Interferometric detection of proteins

Ming Zhao, Purdue University


We employ common-path phase quadrature interferometry to measure optical phase shifts produced by biomolecules on a solid substrate, and apply it in immunoassay applications. Common-path interferometry produces a local reference wave that shares the same optical path with the signal wave, making the system ultra stable to mechanical vibrations or motions. Phase quadrature interference converts optical phase modulation caused by biomolecules into an intensity shift that is measured in the far-field. Two interferometric detection schemes of biomolecules are developed. The first is spinning-disc interferometry – the BioCD, which uses a laser beam to scan across a spinning disc carrying biomolecules. High-speed scanning moves the detection frequency far from 1/f noise of the system, providing a 50 dB suppression of the noise floor compared to static detection, which enables highly sensitive measurement of captured molecules on the surface. Phase-contrast and in-line quadrature classes of BioCD are described. Phase-contrast SDI detects edge diffraction from patterned protein, producing a signal proportional to the derivative of the surface height profile. In-line SDI detects reflectance change caused by the presence of a biolayer that is proportional to the height of the biolayer. The high throughput, high sensitivity and potential for high multiplexing makes the BioCD a potential platform for label-free multiplexed immunoassays. A related interferometric detection technique that is performed using a microscope is molecular interferometric imaging (MI2). MI2 is based on inline quadrature interference and uses parallel data-acquisition of a pixel array detector, taking full advantage of the parallelism of light. Illumination background normalization is used to remove spatial 1/f noise of illumination and obtain shotnoise or surface roughness limited measurements of surface topology. At the metrology limit, a height resolution of 15 pm per 0.4 micron pixel is achieved with a scaling mass sensitivity of 7 fg/mm, and a molecular resolution of about 12 IgG antibody molecules. MI2 is capable of measuring real-time binding between biomolecules under active flow conditions, with a dynamic range of over 1000 at a scaling mass sensitivity of 1 pg/mm. Real-time binding measurements were used to extract crucial information in understanding the dynamics and reaction kinetics between biomolecules.




Nolte, Purdue University.

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