Magnetic Silica‐Coated Fluorescent Microspheres (MagSiGlow) for Simultaneous Detection of Tumor‐Associated Proteins

Abstract

Multiplexed bead assays for solution‐phase biosensing often encounter cross‐over reactions during signal amplification, leading to false positives and high background signals. Current solutions involve complex, custom‐designed equipment or costly setups, limiting their application in simple laboratory environments. In this study, we introduce a straightforward protocol to adapt a multiplexed single‐bead assay to standard fluorescence imaging plates, enabling the simultaneous analysis of thousands of reactions per plate. Our approach focuses on the design and synthesis of bright fluorescent and magnetic microspheres (MagSiGlow) with multiple fluorescent wavelengths serving as detection markers. This imaging‐based single‐bead assay, combined with a scripted algorithm, allows the detection, segmentation, and co‐localization on average of 7500 microspheres per field of view across five imaging channels in less than one second. We demonstrate the effectiveness of this method with remarkable sensitivity (detection limits of 100 pg/mL). This technique showed over 85% reduction in signal cross‐over to the solution‐based approaches after the concurrent detection of tumor‐associated protein biomarkers. This approach promises to significantly advance high throughput biosensing in diverse laboratory settings.