Single-cell And Single-molecule Electrochemical Analysis Based On Micro/Nanocapillary

Single-molecule sensing can obtain molecular structure,property,kinetics and thermodynamic information.Measuring changes in the content of important biomolecules in different life states at the single-cell level is essential to understanding the life process.At present,methods for studying single cells mainly include methods such as electrochemistry,fluorescence,and mass spectrometry.Among these methods,the micro/nanoelectrode-based electrochemical method is an important technique for real-time quantitative detection of single cells with its high sensitivity.According to the different sensing modes,it can be divided into two types of electrodes.One type is an electrode based on a Faraday current signal that can detect redox biomolecules inside or outside the cell.The other is a micro/nanotube electrode based on ion current signals,the important feature of which is the tip of the submicron to nanometer size opening,the hollow structure and the surface are easy to functional,making it suitable for mass delivery of individual living cells as well as the extraction and detection of individual living cell contents.In this thesis,an electrochemical analysis system for single-cell analysis was first established.Then,based on micro/nanotubes,the ion channel function of the cell membrane and the activation process of the ion channel of the cell membrane under the stimulation of gold nanorods assisted near-infrared light were studied.At the same time,nanopipette combined with precise micromanipulation techniques,analysis of subcellular levels of reactive oxygen species was achieved,and by the use of DNA carriers with binding sites for aptamer for specific sensing of target protein is possible to perform single-molecule sensing.1.Construction of single-cell electrochemical analysis systemDue to the high noise and low sampling frequency of traditional electrochemical workstations,it is hard to realize the application of micro/nanocapillary in single-cell electrochemical analysis.Therefore,we built a single-cell electrochemical analysis system which mainly includes:optical imaging device,micro-manipulation positioning device,perfusion drug delivery system and data acquisition system.In order to study the ion channel current under near-infrared light stimulation,we added a self-built near-infrared light source system to the single-cell electrochemical analyzer.Since the system needs to detect cell membrane ion channel currents as small as pA,it is susceptible to external influences.In order to improve the detection sensitivity,we perform anti-vibration,anti-interference and noise reduction processing on the system.Finally,we studied HEK-293 cells transfected with hERG potassium channels,which proved that the system can be used for single cell analysis.2.Study of gold nanorods assisted near-infrared activated membrane ion channel based on patch clamp techniqueMembrane ion channels are ultimately responsible for the propagation and integration of electrical signals in the nervous,muscular,and other systems.Their activation or malfunctioning plays a significant role in physiological and pathophysiological processes.As a consequence,these channels have engendered very high interest as future drug targets.The patch-clamp technique as a standard method to analyze plasma membrane ion channels and provides a direct measure of an ion channel's function.Measuring ionic flux provides a relatively easy way of probing protein function in comparison to other classes of membrane protein.Herein,patch-clamp technique was used for the detection of gold nanorods assisted near-infrared activated membrane ion channels detection.Gold nanorods(GNRs)used as an extrinsic NIR absorber to control the thermosensitive ion channels in breast cancer cells.The GNRs are designed conjugated with TRPV1 monoclonal antibody(pGNRs-TRPV1),which enables specific binding to TRPV1 on the plasma membrane of cells efficiently.By virtue of the fast heating capability in conjunction with the precise targeting to the thermosensitive ion channel,GNRs-TRPV1 can specifically and rapidly activate the intracellular Ca2+influx of breast cancer cells in a reversible and safe manner.The increase of the intracellular Ca2+concentration caused cell apoptosis by the activation of TRPV1.This novel optogenetic therapy has potential applications for tumor cells with high expression of TRPV1.The successful application of whole-cell patch clamp method in infrared light-activated ion channel research provides an effective means for screening suitable ion channel activators.3.Ultrasmall Nanopipette:Toward Continuous Monitoring of Redox Metabolism at Subcellular LevelThe fast-developing nanoelectrochemical method,as a label-free method,has significant advantages in space-time resolution detection and is suitable for single-cell detection.In order to achieve the redox metabolism monitoring at the subcellular level,we prepared a nanotube-ICR system with a 30 nm ultrafine capillary and applied it to the detection of H2O2,the main component of reactive oxygen species(ROS).The G-quadruplex DNAzyme(G4)is modified to the inner surface of the nanotubule by chemical bonding to have H2O2 recognition ability.G4 catalyzes H2O2 and produces intermediates that oxidize 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate)(ABTS)perfused in nanotubes.These reactions change the charge density in the inner wall of the nano-microtube.The change in charge density depends on the concentration of the analyte,so the H2O2 can be quantitatively analyzed by the I-V curve.At the same time,the nanotube-ICR system constructed by G4 is very stable and can be used repeatedly and has good reproducibility.We used fluorescent dyes to study the effects of nanotubes on cell viability and cell function.The results show that ultra-small-end nanotubes can be repeatedly inserted into a single cell for long-term measurement without impairing cell function.Combined with high-precision three-dimensional positioning system,sub-micron regulation is realized,and ROS analysis on single-cell three-dimensional scale is realized.At the same time,changes in the levels of aerobic metabolism of different cells under the stimulation of the drug capsaicin can be continuously monitored.4.Single cytochrome C molecule detection using aptamer modified DNA carriersIn traditional biological research,most experiments are to detect the combined average effect of molecules.By manipulating and detecting single molecules such as nucleic acids,proteins and polypeptides,the molecular structure,properties,kinetics and thermodynamic information can be obtained.Glass capillaries have emerged as an alternative to both biological and silicon based nanopores.However,detection of proteins using nanopores is often challenging due to their heterogeneous charge,fast translocation times,poor signal-noise ratio especially for small proteins(<15 kDa),and nonspecific adsorption to the pore.Here,we demonstrate a fully flexible,scalable and low-cost detection platform to sense multiple protein targets simultaneously by the use of DNA carriers with binding sites for aptamer for specific sensing of target cytochrome c protein.Protein bound to the aptamer produces unique ionic current signatures which facilitates accurate target recognition.This powerful approach allows us to differentiate individual protein sizes via characteristic changes in the sub-peak current.Furthermore,we show that by using DNA carriers it is possible to perform single-molecule screening in serum at ultra-low protein concentrations.