Walter Vastarella

Enzyme/semiconductor nanoclusters combined systems for novel amperometric biosensors

In this work quantum-sized CdS nanocrystals were synthesized using a quaternary water-in-oil microemulsion and immobilized onto gold working electrode by self-assembled monolayers techniques. Formaldehyde dehydrogenase was covalently immobilized onto a protecting membrane, which was stratified on part of the semiconductor nanoparticles modified electrode. The covalent enzyme immobilization has been required to improve the stability of the catalytic oxidation of formaldehyde, which occurs after light stimulation of the semiconductor through the electron/hole recombination. A study about the best electrochemical oxidation potentials under different flow conditions was performed. Preliminary sensor stability and interferences tests were also carried out, for a sensitive and selective detection of formaldehyde. A detection limit of 41ppb of formaldehyde was calculated and an operational stability of 6h was achieved under flow conditions by means of this novel amperometric biosensor based on FDH-semiconductor hybrid systems, not requiring NAD(+)/NADH as charge transfer in the enzymatic reaction.

Determination of phenolic acids using Trametes versicolor laccase

Two biosensors based on Trametes versicolor laccase (TvL) were developed for the determination of phenolic compounds. Commercial oxygen electrode and ferrocene-modified screen-printed graphite electrodes were used for preparation of laccase biosensors. The systems were calibrated for three phenolic acids. Linearity was obtained in the concentration range 0.1-1.0muM caffeic acid, 0.05-0.2muM ferulic acid, 2.0-14.0muM syringic acid for laccase immobilised on a commercial oxygen electrode and 2.0-30.0muM caffeic acid, 2.0-10.0muM ferulic acid, 4.0-30.0muM syringic acid for laccase immobilised on ferrocene-modified screen-printed electrodes. Furthermore, optimal pH, temperature and thermal stability studies were performed with the commercial oxygen electrode. Both electrodes were used for determination of a class of phenolic acids, achieving a cheap and fast tool and an easy to be used procedure for screening real samples of human plasma.

Tyrosinase biosensor based on modified screen printed electrodes: measurements of total phenol content

A disposable tyrosinase biosensor has been developed for the analysis of phenols and polyphenols, by modifying screen printed electrodes (SPEs) with addition of suitable mediators of redox processes directly into the conductive pastes. The percent ratio of mediator in the pastes was chosen depending on the electrochemical sensitivity either in batch or standard flow conditions. Ferrocene modified carbon electrodes have been used on whose surface the enzyme tyrosinase was immobilized in a glutaraldehyde cross-linked matrix of bovine serum albumin. Mixing the mediator to the electrode pastes should overcome transport limitations, due to its dissolution, which occur in commonly used immobilization procedures. Different immobilization techniques of tyrosinase on SPEs in the detection of phenolic compounds were tested and compared. Modified SPEs showed relatively good reproducibility and detection limits in the micromolar range for all phenolic compounds used. Major sensor parameters have been optimized in flow systems putting special attention on operating potential, pH and buffer composition, which strongly affect the detection of polyphenols and operational stability in wine. The resulting biosensors were stored and dried for a minimum of 8 h at 4°C, and showed a shelf stability of about 30 days. The procedure has been applied both on a synthetic wine matrix and on real samples, to determine the ‘pool’ of phenolic composition in terms of phenol concentration.

Biosensors based on gold nanoelectrode ensembles and screen printed electrodes

Gold nanowires were synthesized within polycarbonate membranes according to an electroless deposition method, obtaining nanoelectrode ensembles (NEEs) with special electrochemical features. NEEs were coupled with home-produced carbon graphite screen printed electrodes and the electrochemical properties of the original nanoelectrode ensemble on screen printed substrate (NEE/SPS) assembly has been tested for sensors application. Glucose oxidase has been used as model enzyme in order to verify the feasibility of disposable gold NEE/SPS biosensors. Finally, different immobilisation and electrochemical deposition techniques based on either self assembled monolayers of cysteamine (CYS) or amino-propyl-triethoxysilane (APTES) and conductive polyaniline (PANI) molecular wires were used. Spatial patterning of the enzyme on the polycarbonate surface and of PANI wires on gold nanoelectrodes was obtained. Possible direct electron transfer between the enzyme and the PANI modified gold nanoelectrodes has been evaluated.

A preliminary study on electrochemical biosensors for the determination of total cholinesterase inhibitors in strawberries

Organophosphorous (OP) insecticides reveal acute toxicity because of their capability to affect the nervous system through the inhibition of acetyl cholinesterase function in regulating the neurotransmitter acetylcholine. The present work shows an example of an easy to be handled inhibition electrochemical biosensor, based on thick film technology for low cost production of screen printed electrodes. Anti-cholinesterase activity in specific fruits was determined measuring the inhibition of acetyl cholinesterase enzyme owing to the presence of OP pesticides. Paraoxon was taken as reference pesticide for each measurement. The main fluidic critical parameters were investigated under flow injection analysis, through the comparison of different enzymatic immobilisation methods. Analytical features were evaluated as a function of experimental parameters. The analytical detection was developed in a three step procedure and the pesticides content was measured in strawberries samples taken from the local market. The separation between the acetyl cholinesterase inhibition and the electrochemical detection with the choline oxidase biosensor decreases the total analysis time, allowing improvements in reproducibility and stability of the system. A comparison with reference materials and standard analytical procedures for pesticides will be required in the future for evaluating the reliability of the method.

SCREEN PRINTED ELECTRODES FOR BIOSENSOR APPLICATION: REPRODUCIBILITY, SENSITIVITY AND STABILITY

Screen printed electrodes (SPEs) have been prepared in the Biosensor Lab @ ENEA, according to different layouts and configurations, i.e. front-back or concentric geometry. Different pastes and composition have been tested depending on the application and requirements for flow or batch measurements. In order to optimize the quality of the electrochemical response, graphite pastes and metal doped inks have been used, varying their physical features and composition. Preparation and analytical evaluation of SPEs probes have shown useful for specific detection of contaminants in complex matrices or for online monitoring their toxicological effect. Electrochemical comparison between commercially available probes and home-produced SPEs and statistical evaluation have been performed, to verify reproducibility at medium scale. Higher sensitivity and stability of these new probes have been achieved both in amperometric flow conditions and in voltammetric measurements. The results obtained show the possibility to use our SPEs, especially those based on Gwent Electronic Materials pastes for several reliable sensing applications.

Developing a miniaturized continuous flow electrochemical cell for biosensor applications

The development of a miniaturized electrochemical cell for biosensor application regards both the structuring of an array of electrodes in a fluidic chamber and their connections to the control & processing unit The sensitivity of the chrono-amperometric measurement performed with the cell is increased by: (a) integrating the reference electrode on the same chip with the counter- and working- electrodes, (b) designing a specific pattern of the gold electrodes and (c) serially distributing them along the pipeline reservoir. Borosilicate glass is used as substrate for the electrodes, allowing, due to its transparency, an accurate and easy pad to pad alignment of the up-side-down chip versus a PCB soldered on a standard DIL 40 socket. This alignment is necessary to accomplish the elastomer-based-solderless electric contact, between chip and PCB. The solderless contact significantly improves both reliability and signal processing accuracy. The reservoir and its cover are micromachined out of silicone rubber respectively photosensitive glass in order to easy disassemble the fluidic chamber without any damage. Both thickness and elasticity of the photosensitive glass rend the device less brittle. A plug-in -plug-flow device with improved characteristics has been obtained with a modular structure that allows further extension of the number of electrodes.

Electrochemical and Morphological Investigation on Gold Bio-Nano-Electrodes. Preliminary Results

Gold nano-electrode ensembles were synthesized within nanoporous polycarbonate membranes and coupled with screen printed substrates. A disposable and versatile electrochemical system for biosensing and bio analytical applications has been obtained. Scanning Electron Microscopy and Scanning Probe Microscopy techniques were used to characterize the membrane surface, the nanostructured surfaces and the resulting biosensing devices. Efficiency and response of nano-probes were tested with several enzymatic immobilizations on the sensitive surface. Herein, the model case of a glucose oxidase biosensor based on nanoelectrodes is taken into account, for further evaluations on the accuracy of the biosensor system.

Engineering a continuous flow electrochemical micro-cell for biosensor applications: new achievements

The functional tests of a previously obtained continuous flow micro-cell revealed a rather low value of the sensitivity of the chronoamperometric measurements as well as a poor reliability of both fluidic and electric connections. The engineering of the micro-cell regards the improvement of the following characteristics: (1) integrating the reference electrode on the chip, (2) designing a specific pattern of the electrodes and (3) serially distributing them along the pipeline reservoir. Thus, a plug-flow device is obtained and the preliminary tests confirm its functionality together with an improved sensitivity. The use of photosensitive glass (Foturan®) for obtaining the reservoir and its vertical openings allowed an improvement of the transversal shape and thus of the fluid flow. Better connections are achieved by the development of more reliable and rigid glass fluidic connections as well as of flexible elastomer-based solderless electric connections combined with both a standard DIL40 socket and standard flat cable connectors.