TY - JOUR
T1 - Whole-cell amperometric biosensor for screening of cytochrome P450 inhibitors
AU - Yoetz-Kopelman, Tal
AU - Porat-Ophir, Carmit
AU - Shacham-Diamand, Yosi
AU - Freeman, Amihay
N1 - Publisher Copyright:
© 2015 Elsevier B.V. All rights reserved.
PY - 2016/2/1
Y1 - 2016/2/1
N2 - A fast and cost-effective whole-cell electrochemical biosensor aiming at early-stage screening of potential inhibitors of cytochrome P450 is proposed and its feasibility demonstrated. Sensing is performed by monitoring the decrease in the electrochemical signal generated by the oxidation of the product of the enzymatic reaction of cytochrome P450 BM3 expressed in E. coli cells. The system is self-maintained and does not require enzyme purification or external addition of NADPH or other cofactors. Measurements were performed simultaneously at eight chips at low positive potential of 100 mV vs. Ag/AgCl under continuous stirring. Kinetic analysis of aniline determination by the whole-cell system was performed and the concentration of aniline for the inhibition studies was selected accordingly. Three known inhibitors - imidazole, metyrapone and 1-aminobenzotriazole (ABT) - were tested and their inhibition profiles characterized. Imidazole was found to be the most potent inhibitor of the three. To the best of our knowledge, the system developed enables for the first time rapid and cheap cytochrome P450 inhibitors detection by a disposable whole-cell electrochemical chip, combined with the advantages of a whole-cell system, without neither enzyme purification nor NADPH addition. Furthermore, the system developed also provides capability of performing aniline detection e.g. for environmental monitoring, by means of electrochemical biosensing.
AB - A fast and cost-effective whole-cell electrochemical biosensor aiming at early-stage screening of potential inhibitors of cytochrome P450 is proposed and its feasibility demonstrated. Sensing is performed by monitoring the decrease in the electrochemical signal generated by the oxidation of the product of the enzymatic reaction of cytochrome P450 BM3 expressed in E. coli cells. The system is self-maintained and does not require enzyme purification or external addition of NADPH or other cofactors. Measurements were performed simultaneously at eight chips at low positive potential of 100 mV vs. Ag/AgCl under continuous stirring. Kinetic analysis of aniline determination by the whole-cell system was performed and the concentration of aniline for the inhibition studies was selected accordingly. Three known inhibitors - imidazole, metyrapone and 1-aminobenzotriazole (ABT) - were tested and their inhibition profiles characterized. Imidazole was found to be the most potent inhibitor of the three. To the best of our knowledge, the system developed enables for the first time rapid and cheap cytochrome P450 inhibitors detection by a disposable whole-cell electrochemical chip, combined with the advantages of a whole-cell system, without neither enzyme purification nor NADPH addition. Furthermore, the system developed also provides capability of performing aniline detection e.g. for environmental monitoring, by means of electrochemical biosensing.
KW - Aniline
KW - Cytochrome P450 BM3
KW - E. coli
KW - Electrochemical biosensor
KW - Inhibitors screening
KW - Whole-cell biosensor
UR - http://www.scopus.com/inward/record.url?scp=84944097455&partnerID=8YFLogxK
U2 - 10.1016/j.snb.2015.09.111
DO - 10.1016/j.snb.2015.09.111
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AN - SCOPUS:84944097455
SN - 0925-4005
VL - 223
SP - 392
EP - 399
JO - Sensors and Actuators, B: Chemical
JF - Sensors and Actuators, B: Chemical
ER -