Investigation On The Biosensing Application Of Carbon Nanotubes And DNA Ligase Reaction

With the progress of science and technology, detection of specific sequence of the DNA, and the small molecule and enzymes have been rapidly developed, the gene diseases’mechanism of diagnosis and treatment is gradually uncovered. Due to the special mechanics, electricity and the superiorities, carbon nanotubes have been widely applied to the many other aspects of the chemical, sensing, biomedical and other fields. The molecular recognition platform is available for it can be bound with the nucleic acid, producing and enlarging detection signal. The binding abilities between carbon nanotubes and the single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) are different, and the capability will lead the carbon nanotubes to disperse or not. At the same time, the E.coli DNA ligase controls the dsDNA formed, it can catalyse DNA chains’end of the phosphoric acid and3’ in a hydroxyl end in order to combines with the template DNA complementarity. Accordingly, connection reaction with carbon nanotubes binding will improve the ability to identify the molecule and construction of the new signal output platform.This thesis studies the nucleic acid molecules and carbon nanotubes non-covalent interactions, combination of connected action and carbon nanotube, adoption of the method of time-resolved luminescent measurement and fluorescence anisotropy detection to carried out the highly sensitive and selective of the fluorescence detection of nucleic acids and bio-enzyme, less prone to the light bleaching, not subject to scattered light interference, suitable for in the the the biology of detection of the complex environment. The contents of the study are as follows:1. Lable free and time-resolved luminescent assay of SNP in complex environment:coupling carbon nanotubes quenching-Eu3+complex with DNA Ligase Reaction. We have developed a new approach for label-free fluorescence detection of single nucleotide polymorphism (SNPs) with high sensitivity and fidelity. The design exploits the fluorescence quenching of single-walled carbon nanotubes (SWNTs) for Eu3+/BHHT complex and the difference in noncovalent interactions of the SWNTs with ssDNA and dsDNA. In the assay, two half primer DNA, one being phosphorylated, were first incubated at40℃with a target DNA template. In the presence of DNA ligase, the two captured ssDNA were linked for the perfectly matched DNA target to form a stable duplex, but the duplex could not be formed by the single-base mismatched DNA template. After addition of Eu3+/BHHT and SWNTs, the fluorescence of Eu3+/BHHT will be efficiently quenched unless the perfectly matched DNA target is present.Time-resolved luminescent measurement is4-fold higher than that using steady-state luminescence measurement, demonstrating the efficiency of the approach for direct quantification of SNP in complex biological fluids. This high sensitivity and fidelity for SNP discrimination would provide good groundwork for the future DNA diagnosis.2. Based on the the fluorescence anisotropy and carbon nanotubes (SWNTs) to detection ligase activity. E.coli DNA ligase catalytic the two ssDNA and template DNA to form dsDNA, and enzyme activity is proportional to the dsDNA numbles, SYBR Green I(SG I)selectively imbedded in the dsDNA, but the dsDNA can not be adsorbed on adding carbon nanotubes, after centrifugation, the supernatant liquid was measured lower fluorescence anisotropy value. On the other hand, the probe ssDNA and SG I will be adsorbed to the surface of carbon nanotubes without the E.coli DNA ligase in the system, the molecular of carbon nanotubes adsorbed DNA and SG I weight larger after centrifugation, lead to higher fluorescence anisotropy value.The value of the fluorescence anisotropy reprensent the activity of the E.coli DNA ligase. This method is a homogeneous detection method, high sensitivity, can reduce the interference of the scattered light, activity detection for complex biological environment.