Ultrasensitive Dual\nFluorophore-Conjugated Carbon\nDots for Intracellular pH Sensing in 3D Tumor Models

The dysregulation of pH has been linked to the onset\nof chronic\nconditions, such as cancer and neurological diseases. Consequently,\nthe development of a highly sensitive tool for intracellular pH sensing\nis imperative to investigate the interplay between pH and the biochemical\nchanges accompanying disease pathogenesis. Here, we present the development\nof a ratiometric fluorescent nanoprobe, <b>NpRhoDot</b>, designed\nfor precisely measuring pH levels. We demonstrate its efficacy in\nsensitively reporting intracellular pH in monolayer A549 lung cancer\ncells, primary fibroblast cells, and 3D tumor spheroids derived from\nthe DLD-1 colorectal adenocarcinoma cell line. <b>NpRhoDot</b> leverages a novel design, where stable carbon dots are functionalized\nwith a pH-responsive ratiometric fluorescent probe comprising a naphthalimide-rhodamine\nmoiety, <b>NpRho1</b>. This design confers <b>NpRhoDot</b> with the high pH sensitivity characteristics of organic fluorescent\nprobes, along with excellent photostability up to 1 h and biocompatibility\nof carbon dots. Through one-photon and two-photon fluorescence microscopy,\nwe validate the reliability of <b>NpRhoDot</b> for biosensing\nintracellular pH in monolayer and three-dimensional tumor models from\npH 4 to 7.