A chronoamperometric method to estimate changes in the membrane composition of ion-selective membranes.

A new chronoamperometric method is used to estimate changes in the membrane composition of mobile-site, ionophore-based membranes. The characteristic features of the chronoamperometric curves (initial current, slope, break time) of valinomycin-based, potassium-selective membranes loaded with potassium tetrakis(4-chlorophenyl)borate are correlated with the mobile-site and free ionophore concentration in the membrane. limiting cases for strong and negligible ion pair formation are distinguished. Replicate measurements indicate a relative standard deviation in the calculated values less than 10%. The practical applicability of the method was tested with membranes incorporated into conventional ion-selective electrode bodies or cast onto microfabricated planar sensor structures.

Electrodeposited iridium oxide pH electrode for measurement of extracellular myocardial acidosis during acute ischemia.

In the present paper, fabrication, characterization, and physiological applications of a solid-state pH electrode are described. The pH sensing layer was based on an anodic electrodeposited iridium oxide film (AEIROF). Sputtered platinum electrodes (1 mm diameter) fabricated on flexible Kapton films or platinum wires were used as planar or cylindrical supports. Each electrode site was coated with Nafion to attenuate the interference of anionic redox species and to protect the electrode surface during in vivo measurements. Performance of the AEIROF was evaluated, for the first time, as a pH electrode and proved to have a slightly super-Nernstian response with slope of -63.5 +/- 2.2 mV/pH unit for both wire and planar sputtered platinum electrodes. Linear pH responses were obtained in the pH range 2-10. The electrodes have a working lifetime of at least 1 month with accuracy of about 0.02 pH unit and fast response time. The electrodes showed very low sensitivities for different species, such as Na+, K+, Li+, NH4+, Ca2+, Mg2+, dissolved oxygen, lactate, ascorbate, and urate, which are important for physiological applications. The electrodes were applied in extracellular pH measurements during brief regional ischemia in a swine heart and no-flow ischemia in an isolated rabbit papillary muscle. A first report on extracellular pH, K+, and lactate simultaneous measurements during no-flow ischemia using the AEIROF pH electrode and the previously described K+ and lactate electrodes is presented as well.

Amperometric measuring cell for the determination of putrescine oxidase activity.

A microfabricated, flat form, amperometric microcell (microchip) is used in a simple, two-electrode arrangement for putrescine oxidase enzyme activity determinations. The cell contains a platinum microdisk working electrode and an Ag/AgCl reference electrode covered by a porous, hydrophilic membrane. An electrochemically-prepared size-exclusion layer is applied on the working electrode surface, to avoid the effect of electroactive interferences in the sample. The hydrophilic membrane, resting on the bottom of the cell, is soaked with a small volume of buffered substrate solution and a few mul enzyme containing sample solution is dispensed over the electrodes. During the enzyme activity measurement a catalytic reaction takes place in the membrane-supported liquid film over the working electrode surface. The hydrogen peroxide produced in the reaction is detected amperometrically. The amperometric current-time curves are used for evaluation. In our work putrescine was used as a substrate to determine the unknown putrescine oxidase enzyme activity of the sample. Elevated diamine oxidase enzyme activity in the vaginal milieu can indicate premature rupture of the amniotic membrane. Results with membrane discs, containing all the necessary chemicals in solid or lyophilized form, are very encouraging with respect of a single use, 'reagentless' biosensor for home care.

A conducting salt-based amperometric biosensor for measurement of extracellular lactate accumulation in ischemic myocardium.

In this paper, fabrication, characterization, and physiological application of a miniaturized amperometric lactate biosensor are described. The sensor is based on cross-linked lactate oxidase and tetrathiafulvalene-tetracyano-quinodimethane (TTF-TCNQ) charge transfer complex. The sensor was developed for continuous quantitative measurement of the lactate accumulation in ischemic myocardium under severe depletion of oxygen. The sensor was evaluated in vitro at an applied potential of 0.15 V vs Ag/AgCl; it proved to combine all the performance characteristics desired for the present application, such as proper response in absence of oxygen, good operational stability, good accuracy and precision (103.5 +/- 1.2%), adequate response time (t95% = 80 s), and wide linear dynamic range up to 27 mM (r = 0.9998) in N2-saturated solutions and at 37 degrees C. The prepared sensors (n = 12) showed sensitivity of 380 +/- 90 nA/mM, and a background current of 240 +/- 50 nA. The lower limit of detection is 0.4 +/- 0.15 mM with a S/N ratio equal to 3. Results obtained for direct lactate monitoring in ischemic rabbit papillary muscle under no-flow conditions and PO2 < 6 mm Hg are presented.

Amperometric monitoring of lactate accumulation in rabbit ischemic myocardium.

Fabrication and characterization of miniature, flexible, planar biosensors for monitoring l-lactate accumulation in an ischemic myocardium are described. Three configurations of Au-based electrodes were fabricated by a photolithographic technique on flexible polyimide Kapton((R)) foil. All sensors are based on an immobilized lactate oxidase with amperometric detection of the enzymatically produced hydrogen peroxide at a platinum-electroplated-gold base electrode polarized at 0.5 V versus Ag/AgCl. An inner electropolymeric layer is used to prevent electrode fouling and to reject the interference effects of easily oxidizable molecules. In addition, a diffusion controlling outer layer that greatly enhances the linear dynamic range of the sensor, is obtained by casting a polyurethane external film. The developed sensor was evaluated in vitro and proved to have high selectivity, good operational stability, good accuracy and precision (average recovery = 102.3 +/- 0.4% for control sera), fast response time (t(95) = 20 s) and high upper limit of the linear dynamic range (25-80 mM, with sensitivity of 1.7-0.4 nA mM(-1) respectively at PO(2) = 15 mmHg). Subsequently, the sensor was brought into direct contact with the surface of the rabbit papillary muscle and used for continuous quantitative monitoring of extracellular lactate accumulation during no-flow ischemia.

Development of a diamine biosensor.

An amperometric diamine sensor is developed for clinical applications in diagnosis of bacterial vaginosis (BV). The sensor is based on crosslinked putrescine oxidase (PUO) which catalyzes the conversion of diamines (mainly putrescine and cadaverine) to products including hydrogen peroxide. The hydrogen peroxide is detected anodically at platinum electrode polarized at 0.5 V versus Ag/AgCl. Platinum-plated gold electrodes used as a substrate for the sensor construction, are batch-fabricated on a flexible polyimide foil (Kapton(R), DuPont). A three-electrode cell configuration is used in all amperometric measurements. The sensor construction is based on three layers: an inner layer to reject the interference effect of oxidizable molecules, an outer diffusion controlling layer, and in addition, an enzyme middle layer. The enzyme layer was immobilized by crosslinking PUO with bovine serum albumin (BSA) using glutaraldehyde (GA). An optimization study of the enzyme solution composition was carried out. With the optimized enzyme layer, the biosensor showed a very high sensitivity and fast response time of ca. 20 s. The sensor has a linear dynamic range from (0.5-300 muM) for putrescine that covers the expected biological levels of the analyte. Details on sensor fabrication and characterization are given in the present work.

Miniaturized biosensors employing electropolymerized permselective films and their use for creatine assays in human serum.

Miniaturized, disposable amperometric biosensors for determination of creatinine in human serum are described. The base electrodes are fabricated using micro-electronics techniques, to build a multilayer film structure on a polyimide foil. By using a thin electropolymerized film of poly(1,3-diaminobenzene), the electrochemical interferences from ascorbate, urate, acetaminophen, and other oxidizable species are greatly diminished. The multienzyme system (creatininase, creatinase, sarcosine oxidase) is immobilized on top of the permselective layer using cross-linking of the proteins with glutaraldehyde. The electropolymerization conditions for obtaining almost ideal permselectivity of the inner layer are defined, as well as the optimal enzyme layer preparation. A composite polymeric outer membrane [Nafion + poly-(2-hydroxy-ethyl methacrylate) is used for diffusion control and to protect the enzyme layer from fouling. The reagentless planar sensors for creatinine and creatine have fast response time (t95 = 1 min), linear response up to 1.2 mM in batch-type and 2.0 mM in flow injection analysis and a detection limit of 10-20 muM. They are applied in a differential setup for creatinine assay in control and hospital human serum samples and are suitable for incorporation in a portable analyzer.

Aliphatic polyurethane as a matrix for pH sensors: effects of native sites and added proton carrier on electrical and potentiometric properties.

The potentiometric and impedance characteristics of polymeric membranes, based on aliphatic polyurethane (Tecoflex) as a matrix, are described and interpreted by theory and experiments for H(+) and alkali metal ion-sensitive sensors. Both dummy plasticized membranes and proton carrier-loaded membranes can show pH response. The pH response of dummy membranes is due to protonated natural negative sites in the polyurethane matrix. The electrodes with added proton carrier show improved rejection of Li(+), Na(+), and K(+) responses and give useful analytical responses. Optimal performance requires control of negative site concentration by addition of lipophilic salt (e.g. tetraphenylborate derivatives). Impedance analyses show surface-rate semicircles and, depending on the bathing electrolyte solution, appearance of a diffusional Warburg impedance. In addition to these time-dependence surface region effects, changes in the bulk membrane resistance with soaking time can be well correlated with equilibrium water content of plasticized membranes.

Electroanalytical and biocompatibility studies on carboxylated poly(vinyl chloride) membranes for microfabricated array sensors.

Potassium ion-selective and pH membrane electrodes based on neutral carrier ionophores for K+ (valinomycin) and H+ (TDDA and ETH 5294), respectively, immobilized in carboxylated PVC (PVC-COOH) with normal (classical) and reduced amounts of plasticizer, were investigated with respect to their general analytical performances (linear range, slope, detection limit, selectivity, internal membrane resistance), their biocompatibility and cellular responses. The analytical performance of potassium selective electrodes was not affected by reducing the plasticizer content from 66% (m/m) to about 33% (m/m) while that of pH electrodes was significantly changed at the lower plasticizer concentration level. The adhesive properties of PVC-COOH membranes to an inert substrate such as polyimide-coated Kapton are greatly improved by reducing the plasticizer content of the membrane. In addition, as was reported earlier by this group, improved biocompatibility was observed with these membranes relative to those with increased plasticizer content. A ratio of 1:1 m/m for PVC-COOH to plasticizer is recommended for the construction of planar ISEs without massive use of internal solution.

Ion-selective membranes with low plasticizer content: electroanalytical characterization and biocompatibility studies.

High molecular weight poly(vinyl chloride) and aliphatic polyurethane (Tecoflex)-based ion selective membranes, with normal and reduced amounts of plasticizer, as well as without plasticizer, were tested with respect to their analytical properties, their biocompatibility, and cellular responses. The analytical properties of the membranes did not change significantly within a wide range of polymer to plasticizer ratios. However, the membranes with reduced plasticizer content had better adhesive properties, less anion interference, extended life time, and better biocompatibility. Using the cage implant system, the results showed that an increase of plasticizer weight percent in Tecoflex membranes correlated positively with the increase in host inflammatory response up to 14 days of implantation. The results also demonstrated that both PVC and Tecoflex-based ion-selective membranes with the most common membrane composition (1:2 polymer to plasticizer ratio) exhibited a similar acute inflammatory response, but the PVC-based membrane elicited a reduced chronic inflammatory response when compared with the Tecoflex-based membrane.

Validation of carrier-mediated transport of H(+) and Na(+) through mobile-site membranes.

The response of membranes containing neutral ion carriers of either H(+) or Na(+) to an externally-applied potential step was investigated using previously developed techniques for the analysis of charge and mass transport in ion-selective membranes. The results from constant-resistance membranes, e.g. membranes with unperturbed negative site concentration profiles, showed that tridodecylamine behaved as a carrier for H(+), and there was no evidence for proton hopping from stationary carriers. In addition, the experimental outcome supported the assumption of a failure of the Donnan exclusion principle at very low pH levels in these membranes. The results from membranes containing the Na(+) carrier illustrated the significant concentration polarization of ionic species, which was related to significant changes in bulk membrane resistance.

Responses of H(+) selective solvent polymeric membrane electrodes fabricated from modified PVC membranes.

Potentiometric responses of a novel class of pH sensitive ionophores, namely several phenoxazine derivatives, were tested in different modified PVC matrices. The ionophores were compounded into liquid membranes as usual or were covalently coupled to the polymeric matrix. The general analytical performance of the membranes and other membrane characteristics (i.e., resistance and response time, as measures of membrane decomposition or structural changes) were followed in time. The transient responses of membranes with mobile ionophores in high molecular weight (HMW) and carboxylated PVC (PVC-COOH) were compared to those with immobilized ionophores. The response time of membranes with immobilized ionophores was found to be between those with mobile ionophores in HMW (fast response) and PVC-COOH (sluggish response). Accordingly, the rate of response was correlated primarily to the -COOH content of the membranes.

Impedances of thin and layered systems: cells with even or odd numbers of interfaces.

Impedance data, e.g., system responses, from perturbing small amplitude applied sinusoid signals of near DC to high kilohertz frequencies, give chemical information. Analysis of frequency-dependent imaginary and real impedance proceeds from equivalent analog circuit elements to chemical and physical significance determined from many model systems. Already, it is possible to interpret bulk transport processes, surface kinetic effects, diffusion phenomena, and dependencies on the type of contacts: symmetric ion contact, symmetric metal contact or asymmetric metal-ion interfaces, and cell design; even (battery or sensor) and odd numbered (constrained junction or immiscible liquid) interfaces in a system. These analyses cover the chemical origins, locations and meanings of the lumped resistances, capacitances and transmission lines that are introduced by engineers in their strict analog interpretations of the impedance data. Examples cover simple ohmic, simple diffusive behavior, complex behavior with surface interfacial kinetics or surface resistances, and with finite (nonblocking) or infinite (blocking) DC impedance. High and low frequency responses may show so-called constant phase element character that suggests fractal behavior. Low frequency data occasionally appear in the second quadrant of impedance plane plots. These results are caused by negative capacitances and resistances. In this paper, chemical interpretations of analog circuit elements are mainly based on theory and observations of thin cells of electrolytes and solid and liquid films (membranes) that are ionic or mixed ionic/electronic conductors. The information should carry over into thickened, gelled, and tissue electrolyte phases and serve as a basis for medically-oriented, perhaps diagnostic impedance measurement applications already pioneered by Herman Schwan.

Mediated, anaerobic voltammetry of sulfite oxidase.

The anaerobic voltammetry of the Mo/Fe enzyme, sulfite oxidase (SO), is described for the mediators cytochrome c, [Ru(NH3)6]3+/2+, TMPD+/0, and [Co(bpy)3]3+/2+. Theory derived for steady-state voltammetric catalysis correctly predicts the observed concentration and scan-rate dependencies of the catalytic waves. The instances for which existing ECcat theories may be applied to two catalytic reactions coupled to an interfacial charge transfer are considered. The biomolecular rate constant for the reaction of [Co(bpy)3]3+ with reduced SO is calculated and determined to be approximately 5 X 10(4) L.mol-1.s-1. The appearance of catalytic prepeaks at low sulfite concentrations is noted and the shape of corresponding i/t curves from chronoamperometry is examined. The analytical implications of the novel time dependence of the catalytic current under these conditions are discussed.

Ion transport properties of cyclic and acyclic neutral carrier-containing membranes.

The K(+)-valinomycin permselective membrane provides a standard system for describing the normal current-voltage and current-time responses of the "closed circuit shuttle" carrier mechanism. The responses of a selection of cyclic and acyclic carrier-systems have been measured and compared with the standard system responses. Deviations from expected normal behavior suggest that failure of Donnan exclusion is caused by use of high-permittivity plasticizers and too few fixed sites in the membrane supports. The closed circuit shuttle model has been extended to allow for failure of Donnan exclusion, consumption of free carriers, and additional transport by extracted salts. The predicted limiting currents and current-time responses show many features of failure of Donnan exclusion.

A poly(vinylchloride) membrane electrode for determination of phenytoin in pharmaceutical formulations.

The construction and general performance characteristics of an ion-selective membrane electrode sensitive to the drug phenytoin, based on its ion-pair complex with the quaternary ammonium cation, tricaprylmethylammonium, in a PVC matrix are described. The electrode shows near-Nernstian response over a 10(-1)-10(-4) mol l(-1) range and a detection limit of 1.5 x 10(-5) mol l(-1). The selectivity of the electrode to a number of inorganic and organic anions is reported. OH(-) interference, in the linear range of the calibration curve, is negligible up to pH 11.0. The standard addition method is used to determine phenytoin in pharmaceutical formulations, such as tablets and capsules, with good results. The method is rapid and simple, and does not require prior sample pretreatment.

A quinidine-responsive plastic membrane electrode.

A membrane electrode based on quinidine tetraphenylborate in a PVC matrix is described. The electrode exhibits a rapid and near-Nernstian response in the range 3.5 x 10(-5)-1 x 10(-2) M quinidine sulphate at pH 6-8. In an acidic medium the electrode responds to diprotonated quinidine. In sodium tetraphenylboron (Na TPB) solutions the response is linear in respect of log (TPB-) over the range 10(-4)-10(-2)M . Direct potentiometry and potentiometric titrations are used to determine quinidine in pharmaceutical preparations.