or substituted carbonyl (X 7 -C(O)-), wherein X 6 is at least 1 in number and located in Any position on the benzene ring, X 6 is selected from at least one of halogen, methyl, methoxy, and nitro, n6 is selected from 0, 1, 2; X 7 is selected from alkoxy CH 3 -(CH 2 ) n7 -O- or alkylamino CH 3 -(CH 2 ) n7 -NH-, n7 is selected from 0,1,2,3,4,5.
- the sialic acid ligand immobilizer includes an immobilized protein, a sialic acid ligand molecule with high affinity and a linking molecule, and the immobilized protein is a protein macromolecule used for sealing and immobilization;
- the immobilized protein is selected from protein macromolecules commonly used for sealing and immobilization, such as bovine serum albumin BSA, skimmed milk powder or casein;
- the general structural formula of the linker molecule is R 6 -linker-R 7 , wherein R 6 is a group that can react and link with R 4 , more preferably, the R 6 is selected from a carboxyl group, an ester group, sulfonic acid group, phosphoric acid group or maleimide group; R 7 is a group that can react with active reactive groups (such as amino group, carboxyl group, sulfhydryl group) existing on the surface of the immobilized protein, more preferably, the R 7 is selected from from carboxyl group, ester group, amino group or maleimide group; the linker is selected from alkyl, cycloalkyl, polyethylene glycol chain or aryl; more preferably, the linker is selected from diethyl squaraine Esters, diglycolic acid, maleimide-polyethylene glycol carboxylic acid, 1,4-cyclohexanedicarboxylic acid and other bifunctional compounds.
- R 6 is a group that can react
- colloidal gold label includes gold nanoparticles, sialic acid ligand molecules with moderate affinity and blocking molecules;
- the particle size of the gold nanoparticles is 30-50 nm;
- the blocking molecule is a small molecule thiol polyethylene glycol, more preferably, the general structural formula of the blocking molecule is R 8 -(C 2 H 4 O) n3 -C 2 H 4 -SH, and n3 is selected from From 1, 2, 3, 4, 5, 6, 7, 8, 9, R 8 is selected from hydroxy, methoxy or carboxyl.
- the present invention provides a preparation method of any of the above-mentioned new coronavirus detection test strips, comprising the following steps:
- the colloidal gold marker uses gold nanoparticles as the inner core of the marker, immobilizes sialic acid ligand molecules with medium affinity on the surface of the gold nanoparticles through S-Au bonds, and uses small molecular polyethylene glycol as the blocking molecule;
- the sialic acid ligand immobilizer is a complex in which an immobilized protein is linked with a sialic acid ligand molecule with high affinity through a linking molecule, and the immobilized protein is a protein macromolecule used for blocking and immobilization.
- the preparation method of the colloidal gold label is as follows: disposing a sialic acid ligand molecule with a moderate affinity into an aqueous solution, mixing with the gold nanoparticle solution, stirring at 20-30° C. for 12-24 hours, and passing through the solution. S-Au bonds were formed on the surface of gold nanoparticles and fixed on the surface of gold nanoparticles, then an excess of small molecular mercapto polyethylene glycol was added, and the remaining reaction sites on the surface of gold nanoparticles were further stirred at 20-30 °C for 12-24 hours.
- the sialic acid-gold nanoparticle-labeled complex was obtained by centrifugation and purification;
- the molar ratio of the medium affinity sialic acid ligand molecule to the gold nanoparticle is 1 ⁇ 10 4 :1 ⁇ 2 ⁇ 10 4 :1;
- the preparation method of the sialic acid ligand molecule with medium affinity is: using sialic acid As the starting material, through the protection of the carboxyl group, the R 9 protecting group is introduced into the carboxyl group to obtain Generation of peracetylated chlorosaccharides in the next step of chlorination and acetylation reagents Then, a functional side chain precursor is derived from the 2-position of the sugar ring through a glycosylation reaction After deacetylation, several hydroxyl groups on the sugar ring are exposed to obtain Then, the R5 group was introduced by using the reactivity difference between the primary hydroxyl group of the sugar ring 9-OH and other secondary hydroxyl groups to obtain Finally, a sialic acid ligand molecule with moderate affinity is synthesized through group conversion and deprotection reaction; wherein R 9 is selected from methyl or benzyl, and A 2 is or -(CH 2 )m3-R 10 , R 10 is selected from benzyloxy,
- the preparation method of the gold nanoparticle solution is as follows: heating the aqueous solution of chloroauric acid to boiling, adding sodium citrate, keeping boiling for 15-30 minutes, and cooling to obtain a particle size of 30-50 nm and a surface covered with citric acid
- the sodium gold nanoparticle solution wherein the concentration of the chloroauric acid aqueous solution is 1 mM, and the molar ratio of chloroauric acid and sodium citrate is 1:2-4.
- the preparation method of the sialic acid ligand immobilizer is as follows: mixing the sialic acid ligand molecule with high affinity and the linking molecule in water, and stirring at 20-30° C. for 12-24 hours, which has high affinity
- the sialic acid ligand molecule is covalently linked by the reactive reactive group R 4 on the functional side chain A and the R 6 group of the linking molecule to obtain a sialic acid-linking molecule complex;
- Standing at °C for 15-30 hours, the R 7 group on the sialic acid-linking molecule complex forms a covalent bond with the surface active reactive group of the immobilized protein to obtain the target sialic acid ligand immobilizer;
- the molar ratio of the high-affinity sialic acid ligand molecule, linker molecule, and immobilized protein is 5-10:12-15:1;
- the preparation method of the sialic acid ligand molecule with high affinity is: using sialic acid As the starting material, through the protection of the carboxyl group, the R 11 protecting group is introduced into the carboxyl group to obtain Generation of peracetylated chlorosaccharides in the next step of chlorination and acetylation reagents Then the functional side chain precursor is derived from the glycosylation reaction at the 2-position of the sugar ring After deacetylation and carboxyl protection, an intermediate with all the exposed amino and hydroxyl groups on the sugar ring is obtained Introduce groups in the order of R 2 ⁇ R 1 ⁇ R 3 to obtain Alternatively, the introduction of groups in the order of R 2 ⁇ R 3 ⁇ R 1 can be obtained, respectively.
- the sugar-binding domain pocket of the new coronavirus S protein has high specificity and binding force to the new coronavirus S protein; wherein R 11 is selected from methyl or benzyl, and A 3 is or -(CH 2 )m4-R 12 , R 12 is selected from benzyloxy, 2-naphthylmethoxy or allyloxy, n5 is selected from 0,1,2,3,4,5,6,7, m4 is selected from 2, 3, 4, 5, 6, 7, 8, 9, PG is the abbreviation of protection group; when R 3 is hydroxyl, it is sialic acid The group on the molecule does not need to introduce the relevant reaction process of the R3 group.
- the present invention provides a method for using any of the above-mentioned new coronavirus detection test strips, comprising the following steps:
- the detection T line is red
- the quality control C line is red
- the detection samples include nasopharyngeal swabs, saliva, blood, and excrement.
- the present invention provides a new coronavirus detection test strip, which can be used to directly detect new coronavirus particles, and has the following beneficial effects compared with the existing new coronavirus and related detection methods:
- the new coronavirus detection test strip provided by the present invention directly detects virus particles based on the recognition and combination of sialic acid ligands and the surface S protein of new coronavirus particles;
- the present invention provides a detection test strip that can directly detect new coronavirus particles, which can quickly and conveniently determine whether a sample contains virus particles;
- the new coronavirus detection test strip provided by the present invention can directly detect the new coronavirus particles in the sample, and then quickly determine whether the subject is infectious;
- the new coronavirus detection test strip provided by the present invention does not involve multiple procedures such as sample RNA extraction and PCR, and can be directly sampled and added dropwise, which simplifies the detection process. It avoids the interference caused by manual operation errors; on the other hand, it only takes about 15 minutes to complete the detection, and the color reaction is used to facilitate the judgment of the detection results;
- the new coronavirus detection test strip is a direct detection method, which can directly detect the new coronavirus particles, and can immediately characterize whether there are new coronavirus particles in the sample;
- the new coronavirus detection test strip provided by the present invention is detected by the recognition and combination of sialic acid small molecules and the surface S protein of the new coronavirus.
- the small molecule properties of the molecule have obvious advantages such as good economy, high stability, and mass preparation;
- the new coronavirus detection test strip provided by the present invention can directly detect virus particles, and is rich in sample selection, including nasopharyngeal swabs commonly used in nucleic acid detection, saliva, blood, excrement and other samples that may contain virus particles. ;
- the present invention provides a preparation method of a small molecule-colloidal gold-labeled complex (colloidal gold label) that can be used for colloidal gold detection, and the small-molecule-colloidal gold-labeled complex has advantages in economy and availability. , and also expanded the types and selectable range of gold markers in the colloidal gold method;
- the present invention provides a small molecule-immobilized protein complex (sialic acid ligand immobilized substance) that can be used for colloidal gold detection.
- the small molecule-immobilized protein complex has obvious advantages in economy and availability. The types and selectable ranges of molecules detected by colloidal gold method are described.
- Fig. 1 is the structural representation of the new coronavirus detection test strip of the present invention, Fig. (a) is a top view, and Fig. (b) is a side view;
- Fig. 2 is the structural representation of the colloidal gold marker of the present invention
- Fig. 3 is the preparation flow chart of the sialic acid ligand molecule with medium affinity of the present invention
- Fig. 4 is the preparation flow chart of the colloidal gold marker of the present invention.
- Fig. 5 is the structural representation of sialic acid ligand immobilizer of the present invention.
- Fig. 6 is the preparation flow chart of the high-affinity sialic acid ligand molecule of the present invention.
- Fig. 7 is the preparation flow chart of sialic acid ligand immobilized matter of the present invention.
- Figure 8 is a schematic diagram of the principle of the present invention for directly detecting new coronavirus particles
- Sample addition hole is a colloidal gold marker, is a sialic acid ligand immobilizer, for control.
- the new coronavirus is one of the beta-coronaviruses, and its surface Spike protein (S protein) is a trimer composed of three monomers, each of which is composed of an S1 unit and a conserved S2 unit, of which S1 mainly contains C-terminal binding domain (C-terminus domain, S1-CTD) and N-terminal binding domain (N-terminus domain, S1-NTD), the two together constitute the receptor binding domain (Receptor binding domain, RBD), generally, S1-NTD recognizes sialic acid residues that bind to the surface of host cells, while S1-CTD recognizes peptide molecules that bind to the surface of host cells.
- S protein surface Spike protein
- S protein surface Spike protein
- S protein is a trimer composed of three monomers, each of which is composed of an S1 unit and a conserved S2 unit, of which S1 mainly contains C-terminal binding domain (C-terminus domain, S1-CTD) and N-terminal binding domain (
- the invention provides a test strip for directly detecting new coronavirus particles in a sample to be tested, using sialic acid ligands to detect the identification and binding of S protein molecules on the surface of the new coronavirus, and directly detecting it as a source of pathogenicity and infectivity of new coronary pneumonia It can instantly and quickly determine whether there are new coronavirus particles in the sample of the subject, so as to make a rapid judgment on the infectivity of the subject.
- the structure of the test strip is shown in Figure 1, and Figure (a) is a top view.
- sample pad 2 ⁇ gold marking pad 3 ⁇ testing pad 4 ⁇ water absorbing plate 5 are sample pad 2 ⁇ gold marking pad 3 ⁇ testing pad 4 ⁇ water absorbing plate 5, among which, gold marking pad 3 is sprayed with colloidal gold marker, and detection pad 4 has detection T line 6 and quality control
- the C line 7 is formed by the sialic acid ligand immobilizer and the control substance immobilized on the detection pad 4NC membrane (nitrocellulose membrane) respectively, and the sample pad 2 is provided with a sample addition hole 8;
- Figure (b) is a side view: the main It consists of 5 parts: bottom liner 1 (such as polyvinyl chloride PVC), sample pad 2, gold marking pad 3, detection pad 4, water absorption plate 5, detection pad 4 has detection T line 6 and quality control C line 7 .
- bottom liner 1 such as polyvinyl chloride PVC
- the schematic diagram of the structure of colloidal gold label is shown in Figure 2: Gold nanoparticles are used as the core of the label, and sialic acid ligand molecules with medium affinity are immobilized on the surface of gold nanoparticles through S-Au bonds, and small molecules of polyethylene glycol are used.
- a blocking molecule As a blocking molecule, the flow chart of preparation of sialic acid ligand molecules with moderate affinity is shown in Figure 3, and the preparation flow chart of colloidal gold label is shown in Figure 4; virus particles and colloidal gold labeling occur on the gold labeling pad The recognition and binding of the compound forms a virus-gold-labeled complex.
- the schematic diagram of the structure of the sialic acid ligand immobilizer is shown in Figure 5: it is a complex formed by a high-affinity sialic acid ligand molecule and an immobilized protein through a linker molecule, and is immobilized on the detection pad through the immobilized protein unit.
- the flow chart of the preparation of ligand molecules is shown in Figure 6, and the flow chart of the preparation of sialic acid ligand immobilized substances is shown in Figure 7;
- the ligand exchange reaction exchanges part of the colloidal gold label immobilized on the surface of the virus, so that the immobilized virus-gold label complex develops color.
- Control substance the new coronavirus S protein, which can be obtained through commercialization, and is immobilized on the C-line area of the detection pad, and the colloidal gold label is immobilized by recognizing the sialic acid ligand bound to the surface of the free colloidal gold label to develop color.
- the present invention provides a colloidal gold method similar to the double-antibody sandwich method to detect the new coronavirus particles.
- the gold label is recognized and bound by the protein-sialic acid ligand on the surface to form a virus-gold label complex.
- the virus-gold label complex is recognized and bound to the remaining S protein on the surface of the virus particle under the action of the sialic acid ligand immobilizer. , or exchange part of the colloidal gold label on the virus-gold-labeled complex, so as to fix the virus-gold-labeled complex, and determine the detection result by color change, as shown in Figure 8.
- the positive detection process when the new coronavirus of the sample to be tested flows through the gold-labeled pad, the new coronavirus recognizes and binds to the sialic acid ligand on the surface of the colloidal gold-labeled surface through the surface S protein to form a labeling complex.
- T line Test line
- the remaining S protein on the surface of the new coronavirus recognizes and binds to the immobilized sialic acid ligand, or through a ligand exchange reaction, it is partially immobilized on the surface of the virus particle.
- the gold label is exchanged, and the virus-gold label complex is immobilized, thereby showing red on the T line.
- Control line, C line Control line, C line
- the controlled substance is bound and immobilized, so that the Red on line C;
- the sample to be tested does not contain the new coronavirus, and the labeled complex cannot be formed when the sample flows through the gold labeling pad.
- the immobilized sialic acid ligand cannot immobilize the colloidal gold label, so No red is displayed on the T line, and when the sample continues to flow through the C line, the colloidal gold label is bound and fixed by the control substance, thus showing red on the C line;
- Invalid test result The C line does not show color, and whether the T line shows red or not, it is an invalid result, and another test strip should be taken to test again.
- the compound of formula 1-4 (15.2 g, 22.83 mmol) was dissolved in dichloromethane (60 mL), triethylamine (4.61 g, 45.66 mmol) was added, p-toluenesulfonyl chloride (6.52 g, 34.25 mmol) was added in batches, and the mixture was stirred overnight , TLC detected that the reaction was complete, methanol was added, and after stirring for 10 minutes, the solvent was evaporated, the residue was dissolved in dichloromethane, the organic phase was washed with brine, dried over anhydrous sodium sulfate, evaporated to remove the solvent, and the residue was dissolved in N,N- Dimethylformamide (20 mL) was added with sodium azide (3.70 g, 57.08 mmol), and the mixture was stirred in an oil bath at 50° C.
- the compound of formula 1-6 (8.4 g, 16.41 mmol) was dissolved in dichloromethane (40 mL), triethylamine (3.23 g, 31.91 mmol) was added, p-toluenesulfonyl chloride (4.56 g, 23.93 mmol) was added in batches, and the mixture was stirred overnight , TLC detected that the reaction was complete, methanol was added, and after stirring for 10 minutes, the solvent was evaporated, the residue was dissolved in dichloromethane, the organic phase was washed with brine, dried over anhydrous sodium sulfate, and after concentration, the residue was dissolved in N,N-dimethylmethane Formamide (50 mL), potassium thioacetate (3.54 g, 30.97 mmol) was added, and the mixture was stirred in an oil bath at 50° C.
- the compound of formula 1-7 (7.6 g, 13.00 mmol) was dissolved in methanol (50 mL), sodium methoxide (0.1 M in MeOH) was added, the pH of the reaction solution was adjusted to 8-10, and stirred at room temperature for 15 minutes. TLC detected that the reaction was complete, and added 732 Form H + cation resin, after stirring for 5 minutes, filter off the resin, and concentrate the filtrate to obtain the compound of formula 1 (6.9 g, 97.8%), ESI-MS m/z calcd for [C 21 H 39 N 2 O 12 S] + (M+H) + : 542.60, found: 542.59.
- the preparation process of the compound of formula 5-1 is the same as the compound of formula 1-4 in the embodiment, wherein only the HOPEG 4 OBn reagent is replaced by HOPEG 3 ONap;
- the compound of formula 5-4 (3.5 g, 5.15 mmol) was dissolved in methanol (40 mL), NaOH aqueous solution (1.5 M, 20 mL) was added dropwise, stirred until the reaction was completed by TLC, the reaction solution was neutralized, and the solvent was evaporated. Column separation to obtain the compound of formula 5 (2.7g, 95.8%), ESI-MS m/z calcd for [C 24 H 38 NO 11 S] + (M+H) + : 548.62, found: 548.62;
- the preparation process of the compound of formula 6 is the same as the preparation process of the compound of formula 1, wherein only the HOPEG 4 OBn reagent is replaced by HOC 5 H 10 OBn, and the Ac 2 O reagent in the last step of the step of preparing the compound of formula 1-4 is replaced by a TsCl reagent That is, ESI-MS m/z calcd for [C 23 H 37 N 2 O 10 S 2 ] + (M+H) + : 565.18, found: 565.17.
- the compound of formula 2-3 (19.3 g, 25.47 mmol) was dissolved in methanol (40 mL), NaOH aqueous solution (1.5 M, 20 mL) was added dropwise, stirred until the reaction was complete by TLC detection, the reaction solution was neutralized, the solvent was evaporated, and the residue was dissolved in 2M NaOH aqueous solution (40 mL) was refluxed in an oil bath at 95°C for 10 hours, the reaction solution was neutralized, and the solvent was evaporated.
- the compound of formula 2-5 (18.1 g, 24.4 mmol) was mixed with methyl acrylate (2.46 g, 29.28 mmol) and cuprous iodide (0.93 g, 4.88 mmol) and dissolved in THF (60 mL), and N,N was added dropwise.
- -Diisopropylethylamine DIPEA (4.73gm 36.6mmol) stirred at room temperature for 1 hour, evaporated the reaction solvent, the residue was dissolved in dichloromethane, successively passed through 1M ammonia water, 1N HCl aq , sat.NaHCO 3a.q.
- the compound of formula 2-6 (19.6 g, 23.73 mmol) was dissolved in methanol (100 mL), sodium methoxide (0.1 M in MeOH) was added, the pH of the solution was adjusted to 8-10, stirred at room temperature for 30 minutes, and hydrochloric acid was added dropwise to neutralize the reaction solution, The solvent is evaporated to obtain the crude product of formula 2-7, which is directly used in the next step reaction;
- the compound of formula 2-9 (13.1 g, 17.22 mmol) was dissolved in acetonitrile (50 mL), acetone acetal (5.38 g, 51.66 mmol) and a catalytic amount of p-toluenesulfonic acid (0.3 g, 1.72 mmol) were added, and the mixture was heated at 80° C.
- the crude compound of formula 2-12 obtained in the previous step was dissolved in dichloromethane (60 mL), triethylamine (2.88 g, 28.47 mmol) was added, and after stirring for 5 minutes, p-toluenesulfonyl chloride (4.07 g, 21.35 mmol) was added in batches at room temperature.
- the compound of formula 2-13 (8.2g, 11.55mmol) was dissolved in methanol (30mL), NaOH aqueous solution (1.5M, 10mL) was added, stirred at room temperature for 30 minutes, 732 type H + cation resin was added to neutralize the reaction solution, and the resin was filtered off , Pd/C powder (0.1 g) was added to the filtrate, and the reaction was carried out in an atmosphere of 4 atm H2 for 2 hours, the Pd/C powder was filtered off, and the filtrate was concentrated to obtain the compound of formula 2 (7.1 g, 90.0%), ESI-MS m /z calcd for [C 29 H 44 N 5 O 13 ] + (M+H) + : 670.69, found: 670.68.
- the preparation process of the compound of formula 7-1 is the same as that of the compound of formula 2-4, except that the HOPEG 4 OBn reagent is replaced by HOPEG 3 OBn;
- the process of preparing the compound of formula 7-3 from the compound of formula 7-2 is the same as the process of preparing the compound of formula 1-5 from the compound of formula 1-4, wherein only the Ac 2 O reagent is replaced by TsCl;
- the procedure for preparing the compound of formula 7 from the compound of formula 7-3 is the same as the procedure for preparing the compound of formula 2 from the compound of formula 2-9.
- the preparation process of the compound of formula 8-1 is the same as that of the compound of formula 2-4, except that the HOPEG 4 OBn reagent is replaced with HOC 5 H 10 OAll;
- the process of preparing the compound of formula 8-2 from the compound of formula 8-1 is the same as the process of preparing the compound of formula 7-2 from the compound of formula 7-1, except that the BzCl reagent is replaced by benzyloxycarbonyl chloride CbzCl;
- the process of preparing the compound of formula 8-3 from the compound of formula 8-2 is the same as the process of preparing the compound of formula 5-2 from the compound of formula 5-1;
- the process of preparing the compound of formula 8-4 from the compound of formula 8-3 is the same as the process of preparing the compound of formula 7-4 from the compound of formula 7-3;
- the compound of formula 8-5 (10.1 g, 13.26 mmol) was dissolved in methanol (100 mL), TsOH (114 mg, 0.66 mmol) was added, and the reaction solution was placed in an oil bath at 80 °C for reflux reaction for 12 hours. TLC detected that the reaction was complete, and the reaction solution was cooled. To room temperature, triethylamine was added to neutralize the reaction solution to neutrality, palladium chloride (4.7 g, 26.51 mmol) was added, and the reaction was carried out at room temperature for 1 hour. TLC detected that the reaction was complete. The solvent was evaporated and the residue was dissolved in dichloromethane.
- the process of preparing the compound of formula 8-7 from the compound of formula 8-6 is the same as the process of preparing the compound of formula 7-7 from the compound of formula 7-6;
- the compound of formula 8-7 (4 g, 5.66 mmol) was dissolved in methanol (40 mL), NaOH aqueous solution (1 M, 20 mL) was added dropwise, stirred until the reaction was completed by TLC, the reaction solution was neutralized, the solvent was evaporated, and the residue was dissolved in tetrahydrofuran ( 40mL), Ph3P (4.45g, 16.98mmol) was added, stirred at room temperature for 12 hours, TLC detected that the reaction was complete, the solvent was evaporated, and the residue was directly purified by column chromatography to obtain the compound of formula 8 (3.2g, 84.8%), ESI- MS m/z calcd for [C 36 H 47 N 2 O 10 ] + (M+H) + : 667.77, found: 667.76;
- the preparation method of gold nanoparticle solution with stable sodium citrate and particle size of 30nm is as follows: take 100mL of 1mM HAuCl4 aqueous solution into a two - necked flask, heat it to 100°C, and add sodium citrate (dihydrate) (118mg, 0.3mmol) , After stirring for 15 minutes, take it out and slowly lower it to room temperature.
- PEG 4 -SH mercaptotetraethylene glycol
- the obtained compound of formula 4-1 (1.1g, 1.36mmol) was mixed with bovine serum albumin BSA (66.43kDa, 3g, 45umol) in water, and allowed to stand for 24h.
- the reaction solution was purified by gel column to obtain the compound of formula 4 (3.8 g), dissolved in ultrapure water to make a 1nM solution.
- the length x width are respectively 2cm x 2cm, 1cm x 2cm, 6cm x 2cm, and 1cm x 2cm.
- the sialic acid-gold nanoparticle-labeled complex prepared in Example 7 The 3 solution (10 ⁇ L) was sprayed on the gold-labeled pad, and the sialic acid ligand immobilizer 4 solution (5 ⁇ L) prepared in Example 8 was sprayed on the T line (0.1 ⁇ 2 cm) area of the detection pad to commercialize the new coronavirus S protein.
- the solution (1nM, 5 ⁇ L) was sprayed on the C-line (0.1 ⁇ 2cm) area of the detection pad as the control substance, and the absorbent paper was used as the absorbent pad.
- the layers and positions shown are pasted on the PVC liner, and each layer is overlapped by 0.5cm to prepare a new coronavirus detection test strip.
- the 2019-nCoV is highly pathogenic and infectious.
- the detection experiment was carried out by using 2019-nCoV simulated particles.
- the commercialized 2019-nCoV S protein was immobilized on the surface of SiO 2 nanospheres with a diameter of about 100nm to construct 2019-nCoV simulative particles.
- the T line of the kit is red
- the C line is red.
- test strip A and test strip B Take two new coronavirus detection test strips, numbered test strip A and test strip B, respectively, take the saliva of healthy people, and mark them as saliva A and saliva B respectively, and add saliva A to the new crown prepared in Example 10.
- Virus simulation particle solution add deionized water to saliva B, take 1 drop (about 50 ⁇ L) of the two samples respectively, drop them into the sample holes of test strip A and test strip B respectively, and let stand horizontally for 15 minutes.
- the T line is red, the C line is colored, the T line of the test strip B is not colored, and the C line is colored.
- the embodiment of the present invention provides a test strip that can directly detect new coronavirus particles, which can quickly and conveniently determine whether the sample contains virus particles, and then test whether the subject is infectious. judge quickly.
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Abstract
Bandelette réactive de détection de nouveau coronavirus, et son procédé de préparation et d'utilisation La bandelette réactive comprend une plaque de revêtement (1), et un tampon d'échantillon (2), un tampon de marquage d'or (3), un tampon de détection (4) et une plaque d'absorption d'eau (5), qui sont disposés de manière séquentielle sur la plaque de revêtement (1) d'une extrémité à l'autre extrémité. Une membrane de nitrocellulose est disposée sur la surface du tampon de détection (4), et une ligne T de détection et une ligne C de contrôle de qualité sont disposées sur la membrane de nitrocellulose, la ligne T de détection étant revêtue d'une substance d'immobilisation de ligand d'acide sialique, qui comprend une molécule de ligand d'acide sialique ayant une affinité élevée ; et la ligne C de contrôle de qualité est revêtue d'une substance de contrôle, qui est la protéine du SARS-CoV-2. Une étiquette d'or colloïdal est pulvérisée sur le tampon de marquage d'or (3), et l'étiquette d'or colloïdal est une nanoparticule d'or ayant une molécule de ligand d'acide sialique ayant une affinité modérée immobilisée sur la surface. La bandelette réactive peut être utilisée pour déterminer rapidement et commodément si un échantillon contient des particules de SARS-CoV-2, ce qui permet de déterminer rapidement si un sujet est contagieux.
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WO2023239906A3 (fr) * | 2022-06-10 | 2024-01-18 | Eisai R&D Management Co., Ltd. | Dérivés d'acide sialique et leurs méthodes d'utilisation |
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