Analytical and Bioanalytical Chemistry

, Volume 405, Issue 19, pp 6197–6207 | Cite as

A photoinduced electron transfer-based nanoprobe as a marker of acidic organelles in mammalian cells

Research Paper
Part of the following topical collections:
  1. Optical Nanosensing in Cells


Photoinduced electron transfer (PET)-based molecular probes have been successfully used for the intracellular imaging of the pH of acidic organelles. In this study, we describe the synthesis and characterization of a novel PET-based pH nanoprobe and its biological application for the signaling of acidic organelles in mammalian cells. A fluorescent ligand sensitive to pH via the PET mechanism that incorporates a thiolated moiety was synthesized and used to stabilize gold nanoparticles (2.4 ± 0.6 nm), yielding a PET-based nanoprobe. The PET nanoprobe was unambiguously characterized by transmission electron microscopy, proton nuclear magnetic resonance, Fourier transform infrared, ultraviolet-visible absorption, and steady-state/time-resolved fluorescence spectroscopies which confirmed the functionalization of the gold nanoparticles with the PET-based ligand. Following a classic PET behavior, the fluorescence emission of the PET-based nanoprobe was quenched in alkaline conditions and enhanced in an acidic environment. The PET-based nanoprobe was used for the intracellular imaging of acidic environments within Chinese hamster ovary cells by confocal laser scanning microscopy. The internalization of the nanoparticles by the cells was confirmed by confocal fluorescence images and also by recording the fluorescence emission spectra of the intracellular PET-based nanoprobe from within the cells. Co-localization experiments using a marker of acidic organelles, LysoTracker Red DND-99, and a marker of autophagosomes, GFP-LC3, confirm that the PET-based nanoprobe acts as marker of acidic organelles and autophagosomes within mammalian cells.


A PET based ligand has been used to functionalize gold nanoparticles to develop a pH sensitive nanoprobe. The fluorescence of the nanoprobe, following the PET mechanism, is enhanced in acidic environments and quenched at neutral pH. A combination of spectroscopy and confocal fluorescence microscopy is used for confirmation of the cellular uptake of the nanoprobe by Chinese hamster ovary cells. The PET-based nanoprobe has been used as a marker of acidic organelles and autophagosomes within the CHO cells


Gold nanoparticles Fluorescence Intracellular analysis pH sensing Autophagosomes 

Supplementary material

216_2013_6905_MOESM1_ESM.pdf (55 kb)
ESM 1(PDF 172 kb)


  1. 1.
    Callan JF, de Silva AP, Magri DC (2005) Luminescent sensors and switches in the early 21st century. Tetrahedron 61:8551–8588CrossRefGoogle Scholar
  2. 2.
    Chen X, Tian X, Shin I, Yoon J (2011) Fluorescent and luminescent probes for detection of reactive oxygen and nitrogen species. Chem Soc Rev 40(9):4783–4804CrossRefGoogle Scholar
  3. 3.
    Domaille DW, Que EL, Chang CJ (2008) Synthetic fluorescent sensors for studying the cell biology of metals. Nat Chem Biol 4(3):168–75CrossRefGoogle Scholar
  4. 4.
    Kobayashi H, Ogawa M, Alford R, Choyke PL, Urano Y (2010) New Strategies for Fluorescent Probe Design in Medical Diagnostic Imaging. Chem Rev 110(5):2620–2640CrossRefGoogle Scholar
  5. 5.
    Nagano T (2009) Bioimaging Probes for Reactive Oxygen Species and Reactive Nitrogen Species. J Clin Biochem Nutr 45(2):111–124CrossRefGoogle Scholar
  6. 6.
    Nagano T (2010) Development of fluorescent probes for bioimaging applications. Proceedings of the Japan Academy. Series B, Physical and biological sciences 86(8):837–47CrossRefGoogle Scholar
  7. 7.
    Terai T, Nagano T (2008) Fluorescent probes for bioimaging applications. Curr Opin Chem Biol 12(5):515–521CrossRefGoogle Scholar
  8. 8.
    Bissell RA, de Silva AP, Gunaratne HQN, Lynch PLM, Maguire GEM, Sandanayake K (1992) Molecular fluorescent signaling with fluor spacer receptor systems - approaches to sensing and switching devices via supramolecular photophysics. Chem Soc Rev 21(3):187–195CrossRefGoogle Scholar
  9. 9.
    de Silva AP, Gunaratne HQ, Gunnlaugsson T, Huxley AJ, McCoy CP, Rademacher JT, Rice TE (1997) Signaling Recognition Events with Fluorescent Sensors and Switches. Chem Rev 97(5):1515–1566CrossRefGoogle Scholar
  10. 10.
    de Silva AP, Moody TS, Wright GD (2009) Fluorescent PET (Photoinduced Electron Transfer) sensors as potent analytical tools. Analyst 134(12):2385–2393CrossRefGoogle Scholar
  11. 11.
    Wang YC, Morawetz H (1976) Studies of Intramolecular Excimer Formation in Dibenzyl Ether, Dibenzylamine, and its Derivatives. J Am Chem Soc 98(12):3611–3615CrossRefGoogle Scholar
  12. 12.
    de Silva AP, Vance TP, West ME, Wright GD (2008) Bright molecules with sense, logic, numeracy and utility. Org Biomol Chem 6(14):2468–80CrossRefGoogle Scholar
  13. 13.
    Casey JR, Grinstein S, Orlowski J (2010) Sensors and regulators of intracellular pH. Nat Rev Mol Cell Biol 11(1):50–61CrossRefGoogle Scholar
  14. 14.
    Haas A (2007) The phagosome: Compartment with a license to kill. Traffic 8:311–330CrossRefGoogle Scholar
  15. 15.
    Schindler M, Grabski S, Hoff E, Simon SM (1996) Defective pH regulation of acidic compartments in human breast cancer cells (MCF-7) is normalized in adriamycin-resistant cells (MCF-7adr). Biochemistry 35(9):2811–7CrossRefGoogle Scholar
  16. 16.
    Piwon N, Gunther W, Schwake M, Bosl MR, Jentsch TJ (2000) ClC-5 Cl–channel disruption impairs endocytosis in a mouse model for Dent's disease. Nature 408(6810):369–373CrossRefGoogle Scholar
  17. 17.
    Futerman AH, van Meer G (2004) The cell biology of lysosomal storage disorders. Nat Rev Mol Cell Biol 5(7):554–565CrossRefGoogle Scholar
  18. 18.
    Parkinson-Lawrence EJ, Shandala T, Prodoehl M, Plew R, Borlace GN, Brooks DA (2010) Lysosomal Storage Disease: Revealing Lysosomal Function and Physiology. Physiology 25(2):102–115CrossRefGoogle Scholar
  19. 19.
    Han J, Burgess K (2010) Fluorescent Indicators for Intracellular pH. Chem Rev 110(5):2709–2728CrossRefGoogle Scholar
  20. 20.
    de Silva AP, Rupasinghe R (1985) A New Class of Fluorescent pH Indicators Based on Photoinduced Electron-Transfer. J Chem Soc Chem Commun 23:1669–1670CrossRefGoogle Scholar
  21. 21.
    Johnson I, Spence MTZ (2010) Molecular probes handbook: a guide to fluorescent probes and labeling technologies, 11th edn. Life Technologies, CarlsbadGoogle Scholar
  22. 22.
    Diwu ZJ, Chen CS, Zhang CL, Klaubert DH, Haugland RP (1999) A novel acidotropic pH indicator and its potential application in labeling acidic organelles of live cells. Chem Biol 6(7):411–418CrossRefGoogle Scholar
  23. 23.
    Galindo F, Burguete MI, Vigara L, Luis SV, Kabir N, Gavrilovic J, Russell DA (2005) Synthetic macrocyclic peptidomimetics as tunable pH probes for the fluorescence imaging of acidic organelles in live cells. Angew Chem Int Ed 44(40):6504–8CrossRefGoogle Scholar
  24. 24.
    Burguete MI, Galindo F, Izquierdo MA, O'Connor JE, Herrera G, Luis SV, Vigara L (2010) Synthesis and Evaluation of Pseudopeptidic Fluorescence pH Probes for Acidic Cellular Organelles: In Vivo Monitoring of Bacterial Phagocytosis by Multiparametric Flow Cytometry. Eur J Org Chem 2010(31):5967–5979CrossRefGoogle Scholar
  25. 25.
    Urano Y, Asanuma D, Hama Y, Koyama Y, Barrett T, Kamiya M, Nagano T, Watanabe T, Hasegawa A, Choyke PL, Kobayashi H (2009) Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes. Nature Medicine 15(1):104–109CrossRefGoogle Scholar
  26. 26.
    Tang B, Liu X, Xu K, Huang H, Yang G, An L (2007) A dual near-infrared pH fluorescent probe and its application in imaging of HepG2 cells. Chem Commun 36:3726–3728CrossRefGoogle Scholar
  27. 27.
    De M, Ghosh PS, Rotello VM (2008) Applications of Nanoparticles in Biology. Adv Mater 20(20):1–17Google Scholar
  28. 28.
    Coto-García AM, Sotelo-González E, Fernández-Argüelles MT, Pereiro R, Costa-Fernández JM, Sanz-Medel A (2011) Nanoparticles as fluorescent labels for optical imaging and sensing in genomics and proteomics. Anal Bioanal Chem 399(1):29–42CrossRefGoogle Scholar
  29. 29.
    Lee D-E, Koo H, Sun I-C, Ryu JH, Kim K, Kwon IC (2012) Multifunctional nanoparticles for multimodal imaging and theragnosis. Chem Soc Rev 41(7):2656–2672CrossRefGoogle Scholar
  30. 30.
    Ruedas-Rama MJ, Walters JD, Orte A, Hall EAH (2012) Fluorescent nanoparticles for intracellular sensing: A review. Anal Chim Acta 751:1–23CrossRefGoogle Scholar
  31. 31.
    Wilson R (2008) The use of gold nanoparticles in diagnostics and detection. Chem Soc Rev 37(9):2028–45CrossRefGoogle Scholar
  32. 32.
    Sperling RA, Rivera Gil P, Zhang F, Zanella M, Parak WJ (2008) Biological applications of gold nanoparticles. Chem Soc Rev 37(9):1896–1908CrossRefGoogle Scholar
  33. 33.
    Boisselier E, Astruc D (2009) Gold nanoparticles in nanomedicine: preparations, imaging, diagnostics, therapies and toxicity. Chem Soc Rev 38(6):1759–1782CrossRefGoogle Scholar
  34. 34.
    Giljohann DA, Seferos DS, Daniel WL, Massich MD, Patel PC, Mirkin CA (2010) Gold Nanoparticles for Biology and Medicine. Angew Chem Int Ed 49(19):3280–3294CrossRefGoogle Scholar
  35. 35.
    Saha K, Agasti SS, Kim C, Li X, Rotello VM (2012) Gold Nanoparticles in Chemical and Biological Sensing. Chem Rev 112(5):2739–2779CrossRefGoogle Scholar
  36. 36.
    Marín MJ, Galindo F, Thomas P, Russell DA (2012) Localized Intracellular pH Measurement Using a Ratiometric Photoinduced Electron-Transfer-Based Nanosensor. Angew Chem Int Ed 51(38):9657–9661CrossRefGoogle Scholar
  37. 37.
    Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. (1994) Synthesis of Thiol-derivatised Gold Nanoparticles in a Two-phase Liquid-Liquid System. J Chem Soc Chem Comm 801–802Google Scholar
  38. 38.
    Daniel MC, Astruc D (2004) Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chem Rev 104(1):293–346CrossRefGoogle Scholar
  39. 39.
    Krpetic Z, Nativo P, Porta F, Brust M (2009) A Multidentate Peptide for Stabilization and Facile Bioconjugation of Gold Nanoparticles. Bioconjugate Chemistry 20(3):619–624CrossRefGoogle Scholar
  40. 40.
    Porta F, Krpetic Z, Prati L, Gaiassi A, Scari G (2008) Gold-ligand interaction studies of water-soluble aminoalcohol capped gold nanoparticles by NMR. Langmuir 24(14):7061–4CrossRefGoogle Scholar
  41. 41.
    Schneider G, Decher G, Nerambourg N, Praho R, Werts MH, Blanchard-Desce M (2006) Distance-dependent fluorescence quenching on gold nanoparticles ensheathed with layer-by-layer assembled polyelectrolytes. Nano Letters 6(3):530–6CrossRefGoogle Scholar
  42. 42.
    Lim SY, Kim JH, Lee JS, Park CB (2009) Gold Nanoparticle Enlargement Coupled with Fluorescence Quenching for Highly Sensitive Detection of Analytes. Langmuir 25(23):13302–13305CrossRefGoogle Scholar
  43. 43.
    Hong R, Han G, Fernández JM, Kim BJ, Forbes NS, Rotello VM (2006) Glutathione-mediated delivery and release using monolayer protected nanoparticle carriers. J Am Chem Soc 128(4):1078–9CrossRefGoogle Scholar
  44. 44.
    Tu Y, Wu P, Zhang H, Cai C (2012) Fluorescence quenching of gold nanoparticles integrating with a conformation-switched hairpin oligonucleotide probe for microRNA detection. Chem Commun 48(87):10718–10720CrossRefGoogle Scholar
  45. 45.
    Bardhan R, Grady NK, Cole JR, Joshi A, Halas NJ (2009) Fluorescence Enhancement by Au Nanostructures: Nanoshells and Nanorods. ACS Nano 3(3):744–752CrossRefGoogle Scholar
  46. 46.
    Teixeira R, Paulo PMR, Viana AS, Costa SMB (2011) Plasmon-Enhanced Emission of a Phthalocyanine in Polyelectrolyte Films Induced by Gold Nanoparticles. J Phys Chem C 115(50):24674–24680CrossRefGoogle Scholar
  47. 47.
    Kang KA, Wang J, Jasinski JB, Achilefu S (2011) Fluorescence Manipulation by Gold Nanoparticles: From Complete Quenching to Extensive Enhancement. Journal of Nanobiotechnology 9:16CrossRefGoogle Scholar
  48. 48.
    Marín MJ, Thomas P, Fabregat V, Luis SV, Russell DA, Galindo F (2011) Fluorescence of 1,2-Diaminoanthraquinone and its Nitric Oxide Reaction Product within Macrophage Cells. ChemBioChem 12(16):2471–2477CrossRefGoogle Scholar
  49. 49.
    Klionsky DJ, Emr SD (2000) Cell biology - Autophagy as a regulated pathway of cellular degradation. Science 290(5497):1717–1721CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.School of ChemistryUniversity of East AngliaNorwichUK
  2. 2.Departamento de Química Inorgánica y OrgánicaUniversitat Jaume ICastellón de la PlanaSpain
  3. 3.School of Biological SciencesUniversity of East AngliaNorwichUK
  4. 4.Norwich Medical SchoolUniversity of East AngliaNorwichUK

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