Sequentially Activated Probe Design Strategy for Analyzing Metabolite Crosstalk in a Biochemical Cascade

Interrogating metabolite crosstalk in live cells is important to understand the interplay between metabolic and signal transduction pathways but is challenging due to the lack of efficient analytical techniques. Here we report a sequentially activated probe design strategy resulting in probe HF-6 being capable of imaging the crosstalk between H₂O₂ and formaldehyde in live cells. Fluorescence of HF-6 can only be triggered by first H₂O₂ activation followed by binding with formaldehyde. Facilitated by this sequentially activated mechanism, HF-6 imaging revealed H₂O₂-induced upregulation of formaldehyde in live SH-SY5Y cells, while little change of intracellular H₂O₂ level was observed when cells were stimulated with formaldehyde for limited time. These results establish a link for the crosstalk between H₂O₂ and formaldehyde in redox signaling and provide a starting point to study broader metabolite interactions.