Detection limits of thin layer coulometry with ionophore based ion-selective membranes.

We report here on a significant improvement in lowering the low detection limit of thin layer coulometric sensors based on liquid ion-selective membranes, using a potassium-selective system as a model example. Various possible processes that may result in an elevated residual current reading after electrolysis were eliminated. Self-dissolution of AgCl on the Ag/AgCl inner element may result in a residual ion flux that could adversely affect the lower detection limit. It was here replaced with an Ag/AgI inner pseudoreference electrode where the self-dissolution equilibrium is largely suppressed. Possible residual currents originating from a direct contact between inner element and ion-selective membranes were eliminated by introducing an inert PVDF separator of 50 µm diameter that was coiled around the inner element by a custom-made instrument. Finally, the influence of electrolyte fluxes from the outer solution across the membrane into the sample was evaluated by altering its lipophilic nature and reducing its concentration. It was found that this last effect is most likely responsible for the observed residual current for the potassium-selective membranes studied here. For the optimized conditions, the calibration curves demonstrated a near zero intercept, thereby paving the way to the coulometric calibration-free sensing of ionic species. A linear calibration curve for the coulometric cell with valinomycin potassium-selective membrane was obtained in the range of 100 nM to 10 µM potassium in the presence of a 10 µM sodium background. In the presence of a higher (100 µM) concentration of sodium, a reliable detection of 1-100 µM of potassium was achieved.

Coulometric sodium chloride removal system with Nafion membrane for seawater sample treatment.

Seawater analysis is one of the most challenging in the field of environmental monitoring, mainly due to disparate concentration levels between the analyte and the salt matrix causing interferences in a variety of analytical techniques. We propose here a miniature electrochemical sample pretreatment system for a rapid removal of NaCl utilizing the coaxial arrangement of an electrode and a tubular Nafion membrane. Upon electrolysis, chloride is deposited at the Ag electrode as AgCl and the sodium counterions are transported across the membrane. This cell was found to work efficiently at potentials higher than 400 mV in both stationary and flow injection mode. Substantial residual currents observed during electrolysis were found to be a result of NaCl back diffusion from the outer side of the membrane due to insufficient permselectivity of the Nafion membrane. It was demonstrated that the residual current can be significantly reduced by adjusting the concentration of the outer solution. On the basis of ion chromatography results, it was found that the designed cell used in flow injection electrolysis mode reduced the NaCl concentration from 0.6 M to 3 mM. This attempt is very important in view of nutrient analysis in seawater where NaCl is a major interfering agent. We demonstrate that the pretreatment of artificial seawater samples does not reduce the content of nitrite or nitrate ions upon electrolysis. A simple diffusion/extraction steady state model is proposed for the optimization of the electrolysis cell characteristics.

Enzyme logic gate associated with a single responsive microparticle: scaling biocomputing to microsize systems.

A microsize biocomputing system based on enzyme logic processing biochemical signals was developed. Optical transduction of pH signals generated in situ by the enzyme OR logic gate was achieved with the use of a single optode microparticle.

Surface area effects on the response mechanism of ion optodes: a preliminary study.

The relationship between the surface-to-volume ratio and the response mechanism of polymeric ion probes (ion optodes) is not well understood. In this work, the surface-to-volume ratio of ion optodes was systematically increased in an attempt to characterize this relationship. Several different batches of ion-selective optodes were fabricated via the solvent displacement method using sodium ionophore X, BME-44, and ETH 1001 for sodium-, potassium-, and calcium-selective optodes, respectively. Dilution of the membrane cocktail with varying amounts of an organic solvent provided a convenient tool to control the resulting particle size distribution. Specifically, ion optodes of five different size distributions were fabricated. An apparent shift of the response function on the pH scale was observed for optodes with identical composition that differed in terms of size. There was a strong correlation between the calculated specific surface area and the apparent ion-exchange constant for all three types of ion optodes. However, there was an indication that selectivity does not substantially correlate with the optode size. We hypothesize that the observed effect is caused by surface phenomena which contribute to the overall optode response. The results reported here may raise a word of caution about the application of established response models, which were developed for macroscopic ion optodes, toward probes at micrometer and submicrometer scales.

Fabrication of micrometer and submicrometer-sized ion-selective optodes via a solvent displacement process.

Microsphere-based ion optodes represent a promising and versatile tool to measure ionic activities in confined samples. The reported methods of micro- and nanosphere optode fabrication, however, suffer from various degrees of complexity. We propose a large-scale fabrication of polymeric ion-selective optodes using a solvent displacement method. Plasticized poly(vinyl chloride) along with the optode components was dissolved in a solvent miscible with water. Injection of a polymer solution into a stirred aqueous phase containing a surfactant causes spontaneous emulsification. This technique does not require additional preparation steps and allows one to control the composition of the sensor matrix precisely. Several factors affecting the particle size distribution are examined such as composition of continuous and disperse phases. The concentration of the polymer in the organic solvent and the choice of the solvent nature allowed us to control the particle size distribution within 200 nm-30 microm. The concentration and the nature of the surfactant had a little influence on the particle size distribution. We fabricated three different batches of ion-selective optodes using chromoionophore I, lipophilic ion-exchanger and sodium ionophore X, BME-44, and ETH 5234 for sodium, potassium, and calcium optodes, respectively. The sensors were fully functional with excellent selectivity to interfering ions.

Determination of unbiased selectivity coefficients using pulsed chronopotentiometric polymeric membrane ion sensors.

A new procedure for the determination of selectivity coefficients of neutral carriers using pulsed chronopotentiometric ion selective sensors (pulstrodes) is established. Pulstrode membrane which lacks an ion-exchanger suppresses the zero current ion flux, allowing a Nernstian response slope for even highly discriminated ions. Unlike previously developed methods, unbiased selectivity remains unaltered even with the exposure to the primary ion solution for prolonged time. Studies with potassium-, silver-, and calcium-selective electrodes reveal that pulstrodes yield the same or slightly favorable unbiased selectivity coefficients than reported earlier. In contrast to alternative methods for the determination of unbiased selectivity, this technique offers a unique simplicity and reliability. Therefore the new procedure promises to be a valuable additional tool for the characterization of unbiased selectivity coefficients for the ISEs.

Solid-contact electrochemical polyion sensors for monitoring peptidase activities.

We report here on improved solid-contact electrochemical polyion sensors for the detection of polyion protamine. The polymeric membrane sensors were fabricated with a conducting polymer as an ion-electron transduction layer. We observed that decreasing the magnitude of the applied current pulse caused a significant improvement of the sensor sensitivity to low protamine levels. The protamine sensors exhibited a stable and reversible response to protamine concentrations ranging from 0.05 to 30 mg L-1. The sensors were used for monitoring peptidase activities utilizing galvanostatically controlled solid-contact membrane sensors. The polyion protamine was used as a substrate to detect the activity of the protease trypsin. The enzyme activity was continuously monitored by measuring the protamine concentration as it is cleaved by enzyme into smaller fragments to which the sensor is less sensitive. In the presence of a given level of protamine the initial rate of reaction can be linearly related to the trypsin activity within a 0-5 U mL-1 concentration range. The interference with the enzymatic reaction product arginine was specifically examined.

Reversible detection of proteases and their inhibitors by a pulsed chronopotentiometric polyion-sensitive electrode.

Polymer membrane electrodes operated by pulsed chronopotentiometry have recently been introduced to replace traditional ion-selective electrodes for a number of applications. While ion-selective electrodes for the polycation protamine have been reported, for instance, a pulsed chronopotentiometric readout mode (called here pulstrode) provides improved stability and reproducibility while exhibiting sufficient selectivity for the direct detection of protamine in undiluted whole blood samples. Here, such protamine-sensitive pulstrodes are applied for the real-time detection of the activity of the protease trypsin and its soybean inhibitor. This is possible because small fragments produced by the trypsin digestion are not detectable by the protamine-sensing membrane. The real-time response to the proteolytic reaction is shown to exhibit good reproducibility and reversibility, and the initial reaction rate is dependent on the concentration of the protease and its inhibitor.

Unbiased selectivity coefficients obtained for the pulsed chronopotentiometric polymeric membrane ion sensors.

We report here on the successful observation of the unbiased thermodynamic selectivity of ion-selective sensors working in normal pulse chronopotentiometric mode (pulstrodes). In contrast to ion-selective electrodes, the pulstrodes do not require careful counterbalancing of the transmembrane ionic fluxes to achieve unbiased thermodynamic selectivity. The pulstrodes can work under asymmetric conditions, which are often encountered in practice. The composition of the inner filling solution did not affect the sensor response, indicating that the transmembrane flux of primary ions was indeed effectively suppressed in the absence of ion exchanger. For the K-selective sensor considered here, an improvement of Mg discrimination by a factor of 1000 was demonstrated.

Kinetic modulation of pulsed chronopotentiometric polymeric membrane ion sensors by polyelectrolyte multilayers.

Polymeric membrane ion-selective electrodes are normally interrogated by zero current potentiometry, and their selectivity is understood to be primarily dependent on an extraction/ion-exchange equilibrium between the aqueous sample and polymeric membrane. If concentration gradients in the contacting diffusion layers are insubstantial, the membrane response is thought to be rather independent of kinetic processes such as surface blocking effects. In this work, the surface of calcium-selective polymeric ion-selective electrodes is coated with polyelectrolyte multilayers as evidenced by zeta potential measurements, atomic force microscopy, and electrochemical impedance spectroscopy. Indeed, such multilayers have no effect on their potentiometric response if the membranes are formulated in a traditional manner, containing a lipophilic ion exchanger and a calcium-selective ionophore. However, drastic changes in the potential response are observed if the membranes are operated in a recently introduced kinetic mode using pulsed chronopotentiometry. The results suggest that the assembled nanostructured multilayers drastically alter the kinetics of ion transport to the sensing membrane, making use of the effect that polyelectrolyte multilayers have different permeabilities toward ions with different valences. The results have implications to the design of chemically selective ion sensors since surface-localized kinetic limitations can now be used as an additional dimension to tune the operational ion selectivity.

Pulsed galvanostatic control of solid-state polymeric ion-selective electrodes.

We report on galvanostatically controlled solid-state reversible ion-selective sensors for cationic analytes utilizing a conducting polymer as a transduction layer between the polymeric membrane and electron-conductive substrate. The instrumental control of polymeric membrane ion-selective electrodes based on electrochemically induced periodic ion extraction in alternating galvanostatic/potentiostatic mode was introduced recently creating exciting possibilities to detect clinically relevant polyions such as heparin and protamine and drastically improve the sensitivity of ion-selective sensors limited by the Nernst equation. The present study forms the basis for development of reliable, robust, and possibly maintenance-free sensors that can be fabricated using screen-printing technology. Various aspects of the development of solid-contact galvanostatically controlled ion-selective electrodes with a conducting polymer as a transduction layer are considered in the present work on the example of a model system based on a sodium-selective membrane. The protamine-selective solid-contact sensor was fabricated and characterized, which represents the next step toward commercially viable polyion sensing technology. A substantial improvement of a low detection limit (0.03 mg L-1) was achieved. A simplified diffusion-based theoretical model is discussed predicting the polarization at the interface of the conducting polymer and the membrane, which can cause the disruption of the sensor response function at relatively small current densities.

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.

Selective coulometric release of ions from ion selective polymeric membranes for calibration-free titrations.

Coulometry belongs to one of the few known calibration-free techniques and is therefore highly attractive for chemical analysis. Titrations performed by the coulometric generation of reactants is a well-known approach in electrochemistry, but suffers from limited selectivity and is therefore not generally suited for samples of varying or unknown composition. Here, the selective coulometric release of ionic reagents from ion-selective polymeric membrane materials ordinarily used for the fabrication of ion-selective electrodes is described. The selectivity of such membranes can be tuned to a significant extent by the type and concentration of ionophore and lipophilic ion-exchanger and is today well understood. An anodic current of fixed magnitude and duration may be imposed across such a membrane to release a defined quantity of ions with high selectivity and precision. Since the applied current relates to a defined ion flux, a variety of non-redox active ions may be accurately released with this technique. In this work, the released titrant's activity was measured with a second ionophore-based ion-selective electrode and corresponded well with expected dosage levels on the basis of Faraday's law of electrolysis. Initial examples of coulometric titrations explored here include the release of calcium ions for complexometric titrations, including back titrations, and the release of barium ions to determine sulfate.

Photoresponsive ion-selective optical sensor.

A novel concept of photoresponsive ion-selective optical sensor is described. Photochemical reactions can be utilized to generate and control ion fluxes in an ion-selective optode in the same manner as nonequilibrium electrochemical methods have been used in ion-selective electrodes. In contrast to their equilibrium counterparts, the photoresponsive pH-selective ion optodes are sensitive to both the buffer capacity of the sample and activity of hydrogen ions. Active optical probes are especially attractive for intracellular applications because they can be fabricated as submicron-sized beads. Common optical techniques, such as fluorescence microscopy and flow cytometry, can be combined with active ion probes with only minor modification of the existing experimental setup.

Calcium pulstrodes with 10-fold enhanced sensitivity for measurements in the physiological concentration range.

Ion-selective electrodes ideally operate on the basis of the Nernst equation, which predicts less than 60- and 30-mV potential change for a 10-fold activity change of monovalent and divalent ions measured at room temperature, respectively. Typical concentration ranges in extracellular fluids are quite narrow for the electrolytes of key importance. A range of 2.2-2.6 mM for calcium ions, for instance, translates into just a 2.2-mV potential change. The direct potentiometric measurement of physiological electrolytes is certainly possible with direct potentiometry and is done routinely in clinical analyzers and handheld measuring devices. It places, however, strong demands on the precision of the reference electrode and requires careful temperature control and frequent calibration runs. In this paper, a robust 10-20-fold sensitivity enhancement for calcium measurements is attained by departing from the classical response mechanism and operating in a non-Nernstian response mode. Stable and reproducible super-Nernstian responses of these so-called pulstrodes in a narrow calcium activity range can be controlled by instrumental means in good agreement with theory. The potentials may be measured during a galvanostatic excitation pulse (mode I) or immediately after it (mode II), under open-circuit conditions. Subtraction of the potentials, sampled at different times during a single pulse, allows one to obtain a sensitive differential peak-shaped signal at a critical and fully adjustable analyte activity range. Calcium pulstrodes based on the diamide ionophore AU-1 were characterized and applied to the measurement in model physiological liquids. Super-Nernstian responses exceeding 700 mV/decade were observed in a physiological range of calcium concentration. Such remarkable sensitivity of the pulstrodes, complemented with the well-documented high selectivity of these potentiometric sensors, may provide a significant increase in the accuracy and precision of electrolyte measurements in clinical analysis.

Response characteristics of a reversible electrochemical sensor for the polyion protamine.

We describe here in detail the first reversible electrochemical sensors for the polyion protamine. Potentiometric sensors were proposed in recent years, mainly for the determination of the polyions heparin and protamine. Such potentiometric polyion sensors functioned on the nonequilibrium extraction of polyions into a hydrophobic membrane phase via ion pairing with lipophilic ion exchangers. This made it difficult to design sensors that operate in a truly reversible fashion. The reversible sensors described here utilize the same basic response mechanism as their potentiometric counterparts, but the processes of extraction and ion stripping are now fully controlled electrochemically. Spontaneous polyion extraction is avoided by using membranes containing highly lipophilic electrolytes that possess no ion-exchange properties. Reversible extraction of polyions is induced if a constant current pulse of fixed duration is applied across the membrane, followed by a baseline potential pulse. The key theoretical response principles of this new class of polyion sensors are discussed here and compared to those of its classical potentiometric counterpart. The electrochemical sensing system is characterized in terms of optimal working conditions, membrane composition, selectivity, and influence of sample stirring and organic-phase diffusion coefficient on the response characteristics. Excellent potential stability and reversibility of the sensors are observed, and measurements of heparin concentration in whole blood samples via protamine titration are demonstrated.

Reversible electrochemical monitoring of surface confined reactions at liquid-liquid interfaces by modulation of ion transfer fluxes.

The adsorption of the neutral surfactant Brij35 at a liquid-liquid interface is reversibly monitored via its disturbance of an electrochemically imposed ion flux across the interface, forming a promising experimental tool for the detection of surface confined reactions at such liquids and polymers.

Pulstrodes: triple pulse control of potentiometric sensors.

Ion-selective electrode membranes based on hydrophobic materials doped with chemically selective host molecules are an attractive sensing technology but normally suffer from a limited sensitivity, given by the Nernst equation, and a direct reliance on the reference electrode potential, which makes miniaturization difficult. These fundamental problems are addressed here by imposing a multipulse electrochemical excitation signal onto ion-selective membranes that lack ion-exchange properties. Current pulses are responsible for the generation of ion fluxes in the direction of the membrane, which give reproducible super-Nernstian response slopes that originate from depletion processes at the membrane surface. Membranes may also be measured at zero current after this pulse, giving super-Nernstian response regions at lower concentrations. Difference potentials obtained from subsequent pulses give about 10-fold higher sensitivities than predicted on the basis of the Nernst equation.

Distinguishing free and total calcium with a single pulsed galvanostatic ion-selective electrode.

A pulsed galvanostatic technique is presented to distinguish free and total levels of calcium with a single membrane electrode by varying the magnitude of the applied current. Pulsed chronopotentiometry creates the possibility of accurate control of ion fluxes across ion-selective plasticized polymeric membranes without ion-exchanger properties (note, however, that a low concentration of ion-exchanger impurities is always present in such membranes). During a constant current pulse ions are forced to extract from the aqueous sample into the contacting membrane phase. Each current pulse is followed by a constant potential pulse to remove the extracted ions from the membrane. The induced concentration gradients are reproducible from pulse to pulse. At relatively small applied currents and in the presence of labile complexes in the sample, the sensor responds to the ion activity, in analogy to a conventional ISE. If a larger current is applied, the flux of complexed ions dominates the sensor response because of its increased magnitude, and the observed potential is now a function of the total ionic concentration. This approach allows one to detect, with the same sensor, the levels of free and total ionic concentration by varying the magnitude of the applied current. The technique utilized here gives much more stable signals than with earlier work demonstrating the principle with zero-current potentiometry. This concept is illustrated with calcium selective membranes based on the ionophore ETH 5234.

Pulsed galvanostatic control of ionophore-based polymeric ion sensors.

This paper describes a pulsed galvanostatic technique to interrogate ion-selective electrodes (ISEs) with no intrinsic ion-exchange properties. Each applied current pulse is followed by a longer baseline potential pulse to regenerate the phase boundary region of the ion-selective membrane. The applied current fully controls the magnitude and sign of the ion flux into the membrane, thus offering instrumental control over an effect that has become very important in ion-selective electrode research in recent years. The resulting chronopotentiometric response curves essentially mimic traditional ISE behavior, with apparently Nernstian response slopes and selectivities that can be described with the Nicolsky equation. Additionally, the magnitude and sign of the current pulse may be used to tune sensor selectivity. Perhaps most important, however, appears to be the finding that the extent of concentration polarization near the membrane surface can be accurately controlled by this technique. A growing number of potentiometric techniques are starting to make use of nonequilibrium principles, and the method introduced here may prove to be very useful to advance these areas of research. The basic characteristics of this pulsed galvanostatic technique are here evaluated with plasticized poly(vinyl chloride) membranes containing the sodium-selective ionophore tert-butyl calix[4]arene tetramethyl ester and a lipophilic inert salt.