Colorimetric resonant reflection as a direct biochemical assay technique

Abstract

A novel approach for the detection of molecular interactions is presented in which a colorimetric resonant diffractive grating surface is used as a surface binding platform. A guided mode resonant phenomenon is used to produce an optical structure that, when illuminated with white light, is designed to reflect only a single wavelength. When molecules are attached to the surface, the reflected wavelength (color) is shifted due to the change of the optical path of light that is coupled into the grating. By linking receptor molecules to the grating surface, complementary binding molecules can be detected without the use of any kind of fluorescent probe or particle label. The detection technique is capable of resolving changes of ∼0.1 nm thickness of protein binding, and can be performed with the grating surface either immersed in fluid or dried. The readout system consists of a white light lamp that illuminates a small spot of the grating at normal incidence through a fiber optic probe, and a spectrometer that collects the reflected light through a second fiber, also at normal incidence. A single spectrometer reading is performed in several milliseconds, thus it is possible to quickly measure a large number of molecular interactions taking place in parallel upon a grating surface, and to monitor reaction kinetics in real time. It is expected that this technology will be most useful in applications where large numbers of biomolecular interactions are measured in parallel, particularly when molecular labels will alter or inhibit the functionality of the molecules under study. High throughput screening of pharmaceutical compound libraries with protein targets, and microarray screening of protein–protein interactions for proteomics are examples of applications that require the sensitivity and throughput afforded by this approach. In this paper, we describe the theory behind the sensor, the sensor fabrication approach, and measured sensor performance using example assays.