M. Mascini

Disposable Screen-Printed Electrodes (Spe) Mercury-Free for Lead Detection

ABSTRACT Strategies to modify screen-printed electrodes (SPE) for lead determination are reported. Dithizone was mixed with graphite ink to obtain a modified screen-printed strip to detect ppb levels of lead(II) (detection limit 12 μg/l) using square wave anodic stripping voltammetry (SWASV). In addition, screen-printed electrodes were also modified by casting a few μl of a Nafion® solution onto the working electrode surface. In this case, ppb levels of lead were detected (detection limit 15 μg/1), using potentiometric stripping analysis (PSA). The addition of an ionophore to Nafion® polymer was also investigated, but this did not yield a significant improvement.

Analytical applications of aptamers

Aptamers are single stranded DNA or RNA ligands which can be selected for different targets starting from a library of molecules containing randomly created sequences. Aptamers have been selected to bind very different targets, from proteins to small organic dyes. Aptamers are proposed as alternatives to antibodies as biorecognition elements in analytical devices with ever increasing frequency. This in order to satisfy the demand for quick, cheap, simple and highly reproducible analytical devices, especially for protein detection in the medical field or for the detection of smaller molecules in environmental and food analysis. In our recent experience, DNA and RNA aptamers, specific for three different proteins (Tat, IgE and thrombin), have been exploited as bio-recognition elements to develop specific biosensors (aptasensors). These recognition elements have been coupled to piezoelectric quartz crystals and surface plasmon resonance (SPR) devices as transducers where the aptamers have been immobilized on the gold surface of the crystals electrodes or on SPR chips, respectively.

Detection of coumaphos in honey using a screening method based on an electrochemical acetylcholinesterase bioassay

An analytical protocol based on an electrochemical assay for the detection of acetylcholinesterase (AChE) inhibitors has been optimised for the detection of coumaphos in honey. Coumaphos is a phosphotionate insecticide requiring transformation in the corresponding oxo-form to act as an effective AChE inhibitor. The inhibition assay was based on the electrochemical detection of the product of AChE, choline, via a choline oxidase biosensors obtained using prussian-blue modified screen printed electrodes. A simple procedure for the oxidation of coumaphos via N-bromosuccinimide (NBS) and AChE inhibition was optimised. A calibration curve for coumaphos (8-1000 ng/ml) was obtained in buffer; the intra electrode CV ranged between 8 and 12% whereas the inter electrode CV was comprised between 12 and 14%. A detection limit (LOD) of 8 ng/ml was achieved, with an I(50%) of 105 ng/ml. The assay was then applied to detect coumaphos in honey samples. Despite the solubility of the samples in buffer, the assay was affected by many electrochemical interferences present in this sample matrix A simple C18 based solid phase extraction procedure has been then optimised and used for the assay. This allowed to eliminate all the electrochemical interferences with a satisfactory coumaphos recovery (around 86%) for a final LOD of 33 ng/g. The developed assay applied to detect coumaphos in different honey samples gave data well correlated with LC-MS detection.

Oligopeptides as mimic of acetylcholinesterase: from the rational design to the application in solid-phase extraction for pesticides.

Three different peptides (His-Glu-Pro-Ser, His-Gly-Ser-Ala and Glu-Pro-Ser-Ala) were selected and tested to be used as affinity binding receptors for organophosphate and carbamate pesticides. The peptides were rationally designed by mimicking acetylcholinesterase active site. The simulated binding energy of the three tetrapeptides versus one model of organophosphate (paraoxon) and one of carbamate (carbaryl) pesticide was calculated; a good correlation between shape designed and binding score was obtained. The binding properties of the peptide-pesticide interaction were studied following the variation of UV-visible spectra in different solvents. The binding constants in water, which were nicely correlated with computational data, ranged from 506 (+/-115) to 36(+/-2) M(-1). All the peptides had a 5-fold decrease in binding by changing solvent, going from water to less polar ethanol. The binding affinity suggested the use of these ligands as a preanalytical tool in extraction cartridges. The tetrapeptides efficiency was tested linking the peptides to two different supports. The cartridges prepared using His-Glu-Pro-Ser sequence was, as predicted, able to bind paraoxon and carbaryl with recovery values in the 72-88% range at pH 4.5. Intercolumn, interday RSD was in the 4-7% range. The columns were used for 80 cycles before losing retention ability.

Piezoelectric Sensors Based on Biomimetic Peptides for the Detection of Heat Shock Proteins (HSPs) in Mussels

Abstract We describe a set of label‐free piezoelectric biosensors for fast, qualitative, and direct detection of HSP 70 in crude extract of the mussel mantle. A monoclonal anti‐HSP 70 antibody and three heptapeptides chosen using a computational/combinatorial approach were immobilized covalently to gold particles on quartz surfaces using a self‐assembled monolayer (SAM). A clear frequency shift in the presence of standard solutions of HSP 70 from bovine brain was obtained. The difference in signal intensity among the biomimetic sensors (the peptide sensors) was nicely correlated with the computationally predicted binding scores. Moreover, the peptides exhibited a signal comparable to the monoclonal antibody based immunosensor. Regeneration of the affinity sensors was also investigated obtaining a significant loss of signal only after 10 regeneration cycles with repeatability in the 20%–28% range and reproducibility in the 25%–35% range. Cross‐reactivity of the HSP sensors was tested using bovine serum albumin (BSA) and rabbit IgG. All the affinity sensors exhibited low nonspecific binding, compared with a blank sensor and a negative control sensor. The sensitivity pattern with the real sample (mussel mantle) reflected the one obtained from the standard solution of HSPs 70, demonstrating that all the sensors are able to detect the target analyte in a complex matrix as in a crude extract.

Peptides trapping cocaine: docking simulation and experimental screening by solid phase extraction followed by liquid chromatography mass spectrometry in plasma samples.

Two different hexapeptides were computationally designed and tested as selective SPE sorbent for cocaine. The amino acid residues used for designing the two hexapeptides, tested in SPE experiments, were, according to chemical function and interatomic distances, the most (QHWWDW) and the lowest (ESSIDH) preserved sequences in 4 proteins binding cocaine. The hexapeptide-cocaine complex was docked with different scoring functions combinations and resulting binding scores were compared with the SPE results. The extraction procedure for SPE was optimized considering volume loading, pH effect, and human plasma matrix interferences. Cocaine was loaded onto the modified resin cartridge at 10 ng mL(-1) and the peptide QHWWDW was found to have the highest recovery with the best retention at pH 7.5, in agreement with docking simulation. Retention experiments were carried out also on cocaine metabolites nor-cocaine, benzoylecgonine and ecgonine methyl ester. Except for nor-cocaine the retention of metabolites on resin modified with peptide QHWWDW decreased drastically confirming the peptide selectivity, and validating the simulation data. Compared to standard solutions, only a slight decrease in cocaine recovery was observed loading human plasma samples after a partial protein precipitation.

Selection of peptide ligands for piezoelectric peptide based gas sensors arrays using a virtual screening approach.

Virtual and experimental affinity binding properties of 5 different peptides (cysteinylglycine, glutathione, Cys-Ile-His-Asn-Pro, Cys-Ile-Gln-Pro-Val, Cys-Arg-Gln-Val-Phe) vs. 14 volatile compounds belonging to relevant chemical classes were evaluated. The peptides were selected in order to have a large variability in physicochemical characteristics (including length). In virtual screening a rapid and cost-effective computational methodology for predicting binding scores of small peptide receptors vs. volatile compounds is proposed. Flexibility was considered for both ligands and peptides and each peptide conformer was treated as a possible receptor, generating a dedicated box and then running a docking process vs. all possible conformers of the 14 volatile compounds. The 5 peptides were covalently bound to gold nanoparticles and deposited onto 20 MHz quartz crystal microbalances to realize gas sensors. Gas sensing confirmed that each of the peptide conferred to the gold nanoparticles a particular selectivity pattern able to discriminate the 14 volatile compounds. The largest response was obtained for the pentapeptides Cys-Ile-His-Asn-Pro and Cys-Ile-Gln-Pro-Val while low response was achieved for the dipeptide. The comparative study, carried using a two-tailed T test, demonstrated that virtual screening was able to predict reliably the sensing ability of the pentapeptides. The dipeptide receptor exhibited 29% of virtual-experimental matching vs. 71% of glutathione and up to 93% for the pentapeptides. This virtual screening approach was proved to be a promising tool in predicting the behaviour of sensors array for gas detection.

An Electrochemical Bioassay for Dichlorvos Analysis in Durum Wheat Samples

The use of an acetylcholinesterase inhibition assay for the detection of dichlorvos in durum wheat samples by a simplified extraction procedure is reported. After an incubation step, the residual activity was determined with an amperometric biosensor using a portable potentiostat. The use of electric eel and recombinant acetylcholinesterase was compared. The effect of the matrix extract was evaluated by using various sample:solvent ratios, 1:2.5, 1:5, 1:10, and 1:20. The optimal extraction ratio, considering the electrochemical interferences and the effect on enzyme activity and bioavailability of the pesticide, was 1:10. Calibrations were performed in buffer and durum wheat extract. The calculated detection limits in buffer solution were 10 ng/ ml and 0.045 ng/ml for electric eel and recombinant acetylcholinesterase, respectively, whereas operating in the matrix extract they increased up to 45 ng/ml and 0.07 ng/ml, corresponding to 0.45 mg/kg (extraction ratio 1:10) and 0.07 mg/kg in samples. These characteristics allowed the detection of contaminated samples at the maximum residue limit, which is 2 mg/kg and well below. Fortified samples of durum wheat were obtained with both dichlorvos and the commercial product Didivane, which contains dichlorvos as an active molecule. At all the tested levels, the occurrence of contaminant was detected with an average recovery of 75%. The total assay time, including the extraction step, was 30 min. Because several extractions as well as most of the assay steps can be run simultaneously, the throughput for one operator is 12 determinations per hour.

Fatty acid composition and δ13C of bulk and individual fatty acids as marker for authenticating Italian PDO/PGI extra virgin olive oils by means of isotopic ratio mass spectrometry†

European Regulation (EEC) 2568/91 has been setting the minimum requirements in order to allow labeling of oil as extra virgin. These general requirements, are based on physical-chemical and organoleptic parameters directly linked to the freshness and quality of the product. Isotope ratio mass spectrometry (IRMS) was demonstrated to be a useful tool for the discrimination of the origin of unknown samples, because the obtained data are practically independent of the cultivar employed and the production technique. In this work, the evaluation of the composition of fatty acid methyl esters (FAME) alongside with the determination of stable isotope ratio of C in bulk oils and in main FAME constituents have been investigated as a tool to improve geographical discrimination of Italian Protected Designation of Origin/Protected Geographical Indication (PDO/PGI) samples. For this purpose, authentic PDO/PGI extra virgin olive oils were sampled at oil mills and grouped into different sets according to their areas of provenience. The use of principal component analysis and partial least squares discriminant analysis multivariate analysis techniques demonstrated that discrimination of olive oil samples can be done using geographical and pedoclimatic parameters predominantly by using δ(13) C results of bulk and individual fatty acids. Results showed that δ(13) C values are a more reliable marker of origin with respect to fatty acid composition.

Peptides Trapping Dioxins: A Docking-Based Inverse Screening Approach

A rapid and cost-effective computational methodology for designing and rationalizing the selection of small peptides as receptors for dioxin-like compounds was proposed. The backbone of the dioxin Ah receptor binding site was used to design a series of penta- and hexapeptide libraries, with 1400 elements in total. Peptide flexibility was considered and 10 conformers were found to be a good option to represent peptide conformational space with fair speed-accuracy ratio. Each peptide conformer was treated as a possible receptor, generating a dedicated box and then running a docking process using as ligands a family of 76 dibenzo-p-dioxins and 113 dibenzofurans mono- and polychlorinated. Significant predictions were confirmed by comparing primary structure of top and bottom ranked peptides binding dioxins confirming that scrambled positions of the same amino acids gave completely different predicted binding. The hexapeptide EWFQPW, with the best binding score, was chosen as selective sorbent material in solid-phase extraction. The retention performances were tested using the 2,3,7,8-tetrachlorodibenzo-p-dioxin and two polychlorinated biphenyls in order to verify the hexapeptide specificity. The solid-phase extraction experimental procedure was optimized, and analytical parameters of hexapeptide sorbent material were compared with the resin without hexapeptide and a commercial reversed phase cartridge.