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WIREs Nanomed Nanobiotechnol
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Nanoparticle‐based luminescent probes for intracellular sensing and imaging of pH

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Fluorescence imaging microscopy is an essential tool in biomedical research. Meanwhile, various fluorescent probes are available for the staining of cells, cell membranes, and organelles. Though, to monitor intracellular processes and dysfunctions, probes that respond to ubiquitous chemical parameters determining the cellular function such as pH, pO2, and Ca2+ are required. This review is focused on the progress in the design, fabrication, and application of photoluminescent nanoprobes for sensing and imaging of pH in living cells. The advantages of using nanoprobes carrying fluorescent pH indicators compared to single molecule probes are discussed as well as their limitations due to the mostly lysosomal uptake by cells. Particular attention is paid to ratiometric dual wavelength nanosensors that enable intrinsic referenced measurements. Referencing and proper calibration procedures are basic prerequisites to carry out reliable quantitative pH determinations in complex samples such as living cells. A variety of examples will be presented that highlight the diverseness of nanocarrier materials (polymers, micelles, silica, quantum dots, carbon dots, gold, photon upconversion nanocrystals, or bacteriophages), fluorescent pH indicators for the weak acidic range, and referenced sensing mechanisms, that have been applied intracellularly up to now. WIREs Nanomed Nanobiotechnol 2016, 8:378–413. doi: 10.1002/wnan.1366 This article is categorized under: Diagnostic Tools > In Vitro Nanoparticle-Based Sensing
(a) Protonated and deprotonated form of HPTS and corresponding excitation (at λem = 520 nm) (b) and emission (λexc = 460 nm) spectra (c).
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(a) Schematic illustration of the photon upconversion‐based pH sensor. The pH‐sensitive pHrodo™ Red‐succinimidyl ester is conjugated to the aminogroups of the silane‐coated UCNP. The upconversion resonance energy transfer (UC‐RET)‐sensitized pH‐dependent 590 nm emission of pHrodo™ Red is measured upon 980‐nm excitation. The pH‐independent 550 nm emission of the UCNP is used as reference signal. (b) Transmission electron microscopy image of the hexagonal upconverting nanoparticles with an average size of 28 × 36 nm. The black scale bar is 50 nm. (c) Transmission microscope image of a HeLa cell (left) and fluorescence images of the green emission (550 nm) and red emission (590 nm) of the nanoprobes internalized by the cell. The right image shows the transmission picture superimposed with the green and the red channel. A yellow color indicates a stronger red signal, and therefore, an acidic pH. The white scale bar is 10 µm. (Reprinted with permission from Ref . Copyright 2014 The Royal Society of Chemistry)
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(a) Preparation of dual‐labeled carbon nanodots (A) 4,7,10‐trioxa‐1,13‐tridecanediamine, 220°C, 3 h; (B) FITC and RBITC. (b) Ratiometric cellular imaging. (A) Ratiometric intracellular pH calibration curve. (B)–(F) Ratiometric images of carbon‐nanodot–loaded HeLa cells in PBS (pH 7.4): (B) intact cells, and cells treated with (C) 1 mM NEM, (D) 1 mM NAC, (E) 100 μM H2O2, or (F) 100 μM NaClO for 1 h. Scale bar: 20 µm. (Reprinted with permission from Ref . Copyright 2012 Wiley‐VCH)
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(a) A pH nanosensor consisting of gold nanoparticles stabilized with anthracene (blue) and rhodamine (red) derivatives. (B) Confocal fluorescence microscopy image of a CHO cell incubated with the pH nanosensor (λexc = 364 nm). (c) Fluorescence emission spectra of areas A and B in image (B). (d) pH titration curve of the pH nanosensor obtained from the fluorescence emission intensity ratio I399/I570 as a function of pH (λexc = 364 nm). The error bars show the standard deviation of the measurements. (Reprinted with permission from Ref . Copyright 2011 Wiley‐VCH)
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(a) Spectra of a pH‐sensitive PEBBLE containing a reference dye. Both dyes are effectively excited at 488 nm, and the fluorescein derivative responds to changes in pH while the internal standard remains nearly constant. (b) One 20‐nm PEBBLE next to a primary lysosome in the cell cytoplasm. Original magnification is indicated on the figure and the inset. (Reprinted with permission from Ref . Copyright 1999 American Chemical Society)
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LysoSensor Yellow/Blue DND‐160; pKa ~ 4.2, Exc: λmax = 385/330 nm, Em: λmax = 542/530 nm (protonated/deprotonated form, respectively).
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(a) Basic structure and protonation site of pH‐sensitive tricarbocyanine dyes of type 2. (b) The long‐wave pH indicator AP‐Cy.
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(a) Basic structure and protonation site of pH‐sensitive cyanine dyes of type 1 (n = 1,2). (b) The long‐wave pH indicator CypHer 5.
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Basic structure of (a) seminapthofluoresceins (SNAFL‐1) and (b) seminaphtho‐rhodafluors (SNARF‐1). Indicated are the absorption and emission maxima of the protonated/deprotonated form, respectively.
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(a) Fluorescein and its derivatives (b) fluorescein diacetate (FDA), (c) Oregon Green 488 (OG), and (d) 5‐carboxynaphthofluorescein (CNF).
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Fluorescence decay of diethylaminomethyl pyrene in hydrogel at pH 3 (a) and pH 11 (b), plotted on logarithmic scale. (Reprinted with permission from Ref , Copyright 1995 Elsevier).
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Simplified molecular orbital diagrams showing the relative energy arrangement of HOMO/LUMOs of the fluorophore and HOMO of the donor involved in PET. * represents the excited fluorophore.
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Molecular structure and pH‐dependent emission spectra of 5‐carboxy SNARF‐1 at an excitation wavelength of 514 nm. The signal ratio for the two emission maxima at 580 nm (protonated form) and 640 nm (anionic form) can be calculated at excitation wavelengths between 480 and 550 nm. (Reprinted with permission from Ref , Copyright 2010 Life Technologies)
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Molecular structure and absorption (a) and emission spectra (b) of BCEFC at different pH between 6.1 and 8.1. The signal ratio for the excitation wavelengths 450 and 490 nm is calculated at a fixed emission wavelength between 510 and 535 nm. (Reprinted with permission from Ref , Copyright 2010 Life Technologies)
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(a) TEM picture of the multi‐QD silica particles (550QD inside and 610QD on the surface). (b) Confocal microscope picture of fluorescence emission of self‐referral multi‐QD sensor in water. The particles were excited at 405 nm with emission recorded centered at (i) 529–550 nm and (iii) 593–614 nm. False colors were assigned to each channel: green for 550QD and orange for 610QD. Overlay of (i) and (iii) is shown in (iv). (ii) Light (nonfluorescent) image. (c) Fluorescence emission of (i) 540QDs and (iii) 610QDs in the same multi‐QD sensors. (iv) Overlay of individual QD emissions. (ii) Light microscope image of multi‐QD sensors. (d) Fluorescence emission of mixed populations of 540QD particles and self‐referencing multi‐QD sensors incorporated into T‐REx293 cells. (Reprinted with permission from Ref . Copyright 2010 The Royal Society of Chemistry)
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Fluorophore‐labeled bacteriophage particles for ratiometric intracellular pH sensing. (Reprinted with permission from Ref . Copyright 2008 American Chemical Society)
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(a) Schematic design of pH‐activatable micellar nanoprobes (A). At pH > pKa of ammonium groups (left panel), the neutralized PR segments self‐assemble into the micelle cores, leading to quenching of fluorophores by homoFRET and PeT mechanisms. Upon pH activation (pH < pKa, right panel), formation of charged ammonium groups results in micelle dissociation into unimers with a dramatic increase in fluorescence emission. (B) Structures of the PEO‐b‐(PR‐r‐TMR) copolymers. PeT, photo‐induced electron transfer; PEO, poly (ethylene oxide); PR, ionizable block. (b) Investigation of subcellular activation of nanoprobes 3 and 4 in different endocytic organelles in human H2009 cells (A,B). Representative confocal microscopy images of activated nanoprobe 3 (pHt = 6.3) and 4 (pHt = 5.4) in cells with GFP‐labeled early endosomes (top panel) and late endosomes/lysosomes (bottom panel) at 30 and 45 min, respectively. (C, D) Percentage of positive cells with activated nanoprobe 3 or 4 co‐localizing with early endosomes or late endosomes/lysosomes at different incubation times. (E, F) Schematic illustration of the selective activation of nanoprobe 3 in early endosomes (pH 5.9–6.2) and 4 in late endosomes/lysosomes (pH 5.0–5.5), respectively. (Reprinted with permission from Ref . Copyright 2011 Wiley‐VCH)
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Synthesis of shell cross‐linked ratiometric micelle nanosensors. OGITC, Oregon Green isothiocyanate; FITC, Fluorescein isothiocyanate; and RhBITC, Rhodamine B isothiocyanate. (Reprinted with permission from Ref . Copyright 2014 The Royal Society of Chemistry)
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(a) Synthesis and surface functionalization of pH‐sensitive MSNPs. (b, c) Ratiometric imaging of pH in various intracellular compartments using confocal microscopy. HeLa cells were incubated at 37°C with MSN‐PP and MSN‐TA for 4 h, respectively. The images (overlaid on bright field) of pH sensors in HeLa cells showing (a) MSNP‐PP and (b) MSN‐TA. (Reprinted with permission from Ref . Copyright 2011 The Royal Society of Chemistry)
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Confocal fluorescence microscopy images (overlaid on bright field) of core/shell pH nanosensors in RBL mast cells showing (a) reference dye channel, (b) sensor dye channel, (c) overlaid images, and (d) false‐color ratiometric imaging of pH in various intracellular compartments. (Reprinted with permission from Ref . Copyright 2006 Wiley‐VCH)
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(a) Schematic diagram showing the core/shell architecture of the sensor nanoparticles highlighting the reference dye (TRITC) sequestered in the core coated by a sensor‐dye‐rich (FITC) shell. (b, c) SEM images of 50‐nm core and 70‐nm core/shellparticles. (Reprinted with permission from Ref . Copyright 2006 Wiley‐VCH)
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(a) Design and working principle of pH‐responsive FRET nanoprobes. Schematic illustration showing a dual‐emission nanoprobe that can sense changes of the environmental pH, based on the concept of pH‐responsive FRET of a biocompatible polyelectrolyte, NPCS, conjugated with a donor (Cy3) or an acceptor (Cy5) moiety. (B) Mapping spatial pH changes in living cells. Dual‐emission fluorescence images (scale bar, 20 µm) of cells treated with Cy3−/Cy5− NPCS NPs for distinct durations taken by a confocal laser scanning microscope by irradiating NP suspensions at 543 nm. The fluorescence images were acquired in optical windows between 560–600 nm (Cy3 imaging channel) and 660–700 nm (Cy5 imaging channel). The corresponding pseudo‐colored ratio images were obtained by analyzing the ratio of the signal intensities of Cy5–Cy3 imaging channels. (Reprinted with permission from Ref . Copyright 2010 American Chemical Society)
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(a) Scheme of the pH‐sensitive capsule geometry. The walls of the capsules are composed of onion‐like arranged layers of oppositely charged polymers. The green fluorescence of the capsule wall is a result of its labeling with Alexa Fluor 488, which acts as a pH‐insensitive tag for the capsule. Magnetic properties are given by magnetite nanoparticles located in the capsule shell. The capsule interior is filled with SNARF‐1‐dextran. (b) Overlay of confocal fluorescence microscopy images (green and red channels) of single double‐labeled capsules in acidic and alkaline pH. The fluorescence of the SNARF‐1‐dextran molecules and thus the inside of the capsules changes from green (acidic pH) to red (alkaline pH), whereas the capsule shell shows green fluorescence independent of the pH. (Reprinted with permission from Ref . Copyright 2007 The Royal Society of Chemistry)
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(a) Synthesis of ratiometric polystyrene pH sensing particles. Amino‐modified submicrometer PS particles react sequentially with succinimidyl ester derivatives of Oregon Green and Texas Red at room temperature. (b) Effect of chloroquine on lysosomal pH. The ratio between the fluorescence intensities of Oregon Green and Texas Red (right) and the lysosomal pH (left) are plotted against a time coordinate at increasing chloroquine concentrations. Curve (A) represents a control experiment in chloroquine‐free solution. Curves (B, C, and D) represent the effect of 10, 50, and 100 μM chloroquine, respectively, on the lysosomal pH. The lysosomal pH increase due to exposure to chloroquine is concentration dependent. (Reprinted with permission from Ref . Copyright 2011 American Chemical Society)
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(a) Schematic of the cross‐linked triple‐labeled polyacrylamide nanoprobe. (b) In vitro calibration of the triple‐labeled sensor with both indicators OG and FS, and two dual‐labeled sensors with either OG or FS. Ratiometric calibration has been performed by dividing the reference fluorescence by the indicator fluorescence. (c) Uptake of the triple‐labeled sensor by a HepG2 cell after 24 h and washed and imaged with confocal microscopy. A combined image where the ratios from the intensity images are converted into pH via the calibration curve and color coded on a linear scale according to pH, thereafter overlaid with the differential interference contrast (DIC) image. Scale bar: 10 µm. OG, Oregon Green; FS, fluorescein. (Reprinted with permission from Ref . Copyright 2011 American Chemical Society).
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