Protein - protein and ligand - substrate interactions are central to understanding basic cellular function and for evaluating therapeutics. However, the tools available to quantify these interactions without modifying the proteins or ligands have important limitations. Surface immobilization of the substrate, receptor, or protein or the attachment of a signaling fluorophore influences molecular interactions. It is desirable to eliminate the need for the surface chemistry normally used in binding assays, since it presents persistent problems including: complicated kinetics due to surface bound targets, lack of durability and longevity, high cost, non-specific binding, and difficulty in quantification of the immobilized targets. The ability to perform pure liquid - phase molecular binding analysis in a mu-TAS format would eliminate many of the problems that arise from the surface chemistry, while facilitating small volume samples to be studied. Preliminary observations presented here indicate that molecular interactions, such as protein - protein or ligand - substrate binding, can be studied in the absence of these perturbations, at high sensitivity, in picoliter volumes and in free-solution using on-chip interferometric backscatter detection (OCIBD). The proposed technology uses a simple and inexpensive optical train to facilitate these homogeneous, label-free measurements. OCIBD monitors minute refractive index changes within a microfluidics channel formed in silica or PDMS. OCIBD performed in a non-modified PDMS channel has recently facilitated quantification of reversible and irreversible protein - protein binding without a fluorescent label, the direct measurement of DNA interactions and fluorescent tag perturbations on delta/G. These determinations were performed in picoliter volumes and at attomolar levels and confirmed by Isothermal Titration Calorimetry (ITC). It has been possible to quantify reaction kinetics and binding affinities (Ko) for protein-IgG label-free in free-solution, with detection limits of 4 attomoles for IgG. Recently it has been possible to detect 10,800 molecules of IL-2 with backscattering interferometry. This proposal expands on these results, culminating a multiplexed OCIBD for homogeneous (free-solution) molecular interaction assays.
