Utilization of substrate specific membrane channels in liposome based biosensors: A nitrite biosensor with NirC nitrite channels

Abstract

A liposome-based nitrite biosensor was prepared by encapsulating the nitrite reductase NirB enzyme and embedding the nitrite-specific NirC channels in the liposome structure. The proteoliposomes were coated on the glassy carbon electrode modified with the multi-walled carbon nanotube/chitosan matrix. The absence of channels in the liposome structure inhibits the generation of typical reduction peaks upon the addition of nitrite suggesting that ion channels are necessary for the passage of charged ions into the liposome. The response of the NirB and NirC incorporated liposome-based biosensor (CBLip/MWCNTs/CHIT/GC electrode) to the nitrite is shown to be fast and reproducible. The electrode shows a linear response between 1-500 mu M nitrite concentrations. The sensitivity of the CBLip/MWCNTs/CHIT/GC electrode was 707 mA.M- 1.cm- 2, lower than that of the unentrapped NirB-loaded electrode (1131 mA.M- 1.cm- 2). While the electrode prepared with the unentrapped enzyme lost its activity in 30 days, entrapment of the enzyme in the liposome preserved the electrode activity by 55 %. The electrodes showed unexpected oxidation responses against high concentrations of sulfate and formate, the incorporation of NirC channels contributed to increasing the specificity of the electrode towards nitrite in the presence of these ions. The use of substrate-specific membrane channels in liposome-based biosensors was achieved for the first time in the present study.