SurpHer: a genetically encoded ratiometric sensor for dynamic extracellular pH imaging

Cens Holste, S.; Dos Santos, L.; Charan, M. R.; Nyhegn-Eriksen, O.; Crouigneau, R.; Kragelund, B. B.; Marie, R.; Sandelin, A.; Auxillos, J. Y.; Pedersen, S. F.

doi:10.64898/2026.05.18.725923

bioRxiv · 10.64898/2026.05.18.725923

SurpHer: a genetically encoded ratiometric sensor for dynamic extracellular pH imaging

Cens Holste, S.·Dos Santos, L.·Charan, M. R.·Nyhegn-Eriksen, O.·Crouigneau, R.·Kragelund, B. B.·Marie, R.·Sandelin, A.·Auxillos, J. Y.·Pedersen, S. F.

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Abstract

Extracellular pH is a key microenvironmental factor shaping cell physiology and disease, creating a need for quantitative biosensors that can capture dynamic changes in pHe at the surface of individual living cells. Here, we develop a genetically encoded, ratiometric extracellular pH biosensor through systematic screening of a modular library of membrane-display designs that combine SEpHluorin with a pH-stable reference fluorophore. Screening identified a cell-surface-localised mKate2-SEpHluorin construct, named SurpHer, that exhibits dynamic ratiometric responses across the pHe range of 6 - 7.8. SurpHer shows robust membrane localisation and extracellular pH responsiveness across diverse human cell types including HEK293T, PANC-1 and MDA-MB231 cells. Following stable integration in MDA-MB-231 cells, SurpHer enabled time-course imaging of pHe gradients in a microfluidic platform for modelling tumour microenvironments. SurpHer enables real-time interrogation of the pericellular pH environment of tumor cells and, more broadly, provides a strategy to probe microenvironmental pH dynamics across diverse biological contexts.

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Cens Holste, S., Dos Santos, L., Charan, M. R., Nyhegn-Eriksen, O., Crouigneau, R., Kragelund, B. B., Marie, R., Sandelin, A., Auxillos, J. Y., Pedersen, S. F.. 2026-05-21. SurpHer: a genetically encoded ratiometric sensor for dynamic extracellular pH imaging. https://doi.org/10.64898/2026.05.18.725923

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