Aqueous arrayed imaging reflectometry as a sensitive platform for real-time biomolecular interaction analysis

Abstract

Arrayed imaging reflectometry (AIR) has been previously demonstrated as a highly sensitive biosensing technique, with picomolar limits of detection observed for certain cytokines and growth factors. However, the implementation of AIR has so far been on dry chip surfaces in an end-point sensor format that precludes real-time monitoring of interactions. The simple substrate format used for dry AIR (a thermally grown oxide film on silicon) is unsuitable for imaging in an aqueous medium due to near grazing angles of incidence required to achieve total destructive interference of reflected light, the foundation of AIR. Hence, a new substrate was identified that would allow practically realizable incidence angles. Here, the substrate proposed for AIR imaging under an aqueous environment has been described, and its ability to detect Angstrom level thickness differences has been demonstrated. This substrate consisted of a two-layer stack on silicon: a silicon nitride film followed by a sputtered oxide film, which produced the total destructive interference condition required for AIR for an operating wavelength of 632.8 nm and an angle of incidence of ~52°. The apparatus used for imaging substrates in an aqueous environment was essentially the same as that used for dry AIR, modified by the incorporation of a flow cell. Several chips were patterned with oxide posts such that the background yielded minimum reflectance while the post heights were varied to test the thickness sensitivity of the new sensor configuration. Detectable contrast from substrates bearing oxide posts with a 0.2 nm thickness was observed using pure water or aqueous solutions of sucrose. This substrate can thus be used to monitor biomolecular interactions in real time with a high sensitivity.