Applications Of DNA Concatamers In Biochemical Detection

Recent years, researchers have found that miRNAs play an important role in regulating gene expression. Acting as oncogenes or tumor suppressors, miRNAs can be used as an effective tumor marker for developing novel therapeutic treatments and clinical prognoses. The lengths of mature miRNAs are short and they represent only a small fraction of the mass of a total RNA sample. So it is necessary to develop a rapid, sensitive and selective method for miRNA detection. In order to detect an ultralow amount of target, signal amplification is an essential way to enhance the sensitivity of DNA biosensor. We developed two DNA biosensors for amplification detection of miRNA with ultralow amount, the two DNA biosensors are based on DNA self-assembly to form long concatamers.In chapter two, the principle is based on the two designed auxiliary probes, auxiliary probe 1 and auxiliary probe 2, which can lead to long-range self-assembly and form micrometer-long DNA concatamers on the electrode surface in the presence of the target sequence. Ru(phen)32+, which acts as ECL probe, can intercalate into the double stranded DNA. After the successful intercalation of plentiful Ru(phen)32+, it generates significantly amplified ECL signals for the detection when using TPA as coreactant. The detection limit of the target miRNA is at a femtomolar level. This amplified sensor shows high selectivity for discriminating complementary target and other mismatched RNAs. The biosensor enables the quantification of the expression of miRNA-21 in MCF-7 cells. It also displays very low limits of detection and provides an alternative approach for the detection of RNA or DNA detection in diagnostics and gene analysis.In chapter three, in view of the construction of the chapter two, we reported a simpler label-free electrochemical biosensor for detection of ultralow amounts of miRNA. [Ru(NH3)6]3+ was selected as a signal reporter, which can bind to negatively charged DNA strands. Numerous redox indicators can be gathered to the working electrode via DNA concatamers and eventually produce an amplified electrochemical signal. This biosensor can detect as low as 100 aM target miRNA-21, with sensitivity similar to that of PCR. The main advantage of our work is that miRNA-21 can be directly detected in human serum without enrichment, which can decrease the operation complexity and increase the detection accuracy.