Study On The Conformational Transformation Of Polymers By Nanopore Sensors

Nanopore sensing is a novel single-molecule detection technology.During the past two decades,nanopores have gained prominent progress in a wide variety of research areas including nanopore DNA sequencing,stochastic sensing,single-molecule chemistry,protein unfolding,and early disease diagnosis,etc.In principle,nanopores are suitable for studying synthetic polyelectrolytes because they can also translocate through nanopores like DNA molecules.However,due to the polydiversity of molecular mass and complicated sidechain structures of polyelectrolytes,only moderate successes have been achieved with nanopore analysis of polyelectrolytes.In this thesis,we use single-channel recordings to study the conformational changes of sodium polystyrene sulfonate(PSSNa)when traversing a-hemolysin(a-HL)under different conditions including pH,salt concentrations and different counterions.Besides,based on the bridging effects between Dy3+or Y3+and DNA,and interactions between the trivalent ions and a-HL,we could improve the sensing of oligonucleotides down to two-nucleotide resolution.The main contents and results are as follows.(1)The effect of monovalent counterions on the conformation of PSSNa was investigated in KCl but under two different pH conditions(pH 7.5 and 5.0).Translocation of PSSNa through a-HL is mediated by both electrophoretic and electroosmotic force.When in low salt concentration at pH 7.5,a-HL is only slightly anion-selective and the electroosmotic force is relatively weak.PSSNa adopts "swollen random coil" conformation with large hydration radius and is difficult to enter the vestibule of a-HL.Therefore,the events are mainly Type A.When the salt concentration is increased,the conformation of PSSNa changes to "random coil" with smaller hydration radius.Thus,PSSNa enters the vestibule and then translocates through a-HL after a transient lingering.At pH 5.0,the translocation events are mostly Type B,because under this condition,a-HL is more anion-selective and exhibits strong electroosmotic force.PSSNa can directly pass through the nanopore to give Type B events.When the salt concentration is increased,PSSNa translocation event changes from Type B to Type AB,because PSSNa is more compact in high salt concentration due to the shielding effects,making it difficult to pass through the nanopore.(2)Under the same salt concentration,PSSNa translocation events change from Type B to Type AB when pH is increased.This is because the internal charges of a-HL gradually reduced when pH is increased,and the energy barrier for the translocation of PSSNa through nanopore is gradually increased.(3)The effect of multivalent counterions on the conformation of the negatively charged polymer was investigated.Trivalent metal ions can not only change the conformation of PSSNa by bridging events,but also change the internal charges of a-HL by coordination.The presence of Dy3 or Y3+ can increase the current blockage,dwell time and event frequency of PSSNa translation.Following this trend,we also studied the effect of trivalent ions on the sensing capacity of oligonucleotides by ?-HL.Our data show that the sensitivity of oligonucleotides recognition by ?-HL is improved to the resolution of two nucleotides.This can be used to distinguish different lengths of oligonucleotides.The use of nanopore sensing technology to study the polyelectrolyte conformations can make a nice complement to the traditional methods.The combination of multivalent counterions with ?-HL can improve the resolution of DNA oligonucleotide sensing,which may have broad application for other polymers and molecules.