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

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 mu M concentration range. The apparent Michaelis-Menten constant is 3.37 mM, and maximal current is as high as 3.43 mu A. The surface of the biosensor was characterized by atomic force microscopy operated in the tapping mode.