Detection, Based On Fullerene (c, <sub> 60 </ Sub>) To Build A New Fluorescent Probes For Biologically Active Substances

Various bio-active molecules are indispensable component of organism. They also take part in various life activities. Thus, studies on bio-active molecules including nucleic acid, protein, peptide, and other bio-active ingredients play an important role in life science. In recent years, more and more studies focus on detecting bio-active molecules. Among these methods, using fluorescent probes has many advantages such as high sensitivity, high selectivity and easy to operate. Besides, fluorescent probes can interact with bio-active molecules to form strongly fluorescent substances. With the help of confocal microscopic imaging, visualizing and real-time detecting of bio-active molecules can be achieved. Thus, studies on fluorescent probes for detecting intrecellular bio-active molecules have greatly developed in recent years.Based on the changes in spectrum characters of the fluorescent probes reacting with enzymes or bio-active molecules, we have achieved the detection of these ingredients. The main contents of this thesis were shown as follows:In chapter one, we have summarized two aspects: (1) The structure and properties of fullerene, the applications of fullerene and its derivatives in biology and medicine, and their toxicity to organisms. (2) The development of study on bioactive substances, including the function of trypsin, the physiological and pathological roles of ROS and the development of their detection technologies.In chapter two, a novel fluorescent probe (C60-FL) was designed and synthesized for direct determination of trypsin, which was based on the photoinduced electron transfer (PET). It consists of two separated functional moieties: the fluorescein performs as a fluorophore and an electron donor and the fullerene (C60) acts as an electron acceptor and trypsin substrate analogue. In the presence of trypsin, the probe exhibited fluorescence increase due to the inhibition of electron transformation by combination the C60 section of C60-FL with trypsin. The response of the probe to trypsin was direct and transient. Experimental results showed the increase in fluorescence intensity is proportional to the concentration of trypsin within the range of 4.40×10^-7 to 7.04×10^-5 g mL^-1 under the optimized experimental conditions. The detection limit of the proposed method was 40 ng mL^-1. The method had excellent selectivity for trypsin over other enzymes and most biological species. The remarkable properties of C60-FL help to extend the development of fluorescent probes for investigating enzymes in a biological context. In chapter three, novel fluorescent probes A and B, which were used for the determination of hydrogen peroxide in the mitochondria of living cells, were designed and synthesized through the reaction of fluorescein with fullerene. Based on photoinduced electron transfer (PET), fullerene can quench the fluorescence of fluorescein. In the prescence of hydrogen peroxide, the sulfonic ester bond was cleaved and the fluorescence of fluorescein was recoveried. Therefore, hydrogen peroxide can be detected quantitively. Currently, the experiments are ongoing.