Exploring The Current Pulses Characteristics Of SsDNA And Oligopeptides On Single Aerolysin Nanopore Sensing Interface

Nanopore single-molecule detection technology was developed from the Coulter counter.It mainly detectes and analyzes the blocking current signals formed by small charged molecules passing through the nanopore under the driving force of voltage.Nanopore detection technology has the advantages of no marking,convenient operation,low cost,and high throughput.Now it has been applied in many fields such as DNA sequencing,nucleic acid conformation detection,small biological molecule detection and polypeptide protein detection.It is of great significance to extend nanopore technology to the field of diagnosis and treatment of biomedical diseases.In the first chapter,the principle and origin of nanopore single molecule detection technology were emphatically explained.And the types of bio-nanopore and solid-state nanopore and the preparation methods of solid-state nanopore were introduced in detail.In addition,the application of nanopore detection technology was extensively and comprehensively discussed.Finally,it summarized the research significance and specific content of this topic.In the second chapter,based on the principle of inducing E.coli to express the target protein,the aerolysin protein was prepared using bioengineering technology.The experiment mainly included the steps of plasmid introduction,coating culture,single colony culture,inducing expression,bacteria fragmentation,protein purification,SDS-PAGE electrophoresis,and trypsin treatment.It successfully prepared aerolysin protein with good activity.In the third chapter,based on the aerolysin nanopore sensing interface,the characteristic pulse signals of ssDNA with different chain lengths under the same voltage were discussed.The effect of applied voltage on the characteristics pulse of the ssDNA(including signal frequency,the relative blocking current and dwell time)was also studied.Within the range,as the length of the nucleotide chain increased,its blocking time and relative blocking current increased accordingly.When the voltage increased,the signal frequency of ssDNA increased almost linearly,and its dwell time and relative blocking current decreased to varying degrees.The results of this experiment provide a basis for the more accurate detection of single-stranded nucleotides by nanopore detection technology.In the fourth chapter,the current pulse characteristics of HIV-1 tat Protein(1-9)peptide(MDPVDPNIE)and related designed peptides were explored on the aerolysin nanopore interface.Experiments found that the pulse signal formed by oligopeptide translocation was affected by many factors such as chain length,three-dimensional structure and number of charges.The HIV-1 tat Protein plays a very important role in the transcription of the virus and is of great significance for the treatment of HIV-1.The experiment demonstrates the application prospect of nanopore sensing technology in detecting protein variation.