An amperometric biosensor based on rat cytochrome p450 1A1 for benzo[a]pyrene determination

• Published in: Biosensors & bioelectronics 2011-01, Vol.26 (5), p.2177-2182
• Main Authors: Wu, Yunhua, Liu, Xuequn, Zhang, Li, Wang, Chuntai
• Format: Article
• Language: English
• Subjects: Animals; Benzo(a)pyrene - analysis; Benzo(a)pyrene - chemistry; Benzo[a]pyrene; Biological and medical sciences; Biosensing Techniques - instrumentation; Biosensor; Biosensors; Biotechnology; Conductometry - instrumentation; Cyclic voltammetry; Cytochrome P-450 CYP1A1 - chemistry; Cytochrome P450 1A1; Equipment Design; Equipment Failure Analysis; Escherichia coli; Fundamental and applied biological sciences. Psychology; Methods. Procedures. Technologies; Rats; Various methods and equipments
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2010.09.027

Using the plasmid pCW, high-level expression of rat cytochrome p4501A1 (CYP1A1) has been achieved by making NH 2-terminal translational fusions to bacterial leader sequences ompA (ompA-1A1/pCW). The construct ompA-1A1 was compared with an expression construct in which the Ala codon GCT was placed in the second position and 5′-terminal codons were maximized for A T content (1A1/pCW). Both constructs produced spectrally active, functional protein. However, the ompA-1A1 fusion gave higher levels of expression, and a marked improvement in the recovery of active P450 in bacterial membrane fractions, when compared with the construct 1A1/pCW. The expressed 1A1 from the construct ompA-1A1/pCW in bacterial membrane fractions were collected and immobilized in nano-Na-montmorillonite (nano-SWy-2) and dihexadecylphosphate (DHP) composite film. The direct electrochemistry of CYP1A1 in a nano-SWy-2-DHP film on an edge-plane pyrolytic graphite electrode (EPG) has been obtained and the catalytic activity of the enzyme to benzo[a]pyrene has been investigated by the cyclic voltammetry. The immobilized CYP1A1 displayed a pair of redox peaks with a formal potential of −0.36 mV in pH 7.0 O 2-free phosphate buffers at scan rate of 1 V s −1. The CYP1A1 in the nano-SWy-2-DHP film retained its bioactivity and could catalyze the reduction of dissolved oxygen. Upon the addition of its substrate benzo[a]pyrene (B[a]P) to the air-saturated solution, the reduction peak current of dissolved oxygen increased, which indicates the catalytic behavior of CYP1A1 to B[a]P. By amperometry a calibration linear range for B[a]P was obtained to be 3.31–16.56 μM with a sensitivity of 58.57 μA mM −1. And the apparent Michaelis–Menten constant for the electrocatalytic activity of CYP1A1 was estimated to be 46.27 μM for B[a]P.