Novel Electrochemical Strategies Based On Isothermal Amplification For Nucleic Acids Detection

Ultrasensitive detection of nucleic acids has attracted considerableinterest worldwide owing to the potential applications in different fields suchas pathogen analysis, molecular diagnostics, early screening of cancers,environmental monitoring as well as forensic analysis. Among the differentmethods developed for the ultrasensitive analysis of nucleic acids,isothermal amplification-based strategies without using thermal cyclingprocedures have received a great deal of attention. Electrochemicalbiosensor has been widely used in biomolecular analysis and quantitativedetection due to its simple operation, rapid response, low cost and especiallysuitability for miniaturization. This research has been performed based onelectrochemical biosensor by integrating several isothermal amplificationtechniques for ultrasensitive nucleic acids detection. This dissertationcontains the following two parts:1. A Simple and Highly Sensitive Electrochemical Biosensor formicroRNA Detection Using Target-Assisted Isothermal ExponentialAmplification ReactionA simple and highly sensitive electrochemical biosensor for microRNA(miRNA) detection was successfully developed by integrating atarget-assisted isothermal exponential amplification reaction (EXPAR) withenzyme-amplified electrochemical readout. The binding of target miRNA with the immobilized linear DNA template generated a part duplex andtriggered primer extension reaction to form a double-stranded DNA. Thenone of the DNA strands was cleaved by nicking endonuclease and extendedagain. The short fragments with the same sequence as the target miRNAexcept for the replacement of uridines and ribonucleotides with thyminesand deoxyribonucleotides could be displaced and released. Hybridization ofthese released DNA fragments with other amplification templates and theirextension on the templates led to target exponential amplification.Integrating with enzyme-amplified electrochemical readout, theelectrochemical signal decreases with the increasing target miRNAconcentration. The method could detect miRNA down to98.9fM with alinear range from100fM to10nM. The specificity of the method allowedsingle-nucleotide difference between miRNA family members to bediscriminated. The established biosensor displayed excellent analyticalperformance toward miRNA detection and might present a powerful andconvenient tool for biomedical research and clinic diagnostic application.This dissertation contains the following two parts:2. A novel electrochemical biosensor for nucleic acids detectionbased on homogeneous target-binding-initiated transcriptionamplification in situBy combining RNA in vitro transcription with DNA isothermalamplification in homogeneous system and enzyme-amplifiedelectrochemical readout in heterogeneous phase, the present workdeveloped an ultrasensitive nucleic acids detection method. In the presenceof target DNA, the hairpin probe changed its conformation to formdouble-stranded DNA. While in the presence of KF polymerase, dNTPsand primer，cyclic target-induced primer extension was performed. The extended double-stranded DNA contained the T7promoter of T7RNApolymerase, which could lead to the transcription amplification reaction.The amplification RNA products were hybrided with capture probeself-assembled on the sensor surface and biotinylated detection probe.Under the optimal conditions, the DPV signal was linear with the logarithmof target DNA concentrations in the range of1fM-100pM. Meanwhile, thenovel electrochemical sensor could quantitatively detect salmonella from10to107CFU/mL.