Electrochemistry and biosensing activity of cytochrome c immobilized in macroporous materials


An amperometric biosensor for hydrogen peroxide (H2O2) has been constructed by immobilizing cytochrome c on an indium/tin oxide (ITO) electrode modified with a macroporous material. Cyclic voltammetry showed that the direct and quasi-reversible electron transfer of cytochrome c proceeds without the need for an electron mediator. A surface-controlled electron transfer process can be observed with an apparent heterogeneous electron-transfer rate constant (ks) of 29.2 s−1. The biosensor displays excellent electrocatalytic responses to the reduction of H2O2 to give amperometric responses that increase steadily with the concentration of H2O2 in the range from 5 μM to 2 mM. The detection limit is 0.61 μM at pH 7.4. The apparent Michaelis-Menten constant (Km) of the biosensor is 1.06 mM. This investigation not only provided a method for the direct electron transfer of cytochrome c on macroporous materials, but also established a feasible approach for durable and reliable detection of H2O2.

Biosensor for hydrogen peroxide was developed by immobilizing cytochrome c in the macroporous ordered silica foam (MOSF) through the electrostatic interaction. The achievement of the direct electron transfer between cytochrome c and electrode surface indicated that the MOSF modified electrode displayed good affinity and biocompatibility for cytochrome c.

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  1. 1.

    Halliwell B (1992) Reactive oxygen species and the central-nervous-system. J Neurochem 59(5):1609–1623

    Article  CAS  Google Scholar 

  2. 2.

    Hall ED, Braughler JM (1989) Central nervous-system trauma and stroke II. Physiological and pharmacological evidence for involvement of oxygen radicals and lipid-peroxidation. Free Radical Bio Med 6(3):303–313

    Article  CAS  Google Scholar 

  3. 3.

    Maruyama W, Dostert P, Matsubara K, Naoi M (1995) N-Methyl(R)Salsolinol produces hydroxyl radicals—involvement to neurotoxicity. Free Radical Bio Med 19(1):67–75

    Article  CAS  Google Scholar 

  4. 4.

    Amatore C, Arbault S, Bruce D, de Oliveira P, Erard M, Vuillaume M (2001) Characterization of the electrochemical oxidation of peroxynitrite: relevance to oxidative stress bursts measured at the single cell level. Chem-Eur J 7(19):4171–4179

    Article  CAS  Google Scholar 

  5. 5.

    Miller EW, Albers AE, Pralle A, Isacoff EY, Chang CJ (2005) Boronate-based fluorescent probes for imaging cellular hydrogen peroxide. J Am Chem Soc 127(47):16652–16659

    Article  CAS  Google Scholar 

  6. 6.

    Clarkson DT, Carvajal M, Henzler T, Waterhouse RN, Smyth AJ, Cooke DT, Steudle E (2000) Root hydraulic conductance: diurnal aquaporin expression and the effects of nutrient stress. J Exp Bot 51(342):61–70

    Article  CAS  Google Scholar 

  7. 7.

    Hurdis EC, Romeyn H (1954) Accuracy of determination of hydrogen peroxide by cerate oxidimetry. Anal Chem 26(2):320–325

    Article  CAS  Google Scholar 

  8. 8.

    Matsubara C, Kawamoto N, Takamura K (1992) Oxo[5,10,15,20-Tetra(4-Pyridyl)Porphyrinato] Titanium(Iv)—an ultra-high sensitivity spectrophotometric reagent for hydrogen-peroxide. Analyst 117(11):1781–1784

    Article  CAS  Google Scholar 

  9. 9.

    Zhang GF, Dasgupta PK, Edgemond WS, Marx JN (1991) Determination of hydrogen-peroxide by photoinduced fluorogenic reactions. Anal Chim Acta 243(2):207–216

    CAS  Google Scholar 

  10. 10.

    Rosenzweig Z, Kopelman R (1996) Analytical properties and sensor size effects of a micrometer-sized optical fiber glucose biosensor. Anal Chem 68(8):1408–1413

    Article  CAS  Google Scholar 

  11. 11.

    Luo LR, Zhang ZJ (2006) Sensors based on galvanic cell generated electrochemiluminescence and its application. Anal Chim Acta 580(1):14–17

    Article  CAS  Google Scholar 

  12. 12.

    Rocha FRP, Rodenas-Torralba E, Reis BF, Morales-Rubio A, de la Guardia M (2005) A portable and low cost equipment for flow injection chemiluminescence measurements. Talanta 67(4):673–677

    Article  CAS  Google Scholar 

  13. 13.

    Pinkernell U, Effkemann S, Karst U (1997) Simultaneous HPLC determination of peroxyacetic acid and hydrogen peroxide. Anal Chem 69(17):3623–3627

    Article  CAS  Google Scholar 

  14. 14.

    Lai GS, Zhang HL, Han DY (2009) Amperometric hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase by carbon-coated iron nanoparticles in combination with chitosan and cross-linking of glutaraldehyde. Microchim Acta 165(1–2):159–165

    Article  CAS  Google Scholar 

  15. 15.

    Zhang Y, Luo LQ, Ding YP, Liu XA, Qian ZY (2010) A highly sensitive method for determination of paracetamol by adsorptive stripping voltammetry using a carbon paste electrode modified with nanogold and glutamic acid. Microchim Acta 171(1–2):133–138

    Article  CAS  Google Scholar 

  16. 16.

    Che X, Yuan R, Chai YQ, Ma LP, Li WJ, Li JJ (2009) Hydrogen peroxide sensor based on horseradish peroxidase immobilized on an electrode modified with DNA-L-cysteine-gold-platinum nanoparticles in polypyrrole film. Microchim Acta 167(3–4):159–165

    Article  CAS  Google Scholar 

  17. 17.

    Song J, Xu JM, Zhao PS, Lu LD, Bao JC (2011) A hydrogen peroxide biosensor based on direct electron transfer from hemoglobin to an electrode modified with Nafion and activated nanocarbon. Microchim Acta 172(1):117–123

    Article  CAS  Google Scholar 

  18. 18.

    Xu SX, Zhang XF, Wan T, Zhang CX (2011) A third-generation hydrogen peroxide biosensor based on horseradish peroxidase cross-linked to multi-wall carbon nanotubes. Microchim Acta 172(1):199–205

    Article  CAS  Google Scholar 

  19. 19.

    Lee KP, Gopalan AI, Komathi S (2009) Direct electrochemistry of cytochrome c and biosensing for hydrogen peroxide on polyaniline grafted multi-walled carbon nanotube electrode. Sensor Actuat B-Chem 141(2):518–525

    Article  Google Scholar 

  20. 20.

    Xu Q, Mao C, Liu NN, Zhu JJ, Sheng J (2006) Direct electrochemistry of horseradish peroxidase based on biocompatible carboxymethyl chitosan-gold nanoparticle nanocomposite. Biosens Bioelectron 22(5):768–773

    Article  CAS  Google Scholar 

  21. 21.

    Zeng XD, Li XF, Liu XY, Liu Y, Luo SL, Kong B, Yang SL, Wei WZ (2009) A third-generation hydrogen peroxide biosensor based on horseradish peroxidase immobilized on DNA functionalized carbon nanotubes. Biosens Bioelectron 25(4):896–900

    Article  CAS  Google Scholar 

  22. 22.

    Davis ME (2002) Ordered porous materials for emerging applications. Nature 417(6891):813–821

    Article  CAS  Google Scholar 

  23. 23.

    Zhang L (2008) Direct electrochemistry of cytochrome c at ordered macroporous active carbon electrode. Biosens Bioelectron 23(11):1610–1615

    Article  CAS  Google Scholar 

  24. 24.

    Zhu L, Wang KQ, Lu TH, Xing W, Li J, Yang XG (2008) The direct electrochemistry behavior of Cyt c on the modified glassy carbon electrode by SBA-15 with a high-redox potential. J Mol Catal B-Enzym 55(1–2):93–98

    Article  CAS  Google Scholar 

  25. 25.

    Zhu AW, Tian Y, Liu HQ, Luo YP (2009) Nanoporous gold film encapsulating cytochrome c for the fabrication of a H2O2 biosensor. Biomaterials 30(18):3183–3188

    Article  CAS  Google Scholar 

  26. 26.

    Yablonovitch E (1987) Inhibited spontaneous emission in solid-state physics and electronics. Phys Rev Lett 58(20):2059–2062

    Article  CAS  Google Scholar 

  27. 27.

    Xia YN, Gates B, Yin YD, Lu Y (2000) Monodispersed colloidal spheres: old materials with new applications. Adv Mater 12(10):693–713

    Article  CAS  Google Scholar 

  28. 28.

    Velev OD, Kaler EW (2000) Structured porous materials via colloidal crystal templating: From inorganic oxides to metals. Adv Mater 12(7):531–534

    Article  CAS  Google Scholar 

  29. 29.

    Gao XY, Matsui H (2005) Peptide-based nanotubes and their applications in bionanotechnology. Adv Mater 17(17):2037–2050

    Article  CAS  Google Scholar 

  30. 30.

    Stein A (2003) Advances in microporous and mesoporous solids—Highlights of recent progress. Adv Mater 15(10):763–775

    Article  CAS  Google Scholar 

  31. 31.

    Dimakis VT, Gavalas VG, Chaniotakis NA (2002) Polyelectrolyte-stabilized biosensors based on macroporous carbon electrode. Anal Chim Acta 467(1–2):217–223

    Article  CAS  Google Scholar 

  32. 32.

    Gasparac R, Kohli P, Mota MO, Trofin L, Martin CR (2004) Template synthesis of nano test tubes. Nano Lett 4(3):513–516

    Article  CAS  Google Scholar 

  33. 33.

    Qiao L, Liu Y, Hudson SP, Yang PY, Magner E, Liu BH (2008) A nanoporous reactor for efficient proteolysis. Chem-Eur J 14(1):151–157

    Article  CAS  Google Scholar 

  34. 34.

    Tian RJ, Zhang H, Ye ML, Jiang XG, Hu LH, Li X, Bao XH, Zou HF (2007) Selective extraction of peptides from human plasma by highly ordered mesoporous silica particles for peptidome analysis. Angewandte Chemie-International Edition 46(6):962–965

    Article  CAS  Google Scholar 

  35. 35.

    Wan JJ, Qian K, Qiao L, Wang YH, Kong JL, Yang PY, Liu BH, Yu CZ (2009) TiO2-modified macroporous silica foams for advanced enrichment of multi-phosphorylated peptides. Chem-Eur J 15(11):2504–2508

    Article  CAS  Google Scholar 

  36. 36.

    Qian K, Wan JJ, Qiao L, Huang XD, Tang JW, Wang YH, Kong JL, Yang PY, Yu CZ, Liu BH (2009) Macroporous materials as novel catalysts for efficient and controllable proteolysis. Anal Chem 81(14):5749–5756

    Article  CAS  Google Scholar 

  37. 37.

    Wang HN, Zhou XF, Yu MH, Wang YH, Han L, Zhang J, Yuan P, Auchterlonie G, Zou J, Yu CZ (2006) Supra-assembly of siliceous vesicles. J Am Chem Soc 128(50):15992–15993

    Article  CAS  Google Scholar 

  38. 38.

    George P, Hanania G (1953) A spectrophotometric study of ionizations in methaemoglobin. Biochem J 55(2):236–243

    CAS  Google Scholar 

  39. 39.

    Hawkridg Fm, Kuwana T (1973) Indirect coulometric titration of biological electron-transport components. Anal Chem 45(7):1021–1027

    Article  Google Scholar 

  40. 40.

    Wang SF, Chen T, Zhang ZL, Shen XC, Lu ZX, Pang DW, Wong KY (2005) Direct electrochemistry and electrocatalysis of heme proteins entrapped in agarose hydrogel films in room-temperature ionic liquids. Langmuir 21(20):9260–9266

    Article  CAS  Google Scholar 

  41. 41.

    Vrettos JS, Reifler MJ, Kievit O, Lakshmi KV, de Paula JC, Brudvig GW (2001) Factors that determine the unusually low reduction potential of cytochrome c(550) in cyanobacterial photosystem II. J Biol Inorg Chem 6(7):708–716

    Article  CAS  Google Scholar 

  42. 42.

    Kuznetsov BA, Byzova NA, Shumakovich GP (1994) The effect of the orientation of cytochrome-c molecules covalently attached to the electrode surface upon their electrochemical activity. J Electroanal Chem 371(1–2):85–92

    Article  CAS  Google Scholar 

  43. 43.

    Laviron E (1979) General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems. J Electroanal Chem 101(1):19–28

    Article  CAS  Google Scholar 

  44. 44.

    Zhang X, Wang JF, Wu WJ, Qian SW, Man YH (2007) Immobilization and electrochemistry of cytochrome c on amino-functionalized mesoporous silica thin films. Electrochem Commun 9(8):2098–2104

    Article  CAS  Google Scholar 

  45. 45.

    Jiang X, Zhang L, Dong SJ (2006) Assemble of poly(aniline-co-o-aminobenzenesulfonic acid) three-dimensional tubal net-works onto ITO electrode and its application for the direct electrochemistry and electrocatalytic behavior of cytochrome c. Electrochem Commun 8(7):1137–1141

    Article  CAS  Google Scholar 

  46. 46.

    Zhao GC, Yin ZZ, Zhang L, Wei XW (2005) Direct electrochemistry of cytochrome c on a multi-walled carbon nanotubes modified electrode and its electrocatalytic activity for the reduction of H2O2. Electrochem Commun 7(3):256–260

    Article  CAS  Google Scholar 

  47. 47.

    Dickerso Re, Takano T, Eisenber D, Kallai OB, Samson L, Cooper A, Margolia E (1971) Ferricytochrome C .1. General features of horse and bonito proteins at 2.8 a resolution. J Biol Chem 246(5):1511–1535

    Google Scholar 

  48. 48.

    Li JL, Han T, Wei NN, Du JY, Zhao XW (2009) Three-dimensionally ordered macroporous (3DOM) gold-nanoparticle-doped titanium dioxide (GTD) photonic crystals modified electrodes for hydrogen peroxide biosensor. Biosens Bioelectron 25(4):773–777

    Article  CAS  Google Scholar 

  49. 49.

    Wang B, Zhang JJ, Pan ZY, Tao XQ, Wang HS (2009) A novel hydrogen peroxide sensor based on the direct electron transfer of horseradish peroxidase immobilized on silica-hydroxyapatite hybrid film. Biosens Bioelectron 24(5):1141–1145

    Article  CAS  Google Scholar 

  50. 50.

    Xu SY, Peng B, Han XZ (2007) A third-generation H2O2 biosensor based on horseradish peroxidase-labeled Au nanoparticles self-assembled to hollow porous polymeric nanopheres. Biosens Bioelectron 22(8):1807–1810

    Article  CAS  Google Scholar 

  51. 51.

    Zhang L, Li H, Ni YH, Li J, Liao KM, Zhao GC (2009) Porous cuprous oxide microcubes for non-enzymatic amperometric hydrogen peroxide and glucose sensing. Electrochem Commun 11(4):812–815

    Article  CAS  Google Scholar 

  52. 52.

    Kamin RA, Wilson GS (1980) Rotating-ring-disk enzyme electrode for biocatalysis kinetic-studies and characterization of the immobilized enzyme layer. Anal Chem 52(8):1198–1205

    Article  CAS  Google Scholar 

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This work is supported by NSFC 20925517, STCSM (10XD1406000, 09JC1402600) and Shanghai Leading Academic Discipline B109.

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Corresponding authors

Correspondence to Chengzhong Yu or Baohong Liu.

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Ying Wang and Kun Qian contributed equally to this work.

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Wang, Y., Qian, K., Guo, K. et al. Electrochemistry and biosensing activity of cytochrome c immobilized in macroporous materials. Microchim Acta 175, 87 (2011). https://doi.org/10.1007/s00604-011-0638-8

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  • Macroporous material
  • Cytochrome
  • Hydrogen peroxide
  • Direct electron transfer
  • Biosensor