Label-free and dynamic detection of biomolecular interactions for high-throughput microarray applications.

Direct monitoring of primary molecular-binding interactions without the need for secondary reactants would markedly simplify and expand applications of high-throughput label-free detection methods. A simple interferometric technique is presented that monitors the optical phase difference resulting from accumulated biomolecular mass. As an example, 50 spots for each of four proteins consisting of BSA, human serum albumin, rabbit IgG, and protein G were dynamically monitored as they captured corresponding antibodies. Dynamic measurements were made at 26 pg/mm(2) SD per spot and with a detectable concentration of 19 ng/ml. The presented method is particularly relevant for protein microarray analysis because it is label-free, simple, sensitive, and easily scales to high-throughput.

Interferometric Reflectance Imaging Sensor (IRIS) for Molecular Kinetics with a Low-Cost, Disposable Fluidic Cartridge.

The determination of kinetic information and appropriate binding pairs is fundamental to the proper optimization and selection of ligands used in immunoassays, diagnostics, and therapeutics. However, the ability to estimate such parameters in a multiplexed and inexpensive format remains difficult and modification of the ligand is often necessary. Here, we detail the methods and materials necessary to evaluate hundreds of unlabeled ligands simultaneously using the interferometric reflectance imaging sensor (IRIS). The incorporation of a low-cost fluidic cartridge that integrates on the top of the sensor simplifies reagent handling considerably.

Resonant Cavity Imaging: A Means Toward High-Throughput Label-Free Protein Detection.

The resonant cavity imaging biosensor (RCIB) is an optical technique for detecting molecular binding interactions label free at many locations in parallel that employs an optical resonant cavity for high sensitivity. Near-infrared light centered at 1512.5 nm couples resonantly through a Fabry-Perot cavity constructed from dielectric reflectors (Si/SiO(2)), one of which serves as the binding surface. As the wavelength is swept using a tunable laser, a near-infrared digital camera monitors cavity transmittance at each pixel. A wavelength shift in the local resonant response of the optical cavity indicates binding. Positioning the sensing surface with respect to the standing wave pattern of the electric field within the cavity controls the sensitivity with which the presence of bound molecules is detected. Transmitted intensity at thousands of pixel locations is recorded simultaneously in a 10 s, 5 nm scan. An initial proof-of-principle setup has been constructed. A test sample was fabricated with 25, 100-mum wide square features, each with a different density of 1-mum square depressions etched 12 nm into the SiO(2) surface. The average depth of each etched region was found with 0.05 nm rms precision. In a second test, avidin, bound selectively to biotin conjugated bovine serum albumin, was detected.