Roberto Pilloton

A highly sensitive impedimetric label free immunosensor for Ochratoxin measurement in cocoa beans

In this work the development and optimization of an impedimetric label free immunosensor for the detection of Ochratoxin A (OTA) is reported. Two antibody immobilization methods (oriented and not oriented) were compared highlighting a lower limit of detection (5pg/ml) for the not oriented immobilization but a closer linear range in contrast to oriented anti-OTA immunosensors which showed linearity in the range of 0.01-5ng/mL OTA. The analysis of the Atomic Force Microscopy (AFM) images showed two different nanostructures indicating that the use of oriented immobilization created a more ordered and highly dense antibody surface. Finally the oriented immunosensor was used to quantify OTA in spiked cocoa bean samples and the results were compared with those registered with competitive ELISA kit. The immunosensor was sensitive to OTA lower than 2μg/kg that represents the lower acceptable limit of OTA established by European legislation for the common food products.

Impedimetric Label-Free Immunosensor on Disposable Modified Screen-Printed Electrodes for Ochratoxin A

An impedimetric label-free immunosensor on disposable screen-printed carbon electrodes (SPCE) for quantitative determination of Ochratoxin A (OTA) has been developed. After modification of the SPCE surface with gold nanoparticles (AuNPs), the anti-OTA was immobilized on the working electrode through a cysteamine layer. After each coating step, the modified surfaces were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The capacitance was chosen as the best parameter that describes the reproducible change in electrical properties of the electrode surface at different OTA concentrations and it was used to investigate the analytical parameters of the developed immunosensor. Under optimized conditions, the immunosensor showed a linear relationship between 0.3 and 20 ng/mL with a low detection limit of 0.25 ng/mL, making it suitable to control OTA content in many common food products. Lastly, the immunosensor was used to measure OTA in red wine samples and the results were compared with those registered with a competitive ELISA kit. The immunosensor was sensitive to OTA lower than 2 μg/kg, which represents the lower acceptable limit of OTA established by European legislation for common food products.

A Doped Polyaniline Modified Electrode Amperometric Biosensor for Gluconic Acid Determination in Grapes

In winemaking gluconic acid is an important marker for quantitative evaluation of grape infection by Botrytis cinerea. A screen-printed amperometric bienzymatic sensor for the determination of gluconic acid based on gluconate kinase (GK) and 6-phospho-D-gluconate dehydrogenase (6PGDH) coimmobilized onto polyaniline/poly (2-acrylamido-2-methyl-1-propanesulfonic acid; PANI-PAAMPSA) is reported in this study. The conductive polymer electrodeposed on the working electrode surface allowed the detection of NADH at low potential (0.1 V) with a linear range from 4 × 10−3 to 1 mM (R2 = 0.99) and a sensitivity of 419.44 nA·mM−1. The bienzymatic sensor has been optimized with regard to GK/6PGDH enzymatic unit ratio and ATP/NADP+ molar ratio which resulted equal to 0.33 and 1.2, respectively. Under these conditions a sensitivity of 255.2 nA·mM−1, a limit of detection of 5 μM and a Relative Standard Deviation (RSD) of 4.2% (n = 5) have been observed. Finally, the biosensor has been applied for gluconic acid measurements in must grape samples and the matrix effect has been taken into consideration. The results have been compared with those obtained on the same samples with a commercial kit based on a spectrophotometric enzyme assay and were in good agreement, showing the capability of the bienzymatic PANI-PAAMPSA biosensor for gluconic acid measurements and thus for the evaluation of Botrytis cinerea infection in grapes.

Fabrication of SrTiO3 Layer on Pt Electrode for Label-Free Capacitive Biosensors

Due to their interesting ferroelectric, conductive and dielectric properties, in recent years, perovskite-structured materials have begun to attract increasing interest in the biosensing field. In this study, a strontium titanate perovskite layer (SrTiO3) has been synthesized on a platinum electrode and exploited for the development of an impedimetric label-free immunosensor for Escherichia coli O157:H7 detection. The electrochemical characterization of the perovskite-modified electrode during the construction of the immunosensor, as well as after the interaction with different E. coli O157:H7 concentrations, showed a reproducible decrease of the total capacitance of the system that was used for the analytical characterization of the immunosensor. Under optimized conditions, the capacitive immunosensor showed a linear relationship from to 1 to 7 log cfu/mL with a low detection limit of 1 log cfu/mL. Moreover, the atomic force microscopy (AFM) technique underlined the increase in roughness of the SrTiO3-modified electrode surface after antibody immobilization, as well as the effective presence of cells with the typical size of E. coli.

Sensitive Detection of Escherichia coli O157:H7 in Food Products by Impedimetric Immunosensors

In this work, the development of an impedimetric label-free immunosensor for the detection of Escherichia coli O157:H7 is reported. Different immobilization techniques of monoclonal anti-E. coli were tested, in order to reach the very low limit of detections. The comparison between the immobilization procedures underlined the advantages of the oriented procedure and the use of a dendrimer, which allowed for immobilizing a higher number of antibody units, reaching a very high sensitivity. However, the use of activated ferrocene as electron-transferring mediator, which improved the electrical properties of the system, resulted in a very low limit of detection equal to 3 cfu/mL. This immunosensor was used to analyze milk and meat samples obtaining a good agreement with the results of the ELISA methods.

Development of MoSe2 Nano-Urchins as a Sensing Platform for a Selective Bio-Capturing of Escherichia. coli Shiga Toxin DNA

The present study was aimed to develop “fluorine doped” tin oxide glass electrode with a MoSe2 nano-urchin based electrochemical biosensor for detection of Escherichia coli Shiga toxin DNA. The study comprises two conductive electrodes, and the working electrodes were drop deposited using MoSe2 nano-urchin, and DNA sequences specific to Shiga toxin Escherichia coli. Morphological characterizations were performed using Fourier transforms infrared spectrophotometer; X-ray diffraction technique and scanning electron microscopy. All measurements were done using methylene blue as an electrochemical indicator. The proposed electrochemical geno-sensor showed good linear detection range of 1 fM–100 µM with a low detection limit of 1 fM where the current response increased linearly with Escherichia coli Shiga toxin dsDNA concentration with R2 = 0.99. Additionally, the real sample was spiked with the dsDNA that shows insignificant interference. The results revealed that the developed sensing platform significantly improved the sensitivity and can provide a promising platform for effective detection of biomolecules using minute samples due to its stability and sensitivity.

Morphology-Preferable MoSe2 Nanobrooms as a Sensing Platform for Highly Selective Apta-Capturing of Salmonella Bacteria

The present report employed nanobroom (NB)-shaped two-dimensional molybdenum diselenide (MoSe2) for the preparation of a sensing matrix for the detection of Salmonella paratyphi. An aptamer specific to salmonella was immobilized onto MoSe2NB-modified fluorine-doped tin oxide via glutaraldehyde cross-linking. Structural and morphological characterizations were performed using UV–vis spectroscopy, scanning electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction techniques. Characterizations confirmed the nanobroom morphology and nanosize of the MoSe2 material. Electrochemical studies revealed a good linear detection range of 10–2–10–10 CFU/mL with low detection limit of 1 × 10–10 CFU/mL and with R2 = 0.98. The developed preferable nanobroom-shaped sensing matrix can provide a promising platform for rapid and accurate detection of Salmonella in real samples due to its tremendous stability and sensitivity.