Development Of High-sensitivity (Spectral) Flow Cytometer For Quantitative Characterization Of Individual Nanoparticles

Flow cytometry(FCM)is a well-established technique for the rapid,multiparameter,and high-throughput quantitative analysis or sorting of individual cell and other microscopic particle in aqueous suspension.Information regarding size,internal granularity and surface roughnessof particles can be gathered via light scatter measurements,and biochemical attributes such as the nucleic acid content,enzymatic activity,and antigenic determinants of biological cells can be characterized via fluorescent labeling.In traditional FCM analysis,tens of thousands of cells can be detected per second for rare cell analysis and high-speed cell sorting.Such highthroughput detection requires transit time of sample particles within microseconds,which greatly limits the detection sensitivity of the instrument.Great challenge exists for the conventional FCM,in attempt to detect NPs smaller than 500 nm in size or dim particles with less than several hundreds of fluorescent molecules.The rapid development of nano-technology and the research of life science have put forward higher demand for the characterization of nanoparticles.Based on the inherent multi-dispersibility of nanoparticles,a variety of physical and biochemical characters need to be quantitatively represented at the single particle level.However,there is no technique for the multi-parameter analysis of such nanoparticles at the single-particle level.If the detection sensitivity of FCM could be greatly enhanced,the detection of nanoparticles would have the advantages of rapid,multi-parameter,quantitative and natural state suspension detection.Adopting strategies for single-molecule fluorescence detection in a sheathed flow,we recently developed a high-sensitivity flow cytometry(HSFCM)method.The method uses a unique sheath flow system in which the sample fluid is hydrodynamically focused to a very narrow stream at low flow rate,so that the transit time of analyte particles can be as long as milliseconds.Cooperating with other sensitivity improvement strategies,such as reducing the size of the detection region and using single photon detection module,the detection limit of HSFCM is much lower than that of the traditional FCM.Real-time light-scattering detection of single 7-nm gold nanoparticles,24 nm SiO2 nanoparticles,and fluorescence detection of single R-PE molecule have been achieved.Clearly,the development of advanced flow cytometry enabling rapid and multiparameter characterization of physical and chemical properties of individual nanoparticles is of great importance to nano-biotechnology and bioscience studies.Present study focuses on the further improvement of HSFCM sensitivity and the upgrade of its detection mode for the purpose of expanding its applications.We optimized the optical and fluidics systems of the laboratory-built HSFCM instrument,so the detection sensitivity is further improved.By employing the improved HSFCM,we have developed a rapid and highly sensitive method for the precise quantification of the fluorescence intensity of single quantum dots.In addition,we added a spectral detection module to the HSFCM for the development of a high sensitivity spectral flow cytometer(S-HSFCM).The S-HSFCM inherits the high sensitivity advantage of HSFCM,achieving high S/N ratio detection for single particle fluorescence spectra of nanomaterials and microorganisms.The thesis consists of the following parts:In chapter one,the development history of FCM was briefly introduced.Then,recent advances and development trends on FCM were reviewed.The motivation and the research contents were also included in this part.The second chapter describes the system optimization of HSFCM.The sensitivity and stability of instrument are greatly improved by the upgrading of the optical and fluidics systems.The detected signal strength of the instrument was 2.8 times higher than that of our original setup,and the SNR ratio was?1.7 times higher.The sensitivity enhancement of HSFCM will be of great significance to the quantitative characterization of nanoparticles.The third chapter describes the rapid and quantitative measurement of single quantum dots via HSFCM.Semiconducting quantum dots(QDs)are used in a wide range of biomedical applications due to their intense fluorescence brightness and long-term photostability.Here,we report precise quantification of the fluorescence intensity of single QDs on a laboratory-built high-sensitivity flow cytometer(HSFCM).By analyzing thousands of QDs individually in 1 min,intrinsic polydispersity was quickly revealed in a statistically robust manner.Applications of this technique in QD quality assessment,study of metal ion influence,and evaluation of aggregation upon biomolecule coupling are presented.Moreover,an accurate measurement of the QD particle concentration was achieved via single-particle enumeration.HSFCM is believed to provide a powerful characterization tool for QD synthesis and application development.The fourth chapter describes the development of S-HSFCM.Taking advantage of the unprecedented sensitivity of HSFCM,we developed the S-HSFCM by adding a spectral detection module on HSFCM.Multi-PMT detection module and spectral detection module are placed on both sides of the flow channel perpendicular to the laser incident beam and to the flow of the sample.Single nanoparticle spectrum is acquired by an EMCCD attached to the imaging spectrograph through the trigger generated by the PMT signal of particles' side scatter or fluorescence.The sensitivity of the system was evaluated by analyzing fluorescent SiO2 nanoparticles with intensities quantified in the unit of molecules of equivalent soluble fluorochrome(MESF)per nanoparticle.Auto fluorescence of single bacteria was also examined.S-HSFCM could become a powerful tool for life science and nanobiotechnology studies.The fifth chapter describes the spectra analysis of Synechococcus autofluorescence at the single-cell level by S-HSFCM.Synechococcus are the important and major group of cyanobacteria.They are widely distributed in various aquatic environment including ocean,river and lakes.Synechococcus are abundant in the ocean with high biodiversity,contributing highly to marine primary production and global carbon cycling.The phycobilin pigments in Synechococcus cells result in the unique in vivo fluorescent properties of them.We established a method for high throughput spectra detection of the autofluorescence of single Synechococcus cells via using S-HSFCM.By detecting the autofluorescence spectra of single cell Synechococcus,we identified the pigment components and contents of different Synechococcus species.Given the advantages of high-throughput and high resolution analyses,there would be wide applications of S-HSFCM in the study of Synechoccoccus physiology and ecology.In chapter six,the research progresses were summarized and the future prospects of HSFCM and S-HSFCM were discussed.