Multiplexed flow assay based on absorption-encoded beads with compact read-out

Abstract

For advanced diagnostics there is a strong need to simultaneously detect and quantify multiple biomarkers in a sample. Many different multiplexed assay techniques exist. Particular interesting are so-called flow assays which are based on functionalized micro-beads. Such assays consist of different types of beads which can be distinguished e.g., by size, emission intensity or emission color. Each bead type is functionalized for a specific target. During read-out first the bead type has to be determined and then how much analyte is bound to it. This procedure allows determining the analyte concentration for each target. Unfortunately, current read out instruments are quite complex since multiple excitation sources, complex filter arrangements and multiple detections channels are required. We suggest a new multiplexed bead assay. This assay is based on beads which have different absorption spectra rather than different size or emission spectra. Consider a set of beads which are fabricated by including different mixtures of dyes exhibiting different absorption spectra. Each dye can be separately excited with a different excitation source. Such an assay is perfectly suited to be interrogated with a read-out technique which uses multiple intensity modulated excitation sources. These excitation sources emit at a different wavelength and are modulated at different frequencies. This technique allows measuring the fluorescence emission stimulated by the different excitation lasers with simple optics and a single large area detector. The fluorescence stimulated by different excitation sources will be frequency-encoded and can, therefore, be separated out of the time modulated detector signal during the data evaluation. The combination of this read-out technique with absorption-encoded micro beads allows realizing multiplexed bead assays with a low-cost, compact and rugged instrument.