| Nucleic acid, protein and carbohydrate are the essential material in the organism. The molecular aggregation with various functions in the body is formed through hydrogen bond, electrostatic force, Van der waal's force and hydrophobic force between biomacromolecules and small molecules, and between the biomacromolecules. The affinity between protein and ligand provides the basis for inducing many chemical processes, and the study on it will help us understand the mechanism of these process and search for sensitive biology probe and effective medicine; while the interaction between biomolecules is essential for all biology process. So, it is very important in practice and theory to find new sensitive and adaptive probe for biomacromolecules and discuss the interaction mechanism between biomacromolecules and small molecules, and between the biomacromolecules.Based on the research of analytical chemistry, biochemistry, this thesis studied the interaction mechanism between biomacromolecules and small molecules, and between chitosan and protein or nucleic acid, using the techniques including fluorescence, absorption, resonance light scattering, circular dichroism and transmission electron microscope. Some new assays for protein and nucleic acid are developed. The main conclusions are listed as below:In the first section, we summarize the basic theory and progress of luminescent probes for proteins and nucleic acids, and the application of the chitosan in chemistry and biology fields. 167 references are cited here.In the second section, the interaction between nucleic acid and Tb3+ -protocatechuic acid (PCA) complex is deeply studied. Nucleic acid can quench the fluorescence intensity of the probe and based on it, a new fluorescence method for determination of nucleic acids is established. Under the optimum conditions, the quenched fluorescence intensity is proportional to the concentration of nucleic acid in the range 3.0×l0-8 -1.1×10-6 g/mL and 1.0 ×10-6 -5.0×10-6 g/mL for ctDNA, 1.8×10-8-1.0×10-6 g/mL and 1.0×10-6-8.0×10-6 g/mL for yRNA, the detection limits (S/N=3) are 23 ng/mL and 9.9 ng/mL, respectively. This method has been applied to the determination of synthetic samples and the results are satisfactory. Interaction mechanism is studied by using the techniques including absorption, light scattering, circular dichroism and viscosity determination. It is considered that the major force between the probe and nucleic acid is electrostatic binding. The competition of the oxygen atom of phosphate in nucleic acid framework with PCA for the Tb3+ induces the decrease of fluorescence intensity. The fact that the association constants of RNA with Tb3+ -PCA is much larger than that of DNA also proves that electrostatic interaction is the major force. The interaction between Tb3+ -PCA and DNA makes the double helix of DNA partly undo, the viscosity of this system decrease and the configuration of DNA change.In the third section, the fluorescence enhancement effect of Scopoletin (SLT) by bovine serum albumin (BSA) in the presence of anionic surfactant anionic sodium dodecyl benzene sulfonate (SDBS) is studied. Based on it, a new assay for proteins is developed. The linear ranges for BSA and HSA are 2.0×10-7-2.5×10-5g/mL and 1.2×l0-7-2.2×10-5g/mL, the detection limits (S/N=3) are 1.4×10-8g/ml and 1. l×10-8g/ml, respectively. Actual samples, extracted from fresh plasma, are satisfactorily determined. Under determination conditions, the pH value is lower than the pI of BSA, BSA is positive charged. BSA can bind SLT through hydrophobic force and further associates with anionic surfactant SDBS by electrostatic and hydrophobic forces. Both BSA and SDBS can provide the microenvironment with low polarity and large microviscosity for the SLT, resulting in the decrease of the loss of the energy resulted from the collision between SLT and water molecules. At the same time, the study points that there exists the energy transfer from BSA to SLT. The above two factors lead to the fluorescence enhancement of SLT.In the fourth section, the interaction between nucleic acid and low molecular weight chitosan is studied. Experiment results prove that nucleic acid can greatly enhance the light scattering intensity of chitosan, and based on it, a simple light scattering method for determination nucleic acid is developed. The linear ranges are 2.2×10-8-4.0×l0-6g/mL for DNA, 1.4×10-8-4.0×10-6g/mL for RNA, the detection limits (S/N=3) are 11 ng·mL-1, 6.5 ng·mL-1, respectively. This method has been applied to the determination of actual samples and the results are satisfactory. In this work, interaction mechanism is studied using mult-techniques. Chitosan interacts with nucleic acid not only by electrostatic and hydrogen bond but also by hydrophobic force, which is proved by the light scattering and circular dichroism data using the chitosans with different molecular weight. The interaction between the chitosan and DNA results in the change in the configuration of nucleic acid, the decrease of the melting temperature of DNA and the increase of the light scattering intensity of the system.In the fifth section, we study the interaction between chitosan and protein using the techniques including absorption, fluorescence and fluorescence polarization. It can be seen from the absorbance of BSA at about 200nm that chitosan induces the change in configuration of BSA and the increase of the content of α-helix of BSA. Under lower concentration of chitosan, the increase is rapidly, while under higher concentration of chitosan, it is slower. The effect of chitosan on surface hydrophobicity of BSA is also studied using ANS as the probe. It is considered that the concentration of chitosan influences the association between chitosan and protein. The chitosan molecule exists as different conformation under different concentration. Under lower concentration, the chitosan molecule exists as expanding state, it can adequately interact with protein, resulting in rapidly changing the configuration of BSA and the increasing the association constant between chitosan and protein ; Under higher concentration, the aggregation and self-coiling of chitosan molecules decrease the bind force between chitosan and protein, resulting in slowly changing the configuration of BSA and the decreasing the association constant between chitosan and protein In the sixth section, the chitosan nanoparticles are synthesized. It is found that the size between the chitosan nanoparticles is in correlation with the molecular weight of the chitosan and the spectroscopic properties of the chitosan nanoparticles change in comparision with the chitosan molecule. The association constant and the number of binding site between chitosan nanoparticles and curcumin (CUR) molecule are larger than those between chitosan and CUR molecule, which indicates that the association force between chitosan nanoparticles and CUR molecule is larger than that between chitosan and CUR molecule because the formation of chitosan nanoparticles increases the surface-to-volume ratio of chitosan and the contact area of chitosan with CUR. In the same concentration of chitosan, the small the molecular weight of chitosan, the larger both the surface-to-volume ratio of chitosan nanoparticles formed , and the association constant and the number of binding site between chitosan nanoparticles and CUR molecule.The chief characteristics of this thesis are as follows:1. The interaction mechanism of Tb3+-PCA- nucleic acids system is studied by using spectral techniques including absorption, light scattering, circular dichroism and viscosity measurement. It is considered that the competition of the oxygen atom of phosphate in nucleic acid framework with PCA for the Tb3+ induces the decrease of the fluorescence intensity of this system, which lead to partly undoing the double helix of DNA , and the change in configuration of DNA. Based on it, a new fluorescence method for determination of nucleic acids is proposed.2. It is found that the fluorescence intensity of SLT is enhanced by proteins in the presence of anionic surfactant SDBS, and a new fluorescence method for determination of proteins is proposed. The study of interaction mechanism indicates that the fluorescence enhancement effect originates from both the hydrophobic microenvironment provided by BSA and SDBS, and the intermolecular energy transfer from BSA to SLT.3. It is considered that the chitosan can bind with nucleic acid through not only electrostatic attraction and hydrogen bond, but also hydrophobic force, which results in the increase of light scattering intensity of the system. Based on this, a new method of light scattering for determination nucleic acid is developed.4. It is found that the interaction between chitosan and protein is related to the concentration of the chitosan. Under lower concentration of chitosan, the chitosan molecule exists as expanding state, it can strongly interact with protein, resulting in large change in the configuration of BSA and large association constant between chitosan and protein; Under higher concentration, the aggregation and self-coiling of chitosan molecule induce the decrease of the binding force between chitosan and protein, little change in the configuration of BSA and the decrease of the association constant between chitosan and protein.5. The study indicates that the size of the chitosan nanoparticles is in correlation with the molecular weight of chitosan and their spectroscopic properties change. The forming of chitosan nanoparticles lead to the increase of the association force between chitosan and curcumin.This thesis above not only proposes new assay methods for determination of nucleic acids and proteins, but also discusses the interaction mechanism between both small molecule and biomacromolecule, and between the biomacromolecules. Therefore, this thesis is valuable in theory and practice.
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