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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.)

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.)

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.

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.

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).

Lipase activity against triacylglycerols has been measured using an amperometric enzyme biosensor based on glycerol dehydrogenase/NADH oxidase. A Prussian Blue modified screen-printed electrode was selected as substrate for the two immobilised-enzyme systems due to their higher operative stability reported in previous works. Various parameters such as cofactor (flavin mononucleotide FMN) concentration (1 mM), NAD+ coenzyme concentration (2 mM), pH effect (phosphate buffer pH 6 to 8, Tris buffer pH 8-10) response time and storage stability were evaluated and optimised. The glycerol biosensor was then investigated for lipase activity. The system was challenged against an olive or sunflower oil real samples in order to detect fatty acids and the results were compared with those provided either by the manufacture or reference methods with good agreement.

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.

In the present paper, a comparative study using Co-phthalocyanine and Prussian Blue-modified screen-printed electrodes, has been performed. Both the electrodes have demonstrated an easiness of preparation together with high sensitivity towards thicoholine (LOD=5 x 10(-7) and 5 x 10(-6) M for Co-phthalocyanine and Prussian Blue, respectively) with high potentialities for pesticide measurement. Prussian Blue-modified screen-printed electrodes were then selected for successive enzyme immobilization due to their higher operative stability demonstrated in previous works. AChE and BChE enzymes were used and inhibition effect of different pesticides was studied with both the enzymes. AChE-based biosensors have demonstrated a higher sensitivity towards aldicarb (50% inhibition with 50 ppb) and carbaryl (50% inhibition with 85 ppb) while BChE biosensors have shown a higher affinity towards paraoxon (50% inhibition with 4 ppb) and chlorpyrifos-methyl oxon (50% inhibition with 1 ppb). Real samples were also tested in order to evaluate the matrix effect and recovery values comprised between 79 and 123% were obtained.

Objective: To increase knowledge on the behavior of gastrointestinal stromal tumors (GISTs) and factors influencing therapy., Study Design: The clinicomorphological features of 158 GISTs were analyzed. Survival analysis was performed on the whole series, as well as on a selected group of patients with high risk GIST who did not receive imatinib mesylate. The impact of imatinib mesylate on the prognosis was investigated., Results: Most of the GISTs had a benign behavior. The risk class was a powerful prognostic factor but was unable to predict the outcome in a single case; even patients in the high risk class not receiving imatinib mesylate had a low mortality rate. In this group, it was the mitotic activity that better correlated with prognosis, and a cut point of 10 mitoses per 50 high-power field can be fixed to discriminate cases with favorable or unfavorable outcome. Patients with GISTs presenting as aggressive disease received great benefit from imatinib mesylate therapy., Conclusion: Mitotic activity is important in predicting the outcome of patients with high risk GIST who present at diagnosis without dissemination. This finding can have therapeutic implications.

Purpose: To investigate the prognostic role of thymidylate synthase (TS) polymorphisms in gastric cancer patients treated with radical surgery and fluorouracil-based adjuvant chemotherapy., Experimental Design: Ninety gastric cancer cases were identified among 187 patients previously enrolled in prospective case-control studies for disease susceptibility. Patients were genotyped for a G/C nucleotide change within a triple 28 bp variable number of tandem repeat sequence in the TS 5'-untranslated region (5'-UTR) and a 6 bp deletion in the TS 3'-untranslated region (3'-UTR). According to available functional data, patients with 5'-UTR 2R/2R, 2R/3C, 3C/3C genotypes were classified as low TS producers (5'-UTRlow) and patients with 5'-UTR 3G/3G, 3G/3C, 2R/3G genotypes as high TS producers (5'UTRhigh). Patients with 3'-UTR del6/del6 and del6/ins6 genotypes were classified as low TS producers (3'-UTRlow) and patients with 3'-UTR ins6/ins6 genotype as high TS producers (3'-UTRhigh). The prognostic analysis was based on 5'-UTR/3'-UTR combined genotypes., Results: Ten patients (11%) were 5'-UTRhigh/3'-UTRhigh, 36 patients were 5'-UTRhigh/3'-UTRlow, 19 patients were 5'-UTRlow/3'-UTRhigh, and 25 patients were 5'-UTRlow/3'-UTRlow. 5'-UTRlow/3'-UTRlow patients showed the best outcome and the threshold of statistical significance was achieved in the comparison of disease-free survival and overall survival with 5'-UTRhigh/3'-UTRlow patients and 5'-UTRhigh/3'-UTRhigh patients. The presence of at least one high TS expression genotype showed independent adverse prognostic role in multivariate analysis., Conclusions: The prognostic role of TS polymorphisms in gastric cancer deserves further investigation because the adverse effect of high TS expression genotypes may be a relevant information to improve adjuvant chemotherapeutic strategies.

Purpose: A high interleukin-1beta (IL-1B) and interleukin-1 receptor antagonist (IL-RN) ratio underlies an unfavorable proinflammatory status. Also, it seems to be involved in the mechanisms of cancer cachexia and tumor angiogenesis and metastasis. Two single nucleotide polymorphisms in IL-1B gene (IL-1B-511C/T,IL-1B-31T/C) and a variable number of tandem repeat polymorphisms in IL-RN gene (IL-1RNlong/2) enhance the circulating levels of the two cytokines. The prognostic role of IL-1B/IL-1RN genotypes was investigated in patients with relapsed and metastatic gastric cancer treated with palliative chemotherapy., Patients and Methods: Before starting palliative chemotherapy, 123 prospectively enrolled patients supplied peripheral-blood samples for DNA extraction. Survival data were analyzed according to IL-1RN/IL-1B genotypes., Results: Forty-two patients showed wild-type genotypes (IL-1RNlong/long, IL-1B-511C/C, and IL-1B-31T/T; group A). Forty-five patients showed the IL-1RN2 polymorphism, with wild-type IL-1B genotypes in seven patients and with IL-1B-511C/T and/or IL-1B-31T/C polymorphisms in 38 patients (group B). The remaining 36 patients demonstrated wild-type IL-1RN, with IL-1B-511C/T and/or IL-1B-31T/C polymorphisms (group C). In group A and B patients, the median progression-free survival (PFS) was 25 and 26 weeks, respectively, and median overall survival (OS) was 42 and 43 weeks, respectively. Group C patients showed worse PFS (median, 16 weeks) and OS (median, 28 weeks) than group A (P = .006 for PFS; P = .0001 for OS) and group B patients (P = .01 for PFS; P = .0001 for OS). The long/T/C haplotype was overrepresented in patients with shortened PFS (P = .001) and OS (P = .0005)., Conclusion: In patients with advanced gastric cancer, IL-1B polymorphisms showed adverse prognostic influence when coupled with wild-type IL-1RN genotype. These findings deserve further investigation for potential anticancer activity of recombinant IL-RN.

Objective: Contrast-enhanced real-time low-mechanical-index sonography is a new diagnostic technique for the assessment of macro- and microcirculation. The purpose of our article is to describe contrast-enhancement patterns of different benign focal liver lesions using the second-generation contrast agent SonoVue and to compare these findings with those of gadobenate dimeglumine-enhanced MRI., Conclusion: SonoVue-enhanced real-time low-mechanical-index sonography provides specific contrast-enhancement patterns of different benign focal liver lesions, allowing accurate characterization. Findings on SonoVue-enhanced sonography correlate well with those obtained on gadobenate dimeglumine-enhanced MRI.

Purpose: To assess the feasibility and the accuracy of a new technique in the evaluation of deep venous thrombosis (DVT) in the ilio-caval axis., Materials and Methods: Thirty-eight patients with suspected DVT were prospectively examined with unenhanced and enhanced colour-Doppler ultrasonography, and ascending contrast venography. Thirty-five out of 38 patients underwent US phlebography, consisting in manual injection of Levovist (Schering Ag, Berlin, Germany) through a 21-G cannula in a suitable dorsal vein of the foot ipsilateral to the suspected DVT. Three patients in whom it was not possible to cannulate a dorsal vein of foot underwent contrast-enhanced US after injection of Levovist through an antecubital vein, but where excluded from the present study. A contrast venogram was nonetheless carried out in all out three patients for comparison with the US-phlebography findings., Results: US-phlebography allowed better definition of the presence and extension of deep vein thrombosis in all patients, when compared to conventional color-Doppler US. In the evaluation of suspected acute thrombosis, the comparative efficacy of Doppler-US and US phlebography compared with ascending phlebography as the gold standard, was as follows: sensitivity of 85.7% and 90%, specificity of 71.4 and 100%, accuracy of 80.9 and 95.2%, PPV of 85.7, and 100%, and NPV of 71.4, and 75%, respectively. In the assessment of chronic thrombosis, US and US phlebography achieved a sensitivity of 90% and 100%, a specificity of 75% and 100%, an accuracy of 85.7% and 100%, a PPV of 100, 100%, a NPV of 87.5, 100%, respectively. No complications related to US phlebography were observed., Conclusions: US-phlebography is a promising tool in the assessment of deep vein thrombosis, being highly accurate and feasible. However, further studies based on larger series are mandatory to confirm our promising results and establish a standardised role for this new technique.

Purpose: Recent investigations have demonstrated that hypermethylation is a frequent mechanism for silencing tumor suppressor genes. This is a potentially reversible epigenetic change, and it is the target of a novel class of anticancer compounds with demethylating activity. Better understanding of the clinical implications of hypermethylation will allow the optimal planning of future trials with demethylating drugs. In this perspective, we investigated whether hypermethylation in the CDH1 promoter region is correlated with poor prognosis of patients with surgically resected, node-positive, diffuse gastric cancer., Experimental Design: Consecutive cases of diffuse gastric cancer were considered eligible for study entry. Additional inclusion criteria were radical surgery with a minimum of D1 lymphadenectomy, complete follow-up information, and availability of tumor specimens for methylation-specific PCR and immunohistochemistry analyses., Results: CDH1 promoter hypermethylation was found in 40 of 73 cases (54%), and it was significantly associated with worse prognosis. In patients with and without hypermethylation, the 5-year event-free survival rate was 30% and 62%, respectively, and the 5-year overall survival rate was 35% and 67%, respectively. CDH1 promoter hypermethylation retained its prognostic role for disease-free survival (P < 0.001) and overall survival (P < 0.001) in multivariate analysis. Immunohistochemistry showed a significant association between CDH1 methylation and E-cadherin expression (P < 0.001)., Conclusions: This study shows adverse prognostic effect of CDH1 promoter hypermethylation in patients with diffuse gastric cancer. This form of cancer, and other types with frequent hypermethylation and silencing of critical tumor suppressor genes, would make appropriate targets for the testing of novel compounds with demethylating activity.