Biomedical subjects

Marco Mascini

Publications and source records attributed to Marco Mascini.

28 records · Page 2Linked to original sources

PubMed2004

Oligonucleotide-modified screen-printed gold electrodes for enzyme-amplified sensing of nucleic acids.

An electrochemical genosensor for the detection of specific sequences of DNA has been developed using disposable screen-printed gold electrodes. Screen-printed gold electrodes were firstly modified with a mixed monolayer of a 25-mer thiol-tethered DNA probe and a spacer thiol, 6-mercapto-1-hexanol (MCH). The DNA probe sequence was internal to the sequence of the 35S promoter, which sequence is inserted in the genome of GMOs regulating the transgene expression. An enzyme-amplified detection scheme, based on the coupling of a streptavidin-alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalysed the hydrolysis of the electroinactive alpha-naphthyl phosphate to alpha-naphthol; this product is electroactive and has been detected by means of differential pulse voltammetry. The assay was, firstly, characterised using synthetic oligonucleotides. Relevant parameters, such as the probe concentration and the immobilisation time, the use of the MCH and different enzymatic conjugates, were investigated and optimised. The genosensor response was found to be linearly related to the target concentration between 0 and 25 nmol/L; the detection limit was 0.25 nmol/L. The analytical procedure was then applied for the detection of the 35S promoter sequence, which was amplified from the pBI121 plasmid by polymerase chain reaction (PCR). Hybridisation conditions (i.e., hybridisation buffer and hybridisation time) were further optimised. The selectivity of the assay was confirmed using biotinylated non-complementary amplicons and PCR blanks. The results showed that the genosensor enabled sensitive (detection limit: 1 nmol/L) and specific detection of GMO-related sequences, thus providing a useful tool for the screening analysis of bioengineered food samples.

Alkaline Phosphatase↗

PubMed2004

Electrochemical genosensor for mitomycin C-DNA interaction based on guanine signal.

The interaction of mitomycin C (MC) with fish sperm or calf thymus DNA immobilized onto carbon screen-printed electrodes (CSPE) and carbon paste electrode (CPE) have been studied by using electrochemical techniques as square wave voltammetry (SWV) and differential pulse voltammetry (DPV). After the interaction was occurred between DNA and MC on electrode surface, it was observed that the guanine signal was higher with bare electrode than DNA-modified one. The changes in the experimental parameters such as the concentration of MC, and the accumulation time of MC were studied by using SWV and DPV. In addition, reproducibility, and detection limit parameters were determined using both electrodes. The partition coefficient of MC was also calculated before and after interaction of MC with dsDNA at CPE surface. These results showed that these two different DNA biosensors could be used for the sensitive, rapid and cost effective detection of MC-DNA interaction.

Animals↗

PubMed2004

Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors.

Genosensor technology relying on the use of carbon and gold electrodes is reviewed. The key steps of each analytical procedure, namely DNA-probe immobilisation, hybridisation, labelling and electrochemical investigation of the surface, are discussed in detail with separate sections devoted to label-free and newly emerging magnetic assays. Special emphasis has been given to protocols that have been used with real DNA samples.

Animals↗

PubMed2004

Towards fast and inexpensive molecular diagnostic: the case of TP53.

BACKGROUND: Much research suggests that TP53 mutations have prognostic importance and sometimes are a significant factor in clinical oncology. A considerable effort has been made to develop fast and inexpensive methods for TP53 mutations detection. METHODS: On the basis of describing the role of TP53 as tumor suppressor gene and TP53 mutation spectrum, the authors discuss conventional methods and new technologies for TP53 mutations detection. This discussion is supported by more recent publications in the field of both molecular genetics and analysis technologies. RESULTS: Biosensors and gene chips are of considerable recent interest, due to their tremendous promise for obtaining sequence-specific information in a faster, simpler and cheaper manner compared to traditional methods. CONCLUSIONS: New methods such as biosensors and gene chips appear promising as analytical methods of detecting mutations.

Biomarkers, Tumor↗

PubMed2003

Combination of amplification and post-amplification strategies to improve optical DNA sensing.

The work evaluated a series of approaches to optimise detection of polymerase chain reaction (PCR) amplified DNA samples by an optical sensor based on surface plasmon resonance (SPR) (BiacoreX). The optimised procedure was based on an asymmetric PCR amplification system to amplify predominantly one DNA strand, containing the sequence complementary to a specific probe. The study moved into two directions, aiming to improve the analytical performance of SPR detection in PCR amplified products. One approach concerned the application of new strategies at the level of PCR, i.e. asymmetric PCR to obtain ssDNA amplified fragments containing the target capable of hybridisation with the immobilised complementary probe. The other strategy focused on the post-PCR amplification stage. Optimised denaturing conditions were applied to both symmetrically and asymmetrically amplified fragments. The effective combination of the two strategies allowed a rapid and specific hybridisation reaction. The developed method was successfully applied in the detection of genetically modified organisms.

Biosensing Techniques↗

PubMed2003

Polychlorinated biphenyls (PCBs) detection in food samples using an electrochemical immunosensor.

In this work, a disposable electrochemical immunosensor, based on a competitive assay scheme, was applied to detect polychlorinated biphenyls (PCBs) in food. For this purpose, antibodies against PCBs were directly immobilized onto the carbon surface of a disposable screen-printed electrode. A competition between the PCBs present in the sample and a fixed concentration of an enzyme-labeled PCB was realized and evaluated by electrochemical detection. Alkaline phosphatase was used as the enzyme label, coupled with differential pulse voltammetry (DPV) as the electrochemical technique. The immunosensor was tested on aroclor mixture detection (1242 and 1248) and then on some typologies of food samples to evaluate the possible application for real sample analysis. Samples analyzed were from different matrixes, such as sheep milk, bovine adipose tissue, and bovine muscle. Results obtained were compared with the accredited results according to ISO 17025 methods for PCB detection (HRGC-LRMS) as a confirmatory analysis. Preliminary results show the possibility to use this device as a screening method in food sample analysis. The negligible matrix effect observed may lead to a simplified extraction procedure, and considerable time and consumable savings are the immediate benefits given by the proposed method.

Electrochemistry↗

PubMed2003

Quartz crystal microbalance (QCM) affinity biosensor for genetically modified organisms (GMOs) detection.

A DNA piezoelectric sensor has been developed for the detection of genetically modified organisms (GMOs). Single stranded DNA (ssDNA) probes were immobilised on the sensor surface of a quartz crystal microbalance (QCM) device and the hybridisation between the immobilised probe and the target complementary sequence in solution was monitored. The probe sequences were internal to the sequence of the 35S promoter (P) and Nos terminator (T), which are inserted sequences in the genome of GMOs regulating the transgene expression. Two different probe immobilisation procedures were applied: (a) a thiol-dextran procedure and (b) a thiol-derivatised probe and blocking thiol procedure. The system has been optimised using synthetic oligonucleotides, which were then applied to samples of plasmidic and genomic DNA isolated from the pBI121 plasmid, certified reference materials (CRM), and real samples amplified by the polymerase chain reaction (PCR). The analytical parameters of the sensor have been investigated (sensitivity, reproducibility, lifetime etc.). The results obtained showed that both immobilisation procedures enabled sensitive and specific detection of GMOs, providing a useful tool for screening analysis in food samples.

Affinity Labels↗

PubMed2002

Electrochemical bioassay for the investigation of chlorpyrifos-methyl in vine samples.

This paper reports the optimization of an electrochemical bioassay for the determination of chlorpyrifos-methyl and its application to the analysis of grape and vine leaf samples treated with that pesticide. The analytical method was based on electrochemical determination of the extent of the inhibition exerted by the pesticide on acetylcholinesterase using the substrate acetylthiocholine. Two similar calibration plots were obtained, in the range of 1-300 ng/mL, respectively, for chlorpyrifos-methyl in pure standard form and in the commercial preparation Reldan, with comparable coefficients of variation (CV) in the range of 10% < CV < 20%. After an insecticide treatment, samples were analyzed to evaluate its persistence both in grapes and in vine leaves. Samples were evaluated using different extraction procedures: one based on solvent extraction of pesticide residue from grapes and the other based on aqueous extraction from vine leaves using phosphate buffer. The grape solvent extracts were analyzed using both gas chromatography and electrochemical bioassay, whereas the vine leaf buffer extracts were analyzed using the electrochemical bioassay. Quantitative analysis of chlorpyrifos-methyl determined in the two samples, with the electrochemical bioassay, showed a comparable decrease profile over the experimental period.

Acetylcholinesterase↗

PubMed2002

A comparison between different immobilised glucoseoxidase-based electrodes.

Biosensors obtained by immobilising glucose oxidase 'unentrapped' and 'entrapped in liposomes', both with a classical H2O2 amperometric electrode and with screen-printed electrochemical sensor, were compared. Electrode response, linearity range and the influence of some parameters as phospholipid nature, temperature and measurement techniques were investigated. Experimental results showed that, while with the unentrapped enzyme the output current is linear only up to about 4 mM glucose concentration, the linearity range increases up to about 20 mM using enzyme-loaded liposomes; however the low permeability of the lipid bilayer decreases the electrode sensitivity to very low values (200 nA/M for palmitoylolelyl phosphatidylcholine liposomes). The approach with screen-printed sensors showed a better performance and gave biosensors with higher sensitivity (about 14500 nA/mM). A mathematical model, useful to compare the behaviour of the different analytical systems and to design electrodes with the required properties, was also proposed.

Biosensing Techniques↗

PubMed2002

Disposable electrochemical immunosensor for environmental applications.

An Immunosensor for the detection of polychlorinated biphenyls (PCB) has been developed, using carbon-based screen-printed electrodes as solid-phase and signal transducers. The immunosensor realised is based on a direct competitive immunoassay scheme, in which the antibody against PCB was directly immobilised onto the carbon surface of the screen-printed electrode. Competition between the PCBs present in the sample and a fixed concentration of an enzyme-labelled congener was realised and evaluated by electrochemical detection. The immunosensor developed was tested on Arochlor mixtures (1242 and 1248), and was applied in environmental and food analysis by testing some real samples (from animal tissues and marine sediments). Results obtained demonstrate the ability of this device to detect PCBs in complex matrices.

Animals↗