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M Mascini

Publications and source records attributed to M Mascini.

At least 37 records · Page 2Linked to original sources
PubMed2000

Detection of human apolipoprotein E genotypes by DNA electrochemical biosensor coupled with PCR.

BACKGROUND: Apolipoprotein E (apoE) is an important constituent of several plasma lipoproteins, mainly VLDL, HDL, and chylomicrons. It is involved in the redistribution of lipids in the liver and is implicated in growth and repair of injured neurons in the nervous system. apoE has also been associated with the risk of developing cardiovascular diseases and in familial type III hyperlipoproteinemia. METHODS: We developed a new procedure for detecting genetic polymorphisms of apoE in human blood samples. The procedure is based on coupling of DNA electrochemical sensors with PCR-amplified DNA extracted from human blood. The DNA electrochemical sensor incorporated single-stranded oligonucleotides immobilized on graphite screen-printed electrodes (SPEs) by adsorption at controlled potential. The hybridization reaction on the electrode surface was monitored by chronopotentiometric stripping analysis (PSA), using daunomycin as indicator. RESULTS: With use of two different probes, it was possible to investigate both DNA positions in which the apoE polymorphism takes place and thus to distinguish different genotypes. Real samples containing only complementary sequences gave a good increase in the area of the daunomycin peak ( approximately 600 ms) compared with the peak observed with the buffer. Samples containing 50% complementary sequences gave a much lower increase, and samples containing only mismatch sequences gave a decrease in the daunomycin area. The procedure was validated by comparison with a method based on polyacrylamide gel electrophoresis. CONCLUSION: The coupling of DNA electrochemical sensors with PCR allowed quick discrimination between the different genotypes of apoE.

Apolipoproteins E↗
PubMed1999

Disposable DNA electrochemical sensor for hybridization detection.

A disposable electrochemical sensor for the detection of short DNA sequences is described. Synthetic single-stranded oligonucleotides have been immobilized onto graphite screen printed electrodes with two procedures, the first involving the binding of avidinbiotinylated oligonucleotide and the second adsorption at a controlled potential. The probes were hybridized with different concentrations of complementary sequences. The formed hybrids on the electrode surface were evaluated by differential pulse voltammetry and chronopotentiometric stripping analysis using daunomycin hydrochloride as indicator of hybridization reaction. The probe immobilization step, the hybridization event and the indicator detection, have been optimized. The DNA sensor obtained by adsorption at a controlled potential was able to detect 1 microgram/ml of target sequence in the buffer solution using chronopotentiometric stripping analysis.

Base Sequence↗
PubMed1998

Immunomagnetic separation with mediated flow injection analysis amperometric detection of viable Escherichia coli O157.

The coupling of an immunological separation (using immunomagnetic beads) with amperometric flow injection analysis detection of viable bacteria is presented. Using a solution containing Escherichia coli O157, the electrochemical response with two different mediators [potassium hexacyanoferrate(III) and 2,6-dichlorophenolindophenol] was evaluated in the FIA system. Antibody-derivatized Dynabeads were used to selectively separate E. coli O157 from a matrix. The kinetics and the capacity parameters regarding the attachment of bacteria to the immunobeads were studied. The immunomagnetic separation was then used in conjunction with electrochemical detection to measure the concentration of viable bacteria. A calibration curve of colony-forming units (cfu) against electrochemical response was obtained. The detection limit for this rapid microbiological method was 10(5) cfu mL-1, and the complete assay was performed in 2 h. Some advantages over ELISA methods are the direct detection of viable cells (and not total bacterial load) and the need for only one antibody (not enzyme-labeled), thus making the assay faster (only one washing step is necessary) and less expensive.

2,6-Dichloroindophenol↗
PubMed1998

Surface modifications for the development of piezoimmunosensors.

Four different techniques for the immobilisation of proteins onto the gold electrode of a piezoelectric quartz crystal were investigated. The examined techniques were adsorption, avidin-biotin binding and two different types of covalent binding on self-assembled monolayers (SAM), dithiobis(succinimidylpropionate) (DSP) and a dextran modified thiol monolayer. The reaction of the immobilised proteins (bovine serum albumin (BSA) and anti-human IgG) with their specific antibodies, anti-BSA and hIgG (50 and 200 micrograms/ml) were studied using a quartz crystal microbalance and then compared. Many cycles of measurements were performed on the same crystal regenerating the gold surface with a solution of glycine.HCl, 100 mM, pH 2.1. The interactions of the immobilised reagents with non-specific antibodies were also studied. The adsorption protocol was the quickest, but did not allow regeneration with glycine.HCl. Thiol-dextran coated surfaces did not show any detectable response to non-specific reagents, but needed a very long and complicated protocol. DSP and avidin-biotin coating procedures were easy and not too long. They seemed to have the best characteristics of reproducibility among different crystals and possibility of regeneration of the coated surface, but the percentage of non-specific binding was high.

Adsorption↗
PubMed1994

Characterization of monoclonal antibodies to 2,4-dichlorophenoxyacetic acid using a piezoelectric quartz crystal microbalance in solution.

Piezoelectric quartz crystals (resonance frequency 10 MHz) were used for an investigation of the immunochemical reaction between 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide and several monoclonal antibodies prepared against 2,4-D. The herbicide was immobilized on the gold electrodes of the crystals silanized with 3-aminopropyltriethoxysilane. The activated carboxylic group of 2,4-D was linked either directly to the silanized surface or through hexamethylenediamine or albumin as a macromolecule. The interaction of the immobilized antigen with monoclonal antibody in solution was followed as a change in the resonant frequency of the crystals. The best results were achieved using 2,4-D attached to albumin. The affinity binding of five monoclonal antibodies was characterized by association (ka) and dissociation (kd) kinetic rate constants and by equilibrium association constants (KA) which were obtained from the experimental frequency vs. time curves.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1994

Development of an optical fibre sensor for ammonia, urea, urease and IgG.

An optical fibre sensor utilizing Brilliant Yellow in a thin cellulose acetate membrane as a pH sensor has been developed. By use of gas permeable membrane, urease-immobilized membrane and IgG immobilized membrane we obtained optical sensors for ammonia, urea, urease, IgG suitable for physiological and clinical applications. The system makes use of commercially available instrumentation for absorbance measurement and flow cell for fast analysis.

Ammonia↗
PubMed1992

A flow-through system for the determination of salicylate in blood.

A flow-through system for the determination of salicylate in blood is described. It consists of a Clark-type oxygen probe with the enzyme salicylate hydroxylase chemically bound to polymer supports. This probe, when inserted into a flow-through cell in the presence of salicylate, oxygen and NAD(P)H, produced a current signal proportional to the concentration of salicylate. Salicylate was assayed in the range 10 mumol/l up to 200 mumol/l with a detection limit of 3 mumol/l. Salicylate analysis was optimized by selecting the appropriate pH, buffer, temperature, enzyme immobilization and NAD(P)H concentration. The accuracy of this method was evaluated by recovery studies on 15 sera. Analysis of salicylate in serum sample was performed by diluting samples thirty fold with phosphate buffer to fit the calibration graphs. The response time of the probe was 2 min and 6 min was the time to perform a single analysis. Results compared with the TDx procedure correlated well.

Electrochemistry↗
PubMed1992

Sensitive detection of pesticides using amperometric sensors based on cobalt phthalocyanine-modified composite electrodes and immobilized cholinesterases.

The determination of organophosphate and carbamate pesticides was carried out using cobalt phthalocyanine-modified carbon epoxy composite electrodes coupled with acetylcholinesterase or butyrylcholinesterase. Covalent immobilization of enzymes on Immobilon membranes or nylon nets was examined; the highest sensitivity to inhibitors was found for the nylon net containing low enzyme loading and this was subsequently used for the construction of an amperometric biosensor for pesticides. Analyses were done using acetyl- or butyrylthiocholine as substrates; thiocholine produced by hydrolysis in the enzyme membrane was electrochemically oxidized at +300 mV (vs. Ag/AgCl reference). The decrease of substrate steady-state current caused by the addition of pesticide was used for evaluation. With this approach, 1.5 and 8.4 micrograms l-1 of paraoxon and heptenophos, respectively, can be detected in less than 3 min. These detection limits are similar as those obtained when analyses were performed using free cholinesterase and 10 min incubation with inhibitor.

Biosensing Techniques↗
PubMed1992

Microdialysis and glucose biosensor for in vivo monitoring.

Microdialysis coupled to a glucose biosensor led to a continuous monitor system in vivo for glucose. Several microdialysis probes were used to stabilize the biosensor response. In vivo experiments, especially when the probe was placed subcutaneously, showed that the sensitivity of the biosensor decreased continuously; various kinds of fibers with a molecular weight cut-off (MWCO) ranging from 6,000 to 20,000 were compared. A wall-jet flow cell as detector for glucose showed less interference when compared to a thin layer cell. Glucoday, a new commercial instrument based on this principle, is presented.

Biosensing Techniques↗
PubMed1990

Lactate and glucose electrochemical biosensors for the evaluation of the aerobic and anaerobic threshold in runners.

Lactate and glucose are measured in whole blood of athletes running on a treadmill by using two extracorporeal electrochemical biosensors. The lactate sensor was fixed to an endocrine artificial pancreas (Betalike) which had been used in previous extracorporeal experiments. The lactate sensor gave a signal which resulted in a well defined curve that allowed the evaluation of the aerobic as well as the anaerobic threshold. The results obtained with the glucose sensor supported the theory that muscle anaerobic glycolysis is dependent on muscle glycogen rather than on blood glucose.

Adult↗
PubMed1989

Extracorporeal determination of glucose, lactate and potassium with electrochemical biosensors.

Glucose, lactate and potassium ions have been continuously measured in whole blood by using two extracorporeal electrochemical biosensors and a new flow through potassium electrode. For experiments involving the "glucose clamp", lactate and potassium electrodes have been connected in series to an endocrine artificial pancreas "Betalike" properly modified. The same artificial pancreas has been used in experiments measuring the anaerobic threshold (AT) by continuous monitoring of lactate in athletes. In this case glucose and lactate sensors were connected in series for continuous measurement of both metabolites in blood in real time. The results are in agreement with the biochemical pathways involving such metabolites.

Adult↗
PubMed1989

Glucose electrochemical probe with extended linearity for whole blood.

A glucose probe based on a suitable modification of the silanization process of commercially available polycarbonate membrane was assembled and evaluated. The silanization procedure was carried out in gaseous phase in an anhydrous atmosphere, with dimethyldichlorosilane in a closed vessel. This new procedure seems to be highly reproducible and has the distinct advantage of fast response time and extended linearity up to 0.1 mol l-1 of glucose. Application to serum samples shows the possibility of application in clinical chemistry.

Biocompatible Materials↗
PubMed1987

Lactate and pyruvate electrochemical biosensors for whole blood in extracorporeal experiments with an endocrine artificial pancreas.

Glucose, lactate, and pyruvate can be continuously measured in whole blood by using three extracorporeal electrochemical biosensors. We fixed two newly developed sensors, for lactate and pyruvate, in series with an endocrine artificial pancreas ("Betalike") without affecting its performance. These new sensors for use with whole blood should be able to clarify the fate of the intermediary metabolites of glucose in diabetic patients receiving infusions of drugs or insulin.

Blood Glucose↗
PubMed1986

Clinical uses of enzyme electrode probes.

Clinical applications of enzyme electrochemical sensors are reported; they are based on the coupling of enzymes with potentiometric membrane electrodes (pH, iodide), or potentiometric probes (ammonia, carbon dioxide) or amperometric devices (oxygen, hydrogen peroxide). The most popular and successful immobilization procedures for enzymes are reviewed, namely physical entrapments and chemical methods for binding enzymes to solid support like collagen and nylon net; procedures specifically developed for clinical uses of enzyme probes. The simplicity of the apparatus is evidenced, and it is explained how a single instrument can be useful for all kind of measurements. Practical suggestions for constructing a typical probe are given. Single paragraphs are devoted to the determination of urea, cholesterol, creatinine, amino acids, glucose, lactate, protein, choline and acetylcholine to clarify the sequence of enzymatic and electrochemical reactions in order to elucidate the application range, the sensitivity and the selectivity as well as the relevant interferences for each metabolite either in the enzymatic or in the electrochemical step. The applications performed in vitro, in vivo and ex vivo and the commercial availability of some instruments are reported.

Acetylcholine↗
PubMed1985

Determination of choline-containing phospholipids in human bile and serum by a new enzyme sensor.

A simple method for direct determination both of choline and lecithin (phosphatidylcholine) in human bile and blood sera was developed. An enzyme electrode, based on immobilized choline oxidase on nylon net and an oxygen Clark electrode was assembled. Phosphatidylcholine can be determined by use of phospholipase D as hydrolyzing agent. Reliable results were obtained in the case of determination of lecithin and choline in bile samples.

Alcohol Oxidoreductases↗
PubMed1985

An L-lactate sensor with immobilized enzyme for use in in vivo studies with an endocrine artificial pancreas.

We report the features of a sensor for determining L-lactate. An oxygen sensor, coupled to a nylon net with chemically bound L-lactate oxidase (EC 1.1.3.2), is inserted into an artificial pancreas (Biostator, Miles) "downstream" from the glucose sensor. We used the sensor to continuously monitor the L-lactate concentration in blood after a "glucose clamp" experiment with a diabetic patient. L-Lactate determinations in blood drawn from the patient every 15 min agreed well with results obtained by use of the L-lactate sensor.

Enzymes, Immobilized↗