Detection of Hydrogen Peroxide in vitro and in vivo Using Peroxalate Chemiluminescent Micelles

  • Lee, Il-Jae (Department of BIN Fusion Technology, Chonbuk National University) ;
  • Hwang, On (Department of BIN Fusion Technology, Chonbuk National University) ;
  • Yoo, Dong-Hyuck (Department of BIN Fusion Technology, Chonbuk National University) ;
  • Khang, Gil-Son (Department of Polymer.Nano Science and Technology, Chonbuk National University) ;
  • Lee, Dong-Won (Department of BIN Fusion Technology, Chonbuk National University)
  • Received : 2011.03.02
  • Accepted : 2011.05.10
  • Published : 2011.07.20


Hydrogen peroxide plays a key role as a second messenger in the normal cellular signaling but its overproduction has been implicated in various life-threatening diseases. Peroxalate chemiluminescence is the light emission from a three component reaction between peroxalate, hydrogen peroxide and fluorophores. It has proven great potential as a methodology to detect hydrogen peroxide in physiological environments because of its excellent sensitivity and specificity to hydrogen peroxide. We developed chemiluminescent micelles composed of amphiphilic polymers, peroxalate and fluorescent dyes to detect hydrogen peroxide at physiological concentrations. In this work, we studied the relationship between the chemiluminescence reactivity and stability of peroxalate by varying the substitutes on the aryl rings of peroxalate. Alkyl substitutes on the aryl ring of peroxalate increased the stability against water hydrolysis, but diminished the reactivity to hydrogen peroxide. Chemiluminescent micelles encapsulating diphenyl peroxalate showed significantly higher chemiluminescence intensity than the counterpart encapsulating dimethylphenyl or dipropylphenyl peroxalate. Diphenyl peroxalate-encapsulated micelles could detect hydrogen peroxide generated from macrophage cells stimulated by lipopolysaccharide (LPS) and image hydrogen peroxide generated during LPS-induced inflammatory responses in a mouse.


  1. Azad, N.; Rojanasakul, Y.; Vallyathan, V. Journal of Toxicology and Environmental Health-Part B-Critical Reviews 2008, 11(1), 1.
  2. Miller, E. W.; Albers, A. E.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. Journal of the American Chemical Society 2005, 127(47), 16652.
  3. Lee, J. Y.; Jang, Y. W.; Kang, H. S.; Moon, H.; Sim, S. S.; Kim, C. J. Archives of Pharmacal Research 2006, 29(10), 849.
  4. Chang, M. C. Y.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. Journal of the American Chemical Society 2004, 126(47), 15392.
  5. Carter, W. O.; Narayanan, P. K.; Robinson, J. P. Journal of Leukocyte Biology 1994, 55(2), 253.
  6. Lang, J. D.; McArdle, P. J.; O'Reilly, P. J.; Matalon, S. Chest 2002, 122(6), 314S.
  7. Feder, L. S.; Stelts, D.; Chapman, R. W.; Manfra, D.; Crawley, Y.; Jones, H.; Minnicozzi, M.; Fernandez, X.; Paster, T.; Egan, R. W.; Kreutner, W.; Kung, T. T. American Journal of Respiratory Cell and Molecular Biology 1997, 17(4), 436.
  8. Park, H.; Kim, S.; Song, Y.; Seung, K.; Hong, D.; Khang, G.; Lee, D. Biomacromolecules 2010, 11(8), 2103.
  9. Lee, D.; Khaja, S.; Velasquez-Castano, J. C.; Dasari, M.; Sun, C.; Petros, J.; Taylor, W. R.; Murthy, N. Nature Materials 2007, 6, 765.
  10. Lee, D. W.; Erigala, V. R.; Dasari, M.; Yu, J. H.; Dickson, R. M.; Murthy, N. International Journal of Nanomedicine 2008, 3(4), 471.
  11. Soh, N. Analytical and Bioanalytical Chemistry 2006, 386(3), 532.
  12. Kamyshny, A.; Magdassi, S. Colloids and Surfaces B-Biointerfaces 1998, 11(5), 249.
  13. Hadd, A. G.; Lehmpuhl, D. W.; Kuck, L. R.; Birks, J. W. Journal of Chemical Education 1999, 76(9), 1237.
  14. Motoyoshiya, J.; Sakai, N.; Imai, M.; Yamaguchi, Y.; Koike, R.; Takaguchi, Y.; Aoyama, H. Journal of Organic Chemistry 2002, 67(21), 7314.
  15. Maulding, D. R.; Clarke, R. A.; Roberts, B. G.; Rauhut, M. M. The Journal of Organic Chemistry 1968, 33(1), 250.
  16. Dasari, M.; Lee, D.; Erigala, V. R.; Murthy, N. Journal of Biomedical Materials Research Part A 2009, 89A(3), 561.
  17. Kim, M. S.; Seo, K. S.; Khang, G.; Cho, S. H.; Lee, H. B. Journal of Biomedical Materials Research Part A 2004, 70A(1), 154.
  18. Lim, C. K.; Lee, Y. D.; Na, J.; Oh, J. M.; Her, S.; Kim, K.; Choi, K.; Kim, S.; Kwon, I. C. Advanced Functional Materials 2010, 20(16), 2644.
  19. Sredni-Kenigsbuch, D.; Kambayashi, T.; Strassmann, G. Immunology Letters 2000, 71(2), 97.
  20. Hikosaka, K.; Koyama, Y.; Motobu, M.; Yamada, M.; Nakamura, K.; Koge, K.; Shimura, K.; Isobe, T.; Tsuji, N.; Kang, C. B.; Hayashidani, H.; Wang, P. C.; Matsumura, M.; Hirota, Y. Bioscience Biotechnology and Biochemistry 2006, 70(12), 2853.

Cited by

  1. /NO with Three Different Sets of Fluorescence Signals vol.134, pp.2, 2012,
  2. Amplification of oxidative stress by a dual stimuli-responsive hybrid drug enhances cancer cell death vol.6, pp.2041-1723, 2015,
  3. Hydrogen peroxide-responsive micelles self-assembled from a peroxalate ester-containing triblock copolymer vol.4, pp.2, 2016,
  4. Nanoparticles based on quantum dots and a luminol derivative: implications for in vivo imaging of hydrogen peroxide by chemiluminescence resonance energy transfer vol.52, pp.22, 2016,
  5. Oxalate-curcumin–based probe for micro- and macroimaging of reactive oxygen species in Alzheimer’s disease vol.114, pp.47, 2017,
  6. Possibilities and Challenges for Quantitative Optical Sensing of Hydrogen Peroxide vol.5, pp.4, 2017,
  7. Hydrogen Peroxide-Responsive Nanoprobe Assists Circulating Tumor Cell Identification and Colorectal Cancer Diagnosis vol.89, pp.11, 2017,
  8. Imaging Reactive Oxygen Species-Induced Modifications in Living Systems vol.24, pp.16, 2016,
  9. Quantitative analysis of hydrogen peroxide with special emphasis on biosensors vol.41, pp.3, 2018,
  10. Recent advances in hydrogen peroxide imaging for biological applications vol.4, pp.1, 2014,