Label-free molecular interaction determinations with nanoscale interferometry.
Quantification of protein-protein and ligand-substrate interactions is central to understanding basic cellular function and for evaluating therapeutics. To mimic biological conditions, such studies are best executed without modifying the proteins or ligands (i.e., label-free). While tools for label-free assays exist, they have limitations making them difficult to fully integrate into microfluidic devices. Furthermore, it has been problematic to reduce detection volumes for on-channel universal analyte quantification without compromising sensitivity, as needed in label-free methods. Here we show how backscattering interferometry in rectangular channels (BIRC) facilitates label-free studies within picoliter volumes. The simple and unique optical train was based on rectangular microfluidic channels molded in poly(dimethylsiloxane) and low-power coherent radiation. Quantification of irreversible streptavidin-biotin binding and reversible protein A-human IgG Fc molecular interactions in a 225 pL detection volume was carried out label-free and noninvasively. Detection limits of 47 x 10(-15) mol of biotin reacted with surface-immobilized streptavidin were achieved. In the case of reversible interactions of protein A and the Fc fragment of human IgG, detection limits were determined to be 2 x 10(-15) mol of IgG Fc. These experiments demonstrate for the first time that (1) high-sensitivity universal solute quantification is possible using interferometry performed within micrometer-sized channels formed in inexpensive PDMS chips, (2) label-free reversible molecular interaction can be studied with femtomoles of solute, and (3) BIRC has the potential to quantify binding affinities in a high-throughput format.
Markov
Dmitry A
DA
Department of Chemistry, Vanderbilt University, VU Station B 351822, Nashville, Tennessee 37235-1822, USA.
Swinney
Kelly
K
Bornhop
Darryl J
DJ
D001710:Biotin / Q000737:chemistry; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D007074:Immunoglobulin G / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D018625:Microscopy, Atomic Force; D008857:Microscopy, Interference / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D012828:Silicones / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry
D004129:Dimethylpolysiloxanes; D007074:Immunoglobulin G; D011506:Proteins; D012828:Silicones; D013205:Staphylococcal Protein A; C013830:baysilon; D001710:Biotin; D019809:Streptavidin