A green fluorescent nanosphere, preparation method and application

Abstract

The present invention relates to a green fluorescent nanosphere, a preparation method and uses thereof, comprising: respectively preparing solutions of green fluorescent gold nanoclusters AuNCs, dendritic mesoporous silica nanoparticles DMSN and polyetherimide PEI; adding the PEI solution to the DMSN solution, mixing evenly and carrying out an ultrasonic reaction to obtain a mixed solution of the DMSN solution coated with PEI; centrifuging and redissolving the obtained mixed solution, adding the AuNCs solution, mixing evenly and carrying out an ultrasonic reaction, and obtaining the green fluorescent nanosphere DMSN@AuNCs after multiple centrifugal purifications. The operation of the present invention is simple, the cost is low, the reaction conditions are mild, and it is green and environmentally friendly. The prepared green fluorescent nanosphere DMSN@AuNCs can be used as a fluorescent labeling material in the field of immunological detection; according to the different antibodies and antigens labeled on the DMSN@AuNCs, a variety of fluorescent detection probes can be formed to detect different targets, and it has high popularization and application value in aspects such as high-throughput detection of disease markers.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterials, and particularly relates to a green fluorescent nanosphere, a preparation method and an application thereof. Background Art

[0002] In the prior art, fluorescent gold nanoclusters AuNCs are a type of fluorescent nanomaterial that emerged in recent years. It is a core-shell nanomaterial with a particle size of 0.2 - 3 nm composed of several to hundreds of atoms, and has characteristics such as strong photoluminescence, good photostability, and large Stokes shift. It has been successfully used for the detection of substances such as hydrogen peroxide, heavy metal ions, and tea polyphenols. Among them, fluorescent gold nanoclusters with 6-aza-2-thiothymine ATT and arginine Arg as ligands have better properties such as high quantum yield and super strong green fluorescence, and are expected to become a labeling material for immunoassay detection. However, due to the disadvantages of ultra-small particle size, difficult purification, and the fluorescence intensity being easily affected by metal ions, pH, etc. in the solution, it seriously affects the stability and accuracy of immunoassay detection technology and limits the application range of fluorescent gold nanoclusters. Therefore, increasing the particle size of fluorescent gold nanoclusters and improving their stability are important ways to broaden their application scenarios.

[0003] Silica nanoparticles have characteristics such as strong electronegativity, high dispersion, and good stability. There have been studies on using them as carriers for fluorescent quantum dots to prepare quantum dot microspheres with uniform particle size, high fluorescence intensity, good stability, and high surface functionalization degree, and as fluorescent labeling materials for immunoassay technology, applied to the detection of new coronavirus antigens and antibodies. Compared with silica microspheres, dendritic mesoporous silica nanoparticles (DMSN) not only have a three-dimensional dendritic skeleton and a large central radial emission mesoporous structure, but also have a variable pore surface structure, a larger specific surface area, and a higher loading capacity. As a carrier, it can effectively load macromolecular proteins, small molecule drugs, and luminescent compounds. Therefore, using DMSN as a carrier for fluorescent gold nanoclusters to prepare fluorescent nanospheres can effectively improve the optical properties and stability of fluorescent gold nanoclusters, and at the same time broaden their application range. Summary of the Invention

[0004] In order to overcome the above problems existing in the prior art, the present invention provides a green fluorescent nanosphere, a preparation method and uses thereof to solve the above problems existing in the prior art.

[0005] A preparation method of a green fluorescent nanosphere, the method comprising the following steps:

[0006] S1. Prepare a green fluorescent gold nanocluster AuNCs solution, a dendritic mesoporous silica nanoparticle DMSN solution, and a polyetherimide PEI solution respectively;

[0007] S2. Add the PEI solution to the DMSN solution, mix evenly, and carry out ultrasonic reaction to obtain a mixed solution of DMSN coated with PEI on the surface;

[0008] S3. After centrifuging and redissolving the mixed solution obtained in step S2, add the AuNCs solution, mix well, and carry out ultrasonic reaction. Centrifuge and purify multiple times to remove the unbound AuNCs in the solution, and prepare the green fluorescent nanospheres DMSN@AuNCs.

[0009] In the above aspects and any possible implementation manners, a further implementation manner is provided. The AuNCs solution, DMSN solution, and PEI solution are prepared using an aqueous sodium hydroxide solution with pH = 10, and ultrasonic reaction is carried out under light-shielded conditions.

[0010] In the above aspects and any possible implementation manners, a further implementation manner is provided. The preparation of the dendritic mesoporous silica nanoparticles DMSN in step S1 includes the following steps:

[0011] S11. Add triethanolamine (TEA) to water, stir and heat to obtain a mixed solution;

[0012] S12. Add cetyltrimethylammonium chloride (CTAC) and sodium salicylate to the above mixed solution, react for a period of time, and gradually add tetraethyl orthosilicate (TEOS) dropwise and continue to react for 10 - 14 h;

[0013] S13. Collect the product obtained from the reaction in S12 and wash it by centrifugation with ethanol and water alternately. Finally, obtain the dendritic mesoporous silica nanoparticles DMSN.

[0014] In the above aspects and any possible implementation manners, a further implementation manner is provided, which further includes dispersing the finally obtained dendritic mesoporous silica nanoparticles DMSN in methanol containing hydrochloric acid and extracting at 40 - 60 °C.

[0015] In the above aspects and any possible implementation manners, a further implementation manner is provided. The preparation of the green fluorescent gold nanoclusters AuNCs solution in S1 includes the following steps:

[0016] S21. Mix 6 - azido - 2 - thiothymine (ATT) dissolved in NaOH solution and chloroauric acid in a certain volume ratio, let it stand for reaction to obtain an ATT@AuNCs solution, and purify it by dialysis with pure water;

[0017] S22. Add arginine (Arg) solution to the purified ATT@AuNCs solution, react for 12 - 36 hours, and purify it by dialysis with pure water to prepare the green fluorescent gold nanoclusters AuNCs solution.

[0018] For the aspects and any possible implementation manners described above, a further implementation manner is provided, wherein the volume ratio of 6-aza-2-thiothymine ATT to chloroauric acid Arg is 1:1 - 1:3; and the volume ratio of the chloroauric acid Arg solution to the dialyzed ATT@AuNCs solution is 1:8 - 1:10.

[0019] The present invention also provides a green fluorescent nanosphere, which is obtained by using the preparation method of the present invention.

[0020] The present invention also provides an application of the green fluorescent nanosphere as a detection probe.

[0021] For the aspects and any possible implementation manners described above, a further implementation manner is provided, wherein the green fluorescent nanosphere is used in a fluorescence immunochromatography method based on antigen-antibody specific recognition.

[0022] For the aspects and any possible implementation manners described above, a further implementation manner is provided, wherein the green fluorescent nanosphere is used in a fluorescence immunochromatography method based on antigen-antibody specific recognition, and the method includes the following steps: (1). Determine a specific antibody according to the target in the sample to be chromatographed;

[0023] (2). Then couple the specific antibody with the green fluorescent nanosphere to form a detection probe;

[0024] (3). Add the detection probe to the sample, and obtain a mixture after incubating for a period of time;

[0025] (4). Drop the mixture on the immunochromatography test strip and let it stand for a period of time for chromatographic reaction, and then drop the Arg solution on the detection area and the quality control area of the immunochromatography test strip to realize the detection of the target in the sample.

[0026] Advantages of the present invention

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The preparation method provided by the present invention is simple to operate, low in cost, mild in reaction conditions, and environmentally friendly. The prepared green fluorescent nanosphere DMSN@AuNCs composite material can be used as a fluorescence detection probe in the field of immunological detection; according to different antibodies and antigens labeled on the green fluorescent nanosphere DMSN@AuNCs, a variety of fluorescence detection probes can be formed to detect different target molecules, and it has high popularization and application value in aspects such as multi-residue high-throughput detection. Description of the drawings

[0029] Figure 1 It is a flowchart of the preparation method of the green fluorescent nanosphere DMSN@AuNCs of the present invention;

[0030] Figure 2 Transmission electron microscopy image of the green fluorescent nanospheres DMSN@AuNCs prepared for the present invention;

[0031] Figure 3 Schematic diagram of the fluorescence immunochromatography method based on the specific recognition of antigen and antibody;

[0032] Figure 4 Result diagram of qualitative detection of AFP by the fluorescence immunochromatography method based on green fluorescent nanospheres DMSN@AuNCs. Detailed implementation manners

[0033] To better understand the technical solution of the present invention, the content of the present invention includes but is not limited to the following detailed implementation manners. Similar technologies and methods should be regarded as within the scope of protection of the present invention. To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0034] It should be clear that the embodiments described in the present invention are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0035] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The singular forms "a", "the" and "said" used in the embodiments of the present invention and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0036] As Figure 1 shown, the preparation method of the green fluorescent nanospheres of the present invention includes the following steps: S1. Prepare a green fluorescent gold nanocluster AuNCs solution, a dendritic mesoporous silica nanoparticle DMSN solution and a polyetherimide PEI solution respectively;

[0037] S2. Add an excessive amount of PEI solution to the DMSN solution, mix evenly and carry out an ultrasonic reaction. PEI can be adsorbed on the surface of DMSN through electrostatic interaction. The surface of DMSN modified by PEI shows a positive charge, and a DMSN mixed solution coated with PEI on the surface is obtained.

[0038] S3. After centrifuging and redissolving the mixed solution obtained in step S2, add the AuNCs solution, mix evenly and carry out an ultrasonic reaction, centrifuge multiple times, add water to redissolve and purify the material, and remove the unbound AuNCs in the solution to obtain green fluorescent nanospheres DMSN@AuNCs, as Figure 2 shown.

[0039] The above method steps of the present invention, wherein the green fluorescent gold nanoclusters AuNCs, PEI, and DMSN are assembled by the layer-by-layer method; specifically, a layer of positively charged PEI is first modified on the surface of negatively charged DMSN, and then negatively charged fluorescent gold nanoclusters AuNCs are added and fixed on DMSN by electrostatic interaction through electrostatic interaction, thereby obtaining green fluorescent gold nanospheres.

[0040] Preferably, in the above preparation method, in step S1, an AuNCs solution, a DMSN solution, and a PEI solution are prepared using an aqueous sodium hydroxide solution with pH = 10, and an ultrasonic reaction is carried out under light-shielded conditions.

[0041] Preferably, in the above preparation method, in step S2, the concentration of the DMSN solution is 0.05 mg / mL, and the concentration of PEI is 0.2 mg / mL.

[0042] Preferably, in the above preparation method, in steps S2 and S3, the conditions of the ultrasonic reaction are as follows:

[0043] The temperature is room temperature, the ultrasonic power is 36 w; the time is 40 min.

[0044] Preferably, in the above preparation method, in step S1, the preparation of the dendritic mesoporous silica nanoparticles DMSN solid includes the following steps:

[0045] S11. Add 130 - 140 mg of triethanolamine (TEA) to 30 - 60 mL of water, stir and heat to 80 °C, and the stirring rate is 600 - 1000 rpm;

[0046] S12. Add 750 - 800 mg of cetyltrimethylammonium chloride (CTAC) and 300 - 350 mg of sodium salicylate to the above mixed solution, react for 1 hour, and dropwise add 6 - 10 mL of tetraethyl orthosilicate (TEOS) and continue to react for 10 - 14 h. DMSN is prepared by a surfactant-directed sol-gel process in an aqueous medium using triethanolamine TEA as a catalyst, TEOS as a silica source, CTAC as a cationic surfactant, and sodium salicylate as an anionic surfactant;

[0047] S13. Collect the product obtained from the above reaction and wash it by centrifugation with ethanol and water alternately 3 times to remove excess TEA and TEOS to obtain DMSN solid;

[0048] S14. The residual surfactant is removed by dispersing solid DMSN in methanol containing 8 - 12% v / v hydrochloric acid and extracting for 2 hours at 40 - 60 °C, and then the material is dried in an oven at 60 - 110 °C to obtain high - purity DMSN.

[0049] Preferably, in step S1, the preparation of the green fluorescent gold nanocluster AuNCs solution comprises the following steps:

[0050] S21. 6 - Azido - 2 - thiothymine ATT dissolved in NaOH solution and chloroauric acid are mixed in a certain volume ratio, and after standing and reacting for 1 - 2 hours, an ATT@Au solution is obtained, which is purified by dialysis with pure water;

[0051] S22. Arginine Arg solution is added to the purified ATT@Au solution, and the reaction is carried out at 37 °C for 12 - 36 hours, and then purified by dialysis with pure water to obtain an AuNCs solution. The AuNCs solution is stored in a 4 °C dark environment to prevent photobleaching of the nanoclusters.

[0052] Preferably, the green fluorescent nanospheres DMSN@AuNCs provided by the present invention can be used as a fluorescence detection probe in a fluorescence immunochromatography method based on antigen - antibody specific recognition.

[0053] In the fluorescence immunochromatography method, the following steps are carried out:

[0054] (1). According to the target in the sample to be chromatographed, determine the specific antibody;

[0055] (2). Couple the specific antibody with the green fluorescent nanospheres to form a detection probe;

[0056] (3). Add the detection probe to the sample and incubate for 3 - 5 minutes;

[0057] (4). Drop the mixture into the sample application hole of the immunochromatography test strip, let it stand for chromatographic reaction for 10 - 15 minutes, then drop Arg solution on the detection area and the quality control area of the immunochromatography test strip, observe the result under ultraviolet light, and qualitatively detect the concentration of the target in the sample according to the fluorescence intensity.

[0058] The above - mentioned fluorescence immunochromatography test strip is composed of a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), a blotting paper, a sticky bottom plate, etc.

[0059] The surface charges of fluorescent gold nanoclusters and DMSN are opposite to that of PEI. Therefore, in the present invention, fluorescent gold nanoclusters and DMSN are used as components simultaneously to synthesize green fluorescent nanospheres DMSN@AuNCs, which can achieve the purpose of enriching fluorescent nanoclusters for purification. At the same time, the monoclonal antibody can be labeled by the green fluorescent nanospheres DMSN@AuNCs. Because the reaction conditions are mild, green and pollution-free, and the properties of the organic components participating in the reaction are not affected, the method of the present invention effectively solves the problems such as low labeling efficiency and poor stability of fluorescent gold nanoclusters as fluorescent markers in immunoassays.

[0060] Example 1: Preparation of green fluorescent nanosphere solution

[0061] All glass instruments were soaked in aqua regia for 24 hours and then rinsed thoroughly with distilled water. First, fluorescent gold nanoclusters AuNCs (40 mM), DMSN (1 mg / ml) and PEI (10 mg / ml; pH = 10) solutions were respectively prepared with ultrapure water. Second, 2 mL of DMSN solution (1 mg / ml) was added to 40 ml of aqueous NaOH solution (10 -4 M), and after mixing, 800 μl of PEI (10 mg / ml) solution was added and mixed well, and the reaction was carried out under ultrasonic conditions for 40 min. Then, it was centrifuged at 6000 rpm for 6 min, and the supernatant was discarded. The precipitate was redissolved with ultrapure water, centrifuged and washed 3 times to obtain DMSN modified by PEI. Third, the DMSN material modified by PEI was redissolved with 40 ml of aqueous NaOH solution (10 -4 M), and then 120 μl of AuNCs (40 mM) solution was added and the ultrasonic reaction was carried out for 40 min. Finally, through multiple centrifugation purifications, water was added to redissolve to a total volume of 100 μl, and a green fluorescent nanosphere DMSN@AuNCs solution (solid content about 2%) was prepared, which emitted strong green fluorescence under ultraviolet light irradiation. The material has low production cost and simple process. Due to the huge specific surface area of DMSN, the strong positive surface charge of PEI, the high fluorescence quantum yield of AuNCs and the rich functional groups, the prepared green fluorescent nanospheres have the advantages of stability, high dispersion degree, strong optical signal, easy modification, etc., and are expected to become a kind of fluorescent labeling nanomaterial.

[0062] Example 2: Preparation of detection probe

[0063] First, green fluorescent nanosphere DMSN@AuNCs solution and anti - alpha - fetoprotein AFP monoclonal antibody solution were respectively prepared with ultrapure water for the preparation of detection probes. The anti - AFP monoclonal antibody solution with a concentration of 7.5 μg / ml was added to the DMSN@AuNCs solution (final concentration of 20 μg / ml). After mixing, it was placed in the dark at room temperature and shaken evenly (30 rpm) for 60 min. Then, 50 μl of 1% polyethylene glycol 20000 (PEG20000 ) Shake well for 15 min in the dark, then add 100 μl of bovine serum albumin (BSA) solution (w / v: 10%), shake well for 15 min in the dark to form a fluorescence detection probe for AFP detection; finally, discard the supernatant by centrifugation, and resuspend the fluorescence detection probe with 200 μl of the reconstitution solution, and store it in the dark for later use.

[0064] Example 3: Preparation of immunochromatographic test strip

[0065] The immunochromatographic test strip mainly consists of a nitrocellulose membrane (NC membrane), a sample pad, a conjugate pad, absorbent paper, a sticky bottom plate, etc. The specific operation is as follows: Dilute the anti-AFP antibody and goat anti-mouse IgG to 0.6 mg / mL and 0.2 mg / mL respectively with phosphate buffer solution (PBS), and use a membrane scribing instrument to scribe the two antibody dilution solutions on the NC membrane as the test line (T) and the quality control line (C) at a spraying volume of 1 μl / cm. After drying at 37 °C for 12 h, paste the NC membrane, sample pad, conjugate pad, and absorbent paper on the sticky bottom plate in sequence, and cut it into an immunochromatographic test strip about 3 mm wide for detecting the target AFP in the sample.

[0066] Example 4: Immunochromatography based on green fluorescent nanospheres

[0067] The basic principle of detecting AFP by immunochromatography based on green fluorescent nanospheres is as Figure 3 shown. The specific operation is as follows: Dilute the AFP national standard substance with phosphate buffer solution (PBS) to different concentrations such as 0, 2.2, 4.5, 9.1, 18.2, 36.5 μg / l; respectively take 80 μl of the AFP standard substance dilution solutions with different concentrations as samples containing the target AFP, add 6 μL of the fluorescence detection probe to the samples, after reacting for 3 min, drop them into the sample adding hole of the immunochromatographic test strip, let it stand for chromatography for 10 min, then drop 5 μl of arginine solution (pH = 10, 40 mM) on the NC membrane, and observe the results under ultraviolet light; according to the green fluorescence intensity of the test line on the immunochromatographic test strip, qualitative and semi-quantitative detection of the target AFP in the sample can be carried out. The experimental results ( Figure 4 ) show that as the concentration of the AFP standard substance dilution solution increases, the fluorescence intensity on the test line increases.

[0068] The foregoing description has shown and described several preferred embodiments of the present invention. However, as previously mentioned, it should be understood that the present invention is not limited to the forms disclosed herein, should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications, and environments, and can be altered within the scope of the inventive concept described herein through the above teachings or the techniques or knowledge in the relevant field. Any alterations and changes made by those skilled in the art that do not depart from the spirit and scope of the present invention shall fall within the protection scope of the appended claims of the present invention.

Claims

1. A preparation method of green fluorescent nanospheres, characterized in that, The method includes the following steps: S1. Prepare a green fluorescent gold nanocluster AuNCs solution, a dendritic mesoporous silica nanoparticle DMSN solution, and a polyetherimide PEI solution respectively; the preparation of the dendritic mesoporous silica nanoparticle DMSN in step S1 includes the following steps: S11. Add triethanolamine TEA to water, stir and heat to obtain a mixed solution; S12. Add cetyltrimethylammonium chloride CTAC and sodium salicylate to the above mixed solution, react for a period of time, and gradually add tetraethyl orthosilicate TEOS dropwise and continue to react for 10 - 14 h; S13. Collect the product obtained from the reaction in S12 and wash it by centrifugation with ethanol and water alternately, and finally obtain the dendritic mesoporous silica nanoparticle DMSN; The preparation of the green fluorescent gold nanocluster AuNCs solution in S1 includes the following steps: S21. Mix 6 - azido - 2 - thiothymine ATT dissolved in NaOH solution and chloroauric acid in a certain volume ratio, let it stand and react to obtain an ATT@AuNCs solution, and purify it by dialysis with pure water; S22. Add an arginine Arg solution to the purified ATT@AuNCs solution, react for 12 - 36 hours, and purify it by dialysis with pure water to obtain a green fluorescent gold nanocluster AuNCs solution; S2. Add the PEI solution to the DMSN solution, mix evenly and carry out an ultrasonic reaction to obtain a DMSN mixed solution coated with PEI on the surface; S3. After centrifuging and redissolving the mixed solution obtained in step S2, add the AuNCs solution, mix evenly and carry out an ultrasonic reaction, and centrifuge and purify multiple times to remove the unbound AuNCs in the solution to obtain green fluorescent nanospheres DMSN@AuNCs.

2. The preparation method according to claim 1, characterized in that, In step S1, prepare the AuNCs solution, DMSN solution, and PEI solution with a sodium hydroxide aqueous solution of pH = 10, and carry out an ultrasonic reaction under light - shielding conditions.

3. The preparation method according to claim 1, characterized in that It also includes dispersing the finally obtained dendritic mesoporous silica nanoparticle DMSN in methanol containing hydrochloric acid and extracting it at 40 - 60 °C.

4. The preparation method according to claim 1, wherein The volume ratio of the 6 - azido - 2 - thiothymine ATT and chloroauric acid Arg is 1:1 - 1:3; the volume ratio of the chloroauric acid Arg solution and the ATT@AuNCs solution after dialysis is 1:8 - 1:

10.

5. A green fluorescent nanosphere, characterized in that, The green fluorescent nanospheres are obtained by the preparation method described in any one of claims 1 - 4.

6. An application of using the green fluorescent nanospheres described in claim 5 as an AFP detection probe.

7. Use of the green fluorescent nanospheres according to claim 6 as an AFP detection probe, characterized in that, The green fluorescent nanospheres are used in a fluorescence immunochromatography method based on antigen - antibody specific recognition.

8. Use of the green fluorescent nanospheres according to claim 7 as an AFP detection probe, characterized in that, The green fluorescent nanospheres are used in a fluorescence immunochromatography method based on antigen - antibody specific recognition, including the following steps: (1). Determine the specific antibody according to the target substance in the sample to be chromatographed; (2). Then couple the specific antibody with the green fluorescent nanospheres to form a detection probe; (3). Add the detection probe to the sample, incubate and react for a period of time to obtain a mixture; (4) Drop the mixture onto the immunochromatographic test strip and let it stand for chromatographic reaction for a period of time, and then drop Arg solution onto the detection area and the quality control area of the immunochromatographic test strip to achieve the detection of the target substance in the sample.

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