Enzyme biosensor for androsterone based on 3α-hydroxysteroid dehydrogenase immobilized onto a carbon nanotubes/ionic liquid/NAD+ composite electrode

A 3α-hydrosteroid biosensor for androsterone determination has been prepared by immobilizing the enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD) in a composite electrode platform constituted of a mixture of multi-walled carbon nanotubes (MWCNTs), octylpyridinium hexafluorophosphate (OPPF6) ionic liquid and NAD+ cofactor. This configuration allowed the fast, sensitive and stable electrochemical detection of the NADH generated in the enzyme reaction. All the experimental variables involved in the preparation and performance of the enzyme biosensor were optimized. Amperometry in stirred solutions at +400 mV provided a linear calibration plot for androsterone in the 0.5–10 μM concentration range with a slope value more than 200-times higher than that previously reported. The detection limit achieved was 0.15 μM and a low value of the apparent Michaelis–Menten constant (KappM), 36.0 μM, similar to that reported for the enzyme in solution, was calculated. The 3α-HSD/MWCNTs/OPPF6/NAD+ biosensor provided good results in the determination of androsterone in spiked human serum samples.