AFM-Based Monitoring of Enzymatic Activity of Individual Molecules of Cytochrome CYP102A1.

Herein, we report the use of a nanotechnology-based approach for the study of enzyme-functionalized mica surfaces. Atomic force microscopy (AFM) has been employed for the determination of the catalytic activity of single molecules of heme-containing cytochrome P450 CYP102A1 (CYP102A1) enzyme, which was immobilized on the surface of a mica chip. Height fluctuations in individual molecules of the enzyme were measured under near-native conditions by AFM measurements in liquid using a cantilever with a 10 to 20 nm tip curvature radius. We have found that in the process of enzymatic catalysis, the mean amplitude of height fluctuations in individual enzyme molecules is 1.4-fold higher than that of enzyme molecules in an inactive state. The temperature dependence of the mean amplitude of height fluctuations in cytochrome CYP102A1 has been revealed, and the maximum of this dependence has been observed at 22 °C. The proposed nanotechnology-based approach can be employed in studies of a wide variety of enzymes, which are important for the development of novel diagnostic tests and systems for pharmaceutical analysis. The approach developed in our work will favor further miniaturization of enzyme-based biosensors and the transition from traditional sensors to nanobiosensors.