Electrical detection, mechanisms, and analysis of biological enzyme activity in live cells.

摘要

Biological organisms are likely the most complex entities in the universe. The field of biophysics has scarcely begun to elucidate their inner workings from a physics perspective. Nevertheless, it is clear that electric charges, in the form of cations, anions, and charged groups on enzymes, play vital roles in all the activities that constitute life. These charges both generate and respond to time-varying electric fields. This doctoral dissertation is the outgrowth of efforts to: (1) develop sensors of biological enzyme activity, based on the linear (impedance) and nonlinear (harmonic) responses of cell suspensions to applied sinusoidal electric fields; (2) utilize mathematical tools to analyze the nonlinear behaviors of biological systems; and (3) better understand the biophysics of key biological motors, including the mitochondrial adenosine triphosphate (ATP) producing enzyme ATP synthase and other rotary motors.;In the area of sensor development, our observations of higher harmonics generated by suspensions of live cells are attributed to field-induced conformational changes in membrane proteins. Their frequency- and time-dependences exhibit features that correlate with the consumption of glucose and oxygen. Here, these harmonics are analyzed by incorporating the washboard potential model within the Volterra analysis method, enabling the development of nonlinear harmonic spectroscopy as a powerful new biophysics tool. Finally, a promising new method is developed, to detect cellular and mitochondrial activity at the single-cell level, by performing harmonic spectroscopy measurements using the micropipette electrodes typically employed in patch clamp measurements.;Given the complexity and intrinsic nonlinearity of biological systems, this project has utilized unconventional analysis techniques that incorporate the washboard potential model, the Volterra series method (a generalization of the Fourier series to nonlinear systems), and perturbation theory. Experiments suggest that the energy landscape of F1-ATP synthase (F1) is a washboard potential. Thus, by combining an electric field driven torque model of F0-ATP synthase (F0), recently proposed by Prof. J. Miller, with a washboard potential model, the author of this thesis has generated theoretical predictions of ATP production rate vs. mitochondrial membrane potential, which are found to be in excellent agreement with experiment.