Disassembly-driven turn-on fluorescent nanoprobe with adaptive signal amplification for quantitative carbonic anhydrase detection in whole blood and enhanced cancer imaging

Carbonic anhydrases (CAs), particularly CA Ⅸ, are critical biomarkers in cancer diagnosis and therapy, but quantitative detection in whole blood remains challenging due to intrinsic background fluorescence, limited signal amplification, and interference from blood autofluorescence. Here, we introduce a disassembly-driven fluorescent nanoprobe (SQ-H-SA) leveraging self-assembly, recognition-driven activation, and adaptive signal amplification. SQ-H-SA harnesses squaraine (SQ) dyes that self-assemble into stable, fluorescence-quenched aggregates in aqueous solution; upon CAs binding, the nanoprobe disassembles, enabling SQ monomers to become conformationally shielded within the protein’s hydrophobic pocket, achieving a rapid 45-fold fluorescence enhancement within minutes of interaction with 3 μM CA Ⅸ. SQ-H-SA nanoprobe exhibits good selectivity (with limited interference from serum albumin), sub-nanomolar sensitivity, and large signal-to-background ratio, enabled by ultralow intrinsic fluorescence and protein-assisted amplification. By effectively suppressing blood autofluorescence based on the inner filter effect, SQ-H-SA achieves reliable quantification of endogenous CAs. Notably, SQ-H-SA not only detected elevated CAs levels in untreated lung cancer patients and notable posttreatment reductions consistent with ELISA findings, but also facilitated high-throughput analysis of clinical specimens to improve diagnostic precision in liquid biopsies. Moreover, SQ-H-SA achieves the first selective, cell membrane-specific fluorescence imaging of CA Ⅸ in cancer cells and clinical lung cancer tissues.