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The glycosylation status of proteins is increasingly used as biomarker to improve the reliability in the diagnosis and prognosis of diseases as relevant as cancer. This feeds the need for tools that allow its simple and reliable analysis and are compatible with applicability in the clinic. With this objective in mind, this work reports the first bioelectronic immunoplatforms described to date for the determination of glycosylated haptoglobin (Hp) and the simultaneous determination of total and glycosylated Hp. The bioelectronic immunoplatform is based on the implementation of non-competitive bioassays using two different antibodies or an antibody and a lectin on the surface of commercial magnetic microcarriers. The resulting bioconjugates are labeled with the horseradish peroxidase (HRP) enzyme, and after their magnetic capture on disposable electroplatforms, the amperometric transduction using the H2O2/hydroquinone (HQ) system allows the single or multiple detection. The developed immunoplatform achieves limits of detection (LODs) of 0.07 and 0.46 ng mL-1 for total and glycosylated Hp in buffer solution, respectively. The immunoplatform allows accurate determination using simple and relatively short protocols (approx. 75 min) of total and glycosylated Hp in the secretomes of in vitro-cultured colorectal cancer (CRC) cells with different metastatic potentials, which is not feasible, due to lack of sensitivity, by means of some commercial ELISA kits and Western blot methodology. Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Spanish Ministerio de Ciencia e Innovación (PID2019-103899RB-I00 and RTI2018-095756-B-I00), AES-ISCIII Program co-founded by FEDER funds (PI17CIII/00045 and PI20CIII/00019 grants), TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349). Sí

This paper reports a simple electrochemical strategy for the determination of microRNAs (miRNAs) using a commercial His-Tag-Zinc finger protein (His-Tag-ZFP) that binds preferably (but non-sequence specifically) RNA hybrids over ssRNAs, ssDNAs, and dsDNAs. The strategy involves the use of magnetic beads (His-Tag-Isolation-MBs) as solid support to capture the conjugate formed in homogenous solution between His-Tag-ZFP and the dsRNA homohybrid formed between the target miRNA (miR-21 selected as a model) and a biotinylated synthetic complementary RNA detector probe (b-RNA-Dp) further conjugated with a streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The electrochemical detection is carried out by amperometry at disposable screen-printed carbon electrodes (SPCEs) (- 0.20 V vs Ag pseudo-reference electrode) upon magnetic capture of the resultant magnetic bioconjugates and H2O2 addition in the presence of hydroquinone (HQ). The as-prepared biosensor exhibits a dynamic concentration range from 3.0 to 100 nM and a detection limit (LOD) of 0.91 nM for miR-21 in just ~ 2 h. An acceptable discrimination was achieved between the target miRNA and other non-target nucleic acids (ssDNA, dsDNA, ssRNA, DNA-RNA, miR-122, miR-205, and single central- or terminal-base mismatched sequences). The biosensor was applied to the analysis of miR-21 from total RNA (RNAt) extracted from epithelial non-tumorigenic and adenocarcinoma breast cells without target amplification, pre-concentration, or reverse transcription steps. The versatility of the methodology due to the ZFP's non-sequence-specific binding behavior makes it easily extendable to determine any target RNA only by modifying the biotinylated detector probe.

A magnetic beads (MB)-involved amperometric immunosensor for the determination of ST2, a member of the IL1 receptor family, is reported in this work. The method utilizes a sandwich immunoassay and disposable screen-printed carbon electrodes (SPCEs). Magnetic immunoconjugates built on the surface of carboxylic acid-microsized magnetic particles (HOOC-MBs) were used to selectively capture ST2. A biotinylated secondary antibody further conjugated with a streptavidin peroxidase conjugate (Strep-HRP) was used to accomplish the sandwiching of the target protein. The immune platform exhibits great selectivity and a low limit of detection (39.6 pg mL ) for ST2, allowing the determination of soluble ST2 (sST2) in plasma samples from healthy individuals and patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) in only 45 min once the immunoconjugates have been prepared. The good correlation of the obtained results with those provided by an ELISA kit performed using the same immunoreagents demonstrates the potential of the developed strategy for early diagnosis and/or prognosis of the fatal PDAC disease., This research was funded by Spanish Ministerio de Ciencia e Innovación. PID2019- 103899RB-I00, Spanish Ministerio de Ciencia, Innovación y Universidades, RTI2018-095672-B-I00, Comunidad de Madrid, S2018/NMT-4349, and AES-ISCIII/FEDER, PI20/00625

Metastasis is responsible for about 90% of cancer-associated deaths. In the context of solid tumors, the low oxygen concentration in the tumor microenvironment (hypoxia) is one of the key factors contributing to metastasis. Tumor cells adapt to these conditions by overexpressing certain proteins such as programmed death ligand 1 (PD-L1) and hypoxia-inducible factor 1 alpha (HIF-1α). However, the determination of these tumor hypoxia markers that can be used to follow-up tumor progression and improve the efficiency of therapies has been scarcely addressed using electrochemical biosensors. In this work, we report the first electrochemical bioplatform for the determination of PD-L1 as well as the first one allowing its simultaneous determination with HIF-1α. The target proteins were captured and enzymatically labeled on magnetic microbeads and amperometric detection was undertaken on the surface of screen-printed dual carbon electrodes using the hydrogen peroxide/peroxidase/hydroquinone system. Sandwich immunoassays were implemented for both the HIF-1α and PD-L1 sensors and the analytical characteristics were evaluated providing LOD values of 86 and 279 pg mL−1 for the amperometric determination of PD-L1 and HIF-1α standards, respectively. The developed electrochemical immunoplatforms are competitive versus the only electrochemical immunosensor reported for the determination of HIF-1α and the “gold standard” ELISA methodology for the single determination of both proteins in terms of assay time, compatibility with the simultaneous determination of both proteins making their use suitable for untrained users at the point of attention. The dual amperometric immunosensor was applied to the simultaneous determination of HIF-1α and PD-L1 in cancer cell lysates. The analyses lasted only 2 h and just 0.5 μg of the sample was required.

Harmful algal blooms (HABs) are more frequent as climate changes and tropical toxic species move northward, especially along the Iberian Peninsula, a rich aquaculture area. Monitoring programs, detecting the presence of toxic algae before they bloom, are of paramount importance to protect ecosystems, aquaculture, human health and local economies. Rapid, reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention as an alternative to the legally required but impractical microscopic counting-based techniques. Our electrochemical detection system has improved, moving from conventional sandwich hybridization protocols using different redox mediators and signal probes with different labels to a novel strategy involving the recognition of RNA heteroduplexes by antibodies further labelled with bacterial antibody binding proteins conjugated with multiple enzyme molecules. Each change has increased sensitivity. A 150-fold signal increase has been produced with our newest protocol using magnetic microbeads (MBs) and amperometric detection at screen-printed carbon electrodes (SPCEs) to detect the target RNA of toxic species. We can detect as few as 10 cells L&minus, 1 for some species by using a fast (~2 h), simple (PCR-free) and cheap methodology (~2 EUR/determination) that will allow this methodology to be integrated into easy-to-use portable systems.

Nowadays, electrochemical biosensors are reliable analytical tools to determine a broad range of molecular analytes because of their simplicity, affordable cost, and compatibility with multiplexed and point-of-care strategies. There is an increasing demand to improve their sensitivity and selectivity, but also to provide electrochemical biosensors with important attributes such as near real-time and continuous monitoring in complex or denaturing media, or in vivo with minimal intervention to make them even more attractive and suitable for getting into the real world. Modification of biosensors surfaces with antibiofouling reagents, smart coupling with nanomaterials, and the advances experienced by folded-based biosensors have endowed bioelectroanalytical platforms with one or more of such attributes. With this background in mind, this review aims to give an updated and general overview of these technologies as well as to discuss the remarkable achievements arising from the development of electrochemical biosensors free of reagents, washing, or calibration steps, and/or with antifouling properties and the ability to perform continuous, real-time, and even in vivo operation in nearly autonomous way. The challenges to be faced and the next features that these devices may offer to continue impacting in fields closely related with essential aspects of people’s safety and health are also commented upon.

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.

Proteases are involved in cancer‚ taking part in immune (dis)regulation, malignant progression and tumour growth. Recently, it has been found that expression levels of one of the members of the serine protease family, trypsin, is upregulated in human cancer cells of several organs, being considered as a specific cancer biomarker. Considering the great attention that electrochemical peptide sensors have nowadays, in this work, we propose a novel electroanalytical strategy for the determination of this important biomolecule. It implies the immobilization of a short synthetic peptide sequence, dually labelled with fluorescein isothiocyanate (FITC) and biotin, onto neutravidin-modified magnetic beads (MBs), followed by the peptide digestion with trypsin. Upon peptide disruption, the modified MBs were incubated with a specific fluorescein Fab fragment antibody labelled with horseradish peroxidase (HRP-antiFITC) and magnetically captured on the surface of a screen-printed carbon electrode (SPCE), where amperometric detection was performed using the hydroquinone (HQ)/HRP/H2O2 system. The biosensor exhibited a good reproducibility of the measurements (RSD 3.4%, n = 10), and specificity against other proteins and proteases commonly found in biological samples. This work reports the first quantitative data so far on trypsin expression in human cell lysates. The developed bioplatform was used for the direct determination of this protease in lysates from pancreatic cancer, cervix carcinoma and kidney cells in only 3 h and 30 min using low amounts (~ 0.1 μg) of raw extracts.

This work describes the preparation of an immunoplatform for the sensitive and selective determination of N6-methyladenosine (m6A). The simple and fast protocol involves for the first time the use of micromagnetic immunoconjugates to establish a direct competitive assay between the m6A target and a biotinylated RNA oligomer bearing a single m6A enzymatically labelled with a commercial conjugate of streptavidin-peroxidase (Strep-HRP) as tracer. The cathodic current change measured in the presence of H2O2/hydroquinone (HQ) at screen-printed carbon electrodes (SPCEs) upon surface capturing the magnetic bioconjugates is inversely proportional to the m6A target concentration. After evaluating the effect of key variables, the analytical characteristics were established for the determination of three different targets: the N6-methyladenosine-5′-triphosphate (m6ATP) ribonucleotide, a short synthetic RNA oligomer bearing a single m6A and the positive control provided in a commercial colorimetric kit for m6A-RNA quantification. The obtained results show that this immunoplatform is competitive with other methods reported to date, achieving an improved sensitivity (limit of detection of 0.9 pM for the short synthetic oligomer) using a much simpler and faster protocol (~1 h) and disposable electrodes for the transduction. Furthermore, the applicability for discriminating the metastatic potential of cancer cells by directly analyzing a small amount of raw total RNA without enriching or fragmenting was also preliminary assessed.

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-β 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.

This work reports a new sensitive strategy for the determination of tau protein, a hallmark of Alzheimer's disease (AD), involving a sandwich immunoassay and amperometric detection at disposable screen-printed carbon electrodes (SPCEs) modified with a gold nanoparticles-poly(amidoamine) (PAMAM) dendrimer nanocomposite (3D-Au-PAMAM) covalently immobilized onto electrografted p-aminobenzoic acid (p-ABA). The capture antibody (CAb) was immobilized by crosslinking with glutaraldehyde (GA) on the amino groups of the 3D-Au-PAMAM-p-ABA-SPCE, where tau protein was sandwiched with a secondary antibody labeled with horseradish peroxidase (HRP-DAb). Amperometry at −200 mV (vs the Ag pseudo-reference electrode) upon the addition of hydroquinone (HQ) as electron transfer mediator and H2O2 as the enzyme substrate was used to detect the immunocomplex formation. The great analytical performance of the immunosensor in terms of selectivity and low limit of detection (LOD) (1.7 pg mL−1) allowed the direct determination of the target protein in raw plasma samples and in brain tissue extracts from healthy individuals and post mortem diagnosed AD patients, using a simple and fast protocol.

A bioelectronic system composed of two modified electrodes, one activated in the presence of ketone bodies, a biomarker of diabetic ketoacidosis, and another releasing insulin upon receiving a signal, was designed and tested in vitro to operate as a Sense-and-Act device. The functional integration of biomarker-sensing and insulin-releasing electrodes represents a step to a theranostic system with autonomous operation.

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.

Nowadays, analyzing circulating tumor DNA (ctDNA), a very small part of circulating free DNA (cfDNA) carried by blood, is considered to be an interesting alternative to conventional single-site tumor tissue biopsies, both to assess tumor burden and provide a more comprehensive snapshot of the time-related and spatial heterogeneity of cancer genetic/epigenetic scenery. The determination of ctDNA and/or mapping its characteristic features, including tumor-specific mutations, chromosomal aberrations, microsatellite alterations, and epigenetic changes, are minimally invasive, powerful and credible biomarkers for early diagnosis, follow-up, prediction of therapy response/resistance, relapse monitoring, and tracking the rise of new mutant subclones, leading to improved cancer outcomes This review provides an outline of advances published in the last five years in electrochemical biosensing of ctDNA and surrogate markers. It emphasizes those strategies that have been successfully applied to real clinical samples. It highlights the unique opportunities they offer to shift the focus of cancer patient management methods from actual decision making, based on clinic-pathological features, to biomarker-driven treatment strategies, based on genotypes and customized targeted therapies. Also highlighted are the unmet hurdles and future key points to guide these devices in the development of liquid biopsy cornerstone tools in routine clinical practice for the diagnosis, prognosis, and therapy response monitoring in cancer patients.

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 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.

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.

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 H2O2 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.

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 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 +100 mV (vs. Ag/AgCl), and the reduction of TTF+ at -50 mV 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 5.2 × 10-7 to 2.0 × 10-5 and 4.2 × 10-7 to 2.0 × 10-5 M, respectively. The calculated detection limits were 5.2 × 10-7 and 4.2 × 10-7 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 748T 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. © 2010 Elsevier B.V. All rights reserved., The financial support of the Spanish Ministerio de Educación y Ciencia Research Project CTQ2006-02743BQU

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.

A new approach to bioelectronic Sense-and-Act systems was developed with the use of modified electrodes performing sensing and substance-releasing functions. The sensing electrode was activated by biomolecular/biological signals ranging from small biomolecules to proteins and bacterial cells. The activated sensing electrode generated reductive potential and current, which stimulated dissolution of an Fe(3+)-cross-linked alginate matrix on the second connected electrode resulting in the release of loaded biochemical species with different functionalities. Drug-mimicking species, antibacterial drugs, and enzymes activating a biofuel cell were released and tested for various biomedical and biotechnological applications. The studied systems offer great versatility for future applications in controlled drug release and personalized medicine. Their future applications in implantable devices with autonomous operation are proposed.

A very sensitive magnetoimmunosensor for the determination of β-lactoglobulin (β-LG) is reported in this work. A sandwich configuration involving covalent immobilization of the capture antibody (antiβ-LG) onto activated carboxylic-modified magnetic beads (HOOC-MBs) and incubation of the modified MBs with a horseradish peroxidase labeled antibody (HRP-antiβ-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 β-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 β-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 β-LG in dairy products.

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.

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. © 2013 The Royal Society of Chemistry., The financial support of the Spanish Ministerio de Economía y Competitividad Research Projects, CTQ2012-34238, and the AVANSENS Program from the Comunidad de Madrid (S2009PPQ-1642) are gratefully acknowledged. Felipe Conzuelo acknowledges a FPU fellowship from the Spanish Ministry of Education.

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 Co2+−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., The financial support of the Spanish Ministerio de Economía y Competitividad Research Projects, CTQ2012-34238, and the AVANSENS Program from the Comunidad de Madrid (S2009PPQ-1642) are gratefully acknowledged. Felipe Conzuelo acknowledges an FPU fellowship from the Spanish Ministry of Education.

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.

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.

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.

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.

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, rose, 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.