Alessia Pepe

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.

Detection of benzo(a)pyrene photodegradation products using DNA electrochemical sensors.

The reactivity of photodegradation products of benzo(a)pyrene vs. DNA has been assessed using both genomic and oligonucleotide based DNA electrochemical sensors. The kinetic of a photooxidation reaction of benzo(a)pyrene (BaP) carried out in controlled conditions using a 6 W UV lamp peaked at 365 nm has been studied using LC with fluorimetric detection. Degradation of benzo(a)pyrene by both UV and UV/H(2)O(2) exhibited pseudo-first-order reaction kinetics with half-lives ranging from 3.0 to 9.8h depending on the pH and on the amount of H(2)O(2). The oxidation products of benzo(a)pyrene obtained in different conditions were tested on genomic ssDNA electrochemical sensors obtained via immobilisation of salmon testis ss-DNA on graphite screen-printed electrodes. Guanines oxidation signals obtained using chronopotentiometry were used to detect the interaction of the products with DNA. The dose-response curve obtained with benzo(a)pyrene incubated 24 h at pH 4.7 was different from that of the parent compound indicating a different type of interaction with DNA. A DNA hybridisation sensor was also assembled using a thiolated/biotynilated 24-mer oligonucleotide immobilised on a gold screen-printed electrode and avidin-alkaline phosphatase conjugate. A voltammetric detection of naphtol was used to detect the hybridisation reaction. A net inhibition of the hybridisation reaction was observed after incubation with benzo(a)pyrene oxidation products that was attributed to the formation of stable adducts with the guanines of the biotinylated strand. LC-MS-MS studies of the oxidation products confirmed the presence of chemical species potentially forming adducts with DNA. The data reported demonstrate that DNA electrochemical sensors have the potential to be used to monitor remediation processes and to assess the potential toxicity vs. DNA of chemicals forming stable DNA adducts.

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.

Novel oligopeptides based e-nose for food quality control: Application to extra-virgin olive samples

The potential of an electronic nose to discriminate olive oil samples based on their sensory profiles is proposed. The e-nose was constituted by an array of seven quartz crystal microbalance sensors modified with Gold Nanoparticles (GNPs) conjugated to short peptides. Forty olive oil samples headspaces were characterised by headspace solid-phase microextraction gas chromatography-mass spectrometry analysis to evaluate chemical composition; in parallel, they were chemically and sensory evaluated according to European Regulation EEC 2568/91 and amendments and EU Regulation 640/2008. The steady state sensor responses obtained with the e-nose setup were used to evaluate the discrimination properties of the system by principal component analysis and partial least square method. The results of this study provided a promising perspectives for the use of the electronic nose as a low-cost, easy to use and rapid system for the quality control of extra virgin, virgin and lampante (non-edible) olive oil. This system ...

Nano carbon black-based screen printed sensor for carbofuran, isoprocarb, carbaryl and fenobucarb detection: application to grain samples

An electrochemical screening assay for the detection of phenyl carbamates (i.e. carbaryl, carbofuran, isoprocarb and fenobucarb) was developed and applied to grains samples (i.e. durum wheat, soft wheat and maize). Nano carbon black (CB) was strategically employed to realize an effective, reproducible, fouling resistant, low cost, delocalisable screen printed sensor (CB-SPE). CB-SPEs morphology (SEM and FEM) and electrochemical property (CV and EIS) were studied. The final pesticides analysis protocol consist of: (i) extraction of the analyte (just by mixing), (ii) alkaline hydrolysis (10 min R.T.), (iii) DPV detection directly of 100 µL of extract on the CB-SPE surface. Linear range between 1.0 × 10-7 and 1.0 × 10-4 mol L-1, good determination coefficients (R2 ≥ 0.9971) and satisfactory sensitivity (≥ 3.90 × 10-1 A M-1 cm-2) and LODs (≤ 8.0 × 10-8 mol L-1) were obtained for all the analytes. Excellent recoveries (78-102%) and accuracy (relative error vs. HPLC-MS/MS between 9.0% and -7.8%) resulted from the analysis of grains samples. The proposed CB-SPE based approach has demonstrated to be able to detect carbaryl at Maximum residue limits levels (MRLs), allowing class selective detection of commonly employed phenyl carbamates in food samples.

Determination of Free Fatty Acids in Cheese by Means of Matrix Solid-Phase Dispersion Followed by Ultra-High Performance Liquid Chromatography and Tandem Mass Spectrometry Analysis

Cheese as milk derivate plays a key role in our diet due to their nutritional and functional properties; free fatty acids (FFAs) and especially conjugated linoleic acids (CLAs) represent important healthy components in a dairy matrix. In this study, with the aim to detect FFAs in dairy products, we have developed a fast and accurate analytical protocol based on a matrix solid-phase dispersion (MSPD) extraction followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The proposed method provides a rapid and selective sample pretreatment and a reliable instrumental analysis. The use of MSPD extraction with a suitable dispersing phase as C18, allowed the retention of triglycerides, which are the main cause of interference and matrix effect in this type of analysis and permits a selective elution of the FFAs. The whole method was validated demonstrating the feasibility of the proposed method: correlation coefficients greater than 0.99 were obtained for all analytes; matrix effects were minimized and recoveries ranged between 75 and 105%, with good reproducibility (≤ 12%).

An Amperometric Sensor for the Selective Determination of Ortho-Diphenols in Olive Oil

An electrochemical flow injection analysis method for the selective determination of ortho-diphenols have been developed and optimized. The method is based on the use of sodium molybdate as an electrochemical mediator. The selectivity of the measurement has been evaluated with respect to different ortho-diphenols and phenols that are usually found in extra-virgin olive oil. After FIA optimization the protocol has been applied to the detection of olive oil samples and results were compared with a reference method.