Iridium oxide pH microelectrode

The manufacture, calibration, and signal conditioning during construction of an iridium/iridium oxide pH microsensor is described. The microsensor was designed to be used extracellularly, primarily in biofilm research. The sensing tip diameters were typically in the range of 3-15 mum. The iridium oxide was formed by potential cycling in dilute sulfuric acid. A pH profule across a denitrifying biofilm was measured as an example of an application. The higher Nernstian slope (70-80 mV/pH for fresh electrodes), increased rigidity, and restriction of the sensing tip to the outermost end of the electrode are features which make the iridium/iridium oxide pH microelectrode superior to a glass microelectrode. (c) 1992 John Wiley & Sons, Inc.     

A glass-membrane pH microelectrode.

By miniaturizing the original MacInnes and Dole glass-membrane pH electrode a new pH microelectrode has been developed. The technique developed utilizes the tip of a high electrical resistance glass pipet that can be sealed with a thin membrane of H⁺-sensitive glass. Single-barreled electrodes have been made with tip diameters ranging from 1.5 to 100 μm and double-barreled electrodes with tip diameters from 2 to 28 μm. The glass-membrane pH microelectrode provided a means for sensing the pH of biological solutions with an electrode having theoretical slope and tip configurational control. The most unique characteristics of the electrode were: the pH sensing surface was quite small, the tip diameter could be controlled, and the problem of electrode insulation was eliminated.     

A submicrometer glass-membrane pH microelectrode.

The glass-membrane pH microelectrode (GMpHME) described previously (Anal. Biochem.73, 501, 1976) had a limitation in the minimum size (tip diameter) that could be manufactured (about 1 μm). In addition, when made at this small size the electrical resistance was usually high (10¹¹ Ω) and the response time long (greater than 5 min). The inability to manufacture the GMpHME with tip diameters less than 1 μm was primarily due to the thickness of the pH glass used to form the H⁺-sensitive membrane. In this report we detail a method for thinning the pH glass in such a way that the manufacture of submicrometer glass-membrane pH microelectrodes is possible. These submicrometer pH electrodes have rapid response times (1 to 3 min) and maintain the desirable characteristics of all GMpHMEs, that is, near theoretic slope and a well-defined sensing surface area.     

Thin-film IrOx pH microelectrode for microfluidic-based microsystems

Microsensors are valuable tools to monitor cell metabolism in cell culture volumes. The present research describes the fabrication and characterization of on-chip thin-film iridium oxide pH microsensors with dimensions of 20 microm x 20 microm and 20 microm x 40 microm suitable to be incorporated into nl volumes. IrOx thin films were formed on platinum microelectrodes by electrochemical deposition in galvanostatic mode. Anodically grown iridium oxide films showed a near super-Nernstian response with a slope of -77.6+/-2 mV/pH at 22 degrees C, and linear responses within the pH range of 4-11. Freshly deposited electrodes showed response times as low as 6s. Long-term studies showed a baseline drift of 2-3 mV/month, which could easily be compensated by calibration. This work demonstrated for the first time the use of planar IrOx pH microelectrodes to measure the acidification rate of CHO and fibroblast cells in an on chip cell culture volume of 25 nl with microfluidic control.     

ECF pH dynamics within the ventrolateral medulla: a microelectrode study

Using pH-sensitive microelectrodes, we evaluated pH dynamics of extracellular fluid (ECF) within the ventrolateral medulla (VLM) beneath the central chemoceptive areas in anesthetized, spontaneously breathing cats. Static ECF pH was acid in the superficial layers (less than 1 mm), compared with the overlying cerebrospinal fluid pH that became alkaline gradually during the experiments. In the deeper VLM areas (1-3 mm), no systematic gradients of ECF pH were observed. We found various, isolated regions where intravertebral artery injections of CO2-saturated saline evoked acidic shift of ECF pH in the time course analogous to ventilatory augmentation. Those responsive regions were found to be scattered not only in the superficial layers but also in the deeper VLM areas, although many nonresponsive regions were also intermingled among them. Occlusions of the principal vessels supplying the tested VLM regions diminished but failed to abolish the ECF pH responses to the CO2 loadings, suggesting a collateral blood flow by fine pial vessels. The present study suggests a possibility that the pH-dependent central chemoreceptors, if any, would be scattered in the deeper VLM areas as well as the superficial layers.     

A carbon dioxide air-gap microelectrode based on a neutral hydrogen ion exchanger pH microelectrode

A relatively simple potentiometric pCO2 gas-sensing microelectrode is described. It is based on an ion-exchanger pH electrode, has a 2- to 5-microns tip, and has an air gap which is formed by means of hydrophobic treatments. The microelectrode exhibits a linear response in the range 10(-4)-10(-2) M with a Nernstian slope of 59 to 62 mV/decade at 25 degrees C. Ninety-five percent of the steady-state response time is about 20-30 s at the flow system when the concentration of sodium bicarbonate in buffer solution (pH 4.5) suddenly shifts from 0.2 to 2 mM and the lifetime is longer than 1 week.     

Combined use of colorimetric and microelectrode methods for evaluating rhizosphere pH

Plant control of rhizosphere pH is important for nutrient mobilization and uptake, and also affects microbial activity and pathogens in the vicinity of the root. Limited information is available on the ability of plant species and genotypes within a species to induce pH changes in the rhizosphere. A growth chamber study was conducted to characterize patterns of pH change within the rhizosphere of selected genotypes in an alkaline environment with a balanced nutrient supply. After germination in incubators, seedlings of 32 genotypes of maize (Zea mays L.), soybean (Glycine max. L.), sorghum (Sorghum bicolor L.), sordan [sorghum (Sorghum bicolor L.), sudangrass (Sorghum sudanese L.) hybrid], wheat (Triticum aestivum L.), oats (Avena sativa L.), and barley (Hordeum vulgare L.) were transferred into aseptic agar medium (pH 7.6) with bromocresol purple indicator. Ability of the embedded roots to induce rhizosphere pH change was followed by photographing the color change of the bromocresol purple indicator. The pH for selected genotypes at different root zones (maturation, elongation, meristematic) was also monitored by a microelectrode at 1-, 2-, 3- and 4-mm distances from the root surface. Rhizosphere acidification for selected genotypes within a species were in the order: soybean, Hawkeye>PI-54169; maize, Pioneer-3737>Pioneer-3732>CM-37; sordan, S-757>S-333; sorghum, SC-33-8-9EYSC-118-15E; barley, Bowman>Primus II; oats, Hytest>SD-84104. The pH patterns within the root system varied from species to species. The highest amount of acidification was found at the elongation and meristematic zones for soybean, while the highest amount of acidification was found at the maturation zone for barley under the same experimental conditions. The agar method allowed the determination of a genotype's capability to induce rhizosphere pH changes while the microelectrode method is necessary for quantifying the spatial variation of specific root developmental zones with high resolution.This work is a part of H.T. Gollany's dissertation in partial fulfillment of the requirements for the PhD degree.     

Direct measurement of pH profiles in gel beads immobilizing Lactobacillus helveticus using a pH sensitive microelectrode

A H⁺-selective liquid membrane microelectrode was prepared and used to measure the pH profile evolution during colonization of gel beads immobilizing Lactobacillus helveticus in a whey permeate medium. A large pH gradient was observed in a highly active periferal layer thickness that decreased from 500 to 300 m for an immobilized cell population that increased from 5.8 × 10⁹ to 3.1 × 10¹⁰ CFU/g. The flat pH profile (pH 4.4) in the central part of the bead was attributed to a high concentration of the inhibitory undissociated form of lactic acid.     

pH regulation in the vertebrate central nervous system: microelectrode studies in the brain stem of the lamprey

Studies of intracellular pH (pHi) in nervous tissue are summarized and recent investigation of intracellular and extracellular pH (pHo) in the isolated brain stem of the lamprey is reviewed. In the lamprey, pHi regulation was studied in single reticulospinal neurons using double-barrel ion-selective microelectrodes (ISMs). In nominally HCO3(-)-free HEPES-buffered media, acute acid loading was followed by a spontaneous recovery of pHi requiring 10-20 min and was associated with a prolonged rise in intracellular Na+. The recovery of pHi was blocked by 1-2 mM amiloride. Amiloride also caused a small rise in pHo. Substitution of external Na+ caused a slow intracellular acidification and extracellular alkalinization. Return of external Na+ reversed these effects. Transition from HEPES to HCO3(-)-buffered media increased the rate of acid extrusion during recovery of pHi. Recovery in HCO3(-)-buffered media was inhibited by 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid and was slowed after exposure to Cl(-)-free media. Following inhibition of acid extrusion by amiloride, transition to HCO3- media restored pHi recovery. These data indicate that lamprey neurons recover from acute acid loads by both Na+-H+ exchange and an independent HCO3(-)-dependent mechanism. Evidence for HCO3(-)-dependent acid extrusion in other vertebrate cells and the protocols of pHi studies using ISMs are discussed.     

Sepal color variation of Hydrangea macrophylla and vacuolar pH measured with a proton-selective microelectrode

Sepal color of hydrangea varies with the environmental conditions. Although chemical and biological studies on this color variation have a long history, little correct knowledge has been generated about color development. All colored sepals contain the same anthocyanin, delphinidin 3-glucoside. Thus, there must be some other system for developing the wide variety of colors. In hydrangea sepals the cells of the epidermis are colorless and only the second layer of cells contain pigment. We prepared protoplasts without any color change during enzyme treatment of sepals and measured the vacuolar pH of each of the colored cells. We could correlate the color of a single hydrangea cell with its vacuolar pH using a combination of micro-spectrophotometry and a proton-selective microelectrode. Values for the vacuolar pH of blue (lambda vismax: 589 nm) and red cells (lambda vismax: 537 nm) were 4.1 and 3.3, respectively, the vacuolar pH of blue cells being significantly higher.     

Effect of phloretin on the carrier-mediated electrically silent ion fluxes through the bilayer lipid membrane: measurements of pH shifts near the membrane by pH microelectrode.

The effect of phloretin on the carrier-mediated electrically silent ion fluxes through the bilayer lipid membrane (BLM) was studied. The measurements were carried out according to our conventional technique, i.e. electrical potential recording in the presence of a protonophore, and by a new method--direct measurements of pH shifts in the unstirred layers of the BLM by pH microelectrode. Both techniques gave similar results. It was shown that the addition of phloretin increased the rate of cation/H+ exchange induced by nigericin and decreased the rate of anion/OH(-)-exchange induced by tributyltin. The effect of phloretin was higher in the presence of cholesterol in the BLM. Cholesterol decreased the nigericin- and tributyltin-induced fluxes under our experimental conditions. The application of an external voltage to the membrane had no effect on the ion fluxes thereby showing that these fluxes were electroneutral. The most probable explanation of these results bases on the effect of the membrane dipole potential on the electroneutral fluxes of ions. The possible mechanism of the dipole potential effect on the carrier-mediated electrically silent ion fluxes was discussed in terms of two competing hypotheses--the translocation through the membrane or the reactions at the membrane surface being the rate-limiting steps of the whole transport process.     

On microelectrode ionophoresis

An equation for ionophoresis in large tip microelectrodes is derived from Nernst-Planck equations for the general case of a completely dissociated electrolyte. The relation between the release of ions and the applied electric current is mainly determined by two parameters: the transference number of the ions under consideration and the diffusional leak of the microelectrode. Also it is shown how the release of ions is affected by the concentration of the electrolyte within the electrode and that of the external solution. The equation describes the ionophoretic release of polyvalent spermine. In addition, new equations for tip potential and for tip resistance are derived.     

Application of microelectrode technique to measure pH and oxidoreduction potential gradients in gelled systems as model food

A microelectrode system is used in order to simultaneously measure pH and oxidoreduction potential (Eh) gradients developed during growth by Escherichia coli and Lactobacillus plantarum immobilized in gelled media used as model food. Unlike E. coli, L. plantarum steadily decreased medium pH independently of depth of measurement and time of incubation. Both bacteria brought about the creation of an Eh gradient throughout the gelled medium. This gradient was much more important for E. coli (700 mV) than for L. plantarum (80 mV) but more transitory.     

Determination of absolute configuration of photo-degraded catechinopyranocyanidin A by modified Mosher's method

Catechinopyranocyanidins A and B (cpcA and cpcB) are two purple pigments present in the seed-coat of red adzuki bean, Vigna angularis, of which cpcA is the major pigment, containing two chiral carbons in the catechin part. Their absolute configurations were determined by comparison of their experimental and quantum chemical calculated electronic circular dichroisms (ECDs). These purple pigments are labile on light irradiation and easily decompose to photo-degraded catechinopyranocyanidins A and B (pdcpcA and pdcpcB), while retaining the stereostructure of the catechin residue. We applied modified Mosher's method for determining the chirality of the secondary alcohol in pdcpcA. Hexamethylation of pdcpcA by diazomethane followed by esterification using (S)- and (R)-MTPACl gave (R)- and (S)-MTPA esters, respectively. By analysis of the NMR spectra of (R)- and (S)-MTPA esters of tetramethylated (+)-catechin, the chirality of pdcpcA was determined to be 2R, 3S, same as the absolute configuration of cpcA.     

Measurements of local pH changes near bilayer lipid membrane by means of a pH microelectrode and a protonophore-dependent membrane potential. Comparison of the methods.

Shifts of pH near the bilayer lipid membrane (BLM) were measured in the absence of pH difference between bulk solutions by two methods, i.e. pH microelectrode and membrane potential recordings in the presence of a protonophore. A quantitative agreement of the results of both methods was obtained. The kinetics of the generation of potential induced by the addition of ammonium chloride was accounted for by the time of the diffusion through the unstirred layers. The thickness of the unstirred BLM layers was determined in the experiment.