Determination of the pH gradient across the stratum corneum

The objective of this work was to determine the pH gradient profile across hairless mouse stratum corneum (SC) using ratiometric laser scanning confocal microscopy imaging and direct pH measurement with a flat surface electrode. Dual-emission ratiometric imaging used the fluorophore, carboxy seminaphthorhodafluor-1, which displays a pH-dependent shift in its emission spectrum. The assay developed was unsuccessful, however, because (i) the pKa of seminaphthorhodafluor (approximately 7.5) makes it insufficiently sensitive to the normal pH gradient (4-7.4) across mammalian SC, and (ii) the unexpectedly high buffering capacity of the skin precluded a meaningful calibration of the system. In the second method, pH measurements with a flat surface electrode were recorded as the hairless mouse SC was progressively tape stripped. In the superficial SC, the pH remained relatively constant (approximately 5.9); further removal of the barrier resulted in a steady increase in pH to approximately 7. In conclusion, improved methodology is clearly required to characterize precisely the pH profile across the SC.