Development of a micro free flow electrophoresis assay for the continuous quantification of tumor necrosis factor α (TNFα)

Description:
Affinity assays are powerful tools in bioanalysis because of their high sensitivity and selectivity: They can detect very low target concentrations and bind to a specific molecule of interest. However, traditional affinity assays like ELISAs measure a discrete sample at a single timepoint, which is a drawback when studying the fundamentally dynamic systems of the natural world. We are developing micro free flow electrophoresis (µFFE) techniques that will allow us to perform continuous quantification of biological targets using high-sensitivity affinity reagents, providing unique and dynamic information about how biological systems like cells behave over time.

Recent improvements to the design and fabrication of µFFE devices have allowed us to perform µFFE separations in cyclic olefin copolymer, a durable, optically clear, and chemically inert plastic with relatively low adhesion to small molecules, peptides and affinity reagents. These technical improvements allow for simpler and more cost-effective assay development. Initial development focuses on a continuous µFFE assay for the quantification of tumor necrosis factor α (TNFα), a peptide involved in mediating the body’s immune response.

Ultimately, we aim to couple this assay with a perfusion chamber. As mast cells in the chamber respond to stimuli, the efflux will flow into a mixing junction, where a fluorescently labelled affinity reagent binds TNFα. The mixture will enter a µFFE device, where the bound and unbound affinity reagent are separated into streams according to their electrophoretic mobility. The measured intensity of the separated streams will change proportionately as cells increase or decrease TNFα secretion.

Speaker: Cecilia Douma - University of Minnesota
Cecilia Douma is a Chemistry Ph.D. candidate in the Bowser Lab at the University of Minnesota, where her research has focused on microfluidics and nucleic acids. She earned her Bachelor of Arts degree from Luther College in Decorah, IA.