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Potassium ion channels are expressed on the cell membranes, implicated in wide variety of cell functions and intimately linked to cancer cell behaviors. This work reports the first bioplatform described to date allowing simple and rapid detection of ion channel activity and the effect of their inhibitors in cancer cells. The methodology involves interrogation of the channel of interest from cells specifically captured on magnetic immunoconjugates using specific detection antibodies that are labeled with horseradish peroxidase enzyme. The channel activity is reflected by an amperometric signal transduction of the resulting magnetic bioconjugates onto screen-printed carbon electrodes. The bioplatform feasibility was proven for the detection of the Kv channels in U87 human glioblastoma cells and their blocking by scorpion venom KAaH1 and KAaH2 peptides. The obtained results confirm the high sensitivity (detection of 5 U87 cells⋅mL

This work reports the first amperometric biosensor for the simultaneous determination of the single or total content of the most relevant human immunoglobulin isotypes (hIgs) of anti-dsDNA antibodies, dsDNA-hIgG, dsDNA-hIgM, dsDNA-hIgA and dsDNA-three hIgs, which are considered relevant biomarkers in prevalent autoimmune diseases such as systemic lupus erythematosus (SLE) as well as of interest in neurodegenerative diseases such as Alzheimer's disease (AD). The bioplatform involves the use of neutravidin-functionalized magnetic microparticles (NA-MBs) modified with a laboratory-prepared biotinylated human double-stranded DNA (b-dsDNA) for the efficient capture of specific autoantibodies that are enzymatically labeled with horseradish peroxidase (HRP) enzyme using specific secondary antibodies for each isotype or a mixture of secondary antibodies for the total content of the three isotypes. Transduction was performed by amperometry (-0.20 V vs. the Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system after trapping the resulting magnetic bioconjugates on each of the four working electrodes of a disposable quadruple transduction platform (SP4CEs). The bioplatform demonstrated attractive operational characteristics for clinical application and was employed to determine the individual or total hIgs classes in serum from healthy individuals and from patients diagnosed with SLE and AD. The target concentrations in AD patients are provided for the first time in this work. In addition, the results for SLE patients and control individuals agree with those obtained by applying ELISA tests as well as with the clinical ranges reported by other authors, using individual detection methodologies restricted to centralized settings or clinical laboratories. The financial support of PID2019-103899RB-I00 and PID2021-122457OB-I00 (Spanish Ministerio de Ciencia e Innovación) Research Projects, PI17CIII/00045 and PI20CIII/00019 Grants from the AESISCIII Program co-founded by FEDER funds and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) are gratefully acknowledged. B.A. acknowledges predoctoral contracts from the Spanish Ministerio de Ciencia, Innovación y Universidades (PRE2019-087596). M.G-A. acknowledges the postdoctoral contract Margarita Salas for the requalification of the Spanish University System. A.M-C. was supported by a FPU predoctoral contract supported by the Spanish Ministerio de Educación, Cultura y Deporte. Sí

A disposable enzyme−free biosensing platform for the sensitive and selective voltammetric determination of miRNAs is reported. The bioplatform implies a sandwich−type hybridization configuration involving the use of two synthetic DNA probes that hybridize contiguously with the target miRNA−21. A thiolated capture probe was immobilized through thiol chemistry on disposable carbon electrodes modified with a hybrid nanomaterial composed of reduced graphene oxide (rGO) and gold nanoparticles (AuNPs). A biotinylated detection probe was conjugated with ferrocene-capped AuNPs modified with streptavidin (Fc−AuNPs−Strep) which were used as labeling nanocarriers. The extent of the hybridization event was followed by differential pulse voltammetric measurement of the Fc oxidation peak. Under the optimized conditions, the developed biosensor provides attractive characteristics for the determination of the synthetic target miRNA, with a linear range between 10 fM and 2 pM and a limit of detection (LOD) of 5 fM, fully discrimination towards a highly homologous miRNA (with just one mismatched base) and a storage stability of at least two months. The biosensor was able to determine accurately the target miRNA directly in scarcely diluted serum from breast cancer (BC) patients with no need for a previous total RNA (RNAt) extraction and in a very small amount of RNAt extracted from breast adenocarcinoma cells without the need for amplification or reverse transcription to complementary DNA.

A non-destructive, rapid and simple to use sensing method for direct determination of glucose in non-processed fruits is described. The strategy involved on-line microdialysis sampling coupled with a continuous flow system with amperometric detection at an enzymatic biosensor. Apart from direct determination of glucose in fruit juices and blended fruits, this work describes for the first time the successful application of an enzymatic biosensor-based electrochemical approach to the non-invasive determination of glucose in raw fruits. The methodology correlates, through previous calibration set-up, the amperometric signal generated from glucose in non-processed fruits with its content in % (w/w). The comparison of the obtained results using the proposed approach in different fruits with those provided by other method involving the same commercial biosensor as amperometric detector in stirred solutions pointed out that there were no significant differences. Moreover, in comparison with other available methodologies, this microdialysis-coupled continuous flow system amperometric biosensor-based procedure features straightforward sample preparation, low cost, reduced assay time (sampling rate of 7 h(-1)) and ease of automation.

This paper describes the reliable determination of progesterone (P4) in undiluted saliva making use of a disposable amperometric immunosensors implemented on low-cost and portable device/potentiostat constructed with commercial-off-the-shelf (COTS) components. The immunosensor allows the fast (45 min), selective and sensitive determination (5 pg mL−1 LOD) of P4 using amperometry in stirred solutions. The immunosensor was coupled to the COTS-based potentiostat and amperometry was made into drops of quiescent solutions. No significant differences were apparent between the analytical performance achieved with the immunosensor for P4 using both a conventional and the COST-based potentiostats. The practical applicability of the immunosensor coupled with the COTS-based potentiostat was demonstrated by determining the endogenous P4 content in different undiluted saliva samples with highly variable endogenous contents of the target hormone. The obtained results were in good agreement with those provided by the conventional ELISA methodology and with the contents reported in the literature for samples with similar characteristics. This validated the combined device for the reliable and minimally invasive determination of the target hormone involving a very simple protocol and taking only 45 min.

A novel amperometric magnetoimmunoassay, based on the use of core-shell magnetic nanoparticles and screen-printed carbon electrodes, was developed for the selective determination of Legionella pneumophila SG1. A specific capture antibody (Ab) was linked to the poly(dopamine)-modified magnetic nanoparticles (MNPs@pDA-Ab) and incubated with bacteria. The captured bacteria were sandwiched using the antibody labeled with horseradish peroxidase (Ab-HRP), and the resulting MNPs@pDA-Ab-Legionella neumophila-Ab-HRP were captured by a magnetic field on the electrode surface. The amperometric response measured at -0.15 V vs. Ag pseudo-reference electrode of the SPCE after the addition of H2O2 in the presence of hydroquinone (HQ) was used as transduction signal. The achieved limit of detection, without pre-concentration or pre-enrichment steps, was 10(4) Colony Forming Units (CFUs) mL(-1). The method showed a good selectivity and the MNPs@pDA-Ab exhibited a good stability during 30 days. The possibility of detecting L. pneumophila at 10 CFU mL(-1) level in less than 3 h, after performing a membrane-based preconcentration step, was also demonstrated.

Forensic analysis is an important branch of modern Analytical Chemistry with many legal and socially relevant implications. Biosensors can play an important role as efficient tools in this field considering their well known advantages of sensitivity, selectivity, easy functioning, affordability and capability of miniaturization and automation. This article reviews the latest advances in the use of biosensors for forensic analysis. The different methodologies for the transduction of the produced biological events are considered and the applications to forensic toxicological analysis, classified by the nature of the target analytes, as well as those related with chemical and biological weapons critically commented. The article provides several Tables where the more relevant analytical characteristics of the selected reported methods are gathered.

Disposable amperometric magnetosensors, involving a mixture of modified-magnetic beads (MBs), for the multiplex screening of cephalosporins (CPHs), sulfonamides (SAs) and tetracyclines (TCs) antibiotic residues in milk are reported for the first time in this work. The multiplexed detection relies on the use of a mixture of target specific modified magnetic beads (MBs) and application of direct competitive assays using horseradish peroxidase (HRP)-labeled tracers. The amperometric responses measured at −0.20 V vs . the Ag pseudo-reference electrode of screen-printed carbon electrodes (SPCE) upon the addition of H 2 O 2 in the presence of hydroquinone (HQ) as redox mediator, were used to monitor the extent of the different affinity reactions. The developed methodology, involving a simple and short pretreatment, allowed discrimination between no contaminated UHT and raw milk samples and samples containing antibiotic residues at the maximum residue limits (MRLs). The usefulness of the multiplexed magnetosensor was demonstrated by analyzing spiked milk samples in only 5 min. The results demonstrated that a clear discrimination of milk samples contaminated with antibiotics at their MRL level or their mixtures, allowing the identification of milk not complying with current legislation. These features make the developed methodology a promising alternative in the development of user-friendly devices for on-site analysis to ensure quality control for dairy products.

A non-invasive, passive and simple to use skin surface based sensing device for determining the blood's ethanol content (BAC) by monitoring transdermal alcohol concentration (TAC) is designed and developed. The proposed prototype is based on bienzyme amperometric composite biosensors that are sensitive to the variation of ethanol concentration. The prototype correlates, through previous calibration set-up, the amperometric signal generated from ethanol in sweat with its content in blood in a short period of time. The characteristics of this sensor device permit determination of the ethanol concentration in isolated and in continuous form, giving information of the BAC of a subject either in a given moment or its evolution during long periods of time (8h). Moreover, as the measurements are performed in a biological fluid, the evaluated individual is not able to alter the result of the analysis. The maximum limit of ethanol in blood allowed by legislation is included within the linear range of the device (0.0005-0.6 g L(-1)). Moreover, the device shows higher sensitivity than the breathalyzers marketed at the moment, allowing the monitoring of the ethanol content in blood to be obtained just 5 min after ingestion of the alcoholic drink. The comparison of the obtained results using the proposed device in the analysis of 40 volunteers with those provided by the gas chromatographic reference method for determination of BAC pointed out that there were no significant differences between both methods.

The preparation and performance of a disposable amperometric magneto-immunosensor, involving the use of a selective capture antibody immobilized on the surface of protein G-functionalized magnetic beads (ProtG-MBs) and screen-printed carbon electrodes (SPCEs), for the specific detection and quantification of tetracyclines (TCs) residues in milk is reported. A direct competitive immunoassay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling was performed. The amperometric response measured at -0.2 V vs. the silver pseudo-reference electrode of the SPCE upon the addition of H(2)O(2) in the presence of hydroquinone (HQ) as redox mediator was used as transduction signal. The developed methodology showed very low limits of detection (in the low ppb level) for 4 tetracycline antibiotics tested in untreated milk samples, and a good selectivity against other antibiotic residues frequently detected in milk and dairy products. The usefulness of the magneto-immunosensor was demonstrated by analyzing UHT whole milk samples spiked with 44 ng mL(-1) tetracycline (TC) as well as a reference milk containing a certified oxytetracycline (OTC) content. These features, together with the short analysis time (30 min), the simplicity, and easy automation and miniaturization of the required instrumentation make the developed methodology a promising alternative in the development of devices for on-site analysis.

A novel electrochemical immunosensor was developed for the determination of the hormone prolactin. The design involved the use of screen-printed carbon electrodes and streptavidin-functionalized magnetic particles. Biotinylated anti-prolactin antibodies were immobilized onto the functionalized magnetic particles and a sandwich-type immunoassay involving prolactin and anti-prolactin antibody labelled with alkaline phosphatase was employed. The resulting bio-conjugate was trapped on the surface of the screen-printed electrode with a small magnet and prolactin quantification was accomplished by differential pulse voltammetry of 1-naphtol formed in the enzyme reaction using 1-naphtyl phosphate as alkaline phosphatase substrate. All variables involved in the preparation of the immunosensor and in the electrochemical detection step were optimized. The calibration plot for prolactin exhibited a linear range between 10 and 2000 ng mL−1 with a slope value of 7.0 nA mL ng−1. The limit of detection was 3.74 ng mL−1. Furthermore, the modified magnetic beads–antiprolactin conjugates showed an excellent stability. The immunosensor exhibited also a high selectivity with respect to other hormones. The analytical usefulness of the immnunosensor was demonstrated by analyzing human sera spiked with prolactin at three different concentration levels.

This review covers recent advances in the development of new designs of electrochemical sensors and biosensors that make use of electrode surfaces modification with carbon nanotubes. Applications based on carbon nanotubes-driven electrocatalytic effects, and the construction and analytical usefulness of new hybrid materials with polymers or other nanomaterials will be treated. Moreover, electrochemical detection using carbon nanotubes-modified electrodes as detecting systems in separation techniques such as high performance liquid chromatography (HPLC) or capillary electrophoresis (CE) will be also considered. Finally, the preparation of electrochemical biosensors, including enzyme electrodes, immunosensors and DNA biosensors, in which carbon nanotubes play a significant role in their sensing performance will be separately considered.

A novel progesterone immunosensor using a colloidal gold-graphite-Teflon-tyrosinase composite biosensor as amperometric transducer is reported. A sequential competitive configuration between the analyte and progesterone labelled with alkaline phosphatase (AP) was used. Phenyl phosphate was employed as the AP-substrate and the enzyme reaction product, phenol, was oxidized by tyrosinase to o-quinone, which is subsequently reduced at -0.1 V at the biocomposite electrode. Variables such as the concentration of phenyl phosphate, the amount of antibody attached to the electrode surface, immersion time in a 2% BSA solution, working pH and incubation times in progesterone and AP conjugate were optimized. A linear calibration graph for progesterone was obtained between 0 and 40 ng mL(-1) with a slope value of -82.3 nA ng(-1) mL, and a detection limit of 0.43 ng mL(-1). The time needed to reach the steady-state current from the addition of phenyl phosphate was 30-40 s. These analytical characteristics improve substantially those reported for other progesterone immunosensors. A lifetime of 14 days with no need to apply any regeneration procedure was also achieved. The usefulness of the immunosensor was evaluated by determining progesterone in milk samples spiked with the analyte at 5.0 and 1.5 ng mL(-1) concentration levels. Following a very simple procedure, involving only sample dilution, mean recoveries (n=7) of 98+/-3% and 99+/-3%, respectively, were obtained.

A highly sensitive surface plasmon resonance (SPR) immunosensor for the important ErbB2 breast cancer biomarker has been developed. Optimization of the assay has been carried out, including signal enhancement employing gold nanoparticles (GNPs). The effect of the signal amplification of the GNPs has been also studied. The assay has been tested with clinically relevant matrices. Results in 50% human serum yielded a LOD of 180 pg mL −1 which is a concentration 83 times lower than the clinical cut-off. Raw lysates from model breast cancer cell lines (SK-BR-3, MCF-7 and MDA-MB-436) have been also assayed and higher quantities of the ErbB2 protein were clearly observed in the ErbB2 over-expressing case (SK-BR-3). The results confirmed that the simple and highly sensitive SPR immunosensor represents a feasible tool for ErbB2 detection.

A highly sensitive disposable amperometric immunosensor based on the use of magnetic beads (MBs) is described for determination of Ara h 1, the major peanut allergen, in only 2h. The approach uses a sandwich configuration involving selective capture and biotinylated detector antibodies and carboxylic acid-modified MBs (HOOC-MBs). The MBs bearing the immunoconjugates are captured by a magnet placed under the surface of a disposable screen-printed carbon electrode (SPCE) and the affinity reactions are monitored amperometrically at -0.20 V (vs a Ag pseudo-reference electrode) in the presence of hydroquinone (HQ) as electron transfer mediator and upon addition of H2O2 as the enzyme substrate. The developed immunosensor exhibits a wide range of linearity between 20.8 and 1000.0 ng mL(-1) Ara h 1, a detection limit of 6.3 ng mL(-1), a great selectivity, a good reproducibility with a RSD of 6.3% for six different immunosensors and a useful lifetime of 25 days. The usefulness of the immunosensor was demonstrated by determining Ara h 1 in different matrices (food extracts and saliva). The results correlated properly with those provided by a commercial ELISA method offering a reliable and promising analytical screening tool in the development of user-friendly devices for on-site determination of Ara h 1.

The synthesis and evaluation of a molecularly imprinted polymer (MIP) as a selective solid-phase extraction sorbent, coupled to voltammetric detection, for the efficient preconcentration and determination of sulfamethazine in milk is reported. The polymer was prepared using sulfamethazine as the template molecule, methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as the cross-linking monomer in the presence of acetonitrile as the solvent. The detection of sulfamethazine was carried out by square wave voltammetry (SVW) at a glassy carbon electrode in a MeOH:acetic acid (9:1) medium. This solvent mixture was also used for the elution of the analyte from the MIP microcolumns. Optimisation of the variables involved in the sulfamethazine binding/extraction process was carried out (solvent used for rebinding, pH of the rebinding solution, eluent volume, analyte and eluent flow rates). Using solid-phase extraction cartridges, containing 0.16–0.18 g of MIP, an eluent volume of 2 ml and a sample volume of 90 ml, resulted in a nominal enrichment factor of 45, which was sufficient to analyse sulfamethazine at the maximum level permitted by the Codex Alimentarius Commission in milk (25 mg 1 −1 ). The selectivity of the MIP was evaluated by considering different substances with molecular structures similar to sulfamethazine. Sulfamethazine was determined in milk samples spiked at two concentration levels: 696 mg 1 −1 in the buttermilk obtained after milk deproteinisation, and 25 mg 1 −1 both in the buttermilk and in the milk before deproteinisation. Recoveries of practically 100% were achieved in all cases.

The preparation of a tyrosinase biosensor based on the immobilization of the enzyme onto a glassy carbon electrode modified with electrodeposited gold nanoparticles (Tyr-nAu-GCE) is reported. The enzyme immobilized by cross-linking with glutaraldehyde retains a high bioactivity on this electrode material. Under the optimized working variables (a Au electrodeposition potential of −200 mV for 60 s, an enzyme loading of 457 U, a detection potential of −0.10 V and a 0.1 mol l −1 phosphate buffer solution of pH 7.4 as working medium) the biosensor exhibited a rapid response to the changes in the substrate concentration for all the phenolic compounds tested: phenol, catechol, caffeic acid, chlorogenic acid, gallic acid and protocatechualdehyde. A R.S.D. of 3.6% ( n = 6) was obtained from the slope values of successive calibration plots for catechol with the same Tyr-nAu-GCE with no need to apply a cleaning procedure to the biosensor. The useful lifetime of one single biosensor was of at least 18 days, and a R.S.D. of 4.8% was obtained for the slope values of catechol calibration plots obtained with five different biosensors. The kinetic constants and the analytical characteristics were calculated for all the phenolic compounds tested. The Tyr-nAu-GCE was applied for the estimation of the phenolic compounds content in red and white wines. A good correlation of the results ( r = 0.990) was found when they were plotted versus those obtained by using the spectrophotometric method involving the Folin–Ciocalteau reagent.

An amperometric immunoassay for the determination of tumor necrosis factor alpha (TNFα) protein biomarker in human serum based on the use of magnetic microbeads (MBs) and disposable screen-printed carbon electrodes (SPCEs) has been developed. The specifically modified microbeads were magnetically captured on the working electrode surface and the amperometric responses were measured at −0.20 V (vs. Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as electron transfer mediator and H2O2 as the enzyme substrate. After a thorough optimization of the assay, extremely low limits of detection were achieved: 2.0 pg mL−1 (36 fM) and 5.8 pg mL−1 (105 fM) for standard solutions and spiked human serum, respectively. The simplicity, robustness and this clinically interesting LOD proved the developed TNFα immunoassay as a good contender for real clinical application.

The preparation of a peroxidase biosensor by immobilization of the enzyme during the electropolymerization of N-methylpyrrole and its use in the determination of organic peroxides in a predominantly nonaqueous medium, such as reversed micelles, is reported. Reversed micelles were formed with ethyl acetate as the continuous phase, 4% of 0.05 mol l −1 phosphate buffer solution of pH 7.4 as the dispersed phase, and 0.1 mol l −1 AOT as the emulsifying agent. Working variables affecting the polymer biosensor preparation, such as the polymerization potential, the constant accumulated charge to stop the polymerization process, the concentration of monomer and enzyme in the polymerization solution and the pH and concentration of the phosphate buffer solution, were optimized and discussed. Concerning the variables regarding the amperometric measurements in the reversed micellar medium, the potential value applied, the pH of the phosphate buffer solution, and the temperature were also optimized. Under the optimized conditions, the steady-state current for 2-butanone peroxide is reached in only 4 s. Linear calibration plots over the ranges 5–85 mol l −1 , and 2–48 mmol l −1 were obtained for 2-butanone peroxide and tert-butylhydroperoxide, respectively. The limits of detection obtained were 0.086 mol l −1 , and 0.03 mmol l −1 , respectively. The poly-N-methylpyrrole-HRP amperometric biosensor was used for the determination of the organic peroxide content in body lotion samples, by employing 2-butanone peroxide as a standard. Optimization of the peroxide extraction step from the sample was carried out, and recoveries approximating 100% were obtained. © 2001 Elsevier Science B.V. All rights reserved.

The electroanalytical capabilities of cylindrical carbon fibre microelectrodes (CFMEs) modified with electrodeposited rhodium for their use as amperometric microsensors in the detection of hydrazine under flow-injection conditions are discussed. Metallized CFMEs were prepared potentiostatically at −0.80 V in an acetate buffer solution of pH 4.5 containing 100 mg l−1 Rh(III), and characterised by cyclic voltammetry. Rh-CFMEs showed electrocatalytic ability towards the oxidation of hydrazine and some other organic compounds. A good stability of the metallised microelectrodes was found when they were used for the amperometric detection of hydrazine under flowing conditions, with no need to regenerate the electrode surface. Moreover, the signal-to-noise ratio was remarkably better at the Rh-CFME than at a Rh-modified glassy carbon electrode of conventional size. A limit of detection of 6.2×10−7 mol l−1 hydrazine (approximately 20 μg l−1) was obtained. The effect of the presence of potential interferents was studied and the flow-injection method was applied to the determination of hydrazine in boiler water samples spiked with 0.64 mg l−1 analyte.

The use of cylindrical carbon fiber microelectrodes (CFMEs) modified with poly(3-methylthiophene) (P3MT) films as amperometric microsensors for continuous monitoring of some amino acids such as tyrosine (Tyr), tryptophan (Trp), L-dopa, and related compounds such as tyramine and ascorbic acid, as well as small peptides such as Trp–Ala and Tyr–Gly, is discussed. Important practical advantages with respect to conventional glassy carbon (GC) electrodes, and also with respect to P3MT-coated GC electrodes of conventional size are demonstrated. The electrocatalytic ability of the modified surface allows the possibility of applying moderate potentials for the amperometric detection of the above mentioned compounds. Flow-injection experiments carried out using a phosphate buffer solution of pH 7.0 as the carrier, and an applied potential of +0.8 V showed that no cleaning or regeneration pretreatment was needed when working with the same polymer modified electrode during the whole working day. The effect of the presence of acetonitrile or methanol in the flowing solution was evaluated. A flow-injection method with amperometric detection was developed for the determination of L-dopa in pharmaceutical preparations. Finally, the modified microelectrodes have also shown suitable for amperometric detection in liquid chromatography (LC). A mobile phase composed of 5 : 95 (v/v) methanol : phosphate buffer solution of pH 7.0 allowed a good separation of mixtures of ascorbic acid, L-dopa, Tyr, tyramine and Trp, with detection limits of around 160 pmol. Furthermore, the possibility of detection of Tyr- and Trp-containing oligopeptides was also demonstrated.

The electrochemical behavior of cylindrical carbon fiber microelectrodes (CFMEs), 8 mm in length, has been tested in lowpermitivity organic solvents such as toluene and ethyl acetate. Near-to-ideal steady-state responses were obtained by cyclic voltammetry for ferrocene solutions in these media, using low potential scan rates and a relatively high concentration of background electrolyte. A good adjustment of the voltammetric responses to that predicted by the theory was found in both solvents. Well-defined oxidation peaks were also obtained by differential-pulse and square wave voltammetry. Square-wave voltammetry (SWV) in ethyl acetate has been used to develop a method for the determination of the food additive vanillin. A linear calibration graph was obtained in the 1.010 ˇ5 to 7.010 ˇ4 mol l ˇ1 concentration range with a slope of (7.10.1)10 3 mA l mol ˇ1 . A limit of detection of 4.210 ˇ6 mol l ˇ1 vanillin, and a relative standard deviation of 2.0% for a concentration level of 5.010 ˇ4 mol l ˇ1 (na10) were obtained. The SWV method was applied to the determination of vanillin in dehydrated pudding powder. This determination was performed directly in the samples extract in ethyl acetate. The obtained results were statistically compared with those provided by a reference spectrophotometric method, and no significant differences between both methods were found. # 1999 Elsevier Science B.V. All rights reserved.

An amperometric tyrosinase electrode has been used for biosensing of dimethyl- and diethyldithiocarbamates based on the inhibition effects of these substances on the catalytic activity of the enzyme. A working medium consisting of reversed micelles, and phenol as the substrate has been used. The tyrosinase electrode was constructed by direct adsorption of the enzyme on the surface of a graphite-disk electrode. Reversible inhibition processes are shown to be involved for ziram, diram and zinc diethyldithiocarbamate. Following a simple regeneration of the enzyme electrode, an acceptable reproducibility for the measurements of the inhibition response was obtained. Experimental variables, such as temperature, phenol concentration and the presence of chloroform, affecting the inhibition processes, were optimized. The type of enzyme inactivation for each inhibitor tested was studied, and the inhibition constants were calculated. Detection limits of 0.074, 1.3 and 1.7 μmol l−1 were achieved for ziram, diram and zinc diethyldithiocarbamate, respectively. Other carbamates belonging to families different from dimethyl- and diethyldithiocarbamates showed no amperometric response at the tyrosinase electrode, except for pyrimidine-derivative carbamates. The developed analytical methodology was applied to determine ziram in spiked apple samples.

An electrochemical DNA genosensor constructed by using rough gold as electrode support is reported in this work. The electrode surface nanopatterning was accomplished by repetitive square-wave perturbing potential (RSWPP). A synthetic 25-mer DNA capture probe, modified at the 5′ end with a hexaalkylthiol, able to hybridize with a specific sequence of lacZ gene from the Enterobacteriaceae bacterial family was assembled to the rough gold surface. A 25 bases synthetic sequence fully complementary to the thiolated DNA capture probe and a 326 bases fragment of lacZ containing a fully matched sequence with the capture probe, which was amplified by a specific asymmetric polymerase chain reaction (aPCR), were employed as target sequences. The hybridization event was electrochemically monitored by using two different indicators, hexaammineruthenium (III) chloride showing an electrostatic DNA binding mode, and pentaamineruthenium-[3-(2-phenanthren-9-yl-vinyl)-pyridine] (in brief RuL) which binds to double stranded DNA (dsDNA) following an intercalative mechanism. After optimization of the different variables involved in the hybridization and detection reactions, detection limits of 5.30 pg μL −1 and 10 pg μL −1 were obtained for the 25-mer synthetic target DNA and the aPCR amplicon, respectively. A RSD value of 6% was obtained for measurements carried out with 3 different genosensors prepared in the same manner.

A novel amperometric magnetoimmunosensor using an indirect competitive format is developed for the sensitive detection of the amino-terminal pro-B-type natriuretic peptide (NT-proBNP). The immunosensor design involves the covalent immobilization of the antigen onto carboxylic-modified magnetic beads (HOOC-MBs) activated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), and further incubation in a mixture solution containing variable concentrations of the antigen and a fixed concentration of an HRP-labeled detection antibody. Accordingly, the target NT-proBNP in the sample and that immobilized on the MBs compete for binding to a fixed amount of the specific HRP-labeled secondary antibody. The immunoconjugate-bearing MBs are captured by a magnet placed under the surface of a disposable gold screen-printed electrode (Au/SPE). The amperometric responses measured at –0.10 V (vs. a Ag pseudo-reference electrode), upon addition of 3,3′,5,5′-tetramethylbenzidine (TMB) as electron transfer mediator and H2O2 as the enzyme substrate, are used to monitor the affinity reaction. The developed magnetoimmunosensor provides attractive analytical characteristics in 10-times diluted human serum samples, exhibiting a linear range of clinical usefulness (0.12–42.9 ng mL−1) and a detection limit of 0.02 ng mL−1, which can be used in clinical diagnosis of chronic heart failure in the elderly and for classifying patients at risk of death after heart transplantation. The magnetoimmunosensor was successfully applied to the analysis of spiked human serum samples.

Some new trends in the development of amperometric enzyme electrodes in reversed micelle systems are shown. The possibility of using these media for on-line applications as well as for enzyme inhibition measurements both in the batch and the flow-injection modes is demonstrated. Reversed micelles were formed of ethyl acetate as organic solvent, dioctyl sulfosuccinate as emulsifying agent and a 4% of 0.05 mol l −1 phosphate buffer of pH 7.4 as aqueous phase. An Eastman AQ/enzyme immobilization approach was used. Flow-injection analysis of various phenolic compounds using the reversed micelle as the carrier solution was accomplished. Despite the hydrodynamic conditions, the immobilized enzyme layer displayed a good stability in the reversed micelle flowing stream. Phenolic compounds with all ortho -positions substituted gave no analytical signal. The possibility of developing reversed micelle enzyme inhibition biosensors is illustrated by monitoring inhibitors of tyrosinase such as benzoic acid, thiourea or propyl gallate, and using phenol as the substrate. The steady-state response towards phenol rapidly decreases upon additions of these compounds at a low concentration level. A reversible inhibition process was shown in all cases, and the type of enzyme inactivation in the reversed micelle of each inhibitor studied. The rapid response of the tyrosinase electrode to the changes of inhibitor concentration in the reversed micelle can be also exploited for on-line applications.

The use of graphite-poly(tetrafluoroethylene) composite electrodes as indicator electrodes in flowing systems is reported. Flow-injection methods with amperometric detection based on the electrochemical oxidation of the herbicides thiram and disulfiram on these electrodes are described. No cleaning or pretreatment of the electrode surface after successive injections of 5.0 × 10 −6 mol l −1 herbicide in 0.1 mol 1 −1 phosphate buffer of pH 7.4 was required. Detection limits of 4.3 × 10 −8 and 2.0 × 10 −8 mol l −1 were achieved for thiram and disulfiram, respectively. The effect of the presence of several species which may be present together with thiram and/or disulfiram in water samples such as ziram, phenol and Zn(II) on the amperometric signals for both herbicides was evaluated. The determination of thiram in spiked tap and well waters at a concentration level of 40 μg 1 −1 thiram was carried out with recoveries of 103 ± 6% for tap water, and 97 ± 7% for well water, respectively. The possibility of performing adsorptive accumulation of the analytes by passing through the flow cell a stream of the herbicides was demonstrated. After accumulation, quantitation by a linear sweep anodic potential ramp was accomplished, as well as an electrochemical cleaning of the electrode surface. Linearity was achieved in a shorter range, and no lower detection limits were attained with respect to the direct flow-injection methods. Nevertheless, using adsorptive preconcentration the selectivity was improved. Finally, a continuous separation scheme of both herbicides was accomplished in order to carry out their simultaneous determination.

The behaviour of a nickel phthalocyanine polymer-coated glassy carbon electrode has been investigated in order to test it as an amperometric detector in flow systems. The oxidation process of the antioxidant tert. -butylhydroxyanisole (BHA) has been studied for this purpose. The monomer Ni(II)-4,9,16,23-tetraaminophthalocyanine was synthesized and characterized. Electropolymerization was accomplished by repetitive cycling between −0.2 and + 0.9 V in a dimethyl sulfoxide solution of the monomer. The characteristics of the BHA oxidation process at the polymeric modified electrode were examined by linear sweep voltammetry. A flow-injection method with amperometric detection based on this oxidation process has been developed. At an applied potential of 0.70 V, calibration graphs were linear within the range 10-0.01 mg l −1 BHA with a limit of detection of 2.7 μg l −1 BHA (1.5 × 10 −8 mol l −1 ). Interferences from BHT ( tert. -butylhydroxytoluene), TBHQ ( tert. -butylhydroquinone), propyl gallate, ascorbic acid, sodium bisulfite and citric acid were tested. The method was applied to the determination of BHA in biscuits samples.

An electroanalytical study of the Irganox 1076 oxidation process at a glassy carbon electrode in an aqueous 60% ethanolic medium using different voltammetric techniques has been carried out. The results obtained by differential pulse voltammetry allowed the development of a method for the determination of this antioxidant using a calibration graph in the 1.0 × 10−6−2.5 × 10−5moll−1 Irganox 1076 concentration range (r = 0.9998). The coefficient of variation was 3.3% for a concentration level of 1.0 × 10−5 mol l−1, with a limit of determination of 7.7 × 10−7 mol l−1 and a limit of detection of 2.3 × 10−7 mol l−1. The effect of other antioxidants and plasticizers has been studied. Good results were obtained by applying the proposed differential pulse voltammetric method to the determination of Irganox 1076 in commercial poly(vinyl chloride) based plastics. A flow-injection method with pulsed amperometric detection based on such an oxidation process is also described. The pulse waveform was optimized in order to ascertain the cleaning of the electrode surface and also to obtain the best sensitivity. Calibration graphs were linear within the ranges 2.0 × 10−7−10.0 × 10−7 mol l−1 (r = 0.998) and 1.0 × 10−6−10.0 × 10−6 mol l−1 (r = 0.999). The coefficient of variation was 2.3% and the limits of detection and determination were 8.0 × 10−8 mol l−1 and 2.7 × 10−7 mol l−1, respectively.

The oxidative voltammetric behaviour of the herbicides thiram and disulfiram at graphite-poly(tetrafluoroethylene) (PTFE) composite electrodes has been studied. Graphite-PTFE pellets were prepared by mixing graphite with powdered PTFE and compression molding the mixture. Based on the signal-to-noise characteristics and on the mechanical strength of the resulting pellet, a mixture containing 40% PTFE was selected. Both thiram and disulfiram are adsorbed onto the composite electrode, this process being used as a preconcentration step. The accumulation potential and accumulation time chosen were 0.0 V and 300 s, respectively. The characteristics of the electrode processes were established. Linear calibration graphs were obtained within the ranges 2.0 × 10 −7 −1.0 × 10 −5 and 2.0 × 10 −7 −8.0 × 10 −6 mol l −1 for thiram and disulfiram, respectively. The effect of the presence of ziram, phenol and Zn(II) on the oxidation signals for thiram and disulfiram was evaluated. As an application, the determination of thiram in spiked strawberries was carried out with good results.

This work describes the preparation of an electrochemical immunosensor for estradiol based on the surface modification of a screen printed carbon electrode with grafted p-aminobenzoic acid followed by covalent binding of streptavidin (Strept) and immobilization of biotinylated anti-estradiol (anti-estradiol-Biotin). The hormone determination was performed by applying a competitive immunoassay with peroxidase-labelled estradiol (HRP-estradiol) and measurement of the amperometric response at -200 mV using hydroquinone (HQ) as redox mediator. The calibration curve for estradiol exhibited a linear range between 1 and 250 pg mL(-1) (r=0.990) and a detection limit of 0.77 pg mL(-1) was achieved. Cross-reactivity studies with other hormones related with estradiol at physiological concentration levels revealed the practical specificity of the developed method for estradiol. A good reproducibility, with RSD=5.9% (n=8) was also observed. The operating stability of a single bioelectrode modified with anti-estradiol-Biotin-Strept was nine days when it was stored at 8°C under humid conditions between measurements. The developed immunosensor was applied to the analysis of certified serum and spiked urine samples with good results.

A voltammetric study of the oxidation process of the antioxidant tert-butylhydroxyanisole (BHA) in miceller and emulsified media is described. The non-ionic surfactant Pluronic F-68, in Britton-Robinson buffer at pH 2.0, was found to be the most suitable surfactant for the determination of BHA in micellar solutions. The characteristics of the oxidation process were established. An emulsified medium formed with ethyl acetate as organic solvent and Pluronic F-68 as emulsifying agent was also used. Linear calibration graphs for BHA were obtained by differential pulse voltammetry with the ranges 1.0×10−6−1.0×10−5 mol l−1 and 1.0×10−5−5.0×10−5 mol l−1. The limits of determination were found to be 1.3×10−6 mol l−1 and 8.5×10.−7 mol l−1 BHA in micellar and emulsified media, respectively. Interferences from BHT (tert-butylhydroxytoluene), TBHQ (tert-butylhydroquinone), citric acid, propyl gallate and propylene glycol were tested. The method was applied to the determination of BHA in chewing gum samples by direct emulsification of an aliquot of the antioxidant extract from the sample in ethyl acetate.

A polarographic study of the reduction of the herbicide simazine in micellar solutions and oil-water emulsions is reported. The anionic surfactant sodium pentanesulphonate was chosen as the most suitable agent for micellar solutions and emulsions. In micellar solutions two reduction peaks were observed in differential-pulse polarography (DPP) below pH 2.0, whereas only one peak was obtained above pH 2.0. Ethyl acetate was chosen as the organic solvent to form simazine emulsions. Unlike in micellar solutions, DP polarograms of simazine in oil-water emulsions showed only one peak even at pH values lower than 2.0, suggesting that pesticide hydrolysis is hindered in the emulsified medium. The limiting current is diffusion-controlled and the electrode process is irreversible. The diffusion coefficient of simazine in emulsified medium was 8.85 × 10 −6 cm 2 s −1 , and the α n a and K ° f values were 0.82 and 8.03 × 10 −14 cm s −1 respectively. Using DPP, simazine can be determined over the concentration ranges 8.0 × 10 −7 −4.0 × 10 −5 mol l −1 . The limit of detection was 2.2 × 10 −7 mol l −1 . The effect of the presence of other herbicides belonging to the s -triazine family on the simazine reduction peak was studied. The method was applied to the determination of simazine in spiked irrigation water with good recoveries.

A polarographic study of the oxidation of tert-butylhydroquinone (TBHQ) in micellar and emulsified media is described. The non-ionic surfactant Pluronic F-68, in Britton- Robinson buffer (pH 9.0), was found to be the most suitable for determining this antioxidant in micellar solutions. The characteristics of the oxidation process were established. Linear calibration graphs were obtained for TBHQ by differential-pulse polarography (DPP) within the ranges 1.0 × 10−6−1.0 × 10−5 and 1.0 × 10−5−1.0 × 10−4 mol l−1 with a limit of determination (10 × standard deviation) of 6.7 × 10−7 mol l−1 TBHQ (0.11 mg l−1). An emulsified medium formed with a mixture of hexane and ethyl acetate (99:1) at pH 8.0 was also used. Linear calibration graphs were obtained by DPP within the ranges 1.0 × 10−6−1.0 × 10−5 mol l−1 and 1.0 × 10−5–1.0 × 10−4 mol l−1 TBHQ. The limit of determination was found to be 8.3 × 1 0−7 mol l−1. Interferences from tert-butylhydroxyanisole, tert-butylhydroxytoluene, citric acid, propyl gallate and propylene glycol were tested. Except for propyl gallate, none of them interfered. The proposed method was applied with good results to the determination of TBHQ in spiked edible oils by direct emulsification of the samples.

The determination of the organochlorine pesticides dieldrin, heptachlor, α-endosulfan, β-endosulfan and endosulfan sulphate and their mixtures in spiked apple samples was carried out in oil-water emulsions formed with ethyl acetate and a mixture of Hyamine 2389 and Triton X-405. Extraction of the pesticides from the apples was accomplished using x-hexane. Clean-up of the extract and fractionation was carried out with a Florisil column, using n-hexane and n-hexane-ethyl acetate (20:1) as eluents. The recoveries for each pesticide individually were about 90% for heptachlor, endosulfan sulphate and both isomers of endosulfan and about 70% for dieldrin at concentration levels of 0.37, 0.93 and 1.27 μg g−1 of apple for heptachlor, dieldrin and endosulfan sulphate, respectively, and 1.22 μg g−1 for each of the endosulfan isomers. Similar results were obtained with binary and ternary mixtures and with a mixture containing all the pesticides studied, indicating the validity of the extraction and separation method developed.

Two flow-injection methods are described for the determination of the antioxidants tert-butylhydroxyanisole (BHA) and tert-butylhydroxytoluene (BHT) based on their oxidation at a glassy carbon electrode. In both instances, the analytical solution is injected into a carrier stream consisting of a solution of perchloric acid and 5% (v/v) methanol. Calibration graphs are linear in the concentration ranges 1.0–10.0, 0.1–1.0 and 0.01–0.1 mg l−1 for BHA and 1.0–8.0, 0.1–1.0 and 0.02–0.1 mg l−1 for BHT. Detection limits for BHA and BHT are 2.5 and 6.0 μg l−1, respectively. The determination of BHA in the presence of BHT is possible because the former is more easily oxidized than the latter. Other compounds usually present in commercial antioxidant mixtures, such as citric acid or propylene glycol, do not interfere, although propyl gallate does. Good results were obtained by applying the proposed flow-injection methods to determine BHA in spiked corn oil and BHT in stabilized diethyl ether samples after extraction of the analytes.

This review covers recent advances in the development of new designs of electrochemical sensors and biosensors that make use of electrode surfaces modification with carbon nanotubes. Applications based on carbon nanotubes-driven electrocatalytic effects, and the construction and analytical usefulness of new hybrid materials with polymers or other nanomaterials will be treated. Moreover, electrochemical detection using carbon nanotubes-modified electrodes as detecting systems in separation techniques such as high performance liquid chromatography (HPLC) or capillary electrophoresis (CE) will be also considered. Finally, the preparation of electrochemical biosensors, including enzyme electrodes, immunosensors and DNA biosensors, in which carbon nanotubes play a significant role in their sensing performance will be separately considered.

A sensitive and selective polarographic method for the determination of pirimicarb is proposed. Linear calibration graphs were obtained by differential-pulse polarography in the 3.0 × 10 −7 –9.0 × 10 −5 mol l −1 range, the limit of determination being 2.8 × 10 −7 mol l −1 . No interferences were observed from other carbamate pesticides such as aminocarb, bendiocarb or carbaryl and only a matrix effect occurs in the presence of methiocarb. The method was applied to the determination of Pirimicarb in water samples with good recoveries.

The construction and performance of integrated amperometric biosensors for the determination of glycerol are reported. Two different biosensor configurations have been evaluated: one based on the glycerol dehydrogenase/diaphorase (GDH/DP) bienzyme system, and another using glycerol kinase/glycerol-3-phosphate oxidase/peroxidase (GK/GPOx/HRP). Both enzyme systems were immobilized together with the mediator tetrathiafulvalene (TTF) on a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode by using a dialysis membrane. The electrochemical oxidation of TTF at +150 mV (vs. Ag/AgCl), and the reduction of TTF + at 0 mV were used for the monitoring of the enzyme reactions for the bienzyme and trienzyme configurations, respectively. Experimental variables concerning both the biosensors composition and the working conditions were optimized for each configuration. A good repeatability of the measurements with no need of cleaning or pretreatment of the biosensors was obtained in both cases. After 51 days of use, the GDH/DP biosensor still exhibited 87% of the original sensitivity, while the GK/GPOx/HRP biosensor yielded a 46% of the original response after 8 days. Calibration graphs for glycerol with linear ranges of 1.0 × 10 −6 to 2.0 × 10 −5 or 1.0 × 10 −6 to 1.0 × 10 −5 M glycerol and sensitivities of 1214 ± 21 or 1460 ± 34 μA M −1 were obtained with GDH/DP and GK/GPOx/HRP biosensors, respectively. The calculated detection limits were 4.0 × 10 −7 and 3.1 × 10 −7 M, respectively. The biosensors exhibited a great sensitivity with no significant interferences in the analysis of wines. The biosensors were applied to the determination of glycerol in 12 different wines and the results advantageously compared with those provided by a commercial enzyme kit.

Simple and sensitive methods for the separation and quantification of beta-carboline alkaloids in foods and beverages by HPLC with electrochemical detection at carbon nanotubes-modified glassy carbon electrodes (CNTs-GCE) are reported. Electrode modification with multi-wall CNTs produced an improved amperometric response to beta-carbolines, in spite of the working medium consisting of methanol:acetonitrile: 0.05 mol L(-1) Na(2)HPO(4) solution of pH 9.0 (20:20:60). On the contrary to that observed at a bare GCE, a good repeatability of the amperometric measurements carried out at +900 mV versus Ag/AgCl (R.S.D. of 3.2% for i(p), n=20) was achieved at the CNTs-GCE. Using an Ultrabase C(18) column and isocratic elution with the above mentioned mobile phase, a complete resolution of the chromatographic peaks for harmalol, harmaline, norharmane, harmane and harmine, was achieved. Calibration graphs over the 0.25-100 microM range with detection limits ranging between 4 and 19 ng mL(-1), were obtained. The HPLC-ED at CNTs-GCE method was applied to the analysis of beer, coffee and cheese samples, spiked with beta-carbolines at concentration levels corresponding to those may be found in the respective samples. The steps involved in sample treatment, such as extraction and clean-up, were optimized for each type of sample. Recoveries ranging between 92 and 102% for beer, 92 and 101% for coffee, and 88 and 100% for cheese, at sub-microg mL(-1) or g(-1) analytes concentration levels were achieved.