Principles, Development and Applications of Self-Referencing Electrochemical Microelectrodes to the Determination of Fluxes at Cell Membranes

Excerpt

Tremendous advances in the approach to single cell studies has occurred in the past two decades. Electrophysiology has been dominated by the powerful derivatives of the original patch clamp technique, addressing the biophysics of single channels or whole cells [2]. Imaging has advanced our understanding of architectural dynamics and intracellular ion activities [3,4]. Indeed, the success of the latter displaced the use of intracellular, chemically-selective electrodes where voltage changes and competing ions make the technique difficult and the interpretation complex. Despite these limitations, electrochemical sensors have continued to demonstrate strength and versatility when applied to the external boundary layers of single cells and tissues. For example, Travis and Wightman [5] demonstrated a correlation between vesicle fusion and cell capacitance for dopamine release. In addition, scientists used electrochemistry of 5-HT and histamine to study quantal corelease [6]. Both of these examples require the placement of a carbon fiber, amperometric microelectrode, in close apposition to the plasma membrane where strong electrochemical signals can be derived over low background. These cases are special in that a discrete cellular event carries a clear electrochemically detectable, phasic, signal into the boundary layer. However, all cells modify the diffusive boundary layers because of physiology, for example transporter activities or respiration. These signals can be weak and often imposed on top of significant background levels of the same chemical–oxygen and calcium flux detection are examples. Problems in measuring changes of such chemical signals are further compounded by the instability of any electrode design. How can these limitations be overcome to make boundary layer measurements with high temporal and spatial fidelity?

This chapter proposes two approaches to making measurements in the boundary layer. Firstly, the gradient can be constrained by a restrictive space, effectively “amplifying” and defining the signal to be detected. Secondly, detection can be coupled directly to the chemical gradients radiating into or out of the cell. Two elegant examples of the first approach are available. Poitry et al. sought to measure the oxygen consumption and nucleotide levels, under closely matching conditions, for single cells [7]. To acquire the oxygen levels, they captured single rod photoreceptorsin glass micropipettes such that they lodged approximately 10 μm from the tip. Next, they inserted a Whalen-style oxygen electrode [8] through the pipette tip and measured oxygen consumption in a static configuration. Kang and Hilgemann [9] recently published results from a similar approach, but for the study of the Na⁺ /Ca²⁺ exchanger in a cardiac muscle macropatch. Here an ion-selective electrode (ISE) was advanced into the body of a pipette supporting the macropatch. As the pipette geometry could be precisely modeled, Kang and Hilgemann were able to derive the stochiometry of this electroneutral transporter [9].