Multicolor Quantum Dot Nanobeads Based Fluorescence-Linked Immunosorbent Assay for Highly Sensitive Multiplexed Detection

Improvement of detection sensitivity and achievement of multiplexed detection using a fluorescence-linked immunosorbent assay (FLISA) platform are necessary developments to meet increasing clinical demands. Herein, a novel multicolor quantum dot nanobead (QDNB)-based FLISA platform for highly sensitive multiplexed detection of lung cancer tumor markers has been established. In this proposed platform, three-color QDNBs (green, yellow and red) act as fluorescent labels, completely eliminating energy transfer between different color QDNBs to allow multiplexed detection, as well as achieving high detection sensitivity through use of a fluorescence amplification strategy. We demonstrated three-plex detection of model lung cancer tumor markers (SCCA, CYFRA21-1 and CEA) using this QDNB-based FLISA platform, achieving 100-fold improvement in detection sensitivity compared to R-phycoerythrin (PE)-based FLISA approaches because of signal amplification caused by QDNBs. Moreover, clinical validation experiments performed on human serum samples demonstrated that results obtained using QDNB-based FLISA are consistent with those obtained using the current clinical goldstandard method, the electrochemiluminescence immunoassay (ECLIA). Therefore, the strategy of using multicolor QDNBs as fluorescent labels creates a versatile route by which to improve detection sensitivity and allow construction of multiplexed fluorescent biosensors.