Ilaria Palchetti

Nucleic Acid and Peptide Aptamers: Fundamentals and Bioanalytical Aspects

In recent years new nucleic acid and protein-based combinatorial molecules have attracted the attention of researchers working in various areas of science, ranging from medicine to analytical chemistry. These molecules, called aptamers, have been proposed as alternatives to antibodies in many different applications. The aim of this Review is to illustrate the peculiarities of these combinatorial molecules which have initially been explored for their importance in molecular medicine, but have enormous potential in other biotechnological fields historically dominated by antibodies, such as bioassays. A description of these molecules is given, and the methods for their selection and production are also summarized. Moreover, critical aspects related to these molecules are discussed.

Electrochemical sensor and biosensor for polyphenols detection in olive oils.

Abstract The aim of the work was to compare different techniques, in evaluating the phenolic content of an extra-virgin olive oil with varying storage time and storage conditions. A disposable screen-printed sensor (SPE) was coupled with differential pulse voltammetry (DPV) to determine the phenolic fractions after extraction with a glycine buffer; DPV parameters were chosen in order to study the oxidation peak of oleuropein, which was used as reference compound. A calibration curve of oleuropein was performed in glycine buffer 10 mM, pH=2, NaCl 10 mM (D.L.=0.25 ppm oleuropein, RSD=7%). Moreover a tyrosinase based biosensor operating in organic solvent (hexane) was also assembled, using an amperometric oxygen probe as transducer. The calibration curves were realised using flow injection analysis (FIA) with phenol as the substrate (D.L.=4.0 ppm phenol, RSD=2%). Both of these methods are easy to operate, require no extraction (biosensor) or rapid extraction procedure (SPE), and the analysis time is short (min). The results obtained with these two innovative procedures were compared with a classical spectrophotometric assay using Folin–Ciocalteau reagent and HPLC analysis. Other extra-virgin olive oil quality parameters were investigated using classical methods in order to better define the alteration process and results are reported.

Determination of anticholinesterase pesticides in real samples using a disposable biosensor

A choline amperometric biosensor based on screen-printed electrodes was assembled and used to assess the inhibitory effect of organophosphorus and carbamic pesticides on acetylcholinesterase activi ...

Electroanalytical biosensors and their potential for food pathogen and toxin detection

The detection and identification of foodborne pathogens continue to rely on conventional culturing techniques. These are very elaborate, time-consuming, and have to be completed in a microbiology laboratory and are therefore not suitable for on-site monitoring. The need for a more rapid, reliable, specific, and sensitive method of detecting a target analyte, at low cost, is the focus of a great deal of research. Biosensor technology has the potential to speed up the detection, increase specificity and sensitivity, enable high-throughput analysis, and to be used for monitoring of critical control points in food production. This article reviews food pathogen detection methods based on electrochemical biosensors, specifically amperometric, potentiometric, and impedimetric biosensors. The underlying principles and application of these biosensors are discussed with special emphasis on new biorecognition elements, nanomaterials, and lab on a chip technology.

Electrochemical nucleic acid-based biosensors: Concepts, terms, and methodology (IUPAC Technical Report)

An electrochemical nucleic acid (NA)-based biosensor is a biosensor that integrates a nucleic acid as the biological recognition element and an electrode as the electrochemical signal transducer. The present report provides concepts, terms, and methodology related to biorecognition elements, detection principles, type of interactions to be addressed, and construction and performance of electrochemical NA biosensors, including their critical evaluation, which should be valuable for a wide audience, from academic, biomedical, environmental, and food-testing, drug-developing, etc. laboratories to sensor producers.

Electrochemical DNA biosensor as a screening tool for the detection of toxicants in water and wastewater samples.

Applications of a disposable electrochemical DNA biosensor to standard solutions and to real samples are reported. The DNA biosensor is assembled by immobilising the double stranded calf thymus DNA on the surface of a disposable carbon screen-printed electrode. The immobilised ds-DNA interacts with the sample for 2 min; then is washed and immersed in a clean buffer where the analytical signal (the oxidation peak area of the guanine base) is obtained by a square-wave voltammetric scan. The results were compared with some currently used toxicity tests and in particular with a commercial luminescent bacteria test, Toxalert(R)100.

Disposable ruthenized screen-printed biosensors for pesticides monitoring

A disposable choline biosensor based on ruthenized carbon screen-printed electrodes has been prepared and its use for monitoring organophosphorus pesticides and carbamates is described. The presence of 0.5% ruthenium on activated carbon mixed to form a simple graphite-based ink for the working electrode surface increased the sensitivity towards hydrogen peroxide. The choline biosensor is based on such an electrode coupled with choline oxidase immobilized by adsorption. The inhibition effect of carbamates and organophosphorus pesticides on acetylcholinesterase was detected using this biosensor. With the optimized procedure described (pH, buffer composition, incubation time, substrate concentration) we are able to detect concentrations of pesticides (carbofuran) as low as 1 nM.

Electrochemical DNA biosensor for analysis of wastewater samples

The application of a disposable electrochemical DNA biosensor to wastewater samples is reported. The DNA biosensor is assembled by immobilising double-stranded calf thymus DNA on the surface of a disposable, carbon screen-printed electrode (SPE). The oxidation signal of the guanine base, obtained by a square wave voltammetric scan, is used as analytical signal. The presence of compounds with affinity for DNA is measured by their effect on the guanine oxidation. The comparison of the results with a toxicity test based on bioluminescent bacteria has confirmed the applicability of the method to real samples.

Different approaches for the detection of thrombin by an electrochemical aptamer-based assay coupled to magnetic beads.

Different assay formats based on the coupling of magnetic beads with electrochemical transduction were compared here for the detection of thrombin by using a thrombin specific aptamer. By using the thrombin-binding aptamer, a direct and an indirect competitive assay for thrombin have been developed by immobilising the aptamer or the protein, respectively. Moreover, another strategy was based on the direct measurement of the enzymatic product of thrombin captured by the immobilised aptamer. All the assays were developed by coupling the electrochemical transduction with the innovative and advantageous use of magnetic beads. The assays based on the immobilisation of the protein were not successful since no binding was recorded between thrombin and its aptamer. With the direct competitive assay, when the aptamer was immobilised onto the magnetic beads, a detection limit of 430nM for thrombin was achieved. A lower detection limit for the protein (175nM) was instead obtained by detecting the product of the enzymatic reaction catalysed by thrombin. All these assays were finally compared with a sandwich assay which reached a detection limit of 0.45nM of thrombin demonstrating the best analytical performances. With this comparison the importance of a deep study on the different analytical approaches for thrombin detection to reach the performances of the best assay configuration has been demonstrated.

Electrochemical nanomaterial-based nucleic acid aptasensors

Recent progress in the development of electrochemical nanomaterial–aptamer-based biosensors is summarized. Aptamers are nucleic acid ligands that can be generated against amino acids, drugs, proteins, and other molecules. They are isolated from a large random library of synthetic nucleic acids by an iterative process of binding, separation, and amplification, called systematic evolution of ligands by exponential enrichment (SELEX). In this review, different methods of integrating aptamers with different nanomaterials and nanoparticles for electrochemical biosensing application are described.