| Nanopipettes have unique advantages such as easy fabrication,low cost,controllable pore size,and easily modifiable surface properties,which make them widely used in analytical chemistry and biological cytology.Nanopipettes provide a confined space capable of revealing the dynamic behavior of individual molecules or individual particles.The glass nanopores were fabricated with nanoscale tips by a laser puller,and modified the inner surface of the tips to obtain single-molecule and single-particle sensors with high selectivity and high sensitivity.Based on this,this thesis mainly explores the sensing application of functionalized glass nanopipettes.The main work includes the following three aspects:(1)Metal-organic frameworks(MOFs)can improve the stability and activity of enzymes encapsulated in them.However,it remains a challenge to explore the effects of the MOF environment on enzymatic activity.Nanocrystalline particles of horseradish peroxidase encapsulated in MOF(HRP@MOF)were modified inside glass nanopores to study the catalytic activity and stability of HRP in the MOF environment.The HRP is encapsulated in zeolitic imidazolate framework-90(ZIF-90)and zeolitic imidazolate framework-8(ZIF-8),and modified in the inner wall of nanopore to construct a catalytic platform for the reaction of ABTS and H2O2.The ABTS molecules are oxidized by HRP encapsulated by ZIF-90 to generate ABTS+,and the change of transmembrane ion current will be monitored in real time.With the increase of H2O2 concentration,the amount of produced ABTS+will increase,thus increasing the nanopore conductance,and the ionic current will also increase accordingly.The effects of MOF structure on enzyme activity and stability are also investigated.The HRP encapsulated in MOF and modified inside nanopore provides a novel and unlabeled design for studying enzymatic catalysis in confined environment,which design has attracted more and more attention in the fields of biochemistry,electrocatalysis.(2)Metal-organic frameworks(MOFs)have unique structural properties,such as high porosity,pore size of angstroms.We developed UiO-66-(NH-SAG)2 modified nanopores to achieve ultra-selective proton transport,In situ growth of UiO-66 and UiO-66-(NH2)2 nanocrystal particles at the orifice of glass nanopores,the ionic currents of functionalized nanopores in LiCl and HCl solution were detected by controlling the monovalent anions.Compared with UiO-66 modified nanopores,the nanopore modified by UiO-66-(NH2)2 can improve the proton selectivity.This is because the pore size of UiO-66-(NH2)2 is smaller than that of Li+,which hinders the transport of lithium ions.When UiO-66-(NH2)2 is further modified with sulfoacetic acid,then successfully prepare UiO-66-(NH-SAG)2 modified nanopore.Li+can hardly pass through,and the sulfonic acid groups,which act as the jumping point of protons,and can promote the transport of protons,thereby achieving ultrahigh selectivity to protons.This provided a new way to achieve ion separation and ionselective transport.(3)The aptamer-functionalized nanopore was constructed as a sensing platform,the kinetics analysis of carcinoembryonic antigen(CEA)and its aptamers were carried by resistive pulse technology.The aptamer-modified nanopore was used to detect a single CEA molecule,and the aptamer immobilized on the inner surface of the nanopore captures the CEA molecule,and the captured CEA molecule interacts with the aptamer,that is,the process of association and dissociation.The translocation of CEA molecules in aptamerfunctionalized nanopores has a deeper degree of blockage and longer duration compared to gold-modified nanopores.Aptamer-functionalized nanopores can be used to analyze the kinetics between aptamer and protein without any labeling.The dissociation constants of the combination and dissociation processes were calculated by evaluating the kinetic processes at the single molecular level.The sensing strategy we designed provides new insights for evaluating the binding capacity of aptamers and proteins. |
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