Sensing and Imaging of Intracellular pH Using Photon Upconversion Based Nanoprobes

  • Sensing of intracellular pH is of particular interest in biomedical research since structure and function of biomolecules strongly depend on the concentration of protons in their environment. We have functionalized photon upconversion nano-particles (UCNPs) with pH responsive dyes to achieve nanoprobes for intracellular pH determination. The sensing mechanism is based on a resonance energy transfer (UC-RET) from the 550 nm emission of hexagonal nanocrystals of NaYF4: Yb3+,Er3+ to the pH-sensitive fluorophore pHrodoTM Red. The nanocrystals were coated with thin shells of aminosilane or highly branched polyethylenimine (PEI) with several nanometer layer thickness for coupling of the pH indicator. The nanoprobes are calibrated by ratiometric dual wavelength readout at 550 nm (reference signal) and 590 nm (sensor signal) and visualized using a scanning confocal fluorescence microscope with 980 nm excitation wavelength. It was found that PEI coating enables a higher coupling of indicatorSensing of intracellular pH is of particular interest in biomedical research since structure and function of biomolecules strongly depend on the concentration of protons in their environment. We have functionalized photon upconversion nano-particles (UCNPs) with pH responsive dyes to achieve nanoprobes for intracellular pH determination. The sensing mechanism is based on a resonance energy transfer (UC-RET) from the 550 nm emission of hexagonal nanocrystals of NaYF4: Yb3+,Er3+ to the pH-sensitive fluorophore pHrodoTM Red. The nanocrystals were coated with thin shells of aminosilane or highly branched polyethylenimine (PEI) with several nanometer layer thickness for coupling of the pH indicator. The nanoprobes are calibrated by ratiometric dual wavelength readout at 550 nm (reference signal) and 590 nm (sensor signal) and visualized using a scanning confocal fluorescence microscope with 980 nm excitation wavelength. It was found that PEI coating enables a higher coupling of indicator molecules on the particle surface, better signal to reference ratios in ratiometric readout and an improved cellular uptake compared to the aminosilane coated particles due to a more positive zeta potential. We studied the cellular uptake efficacy of the nanoprobes and determined to which type of compartment, lysosomes, endosomes or cytosol, the probes are targeted to by measuring the pH of their microenvironment. An in situ control was performed in live cells by a treatment with nigericin, whereby the pH of all intracellular compartments is set at extracellular level. Finally, we will show new strategies for the preparation of UCNP-dye conjugates with improved UC-RET efficiency to achieve higher acceptor (sensor) emission.zeige mehrzeige weniger

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Metadaten
Autor*innen:Michael Schäferling
Koautor*innen:T. Näreoja, T. Deguchi, E. Fazeli, R. Peltomaa, Riika Arppe, T. Soukka
Dokumenttyp:Vortrag
Veröffentlichungsform:Präsentation
Sprache:Englisch
Jahr der Erstveröffentlichung:2017
DDC-Klassifikation:Naturwissenschaften und Mathematik / Chemie / Analytische Chemie
Freie Schlagwörter:Chemical Sensing; Live cell imaging; Nanoprobes; PH; Photon Upconversion
Veranstaltung:1st European / 10th German Biosensor Symposium
Veranstaltungsort:Potsdam, Germany
Beginndatum der Veranstaltung:20.03.2017
Enddatum der Veranstaltung:23.03.2017
Verfügbarkeit des Dokuments:Datei im Netzwerk der BAM verfügbar ("Closed Access")
Datum der Freischaltung:02.05.2017
Referierte Publikation:Nein
Eingeladener Vortrag:Nein
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