| This thesis describes the development and application of a microfluidic device for chemical analysis of single cells. The device, made of poly(dimethylsiloxane) (PDMS), can manipulate nanoliter to picoliter volume of liquid, which contributes to higher effective concentrations of analytes for reactions and measurements. The protein analysis chip utilizes microvalves based on the elastomeric property of PDMS to capture one cell into a picoliter chamber and extract protein molecules by lysing the cell. Different protein species are separated using detergent-assisted microchannel electrophoresis and quantified by laser-induced fluorescence detection. Using this device, phycobiliprotein complexes in a cyanobacterium, Synechococcus sp. strain PCC 7942, were analyzed and the cell-to-cell variations in the concentrations of these protein species and their relative ratios were determined. The DNA analysis chip uses gas permeability of PDMS to enable the addition of nanoliter volumes of reagents for miniaturized multiple displacement amplification reactions. The manipulation and isolation of individual cells into reaction chambers are performed with microvalves. The genomic DNA obtained from single cells of thermophilic Synechococcus living in a hot spring microbial mat was amplified and analyzed using both conventional and quantitative polymerase chain reaction assays. Important targets for studying microbial diversity and ecology were identified by Sanger sequencing of the DNA samples amplified from single cells, which include phylogenetic markers based on 16S rRNA sequences and the genomic region containing characteristic repeats of palindromic sequences that is responsible for bacterial immunity. |
