Morena Silvestrini

Functionalized ensembles of nanoelectrodes as affinity biosensors for DNA hybridization detection

A novel electrochemical biosensor for DNA hybridization detection based on nanoelectrode ensembles (NEEs) is presented. NEEs are prepared by electroless deposition of gold into the pores of a templating track-etched polycarbonate (PC) membrane. The wide surface of the templating membrane surrounding the nanoelectrodes is exploited to bind the capture DNA probes via amide coupling with the carboxylic groups present on the PC surface. The probes are then hybridized with the complementary target labelled with glucose oxidase (GO(x)). The occurrence of the hybridization event is detected by adding, to the supporting electrolyte, excess glucose as the substrate and the (ferrocenylmethyl) trimethylammonium cation (FA(+)) as suitable redox mediator. In the case of positive hybridization, an electrocatalytic current is detected. In the proposed sensor, the biorecognition event and signal transduction occur in different but neighbouring sites, i.e., the PC surface and the nanoelectrodes, respectively; these sites are separated albeit in close proximity on a nanometer scale. Finally, the possibility to activate the PC surface by treatment with permanganate is demonstrated and the analytical performances of biosensors prepared with KMnO(4)-treated NEEs and native NEEs are compared and critically evaluated. The proposed biosensor displays high selectivity and sensitivity, with the capability to detect few picomoles of target DNA.

A Sensitive Electrochemiluminescence Immunosensor for Celiac Disease Diagnosis Based on Nanoelectrode Ensembles

We report here the design of a novel immunosensor and its application for celiac disease diagnosis, based on an electrogenerated chemiluminescence (ECL) readout, using membrane-templated gold nanoelectrode ensembles (NEEs) as a detection platform. An original sensing strategy is presented by segregating spatially the initial electrochemical reaction and the location of the immobilized biomolecules where ECL is finally emitted. The recognition scaffold is the following: tissue transglutaminase (tTG) is immobilized as a capturing agent on the polycarbonate (PC) surface of the track-etched templating membrane. It captures the target tissue transglutaminase antibody (anti-tTG), and finally allows the immobilization of a streptavidin-modified ruthenium-based ECL label via reaction with a suitable biotinylated secondary antibody. The application of an oxidizing potential in a tri-n-propylamine (TPrA) solution generates an intense and sharp ECL signal, suitable for analytical purposes. Voltammetric and ECL analyses evidenced that the ruthenium complex is not oxidized directly at the surface of the nanoelectrodes; instead ECL is generated following the TPrA oxidation, which produces the TPrA•+ and TPrA• radicals. With NEEs operating under total overlap diffusion conditions, high local fluxes of these reactive radicals are produced by the nanoelectrodes in the immediate vicinity of the ECL labels, so that they efficiently generate the ECL signal. The radicals can diffuse over short distances and react with the Ru(bpy)32+ label. In addition, the ECL emission is obtained by applying a potential of 0.88 V versus Ag/AgCl, which is about 0.3 V lower than when ECL is initiated by the electrochemical oxidation of Ru(bpy)3(2+). The immunosensor provides ECL signals which scale with anti-tTG concentration with a linearity range between 1.5 ng·mL–1 and 10 μg·mL–1 and a detection limit of 0.5 ng·mL–1. The sensor is finally applied to the analysis of anti-tTG in human serum samples, showing to be suitable to discriminate between healthy and celiac patients.

Nanoelectrode ensembles for the direct voltammetric determination of trace iodide in water

Procedures for the preparation and characterisation of ensembles of gold nanodisk electrodes (NEE) of 30 nm diameter are presented, in particular focusing on improvements in the signal/background current ratios and detection limits with respect to the electrochemical oxidation of iodide and its analytical determination in water samples. At NEEs iodide undergoes a quasi-reversible diffusion controlled oxidation with a slight shift in E 1/2 values and slightly higher peak to peak separation with respect to conventional gold disk electrodes. The double layer charging current at the NEE is significantly lower than at conventional electrodes so that the detection limit (DL) by cyclic voltammetry with NEEs in tap water is significantly lower than DL at the Au-disk millimetre-sized electrode (DL 0.3 µM at NEE vs. 4 µM for Au-disk). Finally, it is shown that NEEs in combination with square wave voltammetry can be applied for the direct determination of iodide in water samples from the lagoon of Venice, with a detection limit of 0.10 µM.

Ensembles of nanoelectrodes modified with gold nanoparticles: characterization and application to DNA-hybridization detection

A new method to increase the active area (Aact) of nanoelectrode ensembles (NEEs) is described. To this aim, gold nanoparticles (AuNPs) are immobilized onto the surface of NEEs using cysteamine as a cross-linker able to bind the AuNPs to the heads of the nanoelectrodes to obtain the so-called AuNPs-NEEs. The analysis of the cyclic voltammograms recorded in pure supporting electrolyte showed that the presence of the nanoparticles reflects in an, approximately, ten-times increase in the electrochemically active area of the ensemble. The measurement of the amount of electroactive polyoxometalates, which can be adsorbed on the gold surface of NEEs vs. AuNPs-NEEs, confirmed a significant increase of active area for the latter. These evidences indicate that there is a good electronic connection between the AuNPs and the underlying nanoelectrodes. The possibility to exploit AuNPs-NEEs for biosensing application was tested for the case of DNA-hybridization detection. After immobilization on the gold surface of AuNPs-NEEs of a thiolated single-stranded DNA, the hybridization with complementary sequences labeled with glucose oxidase (GOx) was performed. The detection of the hybridization was achieved by adding to the electrolyte solution the GOx substrate (i.e., glucose) and a suitable redox mediator, namely the (ferrocenylmethyl) trimethylammonium (FA+) cation; when the hybridization occurs, an electrocatalytic increase of the oxidation current of FA+ is recorded. Comparison of electrocatalytic current recorded at DNA modified NEEs and AuNPs-NEEs indicate, for the latter, a significant increase in sensitivity in the detection of the DNA-hybridization event.

Biofuntionalization of Nanoelectrode Ensembles: protection of the nanoelectrodes with self-assembled monolayers

In this work we study the possibility to use self-assembled monolayers (SAMs) of thiols to protect the surface of nanoelectrodes ensembles (NEEs) from the undesired adsorption of biomolecules, in particular, proteins. Thioctic acid (TA) and 2mercaptoethanesulfonic acid (MES) were chosen for this aim. The behaviour of SAM-modified NEEs was studied by cyclic voltammetry as a function of the solution pH using ferrocenecarboxylate as anionic redox probe (FcCOO - ) and (ferrocenylmethyl) trimethylammonium (FA + ) as cationic redox probe. It was observed that, when the SAM is negatively charged it repels the negative redox probes from the surface of the nanoelectrodes, while the voltammetry of FA + remain unchanged. The possible protective role of the SAM was confirmed by studying the voltammetric signals recorded with NEEs dipped in a solution of a high adsorbable protein, namely casein, comparing signals recorded at bare NEEs vs. signals at SAM-modified NEEs, in different experimental conditions.

Electroanalytical Applications of Sensors Basedon Pyrolized Photoresist Carbon Electrodes

Pyrolyzed photoresist carbon electrodes (PPCEs) fabricated by photolithographic micro-fabrication and pyrolysis of the epoxy-based photoresist named SU-8 are applied to electroanalysis. Bismuth-modified PPCEs (Bi-PPCEs) are used in the adsorptive cathodic stripping voltammetry (AdCSV) of Ni(II) and in the speciation of inorganic Cr, while PPEs are used in the cyclic voltammetric (CV) study of bilirubin (BR) in dimethyl sulfoxide.