Quantitative determination of dextran sulfate and pentosan polysulfate and their binding with protamine using chronopotentiometry with polyion-selective electrodes.

We report for the first time a chronopotentiometric measurement of polyanions based on localized ion depletion at the sample/membrane interface at a characteristic transition time τ, using polymer membrane polyanion-selective electrodes. Chronopotentiometric transduction of polyions based on the measurement of transition time has analytically more attractive applications compared to the controlled-current reversible pulsed chronopotentiometric transduction based on electromotive force (emf) measurement. This is because traditional polyion-selective electrodes based on emf measurement intrinsically give nonlinear (sigmoidal) calibration curves. While these can be used for indirect determination of polyions via polyanion-polycation titrations, they are not convenient for direct quantitation. However, under chronopotentiometric measurement based on the measurement of transition time, the square root of the transition time τ is linearly related to the concentration of the polyion according to the Sand equation and can be used for a direct calibration-free rapid determination. In this work, we have measured the concentrations of dextran sulfate (DS) and pentosan polysulfate (PPS) using polyanion selective electrodes under chronopotentiometric method where the transition time was measured and controlled-current pulsed chronopotentiometric transductions, where the phase boundary potential (emf) was measured. In addition, the protamine-DS and the protamine-PPS binding ratios have been determined using both transductions. The protamine-PPS binding ratio was determined to be 1.51:1 by the titration method and 1.54:1 by chronopotentiometry. The protamine-DS binding ratio was determined to be 1.37:1 by the titration method and 1.41:1 by chronopotentiometry, showing excellent agreement between the two methods. These simple measurement methods of binding ratios between polysaccharides and polypeptides may become important tools for screening safer and more reliable antidotes for the newer and safer anticoagulants such as Low Molecular Weight Heparins(LMWHs) and also to determine the dosages of antidotes needed to neutralize the anticoagulant activity.

Reversible Electrochemical Sensor for Detection of High-Charge Density Polyanion Contaminants in Heparin.

We present a simple, rapid, and inexpensive electrochemical sensor based on a reversible pulsed chronopotentiometric polyanion-selective membrane electrode for the detection and quantification of oversulfated chondroitin sulfate (OSCS) and other high charge-density polyanions that could potentially be used to adulterate heparin. The membrane is free of ion exchanger and is formulated with plasticized poly(vinyl chloride) (PVC) and an inert lipophilic salt, tridodecylmethylammonium-dinonylnaphthaline sulfonate (TDMA-DNNS). The neutral salt is used to reduce membrane resistance and to ensure reversibility of the sensor. More importantly, TDMA(+) is used as the recognition element for the polyanions. Here an anodic galvanostatic current pulse is applied across the membrane to cause the extraction of the polyanions from the sample into the membrane and potential is measured at the sample-membrane interface. The measured electromotive force (emf) is proportional to the concentration and the charge density of the polyanions. High charge-density polyanion contaminants and impurities in heparin can be detected using this method since the overall equilibrium potential response of polyions increases with increasing charge density of the polyions. Here, first the potential response of pure heparin is measured at a saturation concentration, the concentration beyond which further addition of heparin does not produce a change in potential response. Then the potential response of heparin tainted with different quantities of the high charge-density contaminant is measured at a fixed total polyion concentration (heparin concentration + contaminant concentration). The latter gives a greater negative potential response due to the presence of the high charge-density contaminant. The increase in the negative potential response can be used for detection and quantification of high charge-density contaminants in heparin. We demonstrate here that 0.3% (w/w) OSCS as well as 0.1% (w/w) dextran sulfate can be detected in heparin at 20-mg/mL total polyion concentration. It has also been shown that 1% (w/w) of dextran sulfate can readily be detected in heparin at only 2-mg/mL total polyanion concentration with a linear response (R(2) = 0.994).

Selectivity Enhancement for Chloride Ion by In(III)-Porphyrin-Based Polymeric Membrane Electrode Operated in Pulsed Chronopotentiometric Mode.

A robust selectivity enhancement of an In(III)-porphyrin ionophore-based chloride-selective electrode under pulsed chronopotentiometric measurement mode that enables the detection of chloride ions in the presence of a normally interfering concentration of salicylate ions is described. This enhancement is achieved by the rapid depletion of the surface concentration of the more dilute lipophilic anion during an initial anodic current pulse period due to extraction of this preferred anion into the membrane phase. Measurement of chloride with a detection limit of 8 mM and near Nernstian response slope in the presence of 1 mM salicylate is possible using the pulstrode method.

Detection of protease activities by flash chronopotentiometry using a reversible polycation-sensitive polymeric membrane electrode.

A novel electrochemical method, termed flash chronopotentiometry (FCP), is used to develop a rapid and sensitive method for detecting protease activities. In this method, an appropriate current pulse is applied across a polycation-selective polymer membrane to induce a strong flux of the polycationic peptides from the sample phase into the organic membrane of the electrode. During this current pulse, the cell potential (EMF) is monitored continuously, and is a function of the polypeptide concentration. The imposed current causes a local depletion of the polypeptide at the sample/membrane interface, which yields a drastic potential change in the observed chronopotentiogram at a characteristic time, called the transition time (τ). For a given magnitude of current, the square root of τ is directly proportional to the concentration of the polypeptide. Proteases cleave polypeptides into smaller fragments that are not favorably extracted into the membrane of the sensor. Therefore, a decrease in the transition time is observed during the proteolysis process. The degree of change in the transition time can be correlated to protease activity. To demonstrate this approach, the activities of trypsin and α-chymotrypsin are detected using protamine and synthetic polycationic oligopeptides that possess specific cleavage sites that are recognized by these proteases.

Reversible detection of heparin and other polyanions by pulsed chronopotentiometric polymer membrane electrode.

The first fully reversible polymeric membrane-based sensor for the anticoagulant heparin and other polyanions using a pulsed chronopotentiometry (pulstrode) measurement mode is reported. Polymeric membranes containing a lipophilic inert salt of the form R(+)R(-) (where R(+) and R(-) are tridodecylmethylammonium (TDMA(+)) and dinonylnaphthalene sulfonate (DNNS(-)), respectively) are used to suppress unwanted spontaneous ion extractions under zero-current equilibrium conditions. An anodic galvanostatic current pulse applied across the membrane perturbs the equilibrium lipophilic ion distribution within the membrane phase in such a way that anions/polyanions are extracted into the membrane from the sample. The membrane is then subjected to an open-circuit zero current state for a short period, and finally a 0 V vs reference electrode potentiostatic pulse is applied to restore the membrane to its initial full equilibrium condition. Potentials are sampled as average values during the last 10% of the 0.5 s open circuit phase of the measurement cycle. Fully reversible and reproducible electromotive force (emf) responses are observed for heparin, pentosan polysulfate (PPS), chondroitin sulfate (CS), and oversulfated chondroitin sulfate (OSCS), with the magnitude of the potentiometric response proportional to charge density of the polyanions. The sensor provides an emf response related to heparin concentrations in the range of 1-20 U/mL. The responses to variations in heparin levels and toward other polyanions of the pulstrode configuration are analogous to the already established single-use, nonreversible potentiometric polyion sensors based on membranes doped only with the lipophilic anion exchanger TDMA(+).

Measurement of total calcium by flash chronopotentiometry at polymer membrane ion-selective electrodes.

Ionophore-based ion-selective electrodes are widely used for potentiometric electrolyte measurements, in which case they are known to detect the free ion activity. Total ion concentrations cannot be directly assessed by this methodology if the ion is predominantly present in a complexed form. We present here the direct measurement of total calcium using a calcium ion-selective electrode interrogated in a flash chronopotentiometric transduction mode. A high magnitude of cathodic current pulse is applied across a calcium ion-selective membrane containing the ionophore ETH 5234 but void of ion-exchanger to prevent spontaneous extraction. This induces a defined flux of calcium ions from the sample side to the membrane and results in the release of labile bound calcium and a concomitant depletion at the membrane surface at a critical current or time. This is observed as an inflection point on the potential-time curve and the square root of the transition time is linearly related to the total concentration in the sample. It is shown that the responses to solutions of labile calcium complexes of nitrilotriacetic acid (NTA) are in a good agreement with that of the same concentration of calcium chloride in saline solution with this protocol. Initial applications are aimed towards assaying extracellular calcium. Calcium binding to albumin is shown to be inconsequential with sample dilutions typical for clinical assays. Calcium calibration curves in real and artificial dilute serum are finally shown to correspond to that of calcium chloride, suggesting that the methodology is indeed capable of detecting total calcium under these conditions. The present membrane materials allow detection of up to over 0.5mM total calcium in serum, currently requiring such samples to be diluted about 5-fold. The slopes of the square root of time-concentration dependence for the calibrations of free calcium in a background of NaCl and total serum calcium were found to be 3.857 and 3.717 s(1/2)mM(-1), respectively, deviating by just 3.6%. The lower detection limit (3x SD) was calculated as 12 microM.

Flash chronopotentiometric sensing of the polyions protamine and heparin at ion-selective membranes.

We report here on a highly sensitive and rapid detection technique, multipulse flash chronopotentiometry, for the anticoagulant polyion heparin and its antidote protamine. The technique is based on a localized titration of the polyions at the surface of an appropriately formulated polymeric ion-selective membrane devoid of ion exchange properties to prohibit spontaneous extraction processes. A defined ion flux from the sample side to the membrane is induced electrochemically by applying a current pulse of appropriate amplitude and sign. The resulting depletion of the measured ions at the membrane surface gives rise to a characteristic limiting current or transition time and is observed as an inflection point in the resulting chronopotentiogram. The limiting current and the square root of the transition time are linear functions of the concentration of the polyion and yield sensitive and rapid analytical information attractive for clinical diagnostics applications. The polyion protamine is detected in 10-fold diluted blood samples in a matter of seconds via a cathodic current pulse. The utility of the technique for monitoring heparin/protamine titrations in physiological saline solutions is demonstrated.

Beyond potentiometry: robust electrochemical ion sensor concepts in view of remote chemical sensing.

For about 100 years, potentiometry with ion-selective electrodes has been one of the dominating electroanalytical techniques. While great advances in terms of selective chemistries and materials have been achieved in recent years, the basic manner in which ion-selective membranes are used has not fundamentally changed. The potential readings are directly co-dependent on the potential at the reference electrode, which requires maintenance and for which very few accepted alternatives have been proposed. Fouling or clogging of the exposed electrode surfaces will lead to changes in the observed potential. At the same time, the Nernst equation predicts quite small potential changes, on the order of millivolts for concentration changes on the order of a factor two, making frequent recalibration, accurate temperature control and electrode maintenance key requirements of routine analytical measurements. While the relatively advanced selective materials developed for ion-selective sensors would be highly attractive for low power remote sensing application, one should consider solutions beyond classical potentiometry to make this technology practically feasible. This paper evaluates some recent examples that may be attractive solutions to the stated problems that face potentiometric measurements. These include high-amplitude sensing approaches, with sensitivities that are an order of magnitude larger than predicted by the Nernst equation; backside calibration potentiometry, where knowledge of the magnitude of the potential is irrelevant and the system is evaluated from the backside of the membrane; controlled current coulometry with ion-selective membranes, an attractive technique for calibration-free reagent delivery without the need for standards or volumetry; localized electrochemical titrations at ion-selective membranes, making it possible to design sensors that directly monitor parameters such as total acidity for which volumetric techniques were traditionally used; and controlled potential coulometry, where all ions of interest are selectively transferred into the ion-selective organic phase, forming a calibration-free technique that would be exquisitely suitable for remote sensing applications.

Direct sensing of total acidity by chronopotentiometric flash titrations at polymer membrane ion-selective electrodes.

Polymer membrane ion-selective electrodes containing lipophilic ionophores are traditionally interrogated by zero current potentiometry, which, ideally, gives information on the sample activity of ionic species. It is shown here that a discrete cathodic current pulse across an H (+)-selective polymeric membrane doped with the ionophore ETH 5294 may be used for the chronopotentiometric detection of pH in well-buffered samples. However, a reduction in the buffer capacity leads to large deviations from the expected Nernstian response slope. This is explained by the local depletion of hydrogen ions at the sample-membrane interface as a result of the galvanostatically imposed ion flux in direction of the membrane. This depletion is found to be a function of the total acidity of the sample and can be directly monitored chronopotentiometrically in a flash titration experiment. The subsequent application of a baseline potential pulse reverses the extraction process of the current pulse, allowing one to interrogate the sample with minimal perturbation. In one protocol, total acidity is found to be proportional to the magnitude of applied current at the flash titration end point. More conveniently, the square root of the flash titration end point time observed at a fixed applied current is a linear function of the total acid concentration. This suggests that it is possible to perform rapid localized pH titrations at ion-selective electrodes without the need for volumetric titrimetry. The technique is explored here for acetic acid, MES and citric acid with promising results. Polymeric membrane electrodes based on poly(vinyl chloride) plasticized with o-nitrophenyl octyl ether in a 1:2 mass ratio may be used for the detection of acids of up to ca. 1 mM concentration, with flash titration times on the order of a few seconds. Possible limitations of the technique are discussed, including variations of the acid diffusion coefficients and influence of electrical migration.

Selectivity enhancement of anion-responsive electrodes by pulsed chronopotentiometry.

A large and robust selectivity improvement of ion-selective electrodes is presented for the measurement of abundant ions. An improvement in selectivity by more than two orders of magnitude has been attained for the hydrophilic chloride ions measured in a dilute background of the lipophilic ions perchlorate and salicylate in a pulsed chronopotentiometric measurement mode. This is attributed to a robust kinetic discrimination of the dilute lipophilic ions in this measuring mode, which is not possible to achieve in classical potentiometry. Maximum tolerable concentrations of the interfering ions are found to be on the order of 30 microM before causing substantial changes in potential. As an example of practical relevance, the robust detection of chloride in 72 microM salicylate (reflecting 1:10 diluted blood) with a detection limit of 0.5 mM chloride is demonstrated. Corresponding potentiometric sensors did not give a useful chloride response under these conditions.