Nucleic Acid Analysis In Confined Space

Nucleic acids play an important role in various life activities.According to the difference of sugar scaffold,nucleic acids can be divided into DNA and RNA.The detection of nucleic acid helps us analyze viral infections,diagnose cancer,and establish the analysis of other disease based on biomarkers.Polymerase chain reaction(PCR)can amplify a large amount of nucleic acids in a short time,providing the possibility for further detection of nucleic acids.Although single-cell sequencing has been successfully established,developing simple and more effective technology to obtain nucleic acids in single cells,improve the PCR reaction rate and lower the detection limit of nucleic acids is still urgent.In view of the laboratory’s early development of a ‘single cell nanokit’ using a nano-capillary,we have achieved the sampling and analysis of molecules and single organelles within single cells.This thesis will strive to expand the application of nano-capillary based single cell analysis,using confined space in the nano-capillary tip to improve the PCR efficiency,and then real-time analyze nucleic acids in the capillary.The realization of the work will lay a solid foundation for the establishment of nano-capillary based single-cell nucleic acid sampling and analysis.To achieve the above aim,the main part of this thesis is to establish a two-step nucleic acid amplification method that includes a PCR reaction in the confined space of the nanocapillary tip.The involvement of PCR reaction in the confined space will significantly improve the efficiency of nucleic acid molecule amplification and reduce the detection limit of nucleic acids.For the real time detection,we use methylene blue as an electrochemical indicator and nanocapillary as the electrodes to monitor the amplification process of nucleic acids.Eventually,it should be highly feasible to apply nanocapillary to sample and measure nucleic acid molecules in single cells.Meanwhile,in-situ,high spatial resolution imaging of the charge distribution on the sample surface was performed using the ion current through the nano-capillary.The preliminary data obtained could facilitate the future real-time imaging of the charge distribution at the cellular surface.Thereby,a new technology is provided for further understanding of the charge changes of single cells in various physiological processes.In the first part of this thesis,a two-step nucleic acid amplification method was established that includes a PCR reaction in the confined space of the nano-capillary tip.First,we pour various molecules required for PCR reaction into the capillary with a tip opening of about ～ 119 nm,then sort the DNA template molecule into the capillary tip through a syringe pump.Second,the capillary is placed in the PCR instrument for the first step of PCR;after the reaction in the capillary,the solution is injected into a microliter solution for the second PCR step.Third,the amplified DNA molecules are detected by gel electrophoresis imaging to obtain a single clear target DNA molecule band.Compared with one-step PCR reaction in the microliter solution,two-step PCR reaction using a confined space to increase the collision probability of molecules and improve the amplification efficiency.It can amplify and detect DNA molecules as low as 7500,which is expected to provide a new detection approach for trace DNA.In view of the difficulty of real-time analysis of gel electrophoresis imaging,the second part of this thesis attempts to establish a new real-time PCR electrochemical detection method based on nano-capillary electrodes.During the experiment,the electrical signal of methylene blue(MB)during the PCR reaction is collected on the surface of the electrode inside the nano-capillary tip.Since the diffusion rate of the bound MB with double-stranded DNA is lower than that of free MB molecules,the electrochemical signal decreases in presence of DNA molecule.Through the decrease of the current value,the progress of DNA amplification can be obtained,which lays foundation for the future usage of nano-capillary to achieve the sampling,amplification and analysis of nucleic acid molecules in single cells.The third part of this thesis is to use ion current through the nano-capillary for the electrochemical imaging of surface charge,which can be developed to study the distribution of surface charge at single cells.Pt-graphite electrode interface is chosen as the model in early work,and the charge distribution at this planar surface and the junction is studied.When different voltages are applied on the electrode surface,higher charge density is observed at the interface than other planar areas.Thus,this technology realizes the sensitive imaging of the charge density on the surface,and is expected to study the fluctuation of surface charge at single cells during physiological processes.