Study On New DNA Electrochemical Biosensing Methods By Nuclease-based Signal Amplification

The simple and sensitive detection of trace amount of sequence specific DNA is of vitally importance in the field of application in the clinic, forensic analysis, and pharmacy. DNA electrochemical biosensor has attracted wide attention due to lots of advantages such as simple, low cost, high sensitivity and ease to be miniaturized. In vitro amplification techniques of DNA are commonly used in molecular biology research. DNA electrochemical biosensor takes advantages of these new nucleic acid amplification technologies, and the highly sensitive electrochemical measurement to realize the detection of trace target DNA. In recent years, nanotechnology has gradually entered the field of electroanalysis and biosensors, and led to a breakthrough. Nanomaterials have high surface area, unique electronic conductivity and catalytic properties, thus, can greatly improve the sensitivity, reproducibility and selectivity of the DNA electrochemical biosensor. This thesis focus on the nuclease-based signal amplification methods by combining with functional nanomaterials and hybridization chain reaction to develop DNA electrochemical sensing methods:1. A competitive strategy coupled with endonuclease-assistant target recycling for DNA detection using silver nanoparticles-tagged carbon nanosphere as labelA simple competitive strategy was designed for sensitive detection of sequence-specific DNA by combining endonuclease-assistant target recycling and electrochemical stripping analysis of silver nanoparticles (Ag NPs). The Ag NPs-tagged carbon nanosphere was synthesized via In situ reduction of Ag+adsorbed on negatively charged polyelectrolyte layer and functionalized with streptavidin for binding biotin labeled DNA strand. The labeled strand was captured on DNA sensor surface by competitive hybridization of biotinated primer and its cleaved product. The cleaved product could be amplified in homogeneous solution by endonuclease-assistant target recycling with a Y-shaped junction DNA structure, leading to the correlation of the stripping signal to target concentration. The functionalized nanosphere was characterized with X-ray photoelectron spectroscopy, scanning electron microscopy, and transmission electron microscopy. The proposed method showed a linear range from1×10-16to1×10-12M with a limit of detection of0.07fM (3σ) and good selectivity for base discrimination. The designed strategy provided a sensitive tool for DNA analysis and could be widely applied in bioanalysis and biomedicine.2. Label-free electrochemical DNA sensing with a one-target-multitriggered hybridization chain reaction strategyA one-target-multitriggered hybridization chain reaction (MHCR) strategy was designed for ultrasensitive electrochemical detection of DNA by combining the isothermal strand-displacement polymerase reaction (ISDPR) with DNA self-assembly on a DNA sensor surface. The sensor was constructed by immobilizing hairpin-liked capture probe (CP) on a gold electrode via Au-S bond. The ISDPR was triggered by the hybridization of target DNA to open the CP and primer to anneal the complementary part in the bottom of exposed stem and the extension of the primer in the presence of dNTPs and polymerase. Each target copy could produce a few opened CPs. Afterwards, other part of the exposed stem acted as an initiator to trigger the hybridization chain reaction (HCR) when incubation with two hairpin monomers. Using [Ru(NH3)6]3+as an electrochemically active indicator to interact with the MHCR product, the amperometric response demonstrated a perfect multiplied amplification effect, which showed a high sensitivity for detection of the target DNA with a linear range from0.1fM to10pM, a detection limit down to0.02fM (3σ) and good selectivity for base discrimination. This method did not need any modification or labeling process. The proposed strategy provides a powerful tool for cascade signal amplification and has the wide potential applications in bioanalysis.