Novel Biosensor Strategies Based On Target-triggerd DNA Nanoassembly For Nucleic Acid Detection

Nucleic acid detection promises rapid,sensitive and specific diagnosis of infections,inherited and genetic disease.Point-of-care nucleic acid testing is a new technique that is performed beside the patient without need for complex laboratory operations,owning to its simple operation,rapid response,low cost and portable.Among majority of clinical samples with complex biochemical nature and the low abundance of nucleic acid target,the unique functionalities and capabilities of nanotechnology have received a great deal of attention.This research has been combined nanotechnology and isothermal amplification techniques for ultrasensitive nucleic acids detection.This thesis contains the following two steps:1 A facile and pragmatic electrochemical biosensing strategy for ultrasensitive detection of DNA in real sample based on defective T junction induced transcription amplificationA novel and pragmatic electrochemical sensing strategy was developed for ultrasensitive and specific detection of nucleic acids by combining with defective T junction induced transcription amplification(DTITA).The homogeneous recognition and specific binding of target DNA with a pair of designed probes formed a defective T junction,further triggered primer extension reaction and in vitro transcription amplification to produce numerous single-stranded RNA.These RNA products of DTITA could hybridize with the biotinylated detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the surface of the biosensor.The proposed isothermal DTITA strategy displayed remarkable signal amplification performance and reproducibility.The electrochemical DNA biosensor showed very high sensitivity for target DNA with a low detection limit of 0.4 f M(240 molecules of the synthetic DNA),and can directly detect target pathogenic gene of Group B Streptococci(GBS)from as low as 400 copies of genomic DNA.Moreover,the established biosensor was successfully verified for directly identifying GBS in clinical samples.This proposed strategy presented a simple and pragmatic platform toward ultrasensitive and handy nucleic acids detection,and would become a potential tool for general application in point-of-care setting.2.Target-triggered DNA nanoassembly on quantum dots and DNAzyme-modulated double quenching for ultrasensitive micro RNA biosensingHerein,a simple and novel fluorescence biosensing strategy has been developed for ultrasensitive determination of micro RNA(Mi RNA)by combining target-triggered DNA nanoassembly on quantum dots(QDs)with DNAzyme-modulated double quenching of QDs.In presence of Mi RNA target,the target triggered catalytic hairpin assembly(CHA)amplification and powered highly efficient DNA nanoassembly on the surface of QDs,leading to exhibition of numerous G-quadruplexes close to the QDs.The G-quadruplex folded properly and bound hemin to form a stable G-quadruplex/hemin complex.Then the luminescence of QDs was quenched via photoinduced electron transfer by hemin associated with the particles and the electron acceptor of O2 which was in situ generated with the horseradish peroxidase-mimicked G-quadruplex/hemin DNAzymes toward H2O2.Based on this target-triggered highly efficient DNA nanoassembly and DNAzyme-modulated double quenching mechanism,the proposed biosensing strategy showed admirable signal amplification capability.Using Mi RNA-21 as model analyte,the designed nanosensor could detect Mi RNA down to 37 f M with a wide linear detection range of 1 × 10-13 M to 1.0 × 10-8 M,and exhibited good selectivity,acceptable reproducibility and low matrix effect.This proposed strategy presented a simple,powerful platform toward ultrasensitive Mi RNA detection and had great potential for bioanalysis and clinic diagnostic application.