CN-122012083-A - Preparation and application of multicolor nano fluorescent probe based on fluorescence micromolecule FRET

Abstract

The invention discloses preparation and application of a multicolor nanometer fluorescent probe based on fluorescence micro-molecule FRET, which belongs to the technical field of medical reagents, and particularly relates to a preparation method of a nanometer fluorescent probe, comprising the steps of mixing donor fluorescent micro-molecule solution and acceptor fluorescent micro-molecule solution, adding an organic reagent and siloxane precursor to prepare an organic phase solution, adding the organic phase solution into an alkaline aqueous phase system to form nanometer particles, and finally coating silica on the outer layer to prepare the nanometer fluorescent probe, wherein the volume ratio of the donor fluorescent micro-molecule solution to the acceptor fluorescent micro-molecule solution is 0.5-1:0-25. The donor fluorescent small molecule is HMAT blue fluorescent small molecule, and the acceptor fluorescent small molecule comprises BODIPY488 or nile red.

Inventors

• FANG XIAOHONG
• LI PEIXIANG
• JIANG YIFEI
• WU DAN
• WANG XINGBAO

Assignees

• 浙江工业大学
• 中国科学院杭州医学研究所

Dates

Publication Date

20260512

Application Date

20260413

Claims (10)

1. 1. A preparation method of a nano fluorescent probe comprises the steps of mixing donor fluorescent small molecule solution and acceptor fluorescent small molecule solution, adding an organic reagent and a siloxane precursor to prepare an organic phase solution, adding the organic phase solution into an alkaline aqueous phase system to form nano particles, and finally coating silicon dioxide on the outer layer to prepare the nano fluorescent probe, wherein the volume ratio of the donor fluorescent small molecule solution to the acceptor fluorescent small molecule solution is 0.5-1:0-25.
2. 2. The method of claim 1, wherein the donor fluorescent small molecule solution comprises a donor fluorescent small molecule, and the donor fluorescent small molecule is an HMAT blue fluorescent small molecule.
3. 3. The method of claim 1, wherein the acceptor fluorescent small molecule solution comprises an acceptor fluorescent small molecule comprising BODIPY488 or nile red.
4. 4. The method of claim 1, wherein the organic agent comprises an organic polymer, and wherein the organic polymer comprises PS and/or PSMA.
5. 5. The method of claim 4, wherein the organic polymer further comprises polyacrylamide.
6. 6. The method of claim 1, wherein the organic reagent comprises tetrahydrofuran as a solvent.
7. 7. The method of claim 1, wherein the organic reagent comprises dodecyl ethoxy sulfobetaine.
8. 8. The method according to claim 1, wherein the alkaline aqueous system is aqueous ammonia to adjust the deionized water pH to 10 "11.
9. 9. The nano fluorescent probe prepared by the method of any one of claims 1 to 8.
10. 10. Use of the nano-fluorescent probe according to claim 9 for preparing a diagnostic reagent for medical use.

Description

Preparation and application of multicolor nano fluorescent probe based on fluorescence micromolecule FRET Technical Field The invention belongs to the technical field of medical reagents, and particularly relates to preparation and application of a multicolor nanometer fluorescent probe based on fluorescence small molecule FRET. Background The fluorescence imaging technology is widely applied in the fields of biomedical research, disease diagnosis, molecular detection and the like because of high sensitivity and strong spatial resolution capability, and is suitable for living body systems. With the continuous and deep research on biological systems, single-target imaging has been difficult to meet the research requirements, and multi-target detection and multicolor fluorescence imaging are becoming important development directions. Multicolor fluorescence imaging can simultaneously acquire a plurality of biomarker information in the same sample, so that the analysis flux and the detection efficiency are improved. Under the push of the above-mentioned requirements of multicolor imaging applications, multicolor fluorescence imaging places higher performance demands on the fluorescent probes. On the one hand, the fluorescent probe should have high fluorescent brightness and good light stability to ensure that sufficient signal intensity is obtained under the condition of multichannel imaging, and on the other hand, the fluorescent probe should have good distinguishability between different color signals to reduce spectral crosstalk. The fluorescent probes currently applied to the multicolor imaging field mainly comprise organic small molecule fluorescent dyes, nanoparticle fluorescent probes and the like. The organic micromolecular fluorescent dye has the advantages of multiple structures, clear structure-activity relationship, accurate regulation and control of emission wavelength through molecular structure design, and the like, but most micromolecular dyes have the problems of insufficient light stability, limited fluorescent brightness, easiness in light bleaching, poor stability in complex biological environments, and the like, and are difficult to meet the application requirements of long-time and multi-channel imaging. In contrast, nanoparticle fluorescent probes generally have higher fluorescent brightness and better light stability, and simultaneously have good water solubility and biocompatibility, and can meet the basic requirements of long-time and multi-channel imaging, so that the nanoparticle fluorescent probes gradually become the main-stream probe type in multicolor fluorescent imaging application. However, the emission spectrum of the existing nanoparticle fluorescent probe is generally wider, the selectable emission band is limited, and fine distinction between different channels is difficult to realize in multicolor imaging. Fluorescence Resonance energy transfer (F rster Resonance ENERGY TRANSFER, FRET) is an important method for constructing a multicolor fluorescence probe, and by establishing an energy transfer relationship between a donor and an acceptor fluorescent dye and replacing different acceptor dyes, fluorescence signals with a plurality of different emission wavelengths can be obtained under a single excitation condition, thereby realizing multicolor fluorescence imaging. The FRET mechanism is introduced into a nanoparticle fluorescent probe system, and the construction of the FRET nanoparticle fluorescent probe with high brightness, high light stability and adjustable emission characteristics becomes an important development direction for constructing a multicolor fluorescent probe. However, since the types of polymer fluorescent dyes that can be used to construct FRET nanoparticle fluorescent probes are relatively limited, most of the prior art uses a method of directly coating a small molecule fluorescent dye inside a nanoparticle to construct FRET nanoparticle fluorescent probes. In this way, small-molecule dyes are very prone to leakage, so that the ratio and distance relation between the donor and the acceptor are changed, and problems such as background rise, signal crosstalk and unstable FRET efficiency are easily caused in multicolor imaging. Disclosure of Invention The invention aims to provide the preparation and the application of the multicolor nanometer fluorescent probe based on fluorescence small molecule FRET, which can realize the regulation and control of the emission spectrum, and has good water dispersibility, good biocompatibility, good stability and high fluorescence intensity. The technical scheme adopted by the invention for achieving the purpose is as follows: A preparation method of a nano fluorescent probe comprises the steps of mixing donor fluorescent small molecule solution and acceptor fluorescent small molecule solution, adding an organic reagent and a siloxane precursor to prepare an organic phase solution, adding the organic phase solution int