Acidic pH inside cells indicates cellular dysfunctions such as cancer. Therefore, the development of optical pH sensors for measuring and imaging intracellular pH is a demanding challenge. The available pH-sensitive probes are vulnerable to e.g. photobleaching or autofluorescence background in biological materials. Our approach circumvents these problems due to near infrared excitation and upconversion photoluminescence. We introduce a nanosensor based on upconversion resonance energy transfer (UC-RET) between an upconverting nanoparticle (UCNP) and a fluorogenic pH-dependent dye pHrodo™ Red that was covalently bound to the aminosilane surface of the nanoparticles. The sensitized fluorescence of the pHrodo™ Red dye increases strongly with decreasing pH. By referencing the pH-dependent emission of pHrodo™ Red with the pH-insensitive upconversion photoluminescence of the UCNP, we developed a pH-sensor which exhibits a dynamic range from pH 7.2 to 2.5. The applicability of the introduced pH nanosensor for pH imaging was demonstrated by imaging the two emission wavelengths of the nanoprobe in living HeLa cells with a confocal fluorescence microscope upon 980 nm excitation. This demonstrates that the presented pH-nanoprobe can be used as an intracellular pH-sensor due to the unique features of UCNPs: excitation with deeply penetrating near-infrared light, high photostability, lack of autofluorescence and biocompatibility due to an aminosilane coating.

The unique photophysical properties of upconverting nanoparticles (UCNPs) and semiconductor quantum dots (QDs) render them an attractive donor–acceptor combination for near-infrared (NIR) excited FRET-based optical biosensing.

Red blood cell folate is measured for folate deficiency diagnosis, because it reflects the long-term folate level in tissues, whereas serum folate only represents the dietary intake. Direct homogeneous assay from whole blood would be ideal but conventional fluorescence techniques in blood suffer from high background and strong absorption of light at ultraviolet and visible wavelengths. In this study, a new photon upconversion-based homogeneous assay for whole blood folate is introduced based on resonance energy transfer from upconverting nanophosphor donor coated with folate binding protein to a near-infrared fluorescent acceptor dye conjugated to folate analogue. The sensitized acceptor emission is measured at 740 nm upon 980 nm excitation. Thus, optically transparent wavelengths are utilized for both donor excitation and sensitized acceptor emission to minimize the sample absorption, and anti-Stokes detection completely eliminates the Stokes-shifted autofluorescence. The IC50 value of the assay was 6.0 nM and the limit of detection (LOD) was 1 nM. The measurable concentration range was 2 orders of magnitude between 1.0-100 nM, corresponding to 40-4000 nM folate in the whole blood sample. Recoveries of added folic acid were 112%-114%. A good correlation was found when compared to a competitive heterogeneous assay based on the DELFIA-technology. The introduced assay provides a simple and fast method for whole blood folate measurement.

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