The Studies And Applications Of Nucleic Acids In Triatomic System By Resonance Light Scattering Technique

The Resonance Light Scattering (RLS) technique was set up by Pastemack and co-workers using common fluorimetric instrument in 1993. They studied distinguishing, assembly, and aggregation on bimolecular by RLS technique. People follow the RLS with great interest.Nucleic acid is one of the fundamental materials of life. It is the carrier of inheritance information and the base of gene expression. With the effort of a great deal of scientists, people have made much breakthrough in the studies on nucleic acid during the last century. Today, the study field of nucleic acid has been extended. In the chemical studies on nucleic acid, the field of small molecular is always very brighten, because it can be used as the medical treatment of cancer, the coloring agents and the probe to determinate the structure of nucleic acid. Armed with the advanced equipment and sensitive determination methods, analytical chemistry plays an important role in the studies on the interaction between DNA and small molecular for the quantitative analysis of DNA, and for the investigation of the mechanism.Based on a lot of documents and conditions of our laboratory, this thesis establishes several new determination methods of DNA by means of novel resonance light-scattering technique, studies the RLS signal of nucleic acid reacts with anionic small molecular or antineoplastic in the presence of cationic surfactant cetyltrimethylammoniumbromide (CTMAB), and the interaction mechanisms of the triatomic system are investigated by various spectra. These methods are simple and rapid with excellent reappearance, selectivity, sensitivity and wide linear coverage. The thesis has some reference values to the studies on interaction mechanism between drug and small molecular, the design of the structure of drugs, the determination of the dose of drugs, and the extension of the application of resonance light-scattering technique.This paper has eight chapters.In the first chapter, we summarized the development of nucleic acids, the theory of RLS and the foundation of quantitative analysis, the application of RLS in the nucleic acids investigation and the analysis.In the second chapter, we studied the spectral characteristic of the triatomic system of anionic dye methyl blue-cation surfactant cetyltrimethylammonium bromide (CTMAB)-nucleic acid, and the elementary reaction mechanism with the ways of absorption spectra and relative viscosity. Another new determination method of DNA at nanogram level was established. At pH=9.00, nucleic acids and CTMAB could cooperatively enhance the RLS intensity of methyl blue and produce RLS with the maximum scattering peak located at 334 nm. Under the optimum conditions, the enhanced RLS intensity was proportional to the concentration of yDNA,ctDNA,fsDNA and RNA in the appropriate range. The lowest detection limit was 2.70ng/ml. This method was simple, rapid, high sensitivity and good selectivity. The resistant to most of melt and amino acid in biology system was well. And it had been applied to determination of DNA in synthetic samples with satisfactory results.In the third chapter, we studied in the hexamethylene tetramine(HMTA) buffer(pH=6.75), anionic dye alizarin green (AG) react with deoxyribonucleic acid (DNA) and cation surfactant cetyltrimethylammonium bromide (CTMAB) to form a kind of large aggregate, Which result in significant enhancement of RLS intensity. The spectral characteristics of RLS, the effective factors and optimum conditions of the reaction have been investigated. Mechanistic studies showed that the enhanced RLS comes from the aggregation of AG on DNA through the bridged and synergistic effect of CTMAB. Under the optimum conditions, the enhanced RLS intensity is proportional to the concentration of DNA in the range 50 to 1200μg/L for yeast DNA (yDNA) and 50 to 1000μg/L for fish DNA (fsDNA). The linear equation are△I= 24.156+0.698C(C:μg/L)and△I=29.347+0.752 C(C:μg/L)respectively, The correlation coefficient are 0.9996 and 0.9991respectively, the detection limits are3.1ngm/L and 8.2ngmL respectively. This method is simple, rapid and has been applied satisfactorily to determination of DNA in synthetic samples.In the fourth chapter, we studied in the hexamethylene tetramine(HMTA) buffer(pH=7.30), anionic dye xylenol orange (XO) react with deoxyribonucleic acid(DNA) and cation surfactant cetyltrimethylammonium bromide (CTMAB) to form a kind of large particles of complex, Which result in significant enhancement of RLS intensity. The spectral characteristics of RLS, the effective factors and optimum conditions of the reaction have been investigated. Under the optimum conditions, the enhanced RLS intensity is proportional to the concentration of nucleic acids in the appropriate range. This method is simple, rapid and has been applied satisfactorily to determination of DNA in synthetic samples.In the fifth chapter, we studied in the hexamethylene tetramine(HMTA) buffer(pH=7.60), anionic dye thymol blue (TB) react with nucleic acid and cation surfactant cetyltrimethylammonium bromide (CTMAB) to form a kind of large particles of complex Which result in significant enhancement of RLS intensity. The spectral characteristics of RLS, the effective factors and optimum conditions of the reaction have been investigated. Mechanistic studies showed that the enhanced RLS comes from the aggregation of TB on DNA through the bridged and synergistic effect of CTMAB. Under the optimum conditions, the enhanced RLS intensity is proportional to the concentration of nucleic acids in the appropriate range. The detection limit is 1.30ng/mL, This method is simple, rapid and has been applied satisfactorily to determination of DNA in synthetic samples.In the sixth chapter, we studied in the hexamethylene tetramine(HMTA) buffer(pH=11.00), anionic dye bromocresol green (BCG) react with nucleic acids and cation suffactant cetyltrimethylammonium bromide (CTMAB) to form a kind of large aggregate Which results in significant enhancement of RLS intensity. The spectral characteristics of RLS, the effective factors and optimum conditions of the reaction have been investigated. Mechanistic studies showed that the enhanced RLS comes from the aggregation of BCG on DNA through the bridged and synergistic effect of CTMAB. Under the optimum conditions, the enhanced RLS intensity is proportional to the concentration of DNA and RNA in the corresponding ranges respectively. This method is simple, rapid and has been applied satisfactorily to determination of DNA in synthetic samples.In the seventh chapter, Methotrexate (MTX) as an antifolate, which is widely used as chemotherapeutic drugs. A high-dose MTX therapy has a direct toxicity influence on the non-germinal cells, especially the liver cells. We investigated the interaction of methotrexate (MTX) and nucleic acids in aqueous solution by a resonance light scattering (RLS) technique with a common spectrofluorometer in the present of the cationic surfactant, the optimum dosage of MTX which enhanced the RLS intensities to the maximum is 4.54μg, while the inject dose for adults is 10-30mg and is half for children for routine use. In hexamethylenetetramine (HMTA) buffer (pH9.30), MTX and nucleic acids react with cetyltrimethylammonium bromide (CTMAB) to result in strong enhanced RLS signals. With the enhanced RLS signals, the enhanced RLS intensity is proportional to the concentration of nucleic acids in an appropriate range. The synthetic samples of nucleic acids were analyzed and obtained recovery values between 96 and 102%. The eighth chapter is the conclusion.