Serena Laschi

An optimized digestion method coupled to electrochemical sensor for the determination of Cd, Cu, Pb and Hg in fish by square wave anodic stripping voltammetry

An optimized digestion method coupled to electrochemical detection to monitor lead, copper, cadmium and mercury in fish tissues was developed. Square wave anodic stripping voltammetry (SWASV) coupled to disposable screen-printed electrodes (SPEs) was employed as fast and sensitive electroanalytical method for heavy metals detection. Different approaches in digestion protocols were assessed. The study was focused on Atlantic hake fillets because of their wide diffusion in the human nutrition. Best results were obtained by digesting fish tissue with hydrogen peroxide/hydrochloric acid mixture coupled to solid phase (SP) purification of the digested material. This combined treatment allowed quantitative extraction from fish tissue (muscle) of the target analytes, with fast execution times, high sensitivity and avoiding organic residues eventually affecting electrochemical measurements. Finally, the method has been validated with reference standard materials such as dogfish muscle (DORM-2) and mussel tissues (NIST 2977).

Improvement of analytical performances of a disposable electrochemical immunosensor by using magnetic beads

A comparison of two electrochemical immunosensing strategies for PCBs detection, based on the use of two different solid phases, is here discussed. In both cases, carbon-based screen-printed electrodes (SPEs) are used as transducers in a direct competitive immunoassay scheme, where PCBs in solution compete with the tracer PCB28-alkaline phosphatase (AP) labeled for antibodies immobilized onto the solid-phase. In the standard format (called EI strategy), SPEs are both the solid-phase for immunoassay and electrochemical transducers: in this case the immunochemical reaction occurs onto the working electrode. Finally, the enzymatic substrate is added and an electroactive product is generated and detected by electrochemical measurement. In order to improve the performances of the system, a new approach (called EMI strategy) is developed by using functionalized magnetic beads as solid phase for the competitive assay; only after the immunosensing step they are captured by a magnet onto the working surface of the SPE for the electrochemical detection. Experimental results evidenced that the configuration based on the use of separate surfaces for immunoassay and for electrochemical detection gave the best results in terms of sensitivity and speed of the analysis. The improvement of analytical performances of the immunosensor based on EMI strategy was also demonstrated by the analysis of some spiked samples.

Microfluidic-based electrochemical genosensor coupled to magnetic beads for hybridization detection

This paper describes the development of a rapid and sensitive enzyme-linked electrochemical genosensor using a novel microfluidic-based platform. In this work, hybridization was performed on streptavidin-coated paramagnetic micro-beads functionalized with a biotinylated capture probe. The complementary sequence was then recognized via sandwich hybridization with a capture probe and a biotinylated signaling probe. After labeling the biotinylated hybrid with a streptavidin-alkaline phosphatase conjugate, the beads were introduced in a disposable cartridge composed of eight parallel microchannels etched in a polyimide substrate. The modified beads were trapped with a magnet addressing each microchannel individually. The presence of microelectrodes in each channel allowed direct electrochemical detection of the enzymatic product within the microchannel. Detection was performed in parallel within the eight microchannels, giving rise to the possibility of performing a multiparameter assay. Quantitative determinations of the analyte concentrations were obtained by following the kinetics of the enzymatic reaction in each channel. The chip was regenerated after each assay by removing the magnet and thus releasing the magnetic beads. The system was applied to the analytical detection of PCR amplified samples with a RSD%=6. A detection limit of 0.2 nM was evaluated.

PBDEs in Italian sewage sludge and environmental risk of using sewage sludge for land application.

Polybrominated diphenyl ethers (PBDEs) were determined in sewage sludge samples collected from eight Italian wastewater treatment plants (WWTPs) between June 2009 and March 2010. Total PBDE concentrations ranged from 158.3 to 9427 ng g(-1) dw, while deca-BDE (BDE-209) (concentrations ranging from 130.6 to 9411 ng g(-1) dw) dominated the congener profile in all the samples, contributing between 77% and 99.8% of total PBDE. The suitability of using a magnetic particle enzyme-linked immunoassay (ELISA) to analyse PBDEs in sewage sludge was also tested. The ELISA results, expressed as BDE-47 equivalents, were well correlated with those obtained by GC-NCI-MS, with correlation coefficients (r(2)) of 0.899 and 0.959, depending on the extraction procedure adopted. The risk assessment of PBDEs in sewage sludge addressed to land application was calculated. PEC(soil) values compared to the relative PNEC(soil) for penta and deca-BDE suggests that there is a low risk to the soil environment.

Enzyme-amplified electrochemical hybridization assay based on PNA, LNA and DNA probe-modified micro-magnetic beads.

In the present study, we investigated the properties of PNA and LNA capture probes in the development of an electrochemical hybridization assay. Streptavidin-coated paramagnetic micro-beads were used as a solid phase to immobilize biotinylated DNA, PNA and LNA capture probes, respectively. The target sequence was then recognized via hybridization with the capture probe. After labeling the biotinylated hybrid with a streptavidin-enzyme conjugate, the electrochemical detection of the enzymatic product was performed onto the surface of a disposable electrode. The assay was applied to the analytical detection of biotinylated DNA as well as RNA sequences. Detection limits, calculated considering the slope of the linear portion of the calibration curve in the range 0-2 nM were found to be 152, 118 and 91 pM, coupled with a reproducibility of the analysis equal to 5, 9 and 6%, calculated as RSD%, for DNA, PNA and LNA probes respectively, using the DNA target. In the case of the RNA target, the detection limits were found to be 51, 60 and 78 pM for DNA, PNA and LNA probes respectively.

Screen-Printed Electrochemical Immunosensors for PCB Detection

An electrochemical enzyme immunoassay for polychlorinated biphenyls (PCB) using carbon-based screen-printed disposable electrodes as the solid-phase for reagent immobilization and also as the signal transducer has been optimized. Horseradish peroxidase (HRP) was conjugated with Ag or Ab and used as the enzyme label. Hydrogen peroxide and ferrocenemonocarboxylic acid (FCA) were used as substrate and mediator in order to determine the HRP activity in the range 10–8–10–10 M. Indirect and direct competitive assays for PCB have been presented. The advantage of this approach is the relatively fast analysis time (30 min) in comparison with a test based on microtiter assay plates (14 h); moreover, the use of disposable screen-printed electrodes eliminates the problems of fouling and surface regeneration of the electrochemical device.

A new gravity-driven microfluidic-based electrochemical assay coupled to magnetic beads for nucleic acid detection

In this work, the characterisation and the optimisation of hybridisation assays based on a novel, rapid and sensitive micro‐analytical, gravity‐driven, flow device is reported. This device combines a special chip containing eight polymer microchannels, with a portable, computer‐controlled instrument. The device is used as a platform for affinity experiments using oligonucleotide‐modified paramagnetic particles. In our approach, both hybridisation and labelling events are performed on streptavidin‐coated paramagnetic microparticles functionalised with a biotinylated capture probe. Modified particles, introduced in the microchannel inlet of the chip, accumulate near the electrode surface by virtue of a magnetic holder. After hybridisation with the complementary sequence, the hybrid is labelled with an alkaline phosphatase conjugate. The electrochemical substrate for alkaline phosphatase revelation is p‐aminophenyl phosphate. Solutions and reagents are sequentially passed through the microchannels, until enzyme substrate is added for in situ signal detection. Upon readout, the magnet array is flipped away, beads are removed by addition of regeneration buffer, and the so‐regenerated chip is ready for further analysis. This protocol has been applied to the analytical detection of specific DNA sequences of Legionella pneumophila, with an RSD=8.5% and a detection limit of 0.33 nM.

Strategies for electrochemical detection in immunochemistry

In this review, the current status of research in electrochemical immunosensors is considered. We primarily focus on label-free and enzyme-labeled immunosensors, and the analytical capabilities of these devices are discussed. Moreover, the use of magnetic beads as new materials for immunosensors coupled with electrochemical sensing is also described, together with the application of new molecules such as aptamers as specific biorecognition elements. Examples of the applicability of these devices in solving various analytical problems in clinical, environmental and food fields are reported. Finally, the prospects for the further development of immunosensor technologies are shown.

A comparison study between a disposable electrochemical DNA biosensor and a Vibrio fischeri-based luminescent sensor for the detection of toxicants in water samples.

In the present study, a comparison between a disposable electrochemical DNA biosensor and a Vibrio fischeri-based luminescent sensor for the detection of toxicants in water samples was made. In order to realize this study, a disposable electrochemical DNA biosensor has been reported. The DNA biosensor is assembled by immobilizing double stranded Calf Thymus DNA onto the surface of a disposable carbon screen-printed electrode. The oxidation signal of the guanine base, obtained by a square wave voltammetric scan, is used as analytical signal to detect the DNA damage; the presence of low molecular weight compounds with affinity for nucleic acids is measured by their effect on the guanine oxidation peak. Wastewater samples provided during First European Interlaboratory Exercise on water toxicity in the course of the project SWIFT-WFD were analyzed, and biosensor results were compared with a currently used toxicity test ToxAlert 100 based on the bioluminescence inhibition of Vibrio fischeri. This test have been used because is rapid, easy handling and cost effectively responses for the toxicity assessment in real water samples. The results showed a promising correlation between two tests used for the detection of toxic compounds in water samples.

Electrochemical behavior of colchicine using graphite-based screen-printed electrodes

Miniaturized electrochemical graphite based sensor for the analysis of colchicine, along with its detailed construction was described. The electrochemical behavior of colchicine, both by cyclic and differential pulse voltammetry was investigated, showing an irreversible reduction and oxidation mechanism. The effect of several different electrochemical mediators was studied in order to decrease the oxidation potential of colchicine, but none of them showed a favorable electron transfer between the alkaloid and the electrodes surface. The influences of different biomolecules (calf thymus DNA, bovine serum albumin and beta-tubulin) over colchicines electrochemical behavior were also presented. For quantitative determinations the anodic differential pulse voltammetric method in H(3)PO(4)/HClO(4) 0.01 M (pH 2.05) was selected, evaluating one of the oxidation peaks of colchicine at +970 mV versus Ag pseudo-reference. The electrochemical scanning parameters were optimized by an experimental design using response surface modelling. The method was validated according to ICH guidelines in the concentration range 85-1200 ng mL(-1) (R(2)=0.9966, n=7) colchicine in terms of linearity, accuracy, limits of detection (LOD) (41 ng mL(-1)), limits of quantification (LOQ) and fidelity and it was successfully applied to the determination of colchicine in tablets, without the interference of the excipients. The studied method showed the same sensitivity as the more complex HPLC procedure and micellar electrokinetic chromatography with spectrophotometric detection.