Abstract
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.
Highlights
Noninvasive luminescent monitoring of biological molecules or physicochemical parameters in live cells represents a key approach to understanding cell biology, pathology, and other biomedical-related dual-laser confocal microscope
Such an NIR dual-excitation ratiometric strategy based on dye-sensishort-wavelength excitation hindered their tized upconversion luminescence (UCL) probes can be 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
To meet the demand for intracellular detection with high sensitivity and accuracy, we propose a strategy of NIR dualexcitation ratiometric UCL detection based on dye-sensitized nanoprobes overcoming the limitation aforementioned via a purpose-built NIR dual-laser confocal microscope
Summary
We employed NaGdF4:Yb,Er@NaGdF4:Yb as UCNPs in view of the energy overlap between the excited T1 state of NIR cyanine dye and the Yb3+ ground-state absorption, and the successive dye→Yb3+→Er3+ ET process may realize highly efficient UCL (Figure 1b).[11]. The designed IR808-UCNPs-F127 nanoprobes outperform the previously reported dye-quenched UCL probes that exhibited LOD of 0.5 × 10−6 and 0.32 × 10−6 m with energy acceptor of Cy3 and rhodamine, respectively.[7c,17] under 980 nm laser irradiation, addition of NaClO (0 × 10−6–52 × 10−6 m) caused almost no effect on the UCL intensity of IR808-UCNPs-F127 (Figure 3d). The exogenous ClO− can be estimated by subtracting the intrinsic ClO− from the total value detected, which was close to the externally added amount All these results demonstrate that our designed nanoprobes under NIR dual-excitation ratiometric detection model can provide a feasible platform for intracellular detection with merits of specificity, ultrasensitivity, and high accuracy
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