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

Abstract

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.

Optical properties of biomimetic probes engineered from erythrocytes

“…ICG-loaded EGs have been used to characterize the movement of erythrocytes in choriocapillaries and retinal capillaries of rabbits and monkeys [44]. In addition to imaging, erythrocytes have been doped with fluorescein isothiocyanate to create biosensors for monitoring changes in plasma analytes and extracellular pH [45,46]. …”

Chemical reactivation of fluorescein isothiocyanate immunofluorescence-labeled resin-embedded samples

“…Since fluorescein isothiocyanate (FITC) had a high fluorescence quantum yield 13,14 and was widely used for monitoring pH values [15][16][17] and immunofluorescence labeling of biological tissues, [18][19][20][21][22] we chose FITC as one representative of pH-sensitive fluorescent probe to realize the idea. First, we investigated whether the fluorescence of FITC would be quenched during resin-embedding process, then further tested whether the quenched immunofluorescence could be reactivated.…”

Erythrocyte-derived photo-theranostic agents: hybrid nano-vesicles containing indocyanine green for near infrared imaging and therapeutic applications

“…Gold nanoparticles have been incorporated into human erythrocytes to produce tracers with enhanced contrast for potential dynamic X-ray imaging of blood flow25. Fluorescein isothiocyanate, a non -NIR dye, has been successfully loaded into EGs having diameters of ≈5 μm, with the intention of developing such constructs as plasma analyte or extracellular pH sensors2627. Flower et al recently loaded ICG into micron-sized EGs, and utilized the constructs as a fluorescent analogue of erythrocytes to characterize the movement of erythrocytes in retinal capillaries and choriocapillaris of monkeys and rabbits28.…”