Light activated fluorescent probe having protein label positioning function as well as preparation method and application thereof

Abstract

The invention discloses a light activated fluorescent probe having a protein label positioning function, and further discloses a preparation method and application thereof. The light activated fluorescent probe having the protein label positioning function has excellent photophysical properties, such as high-efficiency light control property and high fluorescence quantum yield, and characteristics, such as simple small molecule dye synthesis; furthermore, in combination with a chemical label technology, the light activated fluorescent probe is capable of positioning target molecular specificity in living cells precisely; real-time super-resolution fluorescence imaging in the living cells can be realized; and therefore, the light activated fluorescent probe has important application value in the field of super-resolution, especially super-resolution imaging in the living cells.

Description

technical field [0001] The invention relates to the technical field of fluorescent probes, in particular to a light-activated fluorescent probe with protein label positioning and its preparation method and application. Background technique [0002] In the field of biological research, light microscopy is a widely used and indispensable imaging tool. Light microscopy, especially fluorescence microscopy, has unique advantages over other microscopes: non-invasive (allows observation of living cells, tissues, and organisms) and high precision (precisely specific observation of targeted molecules through fluorescent labeling) ). We know that conventional optical microscopes such as confocal fluorescence microscopes have never been able to break through the limit resolution of 200nm due to the diffraction effect of light waves, that is, limited by the diffraction limit. In order to study the structural characteristics of intracellular molecules at the nanoscale, improving the re...

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Problems solved by technology

However, there are still many deficiencies in this probe system worthy of our attention: 1) Cy3-Cy5 probe pair is labeled on the antibody by immunofluorescence labeling method, and the antibody does not have transmembrane ability, so it can only be applied to fixed cells Or the surface of the cell membrane, which cannot be used for live cell imaging; 2) The photoconversion mechanism of Cy3-Cy5 probes is not clear, and the requirements for the surrounding environment are very harsh, because the photoconversion needs to be carried out under reducing conditions, which is Need to add mercapto-containing reducing agent and oxygen scavenger; 3) The operation is relatively difficult, because the distance between Cy3 and Cy5 must meet 1-3nm to complete the pairing

In addition, compared with fluorescent proteins, although small molecule fluorescent dyes have many advantages, they also have some disadvantages: 1) They are chemically synthesized and added to cells by exogenous sources, and their specific labeling degree is relatively low, often requiring a lot of washing and fluorescence The background is high; 2) Many small molecule fluorescent probes (such as sulfonated Cy dyes) do not have the ability to spontaneously transmembrane, which limits their application to fixed cells or cell membrane surfaces

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