Microelectrodes single cell measurements

Despite the predominant use of fluorescence and/or patch clamp techniques in single cell measurements, there has been a steady increase in the demand for new electroanalytical tools applicable to single cell studies [4]. Traditionally, such methods have been confined to the development and production of hand crafted sensors including the aforementioned glass capillaries [1-3] for patch clamping, as well as conical microelectrodes for scanning electrochemical microscopy (SECM) [5, 6] and carbon fiber microelectrodes to measure for example, the release of neurotransmitter from single neurons [7, 8]. 

Microelectrodes can be made sufficiently small to measure intracellular ion activities, as shown schematically in Fig. 5 The micro ion-selective electrode is inserted into a single cell by means of a micromanipulator. The intracellular activity of the... 

In biological systems, most of the elements are present as complexes, rather than as free ions, hence direct potentiometric measurements provide little information except for major electrolytes such as Fl+, Na+, K+, Cl- and possibly Ca2+, Mg2+. The introduction of ion-selective microelectrodes (e.g. tips < 1 jtm diameter) has allowed these major electrolyte ions to be determined in single cells. 

Single cells are measured with microelectrodes and clamp and patch techniques (see Chapters 7 and 10). 

Electrochemical methods for NO determination offer several features that are not available with spectroscopic approaches. Perhaps the most important is the capability of microelectrodes to directly measure NO in single cells in situ, in close proximity to the source of NO generation. Figure 2 shows sensors that have been developed for the electrochemical measurement of NO. One is based on the electrochemical oxidation of NO on a platinum electrode (the classical Clark probe for detection of oxygen) and operates in the amperometric mode [17]. The other is based on the electrochemical oxidation of NO on conductive polymeric porphyrin (porphyrinic sensor) [24]. The Clark probe uses a platinum wire as a working electrode (anode) and a silver wire serves as the counterelectrode (cathode). The electrodes are mounted in a capillary tube filled with a sodium chlo-ride/hydrochloric acid solution separated from the analyte by a gas-permeable membrane. A constant potential of 0.9 V is applied, and direct current (analytical signal) is measured from the electrochemical oxidation of NO on the platinum anode. In the porphyrinic sensor, NO is catalytically oxidized on a polymeric metalloporphyrin... 

Fig. 18. Current-time profile obtained from a single porcine neutrophil with a carbon fiber microelectrode in Hanks buffer solution. The electrode is opsonized with IgG. Arrows indicate (a) addition of a few drops of 1 x 10 cells/ml porcine neutrophils, (b) contact of a single cell with the surface of the electrode, (c) addition of a drop of 5 mg/ml SOD, Applied potential -f-O.lV vs Ag/AgCl. During the measurement the contact between the single neutrophil and the electrode was monitored by a TV camera. (From ref. [64]).

A typical example of on-chip single cell impedance measurement is shown in Fig. 1. Two pairs of microelectrodes are fabricated in a microchannel which is typically 20 to 40p,m high and wide. The two electrodes allow a differential impedance measurement to be made. One pair is used for sensing the electrical signal fluctu-... 

The initial electrochemical studies on PC 12 cells have focused on quantitation of catecholamine release. Zeptomole levels of catecholamine have been observed for a single-release event using amperometric detection with carbon fiber microelectrodes [15]. To measure the time course of these release events the electrode, held at a potential where dopamine is readily oxidized, is gently placed against the cell surface. Catecholamines are released from individual vesicles following perfusion of the cell with a solution containing nicotine, elevated potassium chloride, or other chemical stimulants. Such agents serve to directly depolarize the... 

Applications of this approach include measurement of neurotransmitters in dialysates and single cell analysis. Microfabricated instrumentation with recycling electrochemical detector may further improve the detection limit for species that can be oxidized and reduced multiple times. Microfabrication may also make this approach more widely available. The main limitation to greater use of CLC with microelectrodes for EC detection is lack of commercial instrumentation that would allow simple implementation of the principles outlined above. 

Using double-barreled liquid ion-exchange microelectrodes intracellular potassium and chloride concentrations were measured simultaneously with membrane PDs in single cells of the proximal tubules of Necturus kidney. The electrometric method measured an intracellular potassium concentration which constituted about 3/4 of the total K content. There was a remarkable agreement between the calculated and measured E across the luminal and peritubular boundaries suggesting passive transport of K", an electrochemical equilibrium distribution and K -selectivity of both membranes. 

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