A Strategy of NIR Dual-Excitation Upconversion for Ratiometric Intracellular Detection

Intracellular detection is highly desirable for biological research and clinical diagnosis, yet its quantitative analysis with noninvasivity, sensitivity, and accuracy remains challenging. Herein, a near-infrared (NIR) dual-excitation strategy is reported for ratiometric intracellular detection through the design of dye-sensitized upconversion probes and employment of a purpose-built NIR dual-laser confocal microscope. NIR dye IR808, a recognizer of intracellular analyte hypochlorite, is introduced as energy donor and Yb,Er-doped NaGdF₄ upconversion nanoparticles are adopted as energy acceptor in the as-designed nanoprobes. The efficient analyte-dependent energy transfer and low background luminescence endow the nanoprobes with ultrahigh sensitivity. In addition, with the nonanalyte-dependent upconversion luminescence (UCL) excited by 980 nm as a self-calibrated signal, the interference from environmental fluctuation can be alleviated. Furthermore, the dual 808/980 nm excited ratiometric UCL is demonstrated for the quantification of the level of intracellular hypochlorite. Particularly, the intrinsic hypochlorite with only nanomolar concentration in live MCF-7 cells in the absence of exogenous stimuli is determined. Such an NIR dual-excitation ratiometric strategy based on dye-sensitized UCL probes can be easily extended to detect various intracellular analytes through tailoring the reactive NIR dyes, which provides a promising tool for probing biochemical processes in live cells and diagnosing diseases.