Search

A label-free dual electrochemical immunosensor was constructed for the multiplexed determination of human growth (hGH) and prolactin (PRL) hormones. The immunosensor used an electrochemical platform composed of carbon nanotube-screen printed carbon electrodes (CNT/SPCEs) modified with poly(ethylene-dioxythiophene) (PEDOT) and gold nanoparticles, on which the corresponding hGH and PRL antibodies were immobilized. The affinity reactions were monitored by measuring the decrease in the differential pulse voltammetric oxidation response of the redox probe dopamine. The experimental variables involved in the preparation of both AuNP/PEDOT/CNT/SPC modified electrodes and the dual immunosensor were optimized. The immunosensor exhibited an improved analytical performance for hGH and PRL with respect to other electrochemical immunosensor designs, showing wide ranges of linearity and low detection limits of 4.4 and 0.22 pg mL(-1), respectively. An excellent selectivity against other hormones and in the presence of ascorbic and uric acids was found. The usefulness of the dual immunosensor for the simultaneous analysis of hGH and PRL was demonstrated by analyzing human serum and saliva samples spiked with the hormones at different concentration levels.

A novel strategy for the construction of a disposable integrated amperometric immunosensor for the sensitive and rapid determination of lipoprotein(a) (Lp(a)), an important predictor of cardiovascular disease risk, in human serum is reported. The approach uses a sandwich format involving the covalent immobilization of selective capture antibodies (antiLp(a)) on the surface of N-[Nα,Nα-bis(carboxymethyl)-lysine]-12-mercaptododecanamide (HS-NTA)-modified screen-printed carbon electrodes (SPCEs). After a blocking step with skimmed milk, the modified antiLp(a)-SPCEs were incubated with a mixture solution containing the target analyte and a fixed concentration of a specific biotinylated antibody (biotin-antiLp(a)) and a streptavidin-horseradish peroxidase (HRP) (Strep-HRP) conjugate. The amperometric responses of the resulting immunosensor at -0.10 V (vs an Ag pseudo-reference electrode), upon addition of 3,3',5,5'-tetramethylbenzidine (TMB) as electron transfer mediator and H2O2 as the enzyme substrate, were used to monitor the extent of the immunoreactions. The developed methodology exhibited a wide range of linearity between 0.02 and 10 μg mL(-1), a low detection limit (LOD) of 8 ng mL(-1), and a great selectivity against other serum components. The usefulness of the Lp(a) immunosensor was demonstrated by analyzing spiked serum samples as well as a reference serum containing a certified Lp(a) content.

We describe in this work a novel electrochemical immunosensor design making use of carbon nanohorns (CNHs) as a scaffold for the preparation of disposable immunosensing platforms for the determination of fibrinogen (Fib). The approach involved the immobilization of Fib onto activated CNHs deposited on screen-printed carbon electrodes (SPCEs) and the implementation of an indirect competitive assay using anti-Fib labeled with horseradish peroxidase (HRP) and hydroquinone (HQ) as the redox mediator. Both CNHs and the Fib-CNHs covalent assembly were characterized by microscopic and electrochemical techniques. The different variables affecting the analytical performance of the amperometric immunosensing strategy were optimized. The calibration plot for Fib allowed a range of linearity between 0.1 and 100 μg/mL (r(2) = 0.994) and a detection limit of 58 ng/mL to be achieved. The Fib-CNHs/SPCEs exhibited an excellent storage stability of at least 42 days. The developed immunosensor provides, in general, an analytical performance better than that reported for other Fib immunosensors and commercial ELISA kits. This simple and relatively low cost immunosensor configuration permitted the sensitive and selective determination of Fib in human plasma and urine.

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 -0.20V (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: 2.0pg mL(-1) (36fM) and 5.8pg mL(-1) (105fM) 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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A disposable PCR-based amperometric magneto-genosensor for detection and identification of Streptococcus pneumoniae was evaluated. ROC curve analysis used to determine optimal signal cutoff values yielded a sensitivity of 91% and a specificity of 90%. The method was also tested for the direct detection of pneumococci in clinical samples., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A new sense-and-act system was realized by integrating a biocatalytic/bioaffinity electrode responding to immune signals represented by an antibody and a polymer-modified electrode loaded with drug-mimicking species. The release of the drug-mimicking species was achieved specifically in response to a signal antibody, thus demonstrating for the first time an immune-induced drug-releasing process. The present approach promises new options for future applications in controlled drug release and personalized medicine.

We report herein the design of a smart delivery system in which cargo delivery from capped mesoporous silica (MS) nanoparticles is controlled by an integrated enzyme-based "control unit". The system consists of Janus-type nanoparticles having opposing Au and MS faces, functionalized with a pH-responsive β-cyclodextrin-based supramolecular nanovalve on the MS surface and two effectors, glucose oxidase and esterase, immobilized on the Au face. The nanodevice behaves as an enzymatic logical OR operator which is selectively fueled by the presence of D-glucose and ethyl butyrate.

MicroRNAs (miRs) have emerged as important clinical biomarkers with both diagnostic and prognostic value for relevant diseases, such as cancer. MiRs pose unique challenges for detection and are currently detected by northern blotting, real-time PCR, and microarray techniques. These expensive, complicated, and time-consuming techniques are not feasible for on-site miR determination. In this study, amperometric magnetobiosensors involving RNA-binding viral protein p19 as a selective biorecognition element were developed for miR quantification. The p19-based magnetosensors were able to detect 0.4 fmol of a synthetic target and endogenous miR-21 (selected as a model for its role in a wide variety of cancers) in only 2 h in total RNA extracted from cancer cells and human breast-tumor specimens without PCR amplification and sample preprocessing. These results open up formidable perspectives for the diagnosis and prognosis of human cancers and for drug-discovery programs., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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 H2O2 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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

An amperometric immunosensor for the determination of the hormone insulin-like growth factor 1 (IGF1) is reported for the first time in this work. As electrochemical transducer, a multiwalled carbon nanotubes-modified glassy carbon electrode on which poly(pyrrole propionic acid) was electropolymerized was prepared. This approach provided a high content of surface confined carboxyl groups suitable for direct covalent binding of anti-IGF1 monoclonal antibody. A sandwich-type immunoassay using a polyclonal antibody labeled with peroxidase, hydrogen peroxide as the enzyme substrate and catechol as redox mediator was employed to monitor the affinity reaction. All the variables involved in the preparation of the modified electrode were optimized and the electrodes were characterized by electrochemical impedance spectroscopy and cyclic voltammetry. Moreover, the different experimental variables affecting the amperometric response of the immunosensor were also optimized. The calibration graph for IGF1 showed a range of linearity extending from 0.5 to 1000 pg/mL, with a detection limit, 0.25 pg/mL, more than 100 times lower than the lowest values reported for the ELISA immunoassays available for IGF1 (30 pg/mL, approximately). Excellent reproducibility for the measurements carried out with different immunosensors and selectivity against other hormones were also evidenced. A commercial human serum spiked with IGF1 at different levels between 0.01 and 10.0 ng/mL was analyzed with good results., (© 2013 Elsevier B.V. All rights reserved.)

Two different fibrinogen (Fib) amperometric immunosensing designs based on the use of magnetic beads (MBs) and a novel specific nanobody (Nb) expressed in Escherichia coli are described for the first time. The immunological reaction for Fib detection was performed on COOH-MBs or His-Tag-Isolation-MBs as solid support for the immobilization of the antigen or the captured Nb. Direct and indirect competitive magnetoimmunosensing configurations have been tested and compared. In the former one, Fib and biotinylated Fib competed for the immobilized Nb binding sites while the latter configuration involved competition of free Fib in solution and immobilized Fib for binding to a fixed amount of the specific biotinylated Nb. Labeling of the captured biotinylated Nb or antigen was made with streptavidin-HRP. The modified magnetic beads were captured by a neodymium magnet on the surface of screen-printed carbon electrodes (SPCEs). Amperometric detection was accomplished at -0.20 V (vs. Ag pseudo-reference electrode) by measuring the catalytic current arising upon addition of H2O2 and using hydroquinone (HQ) as redox mediator in solution. A better analytical performance was achieved with the indirect competitive immunoassay with a detection limit of 0.044 μg mL(-1) Fib. The usefulness of both approaches was successfully demonstrated by analyzing an international standard for Fib plasma. The assays could be carried out in diluted plasma samples in a total analysis time of 90 min., (© 2013 Elsevier B.V. All rights reserved.)

Novel core-shell Fe 3 O 4 @poly(dopamine) magnetic nanoparticles were prepared through an in situ self-polymerization method. The hybrid nanomaterial showed an average core diameter of 11 ± 3 nm and a polymer thin film thickness of 1.8 ± 0.2 nm. The core-shell nanoparticles were employed as solid supports for the covalent immobilization of horseradish peroxidase (HRP), and the resulting biofunctionalized magnetic nanoparticles were employed to construct an amperometric biosensor for H 2 O 2 . The enzyme biosensor showed a high sensitivity of 442.14 mA M -1 cm -2 , a low limit of detection of 182 nM, a wide linear range from 6.0 × 10 -7 to 8.0 × 10 -4 M and high stability for 1 month.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A novel electrochemical immunosensor for the determination of ceruloplasmin (Cp) in human serum and urine is reported. The immunosensor configuration involves an indirect competitive immunoassay implying covalent immobilization of Cp on activated carboxylic groups at carbon nanotubes-modified screen-printed electrodes (CNTs/SPE). After Cp immobilization and reaction between the target analyte and anti-ceruloplasmin antibodies in solution, the remaining non-conjugated antibody is attached on the Cp-CNTs modified electrode. Monitoring of Cp is performed by means of a secondary antibody labeled with peroxidase (HRP-anti-IgG) and measurement of the amperometric current resulting from the addition of hydrogen peroxide in the presence of hydroquinone as the redox mediator. The experimental variables affecting the analytical performance of the immunosensor were optimized. Calibration curves for Cp provided a linear range between 0.07 and 250 μg/mL (r=0.997). The limit of detection achieved was 21 ng/mL. These analytical characteristics allow the immunosensor to be successfully used for the determination of Cp in spiked human serum and urine at various concentration levels., (© 2013 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs), new stars of human genetics, are naturally occurring, 19-25 base pair, noncoding RNAs that regulate gene expression posttranscriptionally and have been demonstrated to be excellent biomarkers for cancer diagnosis/prognosis. Because of their short length, sequence similarity, and very low concentration, their detection in real samples is challenging. Among other methods for miRNA detection, electrochemical nucleic acid biosensors exhibit relevant advantages in terms of high sensitivity, ease of use, short assay time, nontoxic experimental steps, and adaptability to point-of-care testing. This article gives a brief overview of recent advances in the rapidly developing area of electrochemical biosensors for miRNA detection. The fundamentals of the different strategies developed to achieve novel signal amplification and sensitive electrochemical detection are discussed, and some examples of relevant approaches are highlighted, along with future prospects and challenges.

The design, preparation and analytical performance of a novel integrated amperometric immunosensor based on the immobilization of selective capture antibodies on the surface of Protein G-modified screen-printed dual carbon electrodes (SPdCEs) for the multiplexed determination of sulfonamide and tetracycline antibiotics residues in milk is reported in this work. Protein G was covalently immobilized onto a 4-aminobenzoic acid (4-ABA) film grafted on the disposable electrode, and a direct competitive immunoassay using horseradish peroxidase (HRP)-labeled tracers was performed. The amperometric responses measured at -0.2 V vs. the silver pseudo-reference electrode of the SPdCE upon the addition of H2O2 in the presence of hydroquinone (HQ) as mediator were used to monitor the extent of the immunoreactions. The developed methodology showed very low limits of detection (in the low ppb level) for sulfonamide and 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 dual immunosensor was demonstrated by analyzing spiked milk samples as well as a reference milk containing a certified oxytetracycline (OTC) content. Good recoveries were attained in an analysis time of 30 min., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Branched gold nanoparticles were prepared by a seed-mediated approach using per-6-thio-6-deoxy-β-cyclodextrin capped gold nanospheres as seeds and a growth medium similar to those commonly employed to prepare gold nanorods, containing AgNO3, ascorbic acid and cetyltrimethylammonium bromide. Novel jack-shaped gold nanoparticles (102-105 nm) were obtained at a specific range of Ag(+) ion concentrations (62-102 μM). The crystalline structure of these nanoparticles was confirmed by high-resolution transmission electron microscopy. The influence of the perthiolated β-cyclodextrin on the successful preparation of gold nanojacks was demonstrated. The jack-shaped gold nanoparticles showed strong absorption in the near infrared region and excellent catalytic activity for the electrochemical oxidation of H2O2.

The preparation of a disposable electrochemical immunosensor for the quantification of the hormone leptin is described in this work. The preparation approach involved immobilization of a specific biotinylated anti-leptin antibody on the surface of streptavidin-functionalized magnetic beads (Strept-MBs) and a sandwich-type immunoassay involving the target analyte, monoclonal anti-leptin, and IgG labeled with alkaline phosphatase (AP-IgG). The electrochemical transduction step was accomplished by trapping the MBs bearing the immunoconjugates onto screen-printed carbon electrodes (SPCEs) by means of an Nd magnet and measuring the electrochemical oxidation of the 1-naphthol generated in the AP enzyme reaction upon 1-naphthyl phosphate (1-NPP) additions by differential pulse voltammetry (DPV). A calibration plot with a linear range between 5 and 100 pg mL(-1) as well as a detection limit of 0.5 pg mL(-1) (3sb/m) were achieved. This value is more than 27 times lower than that reported in the only voltammetric immunosensor for leptin described in the literature until now. The usefulness of the immunosensor was demonstrated by analyzing different types of real samples: human serum, infant powdered milk, and breast milk from a nursing mother with two months of breastfeeding.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A new gated nanodevice design able to control cargo delivery using glucose as a trigger and cyclodextrin-modified glucose oxidase as a capping agent is reported.

Here, we employ a model electrochemical DNA sensor to demonstrate that the detection limit and specificity of surface-based sensors often are not dependent on the true affinity of the probe for its target but are simply dependent on the effective probe concentration. Under these circumstances, the observed affinity (and thus the sensor's detection limit and specificity) will depend on the density with which the probes are packed on the surface of the sensor, the surface area, and even the volume of sample employed.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Novel Janus nanoparticles with Au and mesoporous silica faces on opposite sides were prepared using a Pickering emulsion template with paraffin wax as the oil phase. These anisotropic colloids were employed as integrated sensing-actuating nanomachines for enzyme-controlled stimuli-responsive cargo delivery. As a proof of concept, we demonstrated the successful use of the Janus colloids for controlled delivery of tris(2,2'-bipyridyl) ruthenium(II) chloride from the mesoporous silica face, which was grafted with pH-sensitive gatelike scaffoldings. The release was mediated by the on-demand catalytic decomposition of urea by urease, which was covalently immobilized on the Au face., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Reduced graphene nanoparticles were prepared from graphene oxide through a two-step covalent modification approach. Graphene oxide was first enriched with reactive epoxy groups by anchoring (3-glycidyloxypropyl)trimethoxysilane at the hydroxyl groups located on the nanocarbon basal plane. Modified graphene oxide was further cross-linked and partially reduced by treatment with the fourth-generation ethylenediamine core polyamidoamine G-4 dendrimer producing graphene nanoparticles with crumpled paper-like morphology. This graphene derivative was employed as a coating material for glassy carbon electrodes and the nanostructured electrode was tested for the preparation of electrochemical biosensors by immobilizing the enzyme tyrosinase through cross-linking with glutaraldehyde. This bioelectrode showed excellent electroanalytical behavior for catechol with a fast response in about 6 s, linear range of 10 nM to 22 μM, sensitivity of 424 mA M -1 , and low detection limit of 6 nM. The enzyme biosensor also showed high stability when stored at 4 °C under dry and wet conditions.

The preparation, characterization and performance evaluation of an amperometric affinity disposable magnetosensor, based on the use of a recombinant penicillin-binding protein (PBP) and screen-printed carbon electrodes (SPCEs), for the specific detection and quantification of β-lactam antibiotic residues in milk are reported. The PBP was immobilized onto His-Tag-Isolation-modified magnetic beads (His-Tag-Isolation-MBs), and a direct competitive assay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling was performed. The amperometric response obtained at -0.20 V vs. the Ag pseudo-reference electrode of the SPCE after the addition of H2O2 in the presence of hydroquinone (HQ) was used as the transduction signal. The developed methodology showed very low detection limits (in the low ppb level) for the 6 antibiotics tested in untreated milk samples, and a good selectivity against other antibiotic residues frequently detected in milk and dairy products. Due to the bioreceptor employed, this methodology was able to detect only the active form of β-lactam antibiotics with high affinities for both penicillins and cephalosporins. Moreover, the analysis took only 30 min.

Cysteamine core polyamidoamine G-4 dendron branched with β-cyclodextrins was chemisorbed on the surface of Au electrodes and further coated with Pt nanoparticles. Adamantane-modified glucose oxidase was subsequently immobilized on the nanostructured electrode surface by supramolecular association. This enzyme electrode was used to construct a reagentless amperometric biosensor for glucose, making use of the electrochemical oxidation of H2O2 generated in the enzyme reaction. The amperometric response of the biosensor was rapid (6 s) and a linear function of glucose concentration between 5 and 705 μmol L(-1). The biosensor had a low detection limit of 2.0 μmol L(-1), sensitivity of 197 mA mol(-1) L cm(-2), and retained 94% of its initial response after storage for nine days at 4 °C.

A novel strategy for the construction of disposable amperometric affinity biosensors is described in this work. The approach uses a recombinant bacterial penicillin binding protein (PBP) tagged by an N-terminal hexahistidine tail which was immobilized onto Co(2+)-tetradentate nitrilotriacetic acid (NTA)-modified screen-printed carbon electrodes (SPCEs). The biosensor was employed for the specific detection and quantification of β-lactam antibiotics residues in milk, which was accomplished by means of a direct competitive assay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling. The amperometric response measured at -0.20 V versus the Ag pseudoreference electrode of the SPCE upon the addition of H2O2 in the presence of hydroquinone (HQ) as redox mediator was used as the transduction signal. The developed affinity sensor allowed limits of detection to be obtained in the low part-per-billion level for the antibiotics tested in untreated milk samples. Moreover, the biosensor exhibited a good selectivity against other antibiotics residues frequently detected in milk and dairy products. The analysis time was of approximately 30 min.

An integrated amperometric sucrose biosensor involving a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold disk electrode (AuE) and coimmobilization of the enzymes invertase (INV) and fructose dehydrogenase (FDH) as well as the redox mediator tetrathiafulvalene (TTF) by means of a dialysis membrane is reported. Amperometry in stirred solutions at a detection potential of +0.10 V provided a linear calibration plot for sucrose over the 1.2 × 10(-6)-3.0 × 10(-3) mol L(-1) concentration range, with a limit of detection of 3.6 × 10(-7) mol L(-1). The practical usefulness of the biosensor was demonstrated by determining sucrose in condensed milk and in an infant food reference material with good results. Additionally, the biosensor was implemented together with commercial fructose and glucose amperometric biosensors in a continuous flow system to perform the multiplexed quantification of sucrose, fructose and glucose in a single experiment. The operational characteristics of the biosensors in this novel flow system were evaluated and their applicability was demonstrated through the simultaneous determination of the three sugars in the above-mentioned reference material., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Mono-6-formyl-β-cyclodextrin moieties were attached to (3-aminopropyl)triethoxysilane-coated superparamagnetic Fe(3)O(4) nanoparticles by reductive alkylation with NaBH(3)CN. The oligosaccharide-capped core-shell nanoparticles were employed as support for the supramolecular immobilization of two different adamantane-modified enzymes, tyrosinase and xanthine oxidase, through host-guest interactions. The enzyme-modified nanomaterial was further used to magnetically modify carbon paste electrodes for constructing amperometric biosensors toward cathecol and xanthine. The tyrosinase and xanthine oxidase based biosensors showed excellent electroanalytical behaviours, with linear ranges of 100 nM-12 μM cathecol and 5.0-120 μM xanthine, sensitivities of 12 mA/M and 130 mA/M, and low detection limits of 22 nM and 2.0 μM, respectively. The supramolecular nature of the immobilization approach was confirmed by electroanalytical methods., (Copyright © 2012 Elsevier Inc. All rights reserved.)

This work demonstrates the design and fabrication of an all cyclo-olefin polymer based microfluidic device capable of capturing magnetic beads and performing electrochemical detection in a series of gold electrodes. The size of chip is of a microscope slide and features six independent measuring cells for multianalyte detection purposes. The aim of this work is to show that rapid prototyping techniques can be instrumental in the development of novel bioassays, particularly in clinical diagnosis applications. We show the successful determination of troponin-T, a cardiac disease marker, in the clinically relevant range of 0.05-1.0 ng/mL. This methodology achieves a detection limit of 0.017 ng/mL in PBS solutions, and is capable of detecting less than 1 ng/mL in a 1:50 human serum dilution., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A 3α-hydrosteroid biosensor for androsterone determination has been prepared by immobilizing the enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD) in a composite electrode platform constituted of a mixture of multi-walled carbon nanotubes (MWCNTs), octylpyridinium hexafluorophosphate (OPPF(6)) ionic liquid and NAD(+) cofactor. This configuration allowed the fast, sensitive and stable electrochemical detection of the NADH generated in the enzyme reaction. All the experimental variables involved in the preparation and performance of the enzyme biosensor were optimized. Amperometry in stirred solutions at +400 mV provided a linear calibration plot for androsterone in the 0.5-10 μM concentration range with a slope value more than 200-times higher than that previously reported. The detection limit achieved was 0.15 μM and a low value of the apparent Michaelis-Menten constant (K(app)(M)), 36.0 μM, similar to that reported for the enzyme in solution, was calculated. The 3α-HSD/MWCNTs/OPPF(6)/NAD(+) biosensor provided good results in the determination of androsterone in spiked human serum samples., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Glassy carbon electrodes modified with single-walled carbon nanotubes and a three-dimensional network of electropolymerized Au nanoparticles capped with 2-mercaptoethanesulfonic acid, p-aminothiophenol, and 1-adamantanethiol were used as hybrid electrochemical platforms for supramolecular immobilization of a synthesized artificial neoglycoenzyme of xanthine oxidase and β-cyclodextrin through host-guest interactions. The ensemble was further employed for the bioelectrochemical determination of xanthine. The biosensor showed fast amperometric response within 5 s and a linear behavior in the 50 nM to 9.5 μM xanthine concentration range with high sensitivity, 2.47 A/(M cm(2)), and very low detection limit of 40 nM. The stability of the biosensor was significantly improved and the interferences caused by ascorbic and uric acids were noticeably minimized by coating the electrode surface with a Nafion thin film.

The preparation and performance of a disposable amperometric immunosensor, based on the use of a selective capture antibody and screen-printed carbon electrodes (SPCEs), for the specific detection and quantification of sulfonamide residues in milk is reported. The antibody was covalently immobilized onto a 4-aminobenzoic acid (4-ABA) film grafted on the disposable electrode, and 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 mediator was used as transduction signal. The developed methodology showed very low limits of detection (in the low ppb level) for 6 sulfonamide antibiotics tested in untreated milk samples, and a good selectivity against other families of antibiotics residues frequently detected in milk and dairy products. 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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This work reports for the first time an electrochemical immunosensor for the determination of adrenocorticotropin hormone (ACTH). The immunoelectrode design involves the use of amino phenylboronic acid for the oriented immobilization of anti-ACTH antibodies onto screen-printed carbon modified electrode surfaces. A competitive immunoassay between the antigen and the biotinylated hormone for the binding sites of the immobilized antibody was performed. The electroanalytical response was generated by using alkaline phosphatase-labelled streptavidin and 1-naphtyl phosphate as the enzyme substrate. The electrochemical oxidation of the enzyme reaction product, 1-naphtol, measured by differential pulse voltammetry was employed to monitor the affinity reaction. Under the optimized working conditions, an extremely low detection limit of 18 pg/L was obtained. Cross-reactivity was evaluated against other hormones (cortisol, estradiol, testosterone, progesterone, hGH and prolactin) and the obtained results demonstrated an excellent selectivity. The developed immunosensor was applied to a human serum sample containing a certified amount of ACTH with good results., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this paper, an electrochemical magneto-immunosensor for the detection of human growth hormone (hGH) is described for the first time. The immunosensor involves the use of tosyl-activated magnetic microparticles (TsMBs) to covalently immobilize a monoclonal mAbhHG antibody. A sandwich-type immunoassay with a secondary pAbhGH antibody and anti-IgG labelled with alkaline phosphatase (anti-IgG-AP) was employed. TsMBs–mAbhGH–hGH–pAbhGH–anti-IgG-AP conjugates were deposited onto the surface of a screen-printed gold electrode using a small neodymium magnet, and electrochemical detection was performed by square-wave voltammetry upon the addition of 4-aminophenyl phosphate as the AP substrate. All the variables involved in the preparation of immunoconjugates and in the immunoassay protocol were optimized. A calibration curve for hGH was constructed with a linear range between 0.01 and 100 ng/mL (r = 0.998) and a limit of detection of 0.005 ng/mL. This value is nearly three orders of magnitude lower than that obtained using surface plasmon resonance (Treviño et al., Talanta 78:1011-1016, 2009). Furthermore, good repeatability, with RSD = 3% (n = 10) at the 1-ng/mL hGH level, was obtained. Cross-reactivity studies with other hormones demonstrated good selectivity. The magneto-immunosensor was applied to the analysis of human serum spiked with hGH at the 4- and 0.1-ng/mL levels. Mean recoveries of 96 ± 6% and 99 ± 2%, respectively, were obtained.

The preparation and characteristics of a disposable amperometric magnetoimmunosensor, based on the use of functionalized magnetic beads (MBs) and gold screen-printed electrodes (Au/SPEs), for the specific detection and quantification of Staphylococcal protein A (ProtA) and Staphylococcus aureus (S. aureus) is reported. An antiProtA antibody was immobilized onto ProtA-modified MBs, and a competitive immunoassay involving ProtA antigen labelled with HRP was performed. The resulting modified MBs were captured by a magnetic field on the surface of tetrathiafulvalene-modified Au/SPEs and the amperometric response obtained at -0.15 V vs the silver pseudo-reference electrode of the Au/SPEs after the addition of H2O2 was used as transduction signal. The developed methodology showed very low limits of detection (1 cfu S. aureus/mL of raw milk samples), and a good selectivity against the most commonly involved foodborne pathogens originating from milk. These features, together with a short analysis time (2 h), 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.

Nowadays, detecting the presence of bioterrorism and biohazard agents in environmental and food samples is of great concern, due to their toxicity, and because many of them are prone to be used in terrorism attacks. The use of functionalized magnetic beads (MBs) in the development of electrochemical immuno- and genosensors has resulted in innovative and powerful detection strategies that may be applied to environmental, food and clinical analysis. This review describes current research on the combination of functionalized MBs with electrochemical detection for the development of magnetobiosensors applied to rapid, sensitive and specific detection of bioterrorism and biohazard agents., (Copyright © 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

Polyfuntionalized gold nanoparticles were prepared by using 2-mercaptoethanesulfonic acid, p-aminothiophenol and cysteamine core polyamidoamine G-4 dendron as capping ligands. The nanoparticles were electropolymerized on a Au electrode surface through the formation of a bisaniline-cross-linked network. The enzyme tyrosinase was further crosslinked on this nanostructured matrix. The enzyme electrode, poised at -100 mV, was used for the amperometric quantification of cathecol. The biosensor showed a linear response from 50 nM to 10 μM cathecol, with a low detection limit of 20 nM and a sensitivity of 1.94 A M(-1) cm(2). The electrode retained 96% and 67% of its initial activity after 16 and 30 days of storage at 4 °C under dry conditions.

The design of a novel biosensing electrode surface, combining the advantages of magnetic ferrite nanoparticles (MNPs) functionalized with glutaraldehyde (GA) and poly(diallyldimethylammonium chloride) (PDDA)-coated multiwalled carbon nanotubes (MWCNTs) as platforms for the construction of high-performance multienzyme biosensors, is reported in this work. Before the immobilization of enzymes, GA-MNP/PDDA/MWCNT composites were prepared by wrapping of carboxylated MWCNTs with positively charged PDDA and interaction with GA-functionalized MNPs. The nanoconjugates were characterized by scanning electron microscopy (SEM) and electrochemistry. The electrode platform was used to construct a bienzyme biosensor for the determination of cholesterol, which implied coimmobilization of cholesterol oxidase (ChOx) and peroxidase (HRP) and the use of hydroquinone as redox mediator. Optimization of all variables involved in the preparation and analytical performance of the bienzyme electrode was accomplished. At an applied potential of -0.05 V, a linear calibration graph for cholesterol was obtained in the 0.01-0.95 mM concentration range. The detection limit (0.85 μM), the apparent Michaelis-Menten constant (1.57 mM), the stability of the biosensor, and the calculated activation energy can be advantageously compared with the analytical characteristics of other CNT-based cholesterol biosensors reported in the literature. Analysis of human serum spiked with cholesterol at different concentration levels yielded recoveries between 100% and 103%, (© 2011 American Chemical Society)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A disposable magnetogenosensor for the rapid, specific and sensitive detection of Streptococcus pneumoniae is reported. The developed procedure involves the use of streptavidin-modified magnetic beads, a specific biotinylated capture probe that hybridizes with a specific region of lytA, the gene encoding the pneumococcal major autolysin, and appropriate primers for asymmetric polymerase chain reaction (PCR) amplification. Capture probes and amplicons specific for S. pneumoniae were selected by a careful analysis of all lytA alleles available. The selected primers amplify a 235-bp fragment of pneumococcal lytA. A detection limit (LOD) of 5.1 nM was obtained for a 20-mer synthetic target DNA without any amplification protocol, while the LOD for the asymmetric PCR amplicon was 1.1 nM. A RSD value of 6.9% was obtained for measurements carried out with seven different genosensors for 1.1-nM aPCR product. The strict specificity of the designed primers was demonstrated by aPCR amplification of genomic DNA prepared from different bacteria, including some closely related streptococci. Direct asymmetric PCR (daPCR), using cells directly from broth cultures of S. pneumoniae, showed that daPCR products could be prepared with as few as 2 colony-forming units (CFU). Furthermore, this methodology did not show any cross-reaction with closely related streptococci such as Streptococcus mitis (or Streptococcus pseudopneumoniae) even when present in the culture at concentrations up to 10(5) times higher than that of S. pneumoniae. Preliminary data for rapid detection of pneumococcus directly in clinical samples has shown that it is possible to discriminate between non-inoculated blood and urine samples and samples inoculated with only 10(3) CFU mL(-1)  S. pneumoniae.

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is widely distributed in nature, being a major phenolic pollutant and a commonly used antioxidant and building-block for drug development. We have characterized the first complete cluster (gal genes) responsible for growth in GA in a derivative of the model bacterium Pseudomonas putida KT2440. GalT mediates specific GA uptake and chemotaxis, and highlights the critical role of GA transport in bacterial adaptation to GA consumption. The proposed GA degradation via the central intermediate 4-oxalomesaconic acid (OMA) was revisited and all enzymes involved have been identified. Thus, GalD is the prototype of a new subfamily of isomerases that catalyses a biochemical step that remained unknown, i.e. the tautomerization of the OMAketo generated by the GalA dioxygenase to OMAenol. GalB is the founding member of a new family of zinc-containing hydratases that converts OMAenol into 4-carboxy-4-hydroxy-2-oxoadipic acid (CHA). galC encodes the aldolase catalysing CHA cleavage to pyruvic and oxaloacetic acids. The presence of homologous gal clusters outside the Pseudomonas genus sheds light on the evolution and ecology of the gal genes in GA degraders. The gal genes were used for expanding the metabolic abilities of heterologous hosts towards GA degradation, and for engineering a GA cellular biosensor., (© 2010 Blackwell Publishing Ltd.)

Disposable amperometric magnetoimmunosensors, based on the use of functionalized magnetic beads and gold screen-printed electrodes, have been developed for the selective detection and quantification of Streptococcus pneumoniae. A specific antibody prepared against a serotype 37 S. pneumoniae strain, selected by flow cytometry among seven anticapsular or antisomatic antibodies, was linked to Protein A-modified magnetic beads and incubated with bacteria. The same antibody, conjugated with horseradish peroxidase, was attached to the bacteria and the resulting modified magnetic beads were captured by a magnetic field on the surface of tetrathiafulvalene-modified gold screen-printed electrodes. The amperometric response obtained at -0.15 V vs. the silver pseudoreference electrode of the Au/SPE after the addition of H(2)O(2) was used as transduction signal. Different assay formats were examined and the experimental variables optimized. The limits of detection achieved, without pre-concentration or pre-enrichment steps, were 1.5×10(4) cfu mL(-1) (colony forming unit) and 6.3×10(5) cfu mL(-1) for S. pneumoniae strains Dawn (serotype 37) and R6 (non-encapsulated), respectively. The developed methodology shows a good selectivity against closely related streptococci and its usefulness for the analysis of inoculated urine samples has been demonstrated. The total analysis time of 3.5 h from sampling to measurement, the possibility to prepare up to 30 sensors per day and the use of small amounts of test solution for S. pneumoniae identification, constitute important advantages that make the developed methodology a promising alternative for clinical diagnosis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A disposable electrochemical immunosensor using screen-printed carbon electrodes (SPCEs) and protein A-functionalized magnetic beads (MBs) was developed for the determination of testosterone. Anti-testosterone was immobilized onto MBs and a direct competitive immunoassay involving testosterone labeled with peroxidase (HRP) was performed. The resulting conjugate was trapped on the SPCE with a small magnet. Testosterone determination was carried out by amperometry at -0.2V upon H2O2 additions using hydroquinone (HQ) as the redox mediator. The experimental variables involved in the immunosensor response to testosterone were evaluated. Under the optimized conditions, a calibration plot for testosterone was obtained with a linear range between 5.0×10(-3) and 50 ng/mL (r=0.995). The detection limit was 1.7 pg/mL and the EC50 was 0.25±0.04 ng/mL. These characteristics are notably better than those achieved with other reported immunosensors. Furthermore, anti-testosterone/MBs conjugates were shown to be stable for at least 25 days. A good selectivity was also found against other steroid hormones. The usefulness of the immunosensor was demonstrated by analyzing human serum spiked with 1 and 10 ng/mL testosterone., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A novel electrochemical immunosensor using screen-printed carbon electrodes and functionalized magnetic particles was developed for the determination of cortisol. Anti-cortisol antibody was immobilized onto protein A-modified magnetic particles and a direct competitive immunoassay involving cortisol antigen labeled with alkaline phosphatase (AP) was employed. The resulting conjugate was trapped on the surface of the screen-printed electrode with a small magnet. Cortisol quantification was accomplished by using 1-naphthyl phosphate as AP substrate and differential pulse voltammetry for the detection of the formed 1-naphthol. The variables involved in the preparation of the immunosensor and in the electrochemical detection reaction were optimized. The calibration plot obtained for cortisol exhibited a linear range between 5.0 x 10(-3) and 150 ng mL(-1) (r = 0.993). The limit of detection was 3.5 pg mL(-1), and the EC(50) was 0.19 ng mL(-1). These results demonstrate the high sensitivity achieved with this immunosensor design which compares favourably with other previous reports. The immunosensor also exhibited a high selectivity with respect to other corticosteroid compounds closely related to cortisol. The usefulness of the immunosensor for the analysis of real samples was demonstrated by analyzing certified human sera containing cortisol at two different concentration levels.