Nanopore Biosensing Based On Nucleic Acid Modification And Single Cell Analysis Application Research

With the continuous development of the analytical chemistry,the related information of proteins,nucleic acids,enzymes and small molecules obtained in life sciences are increasing needed with the help of biosensing technology.It is of great required to fast and accurately obtain the information of these molecules in biomedicine,clinical diagnosis and therapy.With the continuous improvement of medical diagnosis and the deepening of scientific research,the requirements for detection methods in life sciences are also increasing.Development of high sensitivity,specificity,low-cost,simple and rapid qualitative or quantitative analytical methods that can meet the needs of research and practical applications has become an important topic for researchers in analytical chemistry.In this thesis,based on hybridization chain reaction?HCR?amplification technology,nanopore arrays and novel glass nanopore materi als combined with optical and electrochemical detection techniques,we developed a series of high sensitivity,specificity analysis methods for tumor markers,nucleic acids,proteins,and small molecules detection.Living cell analysis at the single cell level is achieved by further combining with nano biochemical system.The detailed content was described as follows:1.Label-free Nanopore biosensor for sensitive detection of Survivin mRNA based on hybridization chain reaction amplification detectionAs a cancer-related gene,Survivin plays a crucial role in inhibiting cell apoptosis.The expression level of RNA-Survivin mRNA is closely associated with many diseases including various kinds of cancers.It's an important tumor marker in research and clinical.In Chapter 2,a label-free nanopore biosensor for detection of Survivin mRNA target is proposed utilizing hybridization chain reaction?HCR?strategy for signal amplification.The DNA target triggered HCR to form large DNA nanostructure inside the nanopore and out the nanopore membrane,which inducing the ionic current decrease effectively,due to the blockage of the nanopore.The developed method achieves a desirable sensitivity of 10 fM with a wide linear dynamic range from 0.01 to 10 pM and demonstrated good application for real sample analysis.This work has great potential to be applied in the early diagnosis of gene-related diseases and provide a new paradigm for label-free nucleic acid amplification strategy in ultrasensitive nanopore biosensor.2.Glass nanopore sensor based on DNA aptamer for small molecules analysis in single cellDopamine?DA?is a well-known and important neurotransmitter because it is involved in motor and cognitive functions,abnormal levels of DA in brain has been correlated with various diseases,including Schizophrenia,Huntington's disease and Parkinson's disease,hence choose as target example for play important role in complex brain functions.In Chapter 3,we proposed a label-free glass nanopipette biosensor based on modulation of surface charge density and coupled with precise nanoscale-positioning to real-time monitoring intracellular molecules in a single living cell by using a 30-50 nm functionalized glass nanopipettes for captu ring intracellular small molecule targets via the changes of ionic current rectification?ICR?.We choose Dopamine?DA?as an important neurotransmitter which is involved in neuronal signal transduction and PC12 cell often used as an in vitro model to study the physiology of central dopaminergic neurons behavior,as examples to demonstrate a proof-of-concept of this real-time monitoring strategy.We show that the intracellular DA can be sensitive ly and selectively monitored by glass nanopipettes in real-time.We believe the ICR nanopipette sensors represent a novel strategy to real-time monitor neurons behavior at single cell levels and potentially can be extended to other platforms for single cell analysis.3.Dual-glass nanopore sensor based on DNA aptamer for Cellular drug therapy and extracellular secretionThe analysis of single cell system can reveal the heterogeneous properties hidden in the whole cell reaction,which is of great significance for drug development.In Chapter 4,we proposed a dual-glass nanopore sensor for studying the drug L-DOPA treatment and dopamine secretion behavior of PC12 cell response to hypoxic buffer.L-DOPA can promote the synthesis of dopamine and increase the content of dopamine in PC12 cells,which cause the decrease of ICR ion current.The hypoxic effect on PC12 cells induces the release of DA through exocytosis,which also cause the decrease of ICR ion current.Therefore,our sensor offers a novel approach for studying the drug treatment and response to stimuli.It has great application potential in biological analysis,disease diagnosis,and drug screening.4.Dual-glass nanopore sensor based on DNA aptamer for ROS secretion of cellTo further extend the application of our single-cell detection platform.In Chapter 5,we proposed a dual-glass nanopore hydrogen peroxide sensor to detect the secretion of hydrogen peroxide from a single MCF-7 cell.The sensor functionalized with G-quadruplex DNAzyme has peroxidase activity and could catalyze H₂O₂-mediated oxidation of ABTS to produce metastable radical ABTS·-,which could bind to DNA and enhance the charge density of the glass nanopore then cause the decrease of ICR ion current.In combination with a home-made cell culture chip device,real-time monitoring of ROS secretion from single cell can be achieved,further expanding the application of our dual-glass sensor.