Horseradish Peroxidase-Attapulgite Clay Nanocomposites: Fabrication and Application to Sensing the Extracellular H₂O₂ Released from Cells

Authors

Ping WU, Jiangsu Key Laboratory of New Power Batteries, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210046, China;
Chen-xin CAI, Jiangsu Key Laboratory of New Power Batteries, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210046, China;Follow

Corresponding Author

Chen-xin CAI(cxcai@njnu.edu.cn)

Abstract

The nanostructured attapulgite clay was employed as a support matrix for immobilizing horseradish peroxidase (HRP). FTIR and electrochemical methods demonstrated that HRP had been effectively assembled on attapulgite surface with the formation of HRP-attapulgite nanocomposites. The HRP-attapulgite hybrid was deposited on the glassy carbon (GC) electrode forming the HRP-attapulgite/GC electrode. Cyclic voltammetric results showed a pair of well-defined redox peaks, which were ascribed to direct electron transfer (DET) of HRP, with the formal potential E₀′ = -335 mV (vs. SCE) in the phosphate buffer solution (PBS, pH 7.0). The dependence of E₀′ on solution pH indicated that DET reaction of HRP was coupled with proton transfer. The developed electrode revealed a good electrocatalytical activity toward the reduction of H₂O₂. The electrocatalyic current was linear to the concentration of H₂O₂ in the concentration range of 0.3 to 75 μmol·L^-1 with high sensitivity ((53 ± 2) μA·(mmol·L^-1)^-1·cm^-2) and low detection limit ((0.10 ± 0.05) μmol·L^-1). Moreover, the HRP-attapulgite/GC electrode was used to detect the level cellular H₂O₂ released from the Macrophages cells (RAW 264.7). Therefore, an electrochemical approach for detection of cellular H₂O₂ based on HRP-attapulgite hybrid was developed. This study has not only established a novel approach to detect cellular H₂O₂, but also provided a general route for fabricating attapulgite-based biosensing platform via assembling enzymes/proteins on attapulgite surface, expanded the scope of attapulgite applications to the field of bioelectroanalytical chemistry and cellular biology.

Graphical Abstract

Keywords

horseradish peroxidase, attapulgite, hydrogen peroxide, macrophages cell

DOI Link

https://doi.org/10.13208/j.electrochem.130883

Publication Date

2014-06-28

Online Available Date

2013-09-30

Revised Date

2013-09-23

Received Date

2013-08-26

Recommended Citation

Ping WU, Chen-xin CAI. Horseradish Peroxidase-Attapulgite Clay Nanocomposites: Fabrication and Application to Sensing the Extracellular H₂O₂ Released from Cells[J]. Journal of Electrochemistry, 2014 , 20(3): 260-265.
DOI: 10.13208/j.electrochem.130883
Available at: https://jelectrochem.xmu.edu.cn/journal/vol20/iss3/7

References

[1] Finkel T. Redox-dependent signal transduction[J]. FEBS Letters, 2000, 476(1): 52-54.
[2] Wu X M, Hu Y J, Jin J, et al. Electrochemical approach for detection of extracellular oxygen released from Erythrocytes based on graphene film integrated with Laccase and 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)[J]. Analytical Chemistry, 2010, 82(9): 3588-3596.
[3] Tate R M, Repine J E. In free radicals in biology[M]. New York: Academic Press, 1984: 199
[4] Chen D X, Wang Q, Jin J, et al. Low-potential detection of endogenous and physiological uric acid at uricase-thionine-single-walled carbon nanotube modified electrodes[J]. Analytical Chemistry, 2010, 82(6): 2448-2455.
[5] Lei C H, Lisdat F, Wollenberger U, et al. Cytochrome c/clay-modified electrode[J]. Electroanalysis, 1999, 11(4): 274-276.
[6] Lei C H, Wollenberger U, Jung C, et al. Clay-bridged electron transfer between cytochrome P450cam and electrode[J]. Biochemical and Biophysical Research Communications, 2000, 268(3): 740-744.
[7] Chen X L, Hu N F, Zeng Y H, et al. Ordered electrochemically active films of hemoglobin, didodecyldimethylammonium ions, and clay[J].Langmuir, 1999, 15(20): 7022-7030.
[8] Zhou Y L, Hu N F, Zeng Y H, et al. Heme protein-clay films: Direct electrochemistry and electrochemical catalysis[J]. Langmuir, 2002, 18(1): 211-219.
[9] Zhou Y L, Li Z, Hu N F, et al. Layer-by-layer assembly of ultrathin films of Hemoglobin and clay nanoparticles with electrochemical and catalytic activity[J].Langmuir, 2002, 18(22): 8573-8579.
[10] Charradi K, Forano C, Prevot V, et al. Characterization of hemoglobin immobilized in MgAl-Layered double hydroxides by the coprecipitation method[J]. Langmuir, 2010, 26(12): 9997-10004.
[11] Charradi K, Forano C, Prevot V, et al. Direct electron transfer and enhanced electrocatalytic activity of hemoglobin at iron-rich clay modified electrodes[J]. Langmuir, 2009, 25(17): 10376-10383.
[12] Xu J M, Li W, Yin Q F, et al. Direct electrochemistry of cytochrome c on natural nano-attapulgite clay modified electrode and its electrocatalytic reduction for H2O2[J]. Electrochimca Acta, 2007, 52(11): 3601-3606.
[13] Xu J M, Li W, Yin Q F, et al. Direct electron transfer and bioelectrocatalysis of hemoglobin on nano-structural attapulgite clay-modified glassy carbon electrode[J]. Journal of Colloid and Interface Science, 2007, 315(1): 170-176.
[14] Xu J M(徐继明), Han W X(韩文霞), Yin Q F(尹起范), et al. Direct electron transfer of glucose oxidase and glucose biosensor based on nano-structural attapulgite clay matrix[J].Chinese Journal of Chemistry, 2009, 27(11): 2197-2202.
[15] Huang J L, Tsai Y C. Direct electrochemistry and biosensing of hydrogen peroxide of horseradish peroxidase immobilized at multiwalled carbon nanotube/alumina-coated silica nanocomposite modified glassy carbon electrode[J]. Sensors and Actuators B: Chemical, 2009, 140(1): 267-272.
[16] Yin Y J, Lv Y F, Wu P, et al. Direct electrochemistry of redox proteins and enzymes promoted by carbon nanotubes[J]. Sensors, 2005, 5(4): 220-234.
[17] Szymczyk N H, Kerr B A E, Freeman T A. Involvement of hydrogen peroxide in the differentiation and apoptosis of preosteoclastic cells exposed to arsenite[J]. Biochemical pharmacology, 2006, 72(6): 761-769.