A sequentially activated afterglow probe for AND-gated imaging of alpha-fetoprotein in hepatocellular carcinoma

Alpha-fetoprotein (AFP) plays multiple roles in hepatocellular carcinoma (HCC), driving tumor progression and immune evasion. However, clinical measurement of serum AFP levels often fails to reflect intratumoral AFP expression, especially for early-stage or well-differentiated HCC. Current imaging modalities lack the sensitivity and specificity to visualize AFP spatially within tumors. Herein, an AND-gated afterglow probe is engineered to be sequentially activated by two features of HCC microenvironment: acidic pH and elevated AFP. The probe consists of afterglow nanoparticles (PZ-NPs) functionalized with BHQ₃-modified dual-aptamer DNA. Initially quenched via afterglow resonance energy transfer (ARET), the probe is activated sequentially through pH-induced triplex folding and AFP binding, which cause conformational tightening and disrupt ARET to restore afterglow luminescence. Such an activation requires an AND logic of three inputs (i.e. light, H⁺ and AFP), which enables a limit of detection for AFP as low as 0.31nM and ensures high specificity with negligible response to interferents. In orthotopic HCC, the probe achieved an 8.3-fold tumor-to-liver ratio (vs. 1.4-fold for fluorescence) with signals that are colocalized with luciferase-defined tumors and able to detect curcumin-mediated AFP suppression in good consistency with immunohistochemistry results. Ex vivo imaging of resected livers further confirmed tumor-specific activation, supporting AFP-selective in vivo imaging and microenvironment-gated biomarker detection in HCC.