Optimization And Application Of Nano-flow Cytometer

Multiparameter information such as size,concentration and biochemical properties of cells or particles can be obtained via scattering and fluorescence detection by flow cytometer(FCM).It has the advantages of high statistical accuracy,reliable data and fast.Surface acoustic wave(SAW)technology can produce pressure gradient in liquid to act on particles of different sizes.It has the advantages of good biocompatibility,label free,contact free and no damage.At present,flow cytometer is being developed towards miniaturization and automation.In terms of instrument performance,sensitivity,dynamic range and accuracy are the key indicators.However,the traditional commercial flow cytometer is mainly designed to detect cells or cell size particles,which is difficult to meet the requirement of high sensitivity and wide dynamic range for nanoparticle analysis.Therefore,the application has been greatly restricted in the fields of virus detection and complex bio-nanoparticle characterization.If the surface acoustic wave(SAW)technology can be integrated with flow cytometry to aid the focusing of nanoparticles,detection with higher precision and lower coefficient of variation can be expected.Employing strategies for single molecule fluorescence detection in a sheathed flow,our laboratory have recently developed nano-flow cytometer(nFCM).Real time light scattering detection of single 7 nm gold nanoparticles,24 nm SiO2 nanoparticles,and fluorescence detection of single R-phycoerythrin molecule have been achieved successfully in 2014.Compared with the traditional flow cytometry,the sensitivity of scattering signal is increased by 4-5 orders of magnitude,and the sensitivity of the fluorescence signal is increased by 2-3 orders of magnitude.In this dissertation,we have achieved a higher degree of instrument integration by upgrading the software and hardware of the sample introduction,optical and data acquisition and processing systems of the nFCM.The dynamic range was improved and the fluorescence detection sensitivity was increased.Moreover,the focusing effect of sample stream was enhanced through the integration with SAW.It was successfully applied to the quantitative analysis of MS2 pseudovirus.This dissertation consists of the following sections:In chapter one,the working principle,development and application of FCM and SAW were briefly introduced.Then,the motivation and the research contents of present dissertation were discribed.The second chapter describes the software and hardware upgrading of the nFCM instrument.The hardware parts include the selection and upgrading of the sample introduction system,high precision regulation of the optical system and the integration of color CCD for the real-time monitoring of the flow cell.Meanwhile,software was programmed using Labview to enable interactive control of the above hardware systems.The third chapter describes the enhanced performance and application of the upgraded nFCM instrument.Employing a National Instruments DAQ card,precised control of the PMT supply voltage was achieved.By matching the linear output range of the PMT socket with the measuring range of the DAQ data acquisition card,the dynamic range of PMT detection channel was improved.Light scattering detection of 40 nm-1 μm polystyrene particles and fluorescence detection of 2.6k-110k FITC were achieved.The fluorescence detection sensitivity of APD channel was improved by using a high numerical-aperture water immersion objective.The fluorescence detection performance was evaluated by 200 nm polystyrene particles,quantum dots,antibody labeled Alexa fluor 488 molecule and R-phycoerythrin molecule.The enhanced sensitivity nFCM was confirmed and successfully applied to characterize wild-type MS2 virus and quantification of concentration of MS2 pseudo virus with different target sequence lengths.The fourth chapter describes the integration of SAW into nFCM.The SAW device and flow cell were designed and fabricated,and integrated into nFCM.Through the combination of SAW with hydrodynamic fluid focusing,an enhanced focusing of nano particles was achieved,which resulted in a higher precision and lower coefficient of variation.In chapter five,the study results are summarized and the future investigation is prospected.