Research On The Translocation Of Free DNA And DNA Bound To Magnetic Beads In Nanopores

Gene sequencing technology based on nanopores is considered to be the most competitive solution of third-generation gene sequencing technology and it is expected to achieve rapid and low-cost gene sequencing.Compared with biological nanopores,solid-state nanopores are not only controllable in pore size,but also highly stable,and are more suitable for the detection of DNA molecules.At present,the main challenge about solid-state nanopore sequencing is that the translocation rate of DNA molecules driven by electric field force is too fast to obtain effective data points during the detection process and identify the subtle structural features of DNA molecules.To explore the translocation of DNA molecules in solid-state nanopores,nanopore preparation and biomolecular detection experimental platforms were built,silicon nitride and graphene nanopores were successfully prepared.Not only the morphology and regularity of DNA molecule translocation nanopore were explored,but also the effects of bias voltage,DNA molecule concentration and nanopore diameter on the signal perforation of DNA molecules were studied.In addition,binding of magnetic particles coated with streptavidin to biotinylated DNA molecules to slow down DNA molecules.The main research work and results are as follows:1)Etching silicon nitride nanopores by focused ion beam;obtaining single-layer graphene samples by mechanical stripping,transferring graphene films to silicon nitride microporous chips by transfer table,and preparing graphene nanopores by focused ion beam.2)Exploring the transport properties of electrolyte solutions at nanoscale and the physical effects of DNA molecules in nanopores,mainly studying the electric double layer effect around the DNA molecules and the walls of solid nanopores;the "electric phenomena" occurs when DNA molecules pass through nanopores,including electroosmosis,electrophoresis,flow potential and sedimentation potential.3)Comparative analysis of the translocation signals of DNA molecules translocated silicon nitride nanopores and graphene nanopores under the same experimental conditions,the experimental data are basically coincident.According to the experimental results,the DNA molecules were translocated with signals of about 30 nm silicon nitride nanopores,and it was found that the DNA molecules were translocated with six straight folds and a straight posture.4)In-depth study of the effects of bias voltage,concentration of DNA molecules and nanopore diameter on the signaling of DNA molecules were analyzed,and the law of DNA molecular translocation of silicon nitride nanopores was studied.According to experimental results,with bias voltage increasing,the DNA molecule translocation rate is increased,the signal-to-noise ratio is increased,the blocking current amplitude and the capture rate are linearly increased.As the concentration of DNA molecules increases,the molecular molecule perforation duration and blocking current amplitude increase,and the capture rate increases linearly.As the pore size increases,the translocation rate of DNA molecules increases,and the signal-to-noise ratio decreases.5)Experiments on the translocation of DNA molecules bound to magnetic beads,including the mechanism and step of binding DNA molecules to magnetic beads;experimental studies on the interaction between magnetic beads,biotinylated DNA molecules through nanopores and streptavidin through nanopores to determine the via signals of bound magnetic beads;comparative analysis via signals of free DNA molecules and DNA molecules bound to magnetic beads,according to the experimental results,the amplitude of the blocking current of the DNA molecule bound to the magnetic beads is increased,because 2 to 3 DNA molecules are bound to each streptavidin;the duration of the via can be reduced by two orders of magnitude.