Highly Sensitive Hill-Type Small-Molecule pH Probe That Recognizes the Reversed pH Gradient of Cancer Cells

A hallmark of cancer cells is a reversed transmembrane pH gradient, which could be exploited for robust and convenient intraoperative histopathological analysis. However, pathologically relevant pH changes are not significant enough for sensitive detection by conventional Henderson-Hasselbalch-type pH probes, exhibiting an acid-base transition width of 2 pH units. This challenge could potentially be addressed by a pH probe with a reduced acid-base transition width (i.e., Hill-type probe), appropriate pK_a, and membrane permeability. Yet, a guideline to allow rational design of such small-molecule Hill-type pH probes is still lacking. We have devised a novel molecular mechanism, enabled sequential protonation with high positive homotropic cooperativity, and synthesized small-molecule pH probes (PHX1-3) with acid-base transition ranges of ca. 1 pH unit. Notably, PHX2 has a pK_a of 6.9, matching the extracellular pH of cancer cells. Also, PHX2 is readily permeable to cell membrane and allowed direct mapping of both intra-and extracellular pH, hence the transmembrane pH gradient. PHX2 was successfully used for rapid and high-contrast distinction of fresh unprocessed biopsies of cancer cells from normal cells and therefore has broad potentials for intraoperative analysis of cancer surgery.