Label-free Optical Detection of Molecular Interactions by Molecular Interferometric Imaging
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Label-free Optical Detection of Molecular Interactions by Molecular Interferometric Imaging Ming Zhao1, Xuefeng Wang1 and David. D. Nolte1 1 Department of Physics, Purdue University, 525 Northwestern Avenue, West Lafayette, IN 47906, U.S.A. ABSTRACT Molecular interferometric imaging (MI2) is a label-free optical biosensor that combines common-path interferometry with shot-noise limited characteristics of a CCD array detector to detect protein binding to surfaces. In the metrology limit, it has achieved roughness-limited surface height resolution of 15 pm per 0.4 micron pixel, corresponding to a scaling mass sensitivity of 7 fg/mm, and a molecular resolution of about 15 IgG molecules per pixel. We have applied MI2 to detect cytokine interleukin-5 at a concentration detection limit of 50 pg/mL with a sandwich immunoassay. Real-time binding assays with MI2 enable the study of reaction kinetics, with a scaling mass sensitivity of 2 pg/mm under 7x magnification. Real-time MI2 measurements of anti-rabbit IgG against rabbit IgG were compared with results from surface plasmon resonance, with identical association rate constants at 5x103 M-1sec-1. INTRODUCTION Direct optical detection of biomolecules relies on virtual optical dipole transitions that are the origin of refractive index in bulk materials. In the single-molecule limit, the molecule causes dipole scattering with an associated phase lag. During the past decades, many label-free optical detection systems have been developed, the majority of which being evanescent wave approaches such as surface plasmon resonance1, waveguides2 and ring resonators3. These approaches rely on resonance to increase signal produced by biomolecules, but at the cost of difficult substrate fabrication and complex optical read-out to maintain critical parameters close to the resonance condition. We take the alternative approach by working with non-resonant structures and maximum noise floor suppression to increase detection signal-to-noise. Molecular interferometric imaging (MI2) combines ultra-stable common-path interferometry with shotnoise limited detection by a CCD camera, and applies shearing interferometry4 to suppress spatial 1/f noise and achieve shot-noise limited detection of the surface profile. This approach can be used in a conventional reflective light microscope with conventional light sources and Koehler illumination and color filters. When applied as a surface metrology, it has achieved a mass sensitivity limit of about 15 antibodies molecules in a single 0.4 micron pixel, close to single molecule detection. When used in a real-time binding measurement, it has a mass sensitivity of 2 pg/mm2 comparable to conventional SPR sensitivities, but can be applied across broad areas without the need to maintain or track resonance conditions. THEORY Molecular interferometric imaging is based on inline-quadrature interference5 combined with far-field optical imaging. The substrate is a simple thermal oxide on silicon with a thickness at or near the condition of phase-quadr
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