Studies on the matched potential method for determining the selectivity coefficients of ion-selective electrodes based on neutral ionophores: experimental and theoretical verification.

A theory is presented that describes the matched potential method (MPM) for the determination of the potentiometric selectivity coefficients (KA,Bpot) of ion-selective electrodes for two ions with any charge. This MPM theory is based on electrical diffuse layers on both the membrane and the aqueous side of the interface, and is therefore independent of the Nicolsky-Eisenman equation. Instead, the Poisson equation is used and a Boltzmann distribution is assumed with respect to all charged species, including primary, interfering and background electrolyte ions located at the diffuse double layers. In this model, the MPM-selectivity coefficients of ions with equal charge (ZA = ZB) are expressed as the ratio of the concentrations of the primary and interfering ions in aqueous solutions at which the same amounts of the primary and interfering ions permselectively extracted into the membrane surface. For ions with unequal charge (ZA not equal to ZB), the selectivity coefficients are expressed as a function not only of the amounts of the primary and interfering ions permeated into the membrane surface, but also of the primary ion concentration in the initial reference solution and the delta EMF value. Using the measured complexation stability constants and single ion distribution coefficients for the relevant systems, the corresponding MPM selectivity coefficients can be calculated from the developed MPM theory. It was found that this MPM theory is capable of accurately and precisely predicting the MPM selectivity coefficients for a series of ion-selective electrodes (ISEs) with representative ionophore systems, which are generally in complete agreement with independently determined MPM selectivity values from the potentiometric measurements. These results also conclude that the assumption for the Boltzmann distribution was in fact valid in the theory. The recent critical papers on MPM have pointed out that because the MPM selectivity coefficients are highly concentration dependent, the determined selectivity should be used not as "coefficient", but as "factor". Contrary to such a criticism, it was shown theoretically and experimentally that the values of the MPM selectivity coefficient for ions with equal charge (ZA = ZB) never vary with the primary and interfering ion concentrations in the sample solutions even when non-Nernstian responses are observed. This paper is the first comprehensive demonstration of an electrostatics-based theory for the MPM and should be of great value theoretically and experimentally for the audience of the fundamental and applied ISE researchers.

Threshold ionic site concentrations required for Nernstian potentiometric responses of neutral ionophore-incorporated ion-selective liquid membranes.

An equation that can describe the concentrations of ionic sites required for a Nernstian potentiometric response slope of neutral ionophore-incorporated ion-selective liquid membranes is presented. This equation is derived from a model based on electrical diffuse layers on both the membrane and the aqueous sides of the interface, in which the phase boundary potential is correlated to the surface charge density as well as the salt concentrations in the bulk membrane and aqueous solution. To experimentally and accurately confirm the validity of this equation, response characteristics of field effect transistors covered by neutral ionophore-based liquid membranes with varying concentrations of a derivative of tetraphenylborate as an anionic site but free of ionic impurities were examined. The observed membrane potentials and the response slopes for membranes with various concentrations of anionic sites were in good agreement with the values calculated from the theory presented in this paper with the measured complexation stability constants for the relevant systems. This result indicates that the theoretical prediction based on the proposed equation for the anionic site concentration is accessible for the preparation of neutral ionophore-incorporated ion-selective liquid membranes, which show Nernstian response slopes for the primary ions.

Potentiometric responses of polymeric liquid membranes based on hydrophobic chelating agents to metal ions.

The effect of hydrophobicity of acidic chelating agents as sensing materials on the potentiometric responses of polymeric liquid membranes was investigated. The chelating agents tested were 8-quinolinol (HOx), dithizone (HDz), 1-(2-pyridylazo)-2-naphthol (PAN) and their alkylated analogues, 5-octyloxymethyl-8-quinolinol (HO8Q), di(phexylphenyl)thiocarbazone (C6HDz), 7-pentadecyloxy-1-(2-pyridylazo)-2-naphthol (C15PAN) and a series of N-alkylcarbonyl-N-phenylhydroxylamines (CnPHA, n = 3, 6, 9, 12). The distribution coefficients between membrane solvent and water were determined to evaluate the hydrophobicity of the agents. The potential-pH profiles of the membranes containing hydrophobic chelating agents demonstrated the generation of potentiometric responses, while less hydrophobic agents gave no response. A possible model for the generation of membrane potential is proposed. The charge separation is attained by the permselective uptake of metal cations by the chelating agent anion at membrane/solution interface, where the high hydrophobicity of the agent enables the anionic or deprotonated form of the agents to remain at the membrane/solution interface.

Imaging of conformational changes of proteins with a new environment-sensitive fluorescent probe designed for site-specific labeling of recombinant proteins in live cells.

We demonstrate herein a new method for imaging conformational changes of proteins in live cells using a new synthetic environment-sensitive fluorescent probe, 9-amino-6,8-bis(1,3,2-dithioarsolan-2-yl)-5H-benzo[a]phenoxazin-5-one. This fluorescent probe can be attached to recombinant proteins containing four cysteine residues at the i, i + 1, i + 4, and i + 5 positions of an alpha-helix. The specific binding of the fluorescent probe to this 4Cys motif enables fluorescent labeling inside cells by its extracellular administration. The high sensitivity of the fluorophore to its environment enables monitoring of the conformational changes of the proteins in live cells as changes in its fluorescence intensity. The present method was applied to calmodulin (CaM), a Ca2+-binding protein that was well-known to expose hydrophobic domains, depending on the Ca2+ concentration. A recombinant CaM fused at its C-terminal with a helical peptide containing a 4Cys motif was labeled with the fluorescent probe inside live cells. The fluorescence intensity changed reversibly depending on the intracellular Ca2+ concentration, which reflected the conformational change of the recombinant CaM in the live cells.

Optical detection in microscopic domains. 2. Inner filter effects for monitoring nonfluorescent molecules with fluorescence.

In this research, we test whether optical detection techniques show different characteristics in microscopic solution volumes (nano-, pico-, and femtoliter range) compared to the usual macroscopic samples. In part 1 (Lu, H.; et al. Anal Chem. 2000, 72, 1569-1576.) absorption spectra of high quality were obtained, quantitatively obeying both Beer-Lambert's law and the law of superposition, despite the micrometer optical path lengths and the curvatures of the droplets studied. Addition and subtraction of absorbing molecules with diffusional microburets (DMBs), as well as more complex operations (simultaneous addition of one and subtraction of another molecule, and a consuming scheme), have been monitored with good spectral and temporal resolution. Despite the unexpectedly good performance of absorption microspectrometry, fluorescence-based detection schemes are considered more sensitive for microscopic studies (e.g., cell physiology). In this paper, we test whether fluorescence-based schemes can be used to indirectly measure nonfluorescent chemicals in microscopic domains. Absorption by such molecules will cause a corresponding decrease in overall fluorescence intensity of the added standard fluorescent dye. This phenomenon, the inner filter effect (IFE), was tested using Lucifer Yellow CH (LY) as the fluorescent standard dye. Its effective irradiation was absorbed by Orange G (primary IFE) or its emission by Bromophenol Blue (secondary IFE). By utilizing these phenomena, (1) we measured the concentration of absorbing molecules in microscopic samples by adding a standard amount of LY by a DMB, and (2) we monitored DMB delivery of nonfluorescent reagents into droplets preloaded with LY. The results prove that IFEs are sensitive indirect means of detection of absorbing molecules in microscopic domains. The techniques presented are expected to find applications in cellular studies where absorption spectrometry is usually not considered.

Dopamine-selective response in membrane potential by homooxacalix[3]arene triether host incorporated in PVC liquid membrane.

Selectivities of membrane potential changes for catecholamines and inorganic cations were investigated with lipophilic derivatives of calix[6]arene and related hosts incorporated in poly(vinyl chloride) (PVC) matrix liquid membranes. Homooxacalix[3]arene triether displayed an excellent selectivity for dopamine against other catecholamines (adrenaline, noradrenaline) and also against inorganic cations (K+, Na+).

Difference in dilatation between endothelium-preserved and -desquamated segments in the flow-loaded rat common carotid artery.

To study arterial dilatation in response to increased flow, we observed the bilateral common carotid arteries (CCAs) of 10 16-week-old rats that were maintained for 8 weeks after construction of an arteriovenous (AV) fistula between the left CCA and the jugular vein at a level 20 mm distal from the aortic orifice. The flow in the left CCA increased 11-fold and that of the right CCA increased twofold compared with values before surgery. The left CCA showed complete desquamation of endothelial cells in the distal one third of the segment proximal to the AV fistula. In the left CCA the endothelium-preserved area dilated significantly (the luminal radius was 1.34 times larger than control; p less than 0.001, n = 4) with a significant increase of the cross-sectional area of the media (p less than 0.01, n = 4) and showed high wall shear stress (70 +/- 11 dynes/cm2 near the aortic orifice). In contrast, the endothelial cell-desquamated area did not dilate but did show very high wall shear stress (231 +/- 23 dynes/cm2) without any intimal smooth muscle cell proliferation. The right CCA dilated significantly (luminal radius was 1.07 times larger than control; p less than 0.001, n = 4) with a wall shear stress of 30 dynes/cm2 near the brachiocephalic orifice. All CCAs retained their fundamental arterial structure. We conclude that in the rat CCA, arterial dilatation in response to increased flow is a gradual remodeling process related to the presence of endothelial cells that have been influenced by the level of flow increase.

Ion-sensitive field effect transistor as a monovalent cation detector for ion chromatography and its application to the measurement of Na+ and K+ concentrations in serum.

An ion-sensitive field effect transistor, which is a small potentiometric ion-sensing probe, was applied as a detector in the ion chromatography of alkali-metal cations. The cation-sensing transistor was prepared by coating the gate part of the probe to form a poly(vinyl chloride) matrix membrane containing tetranactin, which enables detection of alkali-metal ions such as Na+, K+, Rb+, and Cs+. To be able to analyse amounts less than 1 microliter and make full use of the small ion-sensing probe, we constructed a miniature cation-exchange separation column (support 10 microns polystyrene) with a PTFE tube (20 mm x 1.5 mm I.D. x 2.1 mm O.D). As an application of the system, Na+ and K+ concentrations in serum were determined. The analytical results for these two cations were good agreement with those obtained by flame photometry.

Fine structure of capillary proliferation in myocardium of volume overloaded rats.

We studied the fine structure of capillary endothelial cells of volume over-loaded cardiac hypertrophy in rats using A-V shunt between the left common carotid artery and the left external jugular vein. The duration of experiment was 1, 2 and 4 weeks. Volume overload of the heart was constantly induced, because blood flow index (ml/min/kg) of both carotid arteries was increased throughout the experiment and the volume and the weight of the left ventricle were significantly increased in 2 and 4 weeks. After the making of the large panorama photograph composed of 24 TEM photographs, the number of the endothelial cells of each capillary was directly counted under TEM. In 2-week shunted animals, incidences of over 3 endothelial cells per capillary were definitely increased. Also bulging of the endothelial cells, overlapping of two endothelial cells and increasing of microvilli on the luminal surface were mostly prominent in 2 weeks. Endothelial cells of intramyocardial capillaries are considered to be activated as early as 1 week and proliferated in around 2 weeks after volume-overload. An increase of coronary blood flow might be one of the most important causes of the endothelial proliferation and regulated by dilatation of the capillary lumen.

Increase in endothelial cell density before artery enlargement in flow-loaded canine carotid artery.

To investigate the morphologic basis of blood flow-dependent adaptive vascular enlargement, we quantitated endothelial cell density, dimensions, and structure in canine carotid arteries that were flow-loaded for 4 weeks, i.e., just before the development of significant adaptive enlargement. Increased flow was produced in the right common carotid artery of seven adult beagle dogs by an arteriovenous shunt to the right external jugular vein. The left common carotid artery was used to produce sham-operated controls. Five additional animals were used to produce sham-shunted controls, and two dogs were used as nonoperated controls. The blood flow rate (BFR) and wall shear rate (WSR) were markedly increased immediately after anastomosis in the proximal segment of the shunted artery (BFR = 719 +/- 142 ml/min, WSR greater than 4127 +/- 1002/sec) and after 4 weeks (BFR = 628 +/- 157 ml/min, WSR greater than 2919 +/- 388/sec) compared to the same artery before anastomosis (BFR = 154 +/- 50 ml/min, WSR = 904 +/- 314/sec, p less than 0.01 x 10(-3) for both comparisons) and to the contralateral control artery after 4 weeks (BFR = 365 +/- 110 ml/min, WSR = 2136 +/- 876/sec, p less than 0.01 and p less than 0.05, respectively, compared to the shunted side). In the shunted artery, endothelial cell density was markedly increased (6.15 +/- 0.68 x 10(3) cells/mm2 compared to 3.33 +/- 0.70 x 10(3) cells/mm2 for the controls, p less than 0.001). Endothelial cells on the high flow side were markedly narrowed in both axial and circumferential directions, but were radially thickened; nuclei became prolate-spheroid in shape. On the control side, cells were relatively flat and thin. We conclude that elevated wall shear stress induces an early increase in endothelial cell number and that this increase precedes the development of significant blood flow-dependent vascular enlargement.

Endocardium of the left ventricle in volume-loaded canine heart. A histological and ultrastructural study.

To investigate the effects of volume loading of the heart, the endocardium was studied histologically and ultrastructurally. Thirteen adult female beagle dogs were used. An arterio-venous shunt was constructed between the right common carotid artery and the right external jugular vein in nine animals to induce a volume load. Four animals were used as controls. All were kept for 6-12 months. Heart weight, relative heart weight (heart weight/body weight), cardiac output index, stroke volume index and volumes of both ventricles in the experimental animals were significantly larger than in the controls. Shunted blood volume was significantly correlated with heart weight. The endocardium of the left ventricle in the experimental animals showed elastofibrosis and was significantly thicker than in the controls. In 5 hearts, it was more than 20 microns thick and its endothelial cells showed many long microvilli with a very thick basement membrane (1.5-2.0 microns). The thickness of the endocardium was significantly correlated with the heart weight, relative heart weight and cardiac output index, within 1%, 5% and 5% risk, respectively. These endocardial changes were thought to be induced by hemodynamic changes in the left ventricle of the volume-loaded heart, probably being correlated with changes in cardiac function and morphology.