Ultrasensitive Digital Immunoassay in Homogenous Format by Singlet‐Oxygen Activated Up‐conversion Luminescence
Ruiying Peng, Yan‐Li Ji, Daian Chen, Zhan Wu, Jishan Li, Zhaohui Li, Jian‐Hui Jiang
Abstract
Abstract New diagnostic tools for accurately detecting extremely low levels of protein biomarkers in the bloodstream are essential for a wide range of clinical applications. We develop a singlet‐oxygen activated up‐conversion luminescence assay (SOLA) that enables ultrasensitive digital immunometric detection of protein biomarkers in homogenous format with no wash steps. This approach designs a pair of labeled antibodies, one labeled with a singlet‐oxygen ( 1 O 2 ) nanogenerator of porphyrin‐metal organic framework (pMOF) and the other tagged using an upconversion nanoparticle (UCNP) nanoreporter with luminescence caged by 1 O 2 ‐cleavable quenchers. Formation of sandwiched complex between target protein and antibody pairs mediates close proximity between 1 O 2 ‐generator and UCNP reporter, enabling efficient diffusion of 1 O 2 around the reporter to activate its luminescence through cleaving off the quenchers. This approach is demonstrated to be capable of activating detectable fluorescence from a single UCNP reporter with a single‐molecule protein target. A digital homogeneous immunoassay strategy is also developed for ultrasensitive protein detection, allowing a single‐step, no‐wash format for digital protein detection with high signal‐to‐background ratio, wide dynamic range and atto‐molar detection limit. The SOLA assay may provide an invaluable paradigm for developing highly accessible and ultrasensitive tools for diagnostics and biomarker discovery.