Microchimica Acta

, Volume 175, Issue 3–4, pp 241–250 | Cite as

Overcoming the adverse effects of crosslinking in biosensors via addition of PEG: Improved sensing of hydrogen peroxide using immobilized peroxidase

  • Mohammed ElKaoutit
  • Ignacio Naranjo-Rodriguez
  • Manuel Dominguez
  • Jose Luis Hidalgo-Hidalgo de Cisneros
Original Paper


Glutaraldehyde (GA) is widely used as a crosslinker to immobilize enzymes, for examples in biosensors, but often causes partial denaturation. We find that the proper use of poly(ethylene glycol) (PEG) during the crosslinking process can fully preserve the native state and activity of horseradish peroxidase (HRP). An amperometric biosensor was developed based on these findings for the direct determination of hydrogen peroxide. UV-Vis and FTIR spectroscopy reveal that the HRP entrapped in a polypyrrole matrix retains its native structure. The addition of PEG increases the sensitivity and stability of the biosensor and prevents many of effects caused by intra-crosslinking via GA. The biosensor was operated at a potential of −350 mV (vs Ag/AgCl) without any mediator and gave a linear response to H2O2 in the 5 to 190 μM concentration range. The apparent Michaelis-Menten constant is 3.37 mM, and maximal current is as high as 3.43 μA. The surface of the biosensor was characterized by atomic force microscopy operated in the tapping mode.


Solid phase and AFM images of native and denaturized peroxidase


Direct electrocatalysis Heme enzyme Polyethylene glycol Glutaraldehyde Intra-crosslinkage Conformational study 



We thank to the Junta de Andalucía/FEDER (FQM249/P08-FQM-04006) and Ministerio de Ciencia e Innovación of Spain/FEDER (CTQ2010-19058) for their financial support.

Supplementary material

604_2011_686_MOESM1_ESM.doc (708 kb)
ESM 1 (DOC 707 kb)


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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Mohammed ElKaoutit
    • 1
  • Ignacio Naranjo-Rodriguez
    • 1
  • Manuel Dominguez
    • 2
  • Jose Luis Hidalgo-Hidalgo de Cisneros
    • 1
  1. 1.Departamento de Química Analítica, Facultad de CienciasUniversidad de CádizPuerto RealSpain
  2. 2.Departamento de la Física de la Materia Condensada, Facultad de CienciasUniversidad de CádizPuerto RealSpain

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