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