Single Cell Analysis Of Multiple Metal Ions And Single Molecule Detection Of Cell Membrane Proteins

Multiple biomolecules play important roles in organism,such as genes could regulate the signal transmission process and metabolic pathway,proteomics reflected the gene expression level to some extent.What‘s more,metal ions metal ions play functional roles in neurotransmitter release and transmission,neurite outgrowth regulation,synaptogenesis and synaptic transmission and so on.Biological molecules are related and affected to each other.Abnormal expression of one biomolecule may cause a series of changes.Biomolecules are closely related to the normal cellular functions,the genesis and development of diseases and the diagnosis and treatment of diseases.A number of methods aims to detection of the intracellular biomolecules has been developed so far,including electrochemistry,mass spectrometry and fluorescence spectroscopy and so on.However,it is still difficult to achieve the detection of intracellular biomolecules at single-cells level or single molecules level.Firstly,the dimension of biomolecules is nanoscale,as the result,higher spatial resolution is essential.Secondly,the low sensitivity method is hard to measure the low content biomolecules accurately.In addition,the components intracellular are complex,so how to eliminate their interferences is challenging.Thus,it is emerging to establish a method to detect the multi-biomelecules simultaneously in single cells and a high sensitive method to measure intracellular biomolecules at single molecule level.As the result,aiming at these difficulties and chanllenges,this paper is devoted to develop a sensitive method to detect the multi-components simultaneously in single and promote the sensitivity for a high sensitive measurement of membrane proteins at single molecule level.This paper is including the four section as below:The first chapter is preface.The research progress of intracellular various biomelecules detection was summed up at first,including the important physiological function of the biomolecules and the current detected method.Then we introduced the significance of detection at single-cells level and the simultaneous multiple metal ions detion in single cells,and summaried the advantages of microfluidic system used to single-cell detection.At last,we introduced the research progress of single molecule detection and the signific-ance and methods of single-molecule membrane proteins.In the second chapter,based on the difficulties of simultaneous detection of multiple metal ions in single cells,we introduced a simultaneous quantitative method to detect Na⁺,K+,Ca²⁺,Mg²⁺ in neuron-like cells by microfluidic system,and researched the concentration changes in A?25-35 treated PC-12 cells using this novel method.The method produced a new idea in simultaneous detection of multiple metal ions in single cells.What‘s more,the research results provide us new enlightenment on how the metal ions affected the nerve disease.On the basis of single-cells detection,we promote the detection sensitivity and developed a single-molecule detection of membrane proteins in the third chapter.We chose membrane protein PTK7 as the research target,utilized the aptamer to target the membrane proteins and combined with rolling circle amplification method to achieve the amplification of signal.Thus,we developed a sensitive and hypotoxic method which could imaging PTK7 in site at single molecule level.The sensitivity of this method was not only satisfied the detection of abundant membrane proteins,but also could be used to detect low abundance proteins.At last we summarized the established single-cells and single-molecule detected methods.On the point of promoting the detection sensitivity,the simultaneous detection of multiple metal ions at single-cells level and membrane proteins detection at single-molecules level has been achieved.These two methods are progressive and same in essence.