pH 值失调与癌症和神经系统疾病等慢性疾病的发作有关。因此，开发一种高度灵敏的细胞内 pH 传感工具对于研究 pH 值与疾病发病机制所伴随的生化变化之间的相互作用至关重要。在这里，我们展示了比率荧光纳米探针 NpRhoDot 的开发，该探针专为精确测量 pH 水平而设计。我们证明了其在单层 A549 肺癌细胞、原代成纤维细胞和源自 DLD-1 结直肠腺癌细胞系的 3D 肿瘤球体中灵敏报告细胞内 pH 的功效。 NpRhoDot 采用新颖的设计，其中稳定的碳点通过包含萘酰亚胺-罗丹明部分的 pH 响应比率荧光探针 NpRho1 进行功能化。这种设计赋予 NpRhoDot 有机荧光探针的高 pH 灵敏度特性，以及长达 1 小时的优异光稳定性和碳点的生物相容性。通过单光子和双光子荧光显微镜，我们验证了 NpRhoDot 在单层和三维肿瘤模型（pH 4 至 7）中生物传感细胞内 pH 的可靠性。

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