First electrochemical immunosensor for the rapid detection of mustard seeds in plant food extracts.

This paper describes the first biosensor reported to date for the determination of mustard seed traces. The biosensor consists of an amperometric immunosensing platform able to sensitively and selectively determine Sin a 1 content, the major allergen of yellow mustard and the most abundant protein of these seeds. The immunosensing platform exploits the coupling of magnetic microbeads (MBs) modified with sandwich-type immune complexes, comprising polyclonal and monoclonal antibodies, selective to the target protein for its capturing and detection, respectively. In addition, a HRP-conjugated secondary antibody was used for enzymatic labelling of the monoclonal antibody, and amperometric transduction was made at screen-printed carbon electrodes (SPCEs) using the hydroquinone (HQ)/H2O2 system. The electrochemical immunosensor allows the simple and fast detection (a single 1-h incubation step) of Sin a 1 with a limit of detection of 0.82 ng mL-1 (20.5 pg of protein in 25 µL of sample) with high selectivity against structurally similar non-target allergenic proteins (such as Pin p 1 from pine nut). The developed immunoplatform was successfully used for the analysis of peanut, rapeseed, cashew, pine nut and yellow mustard extracts, giving only positive response for the yellow mustard extract with a Sin a 1 content, in full agreement with that provided by conventional ELISA methodology.

Enlightening the advancements in electrochemical bioanalysis for the diagnosis of Alzheimer's disease and other neurodegenerative disorders.

Neurodegenerative disorders (NDD), and particularly Alzheimer's disease (AD), are one of the greatest challenges facing our current medicine and society because of its increasing incidence and the high burden imposed both on patients' families and health systems. Despite this, their accurate diagnosis, mostly conducted by cerebrospinal fluid (CSF) analysis or neuroimaging techniques, costly, time-consuming, and unaffordable for most of the population, remains a complex task. In this situation, electrochemical biosensors are flourishing as promising alternative tools for the simple, fast, and low-cost diagnosis of NDD/AD. This review article provides the relevant clinical details of NDD/AD along with the closely related genetic (genetic mutations, polymorphisms of ApoE and specific miRNAs) and proteomic (amyloid-ß peptides, total and phosphorylated tau protein) biomarkers circulating mostly in CSF. In addition, the article systematically enlightens a general view of the electrochemical affinity biosensors (mostly aptasensors and immunosensors) reported in the past two years for the determination of such biomarkers. The different developed strategies, analytical performances and applications are comprehensively discussed. Recent advancements in signal amplification methodologies involving smart designs and the use of nanomaterials and rational surface chemistries, as well as the challenges that must be struggled and the prospects in electrochemical affinity biosensing to bring more accessibility to NDD/AD diagnosis, prognosis, and follow-up, are also pointed out.

Electrochemical bioplatforms for the simultaneous determination of interleukin (IL)-8 mRNA and IL-8 protein oral cancer biomarkers in raw saliva.

The development of electrochemical magnetobiosensors for the simultaneous determination of two biomarkers associated with salivary oral cancer, protein IL-8 and its messenger RNA (IL-8 mRNA) associated, in undiluted human saliva samples is reported in this work. The implemented methodology involves the use of functionalized magnetic beads, specific antibodies against IL-8 protein, a specific hairpin DNA sequence for IL-8 mRNA and amperometric detection at disposable dual screen printed carbon electrodes. This methodology exhibits high sensitivity and selectivity for the target analytes providing detection limits of 0.21 nM for IL-8 mRNA and 72.4 pgmL(-1) (far below the clinical established cut-off of 600 pgmL(-1)) for IL-8 protein in undiluted saliva samples. The dual amperometric magnetobiosensor was applied to the direct determination of both biomarkers in spiked raw saliva samples and to determine the endogenous content of IL-8 protein in saliva samples from 7 healthy individuals. The obtained results were statistically in agreement with those provided by a commercial ELISA kit.

Electrochemical magnetoimmunosensing platform for determination of the milk allergen ß-lactoglobulin.

A very sensitive magnetoimmunosensor for the determination of ß-lactoglobulin (ß-LG) is reported in this work. A sandwich configuration involving covalent immobilization of the capture antibody (antiß-LG) onto activated carboxylic-modified magnetic beads (HOOC-MBs) and incubation of the modified MBs with a horseradish peroxidase labeled antibody (HRP-antiß-LG), is used. The resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE) and the amperometric responses are 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. The ß-LG magnetoimmunosensor exhibited a wide range of linearity (2.8-100 ng mL(-1)) and a low detection limit of 0.8 ng mL(-1) (20 pg in 25 µL sample). The magnetoimmunosensing platform was successfully applied for the detection of ß-LG in different types of milk without any matrix effect after just a sample dilution. The results correlated properly with those provided by a commercial ELISA method offering a truthful analytical screening tool. These features make the developed methodology a promising alternative in the development of user-friendly devices for on-site determination of ß-LG in dairy products.

Rapid screening of multiple antibiotic residues in milk using disposable amperometric magnetosensors.

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.

A novel non-invasive electrochemical biosensing device for in situ determination of the alcohol content in blood by monitoring ethanol in sweat.

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.

Disposable amperometric magnetoimmunosensors using nanobodies as biorecognition element. Determination of fibrinogen in plasma.

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.

Development of an integrated electrochemical biosensor for sucrose and its implementation in a continuous flow system for the simultaneous monitoring of sucrose, fructose and glucose.

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.

An amperometric affinity penicillin-binding protein magnetosensor for the detection of ß-lactam antibiotics in milk.

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 ß-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 ß-lactam antibiotics with high affinities for both penicillins and cephalosporins. Moreover, the analysis took only 30 min.

An integrated amperometric biosensor for the determination of lactose in milk and dairy products.

An integrated amperometric biosensor for the determination of lactose is reported. The bioelectrode design is based on the use of a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode on which the enzymes beta-galactosidase (beta-Gal), glucose oxidase (GOD), peroxidase (HRP) and the mediator tetrathiafulvalene (TTF) are coimmobilized by a dialysis membrane. beta-Gal catalyzes the hydrolysis of lactose, and the produced glucose is catalytically oxidized to gluconic acid and H(2)O(2), which is reduced in the presence of HRP. This enzyme reaction is mediated by TTF, and the reduction of TTF(+) at 0.00 V (vs Ag/AgCl) gives rise to an amperometric signal proportional to the lactose concentration. The biosensor exhibits a good repeatability of the measurement carried out with the same biosensor, a good reproducibility of the responses obtained with different biosensors and a useful lifetime of 28 days. A linear calibration plot was obtained for lactose over the 1.5 x 10(-6) to 1.2 x 10(-4) M concentration range, with a limit of detection of 4.6 x 10(-7) M. The effect of potential interferents (sucrose, lactulose, fructose, arabinose, maltose, galactose, glucose and uric and ascorbic acids) on the biosensor response was evaluated. Furthermore, the bioelectrode exhibits a suitable performance in flow-injection systems in connection with amperometric detection. The developed biosensor was applied to the determination of lactose in milk and other foodstuffs (chocolate, butter, margarine, yogurt, cheese and mayonnaise), and the results obtained were validated by comparison with those provided by using a commercial enzyme test kit.

Integrated multienzyme electrochemical biosensors for monitoring malolactic fermentation in wines.

Integrated amperometric biosensors for the determination of L-malic and L-lactic acids were developed by coimmobilization of the enzymes L-malate dehydrogenase (MDH) and diaphorase (DP), or L-lactate oxidase (LOX) and horseradish peroxidase (HRP), respectively, together with the redox 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 +100mV (vs. Ag/AgCl), and the reduction of TTF(+) at -50mV were used for the monitoring of the enzyme reactions involved in L-malic and L-lactic acid determinations, respectively. Experimental variables concerning the biosensors composition and the detection conditions were optimized for each biosensor. Good relative standard deviation values were obtained in both cases for the measurements carried out with the same biosensor, with no need of cleaning or pretreatment of the bioelectrodes surface, and with different biosensors constructed in the same manner. After 7 days of continuous use, the MDH/DP biosensor still exhibited 90% of the original sensitivity, while the LOX/HRP biosensor yielded a 91% of the original response after 5 days. Calibration graphs for L-malic and L-lactic were obtained with linear ranges of 5.2x10(-7) to 2.0x10(-5) and 4.2x10(-7) to 2.0x10(-5)M, respectively. The calculated detection limits were 5.2x10(-7) and 4.2x10(-7)M, respectively. The biosensors exhibited a high selectivity with no significant interferences. They were applied to monitor malolactic fermentation (MLF) induced by inoculation of Lactobacillus plantarum CECT 748(T) into a synthetic wine. Samples collected during MLF were assayed for L-malic and L-lactic acids, and the results obtained with the biosensors exhibited a very good correlation when plotted against those obtained by using commercial enzymatic kits.

Microorganisms recognition and quantification by lectin adsorptive affinity impedance.

Lectin-based screen-printed gold electrodes are reported for the impedimetric label-free detection of bacteria. The selective interaction of lectins with carbohydrate components from microorganisms surface was used as the recognition principle for their detection and identification. Electrochemical impedance spectroscopy (EIS) was employed for the direct label-free transduction of the bacteria-lectin binding. Biotinylated Concanavalin A (Con A) and Escherichia coli were used for the evaluation of the lectin-bacteria complex formation. This complex was formed in solution, and then adsorbed onto the gold SPE surface. No bacteria immobilization was observed on the sensor prepared in the absence of ConA, demonstrating the absence of non-specific bacteria adsorption onto the gold SPE. On the contrary, the changes in electron transfer resistance allowed monitoring of E. coli-biotinylated Con A complex formation without any amplification step. Experimental variables such as the biotinylated-Con A concentration and the bacteria-lectin incubation time were optimized. The electron transfer resistance varied linearly with the logarithmic value of E. coli concentration over four orders of magnitude, 5.0 x 10(3) and 5.0 x 10(7) cfu mL(-1). The selectivity of the approach was evaluated by checking the impedimetric responses of gold SPE modified with the complexes formed between nine lectins and three different bacteria (E. coli, Staphylococcus aureus and Mycobacterium phlei). Different response profiles were found when the different lectins were used as recognition elements. principal component analysis (PCA) allowed classification and distinction among bacteria. Finally, electrochemical monitoring of beta-galactosidase activity for the surface attached bacteria was demonstrated to be useful to distinguish between E. coli and S. aureus, which exhibit a similar affinity towards biotinylated-Con A.

Lectin-modified piezoelectric biosensors for bacteria recognition and quantification.

The use of lectins for microorganism biosensors fabrication is proposed. Lectins are immobilised onto a gold-plated quartz crystal for direct piezoelectric label-free transduction of the bacteria-lectin binding event using an electrochemical quartz crystal microbalance (EQCM). Concanavalin A (Con A) and Escherichia coli were used for the evaluation of the lectin immobilisation method and the biosensor performance. Adsorption on nonpolarised and polarised (-0.200 V) gold-coated quartz crystals and immobilisation through avidin-biotin binding were checked for Con A surface attachment. Lectin-bacteria binding was evaluated in all cases. With a crystal modified with Con A via avidin-biotin immobilisation we obtained a linear calibration plot between 5.0 x 10(6) and 2.0 x 10(7) cfu mL(-1) by measuring frequency changes with E. coli concentration 1 h after bacteria addition. A remarkable increase in sensitivity was achieved when the analytical solution contained free biotinylated Con A, as a consequence of multiple lectin adhesion to Escherichia coli cell wall, which produced an accumulation of Con A-E. coli conjugates in the form of multilayers at the electrode surface. A detection limit of approximately 1.0 x 10(4) cfu mL(-1) was achieved. Moreover nonspecific adsorptions were minimised. Using Con A and lectin from Arachis hypogaea, different response profiles were achieved for Escherichia coli, Staphylococcus aureus and Mycobacterium phlei, thus demonstrating the feasibility of bacteria discrimination. An approach involving filtering of free and lectin-bound bacteria and introduction of a filter in the measuring cell allowed a significant frequency change to be obtained for an E. coli concentration of 1.0 x 10(3) cfu mL(-1) in order to further increase the sensitivity and discriminate between viable and nonviable cells; an approach using electrochemical measurements of bacterial catalase activity was also checked.

Integrated multienzyme electrochemical biosensors for the determination of glycerol in wines.

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 +150mV (vs. Ag/AgCl), and the reduction of TTF(+) at 0mV 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 1.0x10(-6) to 2.0x10(-5) or 1.0x10(-6) to 1.0x10(-5)M glycerol and sensitivities of 1214+/-21 or 1460+/-34microAM(-1) were obtained with GDH/DP and GK/GPOx/HRP biosensors, respectively. The calculated detection limits were 4.0x10(-7) and 3.1x10(-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.

Integrated electrochemical gluconic acid biosensor based on self-assembled monolayer-modified gold electrodes. Application to the analysis of gluconic acid in musts and wines.

An integrated amperometric gluconic acid biosensor constructed using a gold electrode (AuE) modified with a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) on which gluconate dehydrogenase (GADH, 0.84 U) and the mediator tetrathiafulvalene (TTF, 1.5 micromol) were coimmobilized by covering the electrode surface with a dialysis membrane is reported. The working conditions selected were Eapp=+0.15 V and 25+/-1 degrees C. The useful lifetime of one single TTF-GADH-MPA-AuE was surprisingly long. After 53 days of continuous use, the biosensor exhibited 86% of the original sensitivity. A linear calibration plot was obtained for gluconic acid over the 6.0x10(-7) to 2.0x10(-5) M concentration range, with a limit of detection of 1.9x10(-7) M. The effect of potential interferents (glucose, fructose, galactose, arabinose, and tartaric, citric, malic, ascorbic, gallic, and caffeic acids) on the biosensor response was evaluated. The behavior of the biosensor in a flow-injection system in connection with amperometric detection was tested. The analytical usefulness of the biosensor was evaluated by determining gluconic acid in wine and must samples, and the results obtained were validated by comparison with those provided by using a commercial enzyme test kit.

Electrochemical estimation of the polyphenol index in wines using a laccase biosensor.

The use of a laccase biosensor, under both batch and flow injection (FI) conditions, for a rapid and reliable amperometric estimation of the total content of polyphenolic compounds in wines is reported. The enzyme was immobilized by cross-linking with glutaraldehyde onto a glassy carbon electrode. Caffeic acid and gallic acid were selected as standard compounds to carry out such estimation. Experimental variables such as the enzyme loading, the applied potential, and the pH value were optimized, and different aspects regarding the operational stability of the laccase biosensor were evaluated. Using batch amperometry at -200 mV, the detection limits obtained were 2.6 x 10(-3) and 7.2 x 10(-4) mg L(-1) gallic acid and caffeic acid, respectively, which compares advantageously with previous biosensor designs. An extremely simple sample treatment consisting only of an appropriate dilution of wine sample with the supporting electrolyte solution (0.1 mol L(-1) citrate buffer of pH 5.0) was needed for the amperometric analysis of red, rosé, and white wines. Good correlations were found when the polyphenol indices obtained with the biosensor (in both the batch and FI modes) for different wine samples were plotted versus the results achieved with the classic Folin-Ciocalteu method. Application of the calibration transfer chemometric model (multiplicative fitting) allowed that the confidence intervals (for a significance level of 0.05) for the slope and intercept values of the amperometric index versus Folin-Ciocalteu index plots (r = 0.997) included the unit and zero values, respectively. This indicates that the laccase biosensor can be successfully used for the estimation of the polyphenol index in wines when compared with the Folin-Ciocalteu reference method.