Search

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

Early diagnosis in primary care settings can increase access to therapies and their efficiency as well as reduce health care costs. In this context, we report in this paper the development of a disposable immunoplatform for the rapid and simultaneous determination of two protein biomarkers recently reported to be involved in the pathological process of neurodegenerative disorders (NDD), tau protein (tau), and TAR DNA-binding protein 43 (TDP-43). The methodology involves implementation of a sandwich-type immunoassay on the surface of dual screen-printed carbon electrodes (dSPCEs) electrochemically grafted with p-aminobenzoic acid (p-ABA), which allows the covalent immobilization of a gold nanoparticle-poly(amidoamine) (PAMAM) dendrimer nanocomposite (3D-Au-PAMAM). This scaffold was employed for the immobilization of the capture antibodies (CAbs). Detector antibodies labeled with horseradish peroxidase (HRP) and amperometric detection at − 0.20 V (vs. Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system were used. The developed methodology exhibits high sensitivity and selectivity for determining the target proteins, with detection limits of 2.3 and 12.8 pg mL−1 for tau and TDP-43, respectively. The simultaneous determination of tau and TDP-43 was accomplished in raw plasma samples and brain tissue extracts from healthy individuals and NDD-diagnosed patients. The analysis can be performed in just 1 h using a simple one-step assay protocol and small sample amounts (5 μL plasma and 2.5 μg brain tissue extracts).

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.

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.

Early diagnosis in primary care settings can increase access to therapies and their efficiency as well as reduce health care costs. In this context, we report in this paper the development of a disposable immunoplatform for the rapid and simultaneous determination of two protein biomarkers recently reported to be involved in the pathological process of neurodegenerative disorders (NDD), tau protein (tau), and TAR DNA-binding protein 43 (TDP-43). The methodology involves implementation of a sandwich-type immunoassay on the surface of dual screen-printed carbon electrodes (dSPCEs) electrochemically grafted with p-aminobenzoic acid (p-ABA), which allows the covalent immobilization of a gold nanoparticle-poly(amidoamine) (PAMAM) dendrimer nanocomposite (3D-Au-PAMAM). This scaffold was employed for the immobilization of the capture antibodies (CAbs). Detector antibodies labeled with horseradish peroxidase (HRP) and amperometric detection at - 0.20 V (vs. Ag pseudo-reference electrode) using the H

Edible electrochemical biosensors with remarkable prolonged resistance to extreme acidic conditions are described for direct glucose sensing in gastrointestinal (GI) fluids of different pH ranges and compositions. Such direct and stable glucose monitoring is realized using carbon-paste biosensors prepared from edible materials, such as olive oil and activated charcoal, shown to protect the activity of the embedded glucose oxidase (GOx) enzyme from strongly acidic conditions. The enzymatic resistance to low-pH deactivation allowed performing direct glucose monitoring in strong acidic environments (pH 1.5) over a 90-min period, while the response of conventional screen-printed (SP) biosensors decreased significantly following 10-min incubation in the same fluid. The developed edible biosensor displayed a linear response between 2 and 10 mM glucose with sensitivity depending on the pH of the corresponding GI fluid. In addition, coating the electrode surface with pH-responsive enteric coatings (Eudragit® L100 and Eudragit® E PO), of different types and densities, allows tuning the sensor activation in gastric and intestinal fluids at specific predetermined times. The attractive characteristics and sensing performance of these edible electrochemical biosensors, along with their pH-responsive actuation, hold considerable promise for the development of ingestible devices towards the biosensing of diverse target analytes after prolonged incubation in challenging body fluids. Graphical Abstract Edible biosensors allow direct electrochemical sensing in different gastrointestinal fluids and display remarkable prolonged resistance to extreme acidic conditions.

A sensitive and disposable amperometric immunosensor for Saccharomyces cerevisiae was constructed by using carbon screen-printed electrodes modified with propionic acid-functionalized graphene oxide as transduction element. The affinity-based biosensing interface was assembled by covalent immobilization of a specific polyclonal antibody on the carboxylate-enriched electrode surface via a water-soluble carbodiimide/N-hydroxysuccinimide coupling approach. A concanavalin A-peroxidase conjugate was further used as signaling element. The immunosensor allowed the amperometric detection of the yeast in buffer solution and white wine samples in the range of 10–107 CFU/mL. This electroanalytical device also exhibited low detection limit and high selectivity, reproducibility, and storage stability. The immunosensor was successfully validated in spiked white wine samples.

The assembly of a novel disposable amperometric immunosensor for the detection of the red wine spoilage yeast Brettanomyces bruxellensis is reported. The nanostructured sensing interface was prepared by first coating carbon screen printed electrodes with a gold nanoparticles-reduced graphene oxide hybrid nanomaterial, which was then modified with 3-mercaptopropionic acid to further immobilize specific antibodies for B. bruxellensis via a carbodiimide-coupling reaction. The functionalized electrode allowed the amperometric detection of B. bruxellensis in buffered solutions and red wine samples in the range of 10–106 CFU/mL and 102–106 CFU/mL, with low detection limits of 8 CFU/mL and 56 CFU/mL, respectively. The electrochemical immunosensor also exhibited high reproducibility, selectivity, and storage stability.

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.

Electrochemical nucleic-acid-based biosensing strategies involving the use of nanoparticles as electrode modifiers and as advanced labels are attractive options for the determination of substances that are relevant clinically, from an environmental perspective, and to food analysis, as these strategies are able to overcome some of the well-known limitations of conventional methodologies for routine applications. In this article, we provide a selective overview of current strategies for nucleic acid electrochemical biosensing based on nanoparticles, in order to demonstrate the relevance and potential of these strategies to readers familiar with this field and to non-experts. The benefits provided by the use of nanoparticles, including enhanced analytical performance of the resulting electrochemical biosensors, as well as the main challenges to be solved and potential future advances in this field are discussed.

A novel label-free electrochemical strategy for nucleic acid detection was developed by using gold electrodes coated with mesoporous silica thin films as sensing interface. The biosensing approach relies on the covalent attachment of a capture DNA probe on the surface of the silica nanopores and further hybridization with its complementary target oligonucleotide sequence, causing a diffusion hindering of an Fe(CN)6 (3-/4-) electrochemical probe through the nanochannels of the mesoporous film. This DNA-mesoporous silica thin film-modified electrodes allowed sensitive (91.7 A/M) and rapid (45 min) detection of low nanomolar levels of synthetic target DNA (25 fmol) and were successfully employed to quantify the endogenous content of Escherichia coli 16S ribosomal RNA (rRNA) directly in raw bacterial lysate samples without isolation or purification steps. Moreover, the 1-month stability demonstrated by these biosensing devices enables their advanced preparation and storage, as desired for practical real-life applications. Graphical abstract Mesoporous silica thin films as scaffolds for the development of novel label-free electrochemical genosensors to perform selective, sensitive and rapid detection of target oligonucleotide sequences. Application towards E. coli determination.

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.

8 páginas, 4 figuras, 1 tabla -- PAGS nros. 2413-2420, 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 105 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 103 CFU mL−1 S. pneumoniae, This research was supported by a grant from the Dirección General de Investigación Científica y Técnica (SAF2009-10824). CIBER de Enfermedades Respiratorias (CIBERES) is an initiative of Spanish Instituto de Salud Carlos III. The financial support of Santander/Complutense Research Project PR 27/05-13953, and of the Spanish Ministerio de Ciencia e Innovación Research Project CTQ2009-09351BQU, and the AVANSENS Program from the Comunidad de Madrid (S2009PPQ-1642) are also gratefully acknowledged. S.C. acknowledges a “Juan de la Cierva” research contract

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.

The electrochemical behaviour of some nitroaromatic explosives (2,4,6-trinitrotoluene, TNT; 2,6-dinitrotoluene, 2,6-DNT; 2-nitrotoluene, 2-NT; 2-amino-4,6-dinitrotoluene, 2-A-4,6-DNT; 3,5-dinitroaniline, 3,5-DNA; and nitrobenzene, NB) at electrochemically activated carbon-fibre microelectrodes is reported. Electrochemical activation of such electrode material by repeated square-wave (SW) voltammetric scans between 0.0 and +2.6 V versus Ag/AgCl, produced a dramatic increase in the cathodic response from these compounds. This is attributed to the increase of the carbon-fibre surface area, because of its fracture, and the appearance of deep fissures along the main fibre axis into which the nitroaromatic compounds penetrate. Based on the important contribution of adsorption and/or thin layer electrolysis to the total voltammetric response, a SW voltammetric method for rapid detection of nitroaromatic explosives was developed. No interference was found from compounds such as hydrazine, phenolic compounds, carbamates, triazines or surfactants. The limits of detection obtained are approximately 0.03 microg mL(-1) for all the nitroaromatic compounds tested. The method was applied for the determination of TNT in water and soil spiked samples; recoveries were higher than 95% in all cases.

An integrated amperometric fructose biosensor based on a gold electrode (AuE) modified with a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) on which fructose dehydrogenase (FDH) and the mediator tetrathiafulvalene (TTF) are co-immobilized by cross-linking with glutaraldehyde is reported. Variables concerning the behavior of the biosensor were optimized by taking the slope value obtained for the fructose calibration plot in the 0.1-1.0 mM concentration range as a criterion of selection. At an applied potential of +0.20 V, a good repeatability of such slope values (RSD=6.7%, n=10) was obtained with no need to apply a cleaning or pretreatment procedure to the modified electrode. Moreover, results from five different TTF-FDH-MPA-AuEs yielded a RSD of 5.8%. The useful lifetime of one single biosensor was approximately 30 days, exhibiting a 93% of the original response on the 33rd day. A linear calibration graph was obtained for fructose over the 1.0 x 10(-5)-1.0 x 10(-3) M range, with a limit of detection of 2.4 x 10(-6) M. The effect of potential interferents was evaluated. The TTF-FDH-MPA-AuE also performed well in the flow-injection mode. The biosensor was used for the determination of fructose in real samples, and the results compared with those provided by using a commercial enzyme test kit.

A novel magnetoimmunosensor design for interleukin-6 (IL-6) which involved the covalent immobilization of anti-IL-6 antibodies onto carboxyl-functionalized magnetic microparticles and a sandwich-type immunoassay with signal amplification using poly-HRP-streptavidin conjugates is reported. All the variables concerning the preparation and the electroanalytical performance of the immunosensor were optimized. The use of poly-HRP-strept conjugates as enzymatic labels instead of conventional HRP-strept allowed enhanced signal-to-blank current ratios to be obtained. A linear calibration plot between the measured steady-state current and the log of IL-6 concentration was achieved in the 1.75 to 500 pg/mL range, which was not feasible when using HRP-strep as label. A limit of detection of 0.39 pg/mL IL-6 was obtained. The anti-IL-6-MB conjugates exhibited an excellent storage stability providing amperometric responses with no significant loss during at least 36 days. The magnetoimmunosensor showed also an excellent selectivity against potentially interfering substances. The immunosensor was used to determine IL-6 in urine samples spiked at three different concentration levels with clinical relevance. Moreover, IL-6 was measured in three different saliva samples corresponding to a periodontitis patient, a smoker volunteer, and a non-smoker volunteer. The obtained results were statistically in agreement with those provided by a commercial ELISA kit.

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.

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-[N α,N α-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.

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.

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.

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 (Trevino 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.

An amperometric immunosensor for the quantification of Staphylococcus aureus based on the coimmobilization of rabbit immunoglobulin G (RbIgG) and tyrosinase on a mercaptopropionic acid self-assembled monolayer modified gold electrode is reported. A competitive mode in which protein-A-bearing S. aureus cells and antiRbIgG labeled with alkaline phosphatase (AP) compete for the binding sites of immobilized RbIgG was used. Monitoring of the affinity reaction was carried out by the amperometric detection at -0.15 V of phenol generated in the enzyme reaction with AP, at the tyrosinase-modified electrode through the electrochemical reduction of the o-quinone formed. Optimization of the working variables, such as the immunosensor composition and incubation times, the applied potential, the working pH and the concentration of phenyl phosphate used as the AP substrate, was carried out. Under the optimized conditions, both the repeatability of the measurements and the reproducibility of the responses obtained with different immunosensors yielded relative standard deviation values for the steady-state current lower than 10%. The immunosensor showed a dynamic range from 4.4x10(5) to 1.8x10(7) S. aureus cells mL(-1), with a detection limit of 1.7x10(5) cells mL(-1). The limit of detection was remarkably improved by subjecting S. aureus cells to wall lysis by heat treatment. The value obtained was 2.3x10(3) cells mL(-1), which is adequate for the monitoring of S. aureus contamination levels in some foodstuffs. As an application, milk samples spiked with bacteria at the 4.8x10(3) cells mL(-1) level were analyzed.

The voltammetric behaviour and amperometric detection of tetracycline (TC) antibiotics at multi-wall carbon nanotube modified glassy carbon electrodes (MWCNT-GCE) are reported. Cyclic voltammograms of TCs showed enhanced oxidation responses at the MWCNT-GCE with respect to the bare GCE, attributable to the increased active electrode surface area. Hydrodynamic voltammograms obtained by flow-injection with amperometric detection at the MWCNT-GCE led us to select a potential value E(det) = +1.20 V. The repeatability of the amperometric responses was much better than that achieved with bare GCE (RSD ranged from 7 to 12%), with RSD values for i (p) of around 3%, thus demonstrating the antifouling capability of MWCNT modified electrodes. An HPLC method with amperometric electrochemical detection (ED) at the MWCNT-GCE was developed for tetracycline, oxytetracycline (OTC), chlortetracycline and doxycycline (DC). A mobile phase consisting of 18:82 acetonitrile/0.05 mol L(-1) phosphate buffer of pH 2.5 was selected. The limits of detection ranged from 0.09 micromol L(-1) for OTC to 0.44 micromol L(-1) for DC. The possibility to carry out multiresidue analysis is demonstrated. The HPLC-ED/MWCNT-GCE method was applied to the analysis of fish farm pool water and underground well water samples spiked with the four TCs at 2.0 x 10(-7) mol L(-1). Solid-phase extraction was accomplished for the preconcentration of the analytes and clean-up of the samples. Recoveries ranged from 87 +/- 6 to 99 +/- 3%. Under preconcentration conditions, limits of detection in the water samples were between 0.50 and 3.10 ng mL(-1).

The performance of a molecularly imprinted polymer (MIP) as a selective solid-phase extraction sorbent for the clean-up and preconcentration of the antibiotic chloramphenicol is described. The MIP was prepared using chloramphenicol as the template, (diethylamino)ethyl methacrylate as the functional monomer, and ethylene glycol dimethacrylate as the cross-linking monomer, and using tetrahydrofuran as the solvent. Detection of chloramphenicol was carried out by square-wave voltammetry at electrochemically activated carbon fiber microelectrodes. Chloramphenicol was eluted from the MIP microcolumn with methanol. Different experimental variables (sample pH, eluent volume, analyte and eluent flow rates and sample volume) associated with the rebinding/elution process were optimized. For a 250 mL sample, a nominal enrichment factor of 500 was attained, and for a chloramphenicol concentration of 3.0x10(-8) mol L(-1) (9.7 microg L(-1)) a recovery of 96+/-4% was obtained. A range of linearity for chloramphenicol between 3.0x10(-8) and 1.0x10(-5) mol L(-1) was obtained by loading 17 mL of analyte solutions of different concentration, eluting with 0.5 mL methanol, evaporating under a stream of nitrogen and dissolving the residue in phosphate buffer of pH 7.8. The MIP selectivity was evaluated by checking several substances with similar molecular structures to that of chloramphenicol. The applicability of the MIP for both clean up and preconcentration was demonstrated by determining chloramphenicol in ophthalmic solutions and spiked milk at different concentration levels.