Polydopamine functionalized nanoporous graphene foam as nanoreactor for efficient electrode-driven metabolism of steroid hormones

Understanding the enzymatic reaction kinetics of cytochrome P450 (CYP) within confined spaces is essential for the development of efficient CYP bioreactors in vitro. Herein, a facile electrochemical enzymatic nanoreactor (CYP3A4/PNGFs) is constructed by immobilizing CYP3A4 inside polydopamine (PDA) modified nanoporous graphene foams (PNGFs) whose pore diameter is controllable by the template of silica spheres. Taking advantages of the unique 3-D structure of NGFs and the ideal crosslinking agent of PDA, CYP3A4 is easily absorbed into PNGFs with high stability and bioactivity. Meanwhile, the enzymatic activity of CYP3A4 can be effectively regulated by changing the pore diameter of PNGFs. At the pore diameter of about 65 nm, CYP3A4 shows the highest affinity to testosterone with the Michaelis-Menten constant (K_m ^app) of 110.70 ± 18 μM and the relatively higher reaction rate with the I_max value of 11.85 ± 1.20 nA, which is due to the maintained native configuration and larger amount of immobilized enzyme. Furthermore, the constructed CYP3A4/PNGF-1 nanoreactor is successfully applied to the metabolism of three steroid hormones (testosterone, estrone and progesterone), and the order of the calculated K_m ^app values is consistent with literature. The proposed enzymatic reactor provides a new strategy for the efficient imitation of the natural P450 metabolic pathway at low cost, and has potential applications in fast steroid hormone metabolism assays, drug development and toxicity screening.