Optimization Of Fabrication Of Solid-state Nanopore And Reasearch Of Translocation Behaviors Of Rigid Rod-Shaped Virus Through Nanopore

Since 1996 the nanopore detection technology was first proposed,it has attracted the attention of many researchers due to its high sensitivity,high speed,lable-free and single molecule detection.It is precisely because of these advantages,so that the nanopore is considered to have the potential to become a new generation of sequencing technology.In order to achieve the purpose,researchers has done a lot of research to solve the lack of the detection technology ahout control of DNA translocation speed,detection sensitivity and enhance the translocation of DNA shape control.Besides,nanopore also be used to detecte proteins,nanoparticles and polymers due to its single molecule and high sensitivity ability,which shows the potential and value in characterization nanoparticle of nanopore.This thesis focuses on research the dynamic behavior of the translocation of rod-shaped molecules and the the development of nanopore application in the characterization of the virus.Tobacco mosaic virus(TMV)was used as the research object,study on the translocation behavior of rod TMV in solid state nanopores and the salt dependence in the translocation process.The main research contents are as follows:1.Fabrication of solid-state nanoporeBased on the self-supporting silicon nitride chip designed by our lab,the processing parameters of nanopore by Dual Beam Focused Ion Beam(DB-FIB)were studied.The result shown that DB-FIB can fabricate nanopore with diameters range from 25 nm to 65 nm on 100 nm thickness chip in spot modle.In this range,the diameter is proportional to the processing time.It can fabricate more than 65 nm diameter nanopore by presetting the diameter and processing time in circle mode.Similarly,preset diameter and processing time are proportional to the nanopore diameter.2.Translocation of TMV through Solid-State Nanopore at Low Salt ConcentrationOur previous research showed molecular dynamic characteristics of Tobacco Mosaic Virus(TMV)at high salt concentration.Based on these previous results,we explored the translocation of rigid rod-shaped TMV through solid-state nanopore at low salt concentration.In this work,five types of events and a strong current fluctuation during the translocation of TMV were observed.Among these events,three types of events with distinctive characteristics at the capture process were similar to those presented in our previous studies.We observed a novel event with multiple amplitudes which had not been observed in our previous study.Relative data analysis proved that the novel event was generated by several molecules with simultaneous interactions with nanopore.Besides,an approximation formula for conductance change due to rigid rod-shaped TMV translocation was developed to demonstrate that the strong current fluctuation during the translocation of TMV was caused by rotation behaviors of the TMV in the nanopore.The discovery of the novel event shows that the change of physical properties of nanopores and particles would influence the dynamics of particles during translocation,and the approximation formula gives a new method for studying rotation behaviors of particles.3.Salt dependence of TMV translocation through solid-state nanopore at various salt concentrationBased on the research of TMV translocation through solid-state nanopore at high/low salt concentration,the salt dependence of TMV translocation through solid-state nanopore was reaserched.Firstly,the salt dependence of nanopore of similar aperture were reaserched from 1μM to 1M to remove the effect of nanopore to the experiment.Subsequently,we measure translocation of TMV form 1μM to 1M salt concentration.TMV translocation conductance is shown to result in either a decrese when CKCl is bigger than 20 mM or increase when CKCl is less than 20 mM with salt concentration decrease.We measured this change over a range of salt concentrations and determined the transition point where TMV translocation has no net effect on the ionic current.It is concluded that the value of the salt concentration of the TMV translocation current that is increased or decreased with change of salt concentration is 16.108± 1.321 mM Meanwhile,it was shown that the surface charge density of TMV in the salt concentration is(9.056±0.107)×10-9C/nm.