Construction Of Single-molecule Detection Platform And Its Application In Analysis

Single-Molecule Detection(SMD)is a kind of ultra-sensitive detection technology developed rapidly in recent years.SMD has been developed to be used in a variety of applications in analytical chemistry,life medicine,fluid mechanics and pharmacy etc.Compared with traditional detection techniques such as spectrophotometry and chemiluminescence,SMD can realize the detection at single molecule level,which holds a great potential in the field of chemical biology and early diagnosis of diseases.In this thesis,we constructed a single molecule detection platform by using enzymatic amplification in an array of femtoliter-rized reaction vessels.The contents are described as follows:(1)Construction of fiber-based single molecule detection platform.The single molecule detection platform was constructed based on the high density fiber microarray and fluorescence microscope.In this work,some optimization was made including constructing of the darkroom,designing and installing of microscope with accessoreies,assembling of the Z direction stage,etc.which build up the foundation for the determination of enzyme molecules from the single molecule level.(2)The ultralow concentration of enzyme and the corresponding substrate was designed to react on the detection platform to analyze the feasibility for single molecule detection.The quantitative relationship between the digital signal and the concentration of ?-galactosidase was successfully obtained by using this developed platform.(3)Acoording to the above research work,?-galactosidase labeled with streptavidin were further detected by using biotinylated magnetic beads on this platform.In the process of experiment,we optimized the experimental conditions including the activation process of the carboxylated beads,conjugation betwwen BSA and the actived beads,biotinlation of BSA on magnetic beads,loading of beads into the fiber wells as well as the loading number.This newly develeped technology lays the foundation for futher application of this platform for ultrasensitive detection of biological macromolecules such as antigen and antibody.