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.     

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 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.     

pH microelectrode: modified Thomas recessed-tip configuration

Through the use of a glass-membrane pH electrode and a water-tight seal a modified Thomas pH microelectrode has been developed. The modified Thomas electrode has a relatively low electrical resistance (10(11) omega), a small sensing chamber (10 microns3), and a rapid response time (10 s) and can be manufactured in both single- and double-barreled configurations. The modified Thomas electrode is designed to measure the intracellular pH of small cells such as those found in the mammalian kidney tubule.     

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.     

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.     

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.     

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.     

Diffusion of nystatin in plasma membrane is inhibited by a glass-membrane seal.

In perforated patch recording, the pore former nystatin is incorporated into a cell-attached patch, to increase its conductance. The possibility of lateral diffusion of nystatin through the membrane and under the glass-membrane seal was examined by reversing the nystatin gradient. Namely, a cell-attached patch on a cell was examined while placing nystatin into the bath. The reversal potential and current-voltage relationship of single Ca2+ activated K+ channels in the patch were readily changed by varying the K+ concentration in the bath, showing that nystatin was active in the cell membrane outside of the patch. However, the patch itself did not become leaky. The absence of a conductance induced in the patch by the nystatin in the rest of the plasma membrane of the cell suggests that the lateral diffusion of nystatin is inhibited by the glass-membrane seal.     

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.     

A new solid-state microelectrode for measuring intracellular chloride activities.

Solid-state microelectrodes from measuring intracellular Cl activity (alphaiCl) were made by sealing the tips of tapered glass capillaries (tip diameter 0.3 mum), coating them under vacuum with a 0.2-0.3 mum thick layer of spectrscopic grade silver, and sealing them (except for the terminal 2-5 mum of the tip) inside tapered glass shields. 106 microelectrodes had an average slope of 55.0+/- 0.6 m V (S,E,) per decade c hange in alphaCl. Tip resistance was (77.1+/- 3.1) x 10(9) omega(n=30). Electrode response was rapid (10-20 s), was unaffected by HCO3, H2PO4, HPO42 or protein, and remained essentially unchanged over a 24-h period. AlphaiCl in frog sartorius muscle fibers and epithelial cells of bullfrog small intestine was measured in vitro. In both tissues, alphaiCl significantly exceeded the value corresponding to equlibrium ditribution of Cl across the cell membrane.     

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.     

A microelectronic pH sensor.

This paper presents preliminary data on a new integrated circuit microelectronic pH sensor. The device is extremely miniaturized by the use of integrated circuit technology, and uses the intrinsic hydrogen ion selective properties of the gate insulator material. In order to make the device compatible with aqueous solution monitoring, the silicon dioxide-silicon nitride gate insulator structure is used. The integrated circuit chip was designed, processed, and packaged by a variety of techniques which protect all metal parts from the aqueous solution. Test data are reported on leakage current, sensitivity, reproducibility, linearity, stability, response time, and life. The results indicate that this type of pH sensor may have many significant advantages for biomedical research and application.     

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.