| Resonance Rayleigh scattering (RRS) and resonance non-linear scattering (RNLS) catch people's more attention because of their high sensitivity, rapid analysis speed and cheapness instrument. Now they have been widely used to pharmaceuticals analysis, the study of reactions between the micromolecules and biological macromolecules and reactions among inorganic molecules. But they are rarely applied to the study of reactions among biological macromolecules and their binding mode.In this work, the applications of RRS and RNLS to the analysis of proteins and polysaccharides were studied; the reation mechanism, binding mode, binding sites and the main interaction forces were investigated.1. Determination of chondroitin sulfate A by resonance Rayleigh scattering and resonance non-linear scattering using proteins as probesIn pH 2.5-4.0 Britton-Robinson (BR) buffer medium, some proteins such as human serum albumin (HSA), bovine serum albumin (BSA), Chymotrypsin (Chy) andα-Amylase (α-Amy) can react with chondroitin sulfate A (CSA) to form binding products by means of electrostatic, hydrogen bonding and hydrophobic forces. As a result, the resonance Rayleigh scattering (RRS), second-order scattering (SOS) and frequency doubling scattering (FDS) were enhanced greatly and new scattering spectra appeared. The increments of scattering intensity (ΔI) were directly proportional to the concentrations of CSA in certain ranges. The detection limits (3σ) of CSA were 1.1-2.0 ng/mL (RRS),1.9-2.9 ng/mL (SOS) and 3.1-13.2 ng/mL (FDS), separately. Among them, the RRS method exhibited the highest sensitivity and the HSA-CSA system was more sensitive than other reaction systems (DL=1.1 ng/mL). The characteristic of the spectra and optimum conditions of RRS method were investigated. Taking RRS method of HSA-CSA system for example, the reaction mechanism and binding mode were discussed, which indicated that the main interaction forces were electrostatic attraction, hydrogen bonding and hydrophobic interaction. The interaction between acetylamino group of CS and chromophore and fluorophore of aromatic amino acids of HSA (mainly in tryptophan and tyrosine) was the important cause of the change of absorption spectra and the fluorescence quenching. But the increase of molecular volume, formation of hydrophobic interface and fluorescence-RRS resonance energy transfer were the main factors of scattering enhancements.2. Determination of proteins by resonance Rayleigh scattering and resonance non-linear scattering methods with chondroitin sulfate A as a probeIn pH 1.8-2.5 BR buffer medium, negatively charged chondroitin sulfate A (CSA) can react with positively charged proteins, such as HSA, BSA, Chy, a-Amy and lysozyme (Lyso), via electrostatic attraction, hydrogen bonding and hydrophobic interaction. This led to the enhancements of resonance Rayleigh scattering (RRS) and resonance non-linear scattering (RNLS), including second order scattering (SOS) and frequency doubling scattering (FDS), and the appearance of new spectra. The scattering intensities (ΔIRRs,ΔISOS andΔIFDs) were directly linear to the concentrations of proteins in certain ranges. Each method had high sensitivity and the detection limits (3σ) were 4.5-12.0 ng/mL (RRS),8.9-15.8 ng/mL (SOS) and 13.4-31.5 ng/mL (FDS) for proteins. The CSA-BSA system was the most sensitive in which the detection limit for BSA was 4.5 ng/mL. In this article, the spectral characteristics, optimum conditions and influencing factors were investigated. The reaction mechanism and binding mode were discussed. Taking RRS method of CSA-BSA system for example, the effects of coexisting substances on the reaction were investigated. The method can be applied to the determination of protein in serum and urine samples with satisfactory results.3. The study of resonance Rayleigh scattering and resonance non-linear scattering of proteins-hyaluronate systems and their analytical applicationsIn suitable acidity of BR buffer medium, sodium hyaluronate (SH) with negative charge can react with possitively charged proteins, such as HSA, BSA, Chy and a-Amy. via electrostatic attraction, hydrogen bonding and hydrophobic interaction. The intensities of RRS and RNLS were enhanced obviously and new spectra appeared. The enhanced scattering intensities (ΔIRRS,ΔISOS andΔIFDS) were directly proportional to the concentrations of hyaluronate in certain ranges. The detection limits (3σ) of hyaluronate for HSA-SH, BSA-SH. Chy-SH andα-Amy-SH were 3.0,7.7,4.3 and 3.5 ng/mL (RRS): 5.4,16.3,10.5 and 6.8 ng/mL (SOS);8.9,21.0,17.2 and 8.8 ng/mL (FDS), respectively. In this article, the spectral characteristics, optimum conditions of the reactions and influencing factors were investigated. Based on the above researches, a high sensitive, simple and fast method for the determination of hyaluronate has been established.4. Determination of sodium alginate by resonance Rayleigh scattering and resonacne non-linear scattering using proteins as probesIn suitable acidity of BR buffer medium, sodium alginate (Alg) can react with some proeins such as HSA, BSA, Chy and Lyso by means of electrostatic attraction, hydrogen bonding and hydrophobic interaction, which led to the enhancement of RRS and RNLS intensities such as SOS and FDS. The maximum scattering wavelenghs were 304 nm (Chy-Alg),300 nm (HSA-Alg, BSA-Alg) and 301 nm (Lyso-Alg) for RRS. The enhanced scattering intensities (ΔIRRS,ΔISOS andΔIFDS) were directly linear to the concentrations of Alg in certain ranges. The detection limits (3σ) of alginate for HSA-Alg, BSA-Alg, Chy-Alg and Lyso-Alg were 1.9,2.1,2.1 andl.6 ng/mL (RRS); 2.6,2.9,3.0 and 2.6 ng/mL (SOS); 5.0,5.1,5.5 and 5.5 ng/mL (FDS), respectively. The optimum conditions, influence factors and the effect of coexisting substances were investigated. Based on above reseacches, a high sensitive, simple and quick method is developed to determine the sodium alginate.5. Determination of sodium carboxymethylcellulose by resonance Rayleigh scattering and resonance non-linear scattering using chymotrypsin as a probeIn pH 4.4 Hac-NaAc buffer medium, sodium carboxymethylcellulose (CMC) can react with Chy by means of electrostatic attraction and hydrophobic interaction. The intensities of RRS, SOS and FDS were enhanced greatly and new scattering spectra appeared. The enhanced scattering intensities (ΔIRRS,ΔISOS andΔIFDS) were directly proportional to the concentrations of CMC in certain ranges. The detection limits (3σ) of three method were 2.3,4.8,4.3 ng/mL for RRS, SOS and FDS, respectively. The optimum conditions, influence factors and the effect of coexisting substances have been investigated. The method can be applied to determination of CMC in cut tobacco.
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