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Article 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 (O) nanogenerator of porphyrin-metal organic framework (pMOF) and the other tagged using an upconversion nanoparticle (UCNP) nanoreporter with luminescence caged by O-cleavable quenchers. Formation of sandwiched complex between target protein and antibody pairs mediates close proximity between O-generator and UCNP reporter, enabling efficient diffusion of O 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.
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