| In this paper, the interaction of some component units of biomacromolecules, such as aromatic amino acids (component which causing proteins’intrinsic fluorescence) and adenine (one of the nucleic acid bases), with metal ions like Pd (II) and Cu (II) were investigated. Besides, the actions between some biomacrorrolecule, like chitosan with some acidic polysaccharoses, sodium carboxymethyl starch with lysozyme, and pepsin with papsin, were studied by absorption spectroscopy, fluorescence spectroscopy and resonance Rayleigh scattering spectra. The mechanisms of interactions, the binding modes, the binding sites and the forces were investigated and new methods for determination of adenine, chitosan and so on were establisl ed. The main work of this thesis is as follows:1. Fluorescence Spectra Study on the Interaction betweei Pd(II) and Tryptophan, Tyrosine and PhenylalanineWhen Pd(II) reacted with tryptophan, tyrosineand phey lalanine, the fluorescence of the three aromatic amino acids was all quenched. From the change of absorption spectra, the effect of temperature and the quenching constant Ksv, this procedure was judged to be static quenching on account of Pd(II) interacting with the above amino acids to form the groundstate coordination compounds. In present of certain Cl’, Pd(II) coordinated with amino acids by N, N or N, O to form mixed-type ternary complexes. This system could not only be used in the study between Pd(II) and above aromatic amino acids, but also be foundation of determining Pd(II) by fluorescence quenching method using amino acid (especially Trp) as a probe.2. Resonance Interaction of Adenine with Palladium Chloride, Determination of Adenine via Resonance Rayleigh Scattering MethodIn pH1.7-3.5acid medium, palladium chloride could react with adenine (A) to form a ternary complex of [PdCl2·A], which would self-aggregate to form uniformly dispersed nanoparticles-[PdCl2-A]n with an average size of42nm through the squeezing effect of aqueous phase and van der Waals force. This resulted in an enhancement of resonance Rayleigh scattering (RRS), second-order scattering (SOS) and frequency doubling scattering (FDS). The maximum wavelengths were located at311nm,611nm and395nm, respectively. The scattering intensities of the three methods were proportional to the concentration of adenine in certain ranges, and the detection limit of the most sensitive RRS method was5.4×l0-9mol/L (0.73ng/mL). The experimental conditions were optimized and effects of coexisting substances were evaluated. The method showed excellent selectivity because a certain amount of other nucleobase, nucleoside or nucleotide wouldn’t influence the measurement. Accordingly, a novel rapid, convenient, sensitive and selective RRS method for determination of adenine was proposed and applied to detect adenine in tablet and hydrolyzates of ctDNA samples with satisfactory results. The shape of nanoparticles was characterized by atomic force microscopy. The reaction mechanism and the reasons for enhancement of scattering were discussed by infrared spectra; quantum chemical calculations and absorption spectroscopy.3. Resonance Rayleigh Scattering Spectra of Cu2+-Adenine-WO42-System and Its Analytical ApplicationIn pH6.6-7.2Tris-HCl buffer, Cu2+could react with adenine (A) to form a1:1coordination cation [CuA]2+, which only resulted in minor change of absorption spectra. However, when this cation further combined with WO42-to form a1:1ternary ion-association complex [CuA]W04, the absorption spectra changed a lot, and the resonance Rayleigh scattering (RRS), second-order scattering (SOS) and frequency doubling scattering (FDS) enhanced significantly. The maximum wavelengths of RRS, SOS and FDS were located at310,592and395nm, respectively. The enhanced intensities of the three methods were proportional to the concentration of adenine in certain ranges, and the detection limit of the most sensitive RRS method was7.4×10-9mol/L (1.0ng/mL), indicating that this method could detect trace adenine. In this work, the optimum reaction conditions and the influencing factors have been studied, some potential interferences and the composition of ion-association complex have been investigated. Meanwhile, the construction of the product and mechanism of reaction have been discussed by atomic force microscopy, transmission electron microscope and quantum chemical calculation. Accordingly, a novel RRS method for determination of adenine has been proposed and applied to detect adenine in real samples with satisfactory results.4. Resonance Relay Scattering Spectra of Chitosan and Some Acidic Polysaccharoses and Its ApplicationIn acidic medium, positive charged chitosan (CT) could react with some negative charged acidic polysaccharese, e.g. chondroitin sulfate (CSA), sodium hyaluronate (SH), sodium heparin (Hep), sodium alginate (Alg) and sodium carboxymethyl starch (CMS), which not only caused a slight change of the absorption spectrum, but also led to great increase of resonance Rayleigh scattering (RRS) and resonance nonlinear scattering (RNLS). The order of enhanced intensity of all three methods were CT-SH>CT-CMS>CT-Alg>CT-CSA>CT-Hep. The RRS maximum wavelength of CT-SH system was located at341nm, and the CT-CMS system’s was373nm, and the others were all at355nm. The SOS and FDS spectra of all these systems were similar and the maximum wavelengths were565nm (SOS) and389nm (FDS). Among these methods, the RRS method using SH as the probe was the most sensitive. The detection limit of chitosan was3.6ng/mL. The spectral characteristics, the optimum reaction conditions and influencing factors were studies. Taking the CT-SH system for example, the effects of coexisting substances were investigated, and a RRS method for the determination for chitosan in the capsule by a SH probe was proposed. Besides, the main force and the binding mode between the two polysaccharides were discussed by UV-visible absorption spectroscopy, infrared spectroscopy and thermogravimetric analysis. It was concluded that the two polysaccharides combined through electrostatic attraction, hydrogen bonding and hydrophobic interactions, and the morphology of the complexes were characterized by TEM and AFM. Then the reasons for enhanced scattering were discussed.5. The resonance Rayleigh scattering spectra study on the sodium carboxymethyl starch and lysozyme.In the alkaline medium of pH8.7-9.1, the positively charged lysozyme (Lyso) could react with the negatively charged sodium carboxymethyl starch (CMS) to form complexes through electrostatic attraction and hydrophobic interaction, which resulted in changes of absorption spectrum and significant enhancement in the resonance Rayleigh scattering (RRS). The new scattering peak appeared and the maximum wavelength was313nm. Within a certain range, the concentration of sodium carboxymethyl starch and lysozyme were proportional to the enhanced RRS intensity ), respectively. The reaction was sensitive, the detection of sodium carboxymethyl starchlimit or lysozyme was6.16ng/mL or9.65ng/mL, so both mutual determinations could achieve. The RRS spectral characteristics, the optimum conditions, influence factors and effects of coexisting substances of reaction were studied. Also the reaction mechanism and the binding mode between Lyso and CMS were discussed coupling with absorption spectra and circular dichroism spectra. It was concluded that the increase of molecular volume, the hydrophobic interface formation, absorption-scattering resonance energy transfer and variation of confirmation were the main reasons for the enhanced RRS.6. The resonance Rayleigh scattering and resonance non-liner scattering spectra study on pepsin and papsin systemIn a weak acid medium of pH5.0-6.5, the negatively charged pepsin (Pep) combined with the positively charged papain (Pap) through electrostatic attraction and hydrophobic interaction to form a complex, which not only caused the changes of absorption spectra, but also led to increase of resonance Rayleigh scattering (RRS) and resonance nonlinear scattering (RNLS) including second-order scattering (SOS) and frequency doubling scattering (FDS). The maximum scattering wavelengths of new spectra were located at311nm(RRS),566nm(SOS),388nm(FDS). The concentration of Pap was proportional to the enhanced intensities in certain ranges. The reaction was sensitive and the determination of Pap was0.011μg/mL(RRS),0.041μg/mL(SOS),(?)0.22μg/mL(FDS). The RRS and RNLS spectral properties of Pep-Pap complex, the optimum reaction conditions, influencing factors and the effects of coexisting substances were investigated. Also the reaction mechanism and forces were discussed. |
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