Research And Application Of Single Solid-state Nanopores On Biosening

Nanopore analytics,based on Coulter counter and single channel current recording technology,is a burgeoning technology for single molecule sensing.Inspired by the transport of ions and molecules through ion channels on cell membranes,and with the development of surface chemistry,material science and nanotechnology,various synthetic nanochannels appeared and developed rapidly.Among them,single solid-state nanopores have been widely used in sensing detection on single molecule and single cell with the advantages of high hardness and stability,controllable size and modifiable surface.When the analytes to be measured translocate through a single solid-state nanopore,due to their volume blocking effect,ionic conductance in the nanopore will be momently reduced and a large number of pulsed current signals will be generated.We can obtain the informations on structure,size and concentration of the analytes according to these signals.Single glass capillary nanopores with sharp nanotips have negligible damage to integrity of cellular function and cell membrane after insertion into single cells,which can be used in substance transport and extraction,and biomolecular sensing detection in single-cells level.Also,the rapid developments and applications of synthetic nanopores have promoted the emergence of nanofluidics,which studies the transport behavior of subtances in nanoscale channels.What's more,in recent years,with the emergence of various new-type nanomaterials and fine processing technology for nanopores,researches on nanofluidics have made great progress.In this doctoral thesis,we mainly focus on construction of single solid-state glass and silicon nitride(SiNx)nanopore sensing platforms to carry out a series of researches of interest.A short chain single-strand DNA(20 nts)was detected with single glass nanopores by integrating with a tetrahedral DN A nanostructure,which laid a foundation for microRNA detection with glass nanopores.Sharp nanoptips of glass nanopores helped them insert into cells for single-intracellular ATP and glucose detection,which was of great significance to clinical research.A bio-nanofluidic device was constructed by intergrating natural pronton-pumping protein bacteriorhodopsin(bR)with SiNx nanopore to detect the proton pumping of a single bR.The details are as follows:1.Although the diameter of glass capillary nanopore can be easily tuned to less than 10 nm,direct eventreadouts of small biomacromolecules,like short-chain oligonucleotide fragments(within-20 nucleotides)remain great challenge,which limited by the configuration of the conical-shaped nanopore and the instrumental temporal resolution.Here,we exploit a smart strategy for glass nanopore detection of short-chain oligonucleotides by using relatively big-sized tetrahedral DNA nanostructures as a signal amplifier,which can amplify the signals and retard the translocation speed meanwhile.The tetrahedral DNA nanostructure with a hairpin loop sequence in one edge,undergoes a shape transformation upon the complementary combination of the target oligonucleotides(called I0),in which the presence of short-chain target oligonucleotide I0 can be readout due to obvious variation in amplitude of ion current pulse that caused by volume change of the DNA tetrahedral.Therefore,this strategy is promising for extending glass nanopore sensing platform for sensitive detection of microRNA.2.The accurate detection and content change in a certain life stage of key substances such as adenosine triphosphate(ATP)in single-cells are crucial for single-cell biology and cancer theranostics,but it remains a key challenge.Herein,we constructed an ATP-specific nanopore sensing platform and exploited for ATP detection in single-cells.The sensing zone of the glass nanopipette(?90 nm,has minimal damage to cell membrane and cellular functions)was functionalized with ATP-responsive gold nanoparticle(?13 nm)assemblies,which partially disassembles in the presence of ATP,leading to change in current response.This sensing platformed can be exploied for ATP content measurement in single cells(normal and cancer cells)and monitoring their content change of single-cells ATP after the electrostimulus(ES)-induced apoptosis.We found that the content differences between single normal-state normal cells and cancer cells were unobvious and the variation of ATP content differs from cancer cells to normal cells(ATP content decrease for normal cells while increase for cancer cells),well reflecting the different metabolic patterns of the two types of cells,which layed a foundation for the research on single-cells glucose detection by single glass nanopipettes.3.We constructed a cytochrome c(Cyt c)-modified glass nanopore(G-nanopore)sensing platform for specific detection of glucose in single cells based on ionic current rectification(ICR)change of the system.In the presence of glucose oxidase(GOX)and substrate glucose,the enzymatically generated H2O2 will oxidize Fe(?)of Cyt c that modified on the inner wall surface of G-nanopore into Fe(?),resulting in the decrease of ICR ratio(R).The Cyt c functionalized G-nanopores were further applied for intracellular glucose detection.We detected the glucose content in single HeLa and H8 cells in satisfy and starvation state,respectively,and distinguished the difference in glucose levels between them(the averaged glucose concentrations in single satiety H8 and HeLa cells and starvation HeLa cells were calculated to be?1.9 mmol/L,3.4 mol/L,0.8 mmol/L and 1.0 mmol/L respectively).We discovered that the glucose level in single cancer cells was higher than that in single normal cells.However,when the cells were in starvation,the glucose level in single cells decreased,more obvious in single cancer cells.This developed G-nanopore sensing platform could be useful for distinguishing cancer cells from normal cells in heterogeneous tissue or organ,as well as for monitoring cancer cell progression in situ.4.The development of various synthetic nanopores has promoted the emergence of nanofluidics,which is an emerging hot research field to explore as substances(such as ions and molecules)transporting through nanoscale channels showed many unexpected behaviors and exotic properties,but construction and test of nanofluid devices are a major bottleneck hindering the development of the field.Herein,we construct,for the first time,a novel biological nanofluidic device with single protein sensitivity,based on proton-pumping protein,bacteriorhodopsin(bR)and a single SiNx nanopore,by spanning bR membrane over the nanopore.Single-protein level nanofluidic probing of proton-pumping activity and response of bR was achieved under applied voltage of 0 V,by interfacing bR with single SiNx nanopore and using resistive-pulse sensing method.Under the irradiation of green light,an ionic current of?3.51 pA/per bR trimer of the device generated by its light-driven proton pumping can be obtained,corresponding to charge density of 815 ?C/cm2 generated by each bR monomer,which is far exceeding the theoretical value of 1.4?C/cm2.The finding and method can further promote the development of artificial biological and hybrid nanofluidic devices in biosensing and energy conversion applications.