Development Of Condenser System And Electrode Moving System For Electrochemical Luminescence Imaging

In life science, clinical tests and pathological study, the "visual" technology and devices are getting more and more attention, and thus, the various medical image techniques such as X-ray computer tomography (CT), nuclear magnetic resonance (NMR), X-ray fluoroscopic imaging, ultrasound imaging, etc. are developed. These physical image techniques are not enough to meet the needs of modern medical diagnosis since the needed detection systems are very complex and related to the chemical reactions. Chemical imaging techniques such as fluorescence imaging and chemiluminescence imaging have played an important part in the field of life science because they have high sensitivity and high selectivity. However, fluorescence imaging biosensing for the detection of proteins is still interfered by the background fluorescence of the proteins and the reproducibility of chemiluminescence imaging biosensing is limited by the mixing the trigger regents with the detection analytes. Electrogenerated chemiluminescence (ECL) is a method of generating light by using electrochemical reactions to produce highly reactive species at the surface of an electrode that can produce excited states in energetic electron transfer reactions. It combines both advantages of electrochemistry and chemiluminescence. ECL is an imaging technique based on ECL reactions, not only can provide electrochemical signals and luminous intensity, but also take optical image. Therefore, the development of ECL imaging analyzer is a research subject with important scientific significance and practical value.Highly sensitive methods for the determination of gene are playing an increasingly important role in the diagnosis of the genetic diseases and the evaluation of treatment effect. ECL gene biosensor has received an increasingly attractive attention due to its high sensitivity. Since it is simple and can keep high hybridization efficiency of recognition gene, label-free ECL gene biosensor has become an important study direction.The purpose of this thesis is to develop a highly sensitive and label-free ECL method for the detection of HIV-1gene based on a supersandwich DNA structure and Ru(phen)32+as an intercalator, design and set up the light gathering system and mobile system of the elctrodes of ECL imaging analyzer. The research work in this thesis is financially supported by the National Natural Science Foundation of China (Grant No.21027007and No.21275095).This thesis includes two parts. First part, Chapter1, is a general introduction, and the second part, Chapter2to Chapter4, is a research report.In Chapter1, the principles and characteristics of ECL, the signal amplification strategy of ECL biosensors based on supersandwich model, the principles, research development, instruments and applications of ECL imaging system are overall introduced. The purpose of this research work is also presented.In chapter2, a highly sensitive and label-free ECL method for the determination of human immunodeficiency virus-1(HIV-1) gene was developed. The thiolated ss-DNA as a capture probe is firstly self-assembled on the surface of a gold electrode. After the target HIV-1gene is completely hybridized with the capture probe, previously hybridized two auxiliary probes are hybridized with the target HIV-1gene to form long-range supersandwich ds-DNA polymers on the surface of the electrode. Finally, Ru(phen)32+is intercalated into the supersandwich ds-DNA grooves, resulted in greatly increasing ECL intensity in tripropylamine solution since a great amount of Ru(phen)32+are intercalated into supersandwich ds-DNA. The results showed that the increased ECL intensity was directly related to the logarithm of the concentration of HIV-1gene in the range from10×10-13to1.0×10-10mol·L-1with a detection limit of2.2×10-14mol·L The perfect-matched HIV-1gene can be effectively discriminated from the two-base mismatched HIV-1gene. This work demonstrates that the high sensitivity and selectivity of an ECL DNA biosensing method could be greatly improved using supersandwich ds-DNA and ECL indicators.In Chapter3, the overall design of ECL imaging analyzer was introduced, and the principle of ECL imaging system was particularly discussed, especially the light gathering system. The relationship among work distance, amplification factor and the field of view was optimized, and3mm was chosen as the field of view in the subsequent experiments. This system was used in ECL imaging analysis of bipolar electrode, and the detection limit of Ru(bpy)32+in TPrA solution on gold bipolar electrode was1×10-8mol·L-1. Meanwhile this system was applied preliminarily in the imaging of biosensor on bipolar electrode. The results showed that the ECL imaging analyzer had a reasonable design and reliable performance so that they will be used in the characterization and analysis of cells in vivo. In Chapter4, on the basis of the introduction of the hardware above, the design and manufacture procedures of electrode array, and the design of stepping motor-driven stage system were introduced complementally; the operations of ECL imaging analyzer were particularly introduced, and some tests were done with this prototype. The finished ECL imaging analyzer has reached the goal which can be used not only for the simultaneous quantitative determination of the EC and ECL intensity of SPCE, but also for the ECL imaging of SPCE and gold BPEs.In Chapter5, the prospect of ECL biosensor was simply presented. Meanwhile, some problems and something which need to be improved were particularly described.