A self-referencing method for microplate label-free photonic-crystal biosensors

To improve the accuracy of optical biosensors, a reference sensor is often incorporated in close proximity to the sensor performing the detection function in order to compensate for common-mode error sources that result in a detected signal but are not a result of biochemical binding to the sensor surface. These error sources include thermal drift, the refractive index of the test sample, and nonspecific binding. Because photonic-crystal biosensors do not allow lateral propagation of light along their surface, spatial images of biochemical-binding density may be generated in which each individual pixel of the image represents an independent sensor. Using this capability, a small region of a photonic-crystal surface can effectively contain a large number of "active" and "reference" pixels when the immobilized ligand is applied to only a portion of the imaged region. In this paper, the use of a photonic-crystal optical biosensor assay protocol and data-analysis method that results in elimination of common-mode error sources from the detected signal is described. When applied to biosensors embedded within a standard 96-well microplate format, the new method enables the use of nanoliter-scale quantities of immobilized ligand reagent, is insensitive to immobilized ligand nonuniformity, and allows rapid analysis of many biochemical assays in parallel.