Evolution of “On-Barcode” Luminescence Oxygen Channeling Immunoassay by Exploring the Barcode Structure and the Assay System

[Image: see text] The multiplexed luminescence oxygen channeling immunoassay (multi-LOCI) platform we developed recently that combines conventional LOCI and suspension array technology is capable of realizing facile “mix-and-measure” multiplexed assays without tedious washing steps. However, previous work lacks comprehensive studies of the structure–performance relationship of the host–guest-structured barcode, which may obstruct the evolution and further translation of this exciting new technology to practical applications. Accordingly, this work revealed that polyelectrolyte interlayers played a crucial role in tuning the packing density of guest acceptor beads (ABs). More interestingly, we noticed that “sparse” barcodes (barcodes with low ABs packing density) exhibited comparable assay performance with “compact” ones (barcodes with high ABs packing density). The high robustness of barcodes allows for multi-LOCI to be a more universal and flexible assay platform. Furthermore, through optimization of the assay system including the laser power, as well as the concentrations of donor beads and biotinylated detection antibodies, the multi-LOCI platform showed a significant improvement in sensitivity compared with our previous work, with the limit of detection decreasing to as low as ca. 1 pg/mL. Impressively, multi-LOCI that enabled simultaneous detection of multiple analytes exhibited comparable sensitivity with the classical single-plexed LOCI, due to the ingenious structural design of the multi-LOCI barcode and the unique “on-barcode” assay format.