title: Design, calibration and application of broad-range optical nanosensors for determining intracellular pH.
abstract: Particle-based nanosensors offer a tool for determining the pH in the endosomal-lysosomal system of living cells. Measurements providing absolute values of pH have so far been restricted by the limited sensitivity range of nanosensors, calibration challenges and the complexity of image analysis. This protocol describes the design and application of a polyacrylamide-based nanosensor (∼60 nm) that covalently incorporates two pH-sensitive fluorophores, fluorescein (FS) and Oregon Green (OG), to broaden the sensitivity range of the sensor (pH 3.1-7.0), and uses the pH-insensitive fluorophore rhodamine as a reference fluorophore. The nanosensors are spontaneously taken up via endocytosis and directed to the lysosomes where dynamic changes in pH can be measured with live-cell confocal microscopy. The most important focus areas of the protocol are the choice of pH-sensitive fluorophores, the design of calibration buffers, the determination of the effective range and especially the description of how to critically evaluate results. The entire procedure typically takes 2-3 weeks.
authors.affiliation: 1] Department of Micro- and Nanotechnology, Technical University of Denmark (DTU) Nanotech, DTU, Lyngby, Denmark. [2] Center for Nanomedicine and Theranostics, DTU, Lyngby, Denmark.
authors.forename: Rikke V
authors.initials: RV
authors.lastname: Søndergaard
authors.affiliation: 1] Center for Nanomedicine and Theranostics, DTU, Lyngby, Denmark. [2] Department of Chemistry, DTU Chemistry, DTU, Lyngby, Denmark.
authors.forename: Jonas R
authors.initials: JR
authors.lastname: Henriksen
authors.affiliation: 1] Department of Micro- and Nanotechnology, Technical University of Denmark (DTU) Nanotech, DTU, Lyngby, Denmark. [2] Center for Nanomedicine and Theranostics, DTU, Lyngby, Denmark.
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Andresen
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods*; D002138:Calibration; D002264:Carboxylic Acids; D011992:Endosomes / Q000737:chemistry; D004867:Equipment Design; D019793:Fluorescein; D005456:Fluorescent Dyes; D006367:HeLa Cells / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D008247:Lysosomes / Q000737:chemistry; D018942:Macrolides / Q000494:pharmacology; D053758:Nanoparticles / Q000737:chemistry; D036103:Nanotechnology / Q000379:methods*; D012235:Rhodamines; D000099166:Polyacrylamides
chemical_list: D000180:Acrylic Resins; D002264:Carboxylic Acids; D019793:Fluorescein; D005456:Fluorescent Dyes; D018942:Macrolides; D012235:Rhodamines; D000099166:Polyacrylamides; C436864:Oregon Green 488 carboxylic acid; C040929:bafilomycin A1