Photoswitch-Based Fluorescence Encoding of Microspheres in a Limited Spectral Window for Multiplexed Detection

Fluorescence barcoding with multicolor fluorophores is limited by spectral crowding. Herein, we propose a fluorescence encoding method in a single-color channel with photoswitches. The photochromic naphthopyran was used to mediate the fluorescence of polystyrene microspheres through resonance energy transfer. The initial fluorescence intensity (F₀) and the fluorescence after UV light activation (F/F₀) were combined to generate hundreds of 2-dimensional barcodes. The coding capacity was further expanded with the different chemical kinetics of the photoswitches. The photoswitch-based fluorescence barcodes were applied to simultaneously and selectively detect the DNA sequences of COVID-19 (with related mutations) as a proof-of-concept for real applications. The compatibility with the state-of-the-art fluorescence microscopes and simple encoding and decoding make the method very attractive for multiplexed and high-throughput analyses.