Title page for ETD etd-04152010-184626


Type of Document Dissertation
Author Lee, Jeonghoon
Author's Email Address jlee62@tigers.lsu.edu
URN etd-04152010-184626
Title Microfluidic Devices Interfaced to Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Proteomics
Degree Doctor of Philosophy (Ph.D.)
Department Chemistry
Advisory Committee
Advisor Name Title
Murray, Kermit K. Committee Chair
Soper, Steven A. Committee Co-Chair
Gilman, Douglass Committee Member
Poliakoff, Erwin Committee Member
Hughes, Richard Dean's Representative
Keywords
  • MALDI
  • electrospray
  • separation
  • microfabrication
  • integrated chip
  • sample purification
  • cell culture
  • protein
  • bacteria
  • continuous deposition
  • spotting deposition
  • microfluidics
  • mass spectrometry
  • peptides
  • trypsin
  • solid-phase bioreactor
Date of Defense 2010-03-29
Availability unrestricted
Abstract
Microfluidic interfaces were developed for off-line matrix-assisted laser desorption/ionization mass spectrometry (MALDI). Microfluidic interfaces allow samples to be manipulated on-chip and deposited onto a MALDI target plate for analysis. For this research, microfluidic culturing devices and automated digestion and deposition microfluidic chip platforms were developed for the identification of proteins. The microfluidic chip components were fabricated on a poly(methyl methacrylate), PMMA, wafer using the hot embossing method and a molding tool with structures prepared via micromilling. One of the most important components of the chip system was a trypsin microreactor. An open channel microreactor was constructed in a 100 m wide and 100 m deep channel with a 4 cm effective channel length. This device integrated frequently repeated steps for MALDI-based proteomics such as digestion, mixing with a matrix solution, and depositing onto a MALDI target. The microreactor provided efficient digestion of proteins at a flow rate of 1 L/min with a residence time of approximately 24 s in the reaction channel. An electrokinetically driven microreactor was also developed using a micropost structured chip for digestion. The micropost chip had a higher digestion efficiency due to the higher surface area-to-volume ratio in the channel. Also, the electrokinetic flow eliminated the need for an external pumping system and gave a flat flow profile in the microchannel. The post microreactor consisted of a 4 cm 200 m 50 m microfluidic channel with trypsin immobilized on an array of 50 m in diameter micropost support structures with a 50 m edge-to-edge inter-post spacing. This micropost reactor was also used for fingerprint analysis of whole bacterial cells. The entire tryptic digestion and deposition procedure for intact bacteria took about 1 min. A contact deposition solid-phase bioreactor coupled with MALDI-TOF MS allowed for low-volume fraction deposition with a smaller spot size and a higher local concentration of the analyte. A bacterial cell-culturing chip was constructed for growing cells on-chip followed by off-line MALDI analysis. Coupling MALDI-TOF MS whole cell analysis with microfluidic culturing resulted in more consistent spectra as well as reduction of the total processing time. The microfluidic cell culturing was performed in a PMMA chip with a polydimethylsiloxane (PDMS) cover to allow gas permeation into the culture channel, which contained a 2.1 μL volume active culture chamber. After incubation of E. coli in a microfluidic culture device at 37 ℃ for 24 h, the cultured cells were analyzed with MALDI MS. Also, a microfluidic cell culture device containing continuous perfusion of culture medium was developed using a polycarbonate membrane. This microfluidic culturing format was improved with a fluidic manifold and thermostatted microheaters. Fingerprint mass spectra distinguishing E. coli strains tested were obtained after a 6 h incubation time, which was shorter compared to the 24 h incubation time using conventional culturing techniques. In addition, an enhanced identification procedure for bacteria was achieved by integrating on-chip digestion of cultured bacteria.
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