Nucleolin-Targeted Ratiometric Fluorescent Carbon Dots with a Remarkably Large Emission Wavelength Shift for Precise Imaging of Cathepsin B in Living Cancer Cells

As one of the most promising biomarkers for numerous malignant tumors, accurate and reliable reporting of Cathepsin B (CTSB) activity is of great significance to achieve efficient diagnosis of cancers at an early stage and predicting metastasis. Here, we report a vigorous ratiometric fluorescent method integrating a cancer-targeting recognition moiety with a remarkably large emission wavelength shift into a single matrix to report CTSB activity sensitively and specifically. As a proof of concept, we synthesized amine-rich carbon quantum dots (CQDs) with a blue fluorescence, which offered an efficient scaffolding to covalently assemble the nucleolin-targeting recognition nucleic acid aptamer AS1411 and a CTSB-cleavable peptide substrate Gly-Arg-Arg-Gly-Lys-Gly-Gly-Cys–COOH that tethered with a near-infrared (NIR) fluorophore chlorin e6 (Ce6–GRRGKGGC, Ce6–Pep), enabling a cancer-targeting and CTSB stimulus-responsive ratiometric nanoprobe AS1411–Ce6–CQDs. Owing to the efficient fluorescence resonance energy transfer (FRET) process from the CQDs to Ce6 inside the assembly of nanoprobe, the blue fluorescence of CQDs at ∼450 nm was remarkably quenched, along with an obvious NIR fluorescence enhancement of Ce6 at ∼650 nm. After selective entry into cancer cells via nucleolin-mediated endocytosis, the overexpressed CTSB in lysosome could cleave Ce6–Pep and trigger the Ce6 moiety dissociation from AS1411–Ce6–CQDs, thus leading to the termination of FRET process, achieving the efficient ratiometric fluorescence response toward endogenous CTSB with a remarkably large emission wavelength shift of ∼200 nm from NIR to blue emission region. Notably, the nanoprobe AS1411–Ce6–CQDs exhibited an excellent specificity for ratiometric fluorescent sensing of CTSB activity with an ultralow detection limit of 0.096 ng/mL, demonstrating its promising use for early precise cancer diagnosis in the near future.