Novel Devices and Protocols Enabling Isolation and Enumeration of Low Abundant Biological Cells from Complex Matrices
The dimensions of microfluidic devices closely parallel those of biological cells; thusly, they are excellent platforms for the speciation, transport, manipulation, and analysis of cells. Electrokinetic transport of Escherichia coli and Saccharomyces cerevisiae was evaluated in microfluidic devices fabricated in pristine and UV-modified poly(methylmethacrylate) and polycarbonate. The magnitude and direction of transport of the cells was dictated by the buffer composition, conduit surface chemistry, and intrinsic cellular electrical properties. Bakers yeast in all devices migrated toward the cathode, because of their smaller electrophoretic mobility compared to the electroosmotic flow of the polymer. E. coli cells suspended in 20 mM PBS migrated toward the anode, which indicated that the apparent mobility of the E. coli cells changed direction at higher ionic strengths. The observed differential migrations were exploited to sort cells, whereby judicious choice of the buffer concentration and the polymeric material in which the cell sorting was performed was controlled, allowed for cell enumeration via laser-based backscatter signals. A novel microfluidic device that selectively and specifically isolated the exceedingly small numbers of circulating tumor cells (CTCs) from whole blood through a monoclonal antibody (mAB) mediated process by sampling large input volumes (?1 mL) of whole blood directly in short time periods (<37 min) was designed, manufactured and implemented. Upon processing, the CTCs were concentrated into small volumes (190 nL) and the number of cells captured were read without the need for labeling by using an integrated conductivity sensor following an enzyme mediated release of the captured CTCs from the microchannel surface. The microchannel walls were covalently decorated with mABs directed toward breast cancer cells that over-express epithelial cell adhesion molecules. The released CTCs were then enumerated on-device using conductivity detection with 100% detection efficiency and exquisite specificity for CTCs. The CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. Extension of the technique to environmental samples was performed using analogously patterned polyclonal anti-E. coli O157:H7 antibodies directed towards the virolent bacterial strain were used to isolate the enterohemorrhagic bacteria while E. coli K12 were not adsorbed to the antibody containing surface.
Advisor:Mark Benfield; Steve Watkins; Steven A. Soper; David Spivak; Robert Cook
School:Louisiana State University in Shreveport
School Location:USA - Louisiana
Source Type:Master's Thesis
Date of Publication:07/15/2008