Preparation and application of multicolor nano fluorescent probe based on fluorescent micromolecule FRET (Fluorescence Resonance Energy Transfer)

Abstract

The invention discloses preparation and application of a multicolor nano fluorescent probe based on fluorescent micromolecules FRET, belongs to the technical field of medical reagents, and particularly relates to a preparation method of the nano fluorescent probe. Then adding an organic reagent and a siloxane precursor to prepare an organic phase solution, adding the organic phase solution into an alkaline water phase system to form nano particles, and finally coating the outer layer with silicon dioxide to prepare the nano fluorescent probe. The use volume ratio of the donor fluorescent small molecule solution to the receptor fluorescent small molecule solution is (0.5-1): (0-25). According to the invention, donor fluorescent micromolecules are HMAT blue fluorescent micromolecules, and receptor fluorescent micromolecules comprise BODIPY488 or Nile red.

Description

Technical Field

[0001] This invention belongs to the field of medical reagent technology, specifically relating to the preparation and application of multicolor nanofluorescent probes based on the fluorescent small molecule FRET. Background Technology

[0002] Fluorescence imaging technology, due to its high sensitivity, strong spatial resolution, and applicability to living systems, has been widely used in biomedical research, disease diagnosis, and molecular detection. With the deepening of research into biological systems, single-target imaging is no longer sufficient to meet research needs, and multi-target detection and multicolor fluorescence imaging are gradually becoming important development directions. Multicolor fluorescence imaging can simultaneously acquire information on multiple biomarkers in the same sample, thereby improving analytical throughput and detection efficiency.

[0003] Driven by the aforementioned demands for multicolor imaging applications, multicolor fluorescence imaging places higher performance requirements on fluorescent probes. On the one hand, fluorescent probes should have high fluorescence brightness and good photostability to ensure sufficient signal intensity under multi-channel imaging conditions; on the other hand, different color signals should have good distinguishability to reduce spectral crosstalk.

[0004] Currently, fluorescent probes used in multicolor imaging mainly include organic small-molecule fluorescent dyes and nanoparticle fluorescent probes. Organic small-molecule fluorescent dyes have advantages such as diverse structures, well-defined structure-activity relationships, and precise control of emission wavelengths through molecular structure design. However, most small-molecule dyes suffer from insufficient photostability, limited fluorescence brightness, susceptibility to photobleaching, and poor stability in complex biological environments, making them unsuitable for long-term, multi-channel imaging applications. In contrast, nanoparticle fluorescent probes typically possess high fluorescence brightness and good photostability, along with good water solubility and biocompatibility, meeting the basic requirements for long-term, multi-channel imaging. Therefore, they have gradually become the mainstream probe type in multicolor fluorescence imaging applications. However, existing nanoparticle fluorescent probes generally have broad emission spectra, limiting the selectable emission bands and making it difficult to achieve fine differentiation between different channels in multicolor imaging.

[0005] Fluorescence resonance energy transfer (FRET) is an important method for constructing multicolor fluorescent probes. By establishing an energy transfer relationship between donor and acceptor fluorescent dyes and changing different acceptor dyes, multiple fluorescence signals with different emission wavelengths can be obtained under a single excitation condition, thereby achieving multicolor fluorescence imaging. Introducing the FRET mechanism into nanoparticle fluorescent probe systems to construct FRET nanoparticle fluorescent probes with high brightness, high photostability, and tunable emission has become an important development direction for constructing multicolor fluorescent probes.

[0006] However, due to the relatively limited variety of polymeric fluorescent dyes available for constructing FRET nanoparticle fluorescent probes, most existing techniques employ the method of directly encapsulating small-molecule fluorescent dyes inside the nanoparticles. This approach is prone to small-molecule dye leakage, leading to changes in the donor-acceptor ratio and distance, which can cause background enhancement, signal crosstalk, and unstable FRET efficiency in multicolor imaging. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing and applying a multicolor nano-fluorescent probe based on the fluorescent small molecule FRET, which has tunable emission spectrum, good water dispersibility, good biocompatibility, good stability, and high fluorescence intensity.

[0008] The technical solution adopted by the present invention to achieve the above objectives is as follows: A method for preparing a nano-fluorescent probe includes: mixing a donor fluorescent small molecule solution and an acceptor fluorescent small molecule solution, then adding an organic reagent and a siloxane precursor to form an organic phase solution, adding the organic phase solution to an alkaline aqueous system to form nanoparticles, and finally coating the outer layer with silica to obtain the nano-fluorescent probe; the volume ratio of the donor fluorescent small molecule solution to the acceptor fluorescent small molecule solution is 0.5-1:0-25. This invention constructs a nanoparticle fluorescent probe based on fluorescence resonance energy transfer (FRET) of small molecule dyes. Through organic-inorganic hybrid encapsulation and an outer shell structure, the donor and acceptor small molecule fluorescent dyes are stably fixed inside the nanoparticles. This improves FRET stability, enhances overall brightness and photostability, effectively suppresses the migration and leakage of small molecule dyes, and allows for tunable emission spectra by changing different acceptor small molecule fluorescent dyes, making it suitable for multicolor fluorescence imaging applications.

[0009] Preferably, the donor fluorescent molecule solution contains a donor fluorescent molecule, which is HMAT blue fluorescent molecule.

[0010] Preferably, the receptor fluorescent small molecule solution contains a receptor fluorescent small molecule, including BODIPY488 or Nile Red.

[0011] Preferably, the organic reagent includes an organic polymer, which includes PS and / or PSMA.

[0012] More preferably, the organic polymer also includes polyacrylamide.

[0013] Preferably, the organic reagent includes the solvent tetrahydrofuran.

[0014] Preferably, the organic reagent includes dodecyl ethoxysulfonate betaine. In the preparation of the nano-fluorescent probe, the organic polymer in the organic reagent of this invention includes at least PS and / or PSMA, and polyacrylamide may also be added. Dodecyl ethoxysulfonate betaine may also be added as a synergistic agent in the organic system. The organic polymer and dodecyl ethoxysulfonate betaine together constitute the donor HMAT blue fluorescent dye and the acceptor small molecule fluorescent dye, which spatially confine and isolate the dye, inhibit the leakage of small molecule dye, reduce the aggregation-induced quenching of small molecule dye, improve fluorescence performance, and enhance stability.

[0015] Preferably, the alkaline aqueous phase system is deionized water with pH adjusted to 10-11 using ammonia.

[0016] This invention discloses the nanofluorescent probes prepared by the above method.

[0017] This invention discloses the application of the above-mentioned nano-fluorescent probes in the preparation of medical diagnostic reagents.

[0018] This invention discloses a method for preparing HMAT blue fluorescent small molecules, including: the preparation of compound 2, the preparation of compound 3 and the preparation of compound 4.

[0019] Preferably, in the preparation of compound 2, compound 1 is added to DMF, and POCl3 solution is added under a nitrogen atmosphere at a temperature of 0-5°C. Then, the temperature is raised to 70-90°C and the reaction is stirred for 10-24 hours. After the reaction is completed, the mixture is cooled to room temperature, quenched with ice water, and then the pH is adjusted to neutral. The mixture is extracted with dichloromethane, and the organic phase is washed successively with deionized water and saturated brine. The phase is dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound 2.

[0020] More preferably, in the preparation of compound 2, the mass-to-volume ratio of compound 1 to DMF is 0.1-1 g: 10-30 mL.

[0021] More preferably, in the preparation of compound 2, the POCl3 solution contains POCl3 and DMF, and the volume ratio of POCl3 and DMF in the POCl3 solution is 1-2:1-3.

[0022] More preferably, in the preparation of compound 2, the volume ratio of POCl3 solution to DMF is 2-5:10-30.

[0023] More preferably, in the preparation of compound 2, the volume ratio of ice water to DMF is 30-100:10-30.

[0024] More preferably, in the preparation of compound 2, the pH is adjusted using a saturated sodium bicarbonate solution.

[0025] More preferably, in the preparation of compound 2, the eluent in the silica gel column chromatography purification contains petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate in the eluent is 90-95:5-10.

[0026] Preferably, in the preparation of compound 3, compound 2 and hydroxylamine hydrochloride are added to NMP, sealed in an air atmosphere, and stirred at 110-120°C for 3-10 h. After the reaction is completed, the mixture is cooled to room temperature, deionized water is added, and then the mixture is extracted with ethyl acetate. The organic phase is washed successively with deionized water and saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound 3.

[0027] More preferably, in the preparation of compound 3, the mass-to-volume ratio of compound 2 to NMP is 200-600 mg: 5-10 mL.

[0028] More preferably, in the preparation of compound 3, the mass ratio of compound 2 to hydroxylamine hydrochloride is 300-500:150-250.

[0029] More preferably, in the preparation of compound 3, the volume ratio of deionized water to NMP is 20-50:5-10.

[0030] More preferably, in the preparation of compound 3, the eluent in the silica gel column chromatography purification contains petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate in the eluent is 90-95:5-10.

[0031] Preferably, in the preparation of compound 4, compound 3, sodium azide and ammonium chloride are added to DMF, sealed in an air atmosphere, and stirred at 120-140°C for 8-24 hours. After the reaction is completed, the mixture is cooled to room temperature, deionized water is added, and then the mixture is extracted with ethyl acetate. The organic phase is washed successively with deionized water and saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound 4.

[0032] More preferably, in the preparation of compound 4, the mass-to-volume ratio of compound 3 to DMF is 100-200 mg: 2-10 mL.

[0033] More preferably, in the preparation of compound 4, the mass ratio of compound 3 to sodium azide is 100-200:300-400.

[0034] More preferably, in the preparation of compound 4, the mass ratio of compound 3 to ammonium chloride is 100-200:250-350.

[0035] More preferably, in the preparation of compound 4, the volume ratio of deionized water to DMF is 20-50:2-10.

[0036] More preferably, in the preparation of compound 4, the eluent used in silica gel column chromatography purification is ethyl acetate.

[0037] Preferably, in the preparation of HMAT blue fluorescent small molecule, under a nitrogen atmosphere, compound 4, benzoic acid and DCC are added to toluene and stirred at 130-150℃ for 8-24h. The reaction is monitored by thin-layer chromatography (TLC). After the reaction is completed, the mixture is cooled to room temperature, dichloromethane is added, and the mixture is frozen at -20℃ for 0.5-2h. The precipitated DCC byproduct is removed by filtration, and the byproduct is washed with dichloromethane. The washing solution and filtrate are combined, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain HMAT blue fluorescent small molecule.

[0038] More preferably, in the preparation of HMAT blue fluorescent small molecules, the mass-to-volume ratio of compound 4 and toluene is 50-150 mg: 2-10 mL.

[0039] More preferably, in the preparation of HMAT blue fluorescent small molecule, the mass molar ratio of compound 4 and benzoic acid is 50-150 mg: 0.1-0.4 mmol.

[0040] More preferably, in the preparation of the HMAT blue fluorescent small molecule, the mass ratio of compound 4 to DCC is 50-150:20-80.

[0041] More preferably, in the preparation of HMAT blue fluorescent small molecules, the volume ratio of dichloromethane to toluene before freezing is 10-20:2-10.

[0042] More preferably, in the preparation of HMAT blue fluorescent small molecules, the eluent used in silica gel column chromatography purification is ethyl acetate.

[0043] Preferably, in the preparation of HMAT nanoparticle probes, HMAT blue fluorescent small molecule solution, polystyrene (PS) solution, polystyrene maleic anhydride copolymer (PSMA) solution, TEOS solution and TCOS solution are mixed, diluted with tetrahydrofuran and ultrasonically mixed to obtain an organic phase solution; an alkaline solution is added under ultrasonication, shaken for 1-5 min, and then concentrated at 80-90℃ under a nitrogen atmosphere to obtain a nanoparticle dispersion; cooled to room temperature, filtered through a microporous membrane, deionized water is added to the filtrate, the pH is adjusted to 9-11, and then the coating precursor solution is added in 5-10 portions, each time stirred and reacted at 20-40℃ for 0.5-2 h. After the coating reaction is completed, the mixture is filtered through a microporous membrane and then ultrafiltered to obtain the HMAT nanoparticle probe.

[0044] More preferably, in the preparation of the HMAT nanoparticle probe, the HMAT blue fluorescent small molecule solution contains HMAT blue fluorescent small molecules and tetrahydrofuran, and the content of HMAT blue fluorescent small molecules in the HMAT blue fluorescent small molecule solution is 0.2-5 mg / mL.

[0045] More preferably, in the preparation of the HMAT nanoparticle probe, the polystyrene (PS) solution contains PS and tetrahydrofuran, and the content of PS in the polystyrene (PS) solution is 0.2-5 mg / mL.

[0046] More preferably, in the preparation of the HMAT nanoparticle probe, the polystyrene maleic anhydride copolymer (PSMA) solution contains PSMA and tetrahydrofuran, and the content of PSMA in the polystyrene maleic anhydride copolymer (PSMA) solution is 0.2-5 mg / mL.

[0047] More preferably, in the preparation of HMAT nanoparticle probes, the TEOS solution contains TEOS and tetrahydrofuran, and the content of TEOS in the TEOS solution is 0.2-5 mg / mL.

[0048] More preferably, in the preparation of HMAT nanoparticle probes, the TCOS solution contains TCOS and tetrahydrofuran, and the content of TCOS in the TCOS solution is 0.2-5 mg / mL.

[0049] More preferably, in the preparation of the HMAT nanoparticle probe, the volume ratio of the HMAT blue fluorescent small molecule solution to the polystyrene (PS) solution is 10-30:50-150.

[0050] More preferably, in the preparation of the HMAT nanoparticle probe, the volume ratio of the HMAT blue fluorescent small molecule solution to the polystyrene maleic anhydride copolymer (PSMA) solution is 10-30:50-150.

[0051] More preferably, in the preparation of HMAT nanoparticle probes, the volume ratio of HMAT blue fluorescent small molecule solution to TEOS solution is 10-30:150-250.

[0052] More preferably, in the preparation of HMAT nanoparticle probes, the volume ratio of HMAT blue fluorescent small molecule solution to TCOS solution is 10-30:150-250.

[0053] More preferably, in the preparation of the HMAT nanoparticle probe, the volume ratio of the organic phase solution to the HMAT blue fluorescent small molecule solution in the organic phase solution obtained by volume adjustment is 1-3 mL: 10-30 μL.

[0054] More preferably, in the preparation of HMAT nanoparticle probes, the alkaline solution is ammonia water added to deionized water to adjust the pH to 10-11, and the volume ratio of the alkaline solution to the HMAT blue fluorescent small molecule solution is 5-15 mL: 10-30 μL.

[0055] More preferably, in the preparation of the HMAT nanoparticle probe, the volume ratio of the concentrated nanoparticle dispersion to the HMAT blue fluorescent small molecule solution is 1-3 mL: 10-30 μL.

[0056] More preferably, in the preparation of the HMAT nanoparticle probe, the nanoparticle dispersion is filtered and then added to deionized water, and the volume ratio of deionized water to nanoparticle dispersion is 0.5-2:0.5-2.

[0057] More preferably, in the preparation of HMAT nanoparticle probes, the pore size of the microporous filter membrane is 0.1-0.3 μm.

[0058] More preferably, in the preparation of HMAT nanoparticle probes, the coating precursor solution contains TEOS and DMSO, and the volume ratio of TEOS to DMSO in the coating precursor solution is 0.5-2:5-10; the volume ratio of the coating precursor solution used each time to the volume ratio of the nanoparticle dispersion is 5-20 μL:1-3 mL; and the cutoff value of the ultrafiltration tube is 50-200 kDa.

[0059] Preferably, in the preparation of the FRET nanoparticle probe, the donor HMAT blue fluorescent small molecule solution is mixed with the acceptor small molecule fluorescent dye to obtain a composite small molecule solution. Then, the composite small molecule solution, PS solution, PSMA solution, TEOS solution, and TCOS solution are mixed, diluted with tetrahydrofuran, and ultrasonically mixed to obtain an organic phase solution. An alkaline solution is added under ultrasonication, and the mixture is shaken for 1-5 min. Then, the mixture is concentrated at 80-90℃ under a nitrogen atmosphere to obtain a nanoparticle dispersion. The mixture is cooled to room temperature, filtered through a microporous membrane, and the filtrate is added with deionized water to adjust the pH to 9-11. Then, the coating precursor solution is added in 5-10 portions, and the mixture is stirred at 20-40℃ for 0.5-2 h each time. After the coating reaction is completed, the mixture is filtered through a microporous membrane and then ultrafiltered to obtain the FRET nanoparticle probe.

[0060] More preferably, in the preparation of the FRET nanoparticle probe, the donor HMAT blue fluorescent small molecule solution contains HMAT blue fluorescent small molecule and tetrahydrofuran, and the content of HMAT blue fluorescent small molecule in the HMAT blue fluorescent small molecule solution is 0.2-5 mg / mL; the acceptor small molecule fluorescent dye includes acceptor BODIPY488 small molecule fluorescent dye or acceptor Nile Red small molecule fluorescent dye, and the volume ratio of donor HMAT blue fluorescent small molecule solution to acceptor small molecule fluorescent dye in the composite small molecule solution is 0.5-1:1-25.

[0061] More preferably, in the preparation of the FRET nanoparticle probe, the polystyrene (PS) solution contains PS and tetrahydrofuran, and the content of PS in the polystyrene (PS) solution is 0.2-5 mg / mL.

[0062] More preferably, in the preparation of the FRET nanoparticle probe, the polystyrene maleic anhydride copolymer (PSMA) solution contains PSMA and tetrahydrofuran, and the content of PSMA in the polystyrene maleic anhydride copolymer (PSMA) solution is 0.2-5 mg / mL.

[0063] More preferably, in the preparation of the FRET nanoparticle probe, the TEOS solution contains TEOS and tetrahydrofuran, and the content of TEOS in the TEOS solution is 0.2-5 mg / mL.

[0064] More preferably, in the preparation of the FRET nanoparticle probe, the TCOS solution contains TCOS and tetrahydrofuran, and the content of TCOS in the TCOS solution is 0.2-5 mg / mL.

[0065] More preferably, in the preparation of FRET nanoparticle probes, the volume ratio of the composite small molecule solution to the polystyrene (PS) solution is 10-30:50-150.

[0066] More preferably, in the preparation of the FRET nanoparticle probe, the volume ratio of the composite small molecule solution to the polystyrene maleic anhydride copolymer (PSMA) solution is 10-30:50-150.

[0067] More preferably, in the preparation of FRET nanoparticle probes, the volume ratio of the composite small molecule solution to the TEOS solution is 10-30:150-250.

[0068] More preferably, in the preparation of FRET nanoparticle probes, the volume ratio of the composite small molecule solution to the TCOS solution is 10-30:150-250.

[0069] More preferably, in the preparation of the FRET nanoparticle probe, the volume ratio of the organic phase solution to the composite small molecule solution in the organic phase solution obtained by volume adjustment is 1-3 mL: 10-30 μL.

[0070] More preferably, in the preparation of the FRET nanoparticle probe, the alkaline solution is ammonia water added to deionized water to adjust the pH to 10-11, and the volume ratio of the alkaline solution to the composite small molecule solution is 5-15 mL: 10-30 μL.

[0071] More preferably, in the preparation of the FRET nanoparticle probe, the volume ratio of the concentrated nanoparticle dispersion to the composite small molecule solution is 1-3 mL: 10-30 μL.

[0072] More preferably, in the preparation of the FRET nanoparticle probe, the nanoparticle dispersion is filtered and then added to deionized water, and the volume ratio of deionized water to nanoparticle dispersion is 0.5-2:0.5-2.

[0073] More preferably, in the preparation of the FRET nanoparticle probe, the pore size of the microporous filter membrane is 0.1-0.3 μm.

[0074] More preferably, in the preparation of the FRET nanoparticle probe, the coating precursor solution contains TEOS and DMSO, and the volume ratio of TEOS to DMSO in the coating precursor solution is 0.5-2:5-10; the volume ratio of the coating precursor solution used each time to the volume ratio of the nanoparticle dispersion is 5-20 μL:1-3 mL; and the cutoff value of the ultrafiltration tube is 50-200 kDa.

[0075] More preferably, in the preparation of the FRET nanoparticle probe, the polystyrene maleic anhydride copolymer (PSMA) solution contains 0.01-0.1 mg / mL of dodecyl ethoxysulfobetaine and 0.01-0.06 mg / mL of polyacrylamide.

[0076] More preferably, in the preparation of the FRET nanoparticle probe, the content of p-hydroxybenzylacetone in the polystyrene-maleic anhydride copolymer (PSMA) solution is 0.005-0.02 mg / mL. In the preparation of the FRET nanoparticle probe using organic reagents as the organic system, in addition to PS, PSMA, and polyacrylamide, dodecyl ethoxysulfonate betaine and p-hydroxybenzylacetone can also be used. Under the action of the organic polymers of PS, PSMA, and polyacrylamide, as well as dodecyl ethoxysulfonate betaine and p-hydroxybenzylacetone, the prepared FRET nanoparticle probe exhibits superior performance, improved fluorescence performance, and enhanced stability.

[0077] This invention spatially confines and isolates the donor HMAT blue fluorescent dye and the acceptor small-molecule fluorescent dye through an organic polymer and hybrid silica network structure within the nanoparticles. An outer silica shell effectively suppresses leakage of the small-molecule dye and reduces aggregation-induced quenching, thus improving the structural stability of the FRET system. This invention achieves multi-wavelength fluorescence emission under a single excitation condition, simplifying multicolor imaging's dependence on multiple excitation sources and reducing the complexity of the imaging system. The small-molecule FRET nanoparticle fluorescent probe prepared by this invention exhibits high fluorescence brightness and good photostability, suitable for long-term imaging. The emission spectrum of the small-molecule FRET nanoparticle fluorescent probe prepared by this invention can be tunably adjusted by flexibly replacing different acceptor small-molecule fluorescent dyes, resulting in a highly versatile and scalable system. The small-molecule FRET nanoparticle fluorescent probe prepared by this invention has good water dispersibility and biocompatibility, making it suitable for biological multicolor imaging and cell labeling applications. Therefore, this invention represents the preparation and application of a multicolor nanoparticle fluorescent probe based on fluorescent small-molecule FRET that achieves tunable emission spectra, good water dispersibility, good biocompatibility, good stability, and high fluorescence intensity. Attached Figure Description

[0078] Figure 1 The image shows the hydrogen NMR spectrum of compound 1.

[0079] Figure 2 This is the hydrogen NMR spectrum of compound 2.

[0080] Figure 3 The image shows the hydrogen NMR spectrum of compound 3.

[0081] Figure 4 The image shows the proton NMR spectrum of compound 4.

[0082] Figure 5 The image shows the hydrogen NMR spectrum of the blue fluorescent small molecule HMAT.

[0083] Figure 6 The absorption spectra of three fluorescent small molecules, HMAT, BODIPY488, and Nile Red, in tetrahydrofuran are shown.

[0084] Figure 7 The emission spectra of three fluorescent small molecules, HMAT, BODIPY488, and Nile Red, in tetrahydrofuran are shown.

[0085] Figure 8 The diagram shows the overlap between the emission spectrum of the donor HMAT blue fluorescent small molecule dye and the absorption spectrum of the small molecule dye acceptor BODIPY488.

[0086] Figure 9The diagram shows the overlap between the emission spectrum of the donor HMAT blue fluorescent small molecule dye and the absorption spectrum of the small molecule dye Nile Red.

[0087] Figure 10 The fluorescence emission intensity diagrams of HMAT and BODIPY488 at different ratios are shown.

[0088] Figure 11 The fluorescence emission intensity diagrams of HMAT and Nile Red at different ratios are shown.

[0089] Figure 12 This is a graph showing the maximum fluorescence intensity.

[0090] Figure 13 This is a particle size distribution.

[0091] Figure 14 This is a fluorescence leakage characterization diagram of the HMAT nanoparticle probe.

[0092] Figure 15 The image shows the fluorescence leakage characterization of the FRET nanoparticle probe prepared in Example 3.

[0093] Figure 16 The image shows the fluorescence leakage characterization of the FRET nanoparticle probe prepared in Example 4.

[0094] Figure 17 The graph shows the fluorescence intensity of the lower filtrate relative to the fluorescence intensity of the corresponding original nanoparticle aqueous solution.

[0095] Figure 18 This is an image of HMAT nanoparticle probes on cellular microtubules.

[0096] Figure 19 This is an image of cellular microtubules created by the FRET nanoparticle probe in Example 3.

[0097] Figure 20 This is an image of cellular microtubules created by the FRET nanoparticle probe in Example 4. Detailed Implementation

[0098] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0099] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0100] Example 1: A method for preparing HMAT blue fluorescent small molecule The structure of compound 1 in this embodiment is as follows: The structure of compound 2 is as follows: The structure of compound 3 is as follows: The structure of compound 4 is as follows: The structure of the HMAT blue fluorescent small molecule is as follows: .

[0101] Preparation of Compound 2: Compound 1 was added to DMF. Under a nitrogen atmosphere, POCl3 solution was added at 0°C, and the temperature was raised to 80°C with stirring for 15 h. After the reaction was completed, the mixture was cooled to room temperature, quenched with ice water, and the pH was adjusted to neutral. The mixture was extracted with dichloromethane, and the organic phase was washed successively with deionized water and saturated brine. After drying, the mixture was concentrated under reduced pressure and purified by silica gel column chromatography to obtain Compound 2. The mass-to-volume ratio of Compound 1 to DMF was 0.5 g: 20 mL; the POCl3 solution contained POCl3 and DMF, and the volume ratio of POCl3 to DMF in the POCl3 solution was 1.3:2; the volume ratio of POCl3 solution to DMF was 3.3:20; the volume ratio of ice water to DMF was 50:20; the pH was adjusted using saturated sodium bicarbonate solution; in the silica gel column chromatography purification, the eluent contained petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate in the eluent was 95:5.

[0102] Preparation of Compound 3: Compound 2 and hydroxylamine hydrochloride were added to NMP, sealed in air, and stirred at 115°C for 5 h. After the reaction was complete, the mixture was cooled to room temperature, deionized water was added, and the mixture was extracted with ethyl acetate. The organic phase was washed successively with deionized water and saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain Compound 3. The mass-to-volume ratio of Compound 2 to NMP was 400 mg: 6.5 mL; the mass ratio of Compound 2 to hydroxylamine hydrochloride was 400: 212; the volume ratio of deionized water to NMP was 30: 6.5; in the silica gel column chromatography purification, the eluent contained petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 95: 5.

[0103] Preparation of Compound 4: Compound 3, sodium azide, and ammonium chloride were added to DMF, sealed in air, and stirred at 130°C for 16 h. After the reaction was complete, the mixture was cooled to room temperature, deionized water was added, and the mixture was extracted with ethyl acetate. The organic phase was washed successively with deionized water and saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain Compound 4. The mass-to-volume ratio of Compound 3 to DMF was 140 mg: 5 mL; the mass ratio of Compound 3 to sodium azide was 140:374; the mass ratio of Compound 3 to ammonium chloride was 140:308; the volume ratio of deionized water to DMF was 30:5; and ethyl acetate was used as the eluent in the silica gel column chromatography purification.

[0104] Preparation of HMAT blue fluorescent small molecule: Under a nitrogen atmosphere, compound 4, benzoic acid, and DCC were added to toluene and stirred at 140 °C for 12 h. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was complete, the mixture was cooled to room temperature, dichloromethane was added, and the mixture was frozen at -20 °C for 1 h. The precipitated DCC byproduct was removed by filtration. The byproduct was washed with dichloromethane, and the washings and filtrate were combined, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain HMAT blue fluorescent small molecule. The mass-to-volume ratio of compound 4 to toluene was 95 mg:5 mL; the mass-to-molar ratio of compound 4 to benzoic acid was 95 mg:0.22 mmol; the mass ratio of compound 4 to DCC was 95:46; the volume ratio of dichloromethane to toluene before freezing was 15:5; and ethyl acetate was used as the eluent in the silica gel column chromatography purification.

[0105] Example 2: A method for preparing an HMAT nanoparticle probe Preparation of HMAT nanoparticle probes: HMAT blue fluorescent small molecule solution, polystyrene (PS) solution, polystyrene maleic anhydride copolymer (PSMA) solution, TEOS solution and TCOS solution were mixed, diluted to volume with tetrahydrofuran and ultrasonically mixed to obtain an organic phase solution; an alkaline solution was added under ultrasonication, shaken for 2 min, and then concentrated at 90 °C under a nitrogen atmosphere to obtain a nanoparticle dispersion; cooled to room temperature, filtered through a microporous membrane, and deionized water was added to the filtrate to adjust the pH to 10. Then, the coating precursor solution was added in 8 portions, and the reaction was stirred at 25 °C for 1 h each time. After the coating reaction was completed, the mixture was filtered through a microporous membrane and then ultrafiltered to obtain HMAT nanoparticle probes.

[0106] The HMAT blue fluorescent small molecule solution contains HMAT blue fluorescent small molecules and tetrahydrofuran, and the content of HMAT blue fluorescent small molecules in the HMAT blue fluorescent small molecule solution is 1 mg / mL. The polystyrene (PS) solution contains PS and tetrahydrofuran, and the PS content in the polystyrene (PS) solution is 1 mg / mL; The polystyrene maleic anhydride copolymer (PSMA) solution contains PSMA and tetrahydrofuran, and the PSMA content in the polystyrene maleic anhydride copolymer (PSMA) solution is 1 mg / mL; The TEOS solution contains TEOS and tetrahydrofuran, and the TEOS content in the TEOS solution is 1 mg / mL; The TCOS solution contains TCOS and tetrahydrofuran, and the TCOS content in the TCOS solution is 1 mg / mL. The volume ratio of HMAT blue fluorescent small molecule solution to polystyrene (PS) solution is 20:100. The volume ratio of HMAT blue fluorescent small molecule solution to polystyrene maleic anhydride copolymer (PSMA) solution is 20:100. The volume ratio of HMAT blue fluorescent small molecule solution to TEOS solution is 20:200. The volume ratio of HMAT blue fluorescent small molecule solution to TCOS solution is 20:200. After adjusting the volume, the organic phase solution was obtained, and the volume ratio of the organic phase solution to the HMAT blue fluorescent small molecule solution was 2 mL: 20 μL.

[0107] The alkaline solution was prepared by adding ammonia to deionized water and adjusting the pH to 11. The volume ratio of the alkaline solution to the HMAT blue fluorescent small molecule solution was 10 mL: 20 μL.

[0108] The concentration yielded a nanoparticle dispersion with a volume ratio of 2 mL to 20 μL for the nanoparticle dispersion and the HMAT blue fluorescent small molecule solution.

[0109] After filtration, the nanoparticle dispersion is added to deionized water, with a volume ratio of 1:1 between deionized water and nanoparticle dispersion. The pore size of the microporous filter membrane is 0.22 μm; The coating precursor solution contains TEOS and DMSO, with a volume ratio of TEOS to DMSO of 1:9. The volume ratio of the coating precursor solution used each time to the volume of the nanoparticle dispersion is 10 μL: 2 mL. The ultrafiltration tube has a cutoff value of 100 kDa.

[0110] Example 3: A method for preparing FRET nanoparticle probes In this embodiment, the receptor small molecule fluorescent dye for the FRET nanoparticle probe is the receptor BODIPY488 small molecule fluorescent dye.

[0111] Preparation of FRET nanoparticle probes: A composite small molecule solution was prepared by mixing the donor HMAT blue fluorescent small molecule solution with the acceptor small molecule fluorescent dye. Then, the composite small molecule solution, PS solution, PSMA solution, TEOS solution, and TCOS solution were mixed, diluted to volume with tetrahydrofuran, and ultrasonically mixed to obtain an organic phase solution. An alkaline solution was added under ultrasonication and shaken for 2 min. The solution was then concentrated at 90 °C under a nitrogen atmosphere to obtain a nanoparticle dispersion. The solution was cooled to room temperature, filtered through a microporous membrane, and deionized water was added to the filtrate to adjust the pH to 10. The coating precursor solution was then added in 8 portions, and the reaction was carried out at 25 °C for 1 h. After the coating reaction was completed, the solution was filtered through a microporous membrane and then ultrafiltered to obtain the FRET nanoparticle probes. The donor HMAT blue fluorescent small molecule solution contains HMAT blue fluorescent small molecule and tetrahydrofuran, with the content of HMAT blue fluorescent small molecule in the HMAT blue fluorescent small molecule solution being 1 mg / mL; the acceptor small molecule fluorescent dye includes the acceptor BODIPY488 small molecule fluorescent dye, and the volume ratio of the donor HMAT blue fluorescent small molecule solution to the acceptor small molecule fluorescent dye in the composite small molecule solution is 1:20. The polystyrene (PS) solution contains PS and tetrahydrofuran, and the PS content in the polystyrene (PS) solution is 1 mg / mL; The polystyrene maleic anhydride copolymer (PSMA) solution contains PSMA and tetrahydrofuran, and the PSMA content in the polystyrene maleic anhydride copolymer (PSMA) solution is 1 mg / mL; The TEOS solution contains TEOS and tetrahydrofuran, and the TEOS content in the TEOS solution is 1 mg / mL; The TCOS solution contains TCOS and tetrahydrofuran, and the TCOS content in the TCOS solution is 1 mg / mL. The volume ratio of the composite small molecule solution to the polystyrene (PS) solution is 20:100. The volume ratio of the composite small molecule solution to the polystyrene maleic anhydride copolymer (PSMA) solution is 20:100. The volume ratio of the composite small molecule solution to the TEOS solution is 20:200. The volume ratio of the composite small molecule solution to the TCOS solution is 20:200. After adjusting the volume, the organic phase solution was obtained, and the volume ratio of the organic phase solution to the composite small molecule solution was 2 mL: 20 μL.

[0112] The alkaline solution is made by adding ammonia water to deionized water to adjust the pH to 11. The volume ratio of the alkaline solution to the complex small molecule solution is 10 mL: 20 μL.

[0113] The concentration yielded a nanoparticle dispersion with a volume ratio of 2 mL:20 μL between the nanoparticle dispersion and the composite small molecule solution.

[0114] After filtration, the nanoparticle dispersion is added to deionized water, with a volume ratio of 1:1 between deionized water and nanoparticle dispersion. The pore size of the microporous filter membrane is 0.22 μm.

[0115] The coating precursor solution contains TEOS and DMSO, with a volume ratio of TEOS to DMSO of 1:9. The volume ratio of the coating precursor solution used each time to the volume of the nanoparticle dispersion is 10 μL: 2 mL. The ultrafiltration tube has a cutoff value of 100 kDa.

[0116] Example 4: A method for preparing FRET nanoparticle probes In this embodiment, the receptor small molecule fluorescent dye for the FRET nanoparticle probe is the receptor Nile Red small molecule fluorescent dye.

[0117] Preparation of FRET nanoparticle probes: A composite small molecule solution was prepared by mixing the donor HMAT blue fluorescent small molecule solution with the acceptor small molecule fluorescent dye. Then, the composite small molecule solution, PS solution, PSMA solution, TEOS solution, and TCOS solution were mixed, diluted to volume with tetrahydrofuran, and ultrasonically mixed to obtain an organic phase solution. An alkaline solution was added under ultrasonication and shaken for 2 min. The solution was then concentrated at 90 °C under a nitrogen atmosphere to obtain a nanoparticle dispersion. The solution was cooled to room temperature, filtered through a microporous membrane, and deionized water was added to the filtrate to adjust the pH to 10. The coating precursor solution was then added in 8 portions, and the reaction was carried out at 25 °C for 1 h. After the coating reaction was completed, the solution was filtered through a microporous membrane and then ultrafiltered to obtain the FRET nanoparticle probes. The donor HMAT blue fluorescent small molecule solution contains HMAT blue fluorescent small molecule and tetrahydrofuran, with the content of HMAT blue fluorescent small molecule in the HMAT blue fluorescent small molecule solution being 1 mg / mL; the acceptor small molecule fluorescent dye includes the acceptor Nile Red small molecule fluorescent dye, and the volume ratio of the donor HMAT blue fluorescent small molecule solution to the acceptor small molecule fluorescent dye in the composite small molecule solution is 1:15. The polystyrene (PS) solution contains PS and tetrahydrofuran, and the PS content in the polystyrene (PS) solution is 1 mg / mL; The polystyrene maleic anhydride copolymer (PSMA) solution contains PSMA and tetrahydrofuran, and the PSMA content in the polystyrene maleic anhydride copolymer (PSMA) solution is 1 mg / mL; The TEOS solution contains TEOS and tetrahydrofuran, and the TEOS content in the TEOS solution is 1 mg / mL; The TCOS solution contains TCOS and tetrahydrofuran, and the TCOS content in the TCOS solution is 1 mg / mL. The volume ratio of the composite small molecule solution to the polystyrene (PS) solution is 20:100. The volume ratio of the composite small molecule solution to the polystyrene maleic anhydride copolymer (PSMA) solution is 20:100. The volume ratio of the composite small molecule solution to the TEOS solution is 20:200. The volume ratio of the composite small molecule solution to the TCOS solution is 20:200. After adjusting the volume, the organic phase solution was obtained, and the volume ratio of the organic phase solution to the composite small molecule solution was 2 mL: 20 μL.

[0118] The alkaline solution is made by adding ammonia water to deionized water to adjust the pH to 11. The volume ratio of the alkaline solution to the complex small molecule solution is 10 mL: 20 μL.

[0119] The concentration yielded a nanoparticle dispersion with a volume ratio of 2 mL:20 μL between the nanoparticle dispersion and the composite small molecule solution.

[0120] After filtration, the nanoparticle dispersion is added to deionized water, with a volume ratio of 1:1 between deionized water and nanoparticle dispersion. The pore size of the microporous filter membrane is 0.22 μm.

[0121] The coating precursor solution contains TEOS and DMSO, with a volume ratio of TEOS to DMSO of 1:9. The volume ratio of the coating precursor solution used each time to the volume of the nanoparticle dispersion is 10 μL: 2 mL. The ultrafiltration tube has a cutoff value of 100 kDa.

[0122] Example 5: A method for preparing FRET nanoparticle probes The difference between this embodiment and Example 3 is that the polystyrene maleic anhydride copolymer (PSMA) solution also contains dodecyl ethoxysulfonate betaine and polyacrylamide.

[0123] The polystyrene-maleic anhydride copolymer (PSMA) solution contains polystyrene-maleic anhydride copolymer, dodecyl ethoxysulfonate betaine, and polyacrylamide, with tetrahydrofuran as the solvent. The PSMA solution contains 1 mg / mL of polystyrene-maleic anhydride copolymer, 0.09 mg / mL of dodecyl ethoxysulfonate betaine, and 0.05 mg / mL of polyacrylamide.

[0124] Example 6: A method for preparing FRET nanoparticle probes The difference between this embodiment and Example 5 lies in the content of dodecyl ethoxysulfonate betaine and polyacrylamide in the polystyrene maleic anhydride copolymer (PSMA) solution. In the PSMA solution, the content of dodecyl ethoxysulfonate betaine is 0.09 mg / mL, and the content of polyacrylamide is 0.02 mg / mL.

[0125] Example 7: A method for preparing FRET nanoparticle probes The difference between this embodiment and Example 5 lies in the content of dodecyl ethoxysulfonate betaine and polyacrylamide in the polystyrene maleic anhydride copolymer (PSMA) solution. In the PSMA solution, the content of dodecyl ethoxysulfonate betaine is 0.02 mg / mL, and the content of polyacrylamide is 0.05 mg / mL.

[0126] Example 8: A method for preparing FRET nanoparticle probes The difference between this embodiment and Example 5 lies in the content of dodecyl ethoxysulfonate betaine and polyacrylamide in the polystyrene maleic anhydride copolymer (PSMA) solution. In the PSMA solution, the content of dodecyl ethoxysulfonate betaine is 0.02 mg / mL, and the content of polyacrylamide is 0.02 mg / mL.

[0127] Example 9: A method for preparing FRET nanoparticle probes The difference between this embodiment and Example 5 is that the polystyrene-maleic anhydride copolymer (PSMA) solution also contains p-hydroxybenzylacetone. The content of p-hydroxybenzylacetone in the polystyrene-maleic anhydride copolymer (PSMA) solution is 0.017 mg / mL.

[0128] Example 10: A method for preparing FRET nanoparticle probes The difference between this embodiment and Example 9 is that the content of p-hydroxybenzylacetone in the polystyrene-maleic anhydride copolymer (PSMA) solution is different. The content of p-hydroxybenzylacetone in the polystyrene-maleic anhydride copolymer (PSMA) solution is 0.008 mg / mL.

[0129] Comparative Example 1: A method for preparing FRET nanoparticle probes The difference between this comparative example and Example 5 lies in the content of dodecyl ethoxysulfonate betaine and polyacrylamide in the polystyrene maleic anhydride copolymer (PSMA) solution. In the PSMA solution, the content of dodecyl ethoxysulfonate betaine is 0.005 mg / mL, and the content of polyacrylamide is 0.05 mg / mL.

[0130] Comparative Example 2: A method for preparing FRET nanoparticle probes The difference between this comparative example and Example 5 lies in the content of dodecyl ethoxysulfonate betaine and polyacrylamide in the polystyrene maleic anhydride copolymer (PSMA) solution. In the PSMA solution, the content of dodecyl ethoxysulfonate betaine is 0.09 mg / mL, and the content of polyacrylamide is 0.005 mg / mL.

[0131] Comparative Example 3: A method for preparing FRET nanoparticle probes The difference between this comparative example and Example 5 lies in the content of dodecyl ethoxysulfonate betaine and polyacrylamide in the polystyrene maleic anhydride copolymer (PSMA) solution. In the PSMA solution, the content of dodecyl ethoxysulfonate betaine is 0.005 mg / mL, and the content of polyacrylamide is 0.005 mg / mL.

[0132] Experimental example: 1. NMR characterization of compounds The present invention characterized compound 1 by proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 1 As shown.

[0133] The present invention characterized compound 2 by proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 2 As shown.

[0134] The present invention characterized compound 3 by proton NMR spectroscopy, and the results are as follows: Figure 3 As shown.

[0135] The present invention characterized compound 4 by proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 4 As shown.

[0136] The HMAT blue fluorescent small molecule prepared in Example 1 was characterized by proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 5 As shown.

[0137] 2. Absorption and emission spectra This invention tested the absorption and emission spectra of three fluorescent small molecules, HMAT, BODIPY488 and Nile Red, as well as the HMAT nanoparticle probe prepared in Example 2 and the FRET nanoparticle probe prepared in Examples 3-4.

[0138] The absorption spectra of three fluorescent small molecules, HMAT, BODIPY488, and Nile Red, in tetrahydrofuran are as follows: Figure 6 As shown, the emission spectra of three fluorescent small molecules, HMAT, BODIPY488, and Nile Red, in tetrahydrofuran are as follows: Figure 7 As shown. Figure 6-7 The results show that the emission peaks of the three fluorescent dyes, HMAT, BODIPY488, and Nile Red, are spaced far apart, which means they are easier to distinguish in multicolor imaging, have less crosstalk, and are suitable for multicolor imaging.

[0139] The overlap between the emission spectrum of the donor HMAT blue fluorescent small molecule dye and the absorption spectrum of the small molecule dye acceptor BODIPY488 is as follows: Figure 8 As shown, the overlap between the emission spectrum of the donor HMAT blue fluorescent small molecule dye and the absorption spectrum of the small molecule dye Nile Red is as follows: Figure 9 As shown, Figure 8-9 The results showed that the emission spectrum of the blue fluorescent dye HMAT had a good overlap with the absorption spectra of two small fluorescent dyes, BODIPY488 and Nile Red, indicating that HMAT and the above two dyes met the basic conditions for fluorescence resonance energy transfer in the spectrum and could be used to construct a donor-acceptor type FRET system.

[0140] This invention follows the method of Example 3, varying the volume ratio of the donor HMAT blue fluorescent small molecule solution to the acceptor small molecule fluorescent dye in the composite small molecule solution. The ratios were 1:0, 1:1, 1:5, 1:10, 1:15, 1:20, and 1:25. The amount of donor HMAT blue fluorescent small molecule solution used in the preparation was fixed at 1 μL, and the amounts of other reagents were measured based on a 20 μL usage of the donor HMAT blue fluorescent small molecule solution. The method for verifying the usage amounts in this invention is a modification of that in Example 2. Fluorescence emission spectra of the above samples were measured under 405 nm excitation conditions.

[0141] The results are as follows Figure 10As shown, the HMAT nanoparticle probe is denoted as HMAT, the FRET nanoparticle probe using the BODIPY488 small molecule fluorescent dye is denoted as BODIPY488, and the FRET nanoparticle probe using the Nile Red small molecule fluorescent dye is denoted as Nile Red. With increasing BODIPY488 content, the blue fluorescence emission intensity of HMAT gradually decreased, while the green fluorescence emission intensity of BODIPY488 gradually increased, indicating that the energy transfer efficiency between the donor and acceptor in the system gradually improved. When the volume ratio of HMAT to BODIPY488 was 1:20, the system exhibited a strong acceptor emission signal. However, when the amount of BODIPY488 was further increased, the acceptor fluorescence emission intensity decreased instead, suggesting that the excess acceptor dye aggregated and quenched within the limited nanoparticles, leading to a decrease in fluorescence intensity.

[0142] This invention follows the method of Example 4, varying the volume ratio of the donor HMAT blue fluorescent small molecule solution to the acceptor small molecule fluorescent dye in the composite small molecule solution. The ratios were 1:0, 1:3, 1:6, 1:9, 1:12, 1:15, and 1:18. The amount of donor HMAT blue fluorescent small molecule solution used in the preparation was fixed at 0.5 μL, and the amounts of other reagents were measured based on a 20 μL usage of the donor HMAT blue fluorescent small molecule solution. The method for verifying the usage amounts in this invention is a modification of that in Example 2. Fluorescence emission spectra of the above samples were measured under 405 nm excitation conditions.

[0143] The results are as follows Figure 11 As shown, the HMAT nanoparticle probe is denoted as HMAT, the FRET nanoparticle probe using the BODIPY488 small molecule fluorescent dye is denoted as BODIPY488, and the FRET nanoparticle probe using the Nile Red small molecule fluorescent dye is denoted as Nile Red. With increasing Nile Red content, the blue fluorescence emission intensity of HMAT gradually decreased, while the red fluorescence emission intensity of Nile Red gradually increased, indicating that the energy transfer efficiency between the donor and acceptor in the system gradually improved. When the volume ratio of HMAT to Nile Red was 1:15, the system exhibited a strong acceptor emission signal; however, when the amount of Nile Red was further increased, the acceptor fluorescence emission intensity decreased instead, suggesting that the excess acceptor dye underwent aggregation-induced quenching within the limited nanoscale confinement space, leading to a decrease in fluorescence intensity.

[0144] according to Figure 10 and Figure 11The results showed that, under 405 nm excitation conditions, with increasing amounts of the acceptor dye BODIPY488 or Nile Red, the blue fluorescence emission intensity of the donor HMAT gradually decreased, while the green or red fluorescence emission intensity of the corresponding acceptor dye gradually increased, indicating that a donor-acceptor type FRET system was successfully constructed inside the nanoparticles. Furthermore, when the amount of acceptor dye exceeded a certain proportion, its emission intensity decreased instead, presumably due to aggregation-induced quenching of the acceptor dye within the nanoscale confinement space.

[0145] The FRET nanoparticle probes prepared in Examples 3, 5-10, and Comparative Examples 1-3 were characterized in this invention. Fluorescence emission spectra of the above samples were measured under 405 nm excitation conditions, and the maximum fluorescence intensity results are as follows: Figure 12 As shown, S3 is Example 3, S5 is Example 5, S6 is Example 6, S7 is Example 7, S8 is Example 8, S9 is Example 9, S10 is Example 10, D1 is Comparative Example 1, D2 is Comparative Example 2, and D3 is Comparative Example 3. In this invention, the donor HMAT blue fluorescent dye and the acceptor small molecule fluorescent dye are spatially confined and isolated by an organic system including organic polymers inside the nanoparticles and a hybrid silica network structure. A silica shell structure is provided on the outer layer. The organic polymers can be PS and PSMA, and further, dodecyl ethoxysulfonate betaine and polyacrylamide can also be used. In the presence of PS and PSMA, dodecyl ethoxysulfonate betaine and polyacrylamide... Using dodecyl ethoxysulfonate betaine and polyacrylamide can improve the fluorescence intensity of FRET nanoparticle probes. However, it is important to note that dodecyl ethoxysulfonate betaine and polyacrylamide need to be used within a certain dosage range. If the dosage of dodecyl ethoxysulfonate betaine or polyacrylamide is too low, even if the other component is within the specific dosage range, it will not effectively improve the fluorescence intensity of FRET nanoparticle probes. Furthermore, when the dosage of both dodecyl ethoxysulfonate betaine and polyacrylamide is too low, it will also not effectively improve the fluorescence intensity of FRET nanoparticle probes. In the organic system of this invention, p-hydroxybenzylacetone can also be added. Within a certain dosage range, p-hydroxybenzylacetone can further improve the fluorescence intensity of FRET nanoparticle probes.

[0146] 3. DLS characterization This invention characterized the particle size changes of HMAT nanoparticle probes, HMAT-BODIPY488 FRET nanoparticle probes, and HMAT-Nile Red FRET nanoparticle probes before coating, 4 hours after coating, and 8 hours after coating. Before the silica coating growth step, a portion of the nanoparticle dispersion was taken as the uncoated sample; after the silica coating growth was completed and purified, the resulting nanoparticles were taken as the coated sample. Dynamic light scattering (DLS) was used to measure the particle size and potential of the uncoated and coated samples.

[0147] The results are as follows Figure 13 As shown, the average hydrated particle size of the uncoated nanoparticles is approximately 18 nm; after silica coating growth, the average hydrated particle size of the nanoparticles increases to approximately 21 nm. These results demonstrate that a silica coating layer was successfully grown on the surface of hybrid nanoparticles, and the coating process did not cause significant particle aggregation; the resulting coated nanoparticles maintained good dispersibility. Figure 12 The results showed that the average particle size of the nanoparticles before coating was approximately 18 nm. After coating for 4 h and 8 h, the particle size increased to approximately 22 nm and 26 nm, respectively, and the particle size distribution exhibited a single-peak distribution. These results indicate that an outer coating structure was successfully grown on the surface of the original nanoparticles, and the coating thickness gradually increased with the extension of reaction time. The coating process was controllable and did not cause significant agglomeration.

[0148] 4. Characterization of fluorescence leakage This invention characterized the fluorescence leakage of the HMAT nanoparticle probe prepared in Example 2 and the FRET nanoparticle probe prepared in Examples 3-4. 1 mL of each of the above nanoparticle probe aqueous solutions was added to a 100 kDa ultrafiltration centrifuge tube, and centrifuged for 3 min at 1000 g. The lower filtrate was collected. Under the same test conditions, the fluorescence emission intensity of the lower filtrate and the corresponding original nanoparticle aqueous solution were measured.

[0149] The fluorescence leakage characterization results of the HMAT nanoparticle probe are as follows: Figure 14 As shown, the fluorescence leakage characterization results of the FRET nanoparticle probe prepared in Example 3 are as follows: Figure 15 As shown, the fluorescence leakage characterization results of the FRET nanoparticle probe prepared in Example 4 are as follows: Figure 16 As shown in the figure. The results indicate that the fluorescence intensity of the lower filtrate is 5% lower than that of the corresponding original nanoparticle aqueous solution. The leakage of HMAT, BODIPY488 and Nile Red small molecule fluorescent dyes in the nanoparticles is extremely low, indicating that the constructed organic-inorganic hybrid core structure and outer silica shell can prevent the leakage of fluorescent small molecules, and the probe has good sealing performance.

[0150] The present invention also characterized the fluorescence leakage of the FRET nanoparticle probes prepared in Examples 5-10 and Comparative Examples 1-3. The fluorescence intensity of the lower filtrate relative to the fluorescence intensity of the corresponding original nanoparticle aqueous solution is shown in the figure below. Figure 17 As shown, S3 is Example 3, S5 is Example 5, S6 is Example 6, S7 is Example 7, S8 is Example 8, S9 is Example 9, S10 is Example 10, D1 is Comparative Example 1, D2 is Comparative Example 2, and D3 is Comparative Example 3. In this invention, the donor HMAT blue fluorescent dye and the acceptor small molecule fluorescent dye are spatially confined and isolated by an organic system including organic polymers inside the nanoparticles and a hybrid silica network structure. A silica shell structure is provided on the outer layer. The organic polymers can be PS and PSMA, and further, dodecyl ethoxysulfonate betaine and polyacrylamide can also be used. In the presence of PS and PSMA, the use of dodecyl ethoxysulfonate betaine and polyacrylamide can reduce FRET. The fluorescence leakage of the FRET nanoparticle probe is reduced, and its stability is improved. However, it is important to note that dodecylethoxysulfonate betaine and polyacrylamide need to be used within a certain range. If the amount of dodecylethoxysulfonate betaine or polyacrylamide used is too low, even if the other component is within the specific range, it is basically impossible to effectively reduce the fluorescence leakage of the FRET nanoparticle probe, resulting in poor stability. Furthermore, when the amounts of both dodecylethoxysulfonate betaine and polyacrylamide are too low, it is also basically impossible to effectively reduce the fluorescence leakage of the FRET nanoparticle probe, resulting in poor stability. In the organic system of this invention, p-hydroxybenzylacetone can also be added. When p-hydroxybenzylacetone is used within a certain range, it can further reduce the fluorescence leakage of the FRET nanoparticle probe, resulting in good stability.

[0151] 5. Use of nanoparticle probes 5.1 Preparation of streptavidin-coupled nanoparticle probes (1) Take 1 mL of the nanoparticle probe aqueous solution and add 20 μL of 10% polyethylene glycol (PEG3350) solution, and shake thoroughly to mix. Then add 20 μL of 1 M HEPES buffer solution, 60 μL of 1 mg / mL streptavidin (SA) solution and 15 μL of 5 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) solution in sequence, and shake thoroughly to mix after each addition of reagent.

[0152] (2) The above mixed solution was placed at room temperature and reacted on a shaker for 4 h to allow the carboxyl groups on the surface of the FRET multicolor fluorescent nanoparticles to undergo a coupling reaction with the amino groups in the streptavidin molecule.

[0153] (3) To block unreacted active sites, add 20 μL of 10% bovine serum albumin (BSA) solution to the reaction system and continue incubation on a shaker for 30 min.

[0154] (4) After the reaction was completed, the reaction system was purified by using a 100 kDa ultrafiltration tube and washed five times with a washing buffer containing 0.1 wt% PEG, 2 wt% HEPES and 97.9 wt% deionized water to remove free streptavidin and small molecule reagents.

[0155] (5) The purified product was concentrated to about 1 mL, and 25 μL of 10% BSA solution was added as a stabilizer to obtain streptavidin-modified small molecule FRET multicolor fluorescent nanoparticle probes. The obtained product was stored at 4 °C in the dark for later use.

[0156] 5.2 Labeling and Confocal Imaging Detection of Cellular Microtubules Using Streptavidin-Conjugated FRET Nanoparticle Probes (1) African green monkey kidney cells BS-C-1 (commercially available) were selected and seeded in 4-well confocal microplates at a density of 3 × 10⁶ cells per well. 4 One cell was cultured overnight at 37°C and 5% CO2 to allow the cells to adhere to the culture dish; the culture medium for BS-C-1 cells was aspirated from the culture dish and the cells were washed three times with PBS buffer solution. (2) Add 400 μL of extraction solution (1 mM EGTA, 1 mM MgCl2, 0.2 wt% Triton X-100, 0.1 MPIPES) to each well, aspirate the extract after 3 min and rinse 3 times with PBS buffer solution; (3) Add 400 μL of fixative (4% PFA, 0.1 wt% GA) to each well, let stand for 15 min, then remove the fixative and rinse three times with PBS buffer solution; (4) Add 400 μL of drilling solution (0.5 wt% Triton X-100) to each well, let stand for 5 min, aspirate the drilling solution and rinse 3 times with PBS buffer solution; (5) Add 400 μL of blocking solution (5 wt% BSA, 0.5 wt% Triton X-100 in PBS) to each well, let stand for 30 min, then aspirate and wash with PBS buffer solution. (6) Dilute the primary antibody solution against β-tubulin (Anti-beta Tubulin, Abcam, cat. no. Ab179513) at a ratio of 1:200 in the blocking buffer. Add 400 μL of the primary antibody dilution buffer to each well and gently shake on a shaker for 60 min. Wash three times with PBS buffer. (7) Dilute the secondary antibody goat anti-rabbit IgG (H+L) polyclonal antibody-biotin-labeled (Goat Polyclonal Antibody to Rabbit IgG (H&L)-Biotin, Yuantai Bio, cat. no. P50075) solution at a ratio of 1:200 in blocking buffer, add 400 μL of primary antibody dilution buffer to each well and gently shake on a shaker for 60 min, and wash 3 times with PBS buffer solution; (8) Add streptavidin-modified multicolor fluorescent nanoprobes, gently shake for 60 min; rinse thoroughly with PBS buffer solution, and store at 4°C. (10) Confocal imaging: Cells were confocally imaged using 405 excitation light from CSU-W1-SoRa.

[0157] In this invention, the nanoparticle probe can be the FRET nanoparticle probe from Example 3 or Example 4, or the HMAT nanoparticle probe from Example 2. The imaging results of the HMAT nanoparticle probe on cell microtubules are as follows: Figure 18 As shown, the imaging results of FRET nanoparticle probes on cell microtubules in Example 3 are as follows: Figure 19 As shown, the imaging results of FRET nanoparticle probes on cell microtubules in Example 4 are as follows: Figure 20 As shown in the figure. The results indicate that all three probes can clearly label cellular microtubules, revealing a continuous microtubule network structure.

[0158] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0159] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A method for preparing a nanofluorescent probe, comprising: The donor fluorescent small molecule solution and the acceptor fluorescent small molecule solution were mixed, and then organic reagents and siloxane precursors were added to prepare an organic phase solution. This solution was then added to an alkaline aqueous phase system to form nanoparticles. Finally, the outer layer was coated with silica to prepare a nano-fluorescent probe. The volume ratio of donor fluorescent small molecule solution to acceptor fluorescent small molecule solution is 0.5-1:0-25.

2. The preparation method according to claim 1, characterized in that, The donor fluorescent molecule solution contains a donor fluorescent molecule, which is a blue HMAT fluorescent molecule.

3. The preparation method according to claim 1, characterized in that, The receptor fluorescent molecule solution contains receptor fluorescent molecules, including BODIPY488 or Nile Red.

4. The preparation method according to claim 1, characterized in that, The organic reagent includes organic polymers, including PS and / or PSMA.

5. The preparation method according to claim 4, characterized in that, The organic polymer also includes polyacrylamide.

6. The preparation method according to claim 1, characterized in that, The organic reagent includes the solvent tetrahydrofuran.

7. The preparation method according to claim 1, characterized in that, The organic reagent includes dodecyl ethoxysulfonate betaine.

8. The preparation method according to claim 1, characterized in that, The alkaline aqueous phase system is deionized water with pH adjusted to 10-11 using ammonia.

9. The nanofluorescent probe prepared by any one of the methods described in claims 1-8.

10. The application of the nanofluorescent probe according to claim 9 in the preparation of medical diagnostic reagents.

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