WO2022094912A1 - Biological probe, manufacturing method therefor, and application thereof - Google Patents

Biological probe, manufacturing method therefor, and application thereof Download PDF

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WO2022094912A1
WO2022094912A1 PCT/CN2020/127031 CN2020127031W WO2022094912A1 WO 2022094912 A1 WO2022094912 A1 WO 2022094912A1 CN 2020127031 W CN2020127031 W CN 2020127031W WO 2022094912 A1 WO2022094912 A1 WO 2022094912A1
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solution
group
gold
sialic acid
substituted
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PCT/CN2020/127031
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French (fr)
Chinese (zh)
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李伟
王怀雨
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深圳先进技术研究院
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Publication of WO2022094912A1 publication Critical patent/WO2022094912A1/en

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    • 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

Definitions

  • the invention belongs to the technical field of biochemistry, and relates to a biological probe and a preparation method and application thereof.
  • the pathogen causing new coronary pneumonia is a new type of coronavirus (referred to as "new coronavirus").
  • the detection methods of 2019-nCoV mainly include nucleic acid RNA detection and 2019-nCoV IgG/IgM antibody detection.
  • the former extracts nucleic acid substances from the sample to be tested and uses polymerase chain reaction (PCR) to amplify the RNA of the sample to be tested.
  • PCR polymerase chain reaction
  • the real-time quantitative PCR technology is generally used for detection; the latter uses that after a person is infected with the new coronavirus, the immune system will react to produce corresponding IgG and IgM antibodies, by detecting the new coronavirus in human blood.
  • Viral IgG and IgM are used to indirectly indicate whether the subject has a new coronavirus infection, and the colloidal gold method is generally used to detect these two antibodies.
  • Nucleic acid RNA detection generally uses RT-PCR technology to directly detect whether there is new coronavirus RNA in the sample, but 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 storage, manual operation, etc., often accompanied by false negative interference , and a small number of people with positive RNA test results did not show infectivity, so there was also false positive interference;
  • the new coronavirus IgG/IgM antibody test is an indirect detection method, by detecting whether there is a new coronavirus IgG/IgM antibody in the blood. To indirectly characterize whether the subject is infected with the new coronavirus, but it cannot indicate whether there are new coronavirus particles in the subject in real time, so it is impossible to judge whether the subject is infectious.
  • the purpose of the present invention is to provide a biological probe and its preparation method and application.
  • the present invention provides a biological probe comprising a probe carrier and a customized sialic acid ligand molecule connected to the probe carrier;
  • R 1 is selected from hydroxyl, methoxy or substituted methoxy
  • R 2 is selected from acetamido, substituted acetamido, benzamido, substituted benzoyl, alkoxycarbonamido, triazole or substituted triazolyl
  • R is selected from hydroxyl, methoxy, substituted methoxy, acetamido, substituted acetamido, sulfonamide or phosphoramido
  • A is n is selected from 0, 1, 2, 3, 4, 5, 6, 7, and m is selected from 2, 3, 4, 5, 6, 7, 8, 9.
  • the customized sialic acid ligand molecule can be further matched to occupy the sugar-binding domain pocket of the new coronavirus S protein through the derivatization of R 1 , R 2 and R 3 groups, and has high specificity and binding force to the new coronavirus S protein.
  • 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 at least one of halogen atoms, methyl groups, methoxy groups, and nitro groups.
  • 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 3 -Ph-(CH 2 ) n1 -) or substituted carbonyl (X 6 -C(O)-), wherein X 3 is at least 1 in number and located in the benzene Any position of the ring, X 3 is selected from at least one of halogen atom, methyl group, methoxy group and nitro group, n1 is selected from 0, 1, 2; X 6 is selected from alkoxy group CH 3 -(CH 2 ) n2 -O- or alkylamino CH3- ( CH2 ) n2 -NH-, n2 is selected from 0,1,2,3,4,5.
  • the probe carrier is selected from nanomaterials with sensitive LSPR effect or substances with obvious signal changes in a dispersed-aggregated state;
  • the nanomaterials with sensitive LSPR effect include gold-based nanoparticles and composite nanoparticles with a metallic gold layer on the surface; more preferably, the gold-based nanoparticles include gold nanorods and gold nanospheres;
  • the substance with obvious signal change in the dispersion-aggregation state comprises an aggregation-induced luminescence AIE material;
  • the aggregation-induced luminescence AIE material comprises a tetrathienylthiophene compound with a cyclic polyene skeleton, a cyano-substituted dithiophene Compounds of styrene skeleton, tetrastyrene-type compounds, divinylanthracene-type compounds, tristyryl-type compounds, etc.
  • the molecular end of the aggregation-induced luminescence AIE material has a maleimide-derived group that is reactively linked with a sulfhydryl group.
  • the probe carrier is a gold nanorod
  • the surface of the gold nanorods is coated with a bilayer of cetyltrimethylammonium bromide
  • the gold nanorods have a diameter of 20-25 nm, a length of 60-80 nm, and an aspect ratio of 2.5:1 to 3.5:1.
  • the molar ratio of the customized sialic acid ligand molecules to the gold nanorods is 1 ⁇ 10 4 :1 ⁇ 2 ⁇ 10 4 :1.
  • the present invention provides a preparation method of any of the above-mentioned biological probes, comprising the following steps:
  • the probe carrier solution is a solution of gold nanorods, and the surface of the gold nanorods is coated with a bilayer of cetyltrimethylammonium bromide; the diameter of the gold nanorods is 20-25 nm, and the length is 60-80nm; customized sialic acid ligand molecules are immobilized on the surface of gold nanorods by forming S-Au bonds on the surface of gold nanorods;
  • the molar ratio of the customized sialic acid ligand molecules to the gold nanorods is 1 ⁇ 10 4 :1 ⁇ 2 ⁇ 10 4 :1.
  • the preparation method of the gold nanorod solution is as follows: preparing gold nanorods by a gold seed growth method in an aqueous phase, first reducing chloroauric acid with sodium borohydride to prepare a 1-2 nm gold nanoseed solution; The gold nanoseed solution was added to the mixture of chloroauric acid HAuC1 4 solution, silver nitrate AgNO 3 solution, dilute hydrochloric acid HCl, ascorbic acid solution, and cetyltrimethylammonium bromide CTAB solution. ⁇ 25nm, 60 ⁇ 80nm long, gold nanorod solution with surface covered by cetyltrimethylammonium bromide;
  • the molar ratio of the sodium borohydride and the chloroauric acid is 2:1 to 3:1, the time for the sodium borohydride to reduce the chloroauric acid is 5 to 20 minutes, and the sodium borohydride to reduce the chloroauric acid.
  • the temperature is 25 ⁇ 35 °C;
  • the concentration of the chloroauric acid HAuC1 4 solution is 4.5-5.5 mM, preferably 5 mM, the concentration of the silver nitrate AgNO 3 solution is 0.09-0.12 M, preferably 0.1 M, and the concentration of the dilute hydrochloric acid HCl is 1.0- 1.5mM, preferably 1.2mM, the concentration of the ascorbic acid solution is 8-15mM, preferably 10mM, the concentration of the cetyltrimethylammonium bromide CTAB solution is 0.15-0.25M; the gold nanoseeds
  • the volume ratio of solution, chloroauric acid solution, silver nitrate solution, dilute hydrochloric acid, ascorbic acid solution and cetyltrimethylammonium bromide solution is 45 ⁇ 55 ⁇ l: 5.5 ⁇ 6.5mL: 70 ⁇ 80 ⁇ L: 70 ⁇ 80 ⁇ L: 3.6 ⁇ 3.8mL: 25 ⁇ 35mL, the temperature of the standing reaction is 25 ⁇ 40°C, and the time of the standing reaction is 6 ⁇ 24
  • sialic acid As the starting material, through the protection of the carboxyl group, the R4 protecting group is introduced into the carboxyl group to obtain Generation of peracetylated 2-chlorosugars 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 According to the sequence of R 2 ⁇ R 1 ⁇ R 3 , the introduction of groups is carried out to obtain Finally, through the conversion of side chain groups and the removal of sugar ring protecting groups, the customized sialic acid sugar ligand I was synthesized;
  • the R4 protecting group is introduced into the carboxyl group to obtain Generation of peracetylated 2-chlorosugars in the next step of chlorination and acetylation reagents
  • the functional side chain precursor is derived from the glycosylation reaction at the 2-position of the sugar ring
  • an intermediate with all the exposed amino and hydroxyl groups on the sugar ring is obtained
  • the introduction of groups is carried out to obtain
  • the custom sialic acid sugar ligand I was synthesized through the conversion of side chain groups and the removal of sugar ring protecting groups;
  • R is selected from methyl or benzyl ;
  • a 1 is selected from or -(CH 2 ) m -R 5 ;
  • R 5 is selected from benzyloxy, 2-naphthylmethoxy or allyloxy, n is 0, 1, 2, 3, 4, 5, 6, 7, m are 2,3,4,5,6,7,8,9;
  • PG is the abbreviation of protection group protection group
  • R1 when R1 is hydroxyl, it is sialic acid
  • the group on the molecule does not need to introduce the relevant reaction process of the R1 group;
  • R2 is acetamido, it is sialic acid
  • the group on the molecule does not need to introduce the relevant reaction process of the R2 group;
  • R3 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 an application of any of the above-mentioned biological probes in a novel coronavirus detection reagent.
  • the method of using biological probes to detect the new coronavirus includes the following steps: mixing the biological probes with the sample to be tested, mixing evenly, and then standing for 15-30 minutes, and detecting the new coronavirus by observing the color of the mixed system;
  • the method for using biological probes to detect the new coronavirus specifically includes the following steps: 1) Take two identical aqueous solutions of biological probes and label them as solution C (Control solution, C solution) and solution T (Test solution, T solution), add the control solution to the solution C, add the sample to be tested to the solution T, mix well and let stand for 15-30 minutes to observe the results; 2) Result judgment: positive result: solution C does not change color, solution T changes color; negative Result: solution C does not change color, solution T does not change color; invalid result: solution C changes color, no matter whether solution T changes color or not, another biological probe aqueous solution should be taken for re-testing;
  • the biological probe is a custom sialic acid ligand molecule-gold nanorod biological probe
  • the concentration of the customized sialic acid ligand molecule-gold nanorod biological probe aqueous solution is 10-100 ⁇ M;
  • the volume ratio of the sample to be tested and the customized sialic acid ligand molecule-gold nanorod biological probe aqueous solution is 1:5 to 1:20;
  • the detection limit of the new coronavirus in the sample to be tested is 104 /mL;
  • the sample to be tested is saliva, nasopharyngeal swab, blood, and skin secretions;
  • control solution is deionized water.
  • the present invention provides a biological probe, which can be used to directly detect new coronavirus particles, and has the following beneficial effects compared with the existing new coronavirus and related virus detection methods:
  • the biological probe provided by the present invention directly detects virus particles based on the recognition and binding of sialic acid ligands and S protein on the surface of new coronavirus particles;
  • the biological probe provided by the present invention is used for the detection of the new coronavirus through parallel reactions, and it only takes about 15 minutes to visually judge by color reaction;
  • the biological probe provided by the present invention does not involve multiple procedures such as sample RNA extraction and PCR. It can be directly sampled and then added dropwise, which simplifies the detection structure and avoids manual operation errors. interference caused;
  • the biological probe provided by the present invention is a method for directly detecting new coronavirus particles, which can immediately characterize whether there are new coronavirus particles in the sample;
  • the present invention performs detection by recognizing and combining the sialic acid small molecule with the surface S protein of the new coronavirus, and the sialic acid detection small molecule has obvious advantages such as good economy, high stability, and mass preparation;
  • the present invention provides a biological probe, which improves the recognition and binding of the target protein by modifying the structure of the sialic acid ligand molecule on the surface of the probe. Compared with the natural sugar ligand detection molecule, the detection efficiency is greatly improved. specificity and sensitivity.
  • Fig. 1 is a schematic diagram of the method for detecting new coronavirus particles of the present invention, and the change diagram of the optical signal of the probe LSPR after it becomes a black and white picture is not obvious, wherein the left picture is tea red, and the right picture is dark gray;
  • FIG. 2 is a schematic diagram of a biological probe provided by the present invention.
  • Fig. 3 is the flow chart of the biological probe preparation method provided by the present invention.
  • Fig. 4 is the general preparation flow chart of customizing sialic acid ligand molecule of general formula I of the present invention according to R 2 ⁇ R 1 ⁇ R 3 group introduction sequence;
  • Fig. 5 is the general preparation flow chart of the custom sialic acid ligand molecule of general formula I of the present invention according to the introduction sequence of R 2 ⁇ R 3 ⁇ R 1 groups.
  • the direct detection method of the new coronavirus provided by the present invention is based on the surface Spike protein of the new coronavirus, and its core principle is to use the customized sialic acid ligand molecule to detect the surface S protein of the new coronavirus.
  • the binding effect was detected by recognizing the binding effect of gold nanorod probe, and the binding effect was characterized by the change of the optical absorption signal exhibited by the LSPR effect of the gold nanorod probe.
  • the corresponding detection principles and detection reagents are described in detail as follows:
  • the S protein on the surface of the new coronavirus particles in the sample can recognize the customized sialic acid ligand molecules bound to the gold nanorod probe. Since the surface of the new coronavirus has abundant S protein molecules, and The surface of the gold nanorods also has a multivalent sialic acid ligand structure, so the two are multivalently combined with each other to form an agglomeration structure. Because the gold nanorods have the LSPR effect, they have optical absorption signals, which can change the surface environment. It is very sensitive.
  • the detection reagent involved in the present invention includes a uniformly dispersed aqueous solution of customized sialic acid ligand molecules-gold nanorod biological probes, and S-Au bonds are passed on the surface of the gold nanorods stabilized by cetyltrimethylammonium bromide (CTAB).
  • CTLAB cetyltrimethylammonium bromide
  • a custom sialic acid ligand molecule is immobilized. By introducing the corresponding group on the sugar ring, the sialic acid ligand molecule can better match the sugar binding domain pocket of the S protein on the surface of the new coronavirus, so that it has high specific recognition for the new coronavirus S protein. , high affinity binding effect, the key composition of the detection reagent is shown in Figure 2.
  • the preparation method of the customized sialic acid ligand molecule-gold nanorod biological probe is shown in FIG. 3 ; the preparation flow chart of the customized sialic acid ligand molecule is shown in FIG. 4 or FIG. 5 .
  • 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 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 (8.9 g, 11.98 mmol) was dissolved in methanol (30 mL), an aqueous NaOH solution (1.5 M, 10 mL) was added, stirred for 30 minutes at room temperature, and the reaction solution was neutralized by adding 732 type H + cation resin, and the resin was filtered off. , the filtrate was concentrated to obtain the compound of formula 2 (7.5g, 91.2%), ESI-MS m/z calcd for [C 29 H 43 N 4 O 13 S] + (M+H) + : 687.73, found: 687.72;
  • the preparation process of gold nanorod solution includes: adding chloroauric acid HAuCl 4 aqueous solution (0.5mM, 5 mL) to CTAB aqueous solution (0.2 M, 5 mL), adding freshly prepared sodium borohydride NaBH 4 ice aqueous solution (10 mM) under vigorous stirring , 600 ⁇ L), continue to stir for 10 minutes to prepare gold nanoseed solution; add HAuCl 4 aqueous solution (5 mM, 6 mL), silver nitrate AgNO 3 aqueous solution (0.1 mM, 75 ⁇ L) to CTAB aqueous solution (0.2 M, 30 mL), continue to add Hydrochloric acid (1.2 mM, 75 ⁇ L) and sodium ascorbate Vc-Na aqueous solution (10 mM, 3.7 mL), shake the solution until colorless, add the prepared gold nanoseed solution (50 ⁇ L), stir for 5 minutes, and place in a 37°C incubator Let stand for 16 hours, centrito
  • the preparation process of gold nanorod solution includes: adding chloroauric acid HAuCl 4 aqueous solution (0.5mM, 5 mL) to CTAB aqueous solution (0.2 M, 5 mL), adding freshly prepared sodium borohydride NaBH 4 ice aqueous solution (10 mM) under vigorous stirring , 600 ⁇ L), continue to stir for 10 minutes to prepare gold nanoseed solution; add HAuCl 4 aqueous solution (5 mM, 6 mL), silver nitrate AgNO 3 aqueous solution (0.1 mM, 75 ⁇ L) to CTAB aqueous solution (0.2 M, 30 mL), continue to add Hydrochloric acid (1.2 mM, 75 ⁇ L) and sodium ascorbate Vc-Na aqueous solution (10 mM, 3.7 mL), shake the solution until colorless, add the prepared gold nanoseed solution (50 ⁇ L), stir for 5 minutes, and place in a 37°C incubator Let stand for 16 hours, centrito
  • the new coronavirus particles are highly infectious and highly pathogenic, and the detection of the new coronavirus in the present invention only involves the surface S protein of the new coronavirus, not its internal structure. Therefore, the commercialized new coronavirus S protein can be immobilized on the surface of SiO 2 nanoparticles with a particle size of 100 nm. To simulate the new coronavirus particles, the detection experiments are carried out by simulating the particles.
  • the present invention provides a new coronavirus particle detection method based on the LSPR effect of gold nanorods (Gold nanorods, GNR) probes.
  • Gold nanorods Gold nanorods, GNR
  • Custom sialic acid ligand molecules are immobilized on the surface of gold nanorods, which can efficiently identify and bind to the new coronavirus.
  • the Spike protein on the surface triggers the agglomeration of the dispersed gold nanoprobes, and the change of the dispersion state of the gold nanoprobes can be reflected by the color exhibited by the LSPR effect, thereby realizing the detection of the new coronavirus.

Abstract

Disclosed are a biological probe, a preparation method therefor, and an application thereof. The biological probe comprises a probe carrier and a custom sialic acid ligand molecule connected to the probe carrier; a structural general formula of the custom sialic acid ligand molecule is as shown in formula I; wherein R1 is selected from a hydroxy group, a methoxy group, a or substituted methoxy group; R2 is selected from acetamido group, a substituted acetamido group, a benzoylamino group, a substituted benzoyl group, an alkoxy carbonyl amide group, a triazole group, or a substituted triazole group; R3 is selected from a hydroxy group, a methoxy group, a substituted methoxy group, an acetamido group, a substituted acetamido group, a sulfonamide group, or a phosphamide group; A is (formula a) or (formula b), n is selected from 0,1, 2, 3, 4, 5, 6, or 7, and m is selected from 2, 3, 4, 5, 6, 7, 8, or 9. The present biological probe can be used to directly detect a novel coronavirus particle.

Description

一种生物探针及其制备方法和应用A kind of biological probe and its preparation method and application 技术领域technical field
本发明属于生物化学技术领域，涉及一种生物探针及其制备方法和应用。The invention belongs to the technical field of biochemistry, and relates to a biological probe and a preparation method and application thereof.
背景技术Background technique
[根据细则9.2改正28.12.2020]　
引起新冠肺炎的病原体是新型冠状病毒(简称“新冠病毒”)，对新冠病毒进行检测对新冠肺炎疫情防控具有重要意义。[Correction 28.12.2020 in accordance with Rule 9.2]
The pathogen causing new coronary pneumonia is a new type of coronavirus (referred to as "new coronavirus").
目前新冠病毒的检测方法主要包括核酸RNA检测和新冠病毒IgG/IgM抗体检测，前者通过提取待测样品中核酸物质，利用聚合酶链式反应(polymerase chain reaction,PCR)对待测样本RNA进行扩增来鉴定其是否为新冠病毒RNA，目前一般应用实时荧光定量PCR技术来进行检测；后者则利用人感染新冠病毒后，免疫系统会反应产生相应的IgG和IgM抗体，通过检测人血液中的新冠病毒IgG和IgM来间接指示受试者是否有新冠病毒感染，一般应用胶体金法来检测这两种抗体。At present, the detection methods of 2019-nCoV mainly include nucleic acid RNA detection and 2019-nCoV IgG/IgM antibody detection. The former extracts nucleic acid substances from the sample to be tested and uses polymerase chain reaction (PCR) to amplify the RNA of the sample to be tested. To identify whether it is the new coronavirus RNA, the real-time quantitative PCR technology is generally used for detection; the latter uses that after a person is infected with the new coronavirus, the immune system will react to produce corresponding IgG and IgM antibodies, by detecting the new coronavirus in human blood. Viral IgG and IgM are used to indirectly indicate whether the subject has a new coronavirus infection, and the colloidal gold method is generally used to detect these two antibodies.
核酸RNA检测一般应用RT-PCR技术直接检测样本中是否存在新冠病毒RNA，但是需要人工提取样本中RNA病毒，存在样本取样方法、样本运输保存、人工操作等多重因素的影响，常伴随假阴性干扰，且有少数RNA检测结果复阳者并未表现出传染性，因此也存在假阳性干扰；新冠病毒IgG/IgM抗体检测属于一种间接检测方法，通过检测血液中是否存在新冠病毒IgG/IgM抗体来间接表征受试者是否感染新冠病毒，但并不能实时指征受试者体内是否存在新冠病毒颗粒，从而无法对受试者是否具有传染性做出判断。Nucleic acid RNA detection generally uses RT-PCR technology to directly detect whether there is new coronavirus RNA in the sample, but 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 storage, manual operation, etc., often accompanied by false negative interference , and a small number of people with positive RNA test results did not show infectivity, so there was also false positive interference; the new coronavirus IgG/IgM antibody test is an indirect detection method, by detecting whether there is a new coronavirus IgG/IgM antibody in the blood. To indirectly characterize whether the subject is infected with the new coronavirus, but it cannot indicate whether there are new coronavirus particles in the subject in real time, so it is impossible to judge whether the subject is infectious.
此外，在更广泛的病毒检测方面，也有利用糖-金纳米探针进行病毒检测的报道，但是，这些报道中普遍利用天然唾液酸配体作为检测分子修饰于金纳米颗粒表面来构建探针，而天然唾液酸配体分子对病毒表面蛋白的识别结合属于一种低特异、低亲和的识别结合作用，其检测的特异性和灵敏度不够，目前也尚无应用金纳米探针检测新冠病毒颗粒的报道。In addition, in the wider virus detection, there are also reports on the use of sugar-gold nanoprobes for virus detection. However, in these reports, natural sialic acid ligands are commonly used as detection molecules to modify the surface of gold nanoparticles to construct probes. The recognition and binding of natural sialic acid ligand molecules to virus surface proteins is a low-specificity and low-affinity recognition and binding effect, and its detection specificity and sensitivity are not enough. At present, there is no application of gold nanoprobes to detect new coronavirus particles. 's report.
发明内容SUMMARY OF THE INVENTION
为了解决上述背景技术中所提出的问题，本发明的目的在于提供一种生物探针及其制备方法和应用。In order to solve the problems raised in the above background art, the purpose of the present invention is to provide a biological probe and its preparation method and application.
为达到上述目的，本发明所采用的技术方案为：In order to achieve the above object, the technical scheme adopted in the present invention is:
一方面，本发明提供了一种生物探针，所述生物探针包括探针载体和与探针载体连接的定制唾液酸配体分子；In one aspect, the present invention provides a biological probe comprising a probe carrier and a customized sialic acid ligand molecule connected to the probe carrier;
所述定制唾液酸配体分子的结构通式如式I所示：
Figure PCTCN2020127031-appb-000001
其中，R 1选自羟基、甲氧基或取代的甲氧基；R 2选自乙酰氨基、取代的乙酰氨基、苯甲酰氨基、取代的苯甲酰基、烷氧羰酰胺基、三氮唑基或取代的三氮唑基，R 3选自羟基、甲氧基、取代的甲氧基、乙酰氨基、取代的乙酰氨基、磺酰胺基或磷酰胺基；A为
Figure PCTCN2020127031-appb-000002
Figure PCTCN2020127031-appb-000003
n选自0,1,2,3,4,5,6,7，m选自2,3,4,5,6,7,8,9。该定制唾液酸配体分子可通过R 1，R 2和R 3基团的衍生，进一步匹配占据新冠病毒S蛋白的糖结合域口袋，对新冠病毒S蛋白具有很高的特异性和结合力。 The general structural formula of the customized sialic acid ligand molecule is shown in formula I:
Figure PCTCN2020127031-appb-000001
Wherein, R 1 is selected from hydroxyl, methoxy or substituted methoxy; R 2 is selected from acetamido, substituted acetamido, benzamido, substituted benzoyl, alkoxycarbonamido, triazole or substituted triazolyl, R is selected from hydroxyl, methoxy, substituted methoxy, acetamido, substituted acetamido, sulfonamide or phosphoramido; A is
Figure PCTCN2020127031-appb-000002
Figure PCTCN2020127031-appb-000003
n is selected from 0, 1, 2, 3, 4, 5, 6, 7, and m is selected from 2, 3, 4, 5, 6, 7, 8, 9. The customized sialic acid ligand molecule can be further matched to occupy the sugar-binding domain pocket of the new coronavirus S protein through the derivatization of R 1 , R 2 and R 3 groups, and has high specificity and binding force to the new coronavirus S protein.
进一步地，所述取代的甲氧基为X 1-CH 2O-，其中，X 1选自苯基或乙烯基； Further, the substituted methoxy group is X 1 -CH 2 O-, wherein X 1 is selected from phenyl or vinyl;
所述取代的乙酰氨基为X 2-CH 2CONH-，其中，X 2选自甲基、乙基、正丙基、异丙基、羟甲基或羟乙基； The substituted acetamido is X 2 -CH 2 CONH-, wherein X 2 is selected from methyl, ethyl, n-propyl, isopropyl, hydroxymethyl or hydroxyethyl;
所述取代的苯甲酰氨基为X 3-BzNH-，其中，X 3的数量至少为1且位于苯环的任意位置，X3选自卤原子、甲基、甲氧基、硝基中的至少一种； The substituted benzamido is X 3 -BzNH-, wherein the number of X 3 is at least 1 and is located at any position of the benzene ring, and X 3 is selected from at least one of halogen atoms, methyl groups, methoxy groups, and nitro groups. A sort of;
所述烷氧羰酰胺基为X 4-OC(O)NH-，其中，X 4选自苄基、烯丙基、叔丁基或三氯乙基； The alkoxycarbonamide group is X 4 -OC(O)NH-, wherein X 4 is selected from benzyl, allyl, tert-butyl or trichloroethyl;
所述取代的三氮唑基为
Figure PCTCN2020127031-appb-000004
其中，X 5选自取代的苯烷基(X 3-Ph-(CH 2) n1-)或取代的羰基(X 6-C(O)-)，其中X 3的数量至少为1且位于苯环的任意位置，X 3选自卤原子、甲基、甲氧基、硝基中的至少一种，n1选自0,1,2；X 6选自烷氧基CH 3-(CH 2) n2-O-或烷胺基CH 3-(CH 2) n2-NH-，n2选自0,1,2,3,4,5。 The substituted triazolyl is
Figure PCTCN2020127031-appb-000004
wherein X 5 is selected from substituted phenylalkyl (X 3 -Ph-(CH 2 ) n1 -) or substituted carbonyl (X 6 -C(O)-), wherein X 3 is at least 1 in number and located in the benzene Any position of the ring, X 3 is selected from at least one of halogen atom, methyl group, methoxy group and nitro group, n1 is selected from 0, 1, 2; X 6 is selected from alkoxy group CH 3 -(CH 2 ) n2 -O- or alkylamino CH3- ( CH2 ) n2 -NH-, n2 is selected from 0,1,2,3,4,5.
进一步地，所述探针载体选自具有灵敏LSPR效应的纳米材料或在分散-聚集状态下具有明显信号变化的物质；Further, the probe carrier is selected from nanomaterials with sensitive LSPR effect or substances with obvious signal changes in a dispersed-aggregated state;
优选地，所述具有灵敏LSPR效应的纳米材料包括金基纳米颗粒、表面有金属金层的复合纳米颗粒；更优选地，所述金基纳米颗粒包括金纳米棒、金纳米球；Preferably, the nanomaterials with sensitive LSPR effect include gold-based nanoparticles and composite nanoparticles with a metallic gold layer on the surface; more preferably, the gold-based nanoparticles include gold nanorods and gold nanospheres;
优选地，所述在分散-聚集状态下具有明显信号变化的物质包括聚集诱导发光AIE材料；所述聚集诱导发光AIE材料包括具有环状多烯骨架的四噻吩基噻吩类化合物、具有氰取代二苯乙烯骨架的化合物、四苯乙烯型化合物、二乙烯基蒽型化合物、三苯乙烯型化合物等，所述聚集诱导发光AIE材料分子末端具有同巯基反应连接的马来酰亚胺衍生基团。Preferably, the substance with obvious signal change in the dispersion-aggregation state comprises an aggregation-induced luminescence AIE material; the aggregation-induced luminescence AIE material comprises a tetrathienylthiophene compound with a cyclic polyene skeleton, a cyano-substituted dithiophene Compounds of styrene skeleton, tetrastyrene-type compounds, divinylanthracene-type compounds, tristyryl-type compounds, etc., the molecular end of the aggregation-induced luminescence AIE material has a maleimide-derived group that is reactively linked with a sulfhydryl group.
进一步地，所述探针载体为金纳米棒；Further, the probe carrier is a gold nanorod;
优选地，所述金纳米棒表面包覆有十六烷基三甲基溴化铵双分子层；Preferably, the surface of the gold nanorods is coated with a bilayer of cetyltrimethylammonium bromide;
优选地，所述金纳米棒直径为20-25nm，长为60-80nm，长径比为2.5:1～3.5:1。Preferably, the gold nanorods have a diameter of 20-25 nm, a length of 60-80 nm, and an aspect ratio of 2.5:1 to 3.5:1.
进一步地，所述定制唾液酸配体分子与金纳米棒的摩尔比为1×10 4:1～2×10 4:1。 Further, the molar ratio of the customized sialic acid ligand molecules to the gold nanorods is 1×10 4 :1˜2×10 4 :1.
另一方面，本发明提供了一种上述任一所述的生物探针的制备方法，包括以下步骤：On the other hand, the present invention provides a preparation method of any of the above-mentioned biological probes, comprising the following steps:
1)提供探针载体溶液和具有结构通式I的定制唾液酸配体分子；1) provide a probe carrier solution and a customized sialic acid ligand molecule with general structural formula I;
2)将具有结构通式I的定制唾液酸配体分子配制成水溶液，与探针载体溶液混合，25～30℃静置12-24小时制得生物探针；2) preparing the customized sialic acid ligand molecule with general structural formula I into an aqueous solution, mixing it with the probe carrier solution, and standing at 25-30° C. for 12-24 hours to obtain a biological probe;
优选地，所述探针载体溶液为金纳米棒溶液，所述金纳米棒表面包覆有十六烷基三甲基溴化铵双分子层；所述金纳米棒直径为20-25nm，长为60-80nm；定制唾液酸配体分子通过在金纳米棒表面形成S-Au键而固定于金纳米棒表面；Preferably, the probe carrier solution is a solution of gold nanorods, and the surface of the gold nanorods is coated with a bilayer of cetyltrimethylammonium bromide; the diameter of the gold nanorods is 20-25 nm, and the length is 60-80nm; customized sialic acid ligand molecules are immobilized on the surface of gold nanorods by forming S-Au bonds on the surface of gold nanorods;
优选地，所述定制唾液酸配体分子与金纳米棒的摩尔比为1×10 4:1～2×10 4:1。 Preferably, the molar ratio of the customized sialic acid ligand molecules to the gold nanorods is 1×10 4 :1˜2×10 4 :1.
进一步地，所述金纳米棒溶液的制备方法为：在水相中以金种生长法制备金纳米棒，先用硼氢化钠还原氯金酸，制备1～2nm的金纳米种子溶液；再将金纳米种子溶液加入到氯金酸HAuC1 4溶液、硝酸银AgNO 3溶液、稀盐酸HC1、抗坏血酸溶液、十六烷基三甲基溴化铵CTAB溶液的混合液中静置反应，生成宽为20～25nm、长为60～80nm、表面被十六烷基三甲基溴化铵覆盖的金纳米棒溶液； Further, the preparation method of the gold nanorod solution is as follows: preparing gold nanorods by a gold seed growth method in an aqueous phase, first reducing chloroauric acid with sodium borohydride to prepare a 1-2 nm gold nanoseed solution; The gold nanoseed solution was added to the mixture of chloroauric acid HAuC1 4 solution, silver nitrate AgNO 3 solution, dilute hydrochloric acid HCl, ascorbic acid solution, and cetyltrimethylammonium bromide CTAB solution. ~25nm, 60~80nm long, gold nanorod solution with surface covered by cetyltrimethylammonium bromide;
优选地，所述硼氢化钠和氯金酸的摩尔比为2:1～3:1，所述硼氢化钠还原氯金酸的时间为5～20分钟，所述硼氢化钠还原氯金酸的温度为25～35℃；Preferably, the molar ratio of the sodium borohydride and the chloroauric acid is 2:1 to 3:1, the time for the sodium borohydride to reduce the chloroauric acid is 5 to 20 minutes, and the sodium borohydride to reduce the chloroauric acid. The temperature is 25 ~ 35 ℃;
所述氯金酸HAuC1 4溶液的浓度为4.5～5.5mM，优选为5mM，所述硝酸银AgNO 3溶液的浓度为0.09～0.12M，优选为0.1M，所述稀盐酸HC1的浓度为1.0～1.5mM，优选为1.2mM，所述抗坏血酸溶液的浓度为8～15mM，优选为10mM，所述十六烷基三甲基溴化铵CTAB溶液的浓度为0.15～0.25M；所述金纳米种子溶液、氯金酸溶液、硝酸银溶液、稀盐酸、抗坏血酸溶液、十六烷基三甲基溴化铵溶液的体积比为45～55μl：5.5～6.5mL：70～80μL：70～80μL：3.6～3.8mL：25～35mL，所述静置反应的温度为25～40℃，所述静置反应的时间为6～24小时。 The concentration of the chloroauric acid HAuC1 4 solution is 4.5-5.5 mM, preferably 5 mM, the concentration of the silver nitrate AgNO 3 solution is 0.09-0.12 M, preferably 0.1 M, and the concentration of the dilute hydrochloric acid HCl is 1.0- 1.5mM, preferably 1.2mM, the concentration of the ascorbic acid solution is 8-15mM, preferably 10mM, the concentration of the cetyltrimethylammonium bromide CTAB solution is 0.15-0.25M; the gold nanoseeds The volume ratio of solution, chloroauric acid solution, silver nitrate solution, dilute hydrochloric acid, ascorbic acid solution and cetyltrimethylammonium bromide solution is 45~55μl: 5.5~6.5mL: 70~80μL: 70~80μL: 3.6 ~3.8mL: 25~35mL, the temperature of the standing reaction is 25~40°C, and the time of the standing reaction is 6~24 hours.
进一步地，所述具有结构通式I的定制唾液酸配体分子的制备方法为：Further, the preparation method of the customized sialic acid ligand molecule with general structural formula I is:
以唾液酸
Figure PCTCN2020127031-appb-000005
为起始原料，经羧基保护在羧基引入R 4保护基得到
Figure PCTCN2020127031-appb-000006
在氯代和乙酰化试剂作用下一步生成全乙酰化2-氯代糖
Figure PCTCN2020127031-appb-000007
继而经糖基化反应在糖环2位衍生出功能侧链前体
Figure PCTCN2020127031-appb-000008
随后经脱乙酰基、羧基保护后得到糖环上氨基和羟基全部裸露的中间体
Figure PCTCN2020127031-appb-000009
按照R 2→R 1→R 3的顺序进行基团的引入分别得到
Figure PCTCN2020127031-appb-000010
最后通过侧链基团转换和糖环保护基脱除反应，合成获得定制唾液酸糖配体I； sialic acid
Figure PCTCN2020127031-appb-000005
As the starting material, through the protection of the carboxyl group, the R4 protecting group is introduced into the carboxyl group to obtain
Figure PCTCN2020127031-appb-000006
Generation of peracetylated 2-chlorosugars in the next step of chlorination and acetylation reagents
Figure PCTCN2020127031-appb-000007
Then the functional side chain precursor is derived from the glycosylation reaction at the 2-position of the sugar ring
Figure PCTCN2020127031-appb-000008
After deacetylation and carboxyl protection, an intermediate with all the exposed amino and hydroxyl groups on the sugar ring is obtained
Figure PCTCN2020127031-appb-000009
According to the sequence of R 2 →R 1 →R 3 , the introduction of groups is carried out to obtain
Figure PCTCN2020127031-appb-000010
Finally, through the conversion of side chain groups and the removal of sugar ring protecting groups, the customized sialic acid sugar ligand I was synthesized;
或以唾液酸
Figure PCTCN2020127031-appb-000011
为起始原料，经羧基保护在羧基引入R 4保护基得到
Figure PCTCN2020127031-appb-000012
在氯代和乙酰化试剂作用下一步生成全乙酰化2-氯代糖
Figure PCTCN2020127031-appb-000013
继而经糖基化反应在糖环2位衍生出功能侧链前体
Figure PCTCN2020127031-appb-000014
随后经脱乙酰基、羧基保护后得到糖环上氨基和羟基全部裸露的中间 体
Figure PCTCN2020127031-appb-000015
按照R 2→R 3→R 1的顺序进行基团的引入分别得到
Figure PCTCN2020127031-appb-000016
最后通过侧链基团转换和糖环保护基脱除反应，合成获得定制唾液酸糖配体I； or with sialic acid
Figure PCTCN2020127031-appb-000011
As the starting material, through the protection of the carboxyl group, the R4 protecting group is introduced into the carboxyl group to obtain
Figure PCTCN2020127031-appb-000012
Generation of peracetylated 2-chlorosugars in the next step of chlorination and acetylation reagents
Figure PCTCN2020127031-appb-000013
Then the functional side chain precursor is derived from the glycosylation reaction at the 2-position of the sugar ring
Figure PCTCN2020127031-appb-000014
After deacetylation and carboxyl protection, an intermediate with all the exposed amino and hydroxyl groups on the sugar ring is obtained
Figure PCTCN2020127031-appb-000015
According to the sequence of R 2 →R 3 →R 1 , the introduction of groups is carried out to obtain
Figure PCTCN2020127031-appb-000016
Finally, the custom sialic acid sugar ligand I was synthesized through the conversion of side chain groups and the removal of sugar ring protecting groups;
其中，R 4选自甲基或苄基； wherein, R is selected from methyl or benzyl ;
A 1选自
Figure PCTCN2020127031-appb-000017
或-(CH 2) m-R 5；R 5选自苄氧基、2-萘甲氧基或烯丙氧基，n为0,1,2,3,4,5,6,7，m为2,3,4,5,6,7,8,9； A 1 is selected from
Figure PCTCN2020127031-appb-000017
or -(CH 2 ) m -R 5 ; R 5 is selected from benzyloxy, 2-naphthylmethoxy or allyloxy, n is 0, 1, 2, 3, 4, 5, 6, 7, m are 2,3,4,5,6,7,8,9;
PG是保护基protection group的缩写；PG is the abbreviation of protection group protection group;
特别的，当R1为羟基时，其为唾液酸
Figure PCTCN2020127031-appb-000018
分子上的基团，不需要引入R1基团的相关反应过程； In particular, when R1 is hydroxyl, it is sialic acid
Figure PCTCN2020127031-appb-000018
The group on the molecule does not need to introduce the relevant reaction process of the R1 group;
当R2为乙酰氨基时，其为唾液酸
Figure PCTCN2020127031-appb-000019
分子上的基团，不需要引入R2基团的相关反应过程； When R2 is acetamido, it is sialic acid
Figure PCTCN2020127031-appb-000019
The group on the molecule does not need to introduce the relevant reaction process of the R2 group;
当R3为羟基时，其为唾液酸
Figure PCTCN2020127031-appb-000020
分子上的基团，不需要引入R3基团的相关反应过程。 When R3 is hydroxyl, it is sialic acid
Figure PCTCN2020127031-appb-000020
The group on the molecule does not need to introduce the relevant reaction process of the R3 group.
再一方面，本发明提供了一种上述任一所述的生物探针在新冠病毒检测试剂中的应用。In another aspect, the present invention provides an application of any of the above-mentioned biological probes in a novel coronavirus detection reagent.
进一步地，采用生物探针进行新冠病毒检测的方法，包括以下步骤：将生物探针与待测样品混合，混合均匀后静置15-30分钟，通过观察混合体系的颜色来检测新冠病毒；Further, the method of using biological probes to detect the new coronavirus includes the following steps: mixing the biological probes with the sample to be tested, mixing evenly, and then standing for 15-30 minutes, and detecting the new coronavirus by observing the color of the mixed system;
优选地，采用生物探针进行新冠病毒检测的方法具体包括以下步骤：1)取两份完全相同的生物探针水溶液，分别标记为溶液C(Control solution,C液)和溶液T(Test solution,T液)，溶液C中加入对照液，溶液T中加入待测样品，混合均匀后静置15-30分钟，观察结果；2)结果判定：阳性结果：溶液C不变色，溶液T变色；阴性结果：溶液C不变色，溶液T不变色；无效结果：溶液C变色，无论溶液T变色与否，应另取生物探针水溶液重新检测；Preferably, the method for using biological probes to detect the new coronavirus specifically includes the following steps: 1) Take two identical aqueous solutions of biological probes and label them as solution C (Control solution, C solution) and solution T (Test solution, T solution), add the control solution to the solution C, add the sample to be tested to the solution T, mix well and let stand for 15-30 minutes to observe the results; 2) Result judgment: positive result: solution C does not change color, solution T changes color; negative Result: solution C does not change color, solution T does not change color; invalid result: solution C changes color, no matter whether solution T changes color or not, another biological probe aqueous solution should be taken for re-testing;
优选地，所述生物探针为定制唾液酸配体分子-金纳米棒生物探针；Preferably, the biological probe is a custom sialic acid ligand molecule-gold nanorod biological probe;
优选地，所述定制唾液酸配体分子-金纳米棒生物探针水溶液的浓度为10-100μM；Preferably, the concentration of the customized sialic acid ligand molecule-gold nanorod biological probe aqueous solution is 10-100 μM;
优选地，所述待测样品与定制唾液酸配体分子-金纳米棒生物探针水溶液的体积比为1:5～1:20；Preferably, the volume ratio of the sample to be tested and the customized sialic acid ligand molecule-gold nanorod biological probe aqueous solution is 1:5 to 1:20;
优选地，所述待测样品中新冠病毒的检测极限为10 4个/mL； Preferably, the detection limit of the new coronavirus in the sample to be tested is 104 /mL;
优选地，所述待测样品为唾液，鼻咽拭子，血液，皮肤分泌物；Preferably, the sample to be tested is saliva, nasopharyngeal swab, blood, and skin secretions;
优选地，所述对照液为去离子水。Preferably, the control solution is deionized water.
本发明提供了一种生物探针，该生物探针可以用于直接检测新冠病毒颗粒，与现有的新冠病毒及相关的病毒检测方法相比，具有以下有益效果：The present invention provides a biological probe, which can be used to directly detect new coronavirus particles, and has the following beneficial effects compared with the existing new coronavirus and related virus detection methods:
1)本发明提供的生物探针基于唾液酸配体和新冠病毒颗粒表面S蛋白识别结合来直接检测病毒颗粒；1) The biological probe provided by the present invention directly detects virus particles based on the recognition and binding of sialic acid ligands and S protein on the surface of new coronavirus particles;
2)本发明提供的生物探针用于新冠病毒检测通过平行反应，仅需15分钟左右即可通过颜色反应进行目视判断；2) The biological probe provided by the present invention is used for the detection of the new coronavirus through parallel reactions, and it only takes about 15 minutes to visually judge by color reaction;
3)本发明提供的生物探针用于新冠病毒检测与核酸检测方法相比，不涉及样本RNA提取、PCR等多重程序，直接取样后滴加即可，简化了检测构成，避免了人工操作误差引起的干扰；3) Compared with the nucleic acid detection method, the biological probe provided by the present invention does not involve multiple procedures such as sample RNA extraction and PCR. It can be directly sampled and then added dropwise, which simplifies the detection structure and avoids manual operation errors. interference caused;
4)本发明提供的生物探针用于新冠病毒检测与新冠病毒IgG/IgM检测方法相比，属直接检测新冠病毒颗粒的方法，可即时表征样本中是否存在新冠病毒颗粒；4) Compared with the new coronavirus IgG/IgM detection method, the biological probe provided by the present invention is a method for directly detecting new coronavirus particles, which can immediately characterize whether there are new coronavirus particles in the sample;
5)本发明通过唾液酸小分子与新冠病毒表面S蛋白识别结合来进行检测，唾液酸检测小分子具有经济性好、稳定性高、可大量制备等明显优点；5) The present invention performs detection by recognizing and combining the sialic acid small molecule with the surface S protein of the new coronavirus, and the sialic acid detection small molecule has obvious advantages such as good economy, high stability, and mass preparation;
6)本发明提供了一种生物探针，通过对探针表面唾液酸配体分子进行结构定制修饰来提高对靶蛋白的识别结合，同天然糖配体检测分子相比，大幅提高了检测的特异性和灵敏度。6) The present invention provides a biological probe, which improves the recognition and binding of the target protein by modifying the structure of the sialic acid ligand molecule on the surface of the probe. Compared with the natural sugar ligand detection molecule, the detection efficiency is greatly improved. specificity and sensitivity.
附图说明Description of drawings
图1是本发明检测新冠病毒颗粒的方法原理图，变为黑白图后探针LSPR光学信号变化图不明显，其中左图为茶红色，右图为暗灰色；Fig. 1 is a schematic diagram of the method for detecting new coronavirus particles of the present invention, and the change diagram of the optical signal of the probe LSPR after it becomes a black and white picture is not obvious, wherein the left picture is tea red, and the right picture is dark gray;
图2是本发明提供的生物探针示意图；2 is a schematic diagram of a biological probe provided by the present invention;
图3是本发明提供的生物探针制备方法流程图；Fig. 3 is the flow chart of the biological probe preparation method provided by the present invention;
图4是本发明通式I定制唾液酸配体分子按R 2→R 1→R 3基团引入顺序的通用制备流程图； Fig. 4 is the general preparation flow chart of customizing sialic acid ligand molecule of general formula I of the present invention according to R 2 →R 1 →R 3 group introduction sequence;
图5是本发明通式I定制唾液酸配体分子按R 2→R 3→R 1基团引入顺序的通用制备流程图。 Fig. 5 is the general preparation flow chart of the custom sialic acid ligand molecule of general formula I of the present invention according to the introduction sequence of R 2 →R 3 →R 1 groups.
具体实施方式Detailed ways
以定制唾液酸配体分子-金纳米棒生物探针为例，本发明提供的新冠病毒直接检测方法基于新冠病毒表面Spike蛋白，其核心原理在于利用定制唾液酸配体分子对新冠病毒表面S蛋白的识别结合作用来进行检测，并通过金纳米棒探针的LSPR效应所表现的光学吸收信号变化来表征该结合作用。相应的检测原理、检测试剂详细描述如下：Taking the customized sialic acid ligand molecule-gold nanorod biological probe as an example, the direct detection method of the new coronavirus provided by the present invention is based on the surface Spike protein of the new coronavirus, and its core principle is to use the customized sialic acid ligand molecule to detect the surface S protein of the new coronavirus. The binding effect was detected by recognizing the binding effect of gold nanorod probe, and the binding effect was characterized by the change of the optical absorption signal exhibited by the LSPR effect of the gold nanorod probe. The corresponding detection principles and detection reagents are described in detail as follows:
检测原理及过程：Detection principle and process:
待测样品同分散的生物探针混合后，样本中的新冠病毒颗粒表面的S蛋白可识别结合金纳米棒探针的定制唾液酸配体分子，由于新冠病毒表面有丰富的S蛋白分子，而金纳米棒表面同样有多价的唾液酸配体结构，因此二者间进行相互的多价结合，从而形成团聚结构，由于金纳米棒具有LSPR效应，其具有光学吸收信号，对表面环境的改变非常敏感，当检测试剂中金纳米棒探针由均匀分散的状态转变为团聚状态，金纳米棒的LSPR效应所表现出的光学吸收发生明显变化，甚至可通过目视判断该变化来判断检测结果，如图1所示。After the sample to be tested is mixed with the dispersed biological probes, the S protein on the surface of the new coronavirus particles in the sample can recognize the customized sialic acid ligand molecules bound to the gold nanorod probe. Since the surface of the new coronavirus has abundant S protein molecules, and The surface of the gold nanorods also has a multivalent sialic acid ligand structure, so the two are multivalently combined with each other to form an agglomeration structure. Because the gold nanorods have the LSPR effect, they have optical absorption signals, which can change the surface environment. It is very sensitive. When the gold nanorod probe in the detection reagent changes from a uniformly dispersed state to an agglomerated state, the optical absorption exhibited by the LSPR effect of gold nanorods changes significantly, and the detection result can even be judged by visually judging the change. ,As shown in Figure 1.
检测试剂：Detection reagents:
本发明涉及的检测试剂有均匀分散的定制唾液酸配体分子-金纳米棒生物探针水溶液，在十六烷基三甲基溴化铵(CTAB)稳定的金纳米棒表面通过S-Au键固定有定制唾液酸配体分子，唾液酸配体分子通过在糖环上引入相应基团，能更好的匹配新冠病毒表面S蛋白的糖结合域口袋，从而对新冠病毒S蛋白具有高特异识别、高亲和结合作用，检测试剂的关键组成如图2所示。The detection reagent involved in the present invention includes a uniformly dispersed aqueous solution of customized sialic acid ligand molecules-gold nanorod biological probes, and S-Au bonds are passed on the surface of the gold nanorods stabilized by cetyltrimethylammonium bromide (CTAB). A custom sialic acid ligand molecule is immobilized. By introducing the corresponding group on the sugar ring, the sialic acid ligand molecule can better match the sugar binding domain pocket of the S protein on the surface of the new coronavirus, so that it has high specific recognition for the new coronavirus S protein. , high affinity binding effect, the key composition of the detection reagent is shown in Figure 2.
其中定制唾液酸配体分子-金纳米棒生物探针的制备方法如图3所示；定制唾液酸配体分子的制备流程图如图4或图5所示。The preparation method of the customized sialic acid ligand molecule-gold nanorod biological probe is shown in FIG. 3 ; the preparation flow chart of the customized sialic acid ligand molecule is shown in FIG. 4 or FIG. 5 .
为了更好地理解本发明的内容，下面结合具体实施方法对本发明内容作进一步说明，但本发明的保护内容不局限以下实施例。In order to better understand the content of the present invention, the content of the present invention will be further described below in conjunction with specific implementation methods, but the protection content of the present invention is not limited to the following examples.
实施例1：定制唾液酸配体分子1的合成Example 1: Synthesis of custom sialic acid ligand molecule 1
Figure PCTCN2020127031-appb-000021
Figure PCTCN2020127031-appb-000021
天然唾液酸Neu5Ac(10g,32.3mmol)溶于乙醇(100mL)，加入碳酸钾(5.36g,38.8mmol)，搅拌15分钟后，滴入溴苄(6.08g,35.6mmol)，于50℃中搅拌2小时，TLC检测反应完全，旋干反应液，残余物经加甲苯旋蒸三次，抽干，得式1-1中间体粗品，直接用于下步反应；Natural sialic acid Neu5Ac (10 g, 32.3 mmol) was dissolved in ethanol (100 mL), potassium carbonate (5.36 g, 38.8 mmol) was added, and after stirring for 15 minutes, benzyl bromide (6.08 g, 35.6 mmol) was added dropwise, and the mixture was stirred at 50 °C After 2 hours, TLC detected that the reaction was complete, the reaction solution was spin-dried, the residue was spin-evaporated three times with toluene, and drained to obtain the crude intermediate of formula 1-1, which was directly used in the next step reaction;
上步所得式1-1粗品溶于氯乙酰(20mL)，室温下搅拌至反应完全，蒸除溶剂，得式1-2粗品，直接用于下步反应；The crude product of formula 1-1 obtained in the previous step was dissolved in chloroacetyl (20 mL), stirred at room temperature until the reaction was complete, and the solvent was evaporated to obtain the crude product of formula 1-2, which was directly used in the next step reaction;
上步所得式1-2粗品与HOPEG 4OBn(13.79g,48.5mmol)混于干燥二氯甲烷(60mL)，冰浴下加入碳酸银(17.83g,64.7mmol)，反应10h，经TLC检测反应完全，滤除不溶物，残余物浓缩拌样柱分离，得式1-3化合物(19.3g,71.6％in 3 steps),ESI-MS m/z calcd for[C 41H 56NO 17] +(M+H) +:834.88,found:834.88； The crude product of formula 1-2 obtained in the previous step was mixed with HOPEG 4 OBn (13.79 g, 48.5 mmol) in dry dichloromethane (60 mL), and silver carbonate (17.83 g, 64.7 mmol) was added under an ice bath to react for 10 h, and the reaction was detected by TLC. Complete, insoluble matter was filtered off, and the residue was concentrated and separated with a sample column to obtain the compound of formula 1-3 (19.3 g, 71.6% in 3 steps), ESI-MS m/z calcd for [C 41 H 56 NO 17 ] + ( M+H) + :834.88,found:834.88;
式1-3化合物(19.3g,23.14mmol)溶于甲醇(60mL)，加入甲醇钠(0.1M)调节反应液pH 8～10，搅拌30min，TLC检测反应完全，中和反应液至中性，蒸除溶剂，残余物加入甲苯旋蒸三次后，即得化合物1-4(15.2g,98.7％),ESI-MS m/z calcd for[C 33H 48NO 13] +(M+H) +:666.73,found:666.72； The compound of formula 1-3 (19.3 g, 23.14 mmol) was dissolved in methanol (60 mL), sodium methoxide (0.1 M) was added to adjust the pH of the reaction solution to 8-10, stirred for 30 min, TLC detected that the reaction was complete, and the reaction solution was neutralized to neutrality, The solvent was evaporated, and the residue was added to toluene for rotary evaporation three times to obtain compound 1-4 (15.2g, 98.7%), ESI-MS m/z calcd for [C 33 H 48 NO 13 ] + (M+H) + :666.73,found:666.72;
式1-4化合物(15.2g,22.83mmol)溶于二氯甲烷(60mL)，加入三乙胺(4.61g,45.66mmol)，分批加入对甲苯磺酰氯(6.52g,34.25mmol)，搅拌过夜，TLC检测反应完全，加入甲醇，搅拌10分钟后，蒸除溶剂，残余物溶于二氯甲烷，有机相经盐水洗涤，无水硫酸钠干燥，蒸除溶剂，残余物溶于N,N-二甲基甲酰胺(20mL)，加入叠氮化钠(3.70g,57.08mmol)，于50℃油浴中搅拌12小时，TLC检测反应完全，蒸除溶剂，残余物溶于四氢呋喃(40mL)，加入三苯基膦(10.48g,39.95mmol)，至无气泡产生后继续搅拌15分钟，反应液直接蒸除溶剂，残余物溶于乙醇(20mL)， 滴入乙酸酐(3.03g,29.68mmol)，搅拌12小时后，蒸除溶剂，残余物直接拌样柱分离，得化合物1-5(11.6g,71.9％),ESI-MS m/z calcd for[C 35H 51N 2O 13] +(M+H) +:707.79,found:707.80； The compound of formula 1-4 (15.2 g, 22.83 mmol) was dissolved in dichloromethane (60 mL), triethylamine (4.61 g, 45.66 mmol) was added, p-toluenesulfonyl chloride (6.52 g, 34.25 mmol) was added in batches, and the mixture was stirred overnight , TLC detected that the reaction was complete, methanol was added, and after stirring for 10 minutes, the solvent was evaporated, the residue was dissolved in dichloromethane, the organic phase was washed with brine, dried over anhydrous sodium sulfate, evaporated to remove the solvent, and the residue was dissolved in N,N- Dimethylformamide (20 mL) was added with sodium azide (3.70 g, 57.08 mmol), and the mixture was stirred in an oil bath at 50° C. for 12 hours. TLC detected that the reaction was complete. The solvent was evaporated and the residue was dissolved in tetrahydrofuran (40 mL). Triphenylphosphine (10.48 g, 39.95 mmol) was added, and stirring was continued for 15 minutes until no bubbles were generated. The reaction solution was directly evaporated to remove the solvent, the residue was dissolved in ethanol (20 mL), and acetic anhydride (3.03 g, 29.68 mmol) was added dropwise. , after stirring for 12 hours, the solvent was evaporated, and the residue was directly separated with a sample column to obtain compound 1-5 (11.6 g, 71.9%), ESI-MS m/z calcd for [C 35 H 51 N 2 O 13 ] + (M+H) + :707.79,found:707.80;
式1-5化合物(11.6g,16.41mmol)溶于甲醇(40mL)，加入Pd/C催化剂(0.1g)，在4atm压力H 2氛围中搅拌6小时，TLC检测反应完全，滤除Pd/C粉末，滤液浓缩，得式1-6化合物(8.4g,97.2％),ESI-MS m/z calcd for[C 21H 39N 2O 13] +(M+H) +:527.54,found:527.54； The compound of formula 1-5 (11.6 g, 16.41 mmol) was dissolved in methanol (40 mL), Pd/C catalyst (0.1 g) was added, and the mixture was stirred at 4 atm pressure H atmosphere for 6 hours. TLC detected that the reaction was complete, and the Pd/C was filtered off. Powder, the filtrate was concentrated to obtain the compound of formula 1-6 (8.4g, 97.2%), ESI-MS m/z calcd for [C 21 H 39 N 2 O 13 ] + (M+H) + : 527.54, found: 527.54 ;
式1-6化合物(8.4g,16.41mmol)溶于二氯甲烷(40mL)，加入三乙胺(3.23g,31.91mmol)，分批加入对甲苯磺酰氯(4.56g,23.93mmol)，搅拌过夜，TLC检测反应完全，加入甲醇，搅拌10分钟后，蒸除溶剂，残余物溶于二氯甲烷，有机相经盐水洗涤，无水硫酸钠干燥，浓缩后残余物溶于N,N-二甲基甲酰胺(50mL)，加入硫代乙酸钾(3.54g,30.97mmol)，于50℃油浴中搅拌12小时，TLC检测反应完全，蒸除溶剂，残余物经柱层析分离纯化，得式1-7化合物(7.6g,81.5％),ESI-MS m/z calcd for[C 23H 41N 2O 13S] +(M+H) +:585.63,found:585.62； The compound of formula 1-6 (8.4 g, 16.41 mmol) was dissolved in dichloromethane (40 mL), triethylamine (3.23 g, 31.91 mmol) was added, p-toluenesulfonyl chloride (4.56 g, 23.93 mmol) was added in batches, and the mixture was stirred overnight , TLC detected that the reaction was complete, methanol was added, and after stirring for 10 minutes, the solvent was evaporated, the residue was dissolved in dichloromethane, the organic phase was washed with brine, dried over anhydrous sodium sulfate, and after concentration, the residue was dissolved in N,N-dimethylmethane Formamide (50 mL), potassium thioacetate (3.54 g, 30.97 mmol) was added, and the mixture was stirred in an oil bath at 50° C. for 12 hours. TLC detected that the reaction was complete, the solvent was evaporated, and the residue was separated and purified by column chromatography to obtain the formula Compound 1-7 (7.6 g, 81.5%), ESI-MS m/z calcd for [C 23 H 41 N 2 O 13 S] + (M+H) + : 585.63, found: 585.62;
式1-7化合物(7.6g,13.00mmol)溶于甲醇(50mL)，加入甲醇钠(0.1M in MeOH)，调节反应液pH 8～10，室温下搅拌15分钟，TLC检测反应完全，加入732型H +阳离子树脂，搅拌5分钟后，滤除树脂，浓缩滤液，得式1化合物(6.9g，97.8％)，ESI-MS m/z calcd for[C 21H 39N 2O 12S] +(M+H) +:542.60,found:542.59. The compound of formula 1-7 (7.6 g, 13.00 mmol) was dissolved in methanol (50 mL), sodium methoxide (0.1 M in MeOH) was added, the pH of the reaction solution was adjusted to 8-10, and stirred at room temperature for 15 minutes. TLC detected that the reaction was complete, and added 732 Form H + cation resin, after stirring for 5 minutes, filter off the resin, and concentrate the filtrate to obtain the compound of formula 1 (6.9 g, 97.8%), ESI-MS m/z calcd for [C 21 H 39 N 2 O 12 S] + (M+H) + :542.60,found:542.59.
实施例2：定制唾液酸配体分子2的合成Example 2: Synthesis of custom sialic acid ligand molecule 2
Figure PCTCN2020127031-appb-000022
Figure PCTCN2020127031-appb-000022
浓盐酸(1mL)滴入甲醇(100mL)中，分批加入天然唾液酸Neu5Ac(10g,32.3mmol)，搅拌反应至溶液澄清，中和反应液，蒸除溶剂，残余物经加甲苯旋蒸三次，抽干，得式2-1中间体粗品，直接用于下步反应；Concentrated hydrochloric acid (1 mL) was dropped into methanol (100 mL), natural sialic acid Neu5Ac (10 g, 32.3 mmol) was added in batches, the reaction was stirred until the solution was clear, the reaction solution was neutralized, the solvent was evaporated, and the residue was rotary evaporated three times by adding toluene , drained to obtain the crude intermediate of formula 2-1, which was directly used in the next step reaction;
上步所得式2-1粗品溶于氯乙酰(40mL)，室温下搅拌至反应完全，蒸除溶剂，得式2-2粗品，直接用于下步反应；The crude product of formula 2-1 obtained in the previous step was dissolved in chloroacetyl (40 mL), stirred at room temperature until the reaction was complete, and the solvent was evaporated to obtain the crude product of formula 2-2, which was directly used in the next step reaction;
上步所得式2-2粗品与HOPEG 4OBn(13.8g,48.5mmol)混于干燥二氯甲烷(60mL)，冰浴下加入碳酸银(17.83g,64.7mmol)，反应12h，经TLC检测反应完全，滤除不溶物，残余物浓缩拌样柱分离，得式2-3化合物(19.3g,78.8％in 3steps),ESI-MS m/z calcd for[C 35H 52NO 17] +(M+H) +:758.78,found:758.77. The crude product of formula 2-2 obtained in the previous step was mixed with HOPEG 4 OBn (13.8 g, 48.5 mmol) in dry dichloromethane (60 mL), and silver carbonate (17.83 g, 64.7 mmol) was added under an ice bath to react for 12 h, and the reaction was detected by TLC. Complete, insoluble matter was filtered off, and the residue was concentrated and separated with a sample column to obtain the compound of formula 2-3 (19.3g, 78.8% in 3steps), ESI-MS m/z calcd for [C 35 H 52 NO 17 ] + (M +H) + :758.78,found:758.77.
式2-3化合物(19.3g,25.47mmol)溶于甲醇(40mL)，滴入NaOH水溶液(1.5M，20mL)，搅拌至TLC检测反应完全，中和反应液，蒸除溶剂，残余物溶于2M NaOH水溶液(40mL)，于95℃油浴中回流10小时，中和反应液，蒸除溶剂，残余物经甲苯共沸旋蒸三次后溶于盐酸/甲醇(v/v＝1:99,50mL)，搅拌至TLC检测反应完全，中和反应液，蒸除溶剂，得式2-4化合物粗品，直接用于下步反应，ESI-MS m/z calcd for[C 25H 42NO 12] +(M+H) +:548.60,found:548.59； The compound of formula 2-3 (19.3 g, 25.47 mmol) was dissolved in methanol (40 mL), NaOH aqueous solution (1.5 M, 20 mL) was added dropwise, stirred until the reaction was complete by TLC detection, the reaction solution was neutralized, the solvent was evaporated, and the residue was dissolved in 2M NaOH aqueous solution (40 mL) was refluxed in an oil bath at 95°C for 10 hours, the reaction solution was neutralized, and the solvent was evaporated. 50mL), stir until TLC detects that the reaction is complete, neutralize the reaction solution, evaporate the solvent to obtain the crude product of the compound of formula 2-4, which is directly used in the next step reaction, ESI-MS m/z calcd for [C 25 H 42 NO 12 ] + (M+H) + :548.60,found:548.59;
叠氮化钠NaN 3(1.99g,30.56mmol)溶于水(30mL)，缓慢滴入三氟甲磺酸酐(8.62g,30.56mmol)，搅拌15分钟，用甲苯萃取水相，有机相经无水硫酸钠干燥后制得三氟甲磺酰叠 氮TfN 3，将上步所得式2-4化合物粗品溶于乙醇，加入所制TfN 3和硫酸铜CuSO4(0.812g,5.09mmol)，搅拌6小时，蒸除溶剂，所得残余物溶于吡啶(50mL)，冰浴条件下滴入乙酸酐(26g,254.7mmol)，室温下搅拌12小时，冰浴下滴入甲醇淬灭反应，蒸除溶剂，残余物溶于二氯甲烷，先后经1N HCl a.q.、sat.NaHCO 3a.q.和饱和食盐水洗涤，无水硫酸钠干燥，浓缩后柱层析纯化，得式2-5化合物(18.1g,95.8％),ESI-MS m/z calcd for[C 33H 47N 3O 16] +(M+H) +:742.74,found:742.73； Sodium azide NaN 3 (1.99g, 30.56mmol) was dissolved in water (30mL), trifluoromethanesulfonic anhydride (8.62g, 30.56mmol) was slowly added dropwise, stirred for 15 minutes, the aqueous phase was extracted with toluene, and the organic phase was washed without After drying with sodium sulfate, trifluoromethanesulfonyl azide TfN 3 was obtained, the crude compound of formula 2-4 obtained in the previous step was dissolved in ethanol, the prepared TfN 3 and copper sulfate CuSO 4 (0.812g, 5.09mmol) were added, and stirred for 6 After 1 hour, the solvent was evaporated, the obtained residue was dissolved in pyridine (50 mL), acetic anhydride (26 g, 254.7 mmol) was added dropwise under an ice bath, stirred at room temperature for 12 hours, methanol was added dropwise under an ice bath to quench the reaction, and the solvent was evaporated. , the residue was dissolved in dichloromethane, washed with 1N HCl aq , sat.NaHCO 3a.q. and saturated brine successively, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain the compound of formula 2-5 (18.1 g , 95.8%), ESI-MS m/z calcd for [C 33 H 47 N 3 O 16 ] + (M+H) + : 742.74, found: 742.73;
式2-5化合物(18.1g,24.4mmol)与丙烯酸甲酯(2.46g,29.28mmol)、碘化亚铜(0.93g,4.88mmol)混于溶于四氢呋喃THF(60mL)，滴入N,N-二异丙基乙胺DIPEA(4.73gm 36.6mmol)，室温下搅拌1小时，蒸除反应溶剂，残余物溶于二氯甲烷，先后经1M氨水、1N HCl a.q.、sat.NaHCO 3a.q.和饱和食盐水洗涤，无水硫酸钠干燥，浓缩后柱层析纯化，得式2-6化合物(19.6g,97.3％),ESI-MS m/z calcd for[C 37H 52N 3O 18] +(M+H) +:826.82,found:826.81； The compound of formula 2-5 (18.1 g, 24.4 mmol) was mixed with methyl acrylate (2.46 g, 29.28 mmol) and cuprous iodide (0.93 g, 4.88 mmol) and dissolved in THF (60 mL), and N,N was added dropwise. -Diisopropylethylamine DIPEA (4.73gm 36.6mmol), stirred at room temperature for 1 hour, evaporated the reaction solvent, the residue was dissolved in dichloromethane, successively passed through 1M ammonia water, 1N HCl aq , sat.NaHCO 3a.q. Washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain the compound of formula 2-6 (19.6g, 97.3%), ESI-MS m/z calcd for [C 37 H 52 N 3 O 18 ] + (M+H) + :826.82,found:826.81;
式2-6化合物(19.6g,23.73mmol)溶于甲醇(100mL)，加入甲醇钠(0.1M in MeOH)，调节溶液pH 8～10，室温下搅拌30分钟，滴入盐酸中和反应液，蒸除溶剂，得式2-7粗品，直接用于下步反应；The compound of formula 2-6 (19.6 g, 23.73 mmol) was dissolved in methanol (100 mL), sodium methoxide (0.1 M in MeOH) was added, the pH of the solution was adjusted to 8-10, stirred at room temperature for 30 minutes, and hydrochloric acid was added dropwise to neutralize the reaction solution, The solvent is evaporated to obtain the crude product of formula 2-7, which is directly used in the next step reaction;
上步所得式2-7粗品溶于二氯甲烷(100mL)，加入三乙胺(4.8g,47.47mmol)，搅拌5分钟后分批加入对甲苯磺酰氯(6.79g，35.60mmol)，室温下搅拌6小时，加入甲醇淬灭反应，蒸除溶剂，残余物溶于二氯，盐水洗涤，有机相经无水硫酸钠干燥，蒸除溶剂，残余物溶于N，N-二甲基甲酰胺(50mL)，加入NaN 3(4.63g,71.20mmol)，于50℃油浴中搅拌12小时，TLC检测反应完全，蒸除溶剂，残余物溶于二氯甲烷，盐水洗涤，无水硫酸钠干燥，浓缩后柱层析得式2-8化合物(14.3g,88.3％)，ESI-MS m/z calcd for[C 29H 43N 6O 13] +(M+H) +:683.68,found:683.69； The crude product of formula 2-7 obtained in the previous step was dissolved in dichloromethane (100 mL), triethylamine (4.8 g, 47.47 mmol) was added, and p-toluenesulfonyl chloride (6.79 g, 35.60 mmol) was added in batches after stirring for 5 minutes. After stirring for 6 hours, methanol was added to quench the reaction, the solvent was evaporated, the residue was dissolved in dichloride, washed with brine, the organic phase was dried over anhydrous sodium sulfate, the solvent was evaporated, and the residue was dissolved in N,N-dimethylformamide (50 mL), NaN 3 (4.63 g, 71.20 mmol) was added, and the mixture was stirred in an oil bath at 50° C. for 12 hours. TLC detected that the reaction was complete, and the solvent was evaporated. The residue was dissolved in dichloromethane, washed with brine, and dried over anhydrous sodium sulfate. , the compound of formula 2-8 (14.3g, 88.3%) was obtained by column chromatography after concentration, ESI-MS m/z calcd for [C 29 H 43 N 6 O 13 ] + (M+H) + : 683.68, found: 683.69;
式2-8化合物(14.3g，20.95mmol)溶于四氢呋喃(100mL)，分批加入三苯基膦(16.48g,62.84mmol)，搅拌至无气泡产生后，继续搅拌12小时，蒸除溶剂，残余物溶于乙醇，缓慢滴入苯甲酰氯(3.53g,25.14mmol)，室温下搅拌1小时，蒸除溶剂，残余物直接柱层析纯化得式2-9化合物(13.1g,82.2％)，ESI-MS m/z calcd for[C 36H 49N 4O 14] +(M+H) +:761.79,found:761.78； The compound of formula 2-8 (14.3 g, 20.95 mmol) was dissolved in tetrahydrofuran (100 mL), triphenylphosphine (16.48 g, 62.84 mmol) was added in batches, stirred until no bubbles were generated, continued stirring for 12 hours, and the solvent was evaporated. The residue was dissolved in ethanol, slowly added dropwise with benzoyl chloride (3.53 g, 25.14 mmol), stirred at room temperature for 1 hour, evaporated to remove the solvent, and the residue was directly purified by column chromatography to obtain the compound of formula 2-9 (13.1 g, 82.2%) , ESI-MS m/z calcd for [C 36 H 49 N 4 O 14 ] + (M+H) + : 761.79, found: 761.78;
式2-9化合物(13.1g,17.22mmol)溶于乙腈(50mL)，加入丙酮缩二甲醇(5.38g,51.66mmol)和催化量的对甲苯磺酸(0.3g,1.72mmol)，于80℃油浴中回流12小时，TLC检测反应完全，加入三乙胺中和反应液，蒸除溶剂，残余物直接柱层析纯化得式2-10化合物(12.8g,92.8％),ESI-MS m/z calcd for[C 39H 53N 4O 13] +(M+H) +:801.86,found:801.86； The compound of formula 2-9 (13.1 g, 17.22 mmol) was dissolved in acetonitrile (50 mL), acetone acetal (5.38 g, 51.66 mmol) and a catalytic amount of p-toluenesulfonic acid (0.3 g, 1.72 mmol) were added, and the mixture was heated at 80° C. Refluxed in an oil bath for 12 hours, TLC detected that the reaction was complete, triethylamine was added to neutralize the reaction solution, the solvent was evaporated, and the residue was directly purified by column chromatography to obtain the compound of formula 2-10 (12.8 g, 92.8%), ESI-MS m /z calcd for[C 39 H 53 N 4 O 13 ] + (M+H) + :801.86,found:801.86;
式2-10化合物(12.8g,15.98mmol)和新制氧化银(7.41g,31.97mmol)混于N,N-二甲基甲酰胺(50mL)，加入碘甲烷(3.40g,23.97mmol)，室温下搅拌4小时，TLC检测反应完全，滤除不溶物， 蒸除溶剂，残余物溶于二氯甲烷，盐水洗涤，无水硫酸钠干燥，浓缩后柱层析纯化得式2-11化合物(11.6g,89.1％)，ESI-MS m/z calcd for[C 40H 55N 4O 14] +(M+H) +:815.89,found:815.88； The compound of formula 2-10 (12.8 g, 15.98 mmol) and freshly prepared silver oxide (7.41 g, 31.97 mmol) were mixed in N,N-dimethylformamide (50 mL), methyl iodide (3.40 g, 23.97 mmol) was added, room temperature After stirring for 4 hours, TLC detected that the reaction was complete, insoluble matter was filtered off, the solvent was evaporated, the residue was dissolved in dichloromethane, washed with brine, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain the compound of formula 2-11 (11.6 g, 89.1%), ESI-MS m/z calcd for [C 40 H 55 N 4 O 14 ] + (M+H) + : 815.89, found: 815.88;
式2-11化合物(11.6g,14.24mmol)溶于甲醇，加入对甲苯磺酸(0.25g,1.42mmol)，置于80℃油浴中回流6小时，TLC检测反应完全，加入三乙胺中和反应液，蒸除溶剂，残余物溶于二氯甲烷，盐水洗涤，无水硫酸钠干燥，浓缩后溶于甲醇，加入Pd/C粉末(0.1g)，于4atm压力H 2氛围中反应2小时，滤除Pd/C粉末，滤液浓缩后得式2-12化合物粗品，直接用于下步反应，ESI-MS m/z calcd for[C 30H 45N 4O 14] +(M+H) +:685.70,found:685.69； The compound of formula 2-11 (11.6 g, 14.24 mmol) was dissolved in methanol, added p-toluenesulfonic acid (0.25 g, 1.42 mmol), placed in an oil bath at 80°C and refluxed for 6 hours, TLC detected that the reaction was complete, added to triethylamine and the reaction solution, evaporated to remove the solvent, the residue was dissolved in dichloromethane, washed with brine, dried over anhydrous sodium sulfate, concentrated and dissolved in methanol, added Pd/C powder (0.1 g), and reacted under 4 atm pressure H atmosphere for 2 hours, the Pd/C powder was filtered off, and the filtrate was concentrated to obtain the crude compound of formula 2-12, which was directly used in the next step reaction, ESI-MS m/z calcd for [C 30 H 45 N 4 O 14 ] + (M+H ) + :685.70,found:685.69;
上步所得式2-12化合物粗品溶于二氯甲烷(60mL)，加入三乙胺(2.88g,28.47mmol)，搅拌5分钟后分批加入对甲苯磺酰氯(4.07g，21.35mmol)，室温下搅拌6小时，加入甲醇淬灭反应，蒸除溶剂，残余物溶于二氯，盐水洗涤，有机相经无水硫酸钠干燥，蒸除溶剂，残余物溶于乙腈(40mL)，加入KSAc(3.25g,28.47mmol)，于室温下搅拌4小时，TLC检测反应完全，蒸除溶剂，残余物溶于二氯甲烷，盐水洗涤，无水硫酸钠干燥，浓缩后柱层析得式2-13化合物(8.9g,84.2％)，ESI-MS m/z calcd for[C 32H 47N 4O 14S] +(M+H) +:743.79,found:743.78； The crude compound of formula 2-12 obtained in the previous step was dissolved in dichloromethane (60 mL), triethylamine (2.88 g, 28.47 mmol) was added, and after stirring for 5 minutes, p-toluenesulfonyl chloride (4.07 g, 21.35 mmol) was added in batches at room temperature. After stirring for 6 hours, methanol was added to quench the reaction, the solvent was evaporated, the residue was dissolved in dichloride, washed with brine, the organic phase was dried over anhydrous sodium sulfate, the solvent was evaporated, the residue was dissolved in acetonitrile (40 mL), and KSAc ( 3.25g, 28.47mmol), stirred at room temperature for 4 hours, TLC detected that the reaction was complete, evaporated to remove the solvent, the residue was dissolved in dichloromethane, washed with brine, dried over anhydrous sodium sulfate, and concentrated by column chromatography to obtain formula 2-13 Compound (8.9 g, 84.2%), ESI-MS m/z calcd for [C 32 H 47 N 4 O 14 S] + (M+H) + : 743.79, found: 743.78;
式2-13化合物(8.9g,11.98mmol)溶于甲醇(30mL)，加入NaOH水溶液(1.5M,10mL)，室温下搅拌30分钟，加入732型H +阳离子树脂中和反应液，滤除树脂，滤液浓缩后得式2化合物(7.5g，91.2％)，ESI-MS m/z calcd for[C 29H 43N 4O 13S] +(M+H) +:687.73,found:687.72； The compound of formula 2-13 (8.9 g, 11.98 mmol) was dissolved in methanol (30 mL), an aqueous NaOH solution (1.5 M, 10 mL) was added, stirred for 30 minutes at room temperature, and the reaction solution was neutralized by adding 732 type H + cation resin, and the resin was filtered off. , the filtrate was concentrated to obtain the compound of formula 2 (7.5g, 91.2%), ESI-MS m/z calcd for [C 29 H 43 N 4 O 13 S] + (M+H) + : 687.73, found: 687.72;
实施例3：唾液酸-金纳米棒探针3的制备Example 3: Preparation of sialic acid-gold nanorod probe 3
Figure PCTCN2020127031-appb-000023
Figure PCTCN2020127031-appb-000023
将实施例1中所得定制唾液酸配体分子1溶于水制成水溶液(10mM)，取100μL加至CTABB覆盖、长75nm、长径比3:1的金纳米棒溶液中(OD＝15，5mL)，静置15小时后，经8000rpm离心10分钟，弃去上清液，离心管底物聚集物重新分散于去离子水(5mL)，得唾液酸-金纳米棒探针3；The customized sialic acid ligand molecule 1 obtained in Example 1 was dissolved in water to prepare an aqueous solution (10 mM), and 100 μL was added to the gold nanorod solution covered by CTABB, with a length of 75 nm and an aspect ratio of 3:1 (OD=15, 5mL), after standing for 15 hours, centrifuge at 8000rpm for 10 minutes, discard the supernatant, and redisperse the centrifuge tube substrate aggregates in deionized water (5mL) to obtain sialic acid-gold nanorod probe 3;
其中，金纳米棒溶液的制备过程包括：加入氯金酸HAuCl 4水溶液(0.5mM,5mL)至CTAB水溶液(0.2M,5mL)中，剧烈搅拌下加入新制的硼氢化钠NaBH 4冰水溶液(10mM,600μL)，继续搅拌10分钟，制得金纳米种子溶液；将HAuCl 4水溶液(5mM,6mL)、硝酸银AgNO 3水溶液(0.1mM,75μL)加至CTAB水溶液(0.2M,30mL)，继续加入盐酸(1.2mM,75μL)和抗坏血酸钠 Vc-Na水溶液(10mM,3.7mL)，摇晃溶液至无色，加入制得的金纳米种子溶液(50μL)，搅拌5分钟后，于37℃恒温箱中静置16小时，经8000rpm离心10分钟，除去上清液，离心管底部残余物分散于等体积去离子水(约40mL)中，得金纳米棒溶液。 Wherein, the preparation process of gold nanorod solution includes: adding chloroauric acid HAuCl 4 aqueous solution (0.5mM, 5 mL) to CTAB aqueous solution (0.2 M, 5 mL), adding freshly prepared sodium borohydride NaBH 4 ice aqueous solution (10 mM) under vigorous stirring , 600 μL), continue to stir for 10 minutes to prepare gold nanoseed solution; add HAuCl 4 aqueous solution (5 mM, 6 mL), silver nitrate AgNO 3 aqueous solution (0.1 mM, 75 μL) to CTAB aqueous solution (0.2 M, 30 mL), continue to add Hydrochloric acid (1.2 mM, 75 μL) and sodium ascorbate Vc-Na aqueous solution (10 mM, 3.7 mL), shake the solution until colorless, add the prepared gold nanoseed solution (50 μL), stir for 5 minutes, and place in a 37°C incubator Let stand for 16 hours, centrifuge at 8000 rpm for 10 minutes, remove the supernatant, and disperse the residue at the bottom of the centrifuge tube in an equal volume of deionized water (about 40 mL) to obtain a gold nanorod solution.
实施例4：唾液酸-金纳米棒探针4的制备Example 4: Preparation of sialic acid-gold nanorod probe 4
Figure PCTCN2020127031-appb-000024
Figure PCTCN2020127031-appb-000024
将实施例2中所得定制唾液酸配体分子2溶于水制成水溶液(10mM)，取100μL加至CTABB覆盖、长75nm、长径比3:1的金纳米棒溶液中(OD＝15，5mL)，静置15小时后，经8000rpm离心10分钟，弃去上清液，离心管底物聚集物重新分散于去离子水(5mL)，得唾液酸-金纳米棒探针4；The customized sialic acid ligand molecule 2 obtained in Example 2 was dissolved in water to make an aqueous solution (10 mM), and 100 μL was added to the gold nanorod solution covered by CTABB, with a length of 75 nm and an aspect ratio of 3:1 (OD=15, 5mL), after standing for 15 hours, centrifuge at 8000rpm for 10 minutes, discard the supernatant, and redisperse the centrifuge tube substrate aggregates in deionized water (5mL) to obtain sialic acid-gold nanorod probe 4;
其中，金纳米棒溶液的制备过程包括：加入氯金酸HAuCl 4水溶液(0.5mM,5mL)至CTAB水溶液(0.2M,5mL)中，剧烈搅拌下加入新制的硼氢化钠NaBH 4冰水溶液(10mM,600μL)，继续搅拌10分钟，制得金纳米种子溶液；将HAuCl 4水溶液(5mM,6mL)、硝酸银AgNO 3水溶液(0.1mM,75μL)加至CTAB水溶液(0.2M,30mL)，继续加入盐酸(1.2mM,75μL)和抗坏血酸钠Vc-Na水溶液(10mM,3.7mL)，摇晃溶液至无色，加入制得的金纳米种子溶液(50μL)，搅拌5分钟后，于37℃恒温箱中静置16小时，经8000rpm离心10分钟，除去上清液，离心管底部残余物分散于等体积去离子水(约40mL)中，得金纳米棒溶液。 Wherein, the preparation process of gold nanorod solution includes: adding chloroauric acid HAuCl 4 aqueous solution (0.5mM, 5 mL) to CTAB aqueous solution (0.2 M, 5 mL), adding freshly prepared sodium borohydride NaBH 4 ice aqueous solution (10 mM) under vigorous stirring , 600 μL), continue to stir for 10 minutes to prepare gold nanoseed solution; add HAuCl 4 aqueous solution (5 mM, 6 mL), silver nitrate AgNO 3 aqueous solution (0.1 mM, 75 μL) to CTAB aqueous solution (0.2 M, 30 mL), continue to add Hydrochloric acid (1.2 mM, 75 μL) and sodium ascorbate Vc-Na aqueous solution (10 mM, 3.7 mL), shake the solution until colorless, add the prepared gold nanoseed solution (50 μL), stir for 5 minutes, and place in a 37°C incubator Let stand for 16 hours, centrifuge at 8000 rpm for 10 minutes, remove the supernatant, and disperse the residue at the bottom of the centrifuge tube in an equal volume of deionized water (about 40 mL) to obtain a gold nanorod solution.
实施例5：检测实验1Example 5: Detection Experiment 1
新冠病毒颗粒具有高传染高致病，而本发明检测新冠病毒仅涉及新冠病毒表面S蛋白，不涉及其内部结构，因此，可在100nm粒径的SiO 2纳米粒表面固定商业化新冠病毒S蛋白来模拟新冠病毒颗粒，通过模拟颗粒进行检测实验。 The new coronavirus particles are highly infectious and highly pathogenic, and the detection of the new coronavirus in the present invention only involves the surface S protein of the new coronavirus, not its internal structure. Therefore, the commercialized new coronavirus S protein can be immobilized on the surface of SiO 2 nanoparticles with a particle size of 100 nm. To simulate the new coronavirus particles, the detection experiments are carried out by simulating the particles.
制备新冠病毒模拟颗粒：取粒径100nm的SiO 2纳米粒(麦克林牌，115mg，约10 14个)，悬于N,N-二甲基甲酰胺(5mL)中，超声1小时，加入(3-氨基丙基)三甲氧基硅烷(347mg,1.91mmol)，搅拌下加入1N HCl水溶液(0.19mL,0.19mmol)，室温下搅拌6小时，经6000rpm离心20分钟，弃去上清液，离心管底聚集物经离心洗涤两次后，分散于10％二甲基亚砜水溶液 DMSO(50mL)中，加入PBS缓冲液(pH 7.4,50mL)，制得SiO 2纳米粒处理液待用；新冠病毒S蛋白(义翘神州牌，5mg)溶于DMSO:PBS:H 2O＝5:50:45的混合溶剂(1mL)中，加入N-羟基琥珀酰亚胺NHS(2.1mg，18μmol)，缓慢搅拌1小时后，加入制得的SiO 2纳米粒处理液(20mL)中，搅拌2小时，经6000rpm离心20分钟，弃去上清液，离心管底聚集物经离心洗涤一次，分散于PBS缓冲液(pH7.4,200mL)中，制得新冠病毒模拟颗粒储备液，其中，模拟颗粒的数量约为10 11个/mL，置-20℃冷冻保存。 Preparation of new coronavirus simulated particles: take SiO nanoparticles with a particle size of 100 nm ( MacLean brand, 115 mg, about 10 14 ), suspend in N,N-dimethylformamide (5 mL), sonicate for 1 hour, add ( 3-Aminopropyl)trimethoxysilane (347 mg, 1.91 mmol) was added with 1N aqueous HCl (0.19 mL, 0.19 mmol) under stirring, stirred at room temperature for 6 hours, centrifuged at 6000 rpm for 20 minutes, discarded the supernatant, and centrifuged The aggregates at the bottom of the tube were washed twice by centrifugation, dispersed in 10% dimethyl sulfoxide aqueous solution DMSO (50 mL), and PBS buffer (pH 7.4, 50 mL) was added to prepare a SiO 2 nanoparticle treatment solution for later use; Virus S protein (Yiqiao Shenzhou brand, 5 mg) was dissolved in a mixed solvent (1 mL) of DMSO:PBS:H 2 O=5:50:45, and N-hydroxysuccinimide NHS (2.1 mg, 18 μmol) was added, After stirring slowly for 1 hour, add the prepared SiO nanoparticle treatment solution (20 mL), stir for 2 hours, centrifuge at 6000 rpm for 20 minutes, discard the supernatant, and wash the aggregates at the bottom of the centrifuge tube once by centrifugation, and then disperse in PBS. In the buffer solution (pH 7.4, 200 mL), a new coronavirus simulated particle stock solution was prepared, wherein the number of simulated particles was about 10 11 /mL, and it was stored at -20°C for cryopreservation.
取实施例3所得金纳米棒探针3溶液2份各1mL，分别标记C液和T液，C液中加入1滴(约50μL)去离子水对照液，T液中加入1滴新冠病毒模拟颗粒溶液(由模拟颗粒储备液解冻、稀释10 4倍而得)，混匀后静置15分钟，观察C液和T液颜色变化，C液颜色无变化，显茶红色，T液颜色发生明显变化，呈暗灰色。 Take 2 parts of 1 mL of the gold nanorod probe 3 solution obtained in Example 3, label C solution and T solution respectively, add 1 drop (about 50 μL) of deionized water control solution to C solution, and add 1 drop of new coronavirus simulation solution to T solution. The granule solution (obtained by thawing and diluting 10 4 times of the simulated granule stock solution), after mixing, let it stand for 15 minutes, and observe the color changes of C solution and T solution. change, dark grey.
实施例6：检测实验2Example 6: Detection Experiment 2
取实施例4所得金纳米棒探针4溶液2份各1mL，分别标记C液和T液，C液中加入1滴(约50μL)去离子水対照液，T液中加入1滴新冠病毒模拟颗粒溶液(由模拟颗粒储备液解冻、稀释10 4倍而得)，混匀后静置15分钟，观察C液和T液颜色变化，C液颜色无变化，显茶红色，T液颜色发生明显变化，呈紫灰色。 Take 2 parts of 1 mL of the gold nanorod probe 4 solution obtained in Example 4, label C solution and T solution respectively, add 1 drop (about 50 μL) of deionized water solution to C solution, and add 1 drop of new coronavirus simulation solution to T solution. The granule solution (obtained by thawing and diluting 10 4 times of the simulated granule stock solution), after mixing, let it stand for 15 minutes, and observe the color changes of C solution and T solution. change, purple-grey.
实施例7：平行检测实验Example 7: Parallel detection experiment
取实施例4所得金纳米棒探针4溶液3份各1mL，分别标记为C液、T 1液、T 2液，取健康人唾液样本分为2份，一份加入去离子水(10％，即去离子水体积：唾液体积＝1:9)标记为样本S 1，一份加入新冠病毒模拟颗粒溶液(10％，模拟颗粒溶液体积：唾液体积＝1:9)标记为样本S 2，分别向C液、T 1液、T 2液中加入1滴(50mL)去离子水对照液、样本S 1、样本S 2，混匀后静置15分钟，观察C液、T 1液、T 2液颜色变化，其中，C液颜色无变化，呈茶红色，T 1液颜色略有变化，呈茶红色，T 2颜色发生明显变化，呈灰色。 Take three 1 mL portions of the gold nanorod probe 4 solution obtained in Example 4, which are marked as C solution, T 1 solution, and T 2 solution respectively. , namely the volume of deionized water: volume of saliva=1:9) is marked as sample S 1 , and one part is added to the new coronavirus simulated particle solution (10%, volume of simulated particle solution: volume of saliva=1:9) is marked as sample S 2 , Add 1 drop (50mL) of deionized water control solution, sample S 1 , and sample S 2 to solution C, solution T 1 and solution T 2 respectively, mix well and let stand for 15 minutes, observe solution C, solution T 1 , T solution The color of liquid 2 changes, among which, the color of liquid C has no change, which is tea red, the color of liquid T 1 changes slightly, which is tea red, and the color of liquid T 2 changes obviously, which is gray.
综上，本发明提供了一种基于金纳米棒(Gold nanorods,GNR)探针LSPR效应的新冠病毒颗粒检测方法，在金纳米棒表面固定定制唾液酸配体分子，其能高效识别结合新冠病毒表面Spike蛋白，从而引发分散的金纳米探针团聚，金纳米探针的分散状态变化能通过LSPR效应所表现的颜色反映出来，从而实现对新冠病毒的检测。In summary, the present invention provides a new coronavirus particle detection method based on the LSPR effect of gold nanorods (Gold nanorods, GNR) probes. Custom sialic acid ligand molecules are immobilized on the surface of gold nanorods, which can efficiently identify and bind to the new coronavirus. The Spike protein on the surface triggers the agglomeration of the dispersed gold nanoprobes, and the change of the dispersion state of the gold nanoprobes can be reflected by the color exhibited by the LSPR effect, thereby realizing the detection of the new coronavirus.
以上所述仅为本发明的具体实施方式，不是全部的实施方式，本领域普通技术人员通过阅读本发明说明书而对本发明技术方案采取的任何等效的变换，均为本发明的权利要求所涵盖。The above descriptions are only specific implementations of the present invention, not all implementations. Any equivalent transformations taken by those of ordinary skill in the art to the technical solutions of the present invention by reading the specification of the present invention are covered by the claims of the present invention. .

Claims (10)

  1. 一种生物探针，其特征在于，所述生物探针包括探针载体和与探针载体连接的定制唾液酸配体分子；A biological probe, characterized in that the biological probe comprises a probe carrier and a customized sialic acid ligand molecule connected to the probe carrier;
    所述定制唾液酸配体分子的结构通式如式I所示：
    Figure PCTCN2020127031-appb-100001
    其中，R 1选自羟基、甲氧基或取代的甲氧基；R 2选自乙酰氨基、取代的乙酰氨基、苯甲酰氨基、取代的苯甲酰基、烷氧羰酰胺基、三氮唑基或取代的三氮唑基，R 3选自羟基、甲氧基、取代的甲氧基、乙酰氨基、取代的乙酰氨基、磺酰胺基或磷酰胺基；A为
    Figure PCTCN2020127031-appb-100002
    Figure PCTCN2020127031-appb-100003
    n选自0,1,2,3,4,5,6,7，m选自2,3,4,5,6,7,8,9。     The general structural formula of the customized sialic acid ligand molecule is shown in formula I:
    Figure PCTCN2020127031-appb-100001
    Wherein, R 1 is selected from hydroxyl, methoxy or substituted methoxy; R 2 is selected from acetamido, substituted acetamido, benzamido, substituted benzoyl, alkoxycarbonamido, triazole or substituted triazolyl, R is selected from hydroxyl, methoxy, substituted methoxy, acetamido, substituted acetamido, sulfonamide or phosphoramido; A is
    Figure PCTCN2020127031-appb-100002
    Figure PCTCN2020127031-appb-100003
    n is selected from 0, 1, 2, 3, 4, 5, 6, 7, and m is selected from 2, 3, 4, 5, 6, 7, 8, 9.
  2. 根据权利要求1所述的生物探针，其特征在于，所述取代的甲氧基为X 1-CH 2O-，其中，X 1选自苯基或乙烯基； The biological probe according to claim 1, wherein the substituted methoxy group is X 1 -CH 2 O-, wherein X 1 is selected from phenyl or vinyl;
    所述取代的乙酰氨基为X 2-CH 2CONH-，其中，X 2选自甲基、乙基、正丙基、异丙基、羟甲基或羟乙基； The substituted acetamido is X 2 -CH 2 CONH-, wherein X 2 is selected from methyl, ethyl, n-propyl, isopropyl, hydroxymethyl or hydroxyethyl;
    所述取代的苯甲酰氨基为X 3-BzNH-，其中，X 3的数量至少为1且位于苯环的任意位置，X 3选自卤原子、甲基、甲氧基、硝基中的至少一种； The substituted benzamido is X 3 -BzNH-, wherein the number of X 3 is at least 1 and is located at any position of the benzene ring, and X 3 is selected from halogen atoms, methyl groups, methoxyl groups, nitro groups. at least one;
    所述烷氧羰酰胺基为X 4-OC(O)NH-，其中，X 4选自苄基、烯丙基、叔丁基或三氯乙基； The alkoxycarbonamide group is X 4 -OC(O)NH-, wherein X 4 is selected from benzyl, allyl, tert-butyl or trichloroethyl;
    所述取代的三氮唑基为
    Figure PCTCN2020127031-appb-100004
    其中，X 5选自取代的苯烷基X 3-Ph-(CH 2) n1-或取代的羰基X 6-C(O)-，其中X 3的数量至少为1且位于苯环的任意位置，X 3选自卤原子、甲基、甲氧基、硝基中的至少一种，n1选自0,1,2；X 6选自烷氧基CH 3-(CH 2) n2-O-或烷胺基CH 3-(CH 2) n2-NH-，n2选自0,1,2,3,4,5。     The substituted triazolyl is
    Figure PCTCN2020127031-appb-100004
    Wherein, X 5 is selected from substituted phenylalkyl X 3 -Ph-(CH 2 ) n1 - or substituted carbonyl X 6 -C(O)-, wherein the number of X 3 is at least 1 and located at any position on the benzene ring , X 3 is selected from at least one of halogen atom, methyl group, methoxy group and nitro group, n1 is selected from 0,1,2; X 6 is selected from alkoxy group CH 3 -(CH 2 ) n2 -O- or alkylamino CH3- ( CH2 ) n2 -NH-, n2 is selected from 0,1,2,3,4,5.
  3. 根据权利要求1所述的生物探针，其特征在于，所述探针载体选自具有灵敏LSPR效应的纳米材料或在分散-聚集状态下具有明显信号变化的物质；The biological probe according to claim 1, wherein the probe carrier is selected from nanomaterials with sensitive LSPR effect or substances with obvious signal changes in a dispersed-aggregated state;
    优选地，所述具有灵敏LSPR效应的纳米材料包括金基纳米颗粒、表面有金属金层的复合纳米颗粒；更优选地，所述金基纳米颗粒包括金纳米棒、金纳米球；Preferably, the nanomaterials with sensitive LSPR effect include gold-based nanoparticles and composite nanoparticles with a metallic gold layer on the surface; more preferably, the gold-based nanoparticles include gold nanorods and gold nanospheres;
    优选地，所述在分散-聚集状态下具有明显信号变化的物质包括聚集诱导发光AIE材料；所述聚集诱导发光AIE材料包括具有环状多烯骨架的四噻吩基噻吩类化合物、具有氰取代二 苯乙烯骨架的化合物、四苯乙烯型化合物、二乙烯基蒽型化合物、三苯乙烯型化合物，所述聚集诱导发光材料分子末端具有同巯基反应连接的马来酰亚胺衍生基团。Preferably, the substance with obvious signal change in the dispersion-aggregation state comprises an aggregation-induced luminescence AIE material; the aggregation-induced luminescence AIE material comprises a tetrathienylthiophene compound with a cyclic polyene skeleton, a cyano-substituted dithiophene Compounds of styrene skeleton, tetrastyrene-type compounds, divinylanthracene-type compounds, and tristyryl-type compounds, the molecular end of the aggregation-induced light-emitting material has a maleimide-derived group that is reactively linked with a sulfhydryl group.
  4. 根据权利要求3所述的生物探针，其特征在于，所述探针载体为金纳米棒；The biological probe according to claim 3, wherein the probe carrier is a gold nanorod;
    优选地，所述金纳米棒表面包覆有十六烷基三甲基溴化铵双分子层；Preferably, the surface of the gold nanorods is coated with a bilayer of cetyltrimethylammonium bromide;
    优选地，所述金纳米棒直径为20-25nm，长为60-80nm，长径比为2.5:1～3.5:1。Preferably, the gold nanorods have a diameter of 20-25 nm, a length of 60-80 nm, and an aspect ratio of 2.5:1 to 3.5:1.
  5. 根据权利要求4所述的生物探针，其特征在于，所述定制唾液酸配体分子与金纳米棒的摩尔比为1×10 4:1～2×10 4:1。 The biological probe according to claim 4, wherein the molar ratio of the customized sialic acid ligand molecules to the gold nanorods is 1×10 4 :1˜2×10 4 :1.
  6. 权利要求1-5任一所述的生物探针的制备方法，其特征在于，包括以下步骤：The preparation method of the biological probe described in any one of claims 1-5, is characterized in that, comprises the following steps:
    1)提供探针载体溶液和具有结构通式I的定制唾液酸配体分子；1) provide a probe carrier solution and a customized sialic acid ligand molecule with general structural formula I;
    2)将具有结构通式I的定制唾液酸配体分子配制成水溶液，与探针载体溶液混合，25～30℃静置12-24小时制得生物探针；2) preparing the customized sialic acid ligand molecule with general structural formula I into an aqueous solution, mixing it with the probe carrier solution, and standing at 25-30° C. for 12-24 hours to obtain a biological probe;
    优选地，所述探针载体溶液为金纳米棒溶液，所述金纳米棒表面包覆有十六烷基三甲基溴化铵双分子层，所述金纳米棒直径为20-25nm，长为60-80nm；Preferably, the probe carrier solution is a solution of gold nanorods, the surface of the gold nanorods is coated with a bilayer of cetyltrimethylammonium bromide, the diameter of the gold nanorods is 20-25 nm, and the length of the gold nanorods is 20-25 nm. is 60-80nm;
    优选地，所述定制唾液酸配体分子与金纳米棒的摩尔比为1×10 4:1～2×10 4:1。 Preferably, the molar ratio of the customized sialic acid ligand molecules to the gold nanorods is 1×10 4 :1˜2×10 4 :1.
  7. 根据权利要求6所述的制备方法，其特征在于，所述金纳米棒溶液的制备方法为：在水相中以金种生长法制备金纳米棒，先用硼氢化钠还原氯金酸，制备1～2nm的金纳米种子溶液；再将金纳米种子溶液加入到氯金酸溶液、硝酸银溶液、稀盐酸、抗坏血酸溶液、十六烷基三甲基溴化铵溶液的混合液中静置反应，生成宽为20～25nm、长为60～80nm、表面被十六烷基三甲基溴化铵覆盖的金纳米棒溶液；The preparation method according to claim 6, wherein the preparation method of the gold nanorod solution is as follows: preparing gold nanorods by a gold seed growth method in an aqueous phase, first reducing chloroauric acid with sodium borohydride, and preparing 1-2nm gold nano-seed solution; then add the gold nano-seed solution to the mixture of chloroauric acid solution, silver nitrate solution, dilute hydrochloric acid, ascorbic acid solution and cetyltrimethylammonium bromide solution and let stand for reaction , resulting in a gold nanorod solution with a width of 20-25 nm, a length of 60-80 nm, and the surface covered by cetyltrimethylammonium bromide;
    优选地，所述硼氢化钠和氯金酸的摩尔比为2:1～3:1，所述硼氢化钠还原氯金酸的时间为5～20分钟，所述硼氢化钠还原氯金酸的温度为25～35℃；Preferably, the molar ratio of the sodium borohydride and the chloroauric acid is 2:1 to 3:1, the time for the sodium borohydride to reduce the chloroauric acid is 5 to 20 minutes, and the sodium borohydride to reduce the chloroauric acid. The temperature is 25 ~ 35 ℃;
    所述氯金酸溶液的浓度为4.5～5.5mM，所述硝酸银溶液的浓度为0.09～0.12M，所述稀盐酸的浓度为1.0～1.5mM，所述抗坏血酸溶液的浓度为8～15mM，所述十六烷基三甲基溴化铵溶液的浓度为0.15～0.25M；所述金纳米种子溶液、氯金酸溶液、硝酸银溶液、稀盐酸、抗坏血酸溶液、十六烷基三甲基溴化铵溶液的体积比为45～55μl：5.5～6.5mL：70～80μL：70～80μL：3.6～3.8mL：25～35mL，所述静置反应的温度为25～40℃，所述静置反应的时间为6～24小时。The concentration of the chloroauric acid solution is 4.5-5.5mM, the concentration of the silver nitrate solution is 0.09-0.12M, the concentration of the dilute hydrochloric acid is 1.0-1.5mM, the concentration of the ascorbic acid solution is 8-15mM, The concentration of the cetyltrimethylammonium bromide solution is 0.15-0.25M; the gold nanoseed solution, chloroauric acid solution, silver nitrate solution, dilute hydrochloric acid, ascorbic acid solution, cetyltrimethyl The volume ratio of the ammonium bromide solution is 45-55 μl: 5.5-6.5 mL: 70-80 μL: 70-80 μL: 3.6-3.8 mL: 25-35 mL, and the temperature of the standing reaction is 25-40° C. The reaction time is 6 to 24 hours.
  8. 根据权利要求6所述的制备方法，其特征在于，所述具有结构通式I的定制唾液酸配体分子的制备方法为：The preparation method according to claim 6, wherein the preparation method of the customized sialic acid ligand molecule with the general structural formula I is:
    以唾液酸
    Figure PCTCN2020127031-appb-100005
    为起始原料，经羧基保护在羧基引入R 4保护基得到
    Figure PCTCN2020127031-appb-100006
    在氯代和乙酰化试剂作用下一步生成全乙酰化2-氯代糖
    Figure PCTCN2020127031-appb-100007
    继而经糖基化反应在糖环2位衍生出功能侧链前体
    Figure PCTCN2020127031-appb-100008
    随后经脱乙酰基、羧基保护后得到糖环上氨基和羟基全部裸露的中间体
    Figure PCTCN2020127031-appb-100009
    按照R 2→R 1→R 3的顺序进行基团的引入分别得到
    Figure PCTCN2020127031-appb-100010
    最后通过侧链基团转换和糖环保护基脱除反应，合成获得定制唾液酸糖配体I；     sialic acid
    Figure PCTCN2020127031-appb-100005
    As the starting material, through the protection of the carboxyl group, the R4 protecting group is introduced into the carboxyl group to obtain
    Figure PCTCN2020127031-appb-100006
    Generation of peracetylated 2-chlorosugars in the next step of chlorination and acetylation reagents
    Figure PCTCN2020127031-appb-100007
    Then the functional side chain precursor is derived from the glycosylation reaction at the 2-position of the sugar ring
    Figure PCTCN2020127031-appb-100008
    After deacetylation and carboxyl protection, an intermediate with all the exposed amino and hydroxyl groups on the sugar ring is obtained
    Figure PCTCN2020127031-appb-100009
    According to the sequence of R 2 →R 1 →R 3 , the introduction of groups is carried out to obtain
    Figure PCTCN2020127031-appb-100010
    Finally, through the conversion of side chain groups and the removal of sugar ring protecting groups, the customized sialic acid sugar ligand I was synthesized;
    或以唾液酸
    Figure PCTCN2020127031-appb-100011
    为起始原料，经羧基保护在羧基引入R 4保护基得到
    Figure PCTCN2020127031-appb-100012
    在氯代和乙酰化试剂作用下一步生成全乙酰化2-氯代糖
    Figure PCTCN2020127031-appb-100013
    继而经糖基化反应在糖环2位衍生出功能侧链前体
    Figure PCTCN2020127031-appb-100014
    随后经脱乙酰基、羧基保护后得到糖环上氨基和羟基全部裸露的中间 体
    Figure PCTCN2020127031-appb-100015
    按照R 2→R 3→R 1的顺序进行基团的引入分别得到
    Figure PCTCN2020127031-appb-100016
    最后通过侧链基团转换和糖环保护基脱除反应，合成获得定制唾液酸糖配体I；     or with sialic acid
    Figure PCTCN2020127031-appb-100011
    As the starting material, through the protection of the carboxyl group, the R4 protecting group is introduced into the carboxyl group to obtain
    Figure PCTCN2020127031-appb-100012
    Generation of peracetylated 2-chlorosugars in the next step of chlorination and acetylation reagents
    Figure PCTCN2020127031-appb-100013
    Then the functional side chain precursor is derived from the glycosylation reaction at the 2-position of the sugar ring
    Figure PCTCN2020127031-appb-100014
    After deacetylation and carboxyl protection, an intermediate with all the exposed amino and hydroxyl groups on the sugar ring is obtained
    Figure PCTCN2020127031-appb-100015
    According to the sequence of R 2 →R 3 →R 1 , the introduction of groups is carried out to obtain
    Figure PCTCN2020127031-appb-100016
    Finally, through the conversion of side chain groups and the removal of sugar ring protecting groups, the customized sialic acid sugar ligand I was synthesized;
    其中，R 4选自甲基或苄基； wherein, R is selected from methyl or benzyl ;
    A 1选自
    Figure PCTCN2020127031-appb-100017
    或-(CH 2) m-R 5；R 5选自苄氧基、2-萘甲氧基或烯丙氧基，n为0,1,2,3,4,5,6,7，m为2,3,4,5,6,7,8,9；     A 1 is selected from
    Figure PCTCN2020127031-appb-100017
    or -(CH 2 ) m -R 5 ; R 5 is selected from benzyloxy, 2-naphthylmethoxy or allyloxy, n is 0, 1, 2, 3, 4, 5, 6, 7, m are 2,3,4,5,6,7,8,9;
    特别的，当R1为羟基时，其为唾液酸
    Figure PCTCN2020127031-appb-100018
    分子上的基团，不需要引入R1基团的相关反应过程；     In particular, when R1 is hydroxyl, it is sialic acid
    Figure PCTCN2020127031-appb-100018
    The group on the molecule does not need to introduce the relevant reaction process of the R1 group;
    当R2为乙酰氨基时，其为唾液酸
    Figure PCTCN2020127031-appb-100019
    分子上的基团，不需要引入R2基团的相关反应过程；     When R2 is acetamido, it is sialic acid
    Figure PCTCN2020127031-appb-100019
    The group on the molecule does not need to introduce the relevant reaction process of the R2 group;
    当R3为羟基时，其为唾液酸
    Figure PCTCN2020127031-appb-100020
    分子上的基团，不需要引入R3基团的相关反应过程。     When R3 is hydroxyl, it is sialic acid
    Figure PCTCN2020127031-appb-100020
    The group on the molecule does not need to introduce the relevant reaction process of the R3 group.
  9. 权利要求1-5任一项所述的生物探针在新冠病毒检测试剂中的应用。Application of the biological probe described in any one of claims 1-5 in a novel coronavirus detection reagent.
  10. 根据权利要求9所述的应用，其特征在于，采用生物探针进行新冠病毒检测的方法，包括以下步骤：将生物探针与待测样品混合，混合均匀后静置15-30分钟，通过观察混合体系的颜色来检测新冠病毒；The application according to claim 9, wherein the method for detecting a new coronavirus by using a biological probe comprises the following steps: mixing the biological probe with the sample to be tested, mixing evenly, and leaving it to stand for 15-30 minutes. The color of the mixed system to detect the new coronavirus;
    优选地，采用生物探针进行新冠病毒检测的方法具体包括以下步骤：1)取两份完全相同的生物探针水溶液，分别标记为溶液C和溶液T，溶液C中加入对照液，溶液T中加入待测样品，混合均匀后静置15-30分钟，观察结果；2)结果判定：阳性结果：溶液C不变色，溶液T变色；阴性结果：溶液C不变色，溶液T不变色；无效结果：溶液C变色，无论溶液T变色与否，应另取生物探针水溶液重新检测；Preferably, the method for detecting novel coronavirus by using biological probes specifically includes the following steps: 1) Take two identical aqueous solutions of biological probes, label them as solution C and solution T respectively, add control solution to solution C, and add solution T to solution Add the sample to be tested, mix well and let stand for 15-30 minutes to observe the results; 2) Result judgment: positive result: solution C does not change color, solution T changes color; negative result: solution C does not change color, solution T does not change color; invalid result : Solution C changes color, no matter whether solution T changes color or not, take another biological probe aqueous solution to test again;
    优选地，所述生物探针为定制唾液酸配体分子-金纳米棒生物探针；Preferably, the biological probe is a custom sialic acid ligand molecule-gold nanorod biological probe;
    优选地，所述定制唾液酸配体分子-金纳米棒生物探针水溶液的浓度为10-100μM；Preferably, the concentration of the customized sialic acid ligand molecule-gold nanorod biological probe aqueous solution is 10-100 μM;
    优选地，所述待测样品与定制唾液酸配体分子-金纳米棒生物探针水溶液的体积比为1:5～1:20；Preferably, the volume ratio of the sample to be tested to the customized sialic acid ligand molecule-gold nanorod biological probe aqueous solution is 1:5 to 1:20;
    优选地，所述待测样品中新冠病毒的检测极限为10 4个/mL； Preferably, the detection limit of the new coronavirus in the sample to be tested is 104 /mL;
    优选地，所述待测样品为唾液，鼻咽拭子，血液，皮肤分泌物；Preferably, the sample to be tested is saliva, nasopharyngeal swab, blood, and skin secretions;
    优选地，所述对照液为去离子水。Preferably, the control solution is deionized water.
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