Fluorimetric Bioprobe Based on DNA-dye Intercalation - Application in Enviromental and Clinical Monitoring
Abstract: Nucleic acids have many unique structural features that offer reliable means of recognizing and monitoring many compounds of medical or environmental importance. In recent years, there has been an enormous increase in the use of nucleic acids as a tool to determine the chemical compounds of environmental and clinical interest and the major research activities in these areas have been directed towards the design of sequence-selective biosensors based on hybridization. In this thesis, a novel bioassay method for detection of various substances with affinity for oligonucleotides including toxic aromatic compounds, metal ions, antibiotics and anticancer agents is described. The method is based on fluorimetric detection wherein the long wavelength fluorescent intercalating dyes TO-PRO-3® or TOTO-3® and dsDNA form a complex. The presence of competing analytes disrupts this DNA-dye complex resulting in decreased fluorescence intensity. The analytes are quantitated indirectly by measuring the changes in the fluorescence intensity. A broad range of analytes like toxic aromatic compounds, metal ions, anticancer agents and antibiotics have been examined and quantified using this method. For example nano-molar concentrations of antibiotics like sulfathiazole and chloramphenicol could be detected; wide concentrations of clinically important anticancer agents, daunomycin and doxorubicin, were quantitated with good linearity. Several home made fluorescence detection systems have been developed and used. They were based on optical fibers and different detection units like PMT or CCD camera. The assay throughput has been improved by the novel design of the detection system based on CCD camera. Different sensitivities and detection ranges have been achieved by choosing different combinations of nucleic acids and analytes. This rapid and simple method has the potential for high throughput detection of environmental and clinical analytes.
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