Rapid Label-free Protein Detection Arrays on Coated Silicon Wafers

Abstract

We are developing label-free Arrayed Imaging Reflectometry (AIR) for rapid and multiplexed protein detection. AIR is based on the high dynamic range in reflected optical intensity near a point of zero reflectance on an antireflection coated substrate. The reflectance is therefore highly sensitive to changes in film thickness, allowing direct detection of molecular binding when appropriate probe molecules are immobilized on the surface. The simplest implementation of AIR uses a 633 nm HeNe laser and a silicon wafer substrate coated with ∼1400 Å of SiO₂. This system has a reflectance zero for s-polarized HeNe light incident at ∼70°. This interference film is then functionalized with probe molecules designed to bind to a specific target, and this binding can be detected with high sensitivity in the reflectance signal. By expanding the laser beam and collecting the reflected signal with a CCD camera, large arrays of detection spots can be imaged simultaneously. Spot intensity increases relative to the amount of target binding and the target solution concentration can then be calculated. We have demonstrated the detection of the Enteropathogenic E Coli membrane protein Intimin at levels below 10 pM using receptor molecule Tir as a probe, and are currently evaluating various clinical targets using more common antibody probes.