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It was established that the components of tea are oxidized on a glassy carbon electrode modified with carbon nanotubes and electropolymerized quercetin in a phosphate buffer solution (pH 7.0) as a supporting electrolyte under conditions of differential pulse voltammetry. The oxidation potentials of the individual phenolic antioxidants of tea (gallic acid, rutin, quercetin, catechin, epigallocatechin gallate, and tannin) on the modified electrode were found. A method for the chronoamperometric determination of the antioxidant capacity (AOC) of tea was developed based on the oxidation of tea antioxidants at a potential of 0.20 V. The AOC of tea was evaluated using a difference between the oxidation currents of the analyte and a supporting electrolyte after 50 s of electrolysis in terms of gallic acid. The analytical range of gallic acid was 0.25-750 [micro]M with a detection limit of 0.063 [micro]M. Positive correlations of the AOC with antioxidant activity in a reaction with 2,2-diphenyl-1-picrylhydrazyl and the total phenolic content were found (r = 0.700 and 0.647 at [r.sub.crit] = 0.396, respectively). Keywords: chronoamperometry, chemically modified electrodes, electropolymerized materials, antioxidant capacity, food analysis, Tea is one of the most common sources of antioxidants in the dietary intake of human beings. The main antioxidants of tea are phenolic compounds, in particular, flavan-3-ols (catechins), flavonols, [...]

The analytical characteristics of gold nanowires prepared by direct electrochemical synthesis were studied with the use of the amperometric determination of glucose as an example. The applicability of the gold nanowires to the determination of glucose in a neutral medium (a phosphate buffer solution with pH 7.2) over a concentration range from 1 x [10.sup.-4] to 5 x [10.sup.-3] M at a detection potential of +0.35 V was shown. It was found that the sensitivity of a nonenzymatic sensor for the determination of glucose on the gold nanowires was high: 3.7 x [10.sup.-4] A [M.sup.-1] [m.sup.-2]. The limit of detection was 3.3 x [10.sup.-5] M. Keywords: amperometry, nonenzymatic sensor, glucose, gold nanowires, Studies [1-6] on the electrocatalytic oxidation of glucose showed the possibility of its nonenzymatic determination in both neutral and alkaline solutions [7, 8]. The use of noble metal nanostructures as [...]

tert-Butylhydroquinone (TBHQ) and tert-butylhydroxyanisole (BHA) are oxidized on a glassy carbon electrode modified with multiwalled carbon nanotubes in a 0.1 M LiCl[O.sub.4] supporting electrolyte in the medium of 1 mM Brij®35 at 0.27 and 0.47 V, respectively. A method for their chronoamperometric determination is developed. It is shown that a steady-state electrolysis is achieved within 100 s. The analytical range is 2.50-1000 µM for TBHQ and 1.50-100 and 250-1000 µM for BHA with the detection limits (S/N = 3) 0.64 and 0.38 µM, respectively. The relative standard deviation in the determination of TBHQ and BHA in model solutions does not exceed 6%. The method was tested on micellar extracts of linseed oils. The accuracy measure is 100 ± 1%, which points to the absence of matrix effects in the determination of TBHQ and BHA. Keywords: chronoamperometry, sterically hindered phenols, micellar media, surfactants DOI: 10.1134/S1061934815120175, Synthetic phenolic antioxidants, such as tert-butylhydroquinone and ieri-butylhydroxyanisole, are often used as stabilizing additives to prevent oxidation of food [1, 2] and industrial oils [3] and thermo-oxidative degradation of polymers [...]

Methods are developed for the non-enzymatic chronoamperometric determination of urea in blood serum and creatinine in model solutions imitating the composition of blood serum. The selectivity of determination is ensured using columns packed with an anion-exchange resin for urea or synthesized molecularly imprinted polymers for creatinine. Keywords: molecularly imprinted polymers, urea, creatinine, chronoamperometry, voltammetry, electrocatalysis, acrylic acid DOI: 10.1134/S1061934814080048, iThe determination of urea and creatinine in biological fluids, plasma, blood serum, and urine, is used in clinical diagnostics for the evaluation of the kidney function and the efficiency of [...]

A ternary surface monolayer, consisting of coassembled thiolated capture probe, mereaptohexanol and dithiothreitol, is shown to offer dramatic improvements in the signal-to-noise characteristics of electrochemical DNA hybridization biosensors based on common self-assembled monolayers. Remarkably low detection limits down to 40 zmol (in 4 [micro]L samples) as well as only 1 CFU Escherichia coli per sensor are thus obtained without any additional amplification step in connection to the commonly used horseradish peroxidase/3,3',5,5'-tetramethylbenzidine system. Such dramatic improvements in the detection limits (compared to those of common binary alkanethiol interfaces and to those of most electrochemical DNA sensing strategies without target or signal amplification) are attributed primarily to the remarkably higher resistance to nonspecific adsorption. This reflects the highly compact layer (with lower pinhole density) produced by the coupling of the cyclic- and linear-configuration 'backfillers' that leads to a remarkably low background noise even in the presence of complex sample matrixes. A wide range of surface compositions have been investigated, and the ternary mixed monolayer has been systematically optimized. Detailed impedance spectroscopy and cyclic voltammetric studies shed useful insights into the surface coverage. The impressive sensitivity and high specificity of the simple developed methodology indicate great promise for a wide range of nucleic acid testing, including clinical diagnostics, biothreat detection, food safety, and forensic analysis. 10.1021/ac101474k

Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybll, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore remains elusive. Here we show that the ability of sybll to support exocytosis is inhibited by addition of one or two residues to the sybll C terminus depending on their energy of transfer from water to the membrane interface, following a Roltzmann distribution. These results suggest that following stimulation, the SNARE complex pulls the C terminus, of sybll deeper into the veside membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed cytoplasmic leaflets. chromaffin cell | patch clamp capacitance measurement | caged calcium | amperometry | electrochemical detector array doi/ 10.1073/pnas.1006727107

A method for simultaneous determination of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) in food was developed that uses multiple pulse amperometry (MPA) with flow injection analysis (FIA). Determination of these phenolic antioxidants was carried out with a cathodically pretreated boron-doped diamond electrode and an aqueous ethanolic (30% ethanol, v/v) 10 mmol [L.sup.-1] KN[O.sub.3] solution ([pH.sub.cond] = 1.5) as supporting electrolyte. A dual-potential waveform, at [E.sub.det1] = 850 mV/200 ms and [E.sub.det2] = 1150 mV/200 ms versus Ag/AgCl (3.0 mol [L.sup.-1] KCl), was employed. The use of [E.sub.det1] or [E.sub.det2] caused the oxidation of BHA or of BHA and BHT, respectively; hence, concentration subtraction could be used to determine both species. The respective analytical curves presented good linearity in the investigated concentration range (0.050-3.0 [micro]mol [L.sup.-1] for BHA and 0.70-70 [micro]mol [L.sup.-1] for BHT), and the detection limits were 0.030 [micro]mol [L.sup.-1] for BHA and 0.40 [micro]mol [L.sup.-1] for BHT. The proposed method, which is simple, quick, and presents good precision and accuracy, was successfully applied in the simultaneous determination of BHA and BHT in commercial mayonnaise samples, with results similar to those obtained by HPLC, at a 95% confidence level. 10.1021/ac101921f

The development of continuous glucose monitoring systems is a major trend in diabetes-related research. Small, easy-to-wear systems which are robust enough to function over many days without maintenance are the goal. We present a new sensing system for continuous glucose monitoring based on a micrereactor incorporating chaotic mixing channels. Two different types of chaotic mixing channels with arrays of either slanted or herringbone grooves were fabricated in poly(dimethylsiloxane) (PDMS) and compared to channels containing no grooves. Mixing in channels with slanted grooves was characterized using a fluorescence method as a function of distance and at different flow rates, and compared to the mixing behavior observed in channels with no grooves. For electrochemical detection, a thin-film Pt electrode was positioned at the end of the fluidic channel as an on-chip detector of the reaction product, [H.sub.2][O.sub.2]. Glucose determination was performed by rapidly mixing glucose and glucose oxidase (GOx) in solution at a flow rate of 0.5 [micro]L/min and 1.5 [micro]L/min, respectively. A 150 U/mL GOx solution was selected as the optimum concentration of enzyme. In order to investigate the dependence of device response on flow rate, experiments with a premixed solution of glucose and GOx were compared to experiments in which glucose and GOx were reacted on-chip. Calibration curves for glucose (0-20 mM, in the clinical range of interest) were obtained in channels with and without grooves, using amperometdc detection and a 150 U/mL GOx solution for in-chip reaction. 10.1021/ac1000509

An amperometric sensor for lactate quantification is presented. The developed biosensor requires only 0.2 U of lactate oxidase, which is immobilized in a mucin/ albumin hydrogel matrix. By protecting the platinum surface with a Nation membrane, typical interference related to negatively charged species such as ascorbic acid has been minimized to practically undetectable levels. Electrochemical properties associated with the Nation membrane are assessed as a function of Nation concentration. In a phosphate buffer solution of pH 7.0, linear dependence of the catalytic current upon lactate bulk concentration was obtained between 2 and ~1000 [micro]M. A detection limit of 0.8 [micro]M can be calculated considering 3 times the standard deviation of the blank signal divided by the sensitivity of the sensor. The lactate biosensor presents remarkable operational stability and sensitivity (0.537 [+ or -] 0.007) mA.[M.sup.-1], where the error is the standard deviation of the slope calculated from the linear regression of the calibration curve of a fresh biosensor. In this regard, the sensor keeps practically the same sensitivity for 5 months, while the linear range decreases until an upper value of 0.8 mM is reached. Assays performed with whole blood samples spiked with 100 [micro]M lactate gave (89 [+ or -] 6)% of recovery. 10.1021/ac1004426

Room temperature ionic liquids (RTILs) have been applied to a microelectrode array and been demonstrated to form effective, membrane-free amperometric gas sensors. Determining the RTIL [[P.sub.6,6,6,14]][FAP] as the most appropriate choice for extended use, the amperomeWic quantification of oxygen has been demonstrated. The response of the sensor was quantified by beth cyclic voltammetry and chronoamperometry. A range of 02 contents (2-13% v/v) and RTIL layer thicknesses (from ca. 6 to 125 [micro]m) have been investigated. The combination of microelectrode array and RTIL, as well as the absence of membrane and volatile solvent, results in an elegant, easy to calibrate gas sensor with potential utility in standard and nonstandard conditions. 10.1021/ac1006359

Amine-transaminases (ATAs, omega-transaminases, [omega]-TA) are PLP-dependent enzymes that catalyze amino group transfer reactions. In contrast to the widespread and wellknown amino acid-transaminases, ATAs are able to convert substrates lacking an [alpha]-carboxylic functional group. They have gained increased attention because of their potential for the asymmetric synthesis of optically active amines, which are frequently used as building blocks for the preparation of numerous pharmaceuticals. Having already introduced a fast kinetic assay based on the conversion of the model substrate [alpha]-methylbenzylamine for the characterization of the amino acceptor specificity, we now report on a kinetic conductivity assay for investigating the amino donor specificity of a given ATA. The course of an ATA-catalyzed reaction can be followed conductometrlcally since the conducting substrates, a positively charged amine and a negatively charged keto acid, are converted to nonconducting products, a noncharged ketone and a zwitterionic amino acid. The decrease of conductivity for the investigated reaction systems were determined to be 33-52 [micro]S m[M.sup.-1]. In contrast to other ATA-assays previously described, with this approach all transamination reactions between any amine and any keto acid can be monitored without the need for an additional enzyme or staining solutions. The assay was used for the characterization of a ATA from Rhodobacter sphaeroides, and the data obtained were in excellent agreement with gas chromatography analysis. 10.1021/ac9028483

Peroxynitrite ([ONOO.sup.-]) is a highly reactive species implicated in the pathology of several cardiovascular and neurodegenerative diseases. It is generated in vivo by the diffusion-limited reaction of nitric oxide ([NO.sup.*]) and superoxide anion ([sup.*][O.sub.2.sup.-]) and is known to be produced during periods of inflammation. Detection of ONOO is made difficult by its short half-life under physiological conditions (~1 s). Here we report a method for the separation and detection of [ONOO.sup.-] from other electroactive species utilizing a microchip electrophoresis device incorporating an amperometric detection scheme. Microchip electrophoresis permits shorter separation times (~25 s for [ONOO.sup.-]) and higher temporal resolution than conventional capillary electrophoresis (several minutes). This faster analysis allows ONOO to be detected before substantial degradation occurs, and the increased temporal resolution permits more accurate tracking of dynamic changes in chemical systems. 10.1021/ac902821v

Lead-based compounds were used during antiquity as both pigments and medicines in the formulation of makeup materials. Chemical analysis of cosmetics samples found in Egyptians tombs and the reconstitution of ancient recipes as reported by Greco-Roman authors have shown that two non-natural lead chlorides (laurionite Pb(OH)CI and phosgenito [Pb.sub.2][Cl.sub.2]C[O.sub.3]) were purposely synthesized and were used as fine powders in makeup and eye lotions. According to ancient Egyptian manuscripts, these were essential remedies for treating eye illness and skin ailments. This conclusion seems because today we focus only on the well-recognized toxicity of lead salts. Here, using ultramicroelectrodes, we obiain new insights into the biochemical interactions between lead (II) ions and cells, which support the ancient medical use of sparingly soluble lead compounds. Submicromolar concentraions of [Pb.sup.2+] ions are shown to be sufficient for eliciting specific oxidative stress responses of kerafinocytes. These consist essentially of an overproduction of nitrogen monoxide (NO[degrees]). Owing to the biological role of NO in stimulating nonspecific immunological defenses, one may argue that these lead compounds were deliberately manufactured and used in ancient Egyptian formulations to prevent and treat eye illnesses by promoting the action of immune cells. 10.1021/ac902348g

The molecular imprinting technique was first introduced into the microchannel of a microfluidic device to form in situ the imprinted polymer for fast enantioseparation of chiral compounds. The molecularly imprinted polymer (MIP) was in situ chemically polymerized on the microchannel wall using acrylamide as the functional monomer and ethylene glycol dimethacrylate as the cross-linker, and characterized by scanning electron microscopy, atomic force microscopy, and infrared spectroscopy. Under the optimized conditions, such as optimal preparation of MIP, composition and pH of mobile phase, and separation voltage, the model enantiomers, tert-butoxycarbonyl-Dtryptophan (Boc-D-Trp) and Boc-D-Trp, could be baseline separated within 75 s. The linear ranges for amperometric detection of the enantiomers using carbon fiber microdisk electrode at + 1.2 V (vs Ag/AgCI) were from 75 to 4000 pM and 400 to 4000 [micro]M with the detection limits of 20 and 140 [micro]M, respectively. The MIP-microchip electrophoresis provided a powerful protocol for separation and detection of Boc-Trp enantiomers within a short analytical time. The molecular imprinting on microchannel wall opens a promising avenue for fast enantioscreening of chiral compounds. 10.1021/ac902201a

The activation of a lipopolysaccharide (LPS) with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in the presence of a surfactant allows an efficient conjugation with dansyl hydrazine or horseradish peroxidase (HRP) in an aqueous medium maintaining its biological activity. In order to promote the reaction a series of amphiphilic compounds were tested, sodium deoxycholate being the most suitable. The method presents several advantages: it is carried out in a mild environment, good conjugation ratios are obtained, it is suitable for any label bearing amino, hydrazine, or hydrazide groups, and the LPS endotoxic and HRP enzymatic activities are preserved. The HRP conjugate is applied in an amperometric competitive assay for the detection of lipopolysaccharides in an electrode array combined with a multipotentiostat able to carry out simultaneous determinations. The system is able to detect samples in concentrations as low as 100 pg [mL.sup.-1] of LPS.

Pt electrodes of different sizes (2 x [10.sup.-5] -2 x [10.sup.-2] [cm.sup.2]) and geometries (disks and cylinders) were coated with the ultrathin non-conducting form of poly(o-phenylene-diamine), PPD, using amperometric electrosynthesis. Analysis of the ascorbic acid (AA) and [H.sub.2][O.sub.2] apparent permeabilities for these Pt/PPD sensors revealed that the PPD deposited near the electrode insulation (Teflon or glass edge) was not as effective as the bulk surface PPD for blocking AA access to the Pt substrate. This discovery impacts on the design of implantable biosensors where electrodeposited polymers, such as PPD, are commonly used as the permselective barrier to block electroactive interference by reducing agents present in the target medium. The undesirable 'edge effect' was particularly marked for small disk electrodes which have a high edge density (ratio of PPD-insulation edge length to electrode area), but was essentially absent for cylinder electrodes with a length of >0.2 mm. Sample biosensors, with a configuration based on these findings (25 [micro]m diameter Pt fiber cylinders) and designed for brain neurotransmitter L-glutamate, behaved well in vitro in terms of Glu sensitivity and AA blocking.

Etched carbon fiber microelectrodes of different radii have been used for amperometric measurements of single exocytotic events occurring at adrenal chromaffin cells. Frequency, kinetic, and quantitative information on exocytosis provided by amperometric spikes were analyzed as a function of the surface area of the microelectrodes. Interestingly, the percentage of spikes with foot (as well as their own characteristics), a category revealing the existence of sufficient long-lasting fusion pores, was found to be constant whatever the microelectrode diameter was, whereas the probability of overlapping spikes decreased with the electrode size. This confirmed that the prespike foot could not feature accidental superimposition of separated events occurring at different places. Moreover, the features of amperometric spikes investigated here (charge, intensity and kinetics) were found constant for all microelectrode diameters. This demonstrated that the electrochemical measurement does not introduce significant bias onto the kinetics and thermodynamics of release during individual exocytotic events. All in all, this work evidences that information on exocytosis amperometrically recorded with the usual 7 [micro]m diameter carbon fiber electrodes is biologically relevant, although the frequent overlap between spikes requires a censorship of the data during the analytical treatment.

The effect of introducing positive charges (lysines) in human cytochrome c (cyt c) on the redox properties and reaction rates of cyt c with superoxide radicals was studied. The mutated forms of this electron-transfer protein are used as sensorial recognition elements for the amperometric detection of the reactive oxygen radical. The proteins were prepared by site-directed mutagenesis focusing on amino acids near the heme edge. The 11 mutants of human cyt c expressed in the course of this research have been characterized by UV--vis spectroscopy, circular dichroism, and NMR spectroscopy to verify overall structure integrity as well as axial coordination of the heme iron. The mutants are investigated voltammetrically using promoter-modified gold electrodes with respect to redox activity and formal redox potential. The rate constants for the reaction with superoxide have been determined spectrophotometrically. Four mutants show a higher reaction rate with the radical as compared to the wild type. These mutants are used for the construction of superoxide sensors based on thiol-modified gold electrodes and covalently fixed proteins. We found that the E66K mutant-based electrode has a clearly higher sensitivity in comparison with the wild-type-based sensor while retaining the high selectivity and showing a good storage stability.

Chronoamperometric experiments with adsorbed electrocatalysts are commonly performed either for analytical purposes or for studying the catalytic mechanism of a redox enzyme. In the context of amperometric sensors, the current may be recorded as a function of time while the analyte concentration is being increased to determine a linearity range. In mechanistic studies of redox enzymes, chronoamperometry proved powerful for untangling the effects of electrode potential and time, which are convoluted in cyclic voltammetric measurements, and for studying the energetics and kinetics of inhibition. In all such experiments, the fact that the catalyst's coverage and/or activity decreases over time distorts the data. This may hide meaningful features, introduce systematic errors, and limit the accuracy of the measurements. We propose a general and surprisingly simple method for correcting for electrocatalyst desorption and inactivation, which greatly increases the precision of chronoamperometric experiments. Rather than subtracting a baseline, this consists in dividing the current, either by a synthetic signal that is proportional to the instant electroactive coverage or by the signal recorded in a control experiment. In the latter, the change in current may result from film loss only or from film loss plus catalyst inactivation. We describe the different strategies for obtaining the control signal by analyzing various data recorded with adsorbed redox enzymes: nitrate reductase, NiFe hydrogenase, and FeFe hydrogenase. In each case we discuss the trustfulness and the benefit of the correction. This method also applies to experiments where electron transfer is mediated, rather than direct, providing the current is proportional to the time-dependent concentration of catalyst.

Even though platelets are known to play a critical role in hemostasis, mediated in part by their uptake, storage, and release of serotonin, there are many unexplored aspects of this process. Herein, single-cell amperometry is employed to characterize the dynamic secretion of serotonin from platelet dense-body granules. On the basis of a three-dimensional random walk simulation that estimates detection efficiency with varied spacing between the carbon-fiber microelectrode and the platelet, it is clear that the detected charge likely represents complete oxidation of the released granule contents and, thus, is a good method to calculate the serotonin concentration in each granule. Using the measured charge and volume estimates based on transmission electron microscopy (TEM) data, the granular concentration of serotonin is approximately 0.5 M. The simulated spike widths are significantly narrower than most of the measured amperometric spikes, clearly indicating that the stored serotonin is highly associated with an aggregate rather than freely diffusible within the dense-body granule. Additionally, by varying extracellular buffer temperature and pH to adjust the driving forces for serotonin delivery from the dense-body granules to the extracellular space, it is clear that, although platelet chemical messenger storage and secretion is similar to that of other secretory cells, there are some important distinctions.

Pt nanoparticle (NP)-modified polycrystalline boron-doped diamond (pBDD) disk electrodes have been fabricated and employed as amperometric sensors for the determination of dissolved oxygen concentration in aqueous solution, pBDD columns were cut using laser micro-machining techniques and sealed in glass, in order to make disk electrodes which were then characterized electrochemically. Electrodeposition of Pt onto the diamond electrodes was optimized so as to give the maximum oxygen reduction peak current with the lowest background signal. Pt NPs, >0-10 nm diameter, were found to deposit randomly across the pBDD electrode, with no preference for grain boundaries. The more conductive grains were found to promote the formation of smaller nanoparticles at higher density. With the use of potential step chronoamperometry, in which the potential was stepped to a diffusion-limited value, a four electron oxygen reduction process was found to occur at the Pt NP-modified pBDD electrode. Furthermore the chronoamperometric response scaled linearly with dissolved oxygen concentration, varied by changing the oxygen/nitrogen ratio of gas flowed into solution. The sensor was used to detect dissolved oxygen concentrations with high precision over the pH range 4-10.

We present a theoretical model for description of real polyreceptor molecular wire sensors (MWS), whose conductance signal may dramatically reduce upon analyte binding to one of the receptors coupled to the molecular wire but may not vanish as completely as assumed in the ideal MWS model. For the present nonideal MWS model, we establish the exact relationship between analyte concentration and the sensory signal intensity. It turns out that, whereas the Stern--Volmer curve of the ideal MWS always has a positive curvature, the Stern--Volmer curve of the imperfect MWS can have a negative curvature, consistent with experimental data. We find that the MWS still performs better than the corresponding ideal monoreceptor sensor, unless the nonideality of the imperfect MWS is egregiously large. We establish the conditions for the imperfect polyreceptor MWS to have a sensitivity and detection limit superior to the traditional monoreceptor sensor.

It has been previously established that a lesion created by a microcapillary in the membrane of a single aerobic cell (from skin or immune origin) was sufficient to induce a local membrane depolarization and the ensuing release of oxidative bursts. Their kinetic and quantitative features reveal the activity of cell constitutive enzymes, namely, NADPH oxidases and NO synthases, prone to produce rapidly reactive oxygen and reactive nitrogen species. Until now, the spatial resolution provided by microelectrodes has been exploited in this context to characterize the chemical composition of oxidative bursts at several cell types with high collection efficiency. In the present work, spatial features of the oxidative bursts from single human fibroblasts were investigated using a step-by-step geometrical mapping approach. The spatial locations of cell active zones and of the extent of the activated area, when a cell membrane was stressed by a microcapillary's tip of 1-[micro]m radius, have been addressed. On cells of large dimensions such as fibroblasts, ROS and RNS emission originated from a disk surface of the membrane limited to ~15-[micro]m radius around the ~l-[micro]m hole created by the microcapillary. This experimental result was rationalized through a simple physicochemical model designed to portray the extent of the membrane activated area due to ion concentration variations resulting from the pinhole channel created across the cell membrane. This is consistent with the fact that the activation of constitutive enzymatic complexes (NOX and NOS) is hypothesized to be a consequence of local variations of ion concentrations such as [K.sup.+], [Na.sup.+] or possibly [Ca.sup.2+]. Our results showed that the calculated area near the cell membrane where the ion concentration gradients are significant was equivalent to the area of species release measured experimentally.

Standing square-wave chronoamperometry (SSWCA) was applied to the analysis of the microfluid flow generated by the movement of the appendages of the Crustacea Daphnia. This novel approach provided for the first time real-time assessment and analysis of the breathing rate/ fluid flow of individual organisms. An electrochemical tracer was delivered into the fluid inflow of the organism and a carbon fiber microelectrode placed in the fluid outflow's path. The variation of the net concentration/flux of the electroactive tracer, dopamine, at the electrode surface was measured with SSWCA. The observed chronoamperometric peaks (with fine structure) of the outflow are seen as a direct representation of appendage movement and, too, the workings and responses of the organism to its environment, e.g., external stimuli such as food or chemicals. It was concluded that SSWCA follows primarily the variation of the convective component of the Nernst--Plank equation for flux and, to lesser extent, diffusion and migration. In this work, SSWCA can clearly be used to monitor changes in the Daphnia-generated fluid outflow on a different time scale than was previously possible. This new application of SSWCA is faster and likely more accurate than using high-speed video.

An amperometric fluorinated xerogei-derived nitric oxide (NO) microelectrode is described. A range of fluorine-modified xerogel polymers were synthesized via the cohydrolysis and condensation of alkylalkoxy--and fluoro-alkoxysilanes. Such polymers were evaluated as NO sensor membranes to identify the optimum composition for maximizing NO permeability while providing sufficient selectivity for NO in the presence of common interfering species. By taking advantage of both the versatility of sol--gel chemistry and the 'poly(tetrafluoroethylene)-like' high NO permselective properties of the xerogels, the performance of the fluorinated xerogel-derived sensors was excellent, surpassing all miniaturized NO sensors reported to date. In contrast to previous electrochemical NO sensor designs, xerogel-based NO microsensors were fabricated using a simple, reliable dip-coating procedure. An optimal permselective membrane was achieved by synthesizing xerogels of methyltrimethoxysilane (MTMOS) and 20% (heptadecafluoro-1,1,2,2-tetrahydrodecyl)tri-methoxysilane (17FrMS, balance MTMOS) under acid-catalyzed conditions. The resulting NO microelectrode had a conical tip of ~20/[micro]m in diameter and ~55 [micro]m in length and exhibited sensitivities of 7.91 pA x [nM.sup.-1] from 0.2 to 3.0 nM ([R.sup.2] = 0.9947) and 7.60 nA x [micro][M.sup.-1] from 0.5 to 4.0 [micro]M ([R.sup.2] = 0.9999), detection limit of 83 pM (S/N= 3), response time ([t.sub.95%]) of

The physiological application of amperometric adenosine triphosphate (ATP) microbiosensors for characterizing the stimulus-response at rat carotid bodies superfused with high potassium concentrations, during normoxic hypercapnia, and during hypoxia is demonstrated using the peripheral arterial chemoreceptors in the carotid body of rats as a model system. Amperometric microbiosensors based on glucose oxidase (GOD) and hexokinase (HEX) immobilized within a polymer matrix at the surface of Pt disk microelectrodes (diameter: 25 [micro]m) are positioned at a distance of approximately 100 [micro]m above the carotid body surface for detecting extracellular ATP. A linear calibration function of ATP microbiosensors in the physiologically relevant concentration range of 0-40 [micro]M ATP enables quantitative detection of ATP released at the carotid body surface in response to physiological stimuli. It is shown that these stimuli induce extracellular ATP release from the carotid body at levels of 4-10 [micro]M. Other electroactive neurotransmitters such as, e.g., catecholamines are coreleased by the carotid body at hypercapnic, hypoxic and high-potassium stimulus, are simultaneously detected utilizing a dual-electrode assembly with an ATP microbiosensor and a second bare channel providing a colocalized reference measurement for ATP quantification.

In this work, carbon-fiber microelectrode amperometry is used to characterize serotonin exocytosis from murine peritoneal mast cells cocultured with fibroblasts in the presence of Au nanoparticles. In the case of mast cell exposure to 1 nM 28 nm diameter spherical Au nanoparticles, there is a decrease of greater than 30% in the number of successful granule transport and fusion events, greater than 30% increase in the rate of intagranular matrix expansion, and greater than 20% increase in the number of secreted serotonin molecules per granule. These results suggest that nanoparticles interrupt the dense-core biopolymer intragranular matrix and present the potential for systematic studies showing how exocytotic function is influenced by nanoparticle size, shape, and composition.

We report a new reusable electrochemical array for parallel biamperometric measurements that has been designed for use with standard microplates. The 48-channel array uses half of the available 96 wells and has 48 pairs of Pt wire electrodes. Applications to the quantitation of a variety of oxidizable species, including acetaminophen, ascorbic acid, hydroquinone, trolox, and uric acid, are demonstrated in assays that use potassium ferricyanide as an oxidant to produce a mixture of ferri- and ferrocyanide. Hydrogen peroxide quantitation is also demonstrated, based on an assay in which ferrocyanide is oxidized, again to produce a mixture of ferri- and ferrocyanide. Detection limits (signal-to-noise ratio (S/N) = 3) in these assays range from 1 (acetaminophen, [R.sup.2] = 0.994) to 8 [micro]M (ascorbic acid, [R.sup.2] = 0.967), and linearity was observed to analyte concentrations of at least 100 [micro]M. We also demonstrate the application of the biamperometric array to enzymatic assays, using the glucose oxidase reaction as an example; following a 20 min enzyme reaction time, a detection limit of 0.1 mM glucose was obtained. These results indicate that applications to other oxidase-based assays are feasible in this high-throughput format. The new electrochemical array employs standard, inexpensive microplates, and the biamperometric measurements are simple, precise, and rapid, requiring only 2 min for 48 parallel measurements.

An alternative method of a protein immunosensor has been developed at boron-doped diamond (BDD) electrode material. In order to construct the base of the immunosensor, o-aminobenzoic acid (o-ABA) was electropolymerized at an electrode by cyclic voltammetry. The polyo-ABA-modified BDD was characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The XPS result found that carboxyl groups were formed at the electrode surface. The carboxyl groups were then used to covalently attach protein probes. The amperometric sensing of mouse IgG (MIgG)was selected as the model at the poly-o-ABA-modified BDD to compare to the poly-o-ABA-modified glassy carbon (GC) at the same condition. An antimouse IgG from goat (GaMIgG) was covalently immobilized at a poly-o-ABA-modified BDD electrode which used a sandwich-type alkaline phosphatase (ALP) catalyzing amperometric immunoassay with 2-phospho-L-ascorbic acid (AAP) as substrate. The ALP enzyme conjugated at the immunosensor can generate AAP to the electroactive species of ascorbic acid (AA), which can be determined by amperometric detection. The signal was found to be proportional with the quantity of MIgG. The limits of detection (LODs) of 0.30 (3 SD) and 3.50 ng [mL.sup.-1] (3 SD) for MIgG at BDD and GC electrodes were obtained. It also was found that the dynamic range of 3 orders of magnitude (1-1000 ng [mL.sup.-1] ) was obtained at BDD, whereas at GC, the dynamic range was more narrow (10-500 ng m[L.sup.-1]). The method was applied to a real mouse serum sample that contains MIgG.

Five secretory carrier membrane proteins (SCAMP-1, -2, -3, -4, and -5) have been characterized in mammalian cells. Previously, SCAMP-1 and -2 have been implicated to function in exocytosis. RNA inhibitor-mediated deficiency of one or both of these SCAMPs interferes with dense core vesicle (DCV) exocytosis in neuroendocrine PC12 cells as detected by amperometry. Knockdowns of these SCAMPs each decreased the number and frequency of depolarization-induced exocytotic events. SCAMP-2 but not SCAMP-1 depletion also delayed the onset of exocytosis. Both knockdowns, however, altered fusion pore dynamics, increasing rapid pore closure and decreasing pore dilation. In contrast, knockdowns of SCAMP-3 and -5 only interfered with the frequency of fusion pore opening and did not affect the dynamics of newly opened pores. None of the knockdowns noticeably affected upstream events, including the distribution of DCVs near the plasma membrane and calcium signaling kinetics, although norepinephrine uptake/storage was moderately decreased by deficiency of SCAMP-1 and -5. Thus, SCAMP-1 and -2 are most closely linked to the final events of exocytosis. Other SCAMPs collaborate in regulating fusion sites, but the roles of individual isoforms appear at least partially distinct. neuroendocrine secretion; membrane fusion; amperometry

A silicone/quartz capillary microchip (SQCM) coupled with an ultranarrow sampling fracture was for the first time constructed without any micromachining. The SQCM could be used for direct determination of carbohydrates at a detection potential of +0.8 V (vs Ag/AgCl) with a copper microdisk electrode. The ultranarrow sampling fracture could be conveniently formed on a quartz capillary, which was fixed by a frame of poly(dimethylsiloxane) (PDMS). The designed fracture sampling suppressed the leakage of sample, thus simplifying the power supply. Furthermore, it thinned the sample plug for enhancing the resolution. The quartz capillary reduced the adsorption of analytes on the separation channel wall compared with a general PDMS microchip, thus enhanced the separation efficiency up to 239 000 plates/m for carbohydrates. This proposed system could satisfactorily separate eight carbohydrates within 180 s with good reproducibility and sensitively detect them in the linear ranges from 1 [micro]M to 0.5 mM for trehalose and sucrose, 2.5 [micro]M to 0.5 mM for lactose, galactose, glucose, and mannose, and 2.5 [micro]M to 1.5 mM for fructose and xylose with the detection limit down to 90 amol. The designed microchip was successfully applied to detect carbohydrates in a practical acacia honey sample.

A method was developed for selective analysis of the secondary amino acids proline and 4-hydroxyproline from gelatin hydrolysates using anion-exchange high-performance liquid chromatography followed by integrated pulsed amperometric detection (HPLC-IPAD). An extraction scheme was implemented prior to HPLC-IPAD analysis to isolate the secondary amino acids by the removal of primary amino acids through derivatization with o-phthalaldehyde followed by solid-phase extraction with C18 packed columns. The use of the IPAD technique eliminated the need for a second derivatization step to detect secondary amino acids. The removal of interfering primary amino acids prior to chromatographic analysis allowed the use of isocratic mobile-phase conditions to achieve effective and efficient separation of the amino acids. This led to a more precise and accurate quantitation of their content in gelatin hydrolysates. Detection limits approach 10 parts per billion (~2 pmol/injection) with a chromatographic analysis time under 8 min. The ratios of secondary amino acids, in addition to their abundances, were used to distinguish gelatin manufactured from bovine, porcine, and fish raw material sources.

Byline: I. V. Kozhevnikov (1) Abstract: A new calculation method was proposed for determining substances by amperometric titration with two indicator electrodes. The model is based on the modified Dalahay equation for reversible redox systems and electrodes of different surface areas. Author Affiliation: (1) Chuvash State University, Moskovskii pr. 15, Cheboksary, 428015, Russia Article History: Registration Date: 06/10/2004

Electrocatalytic oxidation of hydrazine on a dinuclear ruthenium phthalocyanine ([(RuPc).sub.2]) modified electrode was studied using cyclic voltammetry (CV) and rotating disc electrode (RDE) techniques. At pH = 13, a four-electron oxidation of hydruzine to [N.sub.2] was observed. A suitable mechanism was proposed by analyzing the rate equation and the Tafel slope. The flow injection analysis was performed to characterize the [(RuPc).sub.2]-modified electrode as an amperometric sensor for the detection of hydrazine. The electrode displays an excellent accuracy and precision in phosphate solution at pH 12 and 13. The linearity range was from 30 nM to 1 mM with a correlation coefficient of 0.9998. Key words: ruthenium phthalocyanine, electrocatalysis, surface-modified electrode, hydrazine, amperometric sensor.

We have fabricated electrochemical electrodes in picoliter-sized wells for measuring catecholamine release from individual cells with millisecond resolution. Each well-electrode roughly conforms to the shape of the cell in order to capture a large fraction of released catecholamine with high time resolution. Using this device, we can resolve spikes in amperometric current corresponding to quantal catecholamine release via exocytosis. In addition, we have combined amperometric recording on the chip with patch-clamp recordings of membrane capacitance as an assay of exocytosis. A quantitative comparison of the two methods suggests that a large fraction of catecholamine release is oxidized on the surface of the well-electrode. This technology has applications in cell-based biosensor development, high-throughput screening of drugs, and basic science investigations.

In this contribution, a capillary electrophoresis micro-device with an integrated on-chip contactless four-electrode conductivity detector is presented. A 6-cm-long, 70-[micro]m-wide, and 20-[micro]m-deep channel was etched in a glass substrate that was bonded to a second glass substrate in order to form a sealed channel. Four contactless electrodes (metal electrodes covered by 30-nm silicon carbide) were deposited and patterned on the second glass substrate for on-chip conductivity detection. Contactless conductivity detection was performed in either a two- or a four-electrode configuration. Experimental results confirmed the improved characteristics of the four-electrode configuration over the classical two-electrode detection setup. The four-electrode configuration allows for sensitive detection for varying carrier-electrolyte background conductivity without the need for adjustment of the measurement frequency. Reproducible electrophoretic separations of three inorganic cations ([K.sup.+], [Na.sup.+], [Li.sup.+]) and six organic acids are presented. Detection as low as 5 [micro]M for potassium was demonstrated.

Contactless conductivity detection was carried out on a planar electrophoresis device by capacitive coupling using an ac excitation voltage of 500 Vp-p and a frequency of 100 kHz. It was possible to carry out detection in this way through a cover plate of 1 mm thickness. Better sensitivity is obtained, however, by placing the electrodes into troughs that allow tighter coupling to the separation channel. The 3 x S/N detection limits are 0.49, 0.41, and 0.35 [micro]M for the small inorganic ions [K.sup.+], [Na.sup.+], and [Mg.sup.2+]. The detection of heavy metals is demonstrated with the example of [Mn.sup.2+], [Zn.sup.2+], and [Cr.sup.3+] with detection limits of 2.1, 2.8, and 6.8 [micro]M, respectively. The universal nature of the method is further illustrated by the detection of citric and lactic acids, which are of interest in food and beverage analysis, and detection of three antiinflammatory nonsteroid drugs, 4-acetamidophenol, ibuprofen, and salicylic acid, as examples of species of pharmaceutical interest.

We describe here an electrochemical cell ideal for routine analysis of CE-EC experiments (capillary electrophoresis coupled with electrochemical detection). The cell was modified from a fiber-optic connector, named MT, which allowed frequent change and fast alignment between a pair of 4-strand fiber ribbons. The relative standard deviations of the current response and migration time for 100 [micro]M dopamine were, respectively, 3.7 and 0.5% in five repetitive routines of disconnecting, polishing, and assembling the CE cell. The time required for alignment of the separation capillary and the working electrode was

Dynamic characteristics of the amperometric sensing system for tracking of turbulent chemical plumes have been studied. The correlation analysis requires that such sensors respond rapidly and have long-term stability and minimal or low-flow sensitivity. Moreover, for practical purposes, such sensors must respond to an electrochemical marker that would be found in or at least be compatible with the diet of marine animals. Ascorbic acid is such a compound and can be electrochemically oxidized. Its long-term dynamic behavior on several types of electrodes has been studied. It has been found that a Pt electrode coated with polyaniline satisfies all the above requirements. However, this system has a peculiar dynamic behavior that affects the results of the correlation analysis, namely, at the higher frequencies. The behavior of such system in the virtual plume setup has been characterized and its usability for detection of fluctuating concentration of ascorbic acid in saline solution has been confirmed.

To extend the application of a multichannel electrochemical detector (MED) for immunoassay, a MED system consisting of 8 sets of Pt electrodes in an arrangement fitted with the dimensions of a row of microtiter wells in a microtiter plate and a microcomputer-assisted 16-channel potentiostat was constructed. With this developed MED system, electroactive enzymatic products produced in eight microtiter wells can be analyzed simultaneously with a developed amperometric procedure. To demonstrate the applicability of the MED system for immunoassay, an immunosystem containing rabbit-IgG and alkaline phosphatase-conjugated goat anti rabbit-IgG was studied. From the dynamic range of 10-1000 ng/mL (0.064-6.4 pM) and the detection limit of 1.0 ng/mL (6.4 pM) obtained, the developed MED shows a tremendous improvement in sensitivity, detection limit, and efficiency compared with that obtained from conventional electrochemical immunoassay.

The electrode response time and the measured concentrations during dynamic catecholamine changes were compared using constant potential amperometry and fast-scan cyclic voltammetry. The amperometric response to a rectangular pulse of catecholamine is more rectangular than the cyclic voltammetric response; however, the response times are very similar when, during cyclic voltammetry, the temporal lag due to adsorption and desorption of catecholamine to the electrode is removed by deconvolution. Deconvolution of cyclic voltammetry data was applied to stimulated dopamine release in vivo, allowing for modeling of release and uptake kinetics and to measure catecholamine release from single cells, resulting in better resolution of peaks from single vesicles. In vitro postcalibrations were performed to calculate concentrations of catecholamine measured with cyclic voltammetry and amperometry. The addition of 600 [micro] M ascorbic acid to the postcalibration buffer, allowing a catalytic reaction to regenerate dopamine, resulted in similar calculated concentrations for stimulated release of dopamine using amperometry and cyclic voltammetry. Using deconvoluted cyclic voltammetry to remove the response time lag and adding ascorbic acid to the calibration buffer, the shape and concentration of dynamic catecholamine changes are very similar when measured with constant potential amperometry and cyclic voltammetry.

A new method for the determination of the diffusion coefficients of both the substrate, [D.sub.S], and the product, [D.sub.P], of an electrode process has been developed. The method proposed is based on the analysis of the transient currents and can be applied to some reactions of the type [S.sup.z.sub.s] = [P.sup.z.sub.p] + ne and, in contrast to the concept based on the steady-state current, to any ratio of the concentrations of supporting electrolyte and substrate. The diffusion coefficients can be evaluated sequentially from the two parts of the double-potential step chronoamperogram, since the magnitude of the normalized chronoamperometric current of the first step depends on the [D.sub.S] value, while that of the second step is controlled by both [D.sub.S] and [D.sub.P] values. The corresponding, easy-to-use equations and procedures are given in the paper. The equations were derived on the basis of the numerical simulation data. The proposed methods of determination of diffusion coefficients for the substrates and products have been examined experimentally with the charged and uncharged ferrocene derivatives under diffusional and mixed diffusion-migration conditions. 0nly the chronoamperometric Ds values obtained for the substrates could be compared to those determined from the steady-state diffusional current. It was found that for the systems investigated the agreement between these two methods was good. In this limited comparison, the standard deviations for the transient techniques were slightly larger than those obtained for the excess supporting electrolyte steady-state voltammetry.

A one-step enzyme-amplified amperometric sandwich hybridization test for RNA and DNA is described. The test utilizes a carbon electrode, modified with a film of co-electrodeposited avidin and redox polymer; the redox polymer electrically 'wiring' horseradish peroxidase (HRP) reaction centers upon contact. The film is made specific for the particular RNA or DNA sequence tested by conjugating its avidin with a biotinylated oligonucleotide, complementary to the assayed sequence. This oligonucleotide-modified redox polymer film, prepared prior to the test, forms the base of the sandwich. The center layer of the sandwich, added in the test, is the analyte RNA or DNA; its top is a second complemetary oligonucleotide, which is HRP-labeled, and is cohybridized in the test. The test consists of mixing the analyte DNA or RNA solution, the HRP-labeled oligonucleotide solution, and a hydrogen peroxide solution, immersing the base-layer carrying electrode applying a potential of 0 V versus Ag/AgCl, and measuring the [H.sub.2O.sub.2] electroreduction current. Completion of the sandwich brings the HRP label into electrical contact with the redox polymer, convening the nonelectrocatalytic base layer into an electrocatalyst for the electroreduction of [H.sub.2O.sub.2] to water. Flow of [H.sub.2O.sub.2] electroreduction current when the electrode is poised near Ag/AgCl potential indicates the presence of the analyte RNA or DNA. The current density for the maximally sandwich-covered electrode was 250 [micro] A [cm.sup.-2], exceeding more than a 100-fold the current density flowing upon nonspecific binding of the HRP-labeled oligonucleotide. High concentrations of irrelevant DNA and diluted serum did not interfere with the assay. When the electrodes were rotated in order to make the solution-phase mass transport rapid, the test was completed in ~30 min. The test was applied in probing for the presence of a 60-base E. coli mRNA sequence.

The first enzyme-based biosensors capable of determining glycolic acid in various complex matrixes, such as cosmetics, instant coffee, and urine, are presented in this paper. Two separate designs, both based on a three-membrane configuration consisting of an inner cellulose acetate membrane (CA) and an outer polycarbonate membrane (PC), which sandwich a membrane bearing the biomolecule(s), are proposed. Glycolate oxidase was immobilized onto a modified polyethersulfonate membrane by means of chemical bonding, and glycolate oxidase/catalase enzyme mixture was immobilized into a mixed-ester cellulose acetate membrane through physical adsorption. The membrane assemblies were mounted on an amperometric flow cell (hydrogen peroxide detection at a platinum anode poised at +0.65 V vs Ag/AgCl/3 KCl) or on an oxygen electrode, respectively. Both configurations were optimized with respect to various working parameters. The proposed biosensors are interference-free to common electroactive species and were successfully applied for the determination of glycolic acid in various samples, showing an excellent agreement with a reference photometric method. The validity of the proposed method in samples, in which the reference method was not applicable, was tested with recovery studies. Values of 102 [+ or -] % were obtained. Inherent interference of oxalic acid was manipulated by using a primary amine-containing buffer and the enzyme catalase. Both systems were designed in order to be compatible with the current technology of the most widely used commercial analyzers.

The preparation and characterization of an amperometric 2,4,6-trinitrotoluene (TNT) biosensor based on the surface immobilization of a maltose binding protein (MBP) nitroreductase (NR) fusion (MBP-NR) onto an electrode modified with an electropolymerized film of N-(3-pyrrol-1-ylpropyl)-4,4'-bipyridine (PPB) are described. The MBP domain of MBP-NR exhibits a high and specific affinity toward electropolymerized films of PPB with the immobilized enzyme retaining virtually all of its enzymatic activity. Under similar conditions, the wild-type NR enzyme (i.e., without the MBP domain) loses most of its enzymatic activity. The kinetics of the catalytic reaction between the biosensor and TNT and 2,4-dinitrotoluene (DNT) were characterized using rotated disk electrode and cyclic voltammetry techniques, and values of 1.4 x [10.sup.4] and 7.1 x [10.sup.4] [M.sup.-1] [s.sup.-1] were obtained for TNT and DNT, respectively. The apparent Michaelis-Menten constants ([K.sub.M]) for MBP-NR in solution and on the surface, using TNT as substrate, were determined to be 27 and 95/[micro] M, respectively. The corresponding value for 'wild-type' NR in solution containing TNT was 78/[micro] M, which is very close to the value obtained for MBP-NR on the surface. The limits of detection for both TNT and DNT were estimated to be 2/[micro] M, and the sensitivities were determined to be 205 and 222 nA/[micro] M, respectively.

Demanding analytical applications such as on-line process analysis and clinical analysis require robust, reliable, and maintenance-free ion sensors of high potential stability. In this work the stability of the electrode potential of all-solid-state ion-selective electrodes using conducting polymers as ion-to-electron transducers is critically evaluated by using chronopotentiometry and electrochemical impedance spectroscopy. This study is focused on the relationship between the potential stability of the electrode and the capacitance of the solid contact where ion-to-electron transduction takes place. The influence of this capacitance on the potential stability of all-solid-state ion-selective electrodes is studied experimentally by using conducting polymer layers of different thickness as solid contacts in potassium ion-selective electrodes based on a solvent polymeric membrane. Because of its excellent environmental stability, the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) is used as a model compound for the solid contact material. Chronopotentiometry is found to be a convenient and fast experimental method to critically evaluate the potential stability of different types of ion-selective electrodes.

A robust, rapid, and sensitive high-performance anion-exchange chromatographic method for the separation and quantitative determination of lactulose in heated milks, along with other common milk carbohydrates, has been developed. Complete separation of galactose, glucose, N-acetylgalactosamine, lactose, lactulose, and epilactose was isocratically accomplished in about 22 min by an anion-exchange column eluted with 10 mM NaOH spiked with 2 mM Ba(OAc)(sub 2). The within-day repeatability was lower than 2.1% for 10 repetitive injections. Under optimized conditions, there was no need either of post-column addition of strong bases to the eluent for enhancing detection sensitivity or, even more important, for column regeneration between chromatographic runs. Upon 100-fold sample dilution, the amperometric response of lactulose in milk samples was found to be linear up to 100 (mu)M (r = 0.99935) with a limit of detection equal to 1.2 (mu)M (S/N = 3). The lactulose content in ultrahigh-temperature (UHT) and sterilized milks was evaluated by a calibration graph using 2-deoxyglucose as the internal standard, making the proposed method very useful in discriminating among heat-treated milks. Whereas the mean value of lactulose in skimmed, partially skimmed, and whole UHT milks ranged from 10 to 90 mg/100 mL, lactulose content in bottle-sterilized whole milk (two samples) was higher than 140 mg/100 mL. The presence of epilactose, which is another isomer of lactose, was also ascertained in sterilized milk.