AND-Logic-Based Dual-Lock Hemicyanine Fluorescence Probe for Accurate Hypoxia Assessment in Tumors

The hypoxia tumor microenvironment (TME) critically influences treatment efficacy, driving a significant demand for real-time in vivo hypoxia assessment. However, conventional single-stimulus responsive probes suffer from insufficient imaging precision. To address this, we developed a new AND-logic-based pH/nitroreductase (NTR) dual-lock hemicyanine, namely LET-16, for accurate hypoxia assessment in tumors. LET-16 responds to acidic TME, forming a positively charged open-ring structure that enhances cell entry. Inside tumor cells, it reacts with overexpressed NTR, thus emitting a fluorescence signal. LET-16 demonstrates excellent dual-responsive activation both in vitro and in vivo, enabling precise imaging of tumor hypoxia. This work not only provides a robust tool for accurate hypoxia assessment but also offers a strategy for engineering versatile hemicyanine scaffolds adaptable to complex diagnostic needs, such as multianalyte detection and advanced logic-gate responses.