Abstract

Nanoliter-Scale Protein Quantification in Biological Matrices using DNA Annealing and Melting

Christopher Easley, C. Harry Knowles Professor, Auburn University

Recent breakthroughs in cellular co-culture and organ-on-a-chip platforms have revealed that the scale and operation in microfluidic systems is well-matched with that of organized biological tissues.  As shown by our group and others, microfluidic devices permit unique and valuable approaches for hormone secretion sampling, particularly from small amounts of endocrine tissue.  These microanalytical systems possess great value in improving our understanding of biology.  Unfortunately, the development of compatible, simple-readout protein assays has lagged behind these fluidic advancements.  To address this need, we have developed several versions of DNA-driven proximity immunoassays that are well-matched with microfluidic sampling volumes, i.e. the picoliter to nanoliter scale.  By coupling our proximity fluorescence assays to a customized nanoliter rotary mixer, we have achieved continuous sampling, mixing times as short as 2.2 s, then detection of proteins at the 100 attomole range—using only direct fluorescence.  Since proximity assays are defined by protein-triggered DNA annealing, thermally-resolved fluorescence readout (standard qPCR instrument) has recently revealed an additional layer of control.  In essence, we can quantitatively translate protein quantities into DNA melting transitions, a method we have termed the thermofluorimetric proximity assay (TFPA).  Precise temperature control allows straightforward discrimination between signal and background, even facilitating removal of autofluorescent background in complex samples such as mouse or human serum.  Finally, validating the scalability of TFPA, we show that merely 1 attomole of protein in 100 pL microchannels can be detected in 20 min.  Because our assays are applicable to any protein with two aptamers or antibody probes, they should allow us to begin tapping the true potential of microfluidic systems for sampling and quantifying hormone secretion from