A Two-Photon-Active Zr-Based Metal–Organic Framework-Based Orthogonal Nanoprobe for Recognition of Cellular Senescence

A luminescent nanoprobe capable of orthogonal sensing of two independent events is highly significant for unbiased disease-related detection such as the detection of senescent cells. Moreover, it is invaluable that the nanoprobe possesses a two-photon excitable characteristic that is highly suitable for imaging living cells and tissues. Herein, we present a two-photon-excitable multiluminescent orthogonal-sensing nanoprobe (OS nanoprobe) capable of detecting both pH elevation and β-galactosidase (β-gal) overexpression in senescent cells. In the design, Zr-based dual-emissive metal-organic frameworks prepared from two mixed amino linkers, referred to as NH₂-MU, were used as the component for the ratiometric sensing of pH; additionally, fluorogenic resorufin-β-d-galactopyranoside, linked to the NH₂-MU framework, enables β-gal detection. In the OS nanoprobe, the signals for pH and β-gal sensing remain independent while maintaining high colocalization. The two-photon excitable organic linkers of NH₂-MU impart the OS nanoprobe with a bioimaging capability, allowing for the differentiation of senescent human foreskin fibroblast (HFF) cells from younger HFF cells or <i>LacZ</i> positive cells with the 800 nm laser excitation. This study marks the first instance of achieving the multiplexed orthogonal fluorescent sensing of cellular senescence using a two-photon excitation strategy, suggesting the potential of using versatile metal-organic framework (MOFs)-based fluorophores to realize the orthogonal multiplexing of disease-related biomarkers through multiphoton excitation.