Synthesis Of Novel Fluorescent Probe Dyes For Monoclonal Antibody And Its Application In Immunofluorescence Histochemistry

Fluorescent probes have been widely used in molecular biology, microbiology, biochemistry, analytical chemistry, chemical engineering and medicine engineering. At the same time, the request for the properties of fluorescent probes is becoming more and more rigid. Therefore, the synthesis and application research of fluorescent probe dyes become a hot point in interdisciplinary of life science and chemistry.Rhodamine derivatives are prized for their high fluorescence quantum yield, long excitation wavelength, pH insensitivity over a broad rang(low to neutral pH), and the ability of their fluorescence characteristics to be tailored for a particular application. As a result, they are used as fluorescent probe dyes widely.In this thesis, a series of novel fluorescent probe dyes for monoclonal antibody based on rhodamine were designed and synthesized. One of the dyes was investigated for its application in immunofluorescence histochemistry.1. The synthesis conditions of 5(6)-carboxyl-rhodamine110 isomers were studied and optimized. The isomers of 5(6)-carboxyl-rhodamine110 were separated by a new method. By the same method, 5(6)-carboxyl-2' 7'-dimethyl- rhodaminellO isomers complex can be separated successfully. The structures of the isomers were determined by ¹HNMR, elemental analysis and MS.2. It was found that there is difference in λ_em,max between isomers of 5, 6-carboxyl-rhodamine. The λ_em,maxof 5-carboxyl-rhodamine is always larger then 6-carboxyl-rhodamine's. The phenomena are new experimental evidence to research the relationship between molecular structure and fluorescence.3. Two kinds of novel rhodamine-based fluorescent probe dyes with linker for monoclonal antibody were designed. Three linker compounds were synthesized. A novel method to synthesize PEG linker was developed.4. Two novel rhodamine-based fluorescent probe dyes with hydrophilic linker for monoclonal antibody were synthesized. One of the dyes' isomers was separated and the structures of the isomers were determined. The synthesis of rhodamine-basedfluorescent probe dyes with amplifier linker for monoclonal antibody was researched. 5. One of the novel dyes(5,6-R110-L₃) was investigated for application in immunofluorescence histochemistry. It was found that the novel dye(5,6-R110-L3) is an excellent green fluorescent probe dye that can be used to label a variety of monoclonal antibody and other biopolymers.