Microsphere sedimentation arrays for multiplexed bioanalytics

Abstract

A new measurement platform to analyze multiple assays in parallel in a single well of a standard microplate is presented. The approach is based on optically addressable encoded microbeads as carriers for a series of bioassays, a fluorescence microscope equipped with a LED as excitation source, a fast-gateable CCD-camera and automated image analysis. Bead arrays are suspended in the analyte solution and targets are bound to receptors immobilized at the bead surface. Gravitational or magnetical sedimentation of the beads to the transparent bottom of the microplate leads to in situ formation of randomly ordered arrays. Beads are encoded by three features: size, luminescence intensity and luminescence decay time. Luminescence lifetime imaging in the microsecond range is introduced as a new technique for bead identification. Twenty-seven distinguishable bead classes were produced and characterized. The coefficients of variation were 5.4% for size, 9% intensity and 1.3% for decay time. Based on these results, the number of potential codes is calculated to be about 140. Multiplexed DNA assays at detection limits of 2 nM were carried out to demonstrate the feasibility of the concept presented here.