(178d) Microelectrode Cholesterol Sensing at Single Cells, Animal Tissues, and the Human Mucosa for Pre-Clinical Studies and Patient Evaluations

This research group has been investigating cholesterol diffusion rate using oxidase-modified microelectrodes positioned in contact with the cell plasma membrane (PM). The microelectrode-membrane interface establishes a thin aqueous layer between the two hydrophilic surfaces. Although only sparingly soluble, aqueous phase cholesterol between the microelectrode surface and the PM surface is enzymatically consumed perturbing the equilibrium between membrane cholesterol and aqueous cholesterol thus causing unidirectional efflux of cholesterol from the membrane only at the site of microelectrode contact. Lateral radial diffusion of cholesterol in the PM to the electrode contact site provides a steady-state efflux rate and microelectrode current response. In vitro analysis at cells and mouse tissues is conducted by repositioning the microelectrode between the contact position and a position away from the membrane surface so that the steady-state current response for contact can be referenced to the baseline current collected when the microelectrode is not contacting the membrane. For in vivo analysis at the human mucosa, a double potential pulse method was developed for data acquisition. Enzymatically generated hydrogen peroxide is allowed to accumulate between the microelectrode and the membrane during a 5 s hold time after which the hydrogen peroxide is consumed by oxidation during a 0.25 s potential pulse. A second replicate pulse is applied 0.5 s after the first pulse to gauge the background. The difference charge between pulse one and pulse two is collected serially and is averaged to produce the final signal. Key attributes of the method are that baseline drift of signal is avoided through the continuous background subtraction, differences in microelectrode properties are normalized by taking of the difference charge, and only hydrogen peroxide that accumulates at the electrode surface is observed in the difference charge measurement. Because the time at rest potential between pulses (quiet time: 0.5 s) is held longer that the pulse time (0.25 s), the difference charge is blind species that react at the electrode during the pulses through a diffusional flux, including any bulk hydrogen peroxide. These microelectrode cholesterol measurements have been used to demonstrate increased PM cholesterol diffusion rates at cell line models of cystic fibrosis (CF) and at trachea and nasal tissues for mouse models of the CF disease state relative to WT controls. More recently, the double potential pulse-microelectrode method was implemented to show that CF human bronchial epithelial cells exhibit increased PM cholesterol diffusion and that cholesterol diffusion at the oral mucosa of CF subjects is higher compared to healthy control subjects. These data demonstrate the clinical utility of this approach for both pre-clinical studies and for in vivo assessment of CF.