Low‐Background His‐Tag‐Targeting Probes for Turn‐On Fluorescence Detection of Cell Surface Proteins and Their Binding Interactions

Abstract Turn‐on fluorescent probes consisting of dye‐ligand conjugates serve as a powerful tool for detecting cell surface proteins (CSPs) and their interactions with binding partners. However, generating such probes from protein‐based ligands remains challenging. This challenge became particularly evident during the COVID‐19 pandemic, which highlighted the need for assays to evaluate inhibitors of the interaction between the SARS‐CoV‐2 virus receptor‐binding domain (RBD) and the angiotensin‐converting enzyme 2 (ACE2) receptor. To sense this interaction in a cellular environment using turn‐on probes, a tri‐nitrilotriacetic acid (tri‐NTA) unit was conjugated to quinoline‐based cyanine (QBC) dyes. This design leverages the high affinity of tri‐NTA for His‐tag, along with the low‐background and confinement‐sensitive optical responses of QBC dyes, to create probes that fluoresce upon binding to His‐tagged proteins on cell surfaces. Herein, it is shown that this approach enables the development of an exceptionally simple cell‐based assay with which inhibitors of the RBD‐ACE2 interaction can be readily sensed by combining a turn‐on probe, His‐tagged RBD, ACE2‐expressing cells, and recording changes in the probe's emission spectra. The potential of this method is further demonstrated by using such probes to detect lectin binding to cell surface glycans and to image a bacterial CSP under wash‐free conditions.