Description:

Chemical signals detected by the self-referencing technique are inevitably distance dependent, resulting from diffusion of the analyte through the extracellular matrix. Obviously this necessitates the reproducible placement of electrodes at specific distances from the cell. Unfortunately, manual placement of the probe can be quite difficult and is limited to the high contrast boundary in the x-y plane of the cell. Providing an instrumental method for determining distance would not only allow analysis of the cell from the z axis, it would additionally provide a reproducible means for quantifying the distance at which measurements are taken, allowing comparison of multiple points on a single cell or among multiple cells. Our goal is the implementation of impedance feedback to measure and control the sample-probe distance. In the short-term this technology will be applied to the control of distance for flux measurements. In the long-term this technology will allow us to add scanning microscopy to our analytical toolbox.

Progress:

Here we report on having successfully measured the increase in the uncompensated resistance of an electrochemical cell upon approach to single, living, biological cells, while simultaneously measured the metabolic oxygen consumption. This was accomplished by applying an AC and a DC excitation signal to the electrode. A lock-in approach monitored the high frequency component, whose amplitude was dependent on the target-probe separation. This provided positional information with submicron resolution. The DC component polarized the electrode reactive surface to –0.6 V forming a conventional oxygen measuring microelectrode. The two signals were shown not to interfere with one another. Furthermore, it was shown that the sample-probe distance can be measured for approaches to single cells on the order of 10-15 µm diameter and 5 µm height.