1680 IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 13, NO. 6, NOVEMBER/DECEMBER 2007 Molecular Interferometric Imaging for Biosensor Applications Ming Zhao, Xuefeng Wang, Gregory M. Lawrence, Patricio Espinoza, and David D. Nolte (Invited Paper) Abstract—Molecular interferometric imaging (MI2) is a common-path interferometric imaging technique for detecting protein binding to surfaces. The experimental metrology limit is 10 pm/pixel longitudinal resolution at 0.4-μm diffraction-limited lateral resolution, corresponding to 1.7 attogram of protein, which is only 8 antibody molecules per pixel, near to single-molecule detection. The scaling mass sensitivity at the metrology limit is 5 fg/mm. We demonstrate a protein microarray application in a 128-multiplex immunoassay. Assay applications include prostate specific antigen (PSA) at a detection limit of 60 pg/mL and the cy- tokine interleukin-5 (IL-5) at a detection limit of 50 pg/mL. Real- time binding assays using MI2 enable the study of reaction kinetics of antibodies exposed to antigen, and the binding of antibody Fc regions to protein G. Index Terms—Biomedical optics, biosensor, immunoassay, inter- ferometry, label-free, protein microarray. I. INTRODUCTION I N THE field of label-free biosensors [1], there is currently a gap between the number of analytes that can be mea- sured in a biological sample using existing systems, compared to the number of measurements required to understand the pro- teomic signature of health and disease. The measurement prob- lem of proteomics is immense. A single cell can have as many as 10 000 expressed proteins, and each protein interacts with three or four others, on average, in cascaded networks of protein interactions [2], [3]. Optical biosensors [4] have a potential ad- vantage for this problem because of the intrinsic parallelism of light that allows multiple channels to be illuminated and de- tected simultaneously. Some imaging biosensors rely on this parallelism to some degree, but most do not tap the full resource that this parallelism represents. In principle, it is possible to have over a million independent optical modes per square millimeter. There are two challenges to utilizing this millionfold resource for optical biosensors. The first challenge is the preparation of the assay. In the case of mi- croarrays, the individual spatial modes need to be patterned with Manuscript received September 19, 2007; revised October 15, 2007. This work was supported by QuadraSpec, Inc. through the Purdue Research Foundation. M. Zhao, X. Wang, and D. D. Nolte are with the Department of Physics, Purdue University, West Lafayette, IN 47907 USA (e-mail: zhaom@physics. purdue.edu; wang137@physics.purdue.edu; nolte@physics.purdue.edu). G. Lawrence and P. Espinoza are with QuadraSpec, Inc., West Lafayette, IN 47906 USA (e-mail: glawrence@quadraspec.com; PEspinoza@quadraspec. com). Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identifier 10.1109/JSTQE.2007.911002 recognition molecules, either antibodies, peptides, or proteins. This represents a technological challenge that still has far to go. The second challenge, which is the readout of the million opti- cal modes, has already been partially met. High-end pixel arrays today have 10 million pixels, which is sufficient to measure the full-mode density of an area 3 mm on a side. An important part of this challenge is the generation of a robust signal proportional to the amount of protein bound to the surface of the biosensor within a single pixel. The most robust means of performing direct optical detection is common- path phase-quadrature interferometry [5], in which a signal carrying optical phase information from thin protein layers is combined with a reference wave that has a fixed relative phase of 90 ◦ to produce an intensity shift proportional to the protein-induced phase. Interferometry performs the function of a phase-to-intensity transducer. The common-path approach establishes and maintains quadrature independent of mechan- ical vibrations, making the system highly stable and of low noise. Common-path interferometric approaches to protein detec- tion have used spinning-disk interferometry in the form of laser scanning on the biological compact disk (BioCD) [6]. The BioCD uses single-mode illumination by a focused laser that scans bound protein on a spinning disk. There are sev- eral approaches to establishing and maintaining phase quadra- ture on a BioCD, including microdiffraction [7], [8], adaptive optics [9], phase contrast [10], [11], and in-line [12]. The ad- vantages to high-speed sampling on a spinning platform include suppression of temporal 1/f noise, fast scan times, large-area mi- croarrays, and high multiplexing. Conversely, the single-mode illumination used on the BioCD does not access the intrin- sic parallel advantage of optical detection afforded by pixel arrays. In this paper, we describe a full-field protein imaging ap- proach called molecular interferometric imaging (MI2) that uti- lizes the full parallel advantage of a pixel detector while rely- ing on common-path in-line phase quadrature identical to the in-line BioCD. The signal and reference waves are produced locally from a single optical mode and share the same optical path to maintain a stable relative phase. The shot-noise-limited sensitivity of the technique approaches the single-molecule range for moderate molecule sizes. The theoretical operation of MI2, based on in-line quadrature interferometry, is described in Section II, followed in Section III with the technical details of the microscope system and silicon substrates. Immunoassays are described in Section IV with applications to prostate-specific 1077-260X/$25.00 © 2007 IEEE