Development Of A Single-molecule Flow Analyzer

With the completion of human genome sequencing project, life science enters the era of functional genomics and proteomics. More and more protein biomarkers reflecting body physiological and pathological conditions are identified and will provide powerful research tools for disease diagnosis, prognosis, treatment monitoring, and drug screening. Although protein expression can more accurately reflect the abnormity of organism phenotypes than message RNA (mRNA), the abundance of protein biomarkers in conventional samples such as tissue or body fluid are generally very low. Moreover there has not been any method that can amplify proteins, thus the practical application of biological markers depends on the development of ultrasensitive protein detection techniques..Single-molecule detection represents the upper limit in biological detection, so we propose to integrate single molecule detection technique and a microsphere-based immunoassay to develop ultrasensitive yet rapid immunochemical protein detection platform for the early diagnosis of diseases.In this dissertation, the study was to develop a single-molecule flow analyzer based on flow cytometer mainly used in DNA fragments sizing. The single-molecule flow analyzer will provide hardware support for the establishment of ultrasensitive immunochemical protein detection platform.This dissertation is composed of three chapters:In Chapter one, an overview of single-molecule detection technologies is given, especially the principle, key issues, and applications of single-molecule fluorescence detection technology. In the end of this chapter, objective, significance and main contents of this dissertation are introduced.Chapter two describes the design and construction of the single-molecule flow analyzer, including: 1) Function introduction of main components used in apparatus construction; 2) Design of the optical, flow, and detection systems; 3) Design and manufacture of sample holder and special parts; 4) Simulated apparatus comstruction with AutoCAD software to determine the relative position of all optical elements and the height of optical path; 5) Introduction of data acquisition and processing software; 6) Completion of single-molecule flow analyzer construction.Chapter three describes the adjustment and performance assessment of single-molecule flow analyzer. Adjustment works mainly include optical path alignment, flow velocity adjustment, and data acquisition parameter optimization. Performance of the apparatus in terms of sensitivity, reproducibility and resolution were assessed. Experimental results demonstrated that the house-constructed single-molecule flow analyzer has achieved single molecule detection sensitivity with good reproducibility and stability. Moreover, the single-molecule flow analyzer could resolve DNA fragments with different sizes which demonstrated good resolution. The main innovations of this thesis:(1) A single-molecule flow analyzer based on laser-induced fluorescence and hydrodynamic focusing was developed in our lab for which the single molecule detection sensitivity was demonstrated by the successful detection of single DNA fragments and single Phycoerythrin molecules.(2) The single-molecule flow analyzer was applied to DNA fragment sizing with linear correlation between DNA fragment length and mean burst size. Compared with gel electrophoresis separation, analysis speed and sensitivity were improved 100 and 100000 times respectively.(3) Compared with single-molecule fluorescence microscopy, sample analylized in flow can greatly increase the speed and statistic accuracy. Therefore, single-molecule flow analysis is more suitable for rapid and ultrasensitive analysis of biological samples.In addition to applications in ultrasensitive detection of proteins and DNA fragments sizing, single-molecule flow analyzer has potential applications in following fields:â‘ Detection of physiological and chemical characteristics of biological particles such as single virus, bacteria, sub-cellular organelle, and cell;â‘¡Ultrasensitive detection of capillary electrophoresis;â‘¢Combined with microsphere-based suspension array, single-molecule flow analyzer could achieve simultaneous detection of multiple biomarkers. Consequently, single-molecule flow analyzer will play an important role in life science fields.