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ABSTRACT: 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.
Analytica chimica acta 07/2012; 736:92-9. · 4.31 Impact Factor
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ABSTRACT: The water soluble 4-sulfocalix[n]arenes (with n = 4,6,8) have been investigated as potential synthetic receptors for cyclodiene organochlorine pesticides. Steady state fluorescence
experiments in ethanol solution have shown that only the cavitands with n equal to 6 and 8 form complexes, of comparable stability, with heptachlor. Electrochemical data, obtained in water solution,
confirmed the ability of 4-sulfocalix[6]arene to bind the heptachlor, unlike the smaller calixarene. Moreover, a significant
increase in the stability constant is observed in water solutions. This stability is caused by the sterical hindrance of pesticides
with respect to the cavity dimension of the calixarene. This results in a selective interaction of this molecule with other
organochlorine pesticides. Binding experiments, carried out with endosulfan have shown that, despite of chemical similarity,
4-sulfocalix[6]arene and 4-sulfocalix[8]arene behave in a very different way: the former is unable to bind this pesticide,
while the latter shows a binding constant of 4.7 × 105 with endosulfan. To investigate the molecular features of the interactions, molecular dynamic simulations of 4-sulfocalix[6]arene
in presence of heptachlor in water solution have been performed. These simulations show that different configurations of heptachlor
inside the calixarene cavity are equally populated and easily interconverting, suggesting that a non specific hydrophobic
interaction plays a key role in the complex stability. These studies have permitted to individuate versatile synthetic receptors
for organochlorine pesticides.
Microchimica Acta 04/2012; 163(3):195-202. · 3.03 Impact Factor
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ABSTRACT: A disposable tri-enzymatic biosensor is presented for the determination of α-amylase in human saliva. It is based on the quantity
of maltose generated by hydrolysis of maltopentose in the presence of salivary α-amylase. The biosensor is fabricated by co-immobilization
of the enzymes α-glucosidase, glucose oxidase, and mutarotase on screen-printed electrodes modified with Prussian Blue. The
assay can be performed with a “drop” of sample, this allowing for ease and simplicity. A linear relationship is found for
the range from 5 to 250 units per mL, with an LOD of 5 units per mL. The biosensor is stable for at least one month and over
this time retains 80% of its original activity. The system was then evaluated for matrix effects of human saliva and compared
to a spectrometric method using a commercially available kit.
KeywordsBiosensor-Alpha amylase-Biomarker-Saliva-Stress
Microchimica Acta 04/2012; 170(3):243-249. · 3.03 Impact Factor
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ABSTRACT: The present review reports the research carried out during last 9 years on biosensors based on cholinesterase inhibition for
nerve agents, organophosphorus and carbammic insecticides, and aflatoxin B1 detection. Relative applications in environmental and food areas are also reported. Special attention is paid to the optimization
of parameters such as enzyme immobilization, substrate concentration, and incubation time in the case of reversible inhibition
by aflatoxin B1 or irreversible inhibition by organophosphorus and carbamic insecticides, and nerve agents in order to optimize and improve
the analytical performances of the biosensor. Evaluation of selectivity of the system is also discussed.
KeywordsBiosensors-Inhibition-Insecticides-Cholinesterase-Nerve agents-Aflatoxin
Microchimica Acta 04/2012; 170(3):193-214. · 3.03 Impact Factor
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ABSTRACT: Paper-based artworks are among the most valuable assets for transmission of knowledge. Historical paper is composed of different polysaccharides (e.g. cellulose), binders, and glues. During aging all of these components undergo several degradation processes, as a result of external and intrinsic causes, and these can compromise the state of conservation of the document. In this work, application of a new biotechnological strategy for paper artefact preservation is reported. By making use of innovative and non-invasive materials, for example appropriate hydrogels, in combination with selective electrochemical biosensors, it is possible to simultaneously verify the degradation condition of the paper artwork and then to efficiently clean it, while monitoring the process of removal of both pollution and degradation products. In this paper, we focus on specific examples in which such techniques have been applied to paper artworks and that illustrate the advantages and potential of this biotechnology compared with the traditional paper-cleaning methods currently in use.
Analytical and Bioanalytical Chemistry 03/2012; 403(6):1485-9. · 3.78 Impact Factor
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ABSTRACT: 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.
Journal of diabetes science and technology 01/2012; 6(5):1172-81.
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ABSTRACT: Here, we demonstrate a strategy to convert the graded Michaelis-Menten response typical of unregulated enzymes into a sharp, effectively all-or-none response. We do so using an approach analogous to the "branch point effect", a mechanism observed in naturally occurring metabolic networks in which two or more enzymes compete for the same substrate. As a model system, we used the enzymatic reaction of glucose oxidase (GOx) and coupled it to a second, nonsignaling reaction catalyzed by the higher affinity enzyme hexokinase (HK) such that, at low substrate concentrations, the second enzyme outcompetes the first, turning off the latter's response. Above an arbitrarily selected "threshold" substrate concentration, the nonsignaling HK enzyme saturates leading to a "sudden" activation of the first signaling GOx enzyme and a far steeper dose-response curve than that observed for simple Michaelis-Menten kinetics. Using the well-known GOx-based amperometric glucose sensor to validate our strategy, we have steepen the normally graded response of this enzymatic sensor into a discrete yes/no output similar to that of a multimeric cooperative enzyme with a Hill coefficient above 13. We have also shown that, by controlling the HK reaction we can precisely tune the threshold target concentration at which we observe the enzyme output. Finally, we demonstrate the utility of this strategy for achieving effective noise attenuation in enzyme logic gates. In addition to supporting the development of biosensors with digital-like output, we envisage that the use of all-or-none enzymatic responses will also improve our ability to engineer efficient enzyme-based catalysis reactions in synthetic biology applications.
Analytical Chemistry 12/2011; 84(2):1076-82. · 5.86 Impact Factor
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ABSTRACT: 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%).
Analytica chimica acta 11/2011; 707(1-2):171-7. · 4.31 Impact Factor
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Analytical Letters 10/2011; · 1.02 Impact Factor
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ABSTRACT: Surface-confined DNA probes are increasingly used as recognition elements (or presentation scaffolds) for detection of proteins, enzymes, and other macromolecules. Here we demonstrate that the density of the DNA probe monolayer on the gold electrode is a crucial determinant of the final signalling of such devices. We do so using redox modified single-stranded and double-stranded DNA probes attached to the surface of a gold electrode and measuring the rate of digestion in the presence of a non-specific nuclease enzyme. We demonstrate that accessibility of DNA probes for binding to their macromolecular target is, as expected, improved at lower probe densities. However, with double-stranded DNA probes, even at the lowest densities investigated, a significant fraction of the immobilized probe is inaccessible to nuclease digestion. These results stress the importance of the accessibility issue and of probe density effects when DNA-based sensors are used for detection of macromolecular targets.
Analytical and Bioanalytical Chemistry 09/2011; 402(1):413-21. · 3.78 Impact Factor
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ABSTRACT: 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.
ChemSusChem 06/2011; 4(6):792-6. · 6.83 Impact Factor
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Antonio Pietroiusti,
Micol Massimiani,
Ivana Fenoglio,
Massimiliano Colonna,
Federica Valentini, Giuseppe Palleschi,
Antonella Camaioni,
Andrea Magrini,
Gregorio Siracusa,
Antonio Bergamaschi,
Alessandro Sgambato,
Luisa Campagnolo
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ABSTRACT: Several in vitro and in vivo studies suggest local and systemic effects following exposure to carbon nanotubes. No data are available, however, on their possible embryotoxicity in mammals. In this study, we tested the effect of pristine and oxidized single-wall carbon nanotubes (SWCNTs) on the development of the mouse embryo. To this end, SWCNTs (from 10 ng to 30 μg/mouse) were administered to female mice soon after implantation (postcoital day 5.5); 10 days later, animals were sacrificed, and uteri, placentas, and fetuses examined. A high percentage of early miscarriages and fetal malformations was observed in females exposed to oxidized SWCNTs, while lower percentages were found in animals exposed to the pristine material. The lowest effective dose was 100 ng/mouse. Extensive vascular lesions and increased production of reactive oxygen species (ROS) were detected in placentas of malformed but not of normally developed fetuses. Increased ROS levels were likewise detected in malformed fetuses. No increased ROS production or evident morphological alterations were observed in maternal tissues. No fetal and placental abnormalities were ever observed in control animals. In parallel, SWCNT embryotoxicity was evaluated using the embryonic stem cell test (EST), a validated in vitro assay developed for predicting embryotoxicity of soluble chemical compounds, but never applied in full to nanoparticles. The EST predicted the in vivo data, identifying oxidized SWCNTs as the more toxic compound.
ACS Nano 06/2011; 5(6):4624-33. · 10.77 Impact Factor
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ABSTRACT: 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.
Analytical Chemistry 10/2010; · 5.86 Impact Factor
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Daniela Romanazzo,
Francesco Ricci,
Giulia Volpe,
Christopher T Elliott,
Silvia Vesco,
Katy Kroeger,
Danila Moscone,
Joerg Stroka,
Hans Van Egmond,
Markus Vehniäinen, Giuseppe Palleschi
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ABSTRACT: 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.
Biosensors & bioelectronics 08/2010; 25(12):2615-21. · 5.43 Impact Factor
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ABSTRACT: In this work, the electrochemical behavior of carbon black paste electrode prepared using a nanostructured commercial carbon black (N220) was investigated. The sensor was challenged with several potentially interesting analytes by means of cyclic voltammetry technique and the results compared with graphite carbon paste electrode. Shifting in peak potential and/or increase in the peak currents for some analytes such as ferricyanide, ascorbic acid, acetoaminophen, epinephrine, and DOPAC were observed. The carbon black paste was combined with tyrosinase enzyme to produce a biosensor which was challenged in amperometric mode with catechol. The highest sensitivity, equal to 625 nA/μM, coupled with lowest detection limit of 0.008 μM was observed for this formulation relative to those made with graphite and even when compared with carbon nanotubes tyrosinase paste electrode previously reported. In this way, the carbon black could be considered a good electrode material for constructing other electrochemical biosensors with the advantage to be a nanostrutured material at low cost.
Analytical Letters 07/2010; 43(10-11):1688-1702. · 1.02 Impact Factor
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ABSTRACT: 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.
Chemical Communications 03/2010; 46(10):1742-4. · 6.17 Impact Factor
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ABSTRACT: 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.
Journal of Agricultural and Food Chemistry 08/2009; 57(16):7200-4. · 2.82 Impact Factor
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ABSTRACT: 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.
Journal of Visualized Experiments 01/2009;
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ABSTRACT: 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.
Biosensors & bioelectronics 11/2008; 24(7):1962-8. · 5.43 Impact Factor
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ABSTRACT: Sensor technology based on immunological ELISA analyses combined with various electrochemical detection systems is being developed
to quantify phycotoxins in algae and seafood. The use of disposable screen-printed electrodes for the immunosensor development
is illustrated. Laboratory responses on contaminated mussels were obtained by domoic acid and saxitoxin sensors with detection
limit of 5 and 0.1 ng/ml respectively. Application to algal extracts was also performed to detect domoic acid concentration
in phytoplankton populations along Latium (Middle Tyrrhenian Sea, Mediterranean Sea) coast.
06/2008: pages 301-310;
