Microchimica Acta

, 186:805 | Cite as

Two-photon excited fluorescent silica nanoparticles loaded with iron(II) as a probe for determination and imaging of hydrogen peroxide in living cells

  • Di Zhao
  • Hongmin MengEmail author
  • Ming-Qing Shi
  • Na Li
  • Guo-Jiang MaoEmail author
Original Paper


A method is described for determination and optical imaging of hydrogen peroxide (H2O2) by using the two-photon (TP) excited fluorescence of silica (SiO2) nanoparticles containing Fe(II) ions. In the presence of H2O2, hydroxyl radicals (•OH) are produced via the Fenton reaction. This leads to quenching of the green fluorescence of a TP-excitable organic dye loaded into the SiO2NPs. Fluorescence is excited at 370 nm and has an emission peaking at 447 nm. The degree of quenching increases linearly in the 2.5 to 100 μM H2O2 concentration range. The nanoprobe is highly selective and sensitive, with a detection limit of 336 nM. The nanoprobe is biocompatible and was successfully used to image changes in the H2O2 concentration in HeLa cells via TP fluorescence imaging.

Graphical abstract

Schematic rpresentation of the detection of H2O2 by using the two-photon excited fluorescence of silica nanoparticles (TP-SiO2NPs) containing Fe2+. H2O2 triggers the Fenton reaction to produce hydroxyl radicals (•OH), which quench the green fluorescence of the SiO2NPs.


Quenching Fenton reaction Methylene blue Fluorescent nanoprobe Hydroxyl radical HeLa cells Two-photon imaging 



This work was supported by National Natural Science Foundation of China (21605038, 21505032), China Postdoctoral Science Foundation (2019 T120623, 2016 M602245), the Key scientific research project of higher education of the Henan province (16A150013).

Compliance with ethical standards

Conflict of interest

There are no conflicts to declare.

Supplementary material

604_2019_3926_MOESM1_ESM.docx (4.4 mb)
ESM 1 (DOCX 4.42 mb)


  1. 1.
    Han J, Chu C, Cao G, Mao W, Wang S, Zhao Z, Gao M, Ye H, Xu X (2018) A simple boronic acid-based fluorescent probe for selective detection of hydrogen peroxide in solutions and living cells. Bioorg Chem 81:362–366CrossRefGoogle Scholar
  2. 2.
    Gerogianni PS, Chatziathanasiadou MV, Diamantis DA, Tzakos AG, Galaris D (2018) Lipophilic ester and amide derivatives of rosmarinic acid protect cells against H2O2-induced DNA damage and apoptosis: the potential role of intracellular accumulation and labile iron chelation. Redox Biol 15:548–556CrossRefGoogle Scholar
  3. 3.
    Hecquet CM, Malik AB (2009) Role of H2O2- activated trpm2 calcium channel in oxidant-induced endothelial injury. Thromb Haemost 101(4):619–625CrossRefGoogle Scholar
  4. 4.
    Pieri L (2003) Redox modulation of protein kinase/phosphatase balance in melanoma cells: the role of endogenous and γ-glutamyltransferase-dependent H2O2 production. Biochim Biophys Acta 1621(1):76–83CrossRefGoogle Scholar
  5. 5.
    Botteaux A, Hoste C, Dumont JE, Van Sande J, Allaoui A (2009) Potential role of Noxes in the protection of mucosae: H2O2 as a bacterial repellent. Microbes Infect 11(5):537–544CrossRefGoogle Scholar
  6. 6.
    Choi YH (2018) Schisandrin a prevents oxidative stress-induced DNA damage and apoptosis by attenuating ROS generation in C2C12 cells. Biomed Pharmacother 106:902–909CrossRefGoogle Scholar
  7. 7.
    Yang J, Yang J, Liang SH, Xu Y, Moore A, Ran C (2016) Imaging hydrogen peroxide in Alzheimer's disease via cascade signal amplification. Sci Rep 6:35613CrossRefGoogle Scholar
  8. 8.
    Bombicino SS, Iglesias DE, Rukavina-Mikusic IA, Buchholz B, Gelpi RJ, Boveris A, Valdez LB (2017) Hydrogen peroxide, nitric oxide and ATP are molecules involved in cardiac mitochondrial biogenesis in diabetes. Free Radic Biol Med 112:267–276CrossRefGoogle Scholar
  9. 9.
    Zhao P, Wang K, Zhu X, Zhou Y, Wu J (2018) A fluorescent probe for imaging hydrogen peroxide in ovarian cancer cells. Dyes Pigments 155:143–149CrossRefGoogle Scholar
  10. 10.
    Borgmann S (2009) Electrochemical quantification of reactive oxygen and nitrogen: challenges and opportunities. Anal Bioanal Chem 394(1):95–105CrossRefGoogle Scholar
  11. 11.
    Allan Butterfield D, Perluigi M, Reed T, Muharib T, Hughes CP, Robinson RAS, Sultana R (2012) Redox proteomics in selected neurodegenerative disorders: from its infancy to future applications. Antioxid Redox Signal 17:1610–1655CrossRefGoogle Scholar
  12. 12.
    Schoneich C, Sharov VS (2006) Mass spectrometry of protein modifications by reactive oxygen and nitrogen species. Free Radic Biol Med 41(10):1507–1520CrossRefGoogle Scholar
  13. 13.
    Ye S, Hu JJ, Yang D (2018) Tandem Payne/dakin reaction: a new strategy for hydrogen peroxide detection and molecular imaging. Angew Chem Int Ed 57(32):10173–10177CrossRefGoogle Scholar
  14. 14.
    Lin LS, Huang T, Song JB, Ou XY, Wang ZT, Deng HZ, Tian R, Liu YJ, Wang JF, Liu Y, Yu GC, Zhou ZJ, Wang S, Niu G, Yang HH, Chen XY (2019) Synthesis of copper peroxide nanodots for H2O2 self-supplying chemodynamic therapy. J Am Chem Soc 141:9937–9945CrossRefGoogle Scholar
  15. 15.
    Pirsaheb M, Mohammadi S, Salimi A, Payandeh M (2019) Functionalized fluorescent carbon nanostructures for targeted imaging of cancer cells: a review. Microchim Acta 186(4):231CrossRefGoogle Scholar
  16. 16.
    Fenton HJH (1894) LXXIII.—oxidation of tartaric acid in presence of iron. J Chem Soc 65:899–910CrossRefGoogle Scholar
  17. 17.
    Ito S, Mitarai A, Hikino K, Hirama M, Sasaki K (1992) Deactivation reaction in the hydroxylation of benzene with Fenton’s reagent. J Org Chem 57:6937–6941CrossRefGoogle Scholar
  18. 18.
    Casero I, Sicilia D, Rubio S, Pérez-Bendito D (1997) Chemical degradation of aromatic amines by Fenton's reagent. Wat Res Vol 31:1985–1995CrossRefGoogle Scholar
  19. 19.
    Peng J, Shi F, Gu Y, Deng Y (2003) Highly selective and green aqueous–ionic liquid biphasic hydroxylation of benzene to phenol with hydrogen peroxide. Green Chem 5(2):224–226CrossRefGoogle Scholar
  20. 20.
    Wen H, Gu L, Yu H, Qiao X, Zhang D, Ye J (2018) Radical assisted iron impregnation on preparing sewage sludge derived Fe/carbon as highly stable catalyst for heterogeneous Fenton reaction. Chem Eng J 352:837–846CrossRefGoogle Scholar
  21. 21.
    Ganiyu SO, Zhou M, Martínez-Huitle CA (2018) Heterogeneous electro-Fenton and photoelectro-Fenton processes: a critical review of fundamental principles and application for water/wastewater treatment. Appl Catal B-Environ 235:103–129CrossRefGoogle Scholar
  22. 22.
    Qin Y, Song F, Ai Z, Zhang P, Zhang L (2015) Protocatechuic acid promoted alachlor degradation in Fe(III)/H2O2 Fenton system. Environ Sci Technol 49(13):7948–7956CrossRefGoogle Scholar
  23. 23.
    Yue Z, Hong T, Song X, Wang Z (2018) Construction of a targeted photodynamic nanotheranostic agent using upconversion nanoparticles coated with an ultrathin silica layer. Chem Commun 54(75):10618–10621CrossRefGoogle Scholar
  24. 24.
    Mao GJ, Wei TT, Wang XX, Huan SY, Lu DQ, Zhang J, Zhang XB, Tan W, Shen GL, Yu RQ (2013) High-sensitivity naphthalene-based two-photon fluorescent probe suitable for direct bioimaging of H2S in living cells. Anal Chem 85(16):7875–7881CrossRefGoogle Scholar
  25. 25.
    Meng HM, Jin Z, Lv Y, Yang C, Zhang XB, Tan W, Yu RQ (2014) Activatable two-photon fluorescence nanoprobe for bioimaging of glutathione in living cells and tissues. Anal Chem 86(24):12321–12326CrossRefGoogle Scholar
  26. 26.
    Zhao Z, Meng H, Wang N, Donovan MJ, Fu T, You M, Chen Z, Zhang X, Tan W (2013) A controlled-release nanocarrier with extracellular pH value driven tumor targeting and translocation for drug delivery. Angew Chem Int Ed Engl 52(29):7487–7491CrossRefGoogle Scholar
  27. 27.
    Meng HM, Zhang XB, Yang C, Kuai HL, Mao GJ, Gong L, Zhang WH, Feng SL, Chang JB (2016) Efficient two-photon fluorescence nanoprobe for turn-on detection and imaging of ascorbic acid in living cells and tissues. Anal Chem 88:6057–6063CrossRefGoogle Scholar
  28. 28.
    Bai XY, Huang YY, Lu MY, Yang D (2017) HKOH-1: a highly sensitive and selective fluorescent probe for detecting endogenous hydroxyl radicals in living cells. Angew Chem Int Ed 56:12873–12877CrossRefGoogle Scholar
  29. 29.
    Liu F, Bing T, Shangguan D, Zhao MP, Shao N (2016) Ratiometric fluorescent biosensing of hydrogen peroxide and hydroxyl radical in living cells with lysozyme−silver nanoclusters: lysozyme as stabilizing ligand and fluorescence signal unit. Anal Chem 88:10631–10638CrossRefGoogle Scholar
  30. 30.
    Goldstein S, Meyerstein D, Czapski G (1993) The Fenton reagents. Free Radical Bio Med 15(4):435–445CrossRefGoogle Scholar
  31. 31.
    Dunford HB (2002) Oxidations of iron(II)/(III) by hydrogen peroxide: from aquo to enzyme. Coord Chem Rev 233(6):311–318CrossRefGoogle Scholar
  32. 32.
    Ma B, Wang S, Liu F, Zhang S, Duan J, Li Z, Kong Y, Sang Y, Liu H, Bu W, Li L (2019) Self-assembled copper-amino acid nanoparticles for in situ glutathione “and” H2O2 sequentially triggered chemodynamic therapy. J Am Chem Soc 141(2):849–857CrossRefGoogle Scholar
  33. 33.
    Peng T, Wong N, Chen C, Chan Y, Ho H, Sun Z, Hu J, Shen J, El-Nezami H, Yang D (2014) Molecular imaging of peroxynitrite with HKGreen-4 in live cells and tissues. J Am Chem Soc 136(33):11728–11734CrossRefGoogle Scholar
  34. 34.
    Li X, Zhang G, Ma H, Zhang D, Li J, Zhu D (2004) 4,5-Dimethylthio-4′-[2-(9-anthryloxy)ethylthio]tetrathiafulvalene, a highly selective and sensitive chemiluminescence probe for singlet oxygen. J Am Chem Soc 126:11543–11548CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Henan Key Laboratory of Organic Functional Molecule and Drug Innovation, School of Chemistry and Chemical EngineeringHenan Normal UniversityXinxiangPeople’s Republic of China
  2. 2.Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, College of Chemistry and Molecular EngineeringZhengzhou UniversityZhengzhouPeople’s Republic of China

Personalised recommendations