| Resonance Rayleigh scattering (RRS, or resonance light scattering, RLS) and resonance non-linear scattering (RNLS) are new techniques developed in recent years. They have received much attention due to their simplicity and high sensitivity. They have been extensively used to study and determination of biological macromolecules, pharmaceuticals, organic compounds and nanoparticles. However, for biological macromolecules such as proteins and nucleic acids, RRS method has been extensively used to research interactions between them and some small molecules such as dyes and medicines, and seldom used to study interactions between themselves. RRS is more sensitive to weak intermolecular forces (for instance, electrostatic force, hydrophobic force, hydrogen bonding and aromatic stacking interaction) and changes of molecular sizes, images and conformations than other molecular spectral methods such as absorption spectrum, fluorescence spectrum etc., this creates conditions for studying interactions of proteins with nucleic acids using RRS spectrum. When combining with common molecular spectral methods such as UV-Vis absorption spectrum, fluorescence spectrum, circular dichroism spectrum etc., it will provide much richer information for interactions between biological macromolecules. In addition, some high sensitive and selective analytical methods can be developed for determination of nucleic acids, proteins and polypeptides.The main contents and some conclusions of the dissertation are as follows:1. Resonance Rayleigh Scattering and Resonance Non-linear Scattering Spectra of Interaction of Proteins with DNA and Their Analytical Applications(1) Resonance Rayleigh Scattering and Resonance Non-linear Scattering Spectra of Interaction of Papain with DNA and Their Analytical ApplicationsIn weak acidic medium, interaction between papain and DNA resulted in absorption spectral change, fluorescence quenching of papain and remarkable enhancement of RRS. The interaction types and binding modes were discussed by characteristics of RRS, absorption, fluorescence and circular dichroism spectra combining thermodynamic data. There are four interaction types including electrostatic attraction, hydrophobic force, hydrogen bonding and aromatic stacking interaction. Papain interacted with the major groove of ctDNA. Aromatic stacking interaction is the main reason of change of absorption spectrum and fluorescence quenching of papain. Surface enhanced scattering effect, resonance energy transfer effect, increase of molecular volume and conformational change make contribution to RRS enhancement. The enhanced RRS intensity (△I) is directly proportional to the concentration of ctDNA or papain. The detection limit (3σ) is 4.5-15.3 ng-mL-1 for DNA and 5.6 ng-mL-1 for papain. This creates conditions for determination of papain and DNA.(2) Resonance Rayleigh Scattering and Resonance Non-linear Scattering Spectra of Interaction of Insulin with ctDNA and Their Analytical ApplicationsIn pH 7.4 Tris-HCl buffer solution, insulin interacted with ctDNA to form a complex, resulting in the change of absorption and circular dichroism spectra, the fluorescence quenching, enhancement of RRS, SOS and FDS as well as new scattering spectra. The optimum reaction conditions, influencing factors and relative analytical chemistry properties were investigated. The interaction mechanism was discussed combining scattering enhancement with absorption, fluorescence and circular dichroism spectra, as well as atomic force microscopy. There are four interaction types including electrostatic attraction, hydrophobic force, hydrogen bonding and aromatic stacking interaction. It was speculated that insulin entered the major groove of ctDNA, four aromatic residues including Try 16, Try 26, phe 24 and phe 25 were affected. The increase of molecular volume and hydrophobicity, resonance energy transfer and DNA conformation change are the main reasons of scattering enhancement. In addition, a RRS method based on this interaction was proposed to determination of insulin or DNA. When ctDNA is used as a probe to determine PMB, the detection limit (3σ) is 6.0 ng-mL-1. When insulin is used as a probe to determine ctDNA, the detection limit (3σ) is 7.2 ng-mL-1. The RRS method is very simple and sensitive. Therefore, it is significant of this interaction for developing a high sensitive method for determination of insulin or DNA.(3) Resonance Rayleigh Scattering and Fluorescence Rayleigh Scattering Spectra of Interaction of Hemoglobin with sDNA and Their Analytical ApplicationsIn weak acidic medium, interaction between hemoglobin and sDNA resulted in fluorescence quenching and remarkable enhancement of RRS of hemoglobin. And a new RRS spectrum appeared. The optimum reaction conditions, influencing factors and relative property of analytical chemistry were investigated. The quenching constant increased with increasing temperature, and the fluorescence life time decreased after adding sDNA, showing that the fluorescence quenching belongs to dynamic quenching. Surface enhanced scattering effect, resonance energy transfer effect, increase of molecular volume and conformational change make contribution to RRS enhancement. The enhanced RRS intensity (△I) or the quenched fluorescence intensity (△F) is directly proportional to the concentration of sDNA. The detection limits (3σ) of RRS and fluorescence quenching method are 5.5 ng mL-1 and 202.3 ng mL-1, respectively. RRS method was used to determine DNA in synthetic samples with satisfactory.2. Resonance Rayleigh Scattering and Resonance Non-linear Scattering Spectra of Interaction of Polypeptides with DNA and Their Analytical Applications(1) Resonance Rayleigh Scattering and Resonance Non-linear Scattering Spectra of Interaction of Polymyxin B with DNA and Their Analytical ApplicationsA novel assay of DNA or polymyxin B (PMB) with a sensitivity at the nanogram level is proposed based on the measurement of enhanced resonance Rayleigh scattering (RRS) and resonance nonlinear scattering (RNLS) including second order scattering (SOS) and frequency doubling scattering (FDS) resulting from interaction of PMB with DNA. The minor groove binding mechanism was suggested from RRS, absorption and circular dichroism spectra. The optimum reaction conditions, influencing factors and related analytical chemistry properties were tested. The interaction types and reasons of RRS enhancement were discussed. Linear relationships can be established between enhanced scattering intensity and DNA or PMB concentration. When ctDNA is used as a probe to determine PMB, the detection limit (3σ) is 9.8 ng mL-1. When PMB is used as a probe to determine DNA, the detection limit (3a) is in the range of 3.8-9.0 ng mL-1. Samples were analyzed satisfactorily. (2) Resonance Rayleigh Scattering and Resonance Non-linear Scattering Spectra of Interaction of Vancomycin with DNA and Their Analytical ApplicationsAssays were developed for DNA based on enhanced resonance Rayleigh scattering (RRS) and resonance nonlinear scattering (including second order scattering and frequency doubling scattering) that result from the interaction of vancomycin with DNA. Vancomycin possesses the unusual property of promoting the aggregation of DNA which was concluded from the results obtained with RRS, absorption and circular dichroism spectroscopy and from atomic force microscope. The types of interaction and reasons of RRS enhancement are discussed. Linear relationships do exist over a wide range between the intensity of enhanced scattering and the concentrations of DNA. The detection limit (3σ) is in the range from 6.8 to 11.1 ng mL3. Resonance Rayleigh Scattering and Resonance Non-linear Scattering Methods for Determination of Polymyxin B Using Isopoly-tungstic Acid as a ProbeIn pH 1.3-1.6 HCl-NaAc buffer medium, RRS and RNLS of polymyxin B sulfate or isopoly-tungstic acid are very weak. However, when they react with each other, RRS and RNLS will be enhanced significantly. The optimum reaction conditions, influencing factors and relative property of analytical chemistry were investigated. The interaction mechanism is discussed combing RRS enhancement with atomic force microscopy, change of absorption and circular dichroism spectra. Increase of molecular volume, surface enhanced scattering and resonance energy transfer effect are the main reasons of scattering enhancement. RRS,SOS and FDS method for determination of PMB have been established. The three methods all have high sensitivity. The RRS has the highest sensitivity and the detection limit (3σ) for PMB is 5.5 ng-mL-1. The method has better selectivity. It was used to determination of PMB in practical samples with satisfactory results.
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