Identifier

etd-06052013-113438

Degree

Master of Science (MS)

Department

Chemistry

Document Type

Thesis

Abstract

A novel microfluidic device was developed for bacterial cell culturing using mass spectrometry as the detector. One of the challenges in proteomics is to achieve high sensitivity in the identification of proteins in complex samples with widely varying concentrations. The main limitations for proteomic studies are relatively slow and labor-intensive steps such as cell culturing and protein digestion of small sample quantities. Microfluidics is a promising approach to increase throughput and to reduce the time-consuming steps that are necessary for proteomics. When an analytical detection method is combined with microfluidics it can overcome limitations that are important in the analysis of biological samples. In this work a microfluidic device was constructed from poly(methyl methacrylate) PMMA using hot embossing from a brass metal mold prepared from micro-milling and combined with off-line matrix assisted laser desorption-mass spectrometry mass spectrometry (MALDI-MS) for analysis. In this work, E. coli K12 strain was selected as a model for performing the analysis. Microfluidic devices were used to process the sample and mass spectrometry was used as detection method. The microfluidic device used in this study consists of three modules, capture, culture, and digestion chamber, integrated onto a single platform. The cells are captured on the microfluidic chip using polyclonal goat antibody on a modified PMMA surface, and are released using 0.25% trypsin, and transferred to the culture cell, which is filled with the growth medium. The temperature of the culture cell is maintained at 37 ºC using a heater and a PDMS cover slip was used for air perfusion. Samples collected at different culturing durations (4 h, and 10 h) are transferred to a micro-post bioreactor, which contains immobilized trypsin. The effluent from the microfluidic device was spotted onto a MALDI target and analyzed using MALDI time-of-flight mass spectrometry.

Date

2013

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Murray, Kermit

Included in

Chemistry Commons

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