Luminol - Fluorescent Dye System Electrochemiluminescence Energy Transfer Detection Of Dna

With the rapid development of molecular biology, it is particularly important that genetic sequence was detected quickly and accurately in the research field of clinical medicine, immunology, and biology. The developments of many new biological technologies can inject new vigor to the development of high sensitivity and high specificity DNA analysis methods. For the DNA detection, the polymerase chain reaction (PCR) and gene chip methods were mainly used. However, due to their complex technology and expensive equipment, the wide application of those DNA detection technologies was greatiy limited. Various DNA detection technologies by DNA base complementary matching principle have received the high attention of biological analysis workers and were developed. Now DNA detection methods used mainly were electrochemical, electrogenerated chemiluminescence (ECL), fluorescence, method, etc.Compared with fluorescence analysis technology, the ECL analysis technology was characterized by simple instruments, high sensitivity and good selectivity, etc. But similar to the fluorescence energy transfer, when a suitable energy acceptor is present, the excited ECL luminophor can act as an energy donor and transfer its energy to energy acceptor, which will make energy acceptor be excited. In this thesis, the ECL energy transfer method for sensing DNA was developed, in which excited luminol acted as energy donor and fluorescent dyes acted as hybridization indictor and energy acceptor. The aim of this thesis is to explore the new DNA detection method with high sensitivity and good selectivity.The thesis included two parts:review and research work. In part one, first, the definition, characteristics, mechanism and systems of ECL were summarized. Second, energy transfer and its classification were introduced. Third, the principles and methods of DNA detection were reviewed. At last, the research purpose and contents of this thesis were described. Part two was research report and the main contents were described as follows:1. A mobilized, label-free DNA sensing system based on the ECL energy transfer mechanism was developed. ECL reaction of luminol was triggered by step pulse or potential scan signal. Luminol molecules in their excited state could transfer their energy to crystal violet (CV) molecules and CV molecules were excited. While returning to ground state, excited CV molecules will release energy, thereby emitting photons. Because of the difference of the binding site and ability of CV between ssDNA and dsDNA, when excited luminol molecules transferred their energy to the complex of CV and DNA, the ECL signal of CV-ssDNA was different from that of CV-dsDNA. Accordingly, the target DNA sequence could be identified and the target DNA concentration could be detected. The prominent advantage of this developed ECL energy transfer method was that photobleaching in fluorescent method and high potential damage to DNA in electrochemical and ECL methods was avoided. The ECL signal of system was increased when the target DNA concentration ranged from1.0×10-11mol/L to1.0×10-8mol/L, and the detection limit was7.0x10-12mol/L. The recognition capability of this system for target DNA was strong, since it could distinguish single base mismatch target DNA.2. The effect of carbon quantum dots on the ECL energy transfer sensing system for the determination of DNA was investigated. The results showed that the signal-to-noise ratio of luminol-CV ECL system was increased and its reproducibility was improved by the addition of carbon quantum dots. The conditions of detection of DNA were optimized. Under optimal conditions, the linear range for the determination of target DNA was1.0×10-11mol/L-1.0×10-7mol/L, the correlation coefficient was0.9881and the detection limit was4.0×10-12mol/L. The possible interaction mechanism between DNA and carbon quantum dots was also discussed. This method was convenient and showed high sensitivity.3. A label-free DNA sensor based on the ECL energy transfer mechanism was developed. Because of different combining ways between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with optical indicator [Ru(bpy)3]2+, their optical behaviors were also different. Excited luminophor, luminol, could transfer the energy selectively to optical indicator Ru(bpy)32+combined to dsDNA, and make it arrive its excited states. Then excited indicator released energy in photon form in the process of going back to the ground state. Thus the ssDNA and dsDNA could be differentiated by the comparison of the ECL intensity of Au-ssDNA and Au-dsDNA system and concentration of target DNA could be detected. In this proposed method, the background signal in the conventional fluorescence analysis was advoided, and long-strain DNA could be detected. Furthermore, the recognition capability of this system for target DNA was strong. Under optimal conditions, the linear range for the determination of target DNA was1.0×10-14mol/L-1.0×10-11mol/L, the correlation coefficient was0.9921and the detection limit was2.0×10-15mol/L. Reliable means and methods were provided for high sensitivity analysis of the DNA complex system.Combining with the DNA hybrid technology, a new ECL energy transfer method for DNA analysis was established and applied to the sequence recognition and content detection of DNA. The sensor is expected to be used for the high sensitivity and high selectivity early diagnosis of gene disease in clinic, for research of DNA target drug and detection of environmental pollutants.