CN-121991688-A - Nanotube capable of performing multi-step fluorescence resonance energy transfer in tube and preparation method thereof

CN121991688ACN 121991688 ACN121991688 ACN 121991688ACN-121991688-A

Abstract

The invention discloses a nano tube capable of carrying out multi-step fluorescence resonance energy transfer in a tube and a preparation method thereof, comprising the following steps of S1, preparing a supermolecule nano tube with azide functional groups distributed in the space of an inner cavity; S2, covalently and singly or sequentially modifying one or more alkynyl modified fluorescent molecules into the inner cavity of the supermolecule nanotube prepared in the step S1 to prepare the nanotube capable of performing multi-step fluorescence resonance energy transfer in the tube. According to the invention, the reactive functional groups are introduced into the inner cavity of the supermolecular nanotube in advance, and a plurality of fluorescent acceptor molecules are sequentially or combined and introduced into the inner cavity by utilizing click chemistry, so that the ordered arrangement of the fluorescent donor and the plurality of acceptors on the nanoscale is realized, and a multi-step fluorescent resonance energy transfer system with definite directivity, high energy transfer efficiency and obvious antenna effect is obtained.

Inventors

LI YAOJIA
GAO ZHIGUO
CHEN HONGLI
ZHANG YINAN
MA HUIYING
YANG SHUYING
CHAI YONGSHUN

Assignees

河南医药大学

Dates

Publication Date: 20260508
Application Date: 20260109

Claims (10)

1. The preparation method of the nanotube capable of carrying out multi-step fluorescence resonance energy transfer in the tube is characterized by comprising the following steps: S1, preparing a supermolecule nanotube with azide functional groups spatially distributed in an inner cavity; S2, covalently and singly or sequentially modifying one or more alkynyl modified fluorescent molecules into the inner cavity of the supermolecule nanotube prepared in the step S1 through click chemistry reaction to obtain the nanotube capable of performing multi-step fluorescence resonance energy transfer in the tube.
2. The method of claim 1, wherein the fluorescent molecules in step S2 are one or more selected from the group consisting of 1, 8-naphthalimide derivatives, anthocyanin 3 and anthocyanin 5.
3. The method for preparing the nanotube capable of performing in-tube multi-step fluorescence resonance energy transfer according to claim 1, wherein the step S2 of preparing the nanotube capable of performing in-tube multi-step fluorescence resonance energy transfer comprises the following steps: a1, carrying out click chemical reaction on the supermolecule nanotube prepared in the step S1 and the alkynyl modified first fluorescent receptor molecule in the inner cavity of the nanotube under the copper catalysis condition, and covalently anchoring the first fluorescent receptor molecule in the inner cavity to form a preliminary functionalized nanotube; A2, carrying out click chemistry reaction on the preliminary functionalized nanotube prepared in the step A1 and the alkynyl modified second fluorescent receptor molecule in the inner cavity of the nanotube under the copper catalysis condition, and covalently anchoring the second fluorescent receptor molecule in the inner cavity to obtain the nanotube capable of carrying out in-tube two-step fluorescence resonance energy transfer.
4. The method of claim 1, wherein step S2 further comprises A3, performing click chemistry reaction on the primarily functionalized nanotube obtained in step A2 and an alkynyl modified third fluorescent acceptor molecule in a lumen of the nanotube under copper catalysis, and covalently anchoring the third fluorescent acceptor molecule in the lumen to obtain a nanotube capable of performing three-step fluorescence resonance energy transfer in the tube; the first, second and third fluorescent acceptor molecules have different fluorescence emission wavelengths and are selected from two or more of 1, 8-naphthalimide derivatives, anthocyanin 3, anthocyanin 5; Preferably, the first fluorescent receptor molecule is a1, 8-naphthalimide derivative, the second fluorescent receptor molecule is anthocyanin 3, and the third fluorescent receptor molecule is anthocyanin 5; preferably, the copper catalyst in steps A1, A2 and A3 is copper sulfate pentahydrate; preferably, the click chemistry reactions in steps A1, A2 and A3 also add the reducing agent sodium ascorbate; Preferably, after each click chemistry in steps A1, A2 and A3, a purification step is included to remove unreacted fluorescent molecules and catalyst.
5. The method for preparing the nano-tube capable of performing multi-step fluorescence resonance energy transfer in the tube according to claim 1, wherein the preparation method of the nano-tube in the step S1 is characterized in that a supermolecular nano-tube with azide functional groups distributed in the space of the inner cavity comprises the steps of connecting phospholipid and camptothecin by a redox responsive disulfide bond connecting arm to form a phospholipid-camptothecin conjugate, and then forming the supermolecular nano-tube with the azide functional groups distributed in the space of the inner cavity by self-assembly, wherein the self-assembled nano-tube of the camptothecin-phospholipid conjugate is provided with hollow inner cavities, and the azide functional groups are distributed in the space of the inner cavities; the structural formula of the phospholipid-camptothecin conjugate is shown as (I); (I)。
6. The method of claim 4, wherein the self-assembled supramolecular nanotubes have a tubular structure with an outer diameter of 6-7 nm, a lumen diameter of 2-3 nm, and a length of 500-nm to 1 μm, and are stable in aqueous systems and maintain the hollow tubular structure from collapsing.
7. The method for preparing the nanotube capable of performing multi-step fluorescence resonance energy transfer in the tube according to claim 4, comprising the following steps: b1, dissolving the phospholipid-camptothecin conjugate according to claim 1 in an organic solvent to form an organic phase; B2, mixing the organic phase prepared in the step B1 with an aqueous buffer solution to obtain a mixed system; B3, removing the organic solvent in the mixed system prepared in the step B2, and inducing the phospholipid-camptothecin conjugate to self-assemble and mature to form the supramolecular nanotube with the azide functional groups distributed in the inner cavity space.
8. The method for preparing the nanotube capable of performing multi-step fluorescence resonance energy transfer in the tube according to claim 7, wherein the method comprises the following steps: The final concentration of the phospholipid-camptothecin conjugate in step B1 in the aqueous solvent is 0.1-5 mM, preferably about 1.0 mM; Preferably, the organic solvent in step B1 is ethanol; preferably, the aqueous buffer in step B2 is deionized water or a buffer solution; preferably, the organic solvent is removed in the step B3, namely the mixed system obtained in the step B2 is dialyzed; preferably, the time for inducing self-assembly maturation of the phospholipid-camptothecin conjugate in step B3 is between 5 and 7 h.
9. A nanotube capable of performing in-tube multi-step fluorescence resonance energy transfer, prepared by the method for preparing a nanotube capable of performing in-tube multi-step fluorescence resonance energy transfer according to any one of claims 1 to 8, wherein the energy transfer efficiency of each stage is higher than 90%, and the overall antenna effect parameter is higher than 50.
10. Use of a nanotube capable of performing in-tube multistep fluorescence resonance energy transfer according to claim 9 for the preparation of an optical device, a supermolecular light collecting material, an artificial photosynthesis simulation system, a fluorescent biological probe or a biological imaging reagent.

Description

Nanotube capable of performing multi-step fluorescence resonance energy transfer in tube and preparation method thereof Technical Field The invention belongs to the technical field of nano materials and supermolecular chemistry, and particularly relates to a nano tube capable of performing multi-step fluorescence resonance energy transfer in the tube and a preparation method thereof. Background The supermolecule nano structure has the advantages of strong designability, ordered structure, programmable function and the like, and is widely focused in the fields of nano reactors, molecular assembly, energy transfer, biomedical materials and the like. The supermolecule nanotube with hollow cavity structure can provide limited space in nanometer scale, regulate and control molecular arrangement mode and reaction behavior via space constraint effect and provide new way for constructing complicated functional system. Click chemistry reactions, particularly copper-catalyzed azido-alkynyl cycloaddition (CuAAC), have been widely used in functional molecule construction and post-modification processes due to their mild reaction conditions, high selectivity, high functional group tolerance, and the like. In the prior art, click reaction is mostly carried out on the surface of a solution system or a solid carrier, and is used for realizing molecular coupling or material surface functionalization. However, supramolecular self-assembled nanostructures have inherent structural stability problems depending on assemblies that are dynamically non-covalent (e.g., pi-pi stacking, hydrophobic interactions). Any subsequent chemical modification operation is extremely prone to break its delicate non-covalent force balance, resulting in assembly dissociation and morphology collapse, thus losing function. Achieving controllable multicomponent, sequenced click reactions within confined nanospaces, particularly within supramolecular nanotubes, remains a major challenge. Fluorescence resonance energy transfer (fluorescence resonance ENERGY TRANSFER, FRET) is a photophysical process based on non-radiative energy transfer between a donor and an acceptor, and is highly sensitive to molecular spacing and relative spatial arrangement. The multi-step FRET system can realize directional transmission and cascade amplification of energy, and has important application value in the fields of optical sensing, biological imaging, artificial photosynthesis simulation and the like. The existing multi-step FRET system is generally constructed by relying on a complex organic synthesis strategy or a random blending mode, so that accurate positioning of a donor and an acceptor on a nanometer scale is difficult to realize, and the stability and the energy transfer efficiency of the system are limited. The gradual covalent connection of multiple components in a cavity and the construction of an ordered multi-step FRET system are realized on the premise of not damaging the integral structural integrity and self-assembly characteristic of the supermolecular nanotube, and the problem in the prior art is not solved effectively. Therefore, a new technical scheme is needed to be provided, which can realize multi-component clicking reaction in the supermolecule nanotube cavity, and on the basis, a multi-step fluorescence resonance energy transfer system with controllable space and definite energy transfer path is constructed, so as to overcome the defects in the prior art. Disclosure of Invention In view of the above, the present invention is directed to a nanotube capable of performing in-tube multi-step fluorescence resonance energy transfer, a preparation method thereof, and a multi-step Fluorescence Resonance Energy Transfer (FRET) system constructed by using the method, which can be applied to the directions of fluorescent probe construction, energy transfer regulation, and bio-imaging, etc., so as to solve at least one technical problem in the background art. Aiming at the problems of difficult component positioning, difficult energy transmission directivity and efficiency, dependence on complex molecular design or strict space control and the like in the construction process of a multi-step fluorescence resonance energy transfer system in the prior art, the invention provides a multi-component click reaction method based on supermolecule nanotube intracavity finite field chemistry and the multi-step fluorescence resonance energy transfer system constructed by the method. According to the invention, the reactive functional groups are introduced into the inner cavity of the supermolecular nanotube in advance, and a plurality of fluorescent acceptor molecules are sequentially or combined and introduced into the inner cavity by utilizing click chemistry, so that the ordered arrangement of the fluorescent donor and the plurality of acceptors on the nanoscale is realized, and a multi-step fluorescent resonance energy transfer system with definite directivity, hi