A Coordination Complex System For Fluorescent Staining Of Biomolecules And Research About Radical Polymerization Of Acrylamide Monomer In Pure Water

Gel electrophoresis, a technique for the separation of biological species based on the electric field-driven migration of charged objects, is arguably one of the most valuable tools in life sciences. After the electrophoresis, biomolecule bands of different molecular weight separate in gel matrix. The visualization of biomolecule bands in the gel matrix is necessary for getting relative separation information. Thus, the staining step of biomolecule bands, making the biomolecule-loaded zone distinguish from the biomolecule-free gel matrix is very necessary. The research about effective staining of biomolecule bands has tremendous success so far and several criterions such as sensitivity, detection limit, staining speed, simplicity, security, and biomolucules generality always are considered when comparing different staining method.The first chapter introduces basic knowledge of polyacrylamide gel electrophoresis such as the separation mechanism of biomolecules and all kinds of generally used electrophoresis staining methods, for example, organic dyes (ethidium bromide, EB for DNA, Coomassie brilliant blue for protein), silver stain, negative staining and fluoresecent molecules. All have their own pluses and minuses and crossover of different methods may improve the staining effect. Bisedes, we introduce the fundamental of CRP and four widely applied kinds of CRP:iniferter radical polymerization, nitroxide-mediated polymerization (NMP), atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer polymerization (RAFT). Abundantly free radical constantly occur chain transfer and bimolecular termination. Thus, through deactivating abundantly reactive free radical to be dormant can establish a rapid dynamic equilibration between trace amounts of growing free radicals and a large majority of the dormant species and subsequently the chance of chain transfer and bimolecular termination reduce sharply. We compare this four CRP in following aspects:applicable monomer, polymerization condition, polymerization mechanism, polymer molecular weight and its distribution, chain end functional groups, initiator and other additives.Chapter two discuss about a coordination complex system for generic, ultrafast, and sensitive multimode fluorescent of biomolecules. Firstly, we design and synthetize a planar heterocyclic organic small molecule Zl with a nitrogen-based chelation site on the dipyrido moiety. Zl can selectively interact with Zn2+to form [Zn·Z12]2+complex and we research the complex in detail by fluorescence. Then, we introduce this complex to biomolecule staining in gel electrophoresis and remarkably find incubation of the gel matrix after the DNA electrophoresis for only one minute results in the appearance of dark bands. The staining could be accomplished in multimode by successive reaction of the biomolecule-loaded gel with Zn2+and Zl, in either order of loading sequence. The system is generic for staining of DNA, RNA and protein and the detection limit is comparable with EB and Coomassie brilliant blue. Besides, there is a linear relationship between the total gray values of the biomolecule bands and quantities. Image contrast reversal is accomplished through a facile washing or photobleaching procedure.In cahapter three we introduce a kind of radical polymerization of acrylamide monomer in pure water. The polymerization system consists of monomer, CuCl, ligand Me6TREN and solvent water, with no organic initiator comparing with the ATRP system of Matyjaszewski. We can get homopolymer of PNIPAM, polyacrylamide (PAM) and copolymer of P(NIPAM-co-DMAA)(N, N-dimethylcarylamide) through this kind of polymerization. Besides, the polymerization is also feasible with oxygen, leading that both the polymerization conversion and the polymer molecular weight (Mw) decreases. We can adjust the polymerization rate and polymer Mw through adding Cu2+compound and ethanol. Finally, we research the polymerization mechanism by adding different polymerization terminal reagents and demonstrate it is radical polymerization. It is worth emphasizing that when adding the radical polymerization terminal reagent such as2,2,6,6-tetramethylpiperidinyloxy (TEMPO) or benzoquinone to the system, we find the polymerization conversion reduces. We comfirmed the polymer weight indeed decreases and weight of monomer increases after adding through UV-vis spectrum. We speculate there is some interaction among polymer growing free radicals, CuCl2/ligand and TEMPO, lead to decreasing the energy barrier of β-alkyl elimination reaction and making the polymerization equilibrium reaction remove to the monomer direction, subsequently the depolymerization of the polymer.