A FRET-based off-on AIE nanoprobe enables instant and stain-free detection of hypoxic niches in tumor sections

Rationale: Hypoxia is a critical hallmark of solid tumors, significantly influencing their diagnosis, treatment, and prognosis. Currently, instant and accurate detection of hypoxic niches in tumor sectioning remains a major challenge. Conventional tumor section staining methods lack reliability due to dynamic changes in hypoxic conditions during time-consuming sample processing. Methods: Herein, we develop a FRET-based off-on AIE nanoprobe for instant and stain-free detection of tumor hypoxic niches following intravenous administration. A dimeric AIEgen (TNNT) is synthesized by conjugating two tetraphenylethene (TPE) molecules via azobenzene (Azo). TNNT self-assembles into stable nanoassemblies (NAs) with favorable drug delivery and tumor accumulation. Results: Under normoxic conditions, fluorescence is quenched due to FRET between TPE and Azo, effectively "turning off" the AIE signal. Notably, the fluorescence recovers quickly following Azo cleavage under hypoxia in vitro and in vivo. Moreover, the AIE nanoprobe demonstrates an advantage over pimonidazole hydrochloride (HP3, a widely used hypoxia probe) in terms of ex-vivo hypoxia detection. Furthermore, it can also report the varying degrees of hypoxia in tumors of different sizes. Conclusions: This study offers a practical tool for point-of-care tumor hypoxia detection and relevant pathological analysis.