Nanopore Single-Channel Technology For Determination Of Host-Guest Interactions And Detection Of Proteases

Nanopore sensing is a novel single-molecule technology,whose principle is to detect the ionic current signals generated by the analyte passing through the nanopore.After three decades of rapid development,this technology has been widely used in nanopore DNA sequencing,biomacromolecule detection,metal ion detection,and single-molecule chemistry study,etc.As the most extensively studied substance in nanopore research,DNA could generate various types of current signals when translocated through a nanopore,depending on the modification and molecular conformation of DNA.In this thesis,by combining nanopore sensing technology and chemical modification we constructed novel DNA host-guest complex probe and established a new method for the determination of host-guest interactions and the detection of proteases.The main contents and results are as follows.1.Measuring binding constants of cucurbituril-based host-guest interactions at the single-molecule level with nanopores.Different guest compounds(Fc,Ad,KGW,KGY,KGF,KGamF,RG,and KCitV)are covalently attached to the side-chain of DNA molecules by chemical modification.After the addition of cucurbit[7/6]uril,the host-guest DNA hybrids were formed.These DNA hybrids were translocated through aHL nanopore to generate highly characteristic current signature events.After statistical analyses of the events,we could derive the binding constants of the host-guest hybrids through association rate constant and dissociation rate constant of the single-molecule interactions.Compared with traditional methods,this approach can not only measure host-guest interactions at the single molecule level,but also avoid the solubility limitation of the solvent system,which has great potentials for studying other macrocycle-based host-guest interactions.2.Detection of leucine aminopeptidase based on DNA-peptide hybrid probe at the single-molecule level with nanopores.The modified peptide(Nterm-KGFL-Cterm)is covalently attached to the 5' end of the single-stranded DNA molecules to construct the probe DNA-KGFL.When leucine aminopeptidase is present,it excises the leucine moiety in the DNA probe,and the exposed N-terminal phenylalanine has strong interactions with cucurbit[7]uril(CB[7]).After the addition of CB[7],a host-guest hybrid DNA-KGFCCB[7]is formed,which generates highly characteristic current signal when translocated through?HL nanopore.Characteristic current signals could be used to indicate the presence of leucine aminopeptidase and the event frequency could be used to quantify the concentrations of the leucine aminopeptidase.The method is highly sensitive and the detection limit reaches 10-5 U/L,which is among the lowest values reported so far.3.Detection of cathepsin B based on DNA-peptide hybrid probe at the single-molecule level with nanopores.We constructed a DNA probe DNA-KCitVGF which contains a cleavage site for cathepsin B.After the N-terminal phenylalanine in the probe DNA-KCitVGF binds with CB[7],a host-guest hybrid DNA-KGF(?)CB[7]is formed,which generates highly characteristic current signal when translocated through aHL nanopore.When the probe DNA-KCitVGF incubated with cathepsin B,DNA-K was released which could not form the host-guest complex with CB[7].The characteristic signal frequency decreases as the enzyme digests the probe and the signal changes could be used to quantify the concentrations of cathepsin B.The detection limit for cathepsin B is 10-3 U/L,better than most of the currently reported methods.