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Bioorthogonal chemistry in metal clusters: a general strategy for the construction of multifunctional probes for bioimaging in living cells and in vivo.

Multifunctional bioimaging probes based on metal clusters have multiple characteristics of metal clusters and functional conjugates, and their development has broad application prospects in the fields of biomedical imaging and tumor diagnosis. However, current bioconjugation methods on metal clusters are time-consuming and have low reaction efficiency, which hinders the construction of bioimaging probes with multifunctional components. Here, we report a concise and promising design strategy to realize the simple and efficient introduction of functional conjugates through bioorthogonal reactions based on azido-functionalized metal clusters. Based on this strategy, taking the probe FA-CuC@BSA-Cy5 as an example, we demonstrated the design of a copper cluster-based multifunctional near-infrared (NIR) fluorescent probe and its real-time imaging application in vivo. Through the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, the tumor-specific targeting ligand folic acid (FA) and fluorophore (Cy5) can be chemically conjugated to azido-functionalized CuC@BSA-N3 quickly and efficiently under biocompatible conditions. The prepared probe showed numerous advantages of metal clusters, including good stability, ultra-small particle size and low toxicity and rapid renal clearance. At the same time, FA-modified FA-CuC@BSA-Cy5 can specifically target KB cells with high FR expression, and in vivo fluorescence imaging shows higher tumor accumulation. The construction of the azido functional metal cluster platform can be extended to various metal clusters with functional probes and prodrugs, thereby providing more promising candidates for future medical diagnoses.