| Resonance Rayleigh scattering(RRS) and Resonance nonlinear scattering(RNLS) are new analytical techniques developed in 1990s.As its high sensitivity and simple operation,this method has received more and more attention of the scientific workers. It shows advantages on the research of biological molecules.But there are less study on small biological molecules such as nucleotide or nicotinamide coenzymes. Therefore,taking nucleotide and nicotinamide coenzymes as examples,we developed the new methods for the determination of these nucleotide and nicotinamides.The interaction between these enzymes and proteins were also investigated.The RRS method and RNLS method were developed for the determination of these biological molecules.In this thesis,the interactions between these biological molecules and metal ions or dyes were investigated.Besides,the reaction mechanism was investigated by the spectra of resonance Rayleigh scattering and combined with absorption and fluorescence.1.Resonance Rayleigh Scattering Spectra of the Interaction of Cerium(â…£) with Adenylic Nucleotides and Their Analytical ApplicationsIn pH 2-3 Walpole buffer solution,the RRS intensities of cerium(â…£)(Ce(â…£)) and the nucleotides themselves were very weak.However,when Ce(â…£) reacted with nucleotides(AMP,ADP or ATP) to form complexes,the RRS intensity was enhanced greatly and new RRS spectra appeared.The RRS spectral characteristics of the complexes of AMP,ADP and ATP with Ce(â…£) were similar but the relative scattering intensity of the complexes were different.The detection limits were 2.08 ng mL-1 for AMP,2.28 ng mL-1 for ADP and 3.63 ng mL-1 for ATP.In this work,the optimum conditions of the reaction,the influencing factors and the effects of coexisting substances were investigated.In addition,a proposed structure of the complex taking Ce(â…£)-ATP system as an example and the reasons for scattering enhancement were discussed.2.Scattering,Absorption and Fluorescence Spectra of the Interaction of NAD(H) and NADP(H) with Victoria Blue 4RIn pH 7.2 Britton-Robinson(BR) buffer medium,nicotinamide coenzymes such as NAD+,NADP+ and their reduced forms of NADH and NADPH reacted with victoria blue 4R(VB4R) to form ion-association complexes,which resulted in the fading of VB4R.It also led to the great enhancement of resonance Rayleigh scattering (RRS) and appear of new RRS spectra.The characteristics of absorption and RRS spectral of the four reaction products were similar.The maximum fading wavelengths of VB4R by above four nicotinamide coenzymes were all located at 611 nm and the maximum scattering wavelengths were located at 521nm(NAD+,NADP+),524 nm (NADH),526 nm(NADPH).However,the decrements of absorbance(ΔA) and relative intensities of RRS(ΔI) are different.And bothΔA andΔI were directly proportional to the concentrations of the above four nicotinamide coenzymes.It created the conditions for the determination of the above nicotinamide coenzymes by spectrophotometry and RRS methods.In this work,the spectral characteristics,the optimum conditions and the influencing factors of the reaction were studied.The effects of foreign substances were investigated which showed this method had a good selectivity.In addition,the reaction mechanism and the reasons for the scattering enhancement were discussed. 3.Determination of Nicotinamide Coenzymes by Resonance Rayleigh Scattering Method with Pd(â…¡)-PapainIn pH 4.4-4.6 BR buffer medium,coenzymeâ… and coenzymeâ…¡reacted with Pd(â…¡) to form 1:1 chelates,which further reacted with papain to form 1:1:1 complex.As a result,the resonance Rayleigh scattering spectra enhanced greatly and the new spectrum appeared.The products have similar spectral characteristics and their maximum wavelengths were located at 309 nm and 550 nm.In a certain range,the enhancement of the scattering intensity and the concentration of the coenzymes had linear relationship.The detection limits for the coenzymes were in the range of 2.35-3.33×10-8 mol L-1.This method had high sensitivity so that it was suitable for the determination of trace nicotinamide coenzymes.The optimum conditions and influence factors were investigated.The effects of foreign substances showed it had good selectivity.Based on this,a highly sensitive,simple and rapid method for the determination of coenzymes was proposed.4.Study on the Interaction of NADH with Lysozyme and Pd(â…¡) Using Resonance Rayleigh Scattering and Fluorescence spectraIn pH4.6 Britton-Robinson buffer solution,after NADH combined with Pd(â…¡) and lysozyme,it resulted in the fluorescence quenching of NADH and the great enhancement of Resonance Rayleigh Scattering(RRS).The maximum RRS wavelength was located at 309 nm and the enhancement of RRS had a linear relationship with the concentration of NADH in(0.26-31.25)×10-7mol L-1 range.And the detection limit was 5.7 ng mL-1.When NADH was excess,the enhancement of RRS was in a linear relationship with the concentration of lysozyme in(0.035-4.2)×10-7mol L-1 range,and the detection limit was 15.1 ng mL-1.The RRS spectra offered new information for the combination of biological molecule with metal ions and also created the conditions for high sensitively determination of these biological molecules. In this work,the fluorescence quenching of lysozyme by NADH and Pd(â…¡) and the fluorescence quenching type of NADH by lysozyme and Pd(â…¡) were investigated.5.Study on the Resonance Rayleigh Scattering,Second Order Scattering and Frequency Doubling Scattering Methods for the Determination of Proteins with Pd(â…¡)-NADP Chelate and Their Analytical ApplicationsIn pH4.0-5.0 Britton-Robinson buffer medium,NADP reacted with Pd(â…¡) to form anion chelate,which could further react with proteins such as BSA,HSA,HGB,γ-globin to result in the significant enhancement of resonance Rayleigh scattering(RRS) and non-linear scattering such as second order scattering(SOS) and frequency doubling scattering(FDS).The RRS,SOS and FDS spectral characteristics of the four proteins were similar,and the maximum scattering wavelengths were located at 333nm for RRS, 500nm for SOS and 250nm for FDS,respectively.And the scattering intensity increments were directly proportional to the concentration of the proteins in certain ranges.It could be used for determination of these proteins.In this work,the optimum conditions and influencing factors were investigated.And taking HSA as an example, the effects of coexisting substances were examined and the results showed the good selectivity.Based on the above researches,a highly sensitive,simple and fast method for determination of proteins was established.It was applied in the determination of proteins in human serum and urine samples with satisfactory results. |
