Intracellular measurements

By calibration against solutions containing known activities of the ion being determined, measurement of the current can be used to ascertain the activity of the ion in the test solution. Such measurements can be carried out with very small volumes of liquid, and find application in biochemical analyses.36 37 However, the simpler ion-selective electrodes discussed above can be readily adapted for dealing with small volumes, and even for intracellular measurements. 

Fig. 5. Schematic diagram of microelectrode arrangement for intracellular measurements of ion...

One barrel-tip contains the organic membrane phase and an internal reference electrode the other constitutes a second reference electrode. A four-barrel configuration with a 1-pm tip in which three barrels are liquid membrane electrodes for Na, Ca and and the fourth is a reference electrode has been reported Some representative applications of ion-selective electrodes for intracellular measurements are shown in Table 3. 

ZhuGe R, Fogarty KE, Tuft RA, Lifshitz LM, Sayar K, Walsh JV 2000 Dynamics of signaling between Ca2+ sparks and Ca2+-activated K+ channels studied with a novel image-based method for direct intracellular measurement of ryanodine receptor Ca2+ current. J Gen... 

Aylott JW (2003) Optical nanosensors-an enabling technology for intracellular measurements. Analyst 128 309-312... 

The greater conductivity of an ion-exchanger solution containing the potassium salt of anion XVI in solvent 3-o-nitroxylene (Corning No. 476200) [13a] is a reason for the popularity of this system for electrophysiological measurements using ion-selective microelectrodes [216]. Application of this sensor is so widespread that only reviews and books will be mentioned, for microelectrodes in intracellular measurements [23, 32, 78, 86, 211, 217] and for the determination of K in the intercellular liquid [78]. A working valinomycin microelectrode has also been constructed [166]. 

Analytical issues (i) X-ray elemental microanalysis (ii) Ion-selective electrodes for chnical use. (iii) Electron probe and electron energy loss analysis. (iv) Intracellular measurements . (v) Determination of Mg in human tissues and fluids . (vi) Trace elements in hair. (vii) Determination of Ca and Mg in wines . 

Intracellular surface-enhanced Raman imaging has been reviewed by Chourpa and colleagues [23]. For intracellular measurements SERS provides a mechanism for enhancement of the signals from very small amounts of cell constituents, such as DNA, or pharmaceuticals. Typically colloidal silver or... 

For each of the above protocols paired measurements of one or several given parameters of tubule transport are obtained under control conditions and in the presence of a substance under study. Also concentration response curves can be obtained in one single preparation (Schlatter et al. 1983 Wangemann et al. 1986 Wittner et al. 1987). Intracellular measurements are usually required to define the mechanism of action (Greger 1985). Especially the electrical and optical measurements have a very high reproducibility. For screening usually 3 preparations are sufficient. Approximately 10 preparations are required for concentration response curves. 

S. Colombo, A. Beguin, A. Telenti, J. BioUaz, T. Bucbn, B. Rochat, L.A. Decosterd, Intracellular measurements of anti-HIV drugs indinavir, amprenavir, saquinavir, ritonavir, nelfinavir, lopinavir, atazanavir, efavirenz and nevirapine in PBMC by LC-MS-MS, J. Chromatogr. B, 819 (2005)259. 

The size requirements for ISEs utilized for intracellular measurements varies greatly depending on the size of the cells under investigation. Historically, the term micro-ISE has been used to describe electrodes with tip diameters ranging from 200 p.m down to 0.1 p.m. Fabrieation of such devices, particularly those <10 pm in tip diameter is a real art, and highly specialized equipment is required. Over the years, micro-ISEs of the glass, solid-state, and liquid membrane classes have been proposed, although the liquid membrane type are the ones most extensively employed at this time. 

When making actual intracellular measurements, the microelectrodes are mounted in micromanipulators for cell penetration. It has been found that beveling the tip of the micropipet ISE aids in the ability to enter the cell and also enables the fabrication of electrodes with smaller tip diameters of O.I (im. Once inside the cell, single-barrel liquid membrane micro-ISEs (as described above) allow for the measurement of only steady-state ion activities. For excitable cells, where ion levels change rapidly, one cannot differentiate the potential changes resulting from variations in the intracellular activity of a specific ion and the living cell membrane potential. For such situations, double-barrel-type liquid membrane micro-ISEs have been developed (K2). 

When performing potentiometric measurements with a traveling probe, one needs to take into account the effect of ohmic drop. Whereas this effect is the basis of SRET measurements, it becomes a nuisance in potentiometric SECM applications. If the substrate is an electrode involved in a Faradaic process, the current flowing between the substrate and the counterelectrode leads to potential gradients in solution. The tip will be sensitive to the potential distribution, and this may overcome the signal due to the concentration change for the ion of interest. This is particularly pronounced if the reference electrode associated to the tip is located far away in the bulk and of course if the solution conductivity is low. To remedy this situation some researchers have used double barrel electrodes where one channel acts as the ion-sensitive element and the other acts as a reference electrode (81,82). In the life sciences intracellular measurements are usually carried out in this way. Alternatively, it is possible to subtract the ohmic drop from the tip... 

Healey B G, Foran S E and Walt D R 1995 Photodeposition of micrometer-scale polymer patterns on optical imaging fibers Science 269 1078-80 Tsien R Y 1983 Intracellular measurements of ion activities Annu. Rev. Bioeng. 12 91-116... 

Fiber optic chemical sensors and biosensors offer important advantages for in situ monitoring applications because of the optical nature of the detection signal. Recent advances in nanotechnology leading to the development of optical fibers with submicron-sized dimensions have opened up new horizons for intracellular measurements. 

Crow JP (1997) DichlorodUiydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide 1 145-157... 

Commercial silver/silver-chloride reference electrodes are available in a variety of styles and sizes. They are often used as the internal reference electrodes in glass pH and other ion-selective electrodes. Ag/AgCl microelectrodes formed from very thin silver wire have found extensive use, for example, in biomedical applications such as in vivo studies of biological fluids and intracellular measurements, because of the miniaturization possible with these electrodes. 

The phycobiliproteins are hard to beat as extremely bright antibody labels for fluorescence analysis of cell surface antigens by flow cytome-try 15,16 These bacterial photosynthetic macromolecules each have up to 34 individual bilin fluorophores wrapped within the polypeptide structure. An example is R-phycoerythrin (R-PE), which has an extinction coefficient of 2 X 10 L/mol cm and a quantum yield of 0.68. Usually there is room for only one PE molecule per antibody, because the size of an R-PE label is 1.5 times that of an IgG antibody. The large size of the complex reduces the kinetics of binding to cell surface antigens, and some intracellular markers are inaccessible to the R-PE-labeled antibody. For intracellular measurements, lower molecular mass fluorophores (<1 kDa) are usually preferred. 

Carden DE, Diamond D, and Miller AJ (2001) An improved Na+-selective microelectrode for intracellular measurements in plant cells. Journal of Experimental Botany 52 1353-1359. 

---