Compensated Interferometric Reader (CIR),developed by Bornhop group at Vanderbilt university is a label free interferometric biosensor that measures molecular interaction in terms of refractive index (RI) changes with no immobilization or derivitization required for any of the two interacting partners. The mix-and-read free solution assay which allows high degree of flexibility of structures in solution uses droplet generation for higher throughput efficiency from an automated sample introducer to a microfluidic cell (glass capillary). In contrast to back scattering interferometry (BSI) developed by the same group, CRI enables simultaneous differential RI measurements within nano-pico liter volume at an undetectable temperature compensation of 8 degreeC with a sensitivity of 10^-7 RIU. The compensation is possible due to elongating the laser beam along the center of axis of the capillary and measuring the shift of interference pattern from the two adjacent regions of samples interrogated simultaneously by the same laser. The interferometric reader consists of an inexpensive diode laser, two beam conditioning optics, a glass capillary and a CCD array- thus resulting a cost effective, less complex and highly sensitive biosensor reported so far. We have adapted CIR to reveal allosteric and active-site binding towards identifying native and novel chemistries for Lysophospholipids (LPs) receptor modulation, which are having increased medicinal importance in drug-discovery platform. Available classical techniques require non-biological perturbations like labeled ligands and vast protein over-expression and therefore, it remains unknown how native LPs bind to their receptors. We plan to screen the known chemical lipid species, particularly LP’s of varying acyl chain length and saturation, with the six lysophosphatidic acid (LPA) and five sphingosine 1-phosphate (S1P) receptors available. This study can be extended to compare lipid delivery under free vs. carrier-bound conditions since there are a range of carrier proteins or chaperones (albumin, ApoM) that bind to LPs, but their molecular interactions with receptors are poorly understood. Using CIR, the most biologically relevant binding constants (Kd) for each LP species will be determined which will advance our basic understanding of LP receptors by quantifying their binding affinities with various lipid species and their importance in receptor signaling processes.

Basic roles of lysophospholipid receptor signaling studied by compensated interferometric reader (CIR)

2019 Fall National Meeting

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