Study On The Spectral Probes Of Bio-macromolecules And Its Analytical Application

Nucleic acids and proteins are basic of life. Nucleic acids are the genetic material, they play an important role in the action of every stage (growth, up-growth, and so on). Proteins are the most abundant in organism, and they almost attach themselves to all life action. So it is of special value to study the interaction of small molecules and biomolecules, the quantitative analysis of nucleic acids and proteins with high sensitivity and low detection limit are important in life science, biochemical medicament, food analysis and clinical analysis, which is helpful to design the new type of drug and study the toxicity of drug. This project is the forward position and hot point in biochemical and biophysical researches.Based on the fluorescence and resonance light scattering (RLS) as the primary technique, this thesis focus on the development of new probes for nucleic acids and proteins and to establish sensitive methods for the quantitative determination of them, with multiple techniques to study the interaction mechanism. The main conclusions are listed as follows:In the first section, we summarize the recent development of fluorescent probes for nucleic acid, protein and the analytical applications. The progress of nanoparticles in the analysis of nucleic acid and protein is also commented.In the second section, the interaction between CTC, AuNPs and nucleic acids are stuied. From the research, we find that when DNA is added to CTC-AuNPs, resonance light scattering is enhanced. Based on this, a new resonance light scattering method for the determination of nucleic acids is proposed. Under optimum conditions, the enhanced resonance light scattering intensity is proportion to the concentration of nucleic acids in the range of 5.0×10-8-1.5×10-5 g mL-1 for fish sperm DNA (fsDNA) and 6.0×10-8-1.0×10-5 g mL-1 for smDNA, their detection limits (S/N=3) are 1.2×10-9g mL-1,1.5×10-9 g mL-1, respectively. The interaction mechanism is considered that there exists long range assembly effect of the system.In the third section, the fluorescence quenching effect of iCTC-AuNPs-DNA is studied. It is found that DNA can quench the fluorescence intensity of iCTC-AuNPs. Under optimum conditions, there are linear relationships between the quenching extent of fluorescence and the concentration of DNA in the range of 1.0×10-8-2.0×10-6g mL-1 for fsDNA,1.0×10-8-1.6×10-6 g mL-1 for ctDNA. The detection limits (S/N=3) of fsDNA and ctDNA are 3.2×10-9 g mL-1 and 4.0×10-9 g mL-1, respectively. The interaction mechanism investigation indicates that there exist the groove and electrostatic interaction.In the fourth section, it is found that protein can enhance the synchronous fluorescence intensity of MB-SDBS. Under optimum conditions, the enhanced synchronous fluorescence intensity is proportion to the concentration of proteins in the range of 8.0×10-8-4.0×10-5 g mL-1 for BSA,10.0×10-8-3.5×10-5 g mL-1 for EA. The detection limits (S/N=3) of BSA and EA are 8.9×10-9g mL-1,10.0×10-9 g mL-respectively. Samples are satisfactorily determined. The fluorescence enhancement of the system originates from the deaggregation of some MB dimer and the formation of an MB-SDBS-BSA complex, as well as the hydrophobic microenvironment provided by BSA and SDBS.The chief characteristics of this thesis are as follows:1. It is found that fsDNA can enhance the resonance light scattering intensity of CTC-AuNPs system and provide a stable, sensitive and highly stable against fluorescent photobleaching fiuorimetric method for the determination of nucleic acids, the mechanism of the system was also discussed in detail.2. It is found that DNA can obviously quench the fluorescence intensity of iCTC-AuNPs, this method is simple, stable and quick. The fluorescence quenchment mechanism was also discussed in detail.3. It is found that protein can enhance the fluorescence intensity of MB-SDBS system and provide a new fluorimetric method with less influence of bimolecular background for the determination of proteins. The synchourous fluorescence enhancement mechanism was also discussed in detail.