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This work describes the development of an ELIME (Enzyme-Linked ImmunoMagnetic Electrochemical) assay for a sensitive detection of specific IgE (sIgE) towards the aeroallergens G5 and D2 in serum samples. The system, with a detection limit of 0.1 KIU/L, uses magnetic beads as support of the immunological chain and a strip of eight-magnetized screen-printed electrodes, connected to a portable instrument, for a multiplexed detection. The analysis of 46 serum samples performed using both the ELIME and the Immulite 2000 assays, suggests that our method is able to measure very low concentration of sIgE and to discriminate all positive samples from negative ones. [ABSTRACT FROM AUTHOR]

The bulk synthesis of freestanding carbon nanotube (CNT) frameworks is developed through a sulfur-addition strategy during an ambient-pressure chemical vapour deposition process, with ferrocene used as the catalyst precursor. This approach enhances the CNTs’ length and contorted morphology, which are the key features leading to the formation of the synthesized porous networks. We demonstrate that such a three-dimensional structure selectively uptakes from water a mass of toxic organic solvent (i.e. o-dichlorobenzene) about 3.5 times higher than that absorbed by individual CNTs. In addition, owing to the presence of highly defective nanostructures constituting them, our samples exhibit an oil-absorption capacity higher than that reported in the literature for similar CNT sponges. [ABSTRACT FROM AUTHOR]

An amperometric biosensor for the determination of L-lactic acid in probiotic yogurts has been assembled using L-lactate dehydrogenase (EC 1.1.1.27, LDH) entrapped in 1% (v/v) neutralized Nafion® solution deposited on Variamine blue redox mediator modified screen-printed electrodes. The Variamine blue was previously covalently linked to oxidized single-walled carbon nanotubes and used for modifying screen-printed electrodes. The electrochemical cell, containing the L-lactate biosensor operating at an applied working potential of +200 mV vs. Ag|AgCl, was coupled with a microdialysis fiber and connected with a flow system, thus obtaining a microdialysis based sampling experimental set-up. Various analytical parameters, such as the cofactor concentration (2 mM, NAD+), the flow rate (10.5 μL/min), the applied working potential (+200 mV vs. Ag|AgCl), the working buffer (50 mM phosphate buffer +0.1 M KCl), and pH (7.5), were optimized in batch amperometric experiments. The dynamic linear working range was comprised between 2·10-4 and 1·10-3 M. The proposed biosensor was challenged with real samples of yogurt, properly diluted in working buffer, and the performances of the L-lactate biosensor were compared with a commercially available kit for the determination of L-lactic acid in foodstuffs from R-Biopharm GmbH, Germany, showing a good agreement. [ABSTRACT FROM AUTHOR]

An electrochemical biosensor to detect biogenic amines in saliva was developed. This biosensor is based on a Prussian Blue screen-printed electrode, which senses the H2O2 produced by the reaction catalyzed by the diamine oxidase enzyme. The probe was connected with a portable instrument and chronoamperometric drop measurements were performed. After evaluation of the biosensor's ability to interact with different amines, putrescine was chosen as the reference amine. A detection limit of 1 × 10-5 M and a working range between 2 × 10-5 and 3 × 10-4 M were obtained. Once optimized, the saliva treatment, a negligible matrix effect, and an average recovery of 105% were obtained. [ABSTRACT FROM AUTHOR]

The ultrafine (UF) component of airborne pollution may impair cardiovascular autonomic control, a high-risk condition for cardiovascular adverse events. Since engineered nanoparticles, such as single-walled carbon nanotubes (SWCNTs) share physicochemical properties with UF, they might have similar adverse effects. Aim of the study was to evaluate arterial baroreflex function (BRF) at baseline, 24 h after the first instillation, immediately before the second one, and 2 weeks later, in adult Wystar-Kyoto conscious rats undergoing two intratracheal instillations of SWCNT (eight rats) or phosphate buffer saline (PBS) (five rats) at 2-week interval. During each session, 30-min continuous recording of arterial pressure and pulse interval was performed by a telemetered catheter implanted in the abdominal aorta of the rats. BRF was studied by the sequence technique. SWCNTs dispersed in PBS (1 mg/ml) were administered immediately after sonication (1 μg/g body weight). A significant decrease in the number of baroreflex sequences (from 498 ± 27.1 at baseline to 287 ± 40.2 at the recording performed after 4 weeks; P < 0.05) was observed in SWCNT-instilled rats, whereas no significant change was detected in controls. These data suggest that SWCNTs may alter the BRF, thus affecting the autonomic cardiovascular control regulation. [ABSTRACT FROM AUTHOR]

This article describes the conjugation between aflatoxin B1 (AFB1), one of the major mycotoxins, and alkaline phosphatase (AP), one of the most used enzymes for immunoassays. In addition, an application of the ELISA method for aflatoxin B1 determination in corn is presented. Three AFB1-AP conjugates in different toxin-enzyme ratios were prepared and tested. The ELISA results, developed with the most effective conjugate obtained, showed a satisfactory working range between 2.4 and 4000 ng of toxin/g of corn. The detection limit was 2 ng/g in corn samples, and recoveries ranged from 105 to 120%. [ABSTRACT FROM AUTHOR]

The use of a glucose biosensor in conjunction with a microdialysis probe used to sample the interstitial fluid of the patient has been demonstrated to be extremely useful and advantageous for obtaining a continuous glucose monitoring instrument capable of detecting glycemic level in real time for a long period. The first example of this kind of instrument which was cleared for commercialization is the GlucoDay made by Menarini. The approach used by the GlucoDay presents several advantages if compared with other instruments and its features and future prospective are thoroughly discussed. [ABSTRACT FROM PUBLISHER]

In this work are presented methods for detection of aflatoxin B1 and type-A trichothecenes, based on the use of indirect competitive ELISA format coupled with a 96-well screen-printed microplate. Electrochemical immunoassays for AFB1, T-2, and HT-2 were performed and the activity of the alkaline phosphatase or horseradish peroxidase labeled enzymes were measured using intermittent pulse amperometry (IPA) as electrochemical technique. Using standard solutions of the target analyte the LOD of the assays were 0.3 and 0.2 ng ml-1 for T-2 and AFB1 respectively, while the sensitivity was 1.2 ng ml-1 for both. For Aflatoxin B1, a stability study of electrochemical plate was also performed. Moreover, the matrix effect was evaluated using two different extraction treatments from corn. [ABSTRACT FROM AUTHOR]

The aim of the present work was the development of an immunomagnetic electrochemical sensor (IMES) for a simple and fast detection of Bt‐Cry1Ab/Cry1Ac proteins in genetically modified corn flour. The IMES is based on a sandwich format using magnetic beads as immobilization support and disposable screen‐printed electrodes (SPEs) as electrochemical transducers. An alkaline phosphatase labeled antiglobulin (Ab 2 ‐AP) was used to reveal the sandwich complex between monoclonal anti‐Cry1Ab antibodies (MAb), Cry1Ab protein, and polyclonal anti‐Cry1Ab antibodies (PAb). After the immunochemisty steps, the beads were localized onto the surface of the SPEs with the aid of a magnet, prior to the addition of the enzyme substrate (1‐naphthylphosphate) and the measurement of the electroactive product. The current response was found to be directly proportional to the concentration of Cry1Ab protein. The limit of detection was calculated to be 0.1 ng mL -1 with a working range between 0.25 and 4 ng mL -1 and a total analysis time of about 3 h. This method, which was also able to detect Cry1Ac protein, was applied to genetically modified corn samples. After optimization of the extraction procedure certified reference materials (CRMs) with different mass fractions (0.5%, 1%, 2%, 5%) of dried powder prepared from genetically modified (MON 810) maize, were treated and analyzed in replicate in order to assess, in our conditions, the correspondence between the % of genetically modified material and the concentration of Cry1Ab protein (ng g -1 ). Finally, certified reference material with 0% of genitically modified organism (GMO), two maize samples with unknown % of GMO and the two corresponding control samples (with no GMO) were analyzed in order to verify the effectiveness of the IMES‐based assay. [ABSTRACT FROM AUTHOR]

The objective of the present work was to develop and evaluate an SYBR Green real time polymerase chain reaction (PCR) method for the specific detection of Salmonella spp in broth cultures and meat samples experimentally contaminated. Also, a simple and rapid multichannel electrochemical immunosensor (MEI) for this pathogen is under study. The PCR was carried out using primers ttr6 and ttr4 for the amplification of a highly conserved DNA region ( ttr sequence) specific for all Salmonella serovars. A boiling step, for the extraction of DNA, was combined with a real time PCR method based on the double‐stranded DNA (dsDNA) binding dye SYBR Green. The standard curve constructed using the mean threshold cycle and various concentrations of S. enteritidis (ranging fron 10 2 to 10 8 CFU mL -1 ) showed good linearity (R 2 =0.999) with the minimum level of detection of 10 2 CFU mL -1 . The experiments were conducted analyzing 30 Salmonella strains and 20 non‐ Salmonella strains. All Salmonella serotypes tested were ttr ‐positive and all other bacteria yielded no amplification products. The specificity of the reaction was confirmed by the melting temperature (T m ) of the amplicon obtained (T m =80.1±0.1). To verify the effectiveness of the assay, experiments were conducted on experimentally contaminated samples, which were also analyzed for comparison by the standard cultural method. A multichannel electrochemical immunosensor for detection of Salmonella also was developed. It consists of a disposable screen‐printed sensor array, coupled with a multichannel pulse monitor, which was assembled as an immunosensor through the use of specific monoclonal (MAb) and polyclonal (PAb) antibodies in a sandwich format. The limit of detection was calculated to be 2×10 6 UFC mL -1 with a working range between 5×10 6 to 5×10 8 UFC mL -1 and a total analysis time of about 3 h. This immunoelectrochemical system is economical, rapid, and easy to use but it is still under development in order to improve its analytical performance. [ABSTRACT FROM AUTHOR]

In the present work, an indirect competitive electrochemical enzyme linked immunosorbent assay (ELISA) has been used for determination of aflatoxin B 1 (AFB 1 ) in barley. The method involves the use of disposable screen‐printed carbon electrodes (SPCEs) and anti‐aflatoxin B 1 monoclonal antibodies (MAb) for immunosensor development. The specificity of the assay was assessed by studying the cross‐reactivity of the MAb relative to AFB 1 . The results indicated that the MAb could readily distinguish AFB 1 from other toxins, with the exception of AFG 1 . The stability of the coating reagents was evaluated using SPCEs coated with AFB 1 ‐bovine serum albumin (BSA) conjugate. The results showed that the coated electrodes could be used for up to one month after their preparation and storage at 4°C. Prior to evaluating the performance of the electrochemical immunosensor for AFB 1 with spiked samples, the effect of barley extract on assay performance was tested. Using this calibration method, the limit of detection (LOD) was found to be 90 pg mL -1 . The linear range was 0.1–10 ng mL -1 , and recoveries ranged from 100%–125%. The results obtained were confirmed by high performance liquid chromatography (HPLC) coupled with fluorescence detection. These results demonstrated the suitability of the proposed method for routine screening of AFB 1 in barley. [ABSTRACT FROM AUTHOR]

Different ELISA tests to detect and quantify levels of S. aureus in broth cultures were developed and compared. In all cases the assays were a modification of a "sandwich" format based on the use of common IgG as well as specific antibodies to bind protein A, an antigen localized in the cellular wall of S. aureus and partially extracted by boiling. Initially, human IgG was immobilized on the surface of microtitre plate wells in order to bind, by means of the Fc region, protein A that was present either in standard solutions or broth cultures of S. aureus treated by a boiling step. The sandwich format was completed using monoclonal (MAb) antibodies specific for protein A. The amount of bound antibody was evaluated using antiglobulins labelled with alkaline phosphatase (Ab2-AP). Reading the absorbance at 405 nm a detection limit (LOD) of 0.6ng/mL and 2 × 106 CFU/mL was found for protein A and for S. aureus, respectively. In order to improve the performance of the immunoassay, different approaches were pursued: an enzymatic amplification system (Ampli Q); the use of immunomagnetic beads employed both in a colorimetric (ELIMC Enzyme-Linked Immunomagnetic Colorimetric) and in an electrochemical (ELIME = Enzyme-Linked Immunomagnetic Electrochemistry) assay. Using these systems the detection limit decreased by a factor of about 30-fold for Ampli Q and ELIMC, and about 2000-fold for ELIME formats. In addition, a qualitative polymerase chain reaction (PCR) method, using nuc gene primers, was set up and performed in parallel and its various parameters were optimized. This method was able to detect 102 CFU/mL. In terms of minimum detectable concentration of S. aureus and total analysis time, the performance of the PCR assay and ELIME, turned out to be comparable. [ABSTRACT FROM AUTHOR]

An approach to improve the analytical performance of a Prussian Blue (PB)-based hydrogen peroxide transducer is described. In support of this objective, both the stabilizing and anti-interferent properties of nonconducting films were used. Electropolymerization on the top surface of PB modified electrodes is possible due to the high oxidizing ability of Berlin Green, and the growth of nonconductive polymers may be independently monitored by investigating the redox activity of the inorganic polycrystal. The best performance characteristics, which are advantageous over existing H2O2 sensors, were obtained for PB electrodes covered with electropolymerized o-phenylenediamine (1,2-diaminobenzene). The reported transducer remained at the 100% response state for more than 20 h under continuous flow of 0.1-mM hydrogen peroxide (flow rate 1 mlmin-1), which improves the stability level among the selective H2O2 sensors by one order of magnitude. The selectivity factor of the PB-poly (1,2-diaminobenzene) based transducer relative to ascorbate is nominally 600. PB-poly(1,2-diaminobenzene) modified electrode allows the detection hydrogen peroxide in the flow-injection mode down to 10-7 M with sensitivity of 0.3 AM-1cm-2, which is two times lower compared to the uncovered PB-based transducer. [ABSTRACT FROM PUBLISHER]

Diamine oxidase from Lens culinaris has been used for the amperometric detection of biogenic amines in cheese. The enzyme has been immobilised onto polymeric membranes or glass beads and coupled with a Pt based hydrogen peroxide electrode. The resulting enzyme electrode and reactor have been optimised for the response to the six biogenic amines more frequently found in cheese: histamine, tyramine, putrescine, cadaverine, phenylethylamine and tryptamine. Detection limits were found to be in the micromolar range with a linearity up to 3 orders of magnitude. Rejection of the electrochemically active compounds present in cheese was effective by the electropolymerization of 1,4-diaminebenzene onto the Pt surface. The best performances in terms of sensitivity and selectivity were obtained using the enzyme reactor. [ABSTRACT FROM AUTHOR]

Abstract: Novel planar glucose biosensors to be used for continuous monitoring have been developed. The electrodes are produced with the “screen printing” technique, and present a high degree of reproducibility together with a low cost and the possibility of mass production. Prior to enzyme immobilisation, electrodes are chemically modified with ferric hexacyanoferrate (Prussian Blue). This allows the detection of the hydrogen peroxide produced by the enzymatic reaction catalysed by GOD, at low applied potential (ca. 0.0V versus Ag/AgCl), highly limiting any electrochemical interferences. The layer of Prussian Blue (PB) showed a high stability at the working conditions (pH 7.4) and also after 1 year of storage dry at RT, no loss of activity was observed. The assembled glucose biosensors, showed high sensitivity towards glucose together with a long-term operational and storage stability. In a continuous flow system, with all the analytical parameters optimised, the glucose biosensors detected glucose concentration as low as 0.025mM with a linear range up to 1.0mM. These probes were also tested over 50–60h in a continuous flow mode to evaluate their operational stability. A 0.5mM concentration of glucose was continuously fluxed into a biosensor wall-jet cell and the current due to the hydrogen peroxide reduction was continuously monitored. After 50–60h, the drift of the signal observed was around 30%. Because of their high stability, these sensors suggest the possibility of using such biosensors, in conjunction with a microdialysis probe, for a continuous monitoring of glucose for clinical purposes. [Copyright &y& Elsevier]

Describes methods for the construction of Prussian Blued-based carbon paste electrodes. Deposition of prussian blue on graphite powder; Electrodeposition of prussian blue on carbon paste; Modification of solid carbon paste electrodes by enzymes; Sensitivity and stability over wide range of hydrogen-ion concentration.

Enzyme inhibitors like drugs and pollutants are closely correlated to human and environmental health, thus their monitoring is of paramount importance in analytical chemistry. Enzymatic biosensors represent cost-effective, miniaturized and easy to use devices; particularly biosensors based on enzyme inhibition are useful analytical tools for fast screening and monitoring of inhibitors. The present review will highlight the research carried out in the last 9 years (2006–2014) on biosensors based on enzyme inhibition. We underpin the recent advances focused on the investigation in new theoretical approachs and in the evaluation of biosensor performances for reversible and irreversible inhibitors. The use of nanomaterials and microfluidic systems as well as the applications of the various biosensors in real samples is critically reviewed, demonstrating that such biosensors allow the development of useful devices for a fast and reliable alarm system. [ABSTRACT FROM AUTHOR]

Abstract: A bienzyme electrochemical probe has been assembled and used to monitor the inhibition of the enzyme protein phosphatase-2A (PP2A) by okadaic acid (OA), taking advantage of the particular characteristics of a biochemical pathway in which PP2A is involved. This enzyme has significant activity toward glycogen phosphorylase a (PHOS a), which in turn catalyzes the conversion of glycogen to glucose-1-phosphate (G-1-P). In addition, PP2A is strongly inhibited by OA and its derivatives. Due to this combination of properties, PP2A was employed to develop an assay system involving a preliminary phase of off-line enzymatic incubations (OA/PP2A, PP2A/PHOS a, PHOS a/glycogen+phosphate). This off-line step was followed by the electrochemical detection of H2O2, which is the final product of two sequential enzymatic reactions: G-1-P with alkaline phosphatase (AP) producing glucose, then glucose with glucose oxidase (GOD) producing hydrogen peroxide. These two enzymes were coimmobilized on a nylon net membrane that was placed over an H2O2 platinum probe inserted into a flow injection analysis (FIA) system. During a first phase of the study, all analytical parameters were optimized. During a subsequent phase, the inhibition of PP2A enzyme by OA was evaluated. The calibration of the system shows a working range for detection of OA between 30 and 250 pg ml−1. The total analysis time is the sum of 50min for the off-line enzymatic incubations and 4min for the biosensor response. [Copyright &y& Elsevier]

Abstract: A novel continuous lactate monitoring system has been developed modifying the GlucoDay® portable medical device (A. Menarini Diagnostics), already present in the European market, and used to continuously measure glucose levels. Lactate oxidase based biosensors have been developed immobilising the enzyme on nylon net and placing it on a Pt electrode. The biosensor was connected to the portable device provided with a micro-pump and coupled to a microdialysis system. It is capable to record subcutaneous lactate every 3min. In vitro analytical results confirmed that the sensors respond linearly in the interval of concentration between 0.1 and 10mmol/L, covering the whole physiological range. During prolonged monitoring periods, the response of the biosensors remained stable, showing a limited drift of 8%, within 60h. Stability tests are still on route. However, preliminary results have shown a shelf life of about 10 months. In vivo experiments performed on healthy rabbits have demonstrated the good accuracy and reproducibility of the system. A correlation coefficient equal to 0.9547 (N=80) was found, which represents a good correlation between the GlucoDay® and the laboratory reference analyser. A 16h in vivo monitoring on a healthy volunteer has been also performed. [Copyright &y& Elsevier]