| Microdialysis technology that combined perfusion sampling and dialysis technology is a kind of new technology which is making the gradual improvement of the biological in vivo dynamic trace biochemical sampling. It contains several advantages following as continuous in vivo sampling, dynamic observation, quantitative analysis, small sampling volume, lower tissue damage. The biggest advantage of microdialysis technique is that it does not interfere basically with the body’s normal physiological processes while for in vivo, real-time and on-line sampling during in the process of sampling, the tissue fluid composition surrounding the microdialysis probe will not change, so it can be used to collect samples continuously. After the exchange of dialysis fluid, the sample was collected for further analysis by high performance liquid or other instrument. As microdialysis sampling and detection sensitivity improve constantly, the microdialysis technique has the rapid development in biochemical research as a compelling new areas. This technology in life science research reached a climax since the1990s. On-line microdialysis is a trace and dynamic continuous sampling method, which combined with ultraviolet, fluorescence, chemiluminescence or electrochemical method, has been widely applied in these fileds including in vivo analysis and process monitoring and drug-protein interaction and pharmacokinetic studies and so on. On-line microdialysis not only can minimize sample loss, achieve good accuracy and repeatability, and can effectively reduce the time interval between the sample collection and analysis, thus shortening the analysis time.Electrochemiluminescence or Electrogenerated Chemiluminescence (abbreviated as ECL) is that regenerates some special material on the electrode surface by electrochemical method, and these substances or other components of the system reach the excited states formed by electron transfer and the excited state returns to the ground state to produce the luminous phenomenon which is a product of the combination of electrochemical and chemiluminescence methods. This method has the advantages of chemiluminescence with high sensitivity and wide linear range and simple instrumentation. Meanwhile luminescence is excited by electrochemical methods which are easy to control in time and space aspect, good reproducibility and reagent stability. In many electrochemiluminescence system, Ru(bpy)32+electrochemiluminescence has been widely used in scientific research and practical clinical analysis due to the advantages of a wide range of anti-interference ability, reliable result, reagent stability, wide linear range, regenerated reagent, and receive widespread attention. By immobilizing Ru(bpy)32+on an electrode surface to design an ECL biological sensor, solid-state ECL provides several advantages over solution-phase ECL, such as reducing consumption of expensive reagent, simplifying experimental design and enhancing the ECL signal.When the drug enters into the bloodstream, it can be reversibly combined with plasma protein to some extend, and the drug molecules combined with plasma protein are difficult to transport through membrane to the action site owing to their greater molecular weight. And the unbound drugs can freely pass through the capillary wall into the target organs to express efficacy. Some important pharmacokinetic properties such as liver metabolization rate, kidney excretion rate, biomembrane permeation rate, and steady state distribution depend on the amount of free drug part. In this respect, plasma protein binding is an important factor in establishing pharmacokinetic and pharmacodynamic properties of a drug, as only the free fraction of the drug is pharmacologically active. So the study of drug-protein interaction and related parameter determination have important theoretical and practical significance.In this work, we design a novel electrochemiluminescence sensor, immobilized Ru(bpy)32+on the surface of electrode, which is very simple, steady and durable.Using this sensor combined with microdialysis sampling, we realize the sensitive, fast on-line study of drug-protein interaction. This thesis consists of two chapters:Chapter1for the review and Chapter2for research reports.Chapter1:The reviewReview section containes a brief overview of the microdialysis technique, electrochemiluminescence technology and electrochemiluminescence sensor, the construction and application of the new method and significance of studying drug-protein interaction.Chapter2:Research reportsThe research report broadly consists of three parts:1. Immobilized Ru(bpy)32+electrochemiluminescence coupled with microdialysis sampling for on-line studying drug-protein interactionA electrochemiluminescence(ECL) sensor based on the use of Nepem-105D perfluorinated ion exchange solution to form the ion exchange membrane on the surface of platinum electrode for immobilization of Ru(bpy)32+has been developed. A new method was designed combined microdialysis sampling technology and ECL sensor for on-line study the interactions between ciprofloxacin and bovine serum albumin(BSA). Under the optimized experimental conditions the relative ECL intensity is proportional with ciprofloxacin concentration in the range of3.0×10-8-3.0×10-5mol/L with a detection limit of1.0×10-8mol/L. The association constant of compound of ciprofloxacin and BSA and the number of the binding sites with Scatchard analysis are3.51×104(mol/L)-1and1.49, respectively. The results show that the novel method can provide a fast and simple technique for the study of drug-protein interaction.2. On-line microdialysis combined with electrochemiluminescence technology for studying metoclopramide hydrochloride and human serum albumin interactionsRu(bpy)32+electrochemiluminescence sensor combined with microdialysis sampling technology to research on-line metoclopramide hydrochloride and Human Serum Albumin interactions. The experimental results show that, under the optimized experimental conditions, the relative ECL intensity is proportional with metoclopramide hydrochloride concentration in the range of1.0×10-7-1.0×10-5g/ml with a detection limit of3.3×10-8g/ml according to IUPAC recommendations. The regression equation was y=29.0934+3.0996x, where y=ΔI, x=10’c (g/ml), the correlation coefficient R=0.9927. The association constant of compound of metoclopramide hydrochloride and HSA and the number of the binding sites with Scatchard analysis are K=1.24×104(mol/L)-1and n=2.85, respectively.3. Microdialysis sampling electrochemiluminescence sensor for studying hydrochlorothiazide and human serum albumin interactionsCombined with the advanced microdialysis sampling technique, a novel method based on immobilized Ru(bpy)32+electrochemiluminescence sensor was designed to on-line research the interactions between hydrochlorothiazide with human serum albumin. The results show that, under the optimized experimental conditions, the relative intensity is proportional with hydrochlorothiazide concentration in the range of1.0×10-7-1.0×10-5mol/L with a detection limit of3.3×10-8mol/L according to IUPAC recommendations. The linear regression equation is y=30.1296+8.8759x, where y=△I, x=107c (mol/L), the correlation coefficient R=0.9996. The use of new methods hydrochlorothiazide hydrochloride and HSA interaction, including the binding constant and number of binding sites under different temperature conditions and analysis of binding force type with satisfactory results. The experimental results show that the proposed method has potential applications in the pharmacodynamic and pharmacokinetic studies. |
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