Electrochemical DNA Biosensor-Based Nucleic Acids Detection Technology

DNA, deoxyribonucleic acid, is one of the three major macromolecules (along with RNA and proteins) in the body that are essential to all known forms of life. In recent years, there has been an increase in the use of nucleic acids as a tool in the recognition and monitoring of many compounds. Traditional methods for DNA detection are cumbersome and time-consuming. Therefore, it is urgent to establish a rapid, sensitive and simple method for detection of DNA. In order to improve the detection of DNA, the new electrochemical DNA biosensor-based methods have been developed as potential alternatives to break the bottlenecks of the conventional methods, because they have distinct advantages, such as easy to use, rapid response, low cost and inexpensive instrumentation. In our work, two highly sensitive and specific electrochemical biosensors have been developed for detection of DNA, which would become a potential tool for clinical diagnosis, food safety, biothreat detection and environmental monitoring. This dissertation includes the following two parts:1. A novel electrochemical sensing strategy for rapid and ultrasensitive detection of Salmonella by rolling circle amplification and DNA-AuNPs probeA novel electrochemical sensing strategy was developed for ultrasensitive and rapid detection of Salmonella by combining the rolling circle amplification with DNA-AuNPs probe. The target DNA could be specifically captured by probe 1 on the sensing interface. Then the circularization mixture was added to form a typical sandwich structure. In the presence of dNTPs and phi29 DNA polymerase, the RCA was initiated to produce micrometer-long single-strand DNA. Finally, the detection probe (DNA-AuNPs) could recognize RCA product to produce enzymatic electrochemical signal. Under optimal conditions, the calibration curve of synthetic target DNA had good linearity from 10 aM to 10 pM with a detection limit of 6.76 aM (S/N=3). The developed method had been successfully applied to detect Salmonella as low as 6 CFU mL-1 in real milk sample. This proposed strategy showed great potential for clinical diagnosis, food safety and environmental monitoring.2. A new mode for highly sensitive and specific detection of DNA based on exonuclease ?-assisted target recycling amplification and mismatched catalytic hairpin assemblyA novel strategy was developed for highly sensitive and specific detection of DNA by combining exonuclease ?-assisted target recycling amplification with mismatched catalytic hairpin assembly. In this method, the target DNA binded with HO. Then, exonuclease ? could catalyze the stepwise removal of mononucleotides, releasing the target DNA and the output DNA. The released target DNA was free to participate in the next cycle of hybridization and cleavage process (Cycle ?). The output DNA could bind with H1 and unfold the hairpin structure of H1. Following H2 hybridized with the opened H1 and liberated the output DNA, which caused a new catalytic hairpin assembly circuit and the formation of H1-H2 complexes (Cycle ?). Afterwards, the capture probes immobilized on the gold electrode could specifically hybridize with the H1-H2 complexes. The streptavidin-alkaline phosphatase was labeled on the capture probe-H1-H2 complexes by the specific recognition of avidin and biotin, which produced enzymatic electrochemical signal readout. Under optimal conditions, the calibration curve of synthetic target DNA showed good linearity from 100 fM to 5 nM with a detection limit of 92 fM. The developed method had been successfully applied to detect DNA in total DNA of human normal cells and serum samples. This strategy might become a potential alternative approach for DNA detection in the area of clinical diagnosis and therapy, pathogen detection and environmental monitoring in the future.