Fluorescent Protein-Based Turn-On Probe through a General Protection–Deprotection Design Strategy

We demonstrated a general protection–deprotection strategy for the design of fluorescent protein biosensors through the construction of a turn-on Hg²⁺ sensor. A combination of fluorescent protein engineering and unnatural amino acid mutagenesis was used. Unlike previously reported fluorescent protein-based Hg²⁺ sensors that relied on the binding of Hg²⁺ to the sulfhydryl group of cysteine residues, a well-established chemical reaction, oxymercuration, was transformed into biological format and incorporated into our sensor design. This novel Hg²⁺ sensor displayed good sensitivity and selectivity both in vitro and in live bacterial cells. Over 60-fold change in fluorescence signal output was observed in the presence of 10 μM Hg²⁺, while such a change was undetectable when nine other metal ions were tested. This new design strategy could expand the repertoire of fluorescent protein-based biosensors for the detection of small-molecule analytes.