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Purpose: To evaluate the prognostic value of coronary atherosclerotic burden, assessed by coronary artery calcium (CAC) score, and coronary vascular function, assessed by coronary flow reserve (CFR) in patients with suspected coronary artery disease (CAD)., Methods: We studied 436 patients undergoing hybrid 82 Rb positron emission tomography/computed tomography imaging. CAC score was measured according to the Agatston method, and patients were categorized into three groups (0, <400, and ≥400). CFR was calculated as the ratio of hyperemic to baseline myocardial blood flow, and it was considered reduced when <2., Results: Follow-up was 94% complete during a mean period of 47±15 months. During follow-up, 17 events occurred (4% cumulative event rate). Event-free survival decreased with worsening of CAC score category (p < 0.001) and in patients with reduced CFR (p < 0.005). At multivariable analysis, CAC score ≥400 (p < 0.01) and CFR (p < 0.005) were independent predictors of events. Including CFR in the prognostic model, continuous net reclassification improvement was 0.51 (0.14 in patients with events and 0.37 in those without). At classification and regression tree analysis, the initial split was on CAC score. For patients with a CAC score < 400, no further split was performed, while patients with a CAC score ≥400 were further stratified by CFR values. Decision curve analyses indicate that the model including CFR resulted in a higher net benefit across a wide range of decision threshold probabilities., Conclusions: In patients with suspected CAD, CFR provides significant incremental risk stratification over established cardiac risk factors and CAC score for prediction of adverse cardiac events.

Purpose: To assess the relationship between coronary atherosclerotic burden and vascular function in diabetic and nondiabetic patients after balancing for coronary risk factors., Methods: We studied 672 patients without overt coronary artery disease and normal myocardial perfusion on stress 82 Rb PET/CT imaging. To account for differences in baseline characteristics between diabetic patients and nondiabetic patients, we created a propensity score-matched cohort considering clinical variables and coronary risk factors., Results: Before matching, diabetic patients had higher coronary artery calcium (CAC) scores (p < 0.001) and lower coronary flow reserve (CFR; p < 0.001) than nondiabetic patients. After matching, CAC scores were comparable between diabetic and nondiabetic patients, but diabetic patients still had lower hyperaemic myocardial blood flow (p < 0.001) and CFR (p < 0.05). Patients were categorized by ln(CAC score) quartiles. There was a decrease in CFR with increasing CAC score quartile in both diabetic patients (p for trend < 0.01) and nondiabetic patients (p for trend < 0.005). Diabetes was associated with lower CFR across quartile categories (p < 0.002). In a multivariable linear regression analysis, CAC score was inversely related to CFR in both diabetic patients (p < 0.05) and nondiabetic patients (p < 0.001)., Conclusion: Diabetic patients had higher CAC scores than nondiabetic patients, but the difference disappeared when clinical characteristics were taken into account. Of note, diabetic patients also had lower CFR regardless of CAC score than nondiabetic patients after matching. Thus, coronary atherosclerotic burden and vascular function have to be seen as two different entities.

The study of proteins confined on a surface has attracted a great deal of attention due to its relevance in the development of bio-systems for laboratory and clinical settings. In this respect, organic bio-electronic platforms can be used as tools to achieve a deeper understanding of the processes involving protein interfaces. In this work, biotin-binding proteins have been integrated in two different organic thin-film transistor (TFT) configurations to separately address the changes occurring in the protein-ligand complex morphology and dipole moment. This has been achieved by decoupling the output current change upon binding, taken as the transducing signal, into its component figures of merit. In particular, the threshold voltage is related to the protein dipole moment, while the field-effect mobility is associated with conformational changes occurring in the proteins of the layer when ligand binding occurs. Molecular Dynamics simulations on the whole avidin tetramer in presence and absence of ligands were carried out, to evaluate how the tight interactions with the ligand affect the protein dipole moment and the conformation of the loops surrounding the binding pocket. These simulations allow assembling a rather complete picture of the studied interaction processes and support the interpretation of the experimental results.

In this contribution, we propose a label-free immunosensor, based on a novel type of electrolyte-gated field-effect transistor (EGOFET), for ultrasensitive detection of the C-reactive protein (CRP). The recognition layer of the biosensor is fabricated by physical adsorption of the anti-CRP monoclonal antibody onto a poly-3-hexyl thiophene (P3HT) organic semiconductor surface. A supplementary nonionic hydrophilic polymer is used as a blocking agent preventing nonspecific interactions and allowing a better orientation of the antibodies immobilized onto the P3HT surface. The whole biomolecule immobilization procedure does not require any pretreatment of the organic semiconductor surface, and the whole functionalization process is completed in less than 30 min. Surface plasmon resonance (SPR) measurements were performed to assess the amount of biomolecules physisorbed onto the P3HT and to evaluate the CRP binding proprieties of the deposited anti-CRP layer. A partial surface coverage of about 23 % of adsorbed antibody molecules was found to most efficiently sense the CRP. The electrical performance of the EGOFET immunosensor was comparable to that of a bare P3HT EGOFET device, and the obtained CRP calibration curve was linear over six orders of magnitude (from 4 pM to 2 μM). The relative standard deviation of the individual calibration points, measured on immunosensors fabricated on different chips, ranged between 1 and 14 %, and a detection limit of 2 pM (220 ng/L) was established. The novel electronic immunosensor is compatible with low-cost fabrication procedures and was successfully employed for the detection of the CRP biomarker in the clinically relevant matrix serum. Graphical abstract Schematic of the EGOFET immunosensor for CRP detection. The anti-CRP monoclonal antibody layer is physisorbed on the P3HT organic semiconductor and the CRP is directly measured by a label-free electronic EGOFET transducer.

This review aims to provide an update on the development involving dielectric/organic semiconductor (OSC) interfaces for the realization of biofunctional organic field-effect transistors (OFETs). Specific focus is given on biointerfaces and recent technological approaches where biological materials serve as interlayers in back-gated OFETs for biosensing applications. Initially, to better understand the effects produced by the presence of biomolecules deposited at the dielectric/OSC interfacial region, the tuning of the dielectric surface properties by means of self-assembled monolayers is discussed. Afterward, emphasis is given to the modification of solid-state dielectric surfaces, in particular inorganic dielectrics, with biological molecules such as peptides and proteins. Special attention is paid on how the presence of an interlayer of biomolecules and bioreceptors underneath the OSC impacts on the charge transport and sensing performance of the device. Moreover, naturally occurring materials, such as carbohydrates and DNA, used directly as bulk gating materials in OFETs are reviewed. The role of metal contact/OSC interface in the overall performance of OFET-based sensors is also discussed., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A general method to obtain the efficient entrapment of mixtures of glycoenzymes in calcium alginate hydrogel is proposed in this paper. As a proof of principle, three glycoenzymes acting in series (trehalase, glucose oxidase, and horseradish peroxidase) have been coimmobilized in calcium alginate beads. The release of the enzymes from the hydrogel mesh (leakage) is avoided by exploiting the enzyme's aggregation induced by the concanavalin A. The aggregation process has been monitored by dynamic light scattering technique, while both enzyme encapsulation efficiency and leakage have been quantified spectrophotometrically. Obtained data show an encapsulation efficiency above 95% and a negligible leakage from the beads when enzyme aggregates are larger than 300 nm. Operational stability of "as prepared" beads has been largely improved by a coating of alternated shells of polycation poly(diallyldimethylammonium chloride) and of alginate. As a test for the effectiveness of the overall procedure, analytical bioassays exploiting the enzyme-containing beads have been developed for the optical determination of glucose and trehalose, and limit of detection values of 0.2 and of 40 μM, respectively, have been obtained.

Thin-film transistors can be used as high-performance bioelectronic devices to accomplish tasks such as sensing or controlling the release of biological species as well as transducing the electrical activity of cells or even organs, such as the brain. Organic, graphene, or zinc oxide are used as convenient printable semiconducting layers and can lead to high-performance low-cost bioelectronic sensing devices that are potentially very useful for point-of-care applications. Among others, electrolyte-gated transistors are of interest as they can be operated as capacitance-modulated devices, because of the high capacitance of their charge double layers. Specifically, it is the capacitance of the biolayer, being lowest in a series of capacitors, which controls the output current of the device. Such an occurrence allows for extremely high sensitivity towards very weak interactions. All the aspects governing these processes are reviewed here., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Electrolyte-gated organic field-effect transistors are successfully used as biosensors to detect binding events occurring at distances from the transistor electronic channel that are much larger than the Debye length in highly concentrated solutions. The sensing mechanism is mainly capacitive and is due to the formation of Donnan's equilibria within the protein layer, leading to an extra capacitance (CDON) in series to the gating system., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand-protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein-ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters.

Bottom- and top-contact organic thin film transistors (OTFTs) were fabricated, using poly(3-hexylthiophene-2,5-diyl) (P3HT) and poly[2,5-bis(3-tetradecylthiophen-2-yl)thieno[3,2-b]thiophene] (PBTTT-C16) as p-type channel semiconductors. Four different types of OTFTs were fabricated and investigated as gas sensors against three volatile organic compounds, with different associated dipole moments. The OTFT-based sensor responses were evaluated with static and transient current measurements. A comparison between the different architectures and the relative organic semiconductor was made.

An organic field-effect transistor (OFET) integrating bacteriorhodopsin (bR) nanoassembled lamellae is proposed for an in-depth study of the proton translocation processes occurring as the bioelectronic device is exposed either to light or to low concentrations of general anesthetic vapors. The study involves the morphological, structural, electrical, and spectroscopic characterizations necessary to assess the functional properties of the device as well as the bR biological activity once integrated into the functional biointerlayer (FBI)-OFET structure. The electronic transduction of the protons phototranslocation is shown as a current increase in the p-type channel only when the device is irradiated with photons known to trigger the bR photocycle, while Raman spectroscopy reveals an associated C═C isomer switch. Notably, higher energy photons bring the cis isomer back to its trans form, switching the proton pumping process off. The investigation is extended also to the study of a PM FBI-OFET exposed to volatile general anesthetics such as halothane. In this case an electronic current increase is seen upon exposure to low, clinically relevant, concentrations of anesthetics, while no evidence of isomer-switching is observed. The study of the direct electronic detection of the two different externally triggered proton translocation effects allows gathering insights into the underpinning of different bR molecular switching processes.

Among the metal oxide semiconductors, ZnO has been widely investigated as a channel material in thin-film transistors (TFTs) due to its excellent electrical properties, optical transparency and simple fabrication via solution-processed techniques. Herein, we report a solution-processable ZnO-based thin-film transistor gated through a liquid electrolyte with an ionic strength comparable to that of a physiological fluid. The surface morphology and chemical composition of the ZnO films upon exposure to water and phosphate-buffered saline (PBS) are discussed in terms of the operation stability and electrical performance of the ZnO TFT devices. The improved device characteristics upon exposure to PBS are associated with the enhancement of the oxygen vacancies in the ZnO lattice due to Na(+) doping. Moreover, the dissolution kinetics of the ZnO thin film in a liquid electrolyte opens the possible applicability of these devices as an active element in "transient" implantable systems.

The functioning principles of electronic sensors based on organic semiconductor field-effect transistors (OFETs) are presented. The focus is on biological sensors but also chemical ones are reviewed to address general features. The field-induced electronic transport and the chemical and biological interactions for the sensing, each occurring at the relevant functional interface, are separately introduced. Once these key learning points have been acquired, the combined picture for the FET electronic sensing is proposed. The perspective use of such devices in point-of-care is introduced, after some basics on analytical biosensing systems are provided as well. This tutorial review includes also a necessary overview of the OFET sensing structures, but the focus will be on electronic rather than electrochemical detection. The differences among the structures are highlighted along with the implications on the performance level in terms of key analytical figures of merit such as: repeatability, sensitivity and selectivity.

We report on the use of a polyanionic proton conductor, poly(acrylic acid), to gate a poly[2,5-bis(3-tetradecylthiophen-2-yl)thieno[3,2-b]thiophene]-based organic field-effect transistor (OFET). A planar configuration of the OFET is evaluated, and the electrical performance and implementation on a flexible substrate are discussed.

A non-invasive test for oro-ileal transit time (OITT) evaluation was developed, based on the measurement of tauroursodeoxycholic acid (TUDCA) oral fluid concentration profile after its oral administration. Exploiting the fact that TUDCA is actively absorbed only in the ileum, OITT is measured as the time corresponding to TUDCA maximum oral fluid concentration (tmax). To measure oral fluid TUDCA concentration in a point-of-care setting, an ultrasensitive portable immunosensor was developed, based on a competitive chemiluminescent enzyme immunoassay (CL-EIA), using immobilized anti-TUDCA antibody and an ursodeoxycholic acid (UDCA)-peroxidase conjugate as tracer, detected by enhanced chemiluminescence employing a portable charge-coupled device (CCD)-based device. The test was validated in 24 healthy subjects before and after treatment with Loperamide, a drug that increases OITT. The developed CL-EIA was accurate and precise, with a LLOQ of 50 pmol L(-1). The measured OITT for healthy subjects (291 ± 50 min) was fairly well correlated with OITT values obtained by measuring TUDCA in serum (r=0.89). An increased OITT was observed in all the studied subjects after Loperamide treatment. The CL immunosensor can be employed directly in gastroenterology and paediatric units and it can thus represent a new non-invasive simple test for OITT evaluation in a point-of-care setting, with improved diagnostic utility., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A Functional Bio-Interlayer Organic Field-Effect Transistor (FBI-OFET) sensor, embedding a streptavidin protein capturing layer, capable of performing label-free selective electronic detection of biotin at 3 part per trillion (mass fraction) or 15 pM, is proposed here. The response shows a logarithmic dependence spanning over 5 orders of magnitude of analyte concentration. The optimization of the FBI analytical performances is achieved by depositing the capturing layer through a controllable Layer-by-Layer (LbL) assembly, while an easy processable spin-coating deposition is proposed for potential low-cost production of equally highly performing sensors. Furthermore, a Langmuirian adsorption based model allows rationalizing the analyte binding process to the capturing layer. The FBI-OFET device is shown to operate also with an antibody interlayer as well as with an ad hoc designed microfluidic system. These occurrences, along with the proven extremely high sensitivity and selectivity, open to FBI-OFETs consideration as disposable electronic strip-tests for assays in biological fluids requiring very low detection limits.

Anchored, biotinylated phospholipids forming the capturing layers in an electrolyte-gated organic field-effect transistor (EGOFET) allow label-free electronic specific detection at a concentration level of 10 nM in a high ionic strength solution. The sensing mechanism is based on a clear capacitive effect across the PL layers involving the charges of the target molecules., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The detailed action mechanism of volatile general anesthetics is still unknown despite their effect has been clinically exploited for more than a century. Long ago it was also assessed that the potency of an anesthetic molecule well correlates with its lipophilicity and phospholipids were eventually identified as mediators. As yet, the direct effect of volatile anesthetics at physiological relevant concentrations on membranes is still under scrutiny. Organic field-effect transistors (OFETs) integrating a phospholipid (PL) functional bio inter-layer (FBI) are here proposed for the electronic detection of archetypal volatile anesthetic molecules such as diethyl ether and halothane. This technology allows to directly interface a PL layer to an electronic transistor channel, and directly probe subtle changes occurring in the bio-layer. Repeatable responses of PL FBI-OFET to anesthetics are produced in a concentration range that reaches few percent, namely the clinically relevant regime. The PL FBI-OFET is also shown to deliver a comparably weaker response to a non-anesthetic volatile molecule such as acetone., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Differentiation of enantiomers remains one of the most attractive and important research areas in analytical chemistry due to its impact on pharmaceutical, chemical, biotechnology, and food industries. For a long time chiral separation techniques, such as high performance liquid chromatography (HPLC), gas chromatography (GC), and capillary electrophoresis (CE), have represented the gold standard for the separation and determination of enantiomers. These techniques, besides being time consuming and expensive, are also not suitable for real time analysis. Therefore, the development of fast and reliable chiral sensors remains a challenge to achieve on-line analysis of enantiomers in both gas and liquid samples. The scope of this chapter is to provide an overview on the basic functioning principles, as well as on the performance level, of solid-state sensing devices for enantiomers differentiation. Particular attention is paid to work providing a set of analytical figures of merit (sensitivity, repeatability, reproducibility, limit-of-detection, etc.) as well as to studies involving miniaturized (or miniaturizable) analytical devices that can deliver real-time, on-line, and label-free information on chiral compounds.

To evaluate the tomographic features in differentiating benign from malignant splenic diseases, 54 patients with a cytohistological examination and a contrast-enhanced multidetector computed tomography (ce-MDCT) and/or positron emission tomography/computed tomography (PET/CT) were retrospectively selected. Significant associations were observed between ce-MDCT Pattern 3 (focal hyperdense lesion) and Pattern 4 (infarcts/cysts) as well as PET/CT Pattern 3 (focal photopenia/diffuse uptake

Biosystems integration into an organic field-effect transistor (OFET) structure is achieved by spin coating phospholipid or protein layers between the gate dielectric and the organic semiconductor. An architecture directly interfacing supported biological layers to the OFET channel is proposed and, strikingly, both the electronic properties and the biointerlayer functionality are fully retained. The platform bench tests involved OFETs integrating phospholipids and bacteriorhodopsin exposed to 1-5% anesthetic doses that reveal drug-induced changes in the lipid membrane. This result challenges the current anesthetic action model relying on the so far provided evidence that doses much higher than clinically relevant ones (2.4%) do not alter lipid bilayers' structure significantly. Furthermore, a streptavidin embedding OFET shows label-free biotin electronic detection at 10 parts-per-trillion concentration level, reaching state-of-the-art fluorescent assay performances. These examples show how the proposed bioelectronic platform, besides resulting in extremely performing biosensors, can open insights into biologically relevant phenomena involving membrane weak interfacial modifications.

Most of the success of electronic devices fabricated to actively interact with a biological environment relies on the proper choice of materials and efficient engineering of surfaces and interfaces. Organic materials have proved to be among the best candidates for this aim owing to many properties, such as the synthesis tunability, processing, softness and self-assembling ability, which allow them to form surfaces that are compatible with biological tissues. This review reports some research results obtained in the development of devices which exploit organic materials' properties in order to detect biologically significant molecules as well as to trigger/capture signals from the biological environment. Among the many investigated sensing devices, organic field-effect transistors (OFETs), organic electrochemical transistors (OECTs) and microcantilevers (MCLs) have been chosen. The main factors motivating this choice are their label-free detection approach, which is particularly important when addressing complex biological processes, as well as the possibility to integrate them in an electronic circuit. Particular attention is paid to the design and realization of biocompatible surfaces which can be employed in the recognition of pertinent molecules as well as to the research of new materials, both natural and inspired by nature, as a first approach to environmentally friendly electronics.

Aim: To evaluate whether FDG-positron emission tomography (PET)/computed tomography (CT) may be an accurate technique in the assessment of the T stage in patients with colorectal cancer., Methods: Thirty four consecutive patients (20 men and 14 women; mean age: 63 years) with a histologically proven diagnosis of colorectal adenocarcinoma and scheduled for surgery in our hospital were enrolled in this study. All patients underwent FDG-PET/CT preoperatively. The primary tumor site and extent were evaluated on PET/CT images. Colorectal wall invasion was analysed according to a modified T classification that considers only three stages (≤ T2, T3, T4). Assessment of accuracy was carried out using 95% confidence intervals for T., Results: Thirty five/37 (94.6%) adenocarcinomas were identified and correctly located on PET/CT images. PET/CT correctly staged the T of 33/35 lesions identified showing an accuracy of 94.3% (95% CI: 87%-100%). All T1, T3 and T4 lesions were correctly staged, while two T2 neoplasms were overstated as T3., Conclusion: Our data suggest that FDG-PET/CT may be an accurate modality for identifying primary tumor and defining its local extent in patients with colorectal cancer.

The official methods specified in the national bovine and ovine/caprine brucellosis eradication plan are the Rose Bengal and complement fixation tests. In the current phase of the eradication plan, it is often difficult to interpret the results obtained with the official tests. Consequently, additional tests that offer greater sensitivity and specificity are thus required. For this reason, two methods, the indirect chemiluminescence enzyme-linked immunosorbent assay (i-ELISA CL) and the competitive chemiluminescence ELISA (c-ELISA CL) that use a chemiluminescent substrate to determine anti-Brucella antibodies in bovine and ovine serum were validated. The methods are based on the detection of anti-Brucella antibodies in serum by catalysis of a chemiluminescent enzyme substrate (luminol/H2O2/enhancer system) by peroxidase conjugated to secondary anti IgG antibodies in i-ELISA CL and to monoclonal anti-lipopolysaccharide (LPS) antibodies in c-ELISA CL. From the results obtained, a cut-off of 60% for bovine serum and 37.5% for ovine serum, expressed as positivity rate (PR), were established using these cut-off values, for the i-ELISA CL, 100% sensitivity and specificity was obtained for bovine serum and 100% sensitivity and 99.8% specificity for ovine serum. Cut-off values of 30% for bovine serum and 40% for ovine serum, expressed as inhibition rate, were selected for c-ELISA CL, which ensured 100% sensitivity and specificity in both cases.

A simple and rapid multiplexed sandwich chemiluminescent enzyme immunoassay has been developed for the simultaneous detection of Escherichia coli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, and Listeria monocytogenes. To achieve the multiplexed detection of the four pathogens, a new polystyrene 96 well microtiter plate format has been designed, in which each main well contains four subwells in the bottom. The monoclonal antibodies specific for each bacteria were separately immobilized in each subwell. When the samples were added to the main wells, the bacteria able to specifically bind to the corresponding monoclonal antibody were captured in one of the four subwells. Subsequently, a mixture of peroxidase-labeled polyclonal antibodies against the four bacteria was added and the peroxidase activity of the bound polyclonal labeled antibodies in each well was measured by an enhanced luminol-based chemiluminescent cocktail using a low-light charge-coupled imaging device. The assay was simple and fast, and the limit of quantification was in the order of 104-105 CFU/mL for all bacterial species. The accuracy of the method, evaluated by comparison of the results with a conventional culturing methodology, was satisfactory, with recovery values ranging from 90 to 120%. This method can be used as a screening test to evaluate the presence of these pathogen bacteria in different foodstuffs.

Aim: To develop a new formulation with hydroxy propyl methyl cellulose and Shellac coating for extended and selective delivery of butyrate in the ileo-caecal region and colon., Methods: One-gram sodium butyrate coated tablets containing 13C-butyrate were orally administered to 12 healthy subjects and 12 Crohn's disease patients and the rate of 13C-butyrate absorption was evaluated by 13CO2 breath test analysis for eight hours. Tauroursodeoxycholic acid (500 mg) was co-administered as a biomarker of oro-ileal transit time to determine also the site of release and absorption of butyrate by the time of its serum maximum concentration., Results: The coated formulation delayed the 13C-butyrate release by 2-3 h with respect to the uncoated tablets. Sodium butyrate was delivered in the intestine of all subjects and a more variable transit time was found in Crohn's disease patients than in healthy subjects. The variability of the peak 13CO2 in the kinetic release of butyrate was explained by the inter-subject variability in transit time. However, the coating chosen ensured an efficient release of the active compound even in patients with a short transit time., Conclusion: Simultaneous evaluation of breath 13CO2 and tauroursodeoxycholic acid concentration-time curves has shown that the new oral formulation consistently releases sodium butyrate in the ileo-cecal region and colon both in healthy subjects and Crohn's disease patients with variable intestinal transit time. This formulation may be of therapeutic value in inflammatory bowel disease patients due to the appropriate release of the active compound.

Seven monoclonal antibodies (MAbs) specific for Escherichia coli O157:H7, one of the major causes of haemorrhagic colitis in humans, were produced by immunising Balb/c mice with the strain E. coli O157:H7. These monoclonal antibodies do not cross-react with other bacteria such as Salmonella enterica serovar Typhimurium, E. coli O14, E. coli JM109, S. enterica serovar Enteritidis, S. panama, S. saintpaul, S. derby, S. muenchen, S. bredeney, S. hadar, Yersinia enterocolitica, Proteus vulgaris, Shigella flexneri, Listeria ivanovii, L. monocytogenes 13M, L. innocua, Enterobacter cloacae, E. agglomerans, E. amnigenus, Citrobacter freundii, Escherichia fergussoni or Klebsiella pneumoniae. Of the seven MAbs obtained, MAb 8B8C3 was selected to prepare a high-sensitivity sandwich ELISA method specific for O157:H7.

The presence of endocrine-disrupting compounds in influent and effluent water samples from four waste water treatment plants located in Italy was studied. The estrogen-like activity of the water samples was measured using a chemiluminescent recombinant yeast assay which is based on genetically engineered yeast cells that express the human estrogen receptor. This receptor, once activated, elicits the expression of the reporter gene lac-Z and, consequently, the production of beta-galactosidase, which is then measured by chemiluminescence. To control and minimize sample matrix effects, an external control based on a modified yeast strain stably expressing beta-galactosidase was developed and also used in the assay. Rapid and sensitive chemiluminescent enzyme immunoassays were also developed and validated for the quantification of 17beta-estradiol, estrone, and estriol in waste water samples. Results from both methods were compared with a reference high-performance liquid chromatography and electrospray ionization tandem mass spectrometry (HPLC ESI-MS-MS) method developed for the quantification of natural estrogens. The recombinant yeast assay revealed a significant estrogenic activity in the influent samples, ranging from 80 to 400 pmol/L 17beta-estradiol equivalents (EEQ), which was reduced by 70-95% in the effluent samples. The yeast assay also showed a systematic 20-30% overestimation of estrogenic activity relative to the HPLC ESI-MS-MS method, suggesting the presence of other compounds in the samples with estrogenic activity. The chemiluminescent enzyme immunoassays showed the presence of estrogens in the influent samples (mean concentrations: 350-450 pmol/L for estrone, 5-100 pmol/L for 17beta-estradiol, 25-300 pmol/L for estriol), with significantly lower concentrations detected in the respective effluent samples. The waste water treatment was able to reduce natural estrogen concentrations by 40-95%, although a high variability was observed. The enzyme immunoassay data correlated well with data obtained by the HPLC ESI-MS-MS method. Although the recombinant yeast assay represents a useful tool for a first-level screening of estrogenic activity due to its simplicity and high analytical throughput, sample matrix effects observed in waste water of industrial origin were found to strongly affect the yeast cells response, even when properly corrected for using the external control, thereby limiting its use to urban waste water. Its integration with chemiluminescent enzyme immunoassays would improve its performance by reducing false negative results, thereby enabling its use in extensive studies monitoring for the presence of endocrine-disrupting compounds in urban treatment plant effluents.