An AIE-active probe for monitoring calcium-rich biological environment with high signal-to-noise and long-term retention in situ

Fluorescent probe is a first-line method for qualitative and quantitative detection of calcium ions (Ca²⁺) in organisms. However, the high affinity and aggregate-caused quenching (ACQ) characteristics of commercially available probes have restricted the detection limit to low concentrations from nM to μM, unavailable to detect higher Ca²⁺ concentrations from μM to mM in situ. Here, we develop a Ca²⁺ probe of TCM-4COOH with aggregation-induced emission (AIE) activity and desirable affinity, exhibiting a linear response to concentrated Ca²⁺ at mM level. The rapid binding between the TCM-4COOH and Ca²⁺ results in dramatic enhancement in fluorescence with high S/N ratio, and the nature that the chelates are not easy to diffuse from the cells endows the probe with long-term imaging ability in organisms. In the molecular design, the multiple iminodiacetic carboxyl groups ensure the good water solubility and pH biocompatibility of TCM-4COOH, resulting in negligible background fluorescence and high signal-to-noise (S/N) ratio. Moreover, the relatively dispersed carboxyl groups and the electron-withdrawing effect of TCM building block jointly adjust the probe affinity to Ca²⁺, thereby broadening the upper detection limit. In addition, to obtain better cell membrane penetrability, TCM-4COOH was modified with acetoxymethyl ester, which unit can be cleaved by endogenous esterase to release TCM-4COOH, so as to detect intracellular calcium ions. Benefit from the reasonable design of fluorophore and chelating groups, the AIE-active sensor TCM-4COOH can achieve long-term in-situ retention in visualizing calcium-overloaded cells and bone microcracks, especially providing a unique platform to broaden the upper limit of Ca²⁺ detection in biological environments.