CN110872335B

CN110872335B - Sialyloligosaccharide-quantum dot conjugate, preparation method and application thereof

Info

Publication number: CN110872335B
Application number: CN201811007634.4A
Authority: CN
Inventors: 李学兵; 张振兴; 祁晓晓
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2018-08-31
Filing date: 2018-08-31
Publication date: 2021-12-10
Anticipated expiration: 2038-08-31
Also published as: CN110872335A

Abstract

The invention relates to a sialyloligosaccharide-quantum dot conjugate, a preparation method thereof and application thereof in virus detection. The sialyloligosaccharide-quantum dot conjugate is prepared by a modular preparation method; the sialic acid oligosaccharide is used for carrying out specific recognition on the virus or the surface protein thereof, and further the conversion and amplification of signals are realized through energy resonance transfer between quantum dots and gold nanoparticles.

Description

Sialyloligosaccharide-quantum dot conjugate, preparation method and application thereof

Technical Field

The invention relates to the field of biological materials, in particular to a sialyloligosaccharide-quantum dot conjugate, a preparation method thereof and application of the conjugate in virus detection.

Background

Influenza (flu) is a zoonosis caused by influenza virus, and its host involves many animals such as human, pig, bird, horse and dolphin. Research HAs proved that the glycoprotein Hemagglutinin (HA) on the surface of influenza virus can specifically recognize the sugar chain receptor on the surface of host cells, which is the biological basis for influenza virus to infect host and then replicate and propagate. Certain influenza virus variants have high pathogenicity and/or high mortality for a variety of hosts, and pose a serious threat to animal and human health. Therefore, there is a need in the art for rapid and accurate detection of influenza viruses.

Currently, methods for detecting influenza viruses are increasingly diversified. The common methods include etiology detection, immunology detection, molecular biology detection and biochip detection. Etiology detection is the most rigorous and classical method; however, the conventional influenza virus isolation culture process requires one to two weeks, which is cumbersome to operate, takes a long period, and has low safety. The immunological method is simple to operate and can quickly obtain results, and the method becomes one of the most widely applied influenza virus detection methods. Among them, the hemagglutination test and the hemagglutination inhibition test have a simple principle and can discriminate subtypes, but are not highly sensitive and cannot be directly used for detecting a sample having a low virus concentration. The neuraminidase inhibition test can detect influenza viruses and identify NA subtypes of the influenza viruses, but the method has complicated operation steps and more influencing factors. The virus neutralization test is widely applied to detection and identification of viruses, and has high sensitivity and specificity, but the method is complicated in operation, long in time consumption and not suitable for clinical routine detection. The agarose gel diffusion test can detect the antigen of the influenza virus or the serum antibody generated after the infection of the influenza virus, and further analyze the subtype of the influenza virus, however, the method is long in time consumption, and the experimental result is difficult to quantify. The ELISA is simple in operation, strong in specificity and rapid in diagnosis, but cannot analyze the receptor specificity of influenza virus. The molecular biological detection method is relatively simple, easy to operate and capable of rapid diagnosis. Among them, the polymerase chain reaction has very high sensitivity, but is liable to give false positive results. The real-time fluorescent quantitative PCR can simultaneously realize the detection of various subtype influenza viruses, but has complex operation, needs expensive reagents and instruments and is not suitable for large-scale preliminary screening of samples to be detected. The biochip technology has high flux, can simultaneously and accurately detect and analyze a plurality of samples in a short time, but the biochip technology is not widely applied at present because the price of the biochip is high and the requirements on experimental conditions are high.

Nanotechnology developed in recent years offers new possibilities for biological detection. It has been found that various new materials including gold nanomaterials, platinum nanomaterials, magnetic nanomaterials, quantum dots, and the like each have unique physicochemical properties. Compared with the traditional organic fluorescent dye, the quantum dot has the advantages of high fluorescence intensity, difficult quenching and the like, so that a detection system established by using the quantum dot has good stability and high sensitivity.

In the invention, considering that Hemagglutinin (HA) on the surface of influenza virus can specifically recognize and combine different sialyloligosaccharide receptors, synthetic sialyloligosaccharide is respectively modified into quantum dots and gold nanoparticles, and qualitative and/or quantitative detection can be carried out on the influenza virus in a sample to be detected through Fluorescence Resonance Energy Transfer (FRET).

Disclosure of Invention

In a first aspect, the present invention provides a sialyloligosaccharide-quantum dot conjugate, the conjugate having the structure of formula X-I:

wherein, in formula X-I, QD is a quantum dot; m is an integer of 300-600; n is an integer of 0 to 12;

the above-mentioned

Selected from the group consisting of:

in the formulae X-II and X-III, R¹Is hydroxy or acetamido, R²Is hydroxy or L-fucosyl, R³Is hydroxyl or sulfate group, and x is an integer of 1-3.

In a second aspect, the present invention provides a kit comprising the sialyloligosaccharide-quantum dot conjugate of the first aspect, and preferably further comprising sialyloligosaccharide-gold nanoparticles.

In a third aspect, the present invention provides a method of preparing the sialyloligosaccharide-quantum dot conjugate of the first aspect, the method comprising the steps of:

(1) reacting the compound shown as the formula M-I with lipoic acid to obtain a compound shown as a formula M-II;

N₃CH₂(CH₂OCH₂)_nCH₂NH₂formula M-I;

(2) connecting the compound shown in the formula M-III to the compound shown in the formula M-II obtained in the step (1) through a click chemistry reaction to obtain the compound shown in the formula M-IV:

(3) deprotecting the hydroxyl group in the compound represented by the formula M-IV obtained in step (2) to obtain a compound represented by the formula M-V:

(4) sialylating the terminus of the compound of formula M-V obtained in step (3) to give a compound of formula M-VI:

(5) activating the compound of formula M-VI obtained in step (4) to obtain a compound of formula M-VII:

(6) connecting the compound shown in the formula M-VII obtained in the step (5) to the surface of the quantum dot to obtain the sialyloligosaccharide-quantum dot conjugate shown in the formula X-I:

in formula M-I, formula M-II, formula M-III, formula M-IV, formula M-V, formula M-VI, formula M-VII, and formula X-I, n is an integer from 0 to 12;

in the formula X-I, m is an integer of 300-600, and QD is a quantum dot;

in the formulae M-VI, M-VII and X-I, the

Selected from the group consisting of:

In a fourth aspect, the present invention provides the use of the sialyloligosaccharide-quantum dot conjugate of the first aspect or the kit of the second aspect for detecting a virus or a viral protein in a sample.

In a fifth aspect, the present invention provides a method of detecting a virus in a sample, the method comprising:

(i) incubating the sialyloligosaccharide-quantum dot conjugate and the sialyloligosaccharide-gold nanoparticle with a sample; and

(ii) (ii) detecting the FRET signal of the sample after the incubation of step (i).

Drawings

FIG. 1 is a schematic diagram of an embodiment in accordance with the present invention. The quantum dots with the surface modified with the sialyloligosaccharide and the gold nanoparticles with the surface modified with the sialyloligosaccharide have respective characteristic emission spectrums after being excited. When influenza virus exists in the system, due to the combination of sialyloligosaccharide and the influenza virus, the quantum dots are close to the nano gold particles in space, so that fluorescence resonance energy transfer exists between the quantum dots and the nano gold particles, and the emergent light intensity of the quantum dots is inversely related to the concentration of the influenza virus in the system under the excitation wavelength of the quantum dots.

Fig. 2 is a TEM image of a commercially available oil-soluble quantum dot (a) and a 3' SLac-quantum dot (b) prepared according to preparation example 3.

FIG. 3 is a TEM image of free 3 'SLAc-gold nanoparticles and 3' SLAc-quantum dots under the experimental system of example 1 of the present invention (a) and a TEM image of aggregation of 3 'SLAc-gold nanoparticles and 3' SLAc-quantum dots after addition of H7 protein (b).

Fig. 4 shows infrared absorption spectra of free quantum dots and 3' SLac-quantum dots prepared according to preparation example 3 of the present invention.

Fig. 5 shows nuclear magnetic resonance one-dimensional hydrogen spectra (NMR) of free quantum dots and 3' SLac-quantum dots prepared according to preparation example 3 of the present invention.

Fig. 6 shows the amount of modified sialyloligosaccharide on the quantum dot, as analyzed by a Thermogravimetric (TGA) curve.

FIG. 7 shows the detection results of 3 'SLAc-quantum dot and 3' SLAc-gold nanoparticle detection systems with different concentrations.

FIG. 8 shows the results of the detection of different concentrations of H7 protein according to example 1.

FIG. 9 shows the results of the detection of different concentrations of H5N1 influenza virus according to example 2.

FIG. 10 shows the results of the detection of different concentrations of H1N1 influenza virus according to example 3.

Detailed Description

Influenza viruses have a preference for recognition and binding of host sialic acid. For example, avian influenza viruses tend to bind to the sialic acid α 2,3Gal receptor (Sia α 2,3Gal, present in, e.g., avian intestines), while human influenza viruses predominantly bind to the sialic acid α 2,6Gal receptor (Sia α 2,6Gal, present in, e.g., human respiratory epithelial cells). The conjugates of the invention are capable of specifically recognizing influenza viruses that infect sialic acid α 2,3Gal receptor (e.g., conjugates of formula VI) and/or sialic acid α 2,6Gal receptor (e.g., conjugates of formula VII) host cells. The multiple sialyloligosaccharide-quantum dot conjugates can be independently packaged in the same kit, so that the parallel detection of the host specificity of the influenza virus is realized.

The sialyloligosaccharide is connected to the quantum dots through the connecting arms, so that the sialyloligosaccharide-quantum dot conjugate with the specificity recognition performance of an influenza virus host is obtained. The sialic acid oligosaccharide is used for carrying out specificity identification on the virus or the surface protein thereof, and then the conversion and amplification of signals are realized through the energy resonance transfer between the quantum dots and the gold nanoparticles modified by the sialic acid oligosaccharide, so that the detection of the existence of influenza viruses (including A-type and B-type influenza viruses) can be realized, the receptor specificity of the detected influenza viruses can be distinguished, and even the subtype of the virus can be determined through constructing and analyzing fingerprint maps with strong and weak interaction between different sialic acid oligosaccharides and different influenza virus strains.

With presently known sialyloligosaccharide conjugates, the sialic acid residue moiety and the detection moiety (e.g., magnetic nanoparticles, platinum nanoparticles, gold nanoparticles, etc.) are typically attached via an ester linkage. However, both peracid and overbased environments can cause cleavage of the ester bond. The invention firstly proposes that click chemical reaction can be carried out and then deprotection is carried out, so that sialic acid residue is connected with a detection part (quantum dot) through an amido bond, and the prepared sialyloligosaccharide-quantum dot is more stable.

Sialyloligosaccharide-quantum dot conjugate

The sialyloligosaccharide-quantum dot conjugate provided by the invention has a structure shown in a formula X-I:

in formula X-I, QD is a quantum dot; m is an integer of 300-600; n is an integer of 0 to 12;

the above-mentioned

Selected from the group consisting of:

In some preferred embodiments, in said formulae X-II and X-III, R¹Is acetamido, R²Is L-fucosyl, R³Is a hydroxyl group.

In some preferred embodiments, the conjugate has any one of the following structures:

in the formulae X-IV to X-IX, n is an integer of 0 to 12 and m is an integer of 300-600.

Preferably, in the formula X-I, the formula X-IV to the formula X-IX, n is an integer of 3 to 6.

Quantum dots are semiconductor nanocrystals composed of group IIB-VI (e.g., CdSe, CdTe, CdS, and ZnSe) or group IIIA-VA elements (e.g., InP and InAs). The quantum dots have unique optical performance, have wide absorption spectrum and narrow emission spectrum, and can display different colors and different emission waves through different quantum radius sizes. The quantum dots used in the present invention may be quantum dots having an absorption wavelength of 400-800nm, preferably an absorption peak at 520nm, in view of enabling a FRET reaction with gold nanoparticles. For example, the conjugates of the invention can be prepared using composite quantum dots (e.g., CdSeS/ZnS). In addition, the surface of the quantum dot is modified with thioglycolic acid (MAA), so that the quantum dot can be connected with the connecting arm through a disulfide bond.

In some embodiments, the quantum dots have an average diameter of 3nm to 10nm, preferably 4.5 nm. In some embodiments, the sialyloligosaccharide-quantum dot conjugate has an average diameter of 3nm to 10nm, preferably 4.5 nm. Without wishing to be bound by theory, conjugates of this diameter range are more susceptible to energy resonance transfer with gold nanoparticles known in the art and binding to the influenza virus or HA protein surface, the quantum dots can have any shape including, but not limited to, spherical, box, cylindrical, tetrahedral or cubic, and these shapes can be part of a network. The quantum dots may be monodisperse or polydisperse, and the degree of dispersion of the particles may vary with diameter.

When prepared for use in a test kit, the sialyloligosaccharide-quantum dot conjugate of the invention is preferably provided in the form of a solution, preferably in the form of a borate buffer solution, wherein the concentration of the conjugate is 2-5 μ M.

The detection kit may further comprise modified gold nanoparticles capable of binding to influenza virus. For example, sialyloligosaccharide-gold nanoparticles as described in CN 103551562B. As will be appreciated by those skilled in the art, the sialyloligosaccharide-gold nanoparticles used in conjunction with

α

2,3 sialyloligosaccharide-quantum dots (with the sialic acid residue represented by formula X-III) are

α

2,3 sialyloligosaccharide-gold nanoparticles. The sialyloligosaccharide-gold nanoparticles used in combination with the alpha 2,6 sialyloligosaccharide-quantum dots (the sialic acid residue of which is shown in the formula X-II) are alpha 2,6 sialyloligosaccharide-gold nanoparticles.

The sialyloligosaccharide-quantum dot conjugate of the present invention can be prepared by a method comprising the steps of:

N₃CH₂(CH₂OCH₂)_nCH₂NH₂formula M-I;

in formulae M-III and M-IV, Ac is acetyl;

in the formula X-I, m is an integer of 300-600, and QD is a quantum dot;

in the formulae M-VI, M-VII and X-I, the

Selected from the group consisting of:

Preferably, in step (1), the reaction is carried out in the presence of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 4-dimethylaminopyridine. More preferably, the lipoic acid, the 1-ethyl- (3-dimethylaminopropyl) carbodiimide, and the 4-dimethylaminopyridine are dissolved in anhydrous dichloromethane, and the compound of formula M-I is dissolved in dichloromethane.

Preferably, in the step (2), the compound represented by the formula M-II and the compound represented by the formula M-III are dissolved in a dimethylformamide/methanol mixed solution. Preferably, the reaction is carried out under catalysis of cuprous iodide.

Preferably, in step (3), the compound represented by the formula M-IV is dissolved in a methanol solution, and the deprotection reaction is carried out by adjusting the pH of the solution to 9 to 10 with sodium methoxide.

Preferably, in step (4), the compound represented by formula M-V is dissolved in a Tris-HCl buffer containing CMP-N-acetylneuraminic acid, and a sialyltransferase is added. To obtain sialic acid residues of formula X-II, an alpha 2,6 sialyltransferase may be added. To obtain sialic acid residues of the formula X-III, an

alpha

2,3 sialyltransferase may be added.

Preferably, step (5) is carried out in a solution of tris (2-carboxyethyl) phosphine, and after the reaction is completed, alcohol and sodium hydroxide are added for neutralization.

Preferably, step (6) is carried out at room temperature.

Detection method and use

The influenza virus which can be detected by the conjugate and the kit can be influenza A virus and/or influenza B virus and parainfluenza virus, in particular influenza A virus such as H1 (such as H1N1), H3 (such as H3N2), H5 (such as H5N1) and H7 (such as H7N9) and/or influenza B virus such as B/Victoria, B/Yamagata, B/Lee/40 and parainfluenza virus such as hPIV 3. In this regard, the host to which the present invention relates may be human, pig, bird, horse, dolphin, and the like.

In some embodiments, the invention provides the use of the sialyloligosaccharide-quantum dot conjugate of the invention or a kit comprising the sialyloligosaccharide-quantum dot conjugate of the invention for detecting influenza virus or influenza virus HA protein in a sample.

In some embodiments, the sample is a biological fluid obtained from or derived from a subject, a fluid or sample obtained from an environmental source, a fluid from a cell culture, or any combination of the above.

In some embodiments, the sample is a biological fluid selected from the group consisting of: blood, plasma, serum, lactation products, amniotic fluid, sputum, saliva, urine, semen, cerebrospinal fluid, bronchial aspirate, bronchial lavage aspirate, sweat, mucus, liquefied fecal sample, synovial fluid, peritoneal fluid, pleural fluid, pericardial fluid, lymph fluid, tears, tracheal aspirate, homogenate of a tissue sample, or any mixture of the above biological fluids.

In some embodiments, the sample is a fluid or sample obtained from an environmental source, the fluid or sample selected from a fluid or sample obtained from or derived from: food processing products, food and agricultural products, poultry, meat, fish, beverages, dairy products, water (including wastewater), ponds, rivers, reservoirs, swimming pools, soils, food processing and/or packaging plants, agricultural sites, pharmaceutical manufacturing plants, or any combination of the foregoing environmental sources.

The invention also relates to a method for detecting viruses in a sample. Considering that fluorescence energy resonance transfer (FRET) occurs when quantum dots (donors) are spatially adjacent to gold nanoparticles (acceptors), when sialyloligosaccharide-quantum dots and sialyloligosaccharide-modified gold nanoparticles (sialyloligosaccharide-gold nanoparticles) are added into a detection system, if influenza virus or surface protein thereof exists in the system, the sialyloligosaccharide-quantum dots and the sialyloligosaccharide-gold nanoparticles can be simultaneously combined with the influenza virus (or surface protein thereof), so that energy transfer occurs between the quantum dots and the gold nanoparticles when the sialyloligosaccharide-quantum dots and the sialyloligosaccharide-gold nanoparticles are spatially adjacent to each other, and the existence and concentration of the influenza virus can be detected by detecting FRET signals (namely, the fluorescence intensity of the quantum dots is reduced).

In some embodiments, the present invention provides a method of detecting a virus in a sample, the method comprising:

(ii) (ii) detecting the FRET signal after the incubation of step (i).

Preferably, the FRET signal is a decrease in the fluorescence intensity of the quantum dot. Preferably, the detection is performed at an excitation wavelength of 380-400 and an emission wavelength of 400-800nm (more preferably at an emission wavelength of 450-600 nm).

Embodiments of the aspects described herein may be illustrated by the following numbered paragraphs:

1. a sialyloligosaccharide-quantum dot conjugate, the conjugate having the structure of formula X-I:

the above-mentioned

Selected from the group consisting of：

Wherein, in the formulae X-II and X-III, R¹Is hydroxy or acetamido, R²Is hydroxy or L-fucosyl, R³Is hydroxyl or sulfate group, and x is an integer of 1-3.

2. The conjugate of paragraph 1, wherein in the formula X-I, n is an integer from 3 to 6.

3. The conjugate of

paragraphs

1 or 2, wherein R is in the formula X-II or formula X-III¹Is acetamido, R²Is L-fucosyl, R³Is a hydroxyl group.

4. The conjugate of paragraph 1, wherein the conjugate has any one of the following structures:

wherein, in the formulae X-IV, X-V, X-VI, X-VII, X-VIII and X-IX, n is an integer of 0-12 and m is an integer of 300-600.

5. The conjugate of paragraph 4, wherein n is an integer from 3 to 6 in formula X-IV, formula X-V, formula X-VI, formula X-VII, formula X-VIII, and formula X-IX.

6. The conjugate of any of paragraphs 1-5, wherein the quantum dot has an absorption wavelength of 400nm to 800 nm.

7. The conjugate of paragraph 6, wherein the quantum dot has an absorption wavelength of 450nm to 600 nm.

8. The conjugate of any of paragraphs 1-7, wherein the quantum dot is a CdSeS/ZnS quantum dot.

9. The conjugate of any of paragraphs 1-8, wherein the quantum dot surface is modified with thioglycolic acid.

10. The conjugate of any of paragraphs 1-9, wherein the quantum dots have an average diameter of 3nm to 10 nm.

11. The conjugate of paragraph 10, wherein the quantum dots have an average diameter of 4.5 nm.

12. The conjugate of any of paragraphs 1-11, wherein the conjugate has an average diameter of 3nm to 10 nm.

13. The conjugate of paragraph 12, wherein the conjugate has an average diameter of 4.5 nm.

14. A kit comprising the sialyloligosaccharide-quantum dot conjugate of any of paragraphs 1 to 13, and preferably further comprising sialyloligosaccharide-gold nanoparticles.

15. The kit of paragraph 14, wherein the sialyloligosaccharide-quantum dot conjugate is in the form of a borate buffer solution.

16. The kit of paragraph 15, wherein the sialyloligosaccharide-quantum dot conjugate is at a concentration of 2 to 5 μ M.

17. The kit of any of paragraphs 14-16, wherein the sialyloligosaccharide-gold nanoparticles have a diameter of from 10nm to 15 nm.

18. The kit of any of paragraphs 14-17, wherein the sialyloligosaccharide conjugate is an

α

2,3 sialyloligosaccharide-quantum dot and the sialyloligosaccharide-gold nanoparticle is an

α

2,3 sialyloligosaccharide-gold nanoparticle.

19. The kit of any of paragraphs 14-17, wherein the sialyloligosaccharide conjugate is an α 2,6 sialyloligosaccharide-quantum dot and the sialyloligosaccharide-gold nanoparticle is an α 2,6 sialyloligosaccharide-gold nanoparticle.

20. The kit of any of paragraphs 14-17, wherein the kit comprises the

α

2,3 sialyloligosaccharide-quantum dots, the α 2,6 sialyloligosaccharide-quantum dots, the

α

2,3 sialyloligosaccharide-gold nanoparticles, and the α 2,6 sialyloligosaccharide-gold nanoparticles in separate packages.

21. A method of making the sialyloligosaccharide-quantum dot conjugate of any of paragraphs 1 to 13, the method comprising the steps of:

N₃CH₂(CH₂OCH₂)_nCH₂NH₂formula M-I;

wherein, in the formula M-I, the formula M-II, the formula M-III, the formula M-IV, the formula M-V, the formula M-VI, the formula M-VII and the formula X-I, n is an integer of 0 to 12;

in the formula X-I, m is an integer of 300-600, and QD is a quantum dot;

in the formulae M-VI, M-VII and X-I, the

Selected from the group consisting of:

22. The method of paragraph 21 wherein, in step (1), the reaction is carried out in the presence of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 4-dimethylaminopyridine.

23. The method of paragraph 22, wherein said lipoic acid, said 1-ethyl- (3-dimethylaminopropyl) carbodiimide, and said 4-dimethylaminopyridine are dissolved in anhydrous dichloromethane, and said compound of formula M-I is dissolved in dichloromethane.

24. The method of any of paragraphs 21-23, wherein in step (2), the compound of formula M-II and the compound of formula M-III are dissolved in a dimethylformamide/methanol mixed solution. Preferably, the reaction is carried out under catalysis of cuprous iodide.

25. The method according to any one of paragraphs 21-24, wherein, in step (3), the deprotection reaction is carried out by dissolving the compound represented by formula M-IV in a methanol solution and adjusting the solution pH to 9-10 with sodium methoxide.

26. The method according to any one of paragraphs 21-25, wherein in step (4), the compound of formula M-V is dissolved in Tris-HCl buffer containing CMP-N-acetylneuraminic acid and sialyltransferase is added.

27. The method of paragraph 26, wherein in the compound of formula M-VI, the

Is a sialic acid residue of formula X-II, and the sialyltransferase is an α 2,6 sialyltransferase.

28. The method of paragraph 26, wherein in the compound of formula M-VI, the

Is a sialic acid residue of formula X-III, and the sialyltransferase is an

α

2,3 sialyltransferase.

29. The method of any of paragraphs 21-28, wherein step (5) is carried out in a solution of tris (2-carboxyethyl) phosphine, and after completion of the reaction, alcohol and sodium hydroxide are added for neutralization.

27. The method of any of paragraphs 21-26, wherein step (6) is performed at room temperature.

28. Use of the sialyloligosaccharide-quantum dot compound according to any of paragraphs 1 to 13 or the kit according to any of paragraphs 14 to 20 for detecting a virus or a viral protein in a sample.

29. The use of paragraph 28 wherein the virus is selected from the group consisting of influenza a virus, influenza B virus and parainfluenza virus.

30. The use of paragraph 28 wherein the viral protein is an HA protein.

31. The use of any of paragraphs 28-30, wherein the sample is a biological fluid obtained or derived from a subject, a fluid or sample obtained from an environmental source, a fluid from a cell culture, or any combination thereof.

32. A method of detecting a virus in a sample, the method comprising:

(i) incubating the sialyloligosaccharide-quantum dot conjugate of any of paragraphs 1 to 13, sialyloligosaccharide-gold nanoparticles with a sample; and

33. The method of paragraph 32, wherein in step (i) the sialyloligosaccharide-gold nanoparticles have a diameter of from 10nm to 15 nm.

34. The method of paragraph 33, wherein the sialyloligosaccharide conjugate is

α

2,3 sialyloligosaccharide-quantum dots and the sialyloligosaccharide-gold nanoparticle is

α

2,3 sialyloligosaccharide-gold nanoparticle.

35. The method of paragraph 33, wherein the sialyloligosaccharide conjugate is α 2,6 sialyloligosaccharide-quantum dots and the sialyloligosaccharide-gold nanoparticle is α 2,6 sialyloligosaccharide-gold nanoparticle.

36. The method as claimed in any of paragraphs 32-35, wherein in step (ii), the FRET signal is detected at an excitation wavelength of 380-400 and an emission wavelength of 400-800 nm.

37. The method as described in paragraph 36, wherein the FRET signal is detected at an emission wavelength of 450-600 nm.

Examples

Unless otherwise specified, the methods used hereinafter are all conventional methods; the materials and reagents used are commercially available.

Reagents used hereinafter

Tris-HCl buffer (100mM, pH 8.0): tris was dissolved in water at a final concentration of 12.1g/L and the pH was adjusted to 8.0 with HCl.

PBS buffer (10mM, pH 7.4): NaH₂PO₄ 0.24g/L、Na₂HPO₄1.42g/L, KCl 0.2.2 g/L and NaCl 8.0g/L, the solvent is water.

Borate buffer (10mM, pH 7.4): 0.62g/L of boric acid, 3.81g/L of borax and water as a solvent. Strain information for influenza viruses used below is as follows:

strain A/California/04/2009(H1N 1): abbreviation Ca04H1N1, literature: zhang, J.Qi, Y.Shi, Q.Li, F.Gao, Y.Sun, X.Lu, Q.Lu, C.J.Vavricka, D.Liu, J.Yan, G.F.Gao, Protein Cell 2010,1, 459-. The Genbank number of the HA protein amino acid sequence is ACP 41105.1; the receptor recognized by the viral HA protein is known as sialic acid α 2,6 oligosaccharide secreted by the host cell.

Strain A/VietNam/1203/2004(H5N 1): abbreviation vieH5N1, literature: J.Stevens, O.Blixt, T.M.Tumpey, J.K.Taubenberger, J.C.Paulson, I.A.Wilson, Science 2006,312, 404-. The Genbank number of the HA protein amino acid sequence is ABW 90135.1; the receptor recognized by the viral HA protein is known as

sialic acid α

2,3 oligosaccharide secreted by the host cell.

Strain A/Anhui/1/2013(H7N 9): abbreviation AnhH7, literature: liu, W.Shi, Y.Shi, D.Wang, H.Xiao, W.Li, Y.Bi, Y.Wu, X.Li, J.Yan, W.Liu, G.ZHao, W.Yang, Y.Wang, J.Ma, Y.Shu, F.Lei, G.F.Gao, The Lancet,2013,381, 1926-1932; the receptor recognized by the virus HA protein (HA7) and the strain thereof is known as

sialic acid alpha

2,3 or alpha 2,6 oligosaccharide secreted by host cells.

Preparation example 1 Synthesis of linker arm

First, N is synthesized₃CH₂(CH₂OCH₂)₅CH₂NH₂The synthesis route is as follows:

synthesis of Compound 1: in a 250mL round bottom flask hexapolyethylene glycol (1.51g, 5.0mmol) was dissolved in 100mL dry dichloromethane followed by triethylamine (3.4mL, 25.0mmol) and p-toluenesulfonyl chloride (2.4g, 15.0mmol) added slowly in an ice bath and stirred at room temperature for 26 h. TLC monitored the reaction completion. Dilute with 200mL of dichloromethane, then add 1M HCl, then saturated NaHCO₃And 500mL each of saturated NaCl, collecting the organic phase, passing through anhydrous Na₂SO₄Drying, filtering, evaporating the solvent, and separating and purifying the concentrate by silica gel column chromatography (petroleum ether: ethyl acetate: 1:2) to obtain the colorless oily compound 1(2.5g, yield 87.0%).¹H NMR(400MHz,CDCl₃):δ＝7.79(d,J＝8.0Hz,4H,Ar),7.46(d,J＝8.1Hz,4H,Ar),4.20-4.15(m,4H,TsOCH₂CH₂),3.71-3.67(m,4H,TsOCH₂CH₂),3.59(d,J＝15.2Hz,16H,CH₂),2.35(s,6H,CH₃)。¹³C NMR(125MHz,CDCl₃):δ＝144.79,132.90,129.81,127.86,70.70,70.56,70.49,70.50,69.40,68.54,46.19,21.59,8.60。ESI-HRMS:m/z calcd for C₂₆H₃₉O₁₁S₂[M+H]⁺:591.1856,found:591.1867。

Synthesis of Compound 2: in a 50ml round bottom flask, compound 1(1.0g, 1.7mmol) was dissolved in 20ml of DMF, sodium azide (680mg, 7.6mmol) was added, and the reaction was stirred at 80 ℃ for 24 h. TLC monitored the reaction completion. 60mL of water was added for dilution, and the mixture was extracted with ethyl acetate (3X 100 mL). The combined organic phases were washed with saturated NaCl (3X 50mL) and the organic phase was washed with anhydrous Na₂SO₄After drying, filtration and evaporation of the solvent, compound 2 was obtained as a pale yellow oil (490mg, yield 86.7%).¹H NMR(400MHz,CDCl₃):δ＝3.73-3.54(m,20H),3.37(t,J＝5.1Hz,4H,2N₃CH₂)。¹³C NMR(125MHz,CDCl₃):δ＝70.5,70.4,70.2,70.0,69.0,50.3。ESI-HRMS:m/z calcd for C₁₂H₂₄N₆O₅Na[M+Na]⁺:355.1607,found:354.1610。

Synthesis of Compound 3: taking and transformingCompound 2(490mg, 1.47mmol) is dissolved in 10mL ethyl acetate and 2mL 1M HCl and Ph is added₃P (429.7mg, 1.68mmol), reacted at room temperature for 12h, the aqueous phase was recovered, the organic phase was washed with purified water (3X 5mL), the aqueous phases were combined, the solvent was evaporated under reduced pressure, and the concentrate was purified with basic Al₂O₃Purification on a column (ethyl acetate: methanol 10:1) gave compound 3 as a pale yellow oil (440.6mg, 79.9% yield).¹H NMR(500MHz,CD₃OD):δ＝3.62-3.60(m,18H),3.45-3.40(m,2H),3.36-3.31(m,2H,N₃CH₂),3.07-3.03(m,2H,NH₂CH₂)。¹³C NMR(125MHz,CDCl₃):δ＝70.56,70.35,70.33,70.22,70.16,69.79,68.98,50.64,40.75。ESI-HRMS:m/z calcd for C₁₂H₂₇N₄O₅[M+H]⁺:307.1966,found:307.1977。

The resultant path of the linker arm is as follows:

lipoic acid (2.07g, 10mmol), 1-ethyl- (3-dimethylaminopropyl) carbodiimide (3.1g, 20mmol) and 4-dimethylaminopyridine (0.12g, 1mmol) were dissolved in 10mL of anhydrous dichloromethane, and compound 3(100mg, 100mmol) dissolved in 10mL of dichloromethane was added dropwise under nitrogen protection, and the mixture was protected from light at room temperature overnight. After TLC monitoring the disappearance of the starting material, the reaction was concentrated to give an orange yellow slurry. The mixture was purified by silica gel chromatography (eluent was petroleum ether: ethyl acetate 1:1) to give light yellow syrupy compound 4(1.8g, yield 55%).¹H NMR(500MHz,CDCl₃)δ7.26(s,1H),6.12(s,1H),3.68-3.61(m,23H),3.56-3.53(m,3H),3.44(d,J＝4.6Hz,2H),3.38(t,J＝4.8Hz,2H),3.21-3.13(m,1H),3.10(dt,J＝11.0,6.9Hz,1H),2.45(td,J＝12.4,6.5Hz,1H),2.24-2.12(m,3H),1.90(dt,J＝13.5,6.7Hz,1H),1.73-1.61(m,5H),1.46(dd,J＝15.8,6.7Hz,3H)。¹³C NMR(126MHz,CDCl₃)δ77.32,77.06,76.81,70.79,70.26,69.87,56.44,50.69,40.24,39.22,38.48,36.32,34.67,28.93,25.40。ESI-HRMS:m/z calcd for C₂₀H₄₂N₅O₆S₂[M+NH₄]⁺:512.2571,found:512.2580。

Preparation example 2 Synthesis of sialyloligosaccharide and linker moiety

(1) Synthesis of compound 7:

synthesis of Compound 5: acetic anhydride (62mL, 660mmol), D-lactose (25g, 73mmol) and anhydrous sodium acetate (24g, 292mmol) were added to a 250mL three-necked flask and heated to 95-100 ℃ under nitrogen to give a clear solution. The solution is heated to reflux and reacted for 2-3 h. TLC to monitor the reaction, the reaction mixture was poured into crushed ice and stirred vigorously for 2h to give a viscous paste, which was filtered, extracted with ethyl acetate (3X 100mL), the organic phases were combined after precipitation with saturated NaHCO₃The resulting solution was dried over anhydrous sodium sulfate, concentrated and recrystallized from ethanol to give white yellowish solid 5(42.6g, 86% yield).

Synthesis of Compound 6: compound 5(6.8g, 10mmol) and benzylamine (1.2mL, 11mmol) were dissolved in 70mL tetrahydrofuran at 0 deg.C and reacted at room temperature for 30-32 h. After completion of the reaction by TLC, the reaction mixture was concentrated, and the concentrated solution was dissolved in 100mL of dichloromethane, extracted with hydrochloric acid and water in this order, concentrated, and purified by silica gel column chromatography (petroleum ether: ethyl acetate: 1) to obtain a pale yellow paste compound (5.7g, 89% yield). The pasty compound (2.6g, 4.2mmol), 1, 8-diazabicyclo [5.4.0] undec-7-ene (1.2mL, 8.34mmol) and trichloroacetonitrile (4.2mL, 42mmol) were dissolved in 30mL of anhydrous dichloromethane and reacted for 1-2h with magnetic stirring at room temperature. The reaction was monitored by TLC, concentrated, purified by silica gel column chromatography (petroleum ether: ethyl acetate 1:1.5), and dried to give a yellowish solid 6(1.6g, 51% yield).

Synthesis of compound 7: compound 6(500mg, 0.64mmol), propargyl alcohol (0.05mL, 1mmol) and trimethylsilyl trifluoromethanesulfonate (0.01mL, 0.06mmol) were dissolved in 5mL of dichloromethane, reacted at 0 ℃ for one hour under nitrogen protection and then warmed to room temperature for two more hours, and after completion of the reaction was monitored by TLC, the concentrate was purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 1:2) to give 7(178mg, 77% yield) as a yellow solid after drying.

(2) Synthesis of compound 8:

compound 7(1eq) and compound 4(1.5eq) were dissolved in a DMF/MeOH mixture (1:1, 50mL/mmol of compound 1), the mixture was degassed by vacuum and purged with nitrogen. Under nitrogen protection, cuprous iodide (0.5eq) was added to the reaction mixture and reacted at room temperature overnight. After the reaction was monitored by TLC, the reaction mixture was concentrated and purified by gel column chromatography (SephadexG-15). Compound 8 was obtained as a white solid (60% yield).¹H NMR(500MHz,CDCl3)δ7.71(d,J＝3.1Hz,1H),6.17(s,1H),5.35(d,J＝3.0Hz,1H),5.16(dd,J＝21.6,12.3Hz,1H),5.10(dd,J＝10.4,7.9Hz,1H),4.97(d,J＝3.4Hz,1H),4.95-4.93(m,1H),4.92(dd,J＝6.4,2.9Hz,1H),4.89(d,J＝5.8Hz,1H),4.80(s,1H),4.78(s,1H),4.64(d,J＝7.9Hz,1H),4.55(dd,J＝8.2,4.6Hz,2H),4.49(d,J＝6.3Hz,1H),4.15–4.05(m,3H),3.88(dd,J＝7.5,3.5Hz,3H),3.81(t,J＝9.5Hz,1H),3.67–3.59(m,17H),3.59-3.54(m,3H),3.44(dd,J＝10.3,5.2Hz,2H),3.18(ddd,J＝12.3,6.9,5.5Hz,1H),3.11(dt,J＝11.0,6.9Hz,1H),2.46(td,J＝12.4,6.5Hz,1H),2.20(t,J＝7.6Hz,2H),2.15(t,J＝6.1Hz,5H),2.09-2.02(m,10H),1.98(d,J＝9.4Hz,5H),1.90(dt,J＝12.3,6.2Hz,1H),1.75-1.62(m,4H),1.52-1.42(m,2H),1.26(t,J＝7.1Hz,1H)。¹³C NMR(126MHz,CDCl₃)δ170.35,170.15,170.07,169.75,169.74,169.04,101.04,97.87,78.08,77.29,77.04,76.78,76.14,75.47,72.76,72.68,71.30,70.98,70.70,69.09,66.60,61.82,60.82,55.87,20.87,20.80,20.73,20.65,20.52。ESI-HRMS:m/z calcd for C₄₉H₈₀N₅O₂₄S₂[M+NH4]⁺:1186.4629,found:1186.4661。

(3) Synthesis of compound 9:

the compound 8 was dissolved in 5mL of methanol solution, and the pH was adjusted to 9-10 with sodium methoxide, followed by reaction at room temperature. After completion of the reaction monitored by TLC, it was concentrated and purified by silica gel column chromatography (ethyl acetate: methanol: 3:1) and dried to give 9(190mg, 78% yield) as a white solid.¹H NMR(500MHz,D₂O)δ8.04(s,1H),4.91(t,J＝10.1Hz,1H),4.77(dd,J＝26.4,10.3Hz,1H),4.58-4.51(m,2H),4.49(t,J＝7.8Hz,1H),4.36(d,J＝7.8Hz,1H),3.88(dd,J＝19.1,13.5Hz,4H),3.75-3.42(m,25H),3.28(dt,J＝16.9,6.9Hz,3H),3.17-3.03(m,1H),2.40(td,J＝12.2,6.1Hz,1H),2.17(t,J＝7.2Hz,2H),1.89(dt,J＝20.0,6.9Hz,1H),1.65(tt,J＝22.2,11.0Hz,1H),1.53(td,J＝13.8,6.7Hz,3H),1.32(dt,J＝14.7,7.4Hz,2H)。¹³C NMR(126MHz,D₂O)δ176.83,143.48,125.69,102.92,101.28,78.31,75.34,74.81,74.35,72.71,72.50,70.92,69.65,69.63,69.59,69.58,69.47,69.42,68.87,68.72,68.51,61.92,60.99,60.05,56.51,50.02,40.26,38.89,38.07,35.46,33.72,27.85,25.02。ESI-HRMS:m/z calcd for C₃₅H₆₃N₄O₁₇S₂[M+Na]⁺:875.3624,found:875.3610。

(4) Synthesis of compound 10:

a reaction system containing compound 9(10Mm) and CMP-N-acetylneuraminic acid (12.0Mm) was prepared with Tris-HCl buffer (100.0Mm, pH 8.0, 37 ℃). The reaction system is placed in a constant temperature shaking table at 37 ℃, preheated for 5min, and

alpha

2,3 sialyltransferase (Pmaalpha 2,3ST) expressed by escherichia coli and coming from Pasteurella multocida P-1059 strain is added, and after about two hours, the reaction is monitored by TLC to be finished. The product was lyophilized using a-80 ℃ lyophilizer, the resulting mixture was dissolved with a small amount of water and purified by gel column chromatography (SephadexG-15) to give compound 10 in 50% yield.¹H NMR(500MHz,D₂O)δ8.04(d,J＝3.3Hz,1H),4.92(d,J＝12.6Hz,1H),4.82-4.77(m,2H),4.59-4.53(m,2H),4.49(d,J＝8.0Hz,1H),4.44(d,J＝7.9Hz,1H),4.03(dd,J＝9.9,3.1Hz,1H),3.88(dt,J＝13.9,8.0Hz,4H),3.83-3.71(m,5H),3.69-3.40(m,28H),3.30(t,J＝5.3Hz,2H),3.25(dd,J＝11.2,5.9Hz,1H),3.19-3.05(m,3H),2.66(dt,J＝14.3,7.2Hz,2H),2.40(dt,J＝18.8,6.1Hz,2H),2.19-2.11(m,2H),1.94(d,J＝7.9Hz,3H),1.88(dd,J＝13.4,6.6Hz,1H),1.74-1.62(m,3H),1.52(dq,J＝13.6,7.2Hz,4H),1.32(dt,J＝15.1,7.5Hz,3H)。¹³C NMR(126MHz,D2O)δ176.92,174.99,143.47,125.71,102.63,101.27,99.77,78.18,75.14,74.81,74.32,72.85,72.69,68.71,68.06,67.43,62.54,61.91,60.99,60.03,56.49,51.65,50.01,40.24,39.60,38.88,38.04,35.44,33.68,27.79,24.99,22.00。ESI-HRMS:m/z calcd for C₄₆H₇₉N₅NaO₂₅S₂[M+Na]⁺:1188.4398,found:1188.4419。

(5) Synthesis of compound 11:

a reaction system containing compound 9(10Mm) and CMP-N-acetylneuraminic acid (12.0Mm) was prepared with Tris-HCl buffer (100.0Mm, pH 8.0). The reaction system was placed in a 37 ℃ constant temperature shaker, and α 2,6 sialyltransferase (Pm α 2,6ST) from Photobacterium damsel, expressed in escherichia coli, was added thereto, and after about two hours, completion of the reaction was monitored by TLC. Lyophilizing at-80 deg.C, dissolving the mixture with small amount of water, and separating and purifying by gel column chromatography (SephadexG-15) to obtain compound 11 with total yield of about 45%.¹H NMR(500MHz,D2O)δ8.05(s,1H),4.92(d,J＝12.5Hz,1H),4.80(d,J＝12.6Hz,1H),4.56(t,J＝5.0Hz,2H),4.50(d,J＝8.0Hz,1H),4.34(d,J＝7.9Hz,1H),3.92–3.84(m,5H),3.79(dd,J＝12.0,4.8Hz,2H),3.76-3.69(m,3H),3.64-3.51(m,26H),3.51-3.41(m,3H),3.32-3.25(m,3H),3.18-3.06(m,2H),2.62(dd,J＝12.4,4.7Hz,1H),2.40(td,J＝12.3,6.1Hz,1H),2.21-2.13(m,2H),1.99-1.85(m,4H),1.66(dt,J＝16.0,9.9Hz,2H),1.59–1.48(m,3H),1.37-1.28(m,2H)。¹³C NMR(126MHz,D2O)δ176.91,174.87,173.45,143.50,125.71,103.22,101.17,100.26,79.56,74.67,74.61,73.66,72.61,72.50,72.33,71.75,70.75,69.63,69.61,69.58,69.56,69.46,69.41,68.86,68.72,68.33,63.51,62.60,61.93,60.22,56.49,51.76,50.01,40.25,40.08,38.89,38.04,35.44,33.69,27.80,25.00,22.02。ESI-HRMS:m/z calcd for C₄₆H₇₉N₅NaO₂₅S₂[M+Na]⁺:1188.4398,found:1188.4385。

Preparation example 3 preparation of sialyloligosaccharide-quantum dot

40. mu.l of quantum dots (purchased from Wuhanjia Gaojia Quantum dot technology development Co., Ltd., Q1525 oil-soluble quantum dots) (3mg/mL) was taken, and 500. mu.l of ethanol was added. The mixture was centrifuged in a centrifuge (12000rpm, 5min), the solution was removed, and 75. mu.l of methylene chloride was added. The resulting solution was solution A. Each 3. mu.l of TCEP (0.1M) and the prepared sialyloligosaccharide (0.1M) was mixed well and incubated for 20 min. Then 18. mu.l of sodium hydroxide (0.1M) was added. The resulting solution was solution B. The A, B solution was mixed well, and 75. mu.l of ultrapure water was added thereto, followed by standing for 1 to 3 min. And sucking the upper solution. Centrifuge 3 times (12000rpm, 5min) with an ultrafiltration tube. Washing was performed by adding borate buffer every time during centrifugation. Measuring absorbance of the glycosylated water-soluble quantum dot at 450nm by using an ultraviolet spectrophotometer, wherein the concentration of the glycosylated-quantum dot calculated by Lambert beer law is 3.38 mu M (molar absorption coefficient of the quantum dot Q1525 is 2.66 multiplied by 10)⁵). Storing the glycosylated water-soluble quantum dot solution at 4 ℃ for standby.

Transmission electron microscopy imaging was performed on free quantum dots and quantum dots modified with compound 10 (3' SLac-quantum dots), and the results are shown in fig. 2. The electron microscope used was JEM 1400 (japan electronics). To the solution containing 3.38. mu.M 3 'SLAc-quantum dots, 3' SLAc-gold nanoparticles and H7 protein (1.6mg/mL, H7 protein of AnhH7 strain) were added and transmission electron microscopy imaging was performed. As shown in fig. 3, significant aggregation of the conjugate was observed. And performing infrared absorption spectrum characterization on the free quantum dots and the 3' SLAc-quantum dots. The infrared spectrometer used was a Bruker tens 27 infrared spectrometer. As shown in FIG. 4, the quantum dot modified with glycosyl group shows a very obvious new characteristic absorption peak (1087 cm)^-1) This is the result of the stretching vibration of the C-O-C bond on the glycosyl group. The magnetic resonance spectrometer avance400,500,600MHz (Bruker) Nuclear magnetic resonance one-dimensional Hydrogen Spectroscopy (NMR) characterization of Compounds 10 and 3' SLAc-Quantum dots was performed. As shown in figure 5, the conjugate had all the key peaks of sialyloligosaccharide (compound 10), indicating that sialyloligosaccharide was successfully modified onto quantum dots. Thermal weight loss (TGA) analysis was performed on the free quantum dots and the 3' SLac-quantum dots, and the thermal weight loss curve is shown in fig. 6. The weight loss of 1g of the glycosylated quantum dot is 0.29g, and the weight loss value reflects the content of sialyloligosaccharide. Since the relative molecular mass of the 3 'SLAc-quantum dot is 1165g/mol, the amount of the oligosaccharide sialylate surface-modified by the 3' SLAc-quantum dot is about 0.248 mmol/g.

The sialyloligosaccharide-gold nanoparticle 3' SLAc-gold nanoparticle is a compound X-1-1 prepared according to the patent CN103551562B, and can identify influenza virus combined with sialic acid alpha 2,3Gal receptor or surface protein thereof. The sialyloligosaccharide-gold nanoparticle 6' SLAc-gold nanoparticle is a compound X-2-1 prepared according to the CN103551562B patent, and can identify influenza virus combined with sialic acid alpha 2,6Gal receptor or surface protein thereof.

Optimized, the detection system used in the examples is borate buffer containing 3' SLAc-quantum dots (1.042nM) and sialyloligosaccharide-gold nanoparticles (0.648nM), and the system is prepared before use. To 100. mu.l of the assay solution, PBS solution containing 1380nM BSA and 3nM HA7 was added, respectively, and incubated at room temperature for 2-5 min. And measuring the result by using an enzyme-labeling instrument, setting the fluorescence excitation wavelength to be 388nm, the fluorescence absorption wavelength to be 525nm, the scanning step length to be 1nm, and the wavelength range to be 450-600 nm. The results are shown in FIG. 7. This result shows the specificity of the system of the invention for detection with influenza virus.

Example 1 detection of H7 protein

Mu.l of the assay system (borate buffer containing 1.042nM 3 'SLAc-quantum dots and 0.648nM 3' SLAc-gold nanoparticles) and PBS solutions containing different concentrations of H7 protein were added to a 96-well blackboard and incubated at room temperature for 2-5 min. Negative controls were added to PBS containing 1380nM BSA. And measuring the result by using an enzyme-labeling instrument, setting the fluorescence excitation wavelength to be 388nm, the fluorescence absorption wavelength to be 525nm, the scanning step length to be 1nm, and the wavelength range to be 450-600 nm. As shown in FIG. 8, the fluorescence intensity decreased as the molar concentration of H7 protein increased. The linear interval at 525nM is 0-200 nM.

Example 2 detection of H5N1 Virus

Mu.l of the assay system (borate buffer containing 1.042nM 3 'SLAc-quantum dots and 0.648nM 3' SLAc-gold nanoparticles) and PBS solutions containing varying titers of VieH5N1 were added to a 96-well blackboard and incubated at room temperature for 2-5 min. Negative controls were added to PBS containing 1380nM BSA. And measuring the result by using an enzyme-labeling instrument, setting the fluorescence excitation wavelength to be 388nm, the fluorescence absorption wavelength to be 525nm, the scanning step length to be 1nm, and the wavelength range to be 450-600 nm. As shown in FIG. 9, the fluorescence intensity decreased with increasing titer of H5N1, with a linear interval of 2^-7-2¹The titer.

Example 3 detection of H1N1 Virus

Mu.l of the assay system (borate buffer containing 1.042nM 6 'SLAc-quantum dots and 0.648nM 6' SLAc-gold nanoparticles) and PBS solutions containing varying titers of Ca04H1N1 were added to a 96-well blackboard and incubated at room temperature for 2-5 min. Negative controls were added to PBS containing 1380nM BSA. And measuring the result by using an enzyme-labeling instrument, setting the fluorescence excitation wavelength to be 388nm, the fluorescence absorption wavelength to be 525nm, the scanning step length to be 1nm, and the wavelength range to be 450-600 nm. The results are shown in FIG. 10, where the fluorescence intensity decreased with increasing titer of H1N 1.

It can be seen that the method of the invention can be used to detect the presence of influenza virus and to distinguish between host specificities of influenza virus.

Claims

1. A kit comprising a sialyloligosaccharide-quantum dot conjugate and sialyloligosaccharide-gold nanoparticles, the conjugate having any one of the following structures:

wherein, in the formulae X-IV, X-V, X-VI, X-VII, X-VIII and X-IX, n is an integer of 0-12 and m is an integer of 300-600; and is

The quantum dots have an absorption wavelength of 450nm to 600 nm.

2. The kit of claim 1, wherein n is an integer from 3 to 6 in formula X-IV, formula X-V, formula X-VI, formula X-VII, formula X-VIII, and formula X-IX.

3. The kit of claim 1 or 2, wherein the quantum dots are CdSeS/ZnS quantum dots.

4. The kit of claim 1 or 2, wherein the surface of the quantum dot is modified with thioglycolic acid.

5. The kit of claim 1 or 2, wherein the quantum dots have an average diameter of 3nm to 10 nm.

6. The kit of claim 5, wherein the quantum dots have an average diameter of 4.5 nm.

7. The kit of claim 1 or 2, wherein the conjugate has an average diameter of 3nm to 10 nm.

8. The kit of claim 7, wherein the conjugate has an average diameter of 4.5 nm.

9. The kit of claim 8, wherein the sialyloligosaccharide-quantum dot conjugate is in the form of a borate buffer solution.

10. The kit of claim 9, wherein the sialyloligosaccharide-quantum dot conjugate is at a concentration of 2-5 μ Μ.

11. The kit of claim 1 or 2, wherein the sialyloligosaccharide-gold nanoparticles have a diameter of from 10nm to 15 nm.

12. The kit of claim 1 or 2, wherein the sialyloligosaccharide conjugate is an α 2,3 sialyloligosaccharide-quantum dot and the sialyloligosaccharide-gold nanoparticle is an α 2,3 sialyloligosaccharide-gold nanoparticle.

13. The kit of claim 1 or 2, wherein the sialyloligosaccharide conjugate is an α 2,6 sialyloligosaccharide-quantum dot and the sialyloligosaccharide-gold nanoparticle is an α 2,6 sialyloligosaccharide-gold nanoparticle.

14. The kit of claim 1 or 2, wherein the kit comprises α 2,3 sialyloligosaccharide-quantum dots, α 2,6 sialyloligosaccharide-quantum dots, α 2,3 sialyloligosaccharide-gold nanoparticles, α 2,6 sialyloligosaccharide-gold nanoparticles in separate packages.

15. A method of making a kit according to any one of claims 1 to 14, the method comprising the steps of:

N₃CH₂(CH₂OCH₂)_nCH₂NH₂formula M-I;

in the formula X-I, m is an integer of 300-600, and QD is a quantum dot;

in the formulae M-VI, M-VII and X-I, the

Selected from the group consisting of:

in the formulae X-II and X-III, R¹Is hydroxy or acetamido, R²Is hydroxy or L-fucosyl, R³Is hydroxyl or sulfate group, x is an integer of 1-3; wherein the content of the first and second substances,

the conjugate has any one of the following structures:

wherein, in the formulae X-IV, X-V, X-VI, X-VII, X-VIII and X-IX, n is an integer of 0-12 and m is an integer of 300-600;

the quantum dots have an absorption wavelength of 450nm to 600 nm.

16. The process of claim 15, wherein in step (1), the reaction is carried out in the presence of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 4-dimethylaminopyridine.

17. The method of claim 16, wherein said lipoic acid, said 1-ethyl- (3-dimethylaminopropyl) carbodiimide, and said 4-dimethylaminopyridine are dissolved in anhydrous dichloromethane, and said compound of formula M-I is dissolved in dichloromethane.

18. The method according to any one of claims 15 to 17, wherein in the step (2), the compound represented by the formula M-II and the compound represented by the formula M-III are dissolved in a dimethylformamide/methanol mixed solution.

19. The process of claim 18, wherein in step (2), the reaction is catalyzed by cuprous iodide.

20. The process according to any one of claims 15 to 17, wherein, in the step (3), the compound represented by the formula M-IV is dissolved in a methanol solution, and the deprotection reaction is carried out by adjusting the pH of the solution to 9 to 10 with sodium methoxide.

21. The method according to any one of claims 15 to 17, wherein in step (4), the compound represented by formula M-V is dissolved in Tris-HCl buffer containing CMP-N-acetylneuraminic acid, and sialyltransferase is added.

22. The method of claim 21, wherein in the compound of formula M-VI, the

23. The method of claim 21, wherein in the compound of formula M-VI, the

Is a sialic acid residue of formula X-III, and the sialyltransferase is an α 2,3 sialyltransferase.

24. The process according to any one of claims 15 to 17, wherein step (5) is carried out in a solution of tris (2-carboxyethyl) phosphine, and after the reaction is completed, alcohol and sodium hydroxide are added for neutralization.

25. The method of any one of claims 15-17, wherein step (6) is performed at room temperature.

26. Use of a kit according to any one of claims 1 to 14 for non-diagnostic, non-therapeutic purposes in the detection of a virus or viral protein in a sample.

27. The use of claim 26, wherein the virus is selected from the group consisting of influenza a virus, influenza B virus and parainfluenza virus.

28. The use of claim 26, wherein the viral protein is an HA protein.

29. The use of any one of claims 26-28, wherein the sample is a biological fluid obtained or derived from a subject, a fluid or sample obtained from an environmental source, a fluid from a cell culture, or any combination thereof.

30. A method of non-diagnostic, non-therapeutic purposes of detecting a virus in a sample, the method comprising:

(i) incubating sialyloligosaccharide-quantum dot conjugate, sialyloligosaccharide-gold nanoparticles of the kit of any of claims 1 to 14 with a sample; and

(ii) (ii) detecting the FRET signal of the sample after the incubation of step (i) at an emission wavelength of 450-600 nm.

31. The method of claim 30, wherein in step (i), the sialyloligosaccharide-gold nanoparticles have a diameter of from 10nm to 15 nm.

32. The method of claim 31, wherein the sialyloligosaccharide conjugate is an α 2,3 sialyloligosaccharide-quantum dot and the sialyloligosaccharide-gold nanoparticle is an α 2,3 sialyloligosaccharide-gold nanoparticle.

33. The method of claim 31, wherein the sialyloligosaccharide conjugate is an α 2,6 sialyloligosaccharide-quantum dot and the sialyloligosaccharide-gold nanoparticle is an α 2,6 sialyloligosaccharide-gold nanoparticle.

34. The method according to any of claims 30-33, wherein in step (ii), the FRET signal is detected at an excitation wavelength of 380-400nm and an emission wavelength of 450-600 nm.