Positive Charged Polymer As A Probe For The Determination Of Biomacromolecules By Resonance Light Scattering

Since the establishment of resonance light scattering(RLS) technique by Pastemack et al. using common fluorimetric instrument in 1993, it has been applied widely and developed quickly, now it is a necessary part in analytical research.Both protein and nucleic acid are important biomacromolecules. They are the material base of life, and play very important roles in process of life. So it is of special value to the determination of these biomacromolecules. This thesis choose a positive charged polymer, quaternized poly(4-vinyl-N-ethyl)pyridinium( PVP-C₂H₅Br) as a probe, using resonance light scattering technique, established new determination methods of protein and DNA, and use this polymer into the determination of DNA hybridization. The main contents of investigation in the contribution are as follows:1. We studied the reaction between polymer and protein, found out the optimum conditions, and obtained the binding ration and binding constant et al. by calculation, and established a new method for the determination of protein. In the B-R buffer of pH 7.54, RLS of polymer-protein system with the maximum scattering peak located at 470nm. Under the optimum conditions, the enhanced RLS intensity was proportional to the concentration of proteins, over the range of 3.5×10^-9-2.1×10^-7M for BSA and 3.3×10^-9-1.7×10^-7M for HSA, with the limit of determination 1.2×10^-9M for BSA and 1.2×10^-9M for HSA. We also use this method to the determination of synthetic mixtures of proteins, the results were satisfied. 2. We studied the reaction between polymer and calf thymus DNA (ct-DNA), found out the optimum conditions, and obtained the binding ration and binding constant by calculation, established a new method for the determination of DNA. In the PBS buffer of pH7.0 with ion strength 0.0300M, RLS of polymer-DNA system with the maximum scattering peak located at 344.8nm. Under the optimum conditions, the enhanced RLS intensity was proportional to the concentration of DNA over the range of 0.0900 to 2.70μg/mL, with the limit of determination 17ng/mL.3. We use this positive charged into the determination of DNA hybridization, proposed a simple and speedy assay for specific oligonucleotide sequences and single-base mismatch based on the different RLS signals of polymer/ssDNA and polymer/dsDNA, and established a new non-labeled methods for the determination of DNA hybridization. In the PBS buffer of pH7.0,ion strength 0.03M, RLS of polymer-DNA system with the maximum scattering peak located at 470nm. Under the optimum conditions, the enhanced RLS intensity was proportional to the concentration of target DNA over the range of 5.0 to 500nM, with the limit of determination about 2nM.