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In the last decades, there is a growing search for analytical strategies to ensure clinical analysis without the need of laboratory set-up and skilled personnel. Indeed, user-friendly and low-cost devices are highly valued in the era of sustainability for their capability to be applied in low-resource contexts, such as developing countries. To address this issue, herein we report a 96-well paper-based and laboratory setup-free optical platform for the detection of butyrylcholinesterase enzyme (BChE) activity in human serum. We used chromatographic paper to realize a novel analytical tool exploiting its porous structure for reagentless synthesize Prussian Blue Nanoparticles (the sensing element), as well to load all the reagents required for the measurement. The principle of BChE activity detection relies on the reaction between the enzymatic product thiocholine and Prussian Blue, giving the Prussian White with subsequently Prussian Blue's fading, detected by a common office scanner supported by ImageJ software. Using this novel paper-based optical platform, BChE activity was linearly detected in the 2-15 U/mL range with a detection limit down to 0.8 U/mL. The accuracy was successfully demonstrated by recovery study with spiked serum and by comparing the data with the gold standard method., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The synthesis and employment of volatile toxic compounds as chemical weapons with a large-scale destructive power has introduced a new insidious threat over the last century. In this framework, the development of wearable sensing tools represents a critical point within the security field, in order to provide early alarm systems. Herein, a novel wearable electrochemical biosensor was developed for the rapid and on-site detection of mustard agents. Since a chemical attack is typically carried out by spraying these volatile agents into air, the sensor was designed in order to be able to measure mustard agents directly in the aerosol phase, further than in the liquid phase. The electrodes were screen-printed onto a filter paper support, which allowed to harness the porosity of paper to pre-load all the needed reagents into the cellulose network, and hence to realise an origami-like and reagent-free device. Mustard agent detection was carried out by monitoring their inhibitory effects toward the choline oxidase enzyme, through the amperometric measurement of the enzymatic by-product hydrogen peroxide. A carbon black/Prussian blue nanocomposite was used as a bulk-modifier of the conductive graphite ink constituting the working electrode, allowing for the electrocatalysis of the hydrogen peroxide reduction. After having verified the detecting capability toward a mustard agent simulant, the applicability of the resulting origami-like biosensor was demonstrated for the rapid and real-time detection of real sulfur mustard, obtaining limits of detection equal to 1 mM and 0.019 g·min/m 3 for liquid and aerosol phase, respectively., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Herein, we report an impedimetric DNA-based aptamer sensor for a single-step detection of B. anthracis spore simulant (B. cereus spore). Specifically, we designed a miniaturized label-free aptasensor for B. cereus spores based on a gold screen-printed electrode functionalized with B. cereus spores-binding aptamer (BAS-6R). Several parameters were optimized to fabricate the aptasensor such as the concentration of DNA aptamer solution (0.5 µM), the time (48 h), the temperature (4 °C), and the pH (7.5) for aptamer immobilization on the working electrode surface. Once the aptasensor was developed, it was tested against B. cereus spores 14579 evaluating the effect of incubation time and MgCl 2 concentration. Under the optimized conditions (incubation time equal to 3 h and absence of MgCl 2 ), B. cereus spores 14579 were detected with a linear range between 10 4 CFU/ml and 5 × 10 6 CFU/ml and a detection limit of 3 × 10 3 CFU/ml. Furthermore, the study of selectivity toward B. cereus 11778, B. subtilis, Legionella pneumophila, and Salmonella Typhimurium has demonstrated the capability of this sensor to detect B. cereus spores, proving the suitability of the DNA-based sensing element combined with a portable instrument for a label-free measurement on site of B. anthracis spore simulant., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The article presents an original analysis which combines use-wear, 3D modelling and spatial analyses to experimental archaeology in order to investigate Early Upper Palaeolithic flint-knapping gestures and techniques involving the use of macro-lithic tools. In particular, the methodological framework proposed in this paper was applied to the study of Protoaurignacian and Aurignacian macro-tools from Fumane Cave (Verona, Italy). Combining spatial analysis and use wear investigation, both at low and high magnifications, permitted the identification and detailed description of the use-related traces affecting both the hammerstones and retouchers which, at Fumane Cave, were used at different stages during flint tool production. Several experimental activities were performed including core reduction, maintenance, and blank production together with different types of edge retouching. From a methodological perspective, the protocol of analysis permitted to codify specific traces and to produce quantitative data related to their geometry and distribution over the tool's surface, according to the activities and gestures performed. The results obtained allowed a careful investigation of the function and the gestures associated to the use of the macro-lithic tools coming from the Protoaurignacian and Aurignacian levels of Fumane Cave while providing a methodological tool for interpreting different archaeological samples., Competing Interests: The authors have declared that no competing interests exist.

The detection of double stranded DNA (dsDNA) is often associated with the use of laboratory-bound approaches and/or with the prior generation of single stranded DNA (ssDNA), making these methods not suitable for in situ monitoring, i.e., point-of-care diagnostics. Screen-printed technology, coupled to the use of triplex forming oligonucleotides (TFO) as the recognizing probes, offers a great possibility toward the development of portable analytical tools. Moreover, the continuous demand for sustainable processes and waste lowering have highlighted the role of paper-based substrates for manufacturing easy-to-use, low-cost, and sustainable electrochemical devices. In this work, filter paper and copy paper have been utilized to produce E-DNA strips. Gold nanoparticles (AuNPs) have been exploited to immobilize the methylene blue (MB)-tagged TFO and to enhance the charge transfer kinetics at the electrode surface. Both paper-based substrates have been electrochemically characterized, and in addition, the effect of the amount of waxed layers has been evaluated. The paper-based E-DNA strips have been challenged toward the detection of three model targets, obtaining 3 and 7 nM as the detection limit, respectively, for single and double stranded sequences. The repeatability of the manufacturing (homemade) process has been evaluated with a relative standard deviation of approximately 10%. The effectiveness of the filter paper-based platform has been also evaluated in undiluted serum obtaining a similar value of the detection limit (compared to the measurements carried out in buffer solution). In addition, a synthetic PCR amplified dsDNA sequence, related to HIV, has been detected in serum samples.

Chlorine is the most common chemical compound used for treatment of water at ppm levels, although at higher concentration it can generate harmful disinfection by-products. To easily monitor the correct level of free chlorine, cost-effective and miniaturized devices for fast and on-site detection are required. Herein, we report the development of a novel miniaturized and cost-effective screen-printed electrode modified with carbon black nanomaterial for an effective amperometric detection of free chlorine in swimming pool water. The carbon black was used as working electrode modifier for chlorine quantification at low applied potential, overcoming the fouling problem. In addition, the use of stable and cost-effective carbon black dispersion allows for a mass-production of the modified sensor by an automatable drop casting method. This sensor has demonstrated the capability to detect free chlorine in a linear range of 0.05-200 ppm with a detection limit of 0.01 ppm in standard solution as well as in swimming pool water. The achieved results demonstrated for the first time the suitability of the carbon black as electrode nanomodifier for free chlorine detection, and the possibility to produce a miniaturized and cost-effective carbon black-based printed sensor for reliable free chlorine quantification in water samples., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bioassays based on enzyme inhibition are analytical tools widely employed for inhibitor analysis. Beside the conventional analytical techniques such as chromatography and mass spectrometry, these bioassays are cost-effective, easy to use, and suitable for in situ measurement but they are often characterised by a quite narrow linear range. Herein, we report a novel graphical method based on integrated Michaelis-Menten equation, valid for all types of reversible inhibition, which provides an extended linear range. The suitability of this innovative approach was demonstrated in the case of fluoride quantification using a colorimetric bioassay based on acetylcholinesterase inhibition. The "half time reaction", estimated by the progress curve of cholinesterase inhibition, was plotted versus the fluoride inhibitor concentration, observing an extended linear range up to 5 mM, instead of 0.6 mM using initial rate measurements. The applicability of this new concept was further demonstrated in the case of catalase enzyme inhibited by cyanide. Furthermore, it was demonstrated that fixed substrate conversion at level of 10-50% allows determination of inhibitor concentration in a wide linear range with high precision and in short time of analysis. This novel theoretical and practical approach allows for the extension of the linear range without any further experiments, with several advantages including low reagent consumption, reduced waste generation and time of measurement., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The development of portable and user-friendly sensing platforms is a hot topic in the field of analytical chemistry. Among others, electroanalytical approaches exhibit a high amenability for reaching this purpose, i.e. the commercial strips for diabetes care are an obvious success. However, providing fully-integrated and user-friendly methods is the leitmotiv. In this work we evaluate the use of a disposable pipette tip, opportunely configured, to realize the first example of lab-on-a-tip. The combination of a pipette tip, wire electrodes, and cotton wool filter, highlights the suitability of producing a novel one-shot electroanalytical platform that does not require expertise-required tasks. To demonstrate the feasibility of this novel method, copper (Cu 2+ ) is detected in water samples by means of anodic stripping voltammetry. The quantification is performed directly into the tip that contains a cotton wool filter: the filter has the double function of purifying the matrices from gross impurities and releasing all the pre-loaded reagents necessary for the assay. After optimizing the experimental parameters, the lab-on-a-tip was capable of detecting Cu 2+ linearly up to 300 μg/L with a detection limit of 6.3 μg/L. The effectiveness of the platform was confirmed by testing 50, 100, and 150 ppb Cu-spiked river water sample with recovery value comprised between 92 and 103%., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Nowadays, environmentally friendly synthesis pathways for preserving the environment and minimizing waste are strongly required. Herein, we propose filter paper as a convenient scaffold for chemical reactions. To demonstrate this novel approach, Prussian Blue Nanoparticles (PBNPs) were synthesized on filter paper by utilizing few μL of its precursors without external inputs, i.e. pH, voltage, reducing agents, and without producing waste as well. The functional paper, named "Paper Blue", is successfully applied in the sensing field, exploiting the reduction of hydrogen peroxide at low applied potential. The eco-designed "Paper Blue" was combined with wax- and screen-printing to manufacture a reagentless electrochemical point-of-care device for diabetes self-monitoring, by using glucose oxidase as the biological recognition element. Blood glucose was linearly detected for a wide concentration range up to 25 mM (450 mg/dL), demonstrating its suitability for management of diabetes and glucose-related diseases. The Paper Blue-based biosensor demonstrated a correlation coefficient of 0.987 with commercial glucose strips (Bayer Contour XT). The achieved results demonstrated the effectiveness of this approach, which is also extendible to other (bio)systems to be applied in catalysis, remediation, and diagnostics., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The recent goal of sustainability in analytical chemistry has boosted the development of eco-designed analytical tools to deliver fast and cost-effective analysis with low economic and environmental impact. Due to the recent focus in sustainability, we report the use of low-cost filter paper as a sustainable material to print silver electrodes and to load reagents for a reagent-free electrochemical detection of chloride in biological samples, namely serum and sweat. The electrochemical detection of chloride ions was carried out by exploiting the reaction of the analyte (i.e. chloride) with the silver working electrode. During the oxidation wave in cyclic voltammetry the silver ions are produced, thus they react with chloride ions to form AgCl, while in the reduction wave, the following reaction occurs: AgCl + e - -->Ag + Cl - . These reactions at the electrode surface resulted in anodic/cathodic peaks directly proportional to the chloride ions in solution. Chloride ions were detected with the addition of only 10μL of the sample on the paper-based electrochemical cell, obtaining linearity up to 200mM with a detection limit equal to 1mM and relative standard deviation lower than 10%. The accuracy of the sensor was evaluated in serum and sweat samples, with percentage recoveries between 93 ± 10 and 108 ± 8%. Moreover, the results achieved with the paper-based device were positively compared with those obtained by using the gold standard method (Ion Selective Electrode) adopted in routine clinical analyses., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Herein, we evaluated the use of paper towel, waxed paper, and Parafilm M ® (Heathrow Scientific, Vernon Hills, IL, USA) as alternative substrates for screen-printed sensor manufacturing. Morphological study was performed to evaluate the adhesion of the ink on these uncommon substrates, as well as the morphology of the working electrode. The electrochemical characterization was carried out using ferricyanide/ferrocyanide as redox couple. To enhance the electrochemical properties of the developed sensors, the nanomaterial carbon black was used as nanomodifier. The modification by drop casting of the working electrode surface, using a stable dispersion of carbon black, allows to obtain a sensor with improved electrochemical behavior in terms of peak-to-peak separation, current intensity, and the resistance of charge transfer. The results achieved confirm the possibility of printing the electrode on several cost-effective paper-based materials and the improvement of the electrochemical behavior by using carbon black as sustainable nanomaterial., Competing Interests: The authors declare no conflict of interest.

Paper-based microfluidic devices are gaining large popularity because of their uncontested advantages of simplicity, cost-effectiveness, limited necessity of laboratory infrastructure and skilled personnel. Moreover, these devices require only small volumes of reagents and samples, provide rapid analysis, and are portable and disposable. Their combination with electrochemical detection offers additional benefits of high sensitivity, selectivity, simplicity of instrumentation, portability, and low cost of the total system. Herein, we present the first example of an integrated paper-based screen-printed electrochemical biosensor device able to quantify nerve agents. The principle of this approach is based on dual electrochemical measurements, in parallel, of butyrylcholinesterase (BChE) enzyme activity towards butyrylthiocholine with and without exposure to contaminated samples. The sensitivity of this device is largely improved using a carbon black/Prussian Blue nanocomposite as a working electrode modifier. The proposed device allows an entirely reagent-free analysis. A strip of a nitrocellulose membrane, that contains the substrate, is integrated with a paper-based test area that holds a screen-printed electrode and BChE. Paraoxon, chosen as nerve agent simulant, is linearly detected down to 3µg/L. The use of extremely affordable manufacturing techniques provides a rapid, sensitive, reproducible, and inexpensive tool for in situ assessment of nerve agent contamination. This represents a powerful approach for use by non-specialists, that can be easily broadened to other (bio)systems., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Cadmium and lead are highly toxic heavy metals which cause a severe worldwide pollution. In addition to the toxic effect produced by the direct exposure, they can be bioconcentrated and accumulated in living organisms, including humans. Herein, a miniaturized and disposable electrochemical sensor was improved for the simultaneous detection of cadmium and lead ions to study the bioremediation of polluted seawater in presence of the filter-feeding marine organism Styela plicata. A screen-printed electrode modified in situ with a bismuth film was selected using the anodic stripping analysis as detection technique. This sensor was coupled with a portable potentiostat and the detection of cadmium and lead ions was carried out by Square Wave Anodic Stripping Voltammetry, allowing the simultaneous detection of both heavy metals at ppb level (LOD=0.3ppb for lead, 1.5ppb for cadmium). This analytical tool was then applied to assess the bioremediation capacity of S. plicata through a bioremediation experiment, in which the organism has been exposed to seawater artificially polluted with 1000ppb of Cd 2+ and Pb 2+ . The matrix effect of both seawater and acid digested biological samples was evaluated. A bioconcentration phenomenon was observed for both heavy metals through the analysis of S. plicata tissues. In details, Pb 2+ resulted to be about 2.5 times more bioconcentrated than Cd 2+ , giving an effective bioremediation level in seawater of 13% and 40% for Cd 2+ and Pb 2+ , respectively. Thus, our results demonstrate the capability of S. plicata to bioremediate Cd 2+ and Pb 2+ polluted seawater as well as the suitability of the electrochemical sensor for contaminated marine environment monitoring and bioremediation evaluation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Herein, we report the first example of a paper-based screen-printed biosensor for the detection of ethanol in beer samples. Common office paper was adopted to fabricate the analytical device. The properties of this paper-based screen-printed electrode (SPE) were investigated by cyclic voltammetry, electrochemical impedance spectroscopy, and scanning electron microscopy, and they were compared with the well-established polyester-based SPEs as well. Paper demonstrated similar properties when compared with polyester, highlighting suitability towards its utilization in sensor development, with the advantages of low cost and simple disposal by incineration. A nanocomposite formed by Carbon Black (CB) and Prussian Blue nanoparticles (PBNPs), namely CB/PBNPs, was utilized as an electrocatalyst to detect the hydrogen peroxide generated by the enzymatic reaction between alcohol oxidase (AOx) and ethanol. After optimizing the analytical parameters, such as pH, enzyme, concentration, and working potential, the developed biosensor allowed a facile quantification of ethanol up to 10 mM (0.058 % vol ), with a sensitivity of 9.13 μA/mM cm 2 (1574 μA/% vol cm 2 ) and a detection limit equal to 0.52 mM (0.003% vol ). These satisfactory performances rendered the realized paper-based biosensor reliable over the analysis of ethanol contained in four different types of beers, including Pilsner, Weiss, Lager, and alcohol-free. The proposed manufacturing approach offers an affordable and sustainable tool for food quality control and for the realization of different electrochemical sensors and biosensors as well., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Through the years, scientists have developed cutting-edge technologies to make (bio)sensors more convenient for environmental analytical purposes. Technological advancements in the fields of material science, rational design, microfluidics, and sensor printing, have radically shaped biosensor technology, which is even more evident in the continuous development of sensing systems for the monitoring of hazardous chemicals. These efforts will be crucial in solving some of the problems constraining biosensors to reach real environmental applications, such as continuous analyses in field by means of multi-analyte portable devices. This review (with 203 refs.) covers the progress between 2010 and 2015 in the field of technologies enabling biosensor applications in environmental analysis, including i) printing technology, ii) nanomaterial technology, iii) nanomotors, iv) biomimetic design, and (v) microfluidics. Next section describes futuristic cutting-edge technologies that are gaining momentum in recent years, which furnish highly innovative aspects to biosensing devices., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Herein, we report a portable electrochemical biosensor based on butyrylcholinesterase (BChE) immobilized on carbon black (CB)-modified screen-printed electrodes (SPEs) for the detection of organophosphorous pesticides in olive oil. The BChE/CB-SPE biosensor was developed to detect paraoxon in standard solutions as well as in olive oil samples previously treated with the QuEChERS method to extract pesticides from the whole fatty matrix. The biosensor shows a linear concentration range of between 20 and 100 ppb for paraoxon both in standard solutions (phosphate buffer 0.05 M) and in olive oil extracts, with a detection limit of 6 ppb in olive oil extract, corresponding to 10% of inhibition. The accuracy of this biosensor in olive oil samples was assessed with olive oil spiked with paraoxon, obtaining satisfactory recovery values., Competing Interests: The authors declare no conflicts of interest.

We report a screen-printed electrode (SPE) modified with a dispersion of carbon black (CB) and chitosan by drop casting. A cyclic voltammetry technique towards ferricyanide, caffeic acid, hydroquinone, and thiocholine was performed and an improvement of the electrochemical response with respect to bare SPE as well as SPE modified only with chitosan was observed. The possibility to detect thiocholine at a low applied potential with high sensitivity was exploited and an acetylcholinesterase (AChE) biosensor was developed. A dispersion of CB, chitosan, and AChE was used to fabricate this biosensor in one step by drop casting. The enzymatic activity of the immobilized AChE was determined measuring the enzymatic product thiocholine at +300 mV. Owing to the capability of organophosphorus pesticides to inhibit AChE, this biosensor was used to detect these pollutants, and paraoxon was taken as model compound. The enzyme inhibition was linearly related to the concentration of paraoxon up to 0.5 μg L(-1), and a low detection limit equal to 0.05 μg L(-1) (calculated as 10% of inhibition) was achieved. This biosensor was challenged for paraoxon detection in drinking waters with satisfactory recovery values. The use of AChE embedded in a dispersion of CB and chitosan allowed an easy and fast production of a sensitive biosensor suitable for paraoxon detection in drinking waters at legal limit levels. Graphical Abstract Biosensors based on screen-printed electrodes modified with Acetylcholinesterase, Carbon Black, and Chitosan for organophosphorus pesticide detection.

Herein we describe a novel reagentless paper-based electrochemical phosphate sensor, manufactured with a simple and inexpensive approach. By following three easy steps, consisting of wax patterning, paper chemical modification, and electrode screen-printing, the filter paper provides an effective electroanalytical platform to sense phosphate ions in standard solutions and real samples (river water). The electrochemical properties of the paper-based platform were evaluated, firstly, by using ferricyanide as a redox mediator, proving no analyte-entrapment due to the cellulose lattice. Then, the reference colorimetric method for phosphate ions, which is based on the formation of phosphomolybdic complex, was successfully adapted to a reagentless electrochemically paper-based platform. This novel and highly sustainable configuration readily allows for the determination of phosphate ions with high reproducibility and long storage stability, achieving a detection limit of 4 μM over a wide linear range up to 300 μM. This in-house approach would be able to generically develop an affordable in situ and user-friendly sensing device without the addition of any reagent, to be applied for a broad range of analytes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A miniaturized screen-printed electrode (SPE) modified with a carbon black-gold nanoparticle (CBNP-AuNP) nanocomposite has been developed as an electrochemical sensor for the detection of inorganic mercury ions (Hg(2+)). The working electrode surface has been modified with nanocomposite constituted of CBNPs and AuNPs by an easy drop casting procedure that makes this approach extendible to an automatable mass production of modified SPEs. Square wave anodic stripping voltammetry (SWASV) was adopted to perform Hg(2+) detection, revealing satisfactory sensitivity and detection limit, equal to 14 μA ppb(-1) cm(-2) and 3 ppb, respectively. The applicability of the CBNP-AuNP-SPE for the determination of inorganic mercury has been assessed in river water by a simple filtration and acidification of the sample as well as in soil by means of a facile acidic extraction procedure assisted by ultrasound.

In this study, we report a novel assay for the combined on site detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA), through a colorimetric biosensing system for AFB1 and a fluorimetric detection for OTA, exploiting the capability of the portable fibre optic spectrometer to perform both analyses. AFB1 was detected using the acetylcholinesterase (AChE) enzyme that is inhibited by this toxin, and the degree of inhibition was quantified by the Ellman's spectrophotometric method, obtaining a detection limit of 10 µg L(-1). OTA quantification was performed by monitoring its intrinsic fluorescence in methanol, reaching a detection limit of 0.1 µg L(-1). In order to successfully apply the analytical tool in the food analysis, immunoaffinity columns were used. Clean-up and quantification of both AFB1 and OTA in millet samples was obtained by HPLC-dedicated AflaOchra-Test HPLC™ (Vicam™) and Afla-OtaCLEAN™ (LC-Tech) immunoaffinity columns, followed by absorption/fluorescence detection. Millet samples which were fortified with both OTA (50 µg kg(-1)) and AFB1 (20 µg kg(-1)), gave recovery values of 100 ± 6% for OTA, and 110 ± 10% for AFB1, using AflaOchra-Test HPLC™. Single OTA clean-up and quantification in wine samples was obtained, using an OchraTest immunoaffinity column (Vicam™), reaching a detection limit of 0.3 µg L(-1) and recovery values between 80% and 120%. These results demonstrated the possibility of employing a single clean-up and a cost-effective, and easy to use analytical system for both AFB1 and OTA detection at µg kg(-1) (ppb) level. Furthermore, in the case of positive samples, they could be analysed further, using standard chromatographic procedures, without any additional clean-up step, since the same extraction procedure of standard method is proposed in our method., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A highly sensitive electrochemical immunoassay for the initial diagnosis of celiac disease (CD) in saliva samples that overcomes the problems related to its high viscosity and to the low concentration of anti-transglutaminase antigen (tTG) IgA in this medium has been developed for the first time. The system uses magnetic beads (MBs) covered with tTG, which reacts with the anti-tTG IgA antibodies present in positive saliva samples. An anti-human IgA, conjugated with alkaline phosphate (AP) enzyme, was used as the label and a strip of eight magnetized screen-printed electrodes as the electrochemical transducer. In particular, two different immunoassay approaches were optimized and blindly compared to analyze a large number of saliva samples, whose anti-tTG IgA levels were independently determined by the radioimmunoassay (RIA) method. The obtained results, expressed as Ab index, were used to perform a diagnostic test evaluation through the construction of receiver operating characteristic (ROC) curves. The approach, involving a pre-incubation between the anti-human IgA-AP and saliva samples prior to the addition of MBs-tTG, showed a cutoff of 0.022 with 95% clinical sensitivity and 96% clinical specificity. The area under the ROC curve is equal to 1, a result that classifies our test as "perfect." This study demonstrates that it is possible to perform the screening of CD with a rapid, simple, inexpensive, and sensitive method able to detect anti-tTG antibodies in saliva samples, which are easily obtained by non-invasive techniques. This aspect is of fundamental importance to screen a large number of subjects, especially in the pediatric age.

We report a sensor for phosphate detection based on screen-printed electrodes modified with carbon black nanoparticles. The phosphate was measured in amperometric mode via electrochemical reduction of molybdophosphate complex. Carbon black nanoparticles demonstrated the ability to quantify the molybdophosphate complex at a low applied potential. Some analytical parameters such as the working solution (sulfuric acid 0.1M), applied potential (0.125V vs Ag/AgCl), and molybdate concentration (1mM) were optimized. Using these conditions, a linear range of 0.5-100µM was observed with a detection limit of 0.1µM, calculated as three times the standard deviation of the blank divided by the slope of calibration curve. The system was challenged in drinking, river, aquarium, and waste water samples yielding satisfactory recovery values in accordance with a spectrophotometric reference method which demonstrated the suitability of the screen-printed electrode modified with carbon black nanoparticles coupled with the use of molybdate to detect phosphate in water samples., (Copyright © 2015 Elsevier B.V. All rights reserved.)

An automatable flow system for the continuous and long-term monitoring of the phosphate level has been developed using an amperometric detection method based on the use of a miniaturized sensor. This method is based on the monitoring of an electroactive complex obtained by the reaction between phosphate and molybdate that is consequently reduced at the electrode surface. The use of a screen-printed electrode modified with carbon black nanoparticles (CBNPs) leads to the quantification of the complex at low potential, because CBNPs are capable of electrocatalitically enhancing the phosphomolybdate complex reduction at +125 mV versus Ag/AgCl without fouling problems. The developed system also incorporates reagents and waste storage and is connected to a portable potentiostat for rapid detection and quantification of phosphate. Main analytical parameters, such as working potential, reagent concentration, type of cell, and flow rate, were evaluated and optimized. This system was characterized by a low detection limit (6 μM). Interference studies were carried out. Good recovery percentages comprised between 89 and 131.5% were achieved in different water sources, highlighting its suitability for field measurements.

In this work a novel bioassay for mustard agent detection was proposed. The bioassay is based on the capability of these compounds to inhibit the enzyme choline oxidase. The enzymatic activity, which is correlated to the mustard agents, was electrochemically monitored measuring the enzymatic product, hydrogen peroxide, by means of a screen-printed electrode modified with Prussian Blue nanoparticles. Prussian Blue nanoparticles are able to electrocatalyse the hydrogen peroxide concentration reduction at low applied potential (-50 mV vs. Ag/AgCl), thus allowing the detection of the mustard agents with no electrochemical interferences. The suitability of this novel bioassay was tested with the nitrogen mustard simulant bis(2-chloroethyl)amine and the sulfur mustard simulants 2-chloroethyl ethyl sulfide and 2-chloroethyl phenyl sulfide. The bioassay proposed in this work allowed the detection of mustard agent simulants with good sensitivity and fast response, which are excellent premises for the development of a miniaturised sensor well suited for an alarm system in case of terrorist attacks.

We report a modular electrochemical peptide-based sensor targeting the anti-deamidated gliadin peptide (DGP) antibody. A recognition peptide, here DGP, is grafted onto a supporting peptide bearing a redox label. The fabricated peptide-based sensor supports the detection of the target antibody (anti-DGP antibody) in the nanomolar range.

A sensor for the simple and sensitive measurement of hydrogen peroxide has been developed which is based on screen printed electrodes (SPEs) modified with Prussian blue nanoparticles (PBNPs) deposited using piezoelectric inkjet printing. PBNP-modified SPEs were characterized using physical and electrochemical techniques to optimize the PBNP layer thickness and electroanalytical conditions for optimum measurement of hydrogen peroxide. Sensor optimization resulted in a limit of detection of 2 × 10(-7) M, a linear range from 0 to 4.5 mM and a sensitivity of 762 μA ∙ mM(-1) ∙ cm(-2) which was achieved using 20 layers of printed PBNPs. Sensors also demonstrated excellent reproducibility (<5% rsd).

In this work a miniaturized and disposable electrochemical sensor was developed to evaluate the cadmium and lead ion phytoremediation potential by the floating aquatic macrophyte Lemna minor L. The sensor is based on a screen-printed electrode modified "in-situ" with bismuth film, which is more environmentally friendly than the mercury-based sensor usually adopted for lead and cadmium ion detection. The sensor was coupled with a portable potentiostat for the simultaneous measurement of cadmium and lead ions by stripping analysis. The optimized analytical system allows the simultaneous detection of both heavy metals at the ppb level (LOD equal to 0.3 and 2 ppb for lead and cadmium ions, respectively) with the advantage of using a miniaturized and cost-effective system. The sensor was then applied for the evaluation of Pb(2+) or/and Cd(2+) uptake by measuring the amount of the heavy metals both in growth medium and in plant tissues during 1 week experiments. In this way, the use of Lemna minor coupled with a portable electrochemical sensor allows the set up of a model system able both to remove the heavy metals and to measure "in-situ" the magnitude of heavy metal removal.

A simple graphical method for the determination of reversible inhibition type, inhibition constant (Ki) and estimation of fifty percent of inhibition I₅₀ of an enzyme reaction is described. The method consists of plotting experimental data as "degree of inhibition" versus the inhibitor concentration at two or more concentrations of substrate. Diagnosis of inhibition type is based on determination of and the observation of the shift of the inhibition curves. Relationship between I50 and inhibition constant Ki was discussed. A simplified hyperbolae equation of degree of inhibition showing kinetic orders of 1 and zero at low and high concentrations of inhibitors respectively is proposed. The relative error of inhibitor concentration increased drastically when degree of inhibition reached values of 90%. Examples of published inhibition reports as well as an experimental example of amperometric biosensor based on tyrosinase inhibition by benzoic acid were in agreement with the proposed theoretical approach., (© 2013 Published by Elsevier B.V.)

In this paper we propose the construction and application of a portable multi-purpose biosensor array for the simultaneous detection of a wide range of endocrine disruptor chemicals (EDCs), based on the recognition operated by various enzymes and microorganisms. The developed biosensor combines both electrochemical and optical transduction systems, in order to increase the number of chemical species which can be monitored. Considering to the maximum residue level (MRL) of contaminants established by the European Commission, the biosensor system was able to detect most of the chemicals analysed with very high sensitivity. In particular, atrazine and diuron were detected with a limit of detection of 0.5nM, with an RSD% less than 5%; paraoxon and chlorpyrifos were revealed with a detection of 5 μM and 4.5 μM, respectively, with an RSD% less than 6%; catechol and bisphenol A were identified with a limit of detection of 1 μM and 35 μM respectively, with an RSD% less than 5%., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: While most of the common drugs with the potential to interfere with continuous glucose monitoring (CGM) systems are accessible over the counter and can be assumed by CGM patients without medical supervision, many other chemicals are frequently used to treat critically ill patients. Continuous glucose monitoring reading accuracy may also be compromised in patients characterized by abnormally high concentrations of physiological interferents. In this article, 22 species selected from endogenous and exogenous chemicals were screened as possible interferents of GlucoMen®Day (GMD), the new microdialysis-based CGM system from A. Menarini Diagnostics., Method: Interference testing was performed according to the EP7-A2 guideline (Clinical and Laboratory Standards Institute 2005). Interference was evaluated at two levels of glucose, with each interferent additionally tested at two concentrations. Furthermore, two configurations of the GMD disposable sensor kit--one designed for subcutaneous application, the other for direct intravascular CGM--were challenged with interferent-spiked serum and blood samples, respectively., Results: With the exception of dopamine (however, at very high, nonphysiological concentrations), no interference was observed for all the tested substances. Interestingly, none of the common electrochemical interferents (including ascorbic acid, acetaminophen, and salicylic acid, which represent the major specificity issue for the competing CGM systems) significantly affected the system's output., Conclusions: These results provide clear insights into the advantages offered by the use of a microdialysis-based CGM system that additionally relies on the detection of hydrogen peroxide at low operating potential. GlucoMen Day may become the CGM system of choice for those patients who require either regular administration of drugs or their glycemia to be tightly controlled in the intensive care unit or similar environments., (© 2012 Diabetes Technology Society.)

This work reports the optimisation of a new analytical method for lead ion detection in milk; the electrochemical detection scheme is based on the method that was described in Part I. It features the use of a disposable, environmentally friendly bismuth film electrode to replace the traditionally used (toxic) mercury one while here we report an arduous development of sample treatment so that the simple device can be applied as a screening tool in many settings. For this purpose, a milk pre-treatment procedure by means of wet digestion with HCl, HClO(4), and H(2)O(2) combined with an ultrasonic treatment was developed. The detection of lead ions in treated milk was then carried out using a disposable screen-printed electrode modified with Nafion(®) and an "in situ" bismuth film, with the analysis being performed in anodic stripping voltammetry mode. The analytical method developed allows the detection of milk contaminated with lead ions at a concentration of 20 μg Kg(-1) (legal limit) and it can be proposed as a screening method for routine analysis of lead ions in milk with the advantage of employing inexpensive and portable instrumentation. Moreover, dedicated software supported by a portable instrument introduces procedures that are essential to avoid distortion from ambient lead contamination and also makes it possible for an unskilled operator to carry out each step of the analysis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Coeliac disease (CD) is a gluten-induced autoimmune enteropathy found in genetically susceptible subjects. Because of the high number of undetected cases, rapid and cheaper screening methods are needed. Currently, the CD diagnosis involves the detection of anti-transglutaminase IgA antibodies (anti-tTG IgA) in blood serum through the use of ELISA systems with confirmation by histology of the intestinal mucosa. A new, rapid magneto-electrochemical immunosensor for CD diagnosis has been developed and applied to serum sample analysis. The system uses magnetic beads coated with tTG antigen to detect anti-tTG antibodies in positive serum samples and an alkaline phosphatase-conjugated anti-human IgA as label. An electrochemical readout, using magnetized screen-printed electrodes coupled with a portable instrument, is made after the addition of α-naphtyl phosphate, which is enzymatically converted into the electrochemically active α-naphthol product. The work involved the following considerations: (1) optimization of analytical parameters; (2) recovery evaluation, adding known concentrations of anti-tTG IgA to "blank" sera; (3) analysis of 107 blood serum samples; (4) calculation of the ROC curve, resulting in a cut-off of 1.0 U/ml, 100% of clinical sensitivity and 98.36% of clinical specificity; evaluation of the agreement between electrochemical and ELISA kit values (r (2) of 0.943). The system developed could be an useful tool for a correct and rapid CD diagnosis. This method is simple, cheap, rapid, and suitable for screening analyses performed outside of the classical diagnostic laboratory.

Lead determination was carried out in the frame of the European Union project Biocop (www.biocop.org) using a bismuth-modified screen-printed electrode (Bi-SPE) and the stripping analysis technique. In order to choose a sensitive Bi-SPE for lead detection, an analytical comparative study of electrodes modified by Bi using "in situ", "ex situ" and "bulk" procedures was carried out. On the basis of the results obtained, we confirmed that the "in situ" procedure resulted in better analytical performances with respect to not only "ex situ" but also to "Bi(2)O(3) bulk" modified electrodes, allowing for a linear range of lead ion concentration from 0.5 to 100 μg L(-1) and a detection limit of 0.15 μg L(-1). We demonstrated that, before the Bi film deposition, an oxidative electrochemical pre-treatment of the working electrode could be useful because it eliminates traces of lead in the graphite-ink, as shown with stripping measurements. It also improves the electrochemical performance of the electrodes as demonstrated with Electrochemical Impedance Spectroscopy (EIS) measurements. The influence of different analytical parameters, such as the electrolyte solution composition (acetate buffer, chloridric acid, nitric acid, perchloric acid) and the ionic strength was investigated in order to evaluate how to treat the sample before the analysis. The morphology of prepared "in situ" Bi-SPEs was also characterized by Atomic Force Microscopy (AFM). Finally, the Bi-SPEs were used to determine the concentration of lead ions in tap and commercial water samples obtaining satisfactory values of the recovery percentage (81% and 98%)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An efficient electrochemical protocol to monitor hydrogen peroxide consumption during metal-catalyzed oxidation by using screen-printed electrodes modified with Prussian blue is presented. In particular, cyclooctene oxidation to cyclooctene oxide, catalyzed by a vanadium(V)-salophen complex (H(2)salophen=N,N'-o-phenylenebis(salicylideneimine)), in molecular and ionic media was tested. Initially, a protocol for batch analysis was developed for a monophasic system in acetonitrile, and subsequently, an in situ protocol was developed for a biphasic system of 1-butyl-3-methylimidazolium hexafluorophosphate/phosphate buffer. Calibration curves were performed in amperometric mode by applying -50 mV versus an Ag pseudo-reference. The calibration curve of hydrogen peroxide showed a linear correlation from 1 × 10(-6) up to 5 × 10(-3) mol L(-1) with satisfactory inter- and intra-electrode reproducibility (relative standard deviation (RSD) values of 5 and 13%, respectively, for the monophasic system and 11 and 13%, respectively, for the biphasic system). Kinetic studies to investigate the oxidation reaction for both the mono- and biphasic systems have been carried out in amperometric mode as well. Firstly, the decomposition of hydrogen peroxide was examined, which showed that, in 1-butyl-3-methylimidazolium hexafluorophosphate(,) it completely decomposed in 300 min, whereas in acetonitrile, in the same time frame, 20% of the initial amount was still active. In the presence of 1% of the catalyst the decomposition rate increased in both solvents. Finally, the complete oxidation of cyclooctene was followed and the effective conversion was determined. The developed protocols showed high reproducibility, with the advantage that the environmentally friendly biphasic system could also be recycled. The good analytical performance obtained, coupled with a short analysis time, the possibility of in-line automation and the use of ionic liquids instead of molecular solvents, made this system a very attractive choice for monitoring oxidative reactions., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A novel method for aflatoxin B (AFB) determination is proposed. The AFB determination is based on acetylcholinesterase (AChE, from electric eel) inhibition, and the AChE residual activity is determined using the colorimetric method (Ellman's method). To select and optimize the analytical procedures, the investigation on type of AChE inhibition by AFB(1) was carried out. The AChE degree of inhibition by AFB(1) was independent of the incubation time and the enzyme concentrations, showing the reversibility of the inhibition. This reversibility of the inhibition permits a rapid analysis of AFB(1). In fact, only a 3-min analysis is required. For the development of AFB(1) assay, the pH, the reaction time, the temperature, and the substrate concentration were evaluated and optimized. The linear range of 10-60 ng/mL was assessed. To evaluate the selectivity of this method, the cross-reactivity with other aflatoxins, such as AFB(2) (aflatoxin B(2)), AFG(1) (aflatoxin G(1)), AFG(2) (aflatoxin G(2)), and AFM(1) (aflatoxin M(1)), was investigated. The suitability of the assay for AFB(1) quantification in barley was also evaluated. This study shows a new approach to detect aflatoxins based on enzyme inhibition with several advantages, such as the easiness of use, the rapidity, and the cost-effectiveness, demonstrating a possible use as screening method for this type of mycotoxins.

We report a reagentless, electrochemical sensor for the detection of double-stranded DNA targets that employs triplex-forming oligonucleotides (TFOs) as its recognition element. These sensors are based on redox-tagged TFO probes strongly chemisorbed onto an interrogating gold electrode. Upon the addition of the relevant double-stranded DNA target, the probe forms a rigid triplex structure via reverse Hoogsteen base pairing in the major groove. The formation of the triplex impedes contact between the probe's redox moiety and the interrogating electrode, thus signaling the presence of the target. We first demonstrated the proof of principle of this approach by using a well-characterized 22-base polypurine TFO sequence that readily detects a synthetic, double-stranded DNA target. We then confirmed the generalizability of our platform with a second probe, a 19-base polypyrimidine TFO sequence that targets a polypurine tract (PPT) sequence conserved in all HIV-1 strains. Both sensors rapidly and specifically detect their double-stranded DNA targets at concentrations as low as ~10 nM and are selective enough to be employed directly in complex sample matrices such as blood serum. Moreover, to demonstrate real-world applicability of this new sensor platform, we have successfully detected unpurified, double-stranded PCR amplicons containing the relevant conserved HIV-1 sequence.

A reliable and cost-effective electrochemical method for the detection of deoxynivalenol (DON) in cereals and cereal-based food samples based on the use of a novel anti-DON Fab fragment is presented. The analytical system employed, Enzyme-Linked-Immunomagnetic-Electrochemical (ELIME) assay, is based on the use of immunomagnetic beads (IMBs) coupled with eight magnetized screen-printed electrodes (8-mScPEs) as electrochemical transducers. Using standard solutions of DON, a working range between 100 and 4500 ng/ml was obtained with an EC(50) of 380 ng/ml. The ELIME assay was employed to evaluate the cross-reactivity of the Fab fragment towards different trichothecenes revealing a good selectivity towards DON over other trichothecenes with the exception of 3-Ac-DON. The sensor was then applied to cereals and cereal-based food samples (wheat, breakfast cereal and baby-food) and a wide range of sample treatment procedures was tested. Within-laboratory precision (9-24% repeatability for breakfast cereals and 10-33% for baby-food) and recovery data (82-110% for breakfast cereals and 97-108% for baby-food) were calculated by analyzing blank breakfast cereals and baby-foods fortified with DON, demonstrating that the proposed method has the capability for use as a screening assay for DON in such products., (Copyright 2010 Elsevier B.V. All rights reserved.)

Here we demonstrate the use of redox labeled double- and single-stranded oligonucleotides as recognition probes for the reagentless, single-step, electrochemical detection of anti-DNA antibodies directly in blood serum.

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC(1, 2) methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain(3) and screen printed electrodes as sensing platform. Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health(4). The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity(5). The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution. At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution. A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

E-DNA sensors are a reagentless, electrochemical oligonucleotide sensing platform based on a redox-tag modified, electrode-bound probe DNA. Because E-DNA signaling is linked to hybridization-linked changes in the dynamics of this probe, sensor performance is likely dependent on the nature of the self-assembled monolayer coating the electrode. We have investigated this question by characterizing the gain, specificity, response time and shelf-life of E-DNA sensors fabricated using a range of co-adsorbates, including both charged and neutral alkane thiols. We find that, among the thiols tested, the positively charged cysteamine gives rise to the largest and most rapid response to target and leads to significantly improved storage stability. The best mismatch specificity, however, is achieved with mercaptoethanesulfonic and mercaptoundecanol, presumably due to the destabilizing effects of, respectively, the negative charge and steric bulk of these co-adsorbates. These results demonstrate that a careful choice of co-adsorbate chemistry can lead to significant improvements in the performance of this broad class of electrochemical DNA sensors.

Salmonella is one of the main organisms causing outbreaks of foodborne illness, and meat is one of the major vehicles of salmonellosis throughout the world. A novel analytical immunosensor array, based on a 96-well electrochemical plate coupled with immunomagnetic beads (ELIME array), is proposed for the detection of Salmonella in meat samples. After an optimization study, using Salmonella enterica serotype Enteritidis as reference antigen, the ability of the method to interact with a large number of Salmonella serovars commonly present in food was evaluated. The assay was then used to analyze samples of pork, chicken, beef, and turkey experimentally inoculated with Salmonella as well as real samples. The results were compared with those from the International Standard of Organization (ISO) culture method. The comparison showed that the ELIME array is able to detect a low number of Salmonella cells (1-10 CFU per 25 g) after only 6 h of incubation in a pre-enrichment broth. The investigation revealed a very good agreement between culture and ELIME array methods for meat samples, reducing the time for performing the analysis and obtaining the results quickly.

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 microg/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.

A novel biosensor assay format for aflatoxin based on acetylcholinesterase (AChE) inhibition by aflatoxin B(1) (AFB(1)) is proposed. The AChE was present in solution and an amperometric choline oxidase biosensor was used for monitoring its residual activity. To create the biosensor, the choline oxidase was immobilized by cross-linking onto screen-printed electrodes modified with Prussian Blue (PB) and these were used to detect the H(2)O(2) at low potential (-0.05V versus a screen-printed internal silver pseudoreference electrode). For the development of the AFB(1) assay, several parameters such as AChE and substrate concentration, the methanol effect, and pH were evaluated and optimized. The linear working range was assessed to be 10-60ppb. Concentrations as low as 2ppb, which correspond to the legal limit of AFB(1) in food for humans, were detected after a pre-concentration step. The suitability of the method was evaluated using commercial olive oil samples. A recovery equal to 78+/-9% for 10ppb of AFB(1) in olive oil samples was obtained.

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 50 min for the off-line enzymatic incubations and 4 min for the biosensor response.

Trehalose is a disaccharide important in foods, serving as a glucose source in many and also as an additive in the food preparation. Because of its peculiar physico-chemical properties it plays an important role as preservative in drying and deep-freezing treatments. A new biosensor for trehalose determination has been realized by means of a flow system, based on a reactor in which the trehalase enzyme catalyses its hydrolysis into two alpha,d-glucose molecules, and a GOD (glucose oxidase) amperometric biosensor is employed for the glucose determination. The optimum operative conditions have been laid out and a particular attention has been paid to the immobilization procedure of the two enzymes. The electrode used is of the SPE (screen-printed electrode) type and has been activated with the Prussian Blue (PB) and then assembled using GOD immobilized with Nafion. The reactor has been prepared with the trehalase enzyme chemically immobilized on an Immunodyne ABC membrane. As demonstration of its utility, the biosensor has been tested on a real sample of Boletus edulis mushroom.

Different tyrosinase carbon paste modified electrodes to determine bisphenol A (BPA) concentration in aqueous solutions have been constructed. Variables examined were in the carbon paste composition and in particular: (i) the immobilized enzyme amount; (ii) the carbon type (powder, single or multi-walled nanotubes); (iii) the nature of the pasting oil (mineral oil, hexadecane and dodecane). For each biosensor type the amperometric response was evaluated with reference to the linear range and sensitivity. Constant reference has been made to the amperometric signals obtained, under the same experimental conditions, towards the catechol, a specific phenolic substrate for tyrosinase. The most efficient biosensors were those constructed by using the following composition for the carbon paste: 10% of tyrosinase, 45% of single wall carbon nanotubes (SWCN) and 45% of mineral oil. This biosensor formulation displayed the following electrochemical characteristics: a sensitivity equal to 138 microA/mM, LOD of 0.02 microM (based on three times the S/N ratio), linear range of 0.1-12 microM and response time of 6 min. This experimental work represents a first attempt at construction of a new carbon nanotube-tyrosinase based biosensor able to determine the concentration of BPA, one of the most ubiquitous and hazardous endocrine disruptors which can pollute the drinking and surface water, as well as many products of the food chain.

The nerve agents are chemical warfare agents known to be used during terrorist attacks. An inexpensive and portable system to be used by first responders and military personnel is of interest owing to the continuing threat of possible terrorist attacks. Amperometric biosensors based on cholinesterase inhibition show such potentialities. In this work butyrylcholinesterase was immobilized onto screen-printed electrodes modified with Prussian blue and the nerve agent detection was performed by measuring the residual activity of enzyme. The optimized biosensor was tested with sarin and VX standard solutions, showing detection limits of 12 and 14 ppb (10% of inhibition), respectively. The enzymatic inhibition was also obtained by exposing the biosensors to sarin in gas phase. Two different concentrations of sarin gas (0.1 and 0.5 mg m(-3)) at different incubation times (from 30 s up to 10 min) were tested. It is possible to detect sarin at a concentration of 0.1 mg m(-3) with 30-s incubation time, with a degree of inhibition of 34%, which match the legal limits (immediate danger to life and health).

A new method for aflatoxin B (AFB) determination is proposed. The AFB determination is based on acetylcholinesterase (AChE) inhibition, and the AChE residual activity is determined using the colorimetric method (Ellman's method). Cholinesterases (ChEs) from various sources were tested using AFB1 as reference aflatoxin. AChE from electric eel has shown the highest sensitivity to AFB1, and it was chosen for the rest of the work. To select and optimize the analytical procedures, an investigation on the type of AChE inhibition by AFB1 was carried out. The AChE degree of inhibition by AFB1 was independent of the incubation time and the enzyme concentrations, showing the reversibility of the inhibition. This reversibility of the inhibition permits a rapid analysis of AFB1, requiring only 3 min. For the development of the AFB1 assay, the pH, the time of reaction, temperature, and substrate concentration were evaluated and optimized. The linear range of 10-60 ng mL-1 was determined. To evaluate the selectivity of this method, the cross-reactivity with other aflatoxins such as aflatoxin B2, aflatoxin G1, aflatoxin G2, and aflatoxin M1 was investigated. Finally, the suitability of the assay for AFB1 quantification in barley was evaluated. This study shows a new approach to detect aflatoxins based on enzyme inhibition and has advantages such as the ease of use, rapidity, and cost effectiveness. Thus, it could find a possible use as a screening method for this type of mycotoxins.

Glucose biosensors based on the use of planar screen-printed electrodes modified with an electrochemical mediator and with glucose oxidase have been optimised for their application in the continuous glucose monitoring in diabetic patients. A full study of their operative stability and temperature dependence has been accomplished, thus giving useful information for in vivo applications. The effect of dissolved oxygen concentration in the working solution was also studied in order to evaluate its effect on the linearity of the sensors. Glucose monitoring performed with serum samples was performed to evaluate the effect of matrix components on operative stability and demonstrated an efficient behaviour for 72 h of continuous monitoring. Finally, these studies led to a sensor capable of detecting glucose at concentrations as low as 0.04 mM and with a good linearity up to 2.0 mM (at 37 degrees C) with an operative stability of ca. 72 h, thus demonstrating the possible application of these sensors for continuous glucose monitoring in conjunction with a microdialysis probe. Moreover, preliminary in vivo experiments for ca. 20 h have demonstrated the feasibility of this system.

A novel analytical immunosensor array, based on a microtiter plate coupled to a multichannel electrochemical detection (MED) system using the intermittent pulse amperometry (IPA) technique, is proposed for the detection of aflatoxin B1 (AFB1). In the present work, the electrochemical behaviour and electroanalytical performance of the thick-film carbon sensors (also designated as screen-printed electrodes) incorporated in the multichannel electrochemical plate were first evaluated. Then the 96-well screen-printed microplate was modified in accord with a competitive indirect enzyme-linked immunoassay (ELISA) format for aflatoxin B1 detection. The measurements were performed using both spectrophotometric and electrochemical procedures and the results of the calibration curves, detection limit (LOD), sensitivity and reproducibility of the respective assay systems were evaluated. The immunoassay was then applied for analysis of corn samples spiked with AFB1 before and after the extraction treatment, in order to study the extraction efficiency and the matrix effect, respectively. These studies have shown that using this system, AFB1 can be measured at a level of 30 pg/mL and with a working range between 0.05 and 2 ng/mL. Good recoveries (103+/-8%) were obtained, demonstrating the suitability of the proposed assay for accurate determination of the AFB1 concentration in corn samples. The specificity of the assay was assessed by studying the cross-reactivity of PAb relative to AFB1. The results indicated that the PAb could readily distinguish AFB1 from other aflatoxins, with the exception for AFG1.