Improving the Sensitivity of Fluorescence-based Immunoassays by Photobleaching the Autofluorescence of Magnetic Beads

In fluorescence-based assays, usually a target molecule is captured using a probe conjugated to a capture surface, and then detected using a second fluorescently labeled probe. One of the most common capture surfaces is a magnetic bead. However, magnetic beads exhibit strong autofluorescence, which often overlaps with the emission of the reporter fluorescent dyes and limits the analytical performance of the assay. Here, we photobleached several widely used magnetic beads and reduced their autofluorescence to 1% of the initial value. We analyzed their photobleaching decay rates and the stability of the photobleaching over a period of two months. The photobleached beads were stable over time and their surface functionality was retained. In a high sensitivity LX-200™ system using photobleached magnetic beads, human interleukin-8 was detected with a 3-fold improvement in detection limit and signal to noise ratio over results achievable with non-bleached beads. Since many contemporary immunoassays rely on magnetic beads as capture surfaces, pre-bleaching the beads may significantly improve the analytical performance of these assays.