Potentiometric Probe Research Articles

Direct Energy Transfer from a pH Glass Electrode to a Liquid Crystal Display.

Self-powered sensors are attractive because the lack of a dedicated battery makes them environmentally friendly and allows them to be more easily miniaturized. Unfortunately, the development of self-powered potentiometric sensors is challenging because only very limited energy can be harvested from this measurement principle. For the first time, the potential of a high impedance glass pH electrode (130 M Ω) is shown here to be directly read out optically. This is accomplished by a liquid crystal display (LCD) as the electrochromic transducer, which changes its transmission upon imposing an external voltage in the range of 2-3 V. Importantly, owing to its low capacitance of about 50 pF, this process requires a very small transient charge on the order of 100 pC, which may be spontaneously imposable even across pH glass electrodes. For the LCD to be turned on, the cell voltage is boosted by additional Zn2+/Zn elements placed in series. The LCD is found to give a time-dependent absorbance decrease, which is mitigated by adding a high resistance element to attenuate the associated decay. The approach gives repeatable LCD absorbance values that allows one to directly visualize pH with a precision of about 0.01 pH units. The absorbance value depends inversely on pH in a much wider range (pH 1-13) than what is normally observed with optical sensors while based on the same underlying measurement as a potentiometric pH probe.

Prospective of modified polymeric MgO/ZnO nanocomposite sensor for potentiometric determination of chronic myelogenous leukemia medication

Recent attention has been developed towards the use of metal oxide nanocomposites as significant and electroactive nanomaterials in potentiometric and sensing applications. Two potentiometric cytarabine (CTB) coated wire sensors were fabricated using cytarabine-tetraphenyl borate (CTB-TPB), a modified cytarabine-tetraphenyl borate decorated with MgO/ZnO nanocomposite (CTB-TPB-MgO/ZnO) ion associates, polyvinyl chloride (PVC) and o-nitrophenyloctyl ether (o-NPOE) as electroactive materials for conventional and modified sensors, polymeric matrix and fluidizer agent, respectively. The functionalized CTB-TPB-MgO/ZnO polymeric sensor displayed Ultra potentiometric selectivity and high sensitivity for the determination of CTB within 1.0 × 10−10-1.0 × 10−2 mol L−1 solutions, correlation coefficient (r2 = 0.9999), lower detection limit 4.9 × 10-11 mol L−1 and least regression equation EmV = (57.433 ± 0.2) log [CTB] + 673.16 mV. The obtained results were compared to the conventional CTB-TPB type of 1.0 × 10-6-1.0 × 10−2 mol L−1 linear concentration range, correlation coefficient (r2 = 0.9995), lower detection limit 5.0 × 10−7 mol L−1 and least regression equation EmV = (52.633 ± 0.6) log [CTB] + 432.52 mV. It was potentially applied for the quantification of CTB in its commercial injections and spiked serum samples with mean recoveries % 99.85 ± 0.3, 99.87 ± 0.4 and 99.78 ± 0.4 %, respectively. The outcomes were compared with those obtained from various addressed analytical techniques and showed high selectivity and sensitivity for CTB determination rather than other reported analytical techniques. The rationality of the demonstrated potentiometric probe was ensured and assessed with respect to international conference harmonization recommendations. The outcomes proved that the method was accurate and precise for the determination of the investigated drug.

Self-Powered Electrochromic Readout of Potentiometric pH Electrodes.

An absorbance-based colorimetric sensor array that is self-powered by an ion-selective electrode (ISE) in a short-circuited cell is presented. As the cell voltage is maintained at zero, the potential at the ISE serves as the power generator to directly transfer its power to a potential-dependent Prussian blue (PB) film in contact with an electrolyte solution in a separate detection compartment. This allows one to activate the color change of the PB film without the need for an external power supply. The potential of the PB detection element is optimized to change color between 50 and 250 mV (vs Ag/AgCl). Because the potential originates at the ISE, it is proportional to the ion activity in the sample in agreement with the Nernst equation. In this way, a higher cation activity in the sample generates a more positive potential, which enhances the PB absorbance that serves as the analytical signal. A self-powered optical sensor array coupled to poly(vinyl-chloride)-based pH electrodes based on two different ionophores is utilized here as a model. The measuring range is tuned chemically by varying the pH of the inner filling solution of each ISE, giving a measuring range from pH 2 to 10.5. As the optical sensor is driven by a potentiometric probe, the sensor output is independent of solution ionic strength. It is successfully applied for quantitative analysis in unmodified turbid/colored samples that included red wine, coke, coffee, baking soda, and antacid. The colorimetric output correlates well with the reference method, a calibrated pH electrode. Compared to earlier systems where the cell potential is dictated by an external power source, the PB film exhibits excellent reproducibility and a rapid response time of about 44 s.

Role of oxidative metabolism in osseointegration during spinal fusion.

Spinal fusion is a commonly performed orthopedic surgery. Autologous bone graft obtained from the iliac crest is frequently employed to perform spinal fusion. Osteogenic bone marrow stromal (a.k.a. mesenchymal stem) cells (BMSCs) are believed to be responsible for new bone formation and development of the bridging bone during spinal fusion, as these cells are located in both the graft and at the site of fusion. Our previous work revealed the importance of mitochondrial oxidative metabolism in osteogenic differentiation of BMSCs. Our objective here was to determine the impact of BMSC oxidative metabolism on osseointegration of the graft during spinal fusion. The first part of the study was focused on correlating oxidative metabolism in bone graft BMSCs to radiographic outcomes of spinal fusion in human patients. The second part of the study was focused on mechanistically proving the role of BMSC oxidative metabolism in osseointegration during spinal fusion using a genetic mouse model. Patients' iliac crest-derived graft BMSCs were identified by surface markers. Mitochondrial oxidative function was detected in BMSCs with the potentiometric probe, CMXRos. Spinal fusion radiographic outcomes, determined by the Lenke grade, were correlated to CMXRos signal in BMSCs. A genetic model of high oxidative metabolism, cyclophilin D knockout (CypD KO), was used to perform spinal fusion in mice. Graft osseointegration in mice was assessed with micro-computed tomography. Our study revealed that higher CMXRos signal in patients' BMSCs correlated with a higher Lenke grade. Mice with higher oxidative metabolism (CypD KO) had greater mineralization of the spinal fusion bridge, as compared to the control mice. We therefore conclude that higher oxidative metabolism in BMSCs correlates with better spinal fusion outcomes in both human patients and in a mouse model. Altogether, our study suggests that promoting oxidative metabolism in osteogenic cells could improve spinal fusion outcomes for patients.

Optical Sensing with a Potentiometric Sensing Array by Prussian Blue Film Integrated Closed Bipolar Electrodes.

The simultaneous optical readout of a potentiometric sensor array of ion-selective electrodes (ISEs) based on PVC membranes is described here for the first time. The optical array consists of electrochromic Prussian Blue (PB) films in multiple closed ion-selective bipolar electrodes (BPEs), which gives a physical separation between the optical detection and sample compartments. The potential-dependent turnover of PB generates Prussian White (PW). A near-Nernstian response of the PB film is confirmed by colorimetric absorbance experiments as a function of applied potential. In the combined bipolar electrode cell, the overall potential is kept constant with a single potentiostat over the entire array where each PB spot indicates the potential change of an individual connected potentiometric probe. For cation-selective electrodes, the absorbance or blue intensity of the connected PB film is enhanced with increasing target cation activity. The colorimetric absorbance changes are simultaneously followed by a digital camera and analyzed by Mathematica software. A multiple cation-BPE array allows one to achieve simultaneous quantitative analysis of potassium, sodium, and calcium ions, demonstrated here in highly colored fruit juices. Mass transport at the PB thin film is shown not to be rate-limiting. The measuring ranges can be tuned in a wide range by potential control. The PB film exhibits greatly improved reproducibility and stability as compared to previous work with a ferroin redox probe confined in a thin solution layer.

Lithium Sensors Based on Ionic Conducting Ceramics

Tritium is one of the required elements in future fusion reactors. This hydrogen isotope has to be continuously produced in order to supply the fusion reactor. Tritium can be obtained from the fussion reaction of lithium 6. Because of that, future fusion reactors are designed to have a tritium breeding module surrounding the reactor. One of the breeding blanket that has been proposed is based on lead-lithium eutectic melt [1]. Nevertheless, lithium monitoring in the breeding blanket is of great importance for the performance of the reactor. A deviation from the eutectic composition can cause a substantial change on the tritium production.Therefore, in order to control the lithium dosage needed and keep the adequate composition in the melt, on-line lithium measurements will be required. For this purpose, lithium probes must be designed to withstand the high temperatures of the breeding modules (~400 ºC) and the chemical reactivity of lithium. Finally, the constructed sensors need to be tested at different lithium concentrations in order to determine its viability and range of application.Sensors based on solid state electrolytes have several advantages: stable compounds which can withstand the harsh chemical environment of the melts, its ionic conductivity tends to increase frequently with the temperature [2] and the output signal (cell potential or electrochemical current) is easy to measure. Lithium conducting electrolytes for molten metals are under development at the Electrochemical Methods Laboratory at Institut Quimic de Sarria (IQS), Barcelona. Its qualification and performance are being tested. Li-probes for molten metals will be based on the use of ceramic type lithium conducting solid-state electrolytes.In the present work, lithium conducting solid electrolytes Li6BaLa2Ta2O12 [3] and Li6La3Ta1.5Y0.5O12 [4] were synthesized. The crystallographic phases were characterized with XRD, its surface microstructure with SEM and its lithium content was determined. The resulting ceramics were shaped in a disc form. The potentiometric probe consisted in the union of the sintered ceramic disc with an alumina tube using a glass sealant. The design of the sensor involved a two electrode system that permitted to measure the potential difference between a working and a reference electrode. The reference electrode was stablished as a lithium alloy with a fixed and known composition which was placed inside the alumina tube. The working electrode was the alloy where the assembly was submerged. A molybdenum wire was used to allow the electron flow from the electrodes to the measuring system.The sensors response was measured potentiometrically in two different lithium alloys Sn/Li and Pb/Li. The experiments that were performed for each lithium alloy consisted on the measurement of the potential difference between the working and the reference electrode. In each experiment, the lithium concentration was increased in the working electrode while the lithium content in the reference electrode was kept constant. The lithium concentration in the melts (working electrode) covered a range from 3at% up to 30at%. The measurements were performed at three different temperatures: 400 ºC, 500 ºC and 600 ºC. Correlation curves between the measured potential and the lithium composition were evaluated. The slopes of the obtained correlations were in good agreement with the slopes of the Nernst equation.

From Molecular and Emulsified Ion Sensors to Membrane Electrodes: Molecular and Mechanistic Sensor Design.

Selective molecular ion probes are often insoluble in water and require a hydrophobic solvent environment for strong and selective binding, which runs counter to the desire of utilizing them in a homogeneous solution. This Account aims to guide the reader on how such molecules, often coined ionophores, can be harnessed to design exceptionally useful optical and electrochemical sensors. We start here with some historical context on the design of such ionophores and continue with the explanation of the response mechanism of optical and potentiometric sensors and the role of combined components to build a robust ion sensor. This Account is addressed to nonspecialist readers and for this reason avoids extensive use of equations or theoretical considerations. The interested reader should turn to the original literature for further reading. Emulsified optical sensors are introduced as an initial example. Here, multiple reagents are confined in an attoliter sensing nanodroplet of the organic phase, immiscible with the aqueous sample phase. In this case, the ionophore molecules may retain their high affinity and selectivity to the target ion and the aqueous sample phase does not have to be modified. Emulsified optical sensors allow one to achieve the selective chemical sensing of ions, even with optically silent ionophores. Such ionophore-based nanodroplets are also discussed as a useful novel class of complexometric titration reagents and optical end point indicators with unique selectivities. We then turn our attention to potentiometric sensing probes and briefly discuss the unique opportunity of a direct characterization of ion-ionophore complexation properties offered by membrane electrodes. A carbonate-selective membrane electrode containing a highly selective tweezer-type ionophore with trifluoroacetophenone functional groups is then used as an example for the construction of a robust all-solid-state sensor. This potentiometric probe, in combination with a pH electrode, can directly measure PCO2 in freshwater lakes, demonstrating a dramatically improved response time relative to traditional sensors equipped with a gas-permeable membrane. In recent years, new sensing modes and electrode designs have been introduced to expand the application scope of ionophore-based potentiometric sensors. Membrane electrodes containing ionophores are placed under dynamic electrochemistry control to give important progress in the field. We specifically highlight our recent works by membranes that are controlled by chronopotentiometry (controlled current) for speciation analysis, by ion transfer voltammetry on thin sensing films for multianalyte detection, by exhaustive coulometry for potentially calibration-free sensors and with coulometric membrane pumps for the selective delivery of reagents.

Colorimetric Readout for Potentiometric Sensors with Closed Bipolar Electrodes.

We present here a general strategy to translate potential change at a potentiometric probe into a tunable color readout. It is achieved with a closed bipolar electrode where the ion-selective component is confined to one end of the electrode while color is generated at the opposite pole, allowing one to physically separate the detection compartment from the sample. An electrical potential is imposed across the bipolar electrode by solution contact such that the potentiometric signal change at the sample side modulates the potential at the detection side. This triggers the turnover of a redox indicator in the thin detection layer until a new equilibrium state is established. The approach is demonstrated in separate experiments with a chloride responsive Ag/AgCl element and a liquid membrane based calcium-selective membrane electrode, using the redox indicator ferroin in the detection compartment. The principle can be readily extended to other ion detection materials and optical readout principles.

Rapid Throughput Analysis of GABAA Receptor Subtype Modulators and Blockers Using DiSBAC1(3) Membrane Potential Red Dye

Fluorometric imaging plate reader membrane potential dye (FMP-Red-Dye) is a proprietary tool for basic discovery and high-throughput drug screening for G-protein-coupled receptors and ion channels. We optimized and validated this potentiometric probe to assay functional modulators of heterologous expressed GABAA receptor (GABAAR) isoforms (synaptic α1β3γ2, extrasynaptic α4β3δ, and β3 homopentomers). High-resolution mass spectrometry identified FMP-Red-Dye as 5,5'-(1-propen-1-yl-3-ylidene)bis[1,3-dimethyl-2-thio-barbituric acid]. GABAAR-expressing cells equilibrated with FMP-Red-Dye exhibited depolarized equilibrium membrane potentials compared with GABAAR-null cells. The channel blockers picrotoxin, fipronil, and tetramethylenedisulfotetramine, and the competitive antagonist bicuculline reduced fluorescence near the levels in GABAAR-null cells indicating that FMR-Red-Dye, a barbiturate derivative, activates GABAAR-mediated outward Cl- current in the absence of GABA. GABA caused concentration-dependent increases in fluorescence with rank order of potencies among GABAAR isoforms consistent with results from voltage-clamp experiments (EC50 values for α4β3δ, α1β3γ2, and β3 homopentamers were 6 ± 1, 40 ± 11, and >18 mM, respectively), whereas GABAAR-null cells were unresponsive. Neuroactive steroids (NAS) increased fluorescence of GABAAR expressing cells in the absence of GABA and demonstrated positive allosteric modulation in the presence of GABA, whereas benzodiazepines only exhibited positive allosteric modulator (PAM) activity. Of 20 NAS tested, allopregnanolone, (3α,5α,20E)-3-hydroxy-13,24-cyclo-18-norcholan-20-ene-21-carbonitrile, eltanolone, 5β-pregnan-3α,21-diol-20-one, and ganaxolone showed the highest potency. The FMP-Red-Dye-based assay described here provides a sensitive and quantitative method of assessing the activity of GABAAR agonists, antagonists, and PAMs on diverse GABAAR isoforms. The assay has a wide range of applications, including screening for antiseizure agents and identifying channel blockers of interest to insecticide discovery or biosecurity.

Novel dual microelectrode probe for the simultaneous visualization of local Zn2+ and pH distributions in galvanic corrosion processes

Novel dual potentiometric microsensor probe has been developed for the simultaneous detection of Zn2+ concentration and pH distributions in the Scanning Electrochemical Microscopy investigation of corroding galvanized steel. The individual sensors show nearly theoretical behavior over a wide concentration range. The applicability of this probe is first demonstrated on a Fe-Zn galvanic couple, as it shows excellent performance in these simultaneous model experiments. In addition, linear scans recorded over a cut edge of coated galvanized steel evidence the complementary information gathered on the electrochemical behavior of the corroding sample, and adumbrates promising and feasible applications of multi-barrel microelectrodes in corrosion research.

Electric current distribution of a multiwall carbon nanotube

The electric current distribution in a multiwall carbon nanotube (MWCNT) was studied by in situ measuring the electric potential along an individual MWCNT in the ultra-high vacuum transmission electron microscope (TEM). The current induced voltage drop along each section of a side-bonded MWCNT was measured by a potentiometric probe in TEM. We have quantitatively derived that the current on the outermost shell depends on the applied current and the shell diameter. More proportion of the total electronic carriers hop into the inner shells when the applied current is increased. The larger a MWCNT’s diameter is, the easier the electronic carriers can hop into the inner shells. We observed that, for an 8 nm MWCNT with 10 μA current applied, 99% of the total current was distributed on the outer two shells.

Effects of 915 nm GaAs diode laser on mitochondria of human dermal fibroblasts: analysis with confocal microscopy.

Low-level laser therapy (LLLT) is widely used in tissue regeneration and pain therapy. Mitochondria are supposed to be one of the main cellular targets, due to the presence of cytochrome C oxidase as photo-acceptor. Laser stimulation could influence mitochondria metabolism affecting mainly transmembrane mitochondrial potential (Δψm). The aim of our study is to evaluate "in vitro" the early mitochondrial response after irradiation with a 915 GaAs laser. Since some evidences suggest that cellular response to LLLT can be differently modulated by the mode of irradiation, we would like to evaluate whether there are changes in the mitochondrial potential linked to the use of the laser treatments applied with continuous wave (CW) in respect to those applied with pulsed wave (PW). In this study, we analyzed effects of irradiation with a 915-nm GaAs diode laser on human dermal fibroblast. We compared effects of irradiation applied with either CW or PW at different fluences 45-15-5 J/cm(2) on Δψm. Laser scanning microscopy (LSM) was used in living cells to detect ROS (reactive oxygen species) using calcein AM and real-time changes of and Δψm following distribution of the potentiometric probe tetramethylrhodamine methyl ester (TMRM). At higher doses (45-15 J/cm(2)), fibroblasts showed a dose-dependent decrement of Δψm in either the modalities employed, with higher amplitudes in CW-treated cells. This behavior is transient and not followed by any sign of toxicity, even if reactive oxygen species generation was observed. At 5 J/cm(2), CW irradiation determined a little decrease (5%) of the baseline level of Δψm, while opposite behavior was shown when cells were irradiated with PW, with a 10% increment. Our results suggest that different responses observed at cellular level with low doses of irradiation, could be at the basis of efficacy of LLLT in clinical application, performed with PW rather than CW modalities.

Scanning electrochemical microscopy determination of hydrogen flux at liquid|liquid interface with potentiometric probe

Scanning electrochemical microscopy potentiometric determination of local hydrogen concentration and its flux next to the liquid|liquid interface was demonstrated. This method is based on the shift of open circuit potential of Pt-based reversible hydrogen electrode. The detection system was verified with a system generating hydrogen under galvanostatic conditions. Then, it was applied to aqueous|1,2-dichloroethane interface where hydrogen is produced with decamethylferrocene as electron donor.

Abstract 221: MitoROMK Expression Mitigates the Cellular Response to Oxidative Stress

We have recently reported that mitoROMK overexpression protects against, while knockdown of native ROMK exacerbates, oxidative stress-induced cell death and have proposed mitoROMK as the molecular correlate of the cardioprotective mitoKATP channel (Foster, Ho, et al Circ Res 2012). In order to better understand the mechanisms of protection, here we examined how alterations of ROMK expression affect glutathione redox responses during acute oxidative stress, utilizing cytoplasmic or mitochondrially targeted, genetically-encoded GSSG sensors. The tandem protein construct Grx1-roGFP2, a fusion of human glutaredoxin and redox sensitive GFP (Gutscher et al, Nat Methods 2008) was incorporated into adenoviral gene transfer vectors for expression in cardiac-derived H9C2 cells. Cells were subjected to treatment with the Complex III inhibitor Antimycin A (50µM) to induce endogenous mitochondrial reactive oxygen species (ROS) production, and changes in the glutathione redox potential were determined ratiometrically. MitoROMK overexpressing cells showed a significantly lower rate of mitochondrial matrix GSH oxidation secondary to H2O2 scavenging compared to both control and ROMK knockdown cell lines, indicating enhanced ability to maintain ROS balance. Increases in cytoplasmic GSSG were also blunted by mitoROMK overexpression. To determine if ROMK expression affected ROS-induced mitochondrial calcium overload, we also employed the mitochondrially-targeted ratiometric Ca2+ probe GEM-GECO (Zhao et al Science 2011). The same Antimycin A challenge led to large mitochondrial Ca2+ increase in control and knockdown cell lines, but this response was significantly suppressed in mitoROMK overexpressing cells. This could not be explained by differences in baseline mitochondrial membrane potential (ΔΨm; determined using the potentiometric probe tetramethylrhodamine methyl ester), because both control cells and ROMK overexpressors showed similar ΔΨm, while ROMK knockdown cells were more depolarized. The results suggest that enhanced cell survival in mitoROMK overexpressing cells is mediated by improved mitochondrial ROS balance and calcium handling, providing a key molecular link between mitochondrial K+ channels and cardioprotection.

Platinized carbon nanoelectrodes as potentiometric and amperometric SECM probes

Micrometer-sized platinized carbon electrodes have previously been used for the detection of reactive oxygen and nitrogen species (ROS and RNS) in biological systems. Here, we report the preparation and characterization of quartz-sealed platinized carbon nanoelectrodes. Such electrodes can be employed as tips in the scanning electrochemical microscope (SECM). The prepared electrodes were characterized by steady-state voltammetry, scanning electron microscopy, and SECM. In addition to ROS/RNS detection, the high surface area of a platinized nanoelectrode makes it a useful potentiometric probe. Unlike previously fabricated platinized electrodes, carbon electrodes possess a very thin insulating sheath, which is essential for experiments inside biological cells and high-resolution SECM imaging.

Metabolic Determinants of Electrical Failure in Ex-Vivo Canine Model of Cardiac Arrest: Evidence for the Protective Role of Inorganic Pyrophosphate

RationaleDeterioration of ventricular fibrillation (VF) into asystole or severe bradycardia (electrical failure) heralds a fatal outcome of cardiac arrest. The role of metabolism in the timing of electrical failure remains unknown.ObjectiveTo determine metabolic factors of early electrical failure in an Ex-vivo canine model of cardiac arrest (VF+global ischemia).Methods and ResultsMetabolomic screening was performed in left ventricular biopsies collected before and after 0.3, 2, 5, 10 and 20 min of VF and global ischemia. Electrical activity was monitored via plunge needle electrodes and pseudo-ECG. Four out of nine hearts exhibited electrical failure at 10.1±0.9 min (early-asys), while 5/9 hearts maintained VF for at least 19.7 min (late-asys). As compared to late-asys, early-asys hearts had more ADP, less phosphocreatine, and higher levels of lactate at some time points during VF/ischemia (all comparisons p<0.05). Pre-ischemic samples from late-asys hearts contained ∼25 times more inorganic pyrophosphate (PPi) than early-asys hearts. A mechanistic role of PPi in cardioprotection was then tested by monitoring mitochondrial membrane potential (ΔΨ) during 20 min of simulated-demand ischemia using potentiometric probe TMRM in rabbit adult ventricular myocytes incubated with PPi versus control group. Untreated myocytes experienced significant loss of ΔΨ while in the PPi-treated myocytes ΔΨ was relatively maintained throughout 20 min of simulated-demand ischemia as compared to control (p<0.05).ConclusionsHigh tissue level of PPi may prevent ΔΨm loss and electrical failure at the early phase of ischemic stress. The link between the two protective effects may involve decreased rates of mitochondrial ATP hydrolysis and lactate accumulation.

Assessment of Different Functional Parameters of Frozen–Thawed Buffalo Spermatozoa by Using Cytofluorimetric Determinations

Flow cytometry is a useful tool that provides an accurate, objective and rapid evaluation of semen quality. The use of this technique could significantly improve the quality of buffalo semen samples used in artificial insemination. This study was carried out to evaluate, by flow cytometry, frozen-thawed buffalo spermatozoa quality parameters such as sperm viability by SYBR-14/propidium iodide staining; mitochondrial function by JC-1 potentiometric probe; sperm chromatin stability (SCSA) by acridine orange; and acrosome reaction (AR) by FITC-PNA staining. Semen samples from five Italian Mediterranean buffalo bulls were used. Sperm viability was not different between bulls and ranged from 33.4% to 43.6%. A consistent rate (55.1 ± 10.8%) of sperm cells showed high mitochondrial membrane potential (Δψ(high)), with no significant differences between subjects. Sperm chromatin structure assay differed significantly between the five buffalo bulls; moreover, data showed high stability within each buffalo. DNA fragmentation indexes (DFI), such as %-DFI, -DFI, SD-DFI, were 11.2 ± 8.6, 153.3 ± 24.6 and 81.6 ± 21.2, respectively. Regarding AR, the percentage of acrosome-reacted live (ARL) and acrosome-reacted dead (ARD) spermatozoa was 0.3 ± 0.2 and 15.3 ± 5.5, respectively. This functional parameter differed significantly between buffalo bulls and showed high stability. Following to Ca(2+) ionophore A23187 for 3 h, AR significantly differed between subjects and was characterized by an increase in both ARL (10.8%) and ARD population (22.0%). This study indicates that flow cytometry could be a useful tool for a quick multiparametric evaluation of sperm quality in buffalo. In particular, SCSA and AR resulted in sperm functional parameters sensitive enough for the diagnosis of frozen-thawed semen fertilizing potential.

Quantitative measurement of mitochondrial membrane potential in cultured cells: calcium‐induced de‐ and hyperpolarization of neuronal mitochondria

Mitochondrial membrane potential (ΔΨM) is a central intermediate in oxidative energy metabolism. Although ΔΨM is routinely measured qualitatively or semi-quantitatively using fluorescent probes, its quantitative assay in intact cells has been limited mostly to slow, bulk-scale radioisotope distribution methods. Here we derive and verify a biophysical model of fluorescent potentiometric probe compartmentation and dynamics using a bis-oxonol-type indicator of plasma membrane potential (ΔΨP) and the ΔΨM probe tetramethylrhodamine methyl ester (TMRM) using fluorescence imaging and voltage clamp. Using this model we introduce a purely fluorescence-based quantitative assay to measure absolute values of ΔΨM in millivolts as they vary in time in individual cells in monolayer culture. The ΔΨP-dependent distribution of the probes is modelled by Eyring rate theory. Solutions of the model are used to deconvolute ΔΨP and ΔΨM in time from the probe fluorescence intensities, taking into account their slow, ΔΨP-dependent redistribution and Nernstian behaviour. The calibration accounts for matrix:cell volume ratio, high- and low-affinity binding, activity coefficients, background fluorescence and optical dilution, allowing comparisons of potentials in cells or cell types differing in these properties. In cultured rat cortical neurons, ΔΨM is −139 mV at rest, and is regulated between −108 mV and −158 mV by concerted increases in ATP demand and Ca2+-dependent metabolic activation. Sensitivity analysis showed that the standard error of the mean in the absolute calibrated values of resting ΔΨM including all biological and systematic measurement errors introduced by the calibration parameters is less than 11 mV. Between samples treated in different ways, the typical equivalent error is ∼5 mV.

Trk2 transporter is a relevant player in K+ supply and plasma-membrane potential control in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, TRK1 and TRK2 genes encode partially redundant K(+) transporters. Direct involvement in K(+) uptake has been shown for Trk1p since cells growing under limiting environmental K(+) concentrations demand its presence. The biological role of Trk2p is less understood. In our experiments, TRK2 overexpression improved the ability of trk1 cells to grow in low K(+) and led to a higher accumulation of K(+). Using diS-C(3)(3) as a potentiometric probe, we revealed a higher hyperpolarization of trk2 cells compared to the wild type. In addition, the deletion of TRK2 in the trk1 genetic background increased the cell sensitivity to hygromycin B, spermine, and TMA. Our studies reinforced the conclusion that Trk1p is the prominent K(+) uptake transporter and for the first time revealed that though Trk2p is much less effective, its activity contributes significantly to K(+) supply and the maintenance of plasma-membrane potential.

Development of a No-Wash Assay for Mitochondrial Membrane Potential Using the Styryl Dye DASPEI

Mitochondrial dysfunction is a hallmark of several diseases and may also result from drugs with unwanted side effects on mitochondrial biochemistry. The mitochondrial membrane potential is a good indicator of mitochondrial function. Here, the authors have developed a no-wash mitochondrial membrane potential assay using 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI), a rarely used mitochondrial potentiometric probe, in a 96-well format using a fluorescent plate reader. The assay was validated using 2 protonophores (CCCP, DNP), which are known uncouplers, and the neuroleptic thioridazine, which is a suspected mitochondrial toxicant. CCCP and DNP have short-term depolarizing effects, and thioridazine has long-term hyperpolarizing effects on the mitochondrial membrane potential of Chinese hamster ovary (CHO) cells. The assay also detected changes of the mitochondrial membrane potential in CHO cells exposed to cobalt (mimicking hypoxia) and in PC12 cells exposed to amyloid β, demonstrating that the assay can be used in cellular models of hypoxia and Alzheimer's disease. The assay needs no washing steps, has a Z' value >0.5, can be used on standard fluorometers, has good post liquid-handling stability, and thus is suitable for large-scale screening efforts. In summary, the DASPEI assay is simple and rapid and may be of use in toxicological testing, drug target discovery, and mechanistic models of diseases involving mitochondrial dysfunction.