1,721,018 research outputs found
Metodo per la misura, in continuo e non, dell'aldeide formica nell'atmosfera mesdiante l'impiego di un biosensore enzimatico e dispositivo operante secondo tale metodo
No full textMetodo per la determinazione della concentrazione della formaldeide nell’atmosfera mediante un opportuno enzima comprendente le fasi di rimozione della formaldeide dall’aria da analizzare mediante un film costituito da una soluzione acquosa, della totale conversione della formaldeide in acido formico mediante il passaggio continuo o l’iniezione di detta soluzione in un microcamera contenente un enzima opportunamente immobilizzato che converte totalmente e in modo specifico la formaldeide nel corrispondente acido e della misura della variazione della conducibilità della soluzione a seguito della trasformazione della formaldeide in acido formico. L’interazione della formaldeide con l’enzima avviene in modo da garantire la trasformazione di tutta la formaldeide catturata nei prodotti di reazione, trasformazione che determina una variazione della conducibilità elettrica della soluzione in modo direttamente proporzionale alla concentrazione della formaldeide indipendentemente dall’attività del bioelemento, evitando così la necessità di continue calibrazioni. Il sistema che si colloca nel campo tecnico-scientifico della biochimica-biofisica, può essere vantaggiosamente applicato in campo ambientale, biomedico ed industriale
A coulometric biosensor to determine hydrogen peroxide using a monomolecular layer of horseradish peroxidase immobilized on a glass surface
No full textA biosensor to detect hydrogen peroxide, by coulometry, down to submicromolar concentration using a monomolecular layer of horseradish peroxidase was developed. In this device 0.3 pmol of the enzyme were covalently immobilized on the glass surface of the biosensor and the enzyme layer was characterized by atomic force microscopy and activity measurements. The glass surface bearing the peroxidase was faced to a carbon electrode in a cell of 1 microl of active volume. The polarization of the working electrode at -100 mV versus Ag/AgCl, in the presence of 1,4-hydroquinone as mediator, allowed the fast reduction of the injected hydrogen peroxide via the hydroquinone-peroxidase system. This device permitted to measure the total number of H(2)O(2) molecules present in the cell in the concentration range of 0.3-100 microM H(2)O(2), with a sensitivity of 196 nC/microM H(2)O(2), which is close to the theoretical value (193 nC/microM)
Amplificatori dimensionali per l'utilizzo della microscopia a forza atomica in campo diagnostico
No full textI recenti sviluppi delle ricerche mirate all’immobilizzazione selettiva di biomolecole quali DNA, oligonucleotidi e proteine hanno aperto la strada ad un possibile utilizzo della microscopia a forza atomica come nuova tecnica di indagine diagnostica. La strategia generale prevede l’immobilizzazione su un supporto solido di una molecola complementare alla biomolecola di interesse, che possa legare quest’ultima in modo specifico e ne renda così possibile la successiva rilevazione tramite microscopia a forza atomica. Il limite inferiore di rilevabilità a fini diagnostici non risiede tanto nella concentrazione dell’analita (in quanto la microscopia a forza atomica è teoricamente in grado di rilevare la singola biomolecola immobilizzata sulla superficie del supporto) quanto piuttosto nelle dimensioni molecolari dell’analita stesso: essendo le minime dimensioni determinabili dipendenti dalla rugosità matriciale della superficie, l’utilizzo di un amplificatore di rugosità specifica, cioè di un “amplificatore dimensionale” della specifica biomolecola d’interesse, contribuisce ad abbassare la minima dimensione molecolare rilevabile a fini diagnostici dalla microscopia a forza atomica. Scopo del lavoro è stato quello di individuare un set di possibili amplificatori dimensionali di natura inorganica, superficialmente modificabili mediante l’inserimento di opportuni bio-gruppi funzionali tali da renderli atti ad un accoppiamento specifico con l’analita di interesse, e di mettere a punto un sistema modello per la titolazione quantitativa delle biomolecole oggetto di indagini diagnostiche.
Metodi
Il sistema modello preso in considerazione è centrato su un DNA virale quale biomolecola di interesse, per il quale sono state disegnate come molecole partner alcune sequenze complementari costituite da oligonucleotidi di 2030 basi. Come supporti solidi sono stati scelti vetrini silanizzati. Come amplificatori dimensionali sono state utilizzate nanosfere di lattice, di dimensioni 20-40-80 e 200 nm, funzionalizzate superficialmente con gruppi -COOH, sfruttati per il successivo accoppiamento delle nanosfere alle opportune molecole partner. Tutte le molecole partner usate contenevano un linker amminico utilizzato per la loro immobilizzazione chimica tramite carbodiimmidi ai gruppi –COOH presenti sia sulla superficie del supporto sia sulla superficie degli amplificatori dimensionali. Ogni stadio della procedura sperimentale è stato esaminato mediante microscopio a forza atomica, operando in tapping mode in soluzioni tamponate a pH 7.
Risultati
Le nanosfere della dimensione di 80nm sono risultate il miglior compromesso per il loro utilizzo come amplificatori dimensionali. La modulazione delle condizioni di forza ionica, pH e sonicazione per le procedure di lavaggio successive alla immobilizzazione su supporto solido hanno permesso di ridurre al minimo il binding aspecifico e di ottenere una riproduci bile curva di calibrazione per soluzioni contenenti complessi biomolecola-nanosfera in concentrazioni nel range 10-15 – 10-12M.
Conclusioni
Sono state ottenute superfici piatte chimicamente modificate per l’immobilizzazione specifica di complessi oligonucleotide-DNA-oligonucleotide, accoppiati a nanosfere funzionalizzate quali amplificatori dimensionali per la loro determinazione tramite microscopia a forza atomica in soluzione. È stata realizzata una modulazione quantitativa del binding specifico di complessi modello, utilizzabile per la titolazione quantitativa di varie biomolecole di interesse diagnostic
Procedimento per la determinazione in flusso di un analita mediante biomolecole aventi elevata affinita' per l'analita stesso
No full textA sensitive spectrophotometric-based method for the determination of the rate of hydrogen peroroxide generation in biological systems.
No full textA new sensitive spectrophotometric method for the determination of the rate of hydrogen peroxide generation in biological systems has been developed. This method is based on the measurement of the oxidation rate of reduced cytochrome c by H2O2 in the presence of a mediator and permits the detection of H2O2 generation rates as low as 60 nM min-1. The solution of the differential equations of the kinetic process permitted the calculation of the kinetic rate constants and assessment of the conditions required to measure the hydrogen peroxide generation rate
Ab initio study of the mechanism of the binding of triplet O-2 to hemocyanin
No full textAccurate DFT computations have been carried out to investigate the mechanism of oxygen binding to hemocyanins using two different model systems. The simpler model (model 1) is formed only by a Cu+-Cu+ dimer and by the approaching oxygen molecule, while the more complex model (model 2) involves also four ammonia molecules (bonded to the metal atoms) which emulate the real histidine ligands of the protein matrix. The computational results point out the inadequacy of model 1 in describing the oxygen binding process and show that the greater stability of the singlet versus the triplet state of the final complex (experimentally observed) is not an intrinsic property of the oxygenated copper dimer but is due to the presence of the copper ligands which are responsible for the occurrence of an intersystem crossing along the reaction coordinate. Tne ligand effect has been rationalized using a simple hiIO model which considers the interactions occurring between the nitrogen lone pairs of the ligands and the orbitals that describe the bonding in the Cu+(O-O)Cu+ fragment
An ESR assay for alpha-amylase activity toward succinylated starch, amylose and amylopectin
No full textThe esterification of the three polysaccharides, starch, amylose and amylopectin was carried out in pyridine–DMSO by succinic anhydride. The carboxylic groups in the succinylated polysaccharides were measured by FT–IR spectroscopy. The succinic derivatives were tested as a- amylase (1,4-a- D -glucan glucano hydrolase, E.C. 3.2.1.1) substrates. A colorimetric assay of the a-amylase activity indicated that this enzyme is active on succinic esters of starch and amylose and that the activity shows a linear decrease with the number of succinic units introduced into the polysaccharide. Since the colorimetric test was not suitable for the detection of the a-amylase activity when succinylated amylopectin was the substrate, we set-up an assay based on the labeling by a paramagnetic probe of the free carboxylic groups of succinylated polysaccharides. The kinetics of the a-amylase reaction were monitored by ESR spectroscopy through the increase of the mobility of the paramagnetic probe. The spin label used was the commercially available 4-amino-tempo. By this method we demonstrated that a-amylase is active on succinylated amylopectin. The utility of the assay for monitoring a-amylase activity when other methods (i.e. colorimetric tests) fail, is discussed
Deuterium distribution in lactate as tracer of metabolic pathways
No full text2H-NMR spectroscopy has been used to study the distribution of 2H atoms in brain lactate, after intraperitoneal injection of 2H2O. Information on brain metabolism was obtained from the ratio [(3-2H) lactate]/[(2-2H)-lactate] which was found very sensitive to the PO2 in the breathing air. The dependence of this ratio on the rates of metabolic pathways is an example of the possibility to use the distribution of 2H atoms in a molecule to probe metabolic flow through different pathways
Determination of Glucose Oxidase immobilised as monolayer onto a flat surface
No full textAn assay to estimate the amount of glucose oxidase immobilised as a monolayer onto a flat surface is reported. This method is based on the electrochemical detection of the flavin adenine dinucleotide (FAD) cofactor released by the immobilised enzyme in acid solutions. FAD concentration in the acid solution was measured by amperometry, using a flow injection analysis (FIA) system equipped with a wall-jet electrode, and with a sensitivity of (9.2+/-2.0)x10(-2) nA/nM. By this method, the amount of glucose oxidase molecules present in a monolayer deposited on a silanised glass slide was easily detected, in which the detection limit is more than one order of magnitude lower than the maximum loading of the surface with an ordered monolayer of glucose oxidase
Ascorbate oxidation catalyzed by Bis(histidine)copper(II)
No full textThe kinetics of ascorbic acid oxidation by molecular oxygen, catalyzed by bis(histidine)copper(II) (CuL22+), was followed in 0.1 M phosphate buffer at pH 7.0. Saturation of the oxidation rate was observed at increasing O2, ascorbate and CuL22+ concentrations. The oxidation state of the copper ion during the catalysis and the concentration of the ascorbyl radical were followed by ESR and/or by optical spectroscopy. No significant reduction of Cu(II) was observed under vacuum or in the presence of oxygen at ascorbate concentrations <20 mM. Evidence for the binding of ascorbate to CuL22+ was found by ESR, and a stability constant of 20 M-1 was estimated. We suggested a mechanism which is consistent with our experimental findings and explains some of the contradictory data reported in the past by various authors. The saturation of the reaction rate on increasing [CuL22+] is explained in terms of its catalytic effect on ascorbate oxidation and the superoxide dismutase-like activity of this complex. The experimental concentration of the ascorbyl radical, which is an intermediate product, was in good agreement with that calculated on the basis of the proposed mechanism
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