Biomedical subjects

Marco Mascini

Publications and source records attributed to Marco Mascini.

At least 19 recordsLinked to original sources

PubMed2006

Planar electrochemical sensors for biomedical applications.

Electrochemistry has superior properties respect to the other measurement systems because of the rapid, simple and sensitive characteristics. For all these reasons, electrochemical sensors are playing a key role in many scientific sectors. Planar electrochemical sensors present many advantages respect to the three-dimensional devices, but this technology requires the use of specialty chemicals to meet the new functional requirements of planarization and miniaturization. In this paper, some production techniques and procedures to obtain planar electrochemical sensors are reported, together with their applications.

Biological Assay↗

PubMed2006

Biosensor measurements of polar phenolics for the assessment of the bitterness and pungency of virgin olive oil.

Bitterness and pungency, sensory quality attributes of virgin olive oil, are related to the presence of phenolic compounds. Fast and reliable alternatives for the evaluation of sensory attributes and phenolic content are desirable, as sensory and traditional analytical methods are time-consuming and expensive. In this study, two amperometric enzyme-based biosensors (employing tyrosinase or peroxidase) for rapid measurement of polar phenolics of olive oil were tested. The biosensor was constructed using disposable screen-printed carbon electrodes with the enzyme as biorecognition element. The sensor was coupled with a simple extraction procedure and optimized for use in flow injection analysis. The performance of the biosensor was assessed by measuring a set of virgin olive oils and comparing the results with data obtained by the reference HPLC method and sensory scores. The correlations between the tyrosinase- and peroxidase-based biosensors and phenolic content in the samples were high (r = 0.82 and 0.87, respectively), which, together with a good repeatability (rsd = 6%), suggests that these biosensors may represent a promising tool in the analysis of the total content of phenolics in virgin olive oils. The correlation with sensory quality attributes of virgin olive oil was lower, which illustrates the complexity of sensory perception. The two biosensors possessed different specificities toward different groups of phenolics, affecting bitterness and pungency prediction. The peroxidase-based biosensor showed a significant correlation (r = 0.66) with pungency.

Biosensing Techniques↗

PubMed2006

Dendritic-like streptavidin/alkaline phosphatase nanoarchitectures for amplified electrochemical sensing of DNA sequences.

This study describes the development and characterization of a novel dendritic-like signal amplification pathway. Such an analytical strategy relies on the use of streptavidin and biotinylated alkaline phosphatase, which can be simply and conveniently self-assembled to build nanoarchitectures rich in enzyme labels. The performance of this enzyme-based amplification route was demonstrated in connection with the electrochemical sensing of DNA sequences. Compared to the commercially available streptavidin-conjugated alkaline phosphatase labels, a single generation of the streptavidin/biotinylated alkaline phosphatase assembly allowed a 15-20-fold enhancement of the electroanalytical signals. The higher sensitivity allowed by the dendritic-like route was attributed to the lower steric hindrance of the proteins employed for this amplification path. As low as 50 pmol/L of a 388-bp-long amplicon identifying Salmonella spp. was easily detected. The experimental results additionally demonstrated that the sensitivity of the method could be further increased in a linear fashion with the number of protein-enzyme generations.

Alkaline Phosphatase↗

PubMed2006

Point-of-care biosensor systems for cancer diagnostics/prognostics.

With the growing number of fatalities resulting from the 100 or so cancer-related diseases, new enabling tools are required to provide extensive molecular profiles of patients to guide the clinician in making viable diagnosis and prognosis. Unfortunately with cancer-related diseases, there is not one molecular marker that can provide sufficient information to assist the clinician in making effective prognoses or even diagnoses. Indeed, large panels of markers must typically be evaluated that cut across several different classes (mutations in certain gene fragments--DNA; over/under-expression of gene activity as monitored by messenger RNAs; the amount of proteins present in serum or circulating tumor cells). The classical biosensor format (dipstick approach for monitoring the presence of a single element) is viewed as a valuable tool in many bioassays, but possesses numerous limitations in cancer due primarily to the single element nature of these sensing platforms. As such, if biosensors are to become valuable tools in the arsenal of the clinician to manage cancer patients, new formats are required. This review seeks to provide an overview of the current thinking on molecular profiling for diagnosis and prognosis of cancers and also, provide insight into the current state-of-the-art in the biosensor field and new strategies that must be considered to bring this important technology into the cancer field.

Biomarkers, Tumor↗

PubMed2006

Detection of clinically relevant point mutations by a novel piezoelectric biosensor.

Piezoelectric sensing is here applied to point mutation detection in human DNA. The mutation investigated is in the TP53 gene, which results inactivated in most cancer types. TP53 gene maps on chromosome 17 (17p13.1). It contains 11 exons and codifies for the relative protein, involved in cell proliferation. The TP53 gene has a wide mutation spectrum that is related to different tumours. In particular, those occurring in the structurally important L2 and L3 zinc-binding domains, have been linked to patient prognosis and more strongly to radiotherapy and chemotherapy resistance in several major cancers. For this reason, the identification of these mutations represents an important clinical target and biosensors could represent good candidate for fast mutation screening. In this paper, a DNA-based piezoelectric biosensor for the detection of the TP53 gene mutation at codon 248 is reported. A biotinylated probe was immobilised on the sensor surface via dextran-streptavidin modified surfaces. The sensor was optimised using synthetic oligonucleotides. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR)-amplified real samples of DNA extracted from two cell lines, one normal (wild-type) and one mutated, carrying the mutation at codon 248 of the TP53 gene. The results obtained demonstrate that the DNA-based piezoelectric biosensor is able to detect the point mutations in PCR-amplified samples showing the potentialities of this approach for routine analysis.

Biosensing Techniques↗

PubMed2006

Electrochemical biosensor evaluation of the interaction between DNA and metallo-drugs.

Electrochemical techniques were used to study the interaction between a panel of antiproliferative metallo-drugs and double-stranded DNA immobilized on screen-printed electrodes as a model of the analogous interaction occurring in solution. The propensity of a given metal drug to interact with DNA was measured as a function of the decrease of guanine oxidation signal, which was detected by square wave voltammetry. Estimates of variations in experimental parameters, such as the concentration of complexes, time following dissolution (ageing time) and the presence of chloride, are provided.

Biosensing Techniques↗

PubMed2005

DNA-based electrochemical biosensors for monitoring of bis-indoles as potential antitumoral agents, chemistry, X-ray crystallography.

Facile and practical electrochemical DNA bioassay, X-ray diffraction analysis, synthesis and 1H and 13C NMR data of the 5,5'-disubstituted-3,3'-methanediyl-bis-indoles are reported. On the basis of electrochemical measurements we have hypothesized that the analyzed bis-indoles have an effect on human tumor cells due to DNA binding at adenine-thymidine deoxynucleotides rich region in a concentration/substituent dependent manner. Interesting N-H...pi and hydrogen-bonding intermolecular interactions were observed which may differentiate their biological features. The 5,5'-dimethoxy-3,3'-methanediyl-bis-indole (2) was found to reduce considerably the growth of cancer cell lines HOP-92 (lung), A498 (renal) and MDA-MB-231/1TCC (breast). The results indicate that title compounds could be interesting as potential antitumoral chemotherapeutics.

Antineoplastic Agents↗

PubMed2005

Piezoelectric biosensors: strategies for coupling nucleic acids to piezoelectric devices.

The development of a piezoelectric biosensor based on nucleic acids interaction is presented focusing on the methodology for probe immobilization. This is a key step in any DNA biosensor development. Often, the detection limits and, in general, the analytical performances of the biosensor can be improved by optimizing the immobilization of the receptor on the transducer surface. DNA must be attached to the solid support, retaining native conformation, and binding activity. This attachment must be stable over the course of a binding assay and, in addition, sufficient binding sites must be presented to the solution phase to interact with the analyte. In this paper, the optimization of the coating of the gold quartz crystal surface, to immobilize an oligonucleotide probe, is reported. Two immobilization procedures are illustrated in details with a comparison regarding the immobilization of the probe, the detection of the hybridization reaction, and the possibility of regeneration. The two procedures are based on the use of biotinylated or thiolated DNA probes. Specific applications will be also presented.

Biosensing Techniques↗

PubMed2005

Steric factors controlling the surface hybridization of PCR amplified sequences.

This study elucidated the hybridization behavior of surface-bound oligonucleotides to their longer PCR-amplified targets. The screen-printed gold surface of disposable electrodes was the platform onto which thiol-tethered oligonucleotides (21-mer) were immobilized by chemisorption. As a model case, approximately 600-bp amplicons were studied. Surface hybridization was monitored by means of an enzyme-linked assay with electrochemical detection. Use of different surface-tethered probe sequences over a wide range of surface densities was explored to achieve the highest duplex yield. Both the surface coverage by the probe and its relative position on the target strand were found to control the efficiency of capture of the target sequence. Interfacial hybridization occurred with the highest efficiency for a probe coverage of approximately 2.9 x 10(12) molecules/cm2 and when the 3' end of the amplicon was involved. An unusual (bell-shaped) response/amplicon concentration profile was additionally found. It was hypothesised that when the amount of solution-phase target is relatively high, random collisions make reannealing of the approximately 600-bp strands favored over formation of the surface-tethered probe-amplicon complex. This paper also describes a strategy to enhance the sensitivity of enzyme-linked hybridization assays. Such a strategy relies on formation, around the long target sequence, of dendritic-like structures, which could offer multiple anchoring points for the enzyme conjugate. The results shown in this work might have great significance for the practical application of hybridization to oligonucleotide chips.

Base Sequence↗

PubMed2005

Investigations of the antioxidant properties of plant extracts using a DNA-electrochemical biosensor.

In this work, the results of a method based on an electrochemical biosensor to detect DNA damage in vitro for the evaluation of the antioxidant properties of plant extracts are reported. The biosensor consisted of a dsDNA immobilized on a screen-printed electrode surface (SPE). DNA damage was promoted by the generation of the *OH radicals via Fenton-type reaction. The interaction of the radical species with immobilised DNA in the absence and presence of antioxidants was evaluated by means of changes in the guanine oxidation peak obtained by square wave voltammetry. The results demonstrated that the DNA-based biosensor is suitable as a rapid screening test for the evaluation of antioxidant properties of samples.

Antioxidants↗

PubMed2005

Detection of fragmented genomic DNA by PCR-free piezoelectric sensing using a denaturation approach.

Label-free and real-time DNA sequence detection in PCR-amplified DNA samples can now be achieved by different approaches. On the contrary, only few works have been reported dealing with direct sequence detection in nonamplified genomic DNA. Here, a piezoelectric biosensor for direct detection of sequences in nonamplified genomic DNA is described. The system relies on real-time and label-free detection of the hybridization reaction between an immobilized probe and the complementary sequence in solution. The DNA probe is immobilized on the sensing surface (10 MHz quartz crystals), while the complementary sequence is present in the genomic DNA, previously fragmented with restriction enzymes.

Biosensing Techniques↗

PubMed2005

Enzyme-based impedimetric detection of PCR products using oligonucleotide-modified screen-printed gold electrodes.

This paper describes the optimisation and the analytical performances of an enzyme-based electrochemical genosensor, developed using disposable oligonucleotide-modified screen-printed gold electrodes. The immobilisation of a thiol-tethered probe was qualitatively investigated by means of faradic impedance spectroscopy. Impedance spectra confirmed that the thiol moiety unambiguously drives the immobilisation of the oligonucleotide probe. Furthermore, both probe surface densities and hybridisation efficiencies were quantified through chronocoulometric measurements. Electrochemical transduction of the hybridisation process was also performed by means of faradic impedance spectroscopy, after coupling of a streptavidin-alkaline phosphatase conjugate and bio-catalysed precipitation of an insoluble and insulating product onto the sensing interface. Chronocoulometric results allowed discussion of the magnitude of hybridisation signals in terms of probe surface densities and their corresponding hybridisation efficiency. The genosensor response varied linearly (r2 = 0.9998) with the oligonucleotide target concentration over three orders of magnitude, between 12 pmol/L and 12 nmol/L. The estimated detection limit was 1.2 pmol/L (i.e., 7.2 x 10(6) target molecules in 10 microL of sample solution). The analytical usefulness of the impedimetric genosensor was finally demonstrated analysing amplified samples obtained from the pBI121 plasmid and soy and maize powders containing 1 and 5% of genetically modified product. Sensing of such unmodified amplicons was achieved via sandwich hybridisation with a biotinylated signaling probe. The electrochemical enzyme-amplified assay allowed unambiguous identification of all genetically modified samples, while no significant non-specific signal was detected in the case of all negative controls.

Biosensing Techniques↗

PubMed2005

Benzene analysis in workplace air using an FIA-based bacterial biosensor.

A bacterial biosensor based on flow injection analysis (FIA) has been developed for the determination of benzene in workplace air samples. Benzene can be used by the bacteria Pseudomonas putida ML2 as a sole carbon source, and its aerobic degradation can be measured using a dissolved oxygen electrode. The bacterial cells were immobilised between two cellulose acetate membranes and fixed onto a Clark dissolved oxygen probe, which was inserted into a custom-made flow cell. The applicability of the biosensor for the analysis of air samples containing benzene was investigated. Air samples were collected from a controlled exposure room using charcoal adsorption tubes, and benzene extracted with solvent desorption using dimethylformamide (DMF). The biosensor displayed a linear detection range between 0.025 and 0.15 mM benzene based on standard solutions containing a maximum of 2% DMF, with a response time of 6 min. This linear detection range allows the analysis of air containing between 3 and 16 ppm benzene based on a 60-min sampling period. DMF proved to be compatible for use with the biosensor, causing minimal interference with the sensor response and causing no toxic effects on the bacterial cells. The FIA system was easily transported to an in situ location, and a correlation was obtained between the biosensor and gas chromatography (GC) results for the preliminary air samples investigated. Moreover, the biosensor displayed no interference to other benzene related compounds in the BTEX range. The results from this work have shown that the biosensor has potential applications for the analysis of benzene in workplace air samples, with the added advantages over the conventional GC methods of low operation costs, ease of use, and portability for in situ measurements.

Air Pollutants, Occupational↗

PubMed2005

Detection of TP53 mutation using a portable surface plasmon resonance DNA-based biosensor.

A DNA-based surface plasmon resonance (SPR) biosensor has been developed for the detection of TP53 mutation using the inexpensive and commercially available instrument, SPREETA SPR-EVM-BT, from Texas Instruments. A direct immobilisation procedure, based on the coupling of thiol-derivatised oligonucleotide probes (Probe-C6-SH) to bare gold sensor surfaces, was optimized using synthetic oligonucleotides. Hybridisation reactions between the immobilised probe and a short sequence (26 mer) complementary, non-complementary and one-point mutation DNA were then investigated. The main analytical parameters of the sensor system were studied in detail including selectivity, sensitivity, reproducibility and analysis time. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR)-amplified real samples, DNA extracted from both normal, wild-type, (Jurkat) and mutated (Molt 4), carrying the mutation at codon 248 of the TP53 cell lines. The results obtained demonstrate that the DNA-based SPR biosensor was able to distinguish sequences present in the various samples that differ only by one base; and hence, it appears to be a strong candidate technique for the detection of gene mutation.

Biosensing Techniques↗

PubMed2005

New trends in nucleic acid based biosensors: University of Florence (Italy), 25-28 October 2003.

The Workshop on "New Trends in Nucleic Acids Based Biosensors" was held at the University of Florence (Italy), from 25th to 28th October 2003. The workshop was funded by the European Science Foundation (ESF) within the programme "Functional Genomics" (Programme Area: DNA arrays and chips). This report describes the main arguments discussed during the workshop considering the state of art and the future trends in the field of nucleic acid-based biosensors, with particular attention on the aptamer field.

Biosensing Techniques↗

PubMed2005

Direct immobilisation of DNA probes for the development of affinity biosensors.

An immobilisation procedure based on the direct coupling of thiolated probes (Probe-C6-SH) to bare gold sensor surfaces has been compared with a reference immobilisation method, based on the coupling of biotinylated probes onto a streptavidin-coated dextran-modified surface. The instrumentations used were a quartz crystal microbalance (QCM) and the optical instruments Biacore X and Spreeta based on surface plasmon resonance (SPR). The performances of the DNA-based sensors resulting from direct coupling of thiolated DNA probes onto electrodes of quartz crystals or gold SPR-chips have been studied in terms of the main analytical parameters, i.e. selectivity, sensitivity, reproducibility, etc. In particular, the two immobilisation approaches have been applied to the analysis of oligonucleotides, DNA amplified by polymerase chain reaction (PCR) and genomic DNA enzymatically digested.

Animals↗

PubMed2004

Immobilisation of DNA probes for the development of SPR-based sensing.

An immobilisation procedure based on the direct coupling of thiol-derivatised oligonucleotide probes to bare gold sensor surfaces has been used for DNA sensing applications. The instrumentation used relies on surface plasmon resonance (SPR) transduction; in particular the commercially available instruments BIACORE X and SPREETA, have been employed in this study. The performances of the SPR-based DNA sensors resulting from direct coupling of thiol-derivatised DNA probes onto gold chips, have been studied in terms of the main analytical parameters, i.e. selectivity, sensitivity, reproducibility, analysis time, etc. A comparison between the thiol-derivatised immobilisation approach and a reference immobilisation method, based on the coupling of biotinylated oligonucleotide probes onto a streptavidin coated dextran sensor surface, using synthetic complementary oligonucleotides has been discussed. Finally, a denaturation method to obtain ssDNA ready for hybridisation analysis has been applied to polymerase chain reaction (PCR) amplified samples, for the detection of genetically modified organisms (GMOs).

Adsorption↗

PubMed2004

A new approach for the detection of DNA sequences in amplified nucleic acids by a surface plasmon resonance biosensor.

In this paper, a simple and useful approach for DNA sensing based on surface plasmon resonance (SPR) transduction is reported. A new DNA sample pre-treatment has been optimised to allow fast and simple detection of hybridisation reaction between a target sequence in solution and a probe immobilised on the sensing surface. This pre-treatment consisted in a denaturation procedure of double stranded DNA containing the target sequence and was based on an high temperature treatment (95 degrees C, 5 min) followed by a 1 min incubation with small oligonucleotides. The oligonucleotides are designed to prevent the re-hybridising of the denatured strands, while enabling the target sequence to bind the immobilised probe. The important parameters of the procedure, i.e. incubation time, length and concentration of the oligonucleotides, have been studied in detail. The optimised DNA denaturation procedure has been successfully applied to the detection of amplified DNA with a commercially available SPR biosensor (Biacore X). DNA samples extracted from plant and human blood were tested after amplification by polymerase chain reaction (PCR).

Apolipoproteins E↗