A two bead immunoassay in a micro fluidic device using a flat laser intensity profile for illumination

Abstract

Recently it was demonstrated that a collimated Gaussian beam can be converted to a flattop beam using a Keplerian beam reshaper consisting of two aspheric lenses. Here, using the same optical system, we demonstrate that this flattop profile can be maintained when used in a confocal detection system that focuses the laser beam into a diameter of only 33 µm. The intensity profile of the reshaped beam was determined by imaging the excitation of a constant stream of fluorescein inside a microfluidic device. The resulting shape had a 6.38% RSD intensity across the flat profile when imaged with a CCD camera. This flat illumination profile was then used in a two bead multiplex immunoassay where the colour combination and/or the intensities can be used to determine the presence of an antigen. Detection limits of TNF-α and IL-6 were comparable with standard ELISA immunoassays.