DNA Biosensors Based On X-shape DNA Probe And Triplex-AgPtNCs For The Detection Of Single Nucleotide Variant

With the completion of human genome sequencing,the analysis of the diversity of the human genome has become the focus of the study.Single nucleotide variant(SNV)is the most common form of genetic variation,which is highly conserved,widely distributed in the genome,can be used as a new generation of high resolution valuable genetic markers and indicators,and many diseases,such as genetic diseases and cancer are closely related,so it has of great significance in clinical diagnosis and prognosis.Therefore,it is urgent to develop new analytical techniques to achieve high sensitivity detection of SNV in order to improve the accuracy of diagnosis of genetic diseases and tumors.Inspired by the research of nano medical technology,this paper combines the X-shape DNA probe and triplex silver platinum bimetallic nanoclusters(Triplex-AgPtNCs),to design the signal amplification methods of DNA fluorescence and electrochemical biosensor for SNV detection with high sensitivity and high specificity.In chapter 1,this chapter combined the universal X-shape DNA probe,locked nucleic acid(LNA)and Toehold-mediated strand displacement reaction(TMSDR)cycle amplification technology to design an enzyme-free fluorescencebiosensors for beta thalassemia(β-Thal)SNV detection.First of all,X-shape DNA probe with LNA modified has high recognition ability for SNV sequence,even in a large number of wild type sequences,which can detect 0.01% SNV mutation frequency.Secondly,TMSDR amplification system can significantly improve the sensitivity of the sensor;this method can detect the minimum of 6 fmol/L of SNV.In addition,X-shape DNA probe with the functional structure of oligonucleotide probes can bind to specific target areas and has universal application.Therefore,this biosensor will be aneffective method for detection of beta thalassemia or other related diseasesgene detection.In chapter 2,we developed a facile one-step method to synthesize triplex DNA-templated silver platinum bimetallic nanoclusters(Triplex-AgPtNCs).The obtained Triplex-AgPtNCs had good size distribution(about 2 nm in diameter)and showed highly-efficient peroxidase-like catalyze activity,which can catalyze hydrogen peroxide(H2O2)-mediated oxidation of the substrate,3,3’,5,5’-tetramethylbenzidine(TMB).Then we measured the mimic enzyme’s structure and kinetic parameters by the transmission electron microscopy,ultraviolet visible spectrophotometry and fluorescence spectrum methods.The results showed that the Triplex-AgPtNCs have strong peroxide enzyme mimic activity.In addition,this Triplex-AgPtNCs have some advantages,such as simple synthesis,low cost,good stability,easy labeled and so on.They also expected to be a new type of enzyme simulation application in the field of colorimetric analysis and biosensors.In the third chapter,as the prexious chapter,we combined the X-shape DNA probe and Triplex-Ag PtNCs to design a DNA electrochemical biosensor for the detection of SNV.In order to apply the catalytic performance of Triplex-AgPtNCs to electrochemical biosensors,the universal X-shape DNA probe is assembled to the gold electrode surface by Au-S bond.In the presence of target sequence,the universal X-shape DNA probe were decomposed into two parts via the toehold-mediated strand displacementreaction,resulted in the generation of two complex.The one was leave from the surface of GE,with the hybridization of target sequence.Another part stand on the surface of GE linked with Triplex-AgPtNCs.Then the modified GE was immersed into a solution containing the MB for a period of time,and then placed in H2O2 solution to scan the SWV curves.Under the experimental condition,the sensor displayed a good linear relationship in the range of 1 fmol/L~ 10 nmol/L with a detection limit as low as 0.8 fmol/L,and exhibited high discrimination ability for SNV.All the above properties make it a promising candidate for the beta thalassemia or other related diseasesgene detection.