Title page for ETD etd-11092011-205155


Type of Document Master's Thesis
Author Snowden, Brandy Charon
Author's Email Address bsnowd1@lsu.edu
URN etd-11092011-205155
Title Selection and Enumeration of Low EpCAM Expressing Breast Cancer CTCS Using a High Throughput Microfluidic Device
Degree Master of Science (M.S.)
Department Chemistry
Advisory Committee
Advisor Name Title
Soper, Steven A Committee Chair
Murray, Kermit Committee Member
Stanley, George G Committee Member
Keywords
  • microfluidics
  • CTCs
  • breast cancer
Date of Defense 2011-10-10
Availability unrestricted
Abstract
Breast cancer cells overexpress EpCAM that can be used as a marker to select breast cancer CTCs from heterogeneous clinical samples, even when present in low abundance. This study used MDA-MB-231 cells which have a low expression level of EpCAM (1.7 x 103 binding sites per cell.3) Herein, we will report on the use of antiEpCAM antibodies immobilized onto the surface of a capture bed poised within a PMMA microchip that was fabricated into an HTMSU used for selection of breast cancer CTCs seeded into a buffer solution. The microchannel walls within the capture bed were UV modified for 5, 10, 15 and 20 min and covalently decorated with antiEpCAM antibodies of varying concentrations (0.25, 0.5, 1.0 and 2.0 μg/μL) using an EDC-NHS coupling reaction. The HTMSU capture bed consisted of 51 high-aspect ratio microchannels (35 μm width 150 μm depth) that were replicated in PMMA, from a metal mold master. Cultured breast cancer CTCs were fluorescently stained using a fluorescent isothiocyanate staining kit. The microfluidic device was infused with the fluorescently stained breast cancer CTCs at a flow rate of 27.5 uL/min. The capture of the cells was observed and visually quantified using a Zeiss inverted optical microscope.

Percent recoveries of breast cancer CTCs were obtained using HTMSUs that were UV modified for 5, 10, 15 and 20 min. The optimal percent recovery was 81.6% after 10 min of UV modification. Varying concentrations (0.25, 0.5, 1.0 and 2.0 μg/μL) of antiEpCAM antibodies were used and percent recoveries as low as 71.4% and as high as 81.6% were achieved using 0.25 and 1 μg/μL of antiEpCAM antibody respectively.

Lastly, another method for decorating the microfluidic channels with antiEpCAM antibodies was explored. Using 2 min exposure to plasma oxidation, antiEpCAM antibodies were immobilized to the microchannel walls. Following the same cell selection protocol used for the UV modified HTMSU, a mean percent recovery of 50% was obtained. The combination of carboxylate, oxides and anhydride groups formed due to plasma oxidation, instead of exclusively carboxylate groups that form due to UV modification could be a contributing factor to this lower recovery.

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