Design of Histidine Sequence‐Associated Tripeptide Sequences for Recognition of Copper Ions and Their Application to Live Cells

Copper is central to many enzymes in living organisms, and imbalances in copper levels are linked to various diseases. Therefore, developing probes to detect copper ions is essential. Histidine, especially in the polypeptide sequences at the first three N-terminal positions (His1, His2, and His3), uniquely binds to copper ions. This study introduces three groups of tripeptide probes designed to monitor copper ion levels in living cells and organisms. The results show that tripeptides with histidine at the -2 position, specifically HDQL-2 (Asp-His-Gln-Dansyl), HMFM-2 (Met-His-Phe-Dansyl), and HDMB-2 (Asp-His-Met-Dansyl), exhibit a higher affinity for copper ions. These probes responded quickly to copper ions, demonstrating excellent fluorescence turn-off performance and stable detection within a pH range of 6.0-11.0. The detection limits for fluorescence titration, calculated using the 3σ/k equation, were 17.65 nM (HDQL-2), 18.04 nM (HMFM-2), and 15.50 nM (HDMB-2). Peptide probes are ideal for detecting copper ions in living cells via fluorescence imaging because of their low toxicity and good biocompatibility. The fluorescence intensity decreases as copper ion content changes.