For The Determination Of Applications And Synthesis Of Fluorescent Probes In Vivo Activity Substances (free Radicals, Metal Ions, Etc.)

With the progress of biochemistry and molecular biology and the using of chemistry, physics and biology, the course of chemical reaction in organism was elucidated gradually. But more and more experimental evidences suggest that the results obtained from the reaction occurred in test tubes cannot reflect the instance in living object. So it is necessary to explore the real time changes of active substances in order to comprehend the life.Recently, the methods for the detection of active substances in living object, such as Atomic Absorption Spectrometric methods, NMR method and Electron probe microanalysis were developed. But they have many shortages: (1) the sample used must have volatility; (2) need a large amount of cells when used in the detection in cells. (3) can detect the gross of active substances which make the method have low selectivity, or though can detect separately, but cannot describe the distributing of active substances in cells. (4) can not describe the dynamic changes of active substances in and out of cells.Up to date, the dynamic generation of reactive oxygen species cannot be observed in some organs. Besides, enough cognition and sufficient experimental evidences of their generation mechanism, physiological action and dynamic damages on organisms are not acquired. Therefore, the therapy effects of many diseases in clinic are limited and they are main obstacles to the plant cultivation in adversity. Besides,the absorption, trafficking, distributing of ions and their union with protein all need to be explored. So it is one of the most challenging scientific researches in analytical chemistry, in which novel analysis and determination technologies contraposing to reactive oxygen species in vivo need to be developed.Fluorescence analysis and imaging techenology was widely used in biological analysis field because of their high sensitivity and selectivity. Most of biological cells have no or weak fluorescence, which make their sensitivity very low. The interference was lowed largely. Infra-red fluorescence probe was widely used in biological science field because of their special photophysics and photochemical characters, high sensitivity, wide response range and appropriate condition. So we need to design and synthesize fluorescence probe with appropriate spectrum charater. Then we explore the property in chemical circumstance and by integrating with fluorescence imaging technology to detect the concentration changes of metal ions and the distribution in cell on line. We then explored the function of metal ions in life activity and pathology of the shortness of metal ions to achieve the detection of reactive oxygen species in vivo and in situ, explore the pathology of reactive oxygen species burst, which will afford essential basis for biology and medicine study.Fluorescence probe is advantageous because of its high sensitivity. However, fluorescence measurement by increasing fluorescence intensity without much shift of eighter excitation or emission wavelength can be influenced by many factors, such as the location of the probe changes of environment around the probe (e. g., pH, polarity and temprature), and photostability. To reduce the influence of such factors, fluorescence resonance energy transfer (FRET) is utilized, which can be used to detect metal ions and reactive oxygen species sensitively and selectively.Based on the changes in spectrum characters of the fluorescent probes reacting with metal ions and ROSs, we have carried out three aspects of investigation:First, we designed and synthesized DPA-Cy as a novel NIR fluorescence probe for Zn²⁺ and optimized the detecting condition. The reaction mechanism was studied and the proposed methods were then applied to determine Zn²⁺ in macrophage cells.Second, we synthesized fluorescein hydrazide in a simple way and raising the output largely. We then applied it to the detection of NO₂^- and optimized the detecting condition. The method was then used to detect NO. The reaction mechanism was studied and the proposed methods were successfully applied to the determination of NO₂^- in adult serum and rainwater.Third, we desighed and synthesized a new fluorescence probe for Cd2+ based on fluorescence resonance energy transfer and optimized the detecting congdition of it.