| Nucleic acids and proteins are important biology macromolecules. Nucleic acids are the material base of life, they play an important role in the action of growth, upgrowth, and reproduction of organism. There are connection between nucleic acids configuration and its function, which is relational to carcinogenic and anticancer activity. Proteins are most abundant in organism, and they attach themselves almost to all life action, the more complex of organism structure, the more various of proteins kinds and functions. 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.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, using the fluorescence and resonance light scattering (RLS) as the primary technique. The UV-visible spectrometry, circular dichroism (CD), surface tension, electrophoresis by TV, Transmission Electron Microscopy (TEM) techniques are also used to study the interaction mechanism. The main conclusions are listed as below:In the first section, we summarize the recent development of luminescent probes for nucleic acid and protein and the analytical applications, and comment on the basic theory and progress of silver nanoparticles in bioanalysis.In the second section, silver nanoparticles are made by chemical reduction with sodium citrate, and they are utilized in the study of the interaction between morin, nanoAg and nucleic acids. From the research, we find that morin-nanoAg can selectively recognize fish sperm DNA in double-strand nucleic acids. When nanoAg is added to morin, the intrinsic fluorescence of morin is obviously quenched; however, the fluorescence intensity of morin-nanoAg is greatly enhanced when fish sperm DNA (fsDNA) or salmon serum DNA (smDNA) are added to the system. Based on this, a new fluorimetric method for the determination of nucleic acids isproposed. Under optimum conditions, the enhanced fluorescence intensity is proportion to the concentration of nucleic acids in the range of 1.0 × 10-8-2.0×10-5 g/ml for fsDNA, 4.0×10-8-2.0×10-5 g/ml for smDNA, their detection limits (S/N = 3) are 1.2×10-10 g/ml, 1.7×10-10 g/ml, respectively. The study indicates that there are groove binding and molecular forces between morin, nanoAg and fsDNA, which induce the formation of nanocube, the system microenvironment including small polarity and larger microviscosity is also changed, and that lead to the fluorescence enhancement of system.In the third section, the resonance light scattering (RLS) quenching effect of nucleic acid-nanoAg-cationic surfactant is studied. It is found that nucleic acids can quench the RLS intensity of nanoAg-cationic surfactant system, under optimum conditions, the calibration graph for fsDNA are obtained. In nanoAg-CTMAB-fsDNA system, there are linear relationships between the quenching extent of RLS and the concentration of nucleic acids in the range of 4.0×10-9-2.0×10-6 g mL-1 for fish sperm DNA (fsDNA), 7.0×10-9-1.8×10-6 g mL-1 for calf thymus DNA (ctDNA) and 6.0×10-9-1.0×10-6 g mL-1 for yeast RNA (yRNA). The detection limits (S/N=3) of fsDNA, ctDNA and yRNA are 2.7×10-10 g mL-1, 4.8×10-10 g mL-1 and 7.2×10-10 g mL-1, respectively. In nanoAg-CPB-fsDNA system, there are linear relationships between the quenching extent of RLS and the concentration of nucleic acids in the range of 1.0×10-8-4.0×10-6 g mL-1 for fish sperm DNA (fsDNA), 4.0×10-8-4.0×10-6 g mL-1 for calf thymus DNA (ctDNA) and 1.0×10-8-2.0×10-6 g mL-1 for yeast RNA (yRNA). The detection limits (S/N=3) of fsDNA, ctDNA and yRNA are 1.7×10-10 g mL-1, 4.8×10-10 g mL-1 and 1.5×10-10 g mL-1, respectively. The results for the determination of DNA in actual samples isolated from Arabidopsis thaliana are satisfactory. The interaction mechanism investigation indicates that cationic surfactant can react with silver nanoparticles and nucleic acids through electrostatic and hydrophobic forces, which results in well dispersion of nanoAg and a remarkable RLS quenching of nanoAg-CTMAB/CPB. In addition, we compare the difference in RLS between CTMAB and CPB by the techniques of UV-visible spectrometry, circular dichroism (CD) and surface tension.In the fourth section, it is found that the fluorescence intensity of HSA can beenhanced on the surface of silver nanoparticle, which is made firstly by chemical deposited on the surface of APS treated quartz slides, and use collagen protein as isolated medium of nanoAg and albumin. Based on this, a sensitive method for the quantity determination of albumin is developed. Under the optimum conditions, the enhanced fluorescence intensity is proportion to the concentration of albumins in the range of 1.0×10-7-4.0×10-5 g/ml for BSA, 1.0×10-7-7.0×10-5 g/ml for HSA, their-detection limits (S/N=3) are 3.6×10-9 g/ml, 3.6× 10-8 g/ml, respectively. The mechanism study indicates that silver island film enhances the energy of incidence light by plasm resonance, and adjusts the eradiate velocity of nearby fluorescent molecule, which leads to the fluorescence enhancement of system. The chief characteristics of this thesis are as follows:1. We find that morin-nanoAg can selectively recognize fish sperm DNA in double-strand nucleic acids, and provide a simple and sensitive fluorimetric method for the selective determination of fish sperm DNA. The mechanism of system is also studied using the research techniques including RLS, UV-visible spectrometry, CD, TEM, fluorescence life time etc.2. It is found that nucleic acids can obviously quench the RLS intensity of nanoAg-cationic surfactant system, this system has been applied in the determination of DNA in actual samples isolated from Arabidopsis thaliana and the result is satisfactory. Compared with other RLS method, this method is simple, stable, quick and it has a larger linear range and better sensitivity.3. Silver nanoparticles have a surface enhancement effect to fluorescence, that is with an appropriate angle of incidence, the fluorescence of nearby fluorescent molecule can be enhanced if there is an appropriate definite distance between Ag island film and fluorescent molecule. Based on this, the fluorescence enhancement effect of nanoAg-albumin system is studied.
|
PDF Full Text Request