A biological probe and its preparation method and application

Abstract

The present invention discloses a biological probe and a preparation method and application thereof, wherein the biological probe comprises a probe carrier and a customized sialic acid ligand molecule connected to the probe carrier; the structural formula of the customized sialic acid ligand molecule is as shown in Formula I: #imgabs0# wherein R1 is selected from hydroxyl, methoxy or substituted methoxy; R2 is selected from acetylamino, substituted acetylamino, benzylamino, substituted benzoyl, alkoxycarbonylamide, triazole or substituted triazole, R3 is selected from hydroxyl, methoxy, substituted methoxy, acetylamino, substituted acetylamino, sulfonamide or phosphoramide; A is #imgabs1#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 biological probe can be used to directly detect novel coronavirus particles.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry and relates to a biological probe and a preparation method and application thereof. Background Art

[0002] At present, the detection methods of the new coronavirus mainly include nucleic acid RNA detection and new coronavirus IgG / IgM antibody detection. The former extracts nucleic acid substances from the test sample and uses polymerase chain reaction (PCR) to amplify the test sample RNA to identify whether it is new coronavirus RNA. Currently, real-time fluorescence quantitative PCR technology is generally used for detection; the latter uses the fact that after a person is infected with the new coronavirus, the immune system will react to produce corresponding IgG and IgM antibodies. By detecting new coronavirus IgG and IgM in human blood, it can indirectly indicate whether the subject is infected with the new coronavirus. The colloidal gold method is generally used to detect these two antibodies.

[0003] Nucleic acid RNA testing generally uses RT-PCR technology to directly detect whether there is SARS-CoV-2 RNA in the sample, but it requires manual extraction of RNA viruses in the sample, and is affected by multiple factors such as sample sampling method, sample transportation and storage, and manual operation. It is often accompanied by false negative interference, and a small number of people with positive RNA test results did not show infectiousness, so there is also false positive interference; SARS-CoV-2 IgG / IgM antibody testing is an indirect detection method. It indirectly characterizes whether the subject is infected with the SARS-CoV-2 by detecting whether there are SARS-CoV-2 IgG / IgM antibodies in the blood, but it cannot indicate in real time whether there are SARS-CoV-2 particles in the subject's body, and thus cannot judge whether the subject is infectious.

[0004] In addition, in the broader field of virus detection, there are also reports on the use of sugar-gold nanoprobes for virus detection. However, these reports generally use natural sialic acid ligands as detection molecules to modify the surface of gold nanoparticles to construct probes. The recognition and binding of natural sialic acid ligand molecules to viral surface proteins is a low-specificity, low-affinity recognition and binding effect, and its detection specificity and sensitivity are insufficient. There are currently no reports on the use of gold nanoprobes to detect new coronavirus particles. Summary of the invention

[0005] In order to solve the problems raised in the above background technology, the purpose of the present invention is to provide a biological probe and a preparation method and application thereof.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] 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;

[0008] The general structural formula of the customized sialic acid ligand molecule is shown in Formula I: wherein R1 is selected from hydroxy, methoxy or substituted methoxy; R2 is selected from acetylamino, substituted acetylamino, benzylamino, substituted benzoyl, alkoxycarbonylamide, triazolyl or substituted triazolyl; R3 is selected from hydroxy, methoxy, substituted methoxy, acetylamino, substituted acetylamino, sulfonamide or phosphoramide; 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 the sugar binding domain pocket of the SARS-CoV-2 S protein by derivatization of the R1, R2, and R3 groups, and has high specificity and binding force to the SARS-CoV-2 S protein.

[0009] Further, the substituted methoxy group is X1-CH2O-, wherein X1 is selected from phenyl or vinyl;

[0010] The substituted acetylamino group is X2-CH2CONH-, wherein X2 is selected from methyl, ethyl, n-propyl, isopropyl, hydroxymethyl or hydroxyethyl;

[0011] The substituted benzylamino group is X3-BzNH-, wherein the number of X3 is at least 1 and is located at any position of the benzene ring, and X3 is selected from at least one of a halogen atom, a methyl group, a methoxy group, and a nitro group;

[0012] The alkoxycarbonylamide group is X4-OC(O)NH-, wherein X4 is selected from benzyl, allyl, tert-butyl or trichloroethyl;

[0013] The substituted triazole group is wherein X5 is selected from substituted phenylalkyl (X3-Ph-(CH2) n1 -) or substituted carbonyl (X6-C(O)-), wherein the number of X3 is at least 1 and is located at any position of the benzene ring, X3 is selected from at least one of halogen atoms, methyl, methoxy, and nitro, and n1 is selected from 0, 1, and 2; X6 is selected from alkoxy CH3-(CH2) n2 -O- or alkylamino CH3-(CH2) n2 -NH-, n2 is selected from 0, 1, 2, 3, 4, 5.

[0014] Furthermore, the probe carrier is selected from nanomaterials with sensitive LSPR effect or substances with obvious signal changes in the dispersed-aggregated state;

[0015] Preferably, the nanomaterial with sensitive LSPR effect includes gold-based nanoparticles and composite nanoparticles with a metal gold layer on the surface; more preferably, the gold-based nanoparticles include gold nanorods and gold nanospheres;

[0016] Preferably, the substance having an obvious signal change in the dispersed-aggregated state includes an aggregation-induced emission AIE material; the aggregation-induced emission AIE material includes a tetrathienylthiophene compound having a cyclic polyene skeleton, a compound having a cyano-substituted diphenylethylene skeleton, a tetraphenylethylene type compound, a divinyl anthracene type compound, a triphenylethylene type compound, etc., and the molecular end of the aggregation-induced emission AIE material has a maleimide derivative group that reacts with a thiol group.

[0017] Further, the probe carrier is a gold nanorod;

[0018] Preferably, the surface of the gold nanorods is coated with a double molecular layer of hexadecyltrimethylammonium bromide;

[0019] 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.

[0020] Furthermore, the molar ratio of the customized sialic acid ligand molecule to the gold nanorod is 1×10 4 :1~2×10 4 :1.

[0021] In another aspect, the present invention provides a method for preparing any of the above-mentioned biological probes, comprising the following steps:

[0022] 1) providing a probe carrier solution and a customized sialic acid ligand molecule having a general structural formula I;

[0023] 2) preparing a customized sialic acid ligand molecule having a general structural formula I into an aqueous solution, mixing it with a probe carrier solution, and standing at 25-30° C. for 12-24 hours to prepare a biological probe;

[0024] Preferably, the probe carrier solution is a gold nanorod solution, the surface of the gold nanorod is coated with a hexadecyltrimethylammonium bromide bilayer; the gold nanorod has a diameter of 20-25 nm and a length of 60-80 nm; the customized sialic acid ligand molecule is fixed to the surface of the gold nanorod by forming an S-Au bond on the surface of the gold nanorod;

[0025] Preferably, the molar ratio of the customized sialic acid ligand molecule to the gold nanorod is 1×10 4 :1~2×10 4 :1.

[0026] Furthermore, 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 nano seed solution; then adding the gold nano seed solution to a mixture of chloroauric acid HAuC14 solution, silver nitrate AgNO3 solution, dilute hydrochloric acid HCl, ascorbic acid solution, and hexadecyltrimethylammonium bromide CTAB solution for static reaction to generate a gold nanorod solution with a width of 20-25 nm, a length of 60-80 nm, and a surface covered with hexadecyltrimethylammonium bromide;

[0027] Preferably, the molar ratio of sodium borohydride to chloroauric acid is 2:1 to 3:1, the time for reducing chloroauric acid with sodium borohydride is 5 to 20 minutes, and the temperature for reducing chloroauric acid with sodium borohydride is 25 to 35° C.;

[0028] The concentration of the chloroauric acid HAuC14 solution is 4.5-5.5 mM, preferably 5 mM, the concentration of the silver nitrate AgNO3 solution is 0.09-0.12 M, preferably 0.1 M, the concentration of the dilute hydrochloric acid HCl is 1.0-1.5 mM, preferably 1.2 mM, the concentration of the ascorbic acid solution is 8-15 mM, preferably 10 mM, and the concentration of the hexadecyltrimethylammonium bromide CTAB solution is 0.15-0.25 M; the volume ratio of the gold nanoseed solution, the chloroauric acid solution, the silver nitrate solution, the dilute hydrochloric acid, the ascorbic acid solution, and the hexadecyltrimethylammonium 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, the temperature of the static reaction is 25-40°C, and the time of the static reaction is 6-24 hours.

[0029] Furthermore, the preparation method of the customized sialic acid ligand molecule having the general structural formula I is:

[0030] Sialic acid As the starting material, the carboxyl group is protected and the R4 protecting group is introduced into the carboxyl group to obtain In the next step, the fully acetylated 2-chlorosugar is generated by the action of chlorination and acetylation reagents. Then, the functional side chain precursor is derived at the 2nd position of the sugar ring through glycosylation reaction. After deacetylation and carboxyl protection, the intermediate with both amino and hydroxyl groups exposed on the sugar ring was obtained. The groups were introduced in the order of R2→R1→R3 to obtain Finally, the customized sialic acid sugar ligand I was synthesized through side chain group conversion and sugar ring protecting group removal reaction;

[0031] Sialic acid As the starting material, the carboxyl group is protected and the R4 protecting group is introduced into the carboxyl group to obtain In the next step, the fully acetylated 2-chlorosugar is generated by the action of chlorination and acetylation reagents. Then, the functional side chain precursor is derived at the 2nd position of the sugar ring through glycosylation reaction. After deacetylation and carboxyl protection, the intermediate with both amino and hydroxyl groups exposed on the sugar ring was obtained. The groups were introduced in the order of R2→R3→R1 to obtain Finally, the customized sialic acid sugar ligand I was synthesized through side chain group conversion and sugar ring protecting group removal reaction;

[0032] Wherein, R4 is selected from methyl or benzyl;

[0033] A1 is selected from or -(CH2) m -R5; R5 is selected from benzyloxy, 2-naphthylmethoxy or allyloxy, n is 0,1,2,3,4,5,6,7, m is 2,3,4,5,6,7,8,9;

[0034] PG is the abbreviation of protection group;

[0035] In particular, when R1 is hydroxy, it is sialic acid The groups on the molecule do not need to be introduced into the relevant reaction process of the R1 group;

[0036] When R2 is acetylamino, it is sialic acid The groups on the molecule do not need to be introduced into the relevant reaction process of the R2 group;

[0037] When R3 is hydroxy, it is sialic acid The groups on the molecule do not need to be introduced into the relevant reaction process of the R3 group.

[0038] On the other hand, the present invention provides an application of any of the above-mentioned biological probes in a novel coronavirus detection reagent.

[0039] Furthermore, the method of using the biological probe to detect the new coronavirus includes the following steps: mixing the biological probe with the sample to be tested, letting it stand for 15-30 minutes after mixing evenly, and detecting the new coronavirus by observing the color of the mixed system;

[0040] Preferably, the method for detecting the novel coronavirus using a biological probe specifically includes the following steps: 1) taking two identical biological probe aqueous solutions, marked as solution C (Control solution, C solution) and solution T (Test solution, T solution), adding a control solution to solution C, adding a test sample to solution T, mixing them evenly, standing for 15-30 minutes, and observing the results; 2) determining the results: 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, regardless of whether solution T changes color or not, another biological probe aqueous solution should be taken for re-testing;

[0041] Preferably, the biological probe is a customized sialic acid ligand molecule-gold nanorod biological probe;

[0042] Preferably, the concentration of the customized sialic acid ligand molecule-gold nanorod bioprobe aqueous solution is 10-100 μM;

[0043] Preferably, the volume ratio of the sample to be tested to the customized sialic acid ligand molecule-gold nanorod bioprobe aqueous solution is 1:5 to 1:20;

[0044] Preferably, the detection limit of the novel coronavirus in the sample to be tested is 10 4 Pieces / mL;

[0045] Preferably, the sample to be tested is saliva, nasopharyngeal swab, blood, or skin secretion;

[0046] Preferably, the control solution is deionized water.

[0047] The present invention provides a biological probe that can be used to directly detect novel coronavirus particles. Compared with existing novel coronavirus and related virus detection methods, it has the following beneficial effects:

[0048] 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;

[0049] 2) The biological probe provided by the present invention is used for the detection of the new coronavirus through parallel reaction, and visual judgment can be made through color reaction in only about 15 minutes;

[0050] 3) Compared with the nucleic acid detection method, the biological probe provided by the present invention for the detection of the new coronavirus does not involve multiple procedures such as sample RNA extraction and PCR. It can be directly added after sampling, which simplifies the detection structure and avoids interference caused by manual operation errors;

[0051] 4) Compared with the IgG / IgM detection method of the new coronavirus, the biological probe provided by the present invention is a method for directly detecting new coronavirus particles, which can instantly characterize whether there are new coronavirus particles in the sample;

[0052] 5) The present invention detects by binding sialic acid small molecules to the S protein on the surface of the new coronavirus. The sialic acid detection small molecules have obvious advantages such as good economy, high stability, and can be prepared in large quantities;

[0053] 6) The present invention provides a biological probe that improves the recognition and binding of target proteins by customizing the structure of sialic acid ligand molecules on the probe surface. Compared with natural sugar ligand detection molecules, the specificity and sensitivity of detection are greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is a schematic diagram of the method for detecting novel coronavirus particles of the present invention. After changing to black and white, the change of the probe LSPR optical signal is not obvious, where the left picture is tea red and the right picture is dark gray;

[0055] Figure 2 is a schematic diagram of the biological probe provided by the present invention;

[0056] Figure 3 is a flow chart of the method for preparing a biological probe provided by the present invention;

[0057] Figure 4 It is a general preparation flow chart of the customized sialic acid ligand molecule of general formula I of the present invention according to the order of introduction of R2→R1→R3 groups;

[0058] Figure 5 It is a general preparation flow chart of the customized sialic acid ligand molecule of general formula I of the present invention according to the order of introduction of R2→R3→R1 groups. DETAILED DESCRIPTION

[0059] Taking the customized sialic acid ligand molecule-gold nanorod bioprobe as an example, the direct detection method of the new coronavirus provided by the present invention is based on the Spike protein on the surface of the new coronavirus. Its core principle is to use the recognition and binding effect of the customized sialic acid ligand molecule on the S protein on the surface of the new coronavirus for detection, and to characterize the binding effect through the change of the optical absorption signal shown by the LSPR effect of the gold nanorod probe. The corresponding detection principle and detection reagent are described in detail as follows:

[0060] Detection principle and process:

[0061] After the sample to be tested is mixed with the dispersed biological probe, 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 probes. Since there are abundant S protein molecules on the surface of the new coronavirus, and the surface of the gold nanorods also has multivalent sialic acid ligand structures, the two undergo multivalent binding to each other to form an agglomerate structure. Since the gold nanorods have the LSPR effect, they have optical absorption signals and are very sensitive to changes in the surface environment. When the gold nanorod probes in the detection reagent change from a uniformly dispersed state to an agglomerated state, the optical absorption exhibited by the LSPR effect of the gold nanorods changes significantly, and the detection results can even be judged by visually judging the change, such as Figure 1 shown.

[0062] Detection reagents:

[0063] The detection reagent of the present invention comprises a uniformly dispersed customized sialic acid ligand molecule-gold nanorod biological probe aqueous solution, wherein the customized sialic acid ligand molecule is fixed on the surface of the gold nanorod stabilized by cetyltrimethylammonium bromide (CTAB) through an S-Au bond, and the sialic acid ligand molecule can better match the sugar binding domain pocket of the S protein on the surface of the new coronavirus by introducing corresponding groups on the sugar ring, thereby having a high specific recognition and high affinity binding effect on the S protein of the new coronavirus. The key components of the detection reagent are as follows: Figure 2 shown.

[0064] The preparation method of customized sialic acid ligand molecule-gold nanorod biological probe is as follows Figure 3 As shown; the preparation flow chart of customized sialic acid ligand molecules is as follows Figure 4 or Figure 5 shown.

[0065] In order to better understand the content of the present invention, the content of the present invention is further described below in conjunction with specific implementation methods, but the protection content of the present invention is not limited to the following embodiments.

[0066] Example 1: Synthesis of Customized Sialic Acid Ligand Molecule 1

[0067]

[0068] Natural sialic acid Neu5Ac (10 g, 32.3 mmol) was dissolved in ethanol (100 mL), potassium carbonate (5.36 g, 38.8 mmol) was added, and after stirring for 15 minutes, benzyl bromide (6.08 g, 35.6 mmol) was added dropwise, and stirred at 50 ° C for 2 hours. The reaction was complete by TLC detection, and the reaction liquid was dried by rotary evaporation. The residue was evaporated three times by adding toluene and dried to obtain a crude intermediate of formula 1-1, which was directly used in the next step;

[0069] The crude product of formula 1-1 obtained in the previous step was dissolved in chloroacetyl (20 mL), stirred at room temperature until the reaction was complete, and the solvent was evaporated to obtain a crude product of formula 1-2, which was directly used in the next step;

[0070] The crude product of formula 1-2 obtained in the previous step was mixed with HOPEG4OBn (13.79 g, 48.5 mmol) in dry dichloromethane (60 mL), and silver carbonate (17.83 g, 64.7 mmol) was added under ice bath, and the reaction was carried out for 10 h. The reaction was completed by TLC detection, and the insoluble matter was filtered out. The residue was concentrated and separated by column to obtain the compound of formula 1-3 (19.3 g, 71.6% in 3 steps). ESI-MS m / z calcd for [C 41 H 56 NO 17 ] + (M+H) + :834.88,found:834.88;

[0071] The compound of formula 1-3 (19.3 g, 23.14 mmol) was dissolved in methanol (60 mL), sodium methoxide (0.1 M) was added to adjust the pH of the reaction solution to 8-10, and stirred for 30 min. The reaction was completed by TLC detection, and the reaction solution was neutralized to neutrality. The solvent was evaporated, and toluene was added to the residue and evaporated three times to obtain compound 1-4 (15.2 g, 98.7%). ESI-MS m / z calcd for [C 33 H 48 NO 13 ] + (M+H) + :666.73,found:666.72;

[0072] 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. The reaction was complete when detected by TLC. Methanol was added, and the mixture was stirred for 10 minutes. The solvent was evaporated, and the residue was dissolved in dichloromethane. The organic phase was washed with brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The residue was dissolved in N,N-dimethylformamide (20 mL), and sodium azide (3.70 g, 57. 08mmol), stirred in an oil bath at 50℃ for 12 hours, TLC detected that the reaction was complete, evaporated the solvent, dissolved the residue in tetrahydrofuran (40mL), added triphenylphosphine (10.48g, 39.95mmol), and continued stirring for 15 minutes until no bubbles were generated. The reaction solution was directly evaporated to remove the solvent, the residue was dissolved in ethanol (20mL), and acetic anhydride (3.03g, 29.68mmol) was added dropwise. After stirring for 12 hours, the solvent was evaporated, and the residue was directly mixed with a sample column for separation to obtain compound 1-5 (11.6g, 71.9%), ESI-MS m / z calcd for [C 35 H 51 N2O 13 ] + (M+H) + :707.79,found:707.80;

[0073] The compound of formula 1-5 (11.6 g, 16.41 mmol) was dissolved in methanol (40 mL), and Pd / C catalyst (0.1 g) was added. The mixture was stirred for 6 hours in a 4 atm H2 atmosphere. The reaction was complete when detected by TLC. The Pd / C powder was filtered off and the filtrate was concentrated to obtain the compound of formula 1-6 (8.4 g, 97.2%). ESI-MS m / z calcd for [C 21 H 39 N2O 13 ] + (M+H) + :527.54,found:527.54;

[0074] The compound of formula 1-6 (8.4 g, 16.41 mmol) was dissolved in dichloromethane (40 mL), triethylamine (3.23 g, 31.91 mmol) was added, and p-toluenesulfonyl chloride (4.56 g, 23.93 mmol) was added in batches, and stirred overnight. The reaction was complete by TLC detection. Methanol was added and stirred for 10 minutes. The solvent was evaporated and the residue was dissolved in dichloromethane. The organic phase was washed with brine and dried over anhydrous sodium sulfate. After concentration, the residue was dissolved in N,N-dimethylformamide (50 mL), potassium thioacetate (3.54 g, 30.97 mmol) was added, and stirred in an oil bath at 50°C for 12 hours. The reaction was complete by TLC detection. The solvent was evaporated and the residue was separated and purified by column chromatography to obtain the compound of formula 1-7 (7.6 g, 81.5%). ESI-MS m / z calcd for [C 23 H 41 N2O 13 S] + (M+H) + :585.63,found:585.62;

[0075] The compound of formula 1-7 (7.6 g, 13.00 mmol) was dissolved in methanol (50 mL), sodium methoxide (0.1 M in MeOH) was added, the pH of the reaction solution was adjusted to 8-10, and the mixture was stirred at room temperature for 15 minutes. After TLC detection, the reaction was complete, and 732-type H + After stirring for 5 minutes, the resin was filtered off and the filtrate was concentrated to obtain the compound of formula 1 (6.9 g, 97.8%). ESI-MS m / z calculated for [C 21 H 39 N2O 12 S] + (M+H) + :542.60,found:542.59.

[0076] Example 2: Synthesis of Customized Sialic Acid Ligand Molecule 2

[0077]

[0078] Concentrated hydrochloric acid (1 mL) was added dropwise to methanol (100 mL), and natural sialic acid Neu5Ac (10 g, 32.3 mmol) was added in batches. The reaction mixture was stirred until the solution was clear, and the reaction solution was neutralized. The solvent was evaporated, and the residue was evaporated three times with toluene and dried to obtain a crude intermediate of formula 2-1, which was directly used in the next step.

[0079] The crude product of formula 2-1 obtained in the previous step was dissolved in chloroacetyl (40 mL), stirred at room temperature until the reaction was complete, and the solvent was evaporated to obtain a crude product of formula 2-2, which was directly used in the next step;

[0080] The crude product of formula 2-2 obtained in the previous step was mixed with HOPEG4OBn (13.8 g, 48.5 mmol) in dry dichloromethane (60 mL), and silver carbonate (17.83 g, 64.7 mmol) was added under ice bath. The reaction was carried out for 12 h. The reaction was completed by TLC detection. The insoluble matter was filtered out, and the residue was concentrated and separated by column to obtain the compound of formula 2-3 (19.3 g, 78.8% in 3 steps). ESI-MS m / z calcd for [C 35 H 52 NO 17 ] + (M+H) + :758.78,found:758.77.

[0081] The compound of formula 2-3 (19.3 g, 25.47 mmol) was dissolved in methanol (40 mL), and a NaOH aqueous solution (1.5 M, 20 mL) was added dropwise, and the mixture was stirred until the reaction was complete as detected by TLC. The reaction solution was neutralized, and the solvent was evaporated. The residue was dissolved in a 2M NaOH aqueous solution (40 mL), and refluxed in an oil bath at 95°C for 10 hours. The reaction solution was neutralized, and the solvent was evaporated. The residue was azeotropically evaporated with toluene three times and then dissolved in hydrochloric acid / methanol (v / v=1:99, 50 mL). The mixture was stirred until the reaction was complete as detected by TLC. The reaction solution was neutralized, and the solvent was evaporated to obtain a crude compound of formula 2-4, which was directly used in the next step. ESI-MS m / z calcd for [C 25 H 42 NO 12 ] + (M+H) + :548.60,found:548.59;

[0082] Sodium azide NaN3 (1.99 g, 30.56 mmol) was dissolved in water (30 mL), and trifluoromethanesulfonic anhydride (8.62 g, 30.56 mmol) was slowly added dropwise, and stirred for 15 minutes. The aqueous phase was extracted with toluene, and the organic phase was dried over anhydrous sodium sulfate to obtain trifluoromethanesulfonyl azide TfN3. The crude compound of formula 2-4 obtained in the previous step was dissolved in ethanol, and the prepared TfN3 and copper sulfate CuSO4 (0.812 g, 5.09 mmol) were added. Stir for 6 hours, and the solvent was evaporated. The residue was dissolved in pyridine (50 mL), and acetic anhydride (26 g, 254.7 mmol) was added dropwise under ice bath conditions. Stir at room temperature for 12 hours, and methanol was added dropwise under ice bath to quench the reaction. The solvent was evaporated, and the residue was dissolved in dichloromethane and successively treated with 1N HCl a.q. 、sat.NaHCO 3a.q. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain the compound of formula 2-5 (18.1 g, 95.8%). ESI-MS m / z calcd for [C33 H 47 N3O 16 ] + (M+H) + :742.74,found:742.73;

[0083] The compound of formula 2-5 (18.1 g, 24.4 mmol) was mixed with methyl acrylate (2.46 g, 29.28 mmol) and cuprous iodide (0.93 g, 4.88 mmol) and dissolved in tetrahydrofuran (THF) (60 mL). N, N-diisopropylethylamine DIPEA (4.73 gm, 36.6 mmol) was added dropwise. The mixture was stirred at room temperature for 1 hour. The reaction solvent was evaporated and the residue was dissolved in dichloromethane and then treated with 1M ammonia and 1N HCl. a.q. 、sat.NaHCO 3a.q. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain the compound of formula 2-6 (19.6 g, 97.3%). ESI-MS m / z calcd for [C 37 H 52 N3O 18 ] + (M+H) + :826.82,found:826.81;

[0084] The compound of formula 2-6 (19.6 g, 23.73 mmol) was dissolved in methanol (100 mL), sodium methoxide (0.1 M in MeOH) was added, the pH of the solution was adjusted to 8-10, and the mixture was stirred at room temperature for 30 minutes. Hydrochloric acid was added dropwise to neutralize the reaction solution, and the solvent was evaporated to obtain a crude product of formula 2-7, which was directly used in the next step;

[0085] The crude product of formula 2-7 obtained in the previous step was dissolved in dichloromethane (100 mL), triethylamine (4.8 g, 47.47 mmol) was added, and p-toluenesulfonyl chloride (6.79 g, 35.60 mmol) was added in batches after stirring for 5 minutes. The mixture was stirred at room temperature for 6 hours, and methanol was added to quench the reaction. The solvent was evaporated, and the residue was dissolved in dichloromethane and washed with brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated. The residue was dissolved in N, N-dimethylformamide (50 mL), and NaN3 (4.63 g, 71.20 mmol) was added. The mixture was stirred in an oil bath at 50°C for 12 hours. The reaction was complete by TLC detection, and the solvent was evaporated. The residue was dissolved in dichloromethane, washed with brine, dried over anhydrous sodium sulfate, concentrated, and then column chromatography was performed to obtain the compound of formula 2-8 (14.3 g, 88.3%). ESI-MS m / z calcd for [C 29 H 43 N6O 13 ] + (M+H) +:683.68,found:683.69;

[0086] The compound of formula 2-8 (14.3 g, 20.95 mmol) was dissolved in tetrahydrofuran (100 mL), triphenylphosphine (16.48 g, 62.84 mmol) was added in batches, and the mixture was stirred until no bubbles were generated. Stirring was continued for 12 hours, and the solvent was evaporated. The residue was dissolved in ethanol, and benzoyl chloride (3.53 g, 25.14 mmol) was slowly added dropwise. The mixture was stirred at room temperature for 1 hour, and the solvent was evaporated. The residue was directly purified by column chromatography to obtain the compound of formula 2-9 (13.1 g, 82.2%). ESI-MS m / z calcd for [C 36 H 49 N4O 14 ] + (M+H) + :761.79,found:761.78;

[0087] The compound of formula 2-9 (13.1 g, 17.22 mmol) was dissolved in acetonitrile (50 mL), acetone dimethyl acetal (5.38 g, 51.66 mmol) and a catalytic amount of p-toluenesulfonic acid (0.3 g, 1.72 mmol) were added, and refluxed in an oil bath at 80°C for 12 hours. The reaction was complete when detected by TLC. Triethylamine was added to neutralize the reaction solution, the solvent was evaporated, and the residue was directly purified by column chromatography to obtain the compound of formula 2-10 (12.8 g, 92.8%). ESI-MS m / z calcd for [C 39 H 53 N4O 13 ] + (M+H) + :801.86,found:801.86;

[0088] The compound of formula 2-10 (12.8 g, 15.98 mmol) and freshly prepared silver oxide (7.41 g, 31.97 mmol) were mixed in N, N-dimethylformamide (50 mL), iodomethane (3.40 g, 23.97 mmol) was added, and stirred at room temperature for 4 hours. The reaction was complete by TLC detection, and the insoluble matter was filtered off. The solvent was evaporated, and the residue was dissolved in dichloromethane, washed with brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the compound of formula 2-11 (11.6 g, 89.1%), ESI-MS m / z calcd for [C 40 H 55 N4O 14 ] + (M+H) + :815.89,found:815.88;

[0089] The compound of formula 2-11 (11.6 g, 14.24 mmol) was dissolved in methanol, p-toluenesulfonic acid (0.25 g, 1.42 mmol) was added, and the mixture was refluxed in an oil bath at 80°C for 6 hours. The reaction was complete when detected by TLC. Triethylamine was added to neutralize the reaction solution, and the solvent was evaporated. The residue was dissolved in dichloromethane, washed with brine, dried over anhydrous sodium sulfate, concentrated, and dissolved in methanol. Pd / C powder (0.1 g) was added, and the mixture was reacted in a H2 atmosphere at 4 atm pressure for 2 hours. The Pd / C powder was filtered off, and the filtrate was concentrated to obtain a crude compound of formula 2-12, which was directly used in the next step. ESI-MS m / z calcd for [C 30 H 45 N4O 14 ] + (M+H) + :685.70,found:685.69;

[0090] The crude compound of formula 2-12 obtained in the previous step was dissolved in dichloromethane (60 mL), triethylamine (2.88 g, 28.47 mmol) was added, and p-toluenesulfonyl chloride (4.07 g, 21.35 mmol) was added in batches after stirring for 5 minutes. The mixture was stirred at room temperature for 6 hours, and methanol was added to quench the reaction. The solvent was evaporated, and the residue was dissolved in dichloromethane and washed with brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated. The residue was dissolved in acetonitrile (40 mL), KSAc (3.25 g, 28.47 mmol) was added, and stirred at room temperature for 4 hours. The reaction was complete by TLC detection, and the solvent was evaporated. The residue was dissolved in dichloromethane, washed with brine, dried over anhydrous sodium sulfate, concentrated, and then column chromatography was performed to obtain the compound of formula 2-13 (8.9 g, 84.2%), ESI-MS m / z calcd for [C 32 H 47 N4O 14 S] + (M+H) + :743.79,found:743.78;

[0091] The compound of formula 2-13 (8.9 g, 11.98 mmol) was dissolved in methanol (30 mL), and an aqueous solution of NaOH (1.5 M, 10 mL) was added. The mixture was stirred at room temperature for 30 minutes, and 732 H + The reaction solution was neutralized with cationic resin, the resin was filtered off, and the filtrate was concentrated to obtain the compound of formula 2 (7.5 g, 91.2%). ESI-MS m / z calcd for [C 29 H 43 N4O 13 S] + (M+H) + :687.73,found:687.72;

[0092] Example 3: Preparation of sialic acid-gold nanorod probe 3

[0093]

[0094] 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 a CTABB-covered gold nanorod solution (OD=15, 5 mL) with a length of 75 nm and an aspect ratio of 3:1. After standing for 15 hours, the solution was centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the aggregates on the centrifuge tube substrate were redispersed in deionized water (5 mL) to obtain sialic acid-gold nanorod probe 3;

[0095] The preparation process of the gold nanorod solution includes: adding HAuCl4 aqueous solution (0.5mM, 5mL) to CTAB aqueous solution (0.2M, 5mL), adding freshly prepared sodium borohydride NaBH4 icy aqueous solution (10mM, 600μL) under vigorous stirring, and continuing stirring for 10 minutes to obtain a gold nanorod seed solution; adding HAuCl4 aqueous solution (5mM, 6mL) and silver nitrate AgNO3 aqueous solution (0.1mM, 75μL) to CTAB aqueous solution. B aqueous solution (0.2M, 30mL), continue to add hydrochloric acid (1.2mM, 75μL) and sodium ascorbate Vc-Na aqueous solution (10mM, 3.7mL), shake the solution until colorless, add the prepared gold nanoseed solution (50μL), stir for 5 minutes, let stand in a 37℃ constant temperature box for 16 hours, centrifuge at 8000rpm 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 40mL) to obtain a gold nanorod solution.

[0096] Example 4: Preparation of sialic acid-gold nanorod probe 4

[0097]

[0098] The customized sialic acid ligand molecule 2 obtained in Example 2 was dissolved in water to prepare an aqueous solution (10 mM), and 100 μL was added to a CTABB-covered gold nanorod solution (OD=15, 5 mL) with a length of 75 nm and an aspect ratio of 3:1. After standing for 15 hours, the solution was centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the aggregates on the centrifuge tube substrate were redispersed in deionized water (5 mL) to obtain sialic acid-gold nanorod probe 4.

[0099] The preparation process of the gold nanorod solution includes: adding HAuCl4 aqueous solution (0.5mM, 5mL) to CTAB aqueous solution (0.2M, 5mL), adding freshly prepared sodium borohydride NaBH4 icy aqueous solution (10mM, 600μL) under vigorous stirring, and continuing stirring for 10 minutes to obtain a gold nanorod seed solution; adding HAuCl4 aqueous solution (5mM, 6mL) and silver nitrate AgNO3 aqueous solution (0.1mM, 75μL) to CTAB aqueous solution. B aqueous solution (0.2M, 30mL), continue to add hydrochloric acid (1.2mM, 75μL) and sodium ascorbate Vc-Na aqueous solution (10mM, 3.7mL), shake the solution until colorless, add the prepared gold nanoseed solution (50μL), stir for 5 minutes, let stand in a 37℃ constant temperature box for 16 hours, centrifuge at 8000rpm 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 40mL) to obtain a gold nanorod solution.

[0100] Example 5: Detection Experiment 1

[0101] The SARS-CoV-2 particles are highly contagious and pathogenic, but the detection of the SARS-CoV-2 in the present invention only involves the S protein on the surface of the SARS-CoV-2 and does not involve its internal structure. Therefore, commercial SARS-CoV-2 S protein can be fixed on the surface of SiO2 nanoparticles with a particle size of 100 nm to simulate the SARS-CoV-2 particles, and detection experiments can be carried out using the simulated particles.

[0102] Preparation of novel coronavirus simulated particles: Take SiO2 nanoparticles with a particle size of 100 nm (McLean brand, 115 mg, about 10 14 ), suspended in N,N-dimethylformamide (5 mL), ultrasonicated for 1 hour, added with (3-aminopropyl)trimethoxysilane (347 mg, 1.91 mmol), added with 1N HCl aqueous solution (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, washed the aggregates at the bottom of the centrifuge tube twice by centrifugation, dispersed in 10% dimethyl sulfoxide aqueous solution DMSO (50 mL), added with PBS buffer (pH 7.4, 50mL), to prepare SiO2 nanoparticle treatment solution for standby use; SARS-CoV-2 S protein (Sino Biologics brand, 5 mg) was dissolved in a mixed solvent (1 mL) of DMSO:PBS:H2O=5:50:45, N-hydroxysuccinimide NHS (2.1 mg, 18 μmol) was added, and after slow stirring for 1 hour, it was added to the prepared SiO2 nanoparticle treatment solution (20 mL), stirred for 2 hours, centrifuged at 6000 rpm for 20 minutes, the supernatant was discarded, the aggregates at the bottom of the centrifuge tube were washed once by centrifugation, and dispersed in PBS buffer (pH 7.4, 200 mL) to prepare the SARS-CoV-2 simulated particle stock solution, wherein the number of simulated particles was about 10 11 / mL, stored in -20℃ freezer.

[0103] Take 2 portions of the gold nanorod probe 3 solution obtained in Example 3, 1 mL each, and label them as liquid C and liquid T, respectively. Add 1 drop (about 50 μL) of deionized water control solution to liquid C, and add 1 drop of SARS-CoV-2 simulated particle solution (thawed and diluted 10 μL of simulated particle stock solution) to liquid T. 4 After mixing well, let it stand for 15 minutes and observe the color changes of liquid C and liquid T. The color of liquid C did not change and appeared tea red, while the color of liquid T changed significantly and appeared dark gray.

[0104] Example 6: Detection Experiment 2

[0105] Take 2 portions of the gold nanorod probe 4 solution obtained in Example 4, 1 mL each, and label them as solution C and solution T, respectively. Add 1 drop (about 50 μL) of deionized water reference solution to solution C, and add 1 drop of the new coronavirus simulated particle solution (thawed and diluted 10 μL of the simulated particle stock solution) to solution T. 4 After mixing well, let it stand for 15 minutes and observe the color changes of liquid C and liquid T. The color of liquid C did not change and appeared tea red, while the color of liquid T changed significantly and appeared purple gray.

[0106] Example 7: Parallel detection experiment

[0107] Take 3 portions of 1 mL each of the gold nanorod probe 4 solution obtained in Example 4 and mark them as liquid C, liquid T1, and liquid T2, respectively. Take a saliva sample from a healthy person and divide it into 2 portions, add deionized water (10%, i.e., deionized water volume: saliva volume = 1:9) to one portion and mark it as sample S1, add a simulated particle solution of the new coronavirus (10%, simulated particle solution volume: saliva volume = 1:9) to the other portion and mark it as sample S2. Add 1 drop (50 mL) of deionized water control solution, sample S1, and sample S2 to liquid C, liquid T1, and liquid T2, respectively, mix well, and let stand for 15 minutes to observe the color changes of liquid C, liquid T1, and liquid T2. Among them, the color of liquid C did not change and was tea red, the color of liquid T1 changed slightly and was tea red, and the color of liquid T2 changed significantly and was gray.

[0108] In summary, the present invention provides a method for detecting new coronavirus particles based on the LSPR effect of gold nanorods (GNR) probes. Customized sialic acid ligand molecules are fixed on the surface of gold nanorods, which can efficiently identify and bind to the Spike protein on the surface of the new coronavirus, thereby inducing the aggregation of dispersed gold nanoprobes. The changes in the dispersion state of the gold nanoprobes can be reflected by the color expressed by the LSPR effect, thereby realizing the detection of the new coronavirus.

[0109] The above description is only a specific implementation mode of the present invention, not all implementation modes. Any equivalent changes made to the technical solution of the present invention by ordinary technicians in this field after reading the specification of the present invention are covered by the claims of the present invention.

Claims

1. A biological probe, characterized in that: The biological probe includes a probe carrier and a customized sialic acid ligand molecule connected to the probe carrier; The general structural formula of the customized sialic acid ligand molecule is shown in Formula I: Wherein, R1, R2, R3 and A are selected from the following (1) or (2): (1) R1 is selected from hydroxyl group; R2 is selected from acetylamino group; R3 is selected from acetylamino group; A is n is selected from 0, 1, 2, 3, 4, 5, 6, 7, m is selected from 2, 3, 4, 5, 6, 7, 8, 9; (2) R1 is selected from methoxy; R2 is selected from substituted triazole, wherein the substituted triazole is wherein X5 is selected from substituted carbonyl CH3-OC(O)-; R3 is selected from benzamido; 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.

2. The biological probe according to claim 1, characterized in that The probe carrier is selected from nanomaterials having a sensitive LSPR effect.

3. The biological probe according to claim 2, characterized in that: The nanomaterial with sensitive LSPR effect is selected from gold-based nanoparticles or composite nanoparticles with a metal gold layer on the surface.

4. The biological probe according to claim 3, characterized in that: The gold-based nanoparticles are selected from gold nanorods or gold nanospheres.

5. The biological probe according to claim 1, characterized in that: The probe carrier is selected from substances having obvious signal changes in a dispersed-aggregated state.

6. The biological probe according to claim 5, characterized in that: The substance having an obvious signal change in a dispersed-aggregated state includes an aggregation-induced emission (AIE) material; the aggregation-induced emission (AIE) material includes a tetrathienylthiophene compound having a cyclic polyene skeleton, a compound having a cyano-substituted diphenylethylene skeleton, a tetraphenylethylene compound, a divinyl anthracene compound, and a triphenylethylene compound; the molecular end of the aggregation-induced emission material has a maleimide derivative group that reacts with a thiol group.

7. The biological probe according to claim 2, characterized in that: The probe carrier is a gold nanorod; the surface of the gold nanorod is coated with a hexadecyltrimethylammonium bromide double molecular layer.

8. The biological probe according to claim 7, characterized in that: The gold nanorod has a diameter of 20-25 nm, a length of 60-80 nm, and an aspect ratio of 2.5:1 to 3.5:

1.

9. The biological probe according to claim 7, characterized in that: The molar ratio of the customized sialic acid ligand molecule to the gold nanorods is 1×10 4 :1~2×10 4 :

1.

10. The method for preparing a biological probe according to any one of claims 1 to 9, characterized in that: The following steps are involved: 1) providing a probe carrier solution and a customized sialic acid ligand molecule having a general structural formula I; 2) The customized sialic acid ligand molecule having the structural formula I is prepared into an aqueous solution, mixed with the probe carrier solution, and allowed to stand at 25-30° C. for 12-24 hours to prepare the biological probe.

11. The preparation method according to claim 10, characterized in that: The probe carrier solution is a gold nanorod solution. The surface of the gold nanorod is coated with a hexadecyltrimethylammonium bromide double molecular layer. The gold nanorod has a diameter of 20-25 nm and a length of 60-80 nm.

12. The preparation method according to claim 11, characterized in that: The molar ratio of the customized sialic acid ligand molecule to the gold nanorods is 1×10 4 :1~2×10 4 :

1.

13. The preparation method according to claim 11, characterized in that: The method for preparing the gold nanorod solution is as follows: preparing the gold nanorods by a gold seed growth method in an aqueous phase, firstly reducing chloroauric acid with sodium borohydride to prepare a 1-2 nm gold nanoseed solution; The gold nano seed solution is then added to a mixture of chloroauric acid solution, silver nitrate solution, dilute hydrochloric acid, ascorbic acid solution, and hexadecyltrimethylammonium bromide solution and allowed to react to generate a gold nanorod solution with a width of 20 to 25 nm, a length of 60 to 80 nm, and a surface covered with hexadecyltrimethylammonium bromide.

14. The preparation method according to claim 13, characterized in that: The molar ratio of sodium borohydride to chloroauric acid is 2:1 to 3:1, the time for reducing chloroauric acid with sodium borohydride is 5 to 20 minutes, and the temperature for reducing chloroauric acid with sodium borohydride is 25 to 35° C.; The concentration of the chloroauric acid solution is 4.5-5.5 mM, the concentration of the silver nitrate solution is 0.09-0.12 M, the concentration of the dilute hydrochloric acid is 1.0-1.5 mM, the concentration of the ascorbic acid solution is 8-15 mM, and the concentration of the hexadecyltrimethylammonium bromide solution is 0.15-0.25 M; the volume ratio of the gold nano seed solution, the chloroauric acid solution, the silver nitrate solution, the dilute hydrochloric acid, the ascorbic acid solution, and the hexadecyltrimethylammonium 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, the temperature of the static reaction is 25-40° C., and the time of the static reaction is 6-24 hours.

15. The preparation method according to claim 10, characterized in that: The preparation method of the customized sialic acid ligand molecule having the general structural formula I is as follows: Sialic acid As the starting material, the carboxyl group is protected and the R4 protecting group is introduced into the carboxyl group to obtain In the next step, the fully acetylated 2-chlorosugar is generated by the action of chlorination and acetylation reagents. Then, the functional side chain precursor is derived at the 2nd position of the sugar ring through glycosylation reaction. After deacetylation and carboxyl protection, the intermediate with both amino and hydroxyl groups exposed on the sugar ring was obtained. The groups were introduced in the order of R2→R1→R3 to obtain Finally, the customized sialic acid sugar ligand I was synthesized through side chain group conversion and sugar ring protecting group removal reaction; Sialic acid As the starting material, the carboxyl group is protected and the R4 protecting group is introduced into the carboxyl group to obtain In the next step, the fully acetylated 2-chlorosugar is generated by the action of chlorination and acetylation reagents. Then, the functional side chain precursor is derived at the 2nd position of the sugar ring through glycosylation reaction. After deacetylation and carboxyl protection, the intermediate with all amino and hydroxyl groups on the sugar ring exposed was obtained. The groups were introduced in the order of R2→R3→R1 to obtain Finally, the customized sialic acid sugar ligand I was synthesized through side chain group conversion and sugar ring protecting group removal reaction; Wherein, R4 is selected from methyl or benzyl; A1 is selected from or -(CH2) m -R5; R5 is selected from benzyloxy, 2-naphthylmethoxy or allyloxy, n is 0,1,2,3,4,5,6,7, m is 2,3,4,5,6,7,8,9; When R1 is hydroxy, it is sialic acid The groups on the molecule do not need to be introduced into the relevant reaction process of the R1 group; When R2 is acetylamino, it is sialic acid The groups on the molecule do not need to be introduced into the relevant reaction process of the R2 group.

16. Use of the biological probe described in any one of claims 1-9 in the preparation of a novel coronavirus detection reagent.

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