Non-invasive luminescent monitoring of biological molecules or physicochemical parameters in live cells represents a key approach to understand cell biology, pathology and other biomedical related sciences. However, current intracellular analyses by means of target imaging or non-quantitative fluorescence contrast still face challenges, including the inadequate sensitivity of the probes to the target species, and that accurate quantitative detection in live cells was hardly achieved without a self-calibrated reference.  

In a recent study published in Adv. Sci., the research group led by Prof. CHEN Xueyuan from Fujian Institute of Research on the Structure of Matter of the Chinese Academy of Sciences reported a near-infrared (NIR) dual-excitation strategy for ratiometric intracellular detection. 

The researchers designed IR808-UCNPs-F127 nanoprobes for intracellular detection based on the analyte-dependent energy transfer from NIR dye to Yb,Er-doped NaGdF₄ upconversion nanoparticle. Benefiting from the efficient dye-sensitization and low background luminescence, the nanoprobes exhibited ultrasensitive detection for ClO^- with an LOD of 16.1 nM, which is much lower than that of conventional dye-quenched upconversion luminescence (UCL) probes.  

Furthermore, with the non-analyte-dependent UCL excited by 980 nm as a self-calibrated signal, the interference from environmental fluctuation can be alleviated. The researchers realized the dual 808 nm/980 nm-excited ratiometric UCL for quantification the level of intracellular ClO^- by means of a purpose-built NIR dual-laser confocal microscope.  

The researchers also determined the intrinsic hypochlorite with only nanomolar concentration in live MCF-7 cells in the absence of exogenous stimuli.  

Such a 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.