Francesca Malvano

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.

Rapid Assessment of Gray Mold (Botrytis cinerea) Infection in Grapes with a Biosensor System

Botrytis cinerea is the causative agent of gray mold disease, which causes considerable economic losses to winemakers. The extent of gray mold infection of winegrapes is commonly visually estimated, a method that is prone to assessor bias. Here, we used rapid and simple enzyme-based screening consisting of carbon-electrode, screen-printed amperometric biosensors to estimate gluconic acid and glycerol concentration in winegrapes infected with different degrees of B. cinerea. The lower limits of quantification of the screen-printed amperometric biosensors were 3 mg/L for gluconic acid (corresponding to an infection rate of less than 1%) and 35 mg/L for glycerol; the response times with a flow rate of 0.5 mL/min were in a range of 0.5 to 2 min in the linear ranges of the two assays. This study demonstrates the efficacy of amperometric biosensors for rapid analysis of gluconic acid and glycerol in grapes. The measurements confirmed that concentrations of both compounds are highly correlated with the rate of B. cinerea infection (R2 = 0.98). Thus, the biosensor developed to measure gluconic acid in grapes (or must) was more precise and gave a faster response than methods that currently exist for determining the rate of B. cinerea infection of grape berries.

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.

Self Assembled and Electrochemically Deposed Layers of Thiols on Gold Compared with Electrochemical Impedance Spectroscopy and Atomic Force Microscopy

Self-Assembling is based on a spontaneous process in which organic molecules (alkanethiols, silanes) are adsorbed on a substrate (gold, glass, silicon). Although the implementation is extremely easy, it shows a big disadvantage in timing, because the solution has to be in contact overnight with the substrate under mild shaking. An alternative method of molecular deposition is the Electrochemically Deposed Multilayers commonly used in our laboratory for further immobilization of biological molecules in order to obtain specific biosensors for several analytes. It consists in applying a constant potential on gold working electrode (1.3 V vs. Ag/AgCl) for driving molecules in proximity of the electrode and allow them to react on the surface and form a layer similar to self assembled ones. Both the layers, self assembled and electrochemically deposed ones, were tested with Electrochemical Impedance Spectroscopy and Atomic Force Microscopy. The substrate electrochemically covered shows a higher and a more homogeneous deposition than self assembled one and the deposition time is extremely reduced from several hours to a few of seconds (50 s).

An Amperometric Biosensor for the Determination of Lactic Acid During Malolactic Fermentation

A lactate oxidase amperometric biosensor was developed and optimized for the malolactic fermentation monitoring during winemaking process. Lactate oxidase enzyme was immobilized on prussian blue modified screen-printed carbon electrode in order to reduce the electrochemical interferences due to the high content of electroactive compounds abundant in wine and must, such as polyphenols and ascorbic acid. The lactate oxidase biosensor developed showed high sensitivity (852 μA M-1) and a detection limit for lactic acid of 0.005 mM (0.45 mg L-1) The operational stability and the life time of the biosensors were also evaluated equal to 8 h and 30 days respectively. Finally the biosensor in flow injection system was used for lactic acid analysis during malolactic fermentation of a red wine and the results were compared with those registered by ion chromatography with good agreement with two sets of data.