Encapsulation of FITC to monitor extracellular pH: a step towards the development of red blood cells as circulating blood analyte biosensors

A need exists for a long-term, minimally-invasive system to monitor blood analytes. For certain analytes, such as glucose in the case of diabetics, a continuous system would help reduce complications. Current methods suffer significant drawbacks, such as low patient compliance for the finger stick test or short lifetime (i.e., 3–7 days) and required calibrations for continuous glucose monitors. Red blood cells (RBCs) are potential biocompatible carriers of sensing assays for long-term monitoring. We demonstrate that RBCs can be loaded with an analyte-sensitive fluorescent dye. In the current study, FITC, a pH-sensitive fluorescent dye, is encapsulated within resealed red cell ghosts. Intracellular FITC reports on extracellular pH: fluorescence intensity increases as extracellular pH increases because the RBC rapidly equilibrates to the pH of the external environment through the chloride-bicarbonate exchanger. The resealed ghost sensors exhibit an excellent ability to reversibly track pH over the physiological pH range with a resolution down to 0.014 pH unit. Dye loading efficiency varies from 30% to 80%. Although complete loading is ideal, it is not necessary, as the fluorescence signal is an integration of all resealed ghosts within the excitation volume. The resealed ghosts could serve as a long-term (>1 to 2 months), continuous, circulating biosensor for the management of diseases, such as diabetes.