The Application Of Biological Nanochannel Technology In The Early Diagnosis Of Tumors

Over the past two decades,the nanopore has emerged as an attractive,powerful,and sensitive singlemolecule platform that has been used for a wide range of applications including rapid gene sequencing and the detection of various analytes such as metal ions,PEG polymer,DNA and microRNA,peptides and protein.Recently,it was also shown that the sensing capabilities of solid-state nanopore can be enhanced in combination with DNA origami.A hand-held wireless voltage-clamp amplifier that interfaces a sensing probe of nanopore was constructed to enable a miniaturized wireless platform that can be conveniently operated with a smartphone.In particular,an aerolysin nanopore was shown exhibit impressive advantage in the detection of single base differential DNA,which was attributed to its narrow diameter and the strong electrostatic interactions between the aerolysin and the oligonucleotides.All these advancements indicate that the nanopore technique holds a great potential for wide practical applications.This paper mainly carries out the following two aspects of the work:Firstly,using ?-hemolysin nanopores to differentiate microRNA(miRNA)single base mismatches.The accurate discrimination of miRNAs with highly similar sequences would greatly facilitate the screening and early diagnosis of diseases.In the chapter 2,combining biological nanopore with the modification technique of locked nucleic acid(locked nucleic acid,LNA)to detect mi RNAs at a single molecule level.LNA is a novel nucleic acid analog that can enhance thermal stability and improve sensitivity and specificity.Based on this,LNA-modified oligonucleotide probes were designed to bind to ?-hemolysin nanopores to achieve rapid and sensitive detection of let-7b and to clearly distinguish let-7a and let-7c,whose included 1 or 2 base mismatches.The established method was used for the analysis of let-7b in serum samples,showing good selectivity.Secondly,analysis of human guanine glycosylase(hOGG1)activity using ?-hemolysin nanopores.Human guanine glycosylase(hOGG1)plays an important role in maintaining the integrity of living organisms due to its ability to repair DNA oxidative damage.The expression level of hOGG1 is closely related to many diseases including cancer.In Chapter 3,we established a new method for detecting hOGG1 activity with high sensitivity based on nanopores.The rapid and sensitive detection of hOGG1 was achieved by using ?-hemolysin nanocomposites with double-stranded DNA with damaged oxoG: C base pair as the probe to cover the enzyme activity information was converted into DNA concentration signal.The detection method has good selectivity and is successfully used for the analysis of enzyme activity in tumor cell samples.