Self-referenced assay method for photonic crystal biosensors: Application to small molecule analytes

Using an image-based method for label-free detection of biochemical binding density distribution on the surface of a photonic crystal biosensor mbedded within standard format multiwell microplates, a new method for automatic referencing of assay errors due to variability in the bulk efractive index of the analyte test sample is demonstrated. The new method involves application of the immobilized ligand upon the biosensor urface as a small spot, so that uncoated regions of a microplate well may serve as an accurate reference for the “active” regions containing the igand. Due to the high spatial resolution of the image-based detection approach, each well in the microplate may be represented by hundreds of ndependent measurements of biochemical binding density, as measured by the shift in reflected wavelength from the photonic crystal biosensor. A inear plot of detected analyte density as a function of immobilized ligand density is constructed, in which the slope represents the ligand/analyte ffinity and the intercept represents common-mode error effects. Streptavidin–biotin is used as a ligand–analyte model system to demonstrate he ability of this method to separate intentionally introduced bulk refractive index errors from the detection of a small molecule analyte. This eferencing approach will be important in the context of small molecule drug compound library screening, where drug compounds within a large ibrary are often suspended in solutions of inconsistent bulk refractive index. 2006 Elsevier B.V. All rights reserved.