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

Abstract

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.dynamic monitoring ͉ optical biosensor ͉ protein microarray ͉ immunoassay ͉ interference

Integrated imaging instrument for self-calibrated fluorescence protein microarrays

“…Although these items are not trivial, they have been addressed for similar technologies. 16,30 The modular unit for existing fluorescence readers would modify the light source. Wide-field fluorescence microscopes typically use one of the four types of light sources: a xenon arc lamp, a mercury-vapor lamp, a high-power LED, or a supercontinuum source.…”

“…The glass top-layer facilitates the use of well-established surface chemistries. [23][24][25] In addition, the layered design produces an interference signal to enable IRIS measurements 16,17 and shows fluorescence enhancement factors beyond available glass substrates. 26 Cretich and Reddington et al demonstrate an approach to assay development through the implementation of a 2 thickness SiO 2 /Si slide, which is optimized for both IRIS (500 nm SiO 2 ) and fluorescence enhancement (100 nm SiO 2 ) in a single experiment.…”

“…16 A more simplistic system with four unpolarized discrete LEDs, to circumvent imaging issues associated with wide-field laser imaging like speckle, has shown to achieve the same sensitivity with a 500 nm SiO 2 layer and on-chip fiducial marks for intensity monitoring. 17,28 The modeling for the tunable laser can be described by the Fresnel reflection of a two-interface system when assuming a near-normal (paraxial approximation), single-wavelength illumination: 29…”

“…Utilizing optical interference and a silicon oxide on silicon (SiO 2 /Si) slide, IRIS has shown multiplexed, high-throughput, high sensitivity, and dynamic detection capabilities for molecular surface binding. 16,17 Additionally, the layered structure required for IRIS does not have a detrimental effect on the fluorescence readout, and the structure can be adjusted to enhance the fluorescence signal beyond glass slides.…”

Serum tumor marker detection on PEGylated lipid membrane using biosensor based on total internal reflection imaging ellipsometry

“…Microarray biosensor with multiple advantages, such as low sample consumption, fast response time and high throughput, [1][2][3][4] appears to be a promising alternative. A novel microarray biosensor based on total internal reflection imaging ellipsometry (TIRIE) was recently developed for kinetic measurement of biomolecular interaction, which is integrated by imaging ellipsometer and microfluidic reactor system [5,6].…”