| 1. |
- Aquilante, Laura, et al.
(författare)
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Determination of biogenic amines by using amperometric biosensors based on grass PEA amine oxidase and OAT polyamine oxidase
- 2020
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Ingår i: Studia Universitatis Babes-Bolyai Chemia. - : Babes-Bolyai University. - 1224-7154 .- 2065-9520. ; 65:3, s. 9-21
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Tidskriftsartikel (refereegranskat)abstract
- Grass pea amine oxidase (GPAO) and oat polyamine oxidase (OPAO) were immobilized along with horseradish peroxidase (HRP) and an Os-redox polymer (Os-RP) onto the surface of a graphite electrode by crosslinking with poly(ethylenglycol) diglycidyl ether. The resulted reagentless amperometric biosensors were inserted in a flow injection setup and used as electrochemical detectors for the biogenic amines (BA) detection. Both biosensors were operated at low applied potential (-50 mV vs. Ag/AgCl, KCl0.1M) where electrochemical interferences are minimal. The quantification of ten BA (tyramine, putrescine, cadaverine, histamine, cystamine, phenylethylamine, agmatine, tryptamine, spermine, and spermidine) either individual or in mixture (after a preliminary separation by using cation exchange chromatography) was reported. G/(Os-RP)-HRP-GPAO biosensor detected all ten BA, while G/(Os-RP)-HRP-OPAO biosensor detected only spermine and spermidine. Finally, a simple and low-cost method for free and acetylated polyamines determination in human urine samples, by using the highly selective G/(Os-RP)-HRP-OPAO biosensor, was proposed.
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| 2. |
- Fort, Carmen Ioana, et al.
(författare)
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Carbon Aerogel as Electrode Material for Improved Direct Electron Transfer in Biosensors Incorporating Cellobiose Dehydrogenase
- 2016
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Ingår i: Electroanalysis. - : Wiley. - 1040-0397. ; 28:10, s. 2311-2319
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Tidskriftsartikel (refereegranskat)abstract
- High mesoporous carbon aerogels (CA) were prepared by a sol-gel method, followed by supercritical drying with liquid CO2 and pyrolysis under inert atmosphere. The morphological and structural characteristics (surface area, pore size distribution and pore volume) of CA were examined by using N2 adsorption/desorption isotherms, TEM, SEM and AFM measurements. CA, dispersed in a chitosan (Chi) solution, was immobilized on the surface of a glassy carbon (GC) electrode aiming to prepare an amperometric transducer able to improve the direct electron transfer (DET) between Phanerochaete chrysosporium cellobiose dehydrogenase (PcCDH) and the electrode. The resulting GC/CA-Chi/PcCDH biosensor was optimized with regards to the loading of CA and PcCDH, as well as pH of the buffer solution. The electroanalytical parameters of the optimized biosensor (maximum current responses (Imax), apparent Michaelis-Menten constants (KM)) were estimated from amperometric calibration curves for lactose. The remarkably high sensitivity towards lactose (133.7±0.2 µA cm−2 mM−1) at the GC/CA-Chi/PcCDH electrode recommends the GC/CA-Chi electrode as an efficient transducer for PcCDH based biosensors, exploiting DET.
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| 3. |
- Gallego-Parra, Samuel, et al.
(författare)
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Pressure-induced order-disorder transitions in beta-In2S3 : an experimental and theoretical study of structural and vibrational properties
- 2021
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Ingår i: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry. - 1463-9076 .- 1463-9084. ; 23:41, s. 23625-23642
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Tidskriftsartikel (refereegranskat)abstract
- This joint experimental and theoretical study of the structural and vibrational properties of beta-In2S3 upon compression shows that this tetragonal defect spinel undergoes two reversible pressure-induced order-disorder transitions up to 20 GPa. We propose that the first high-pressure phase above 5.0 GPa has the cubic defect spinel structure of alpha-In2S3 and the second high-pressure phase (phi-In2S3) above 10.5 GPa has a defect alpha-NaFeO2-type (R3m) structure. This phase, related to the NaCl structure, has not been previously observed in spinels under compression and is related to both the tetradymite structure of topological insulators and to the defect LiTiO2 phase observed at high pressure in other thiospinels. Structural characterization of the three phases shows that alpha-In2S3 is softer than beta-In2S3 while phi-In2S3 is harder than beta-In2S3. Vibrational characterization of the three phases is also provided, and their Raman-active modes are tentatively assigned. Our work shows that the metastable alpha phase of In2S3 can be accessed not only by high temperature or varying composition, but also by high pressure. On top of that, the pressure-induced beta-alpha-phi sequence of phase transitions evidences that beta-In2S3, a BIII2XV3 compound with an intriguing structure typical of A(II)BIII(2)XVI(4) compounds (intermediate between thiospinels and ordered-vacancy compounds) undergoes: (i) a first phase transition at ambient pressure to a disordered spinel-type structure (alpha-In2S3), isostructural with those found at high pressure and high temperature in other BIII2XV3 compounds; and (ii) a second phase transition to the defect alpha-NaFeO2-type structure (phi-In2S3), a distorted NaCl-type structure that is related to the defect NaCl phase found at high pressure in A(II)BIII(2)XVI(4) ordered-vacancy compounds and to the defect LiTiO2-type phase found at high pressure in A(II)BIII(2)XVI(4) thiospinels. This result shows that In2S3 (with its intrinsic vacancies) has a similar pressure behaviour to thiospinels and ordered-vacancy compounds of the A(II)BIII(2)XVI(4) family, making beta-In2S3 the union link between such families of compounds and showing that group-13 thiospinels have more in common with ordered-vacancy compounds than with oxospinels and thiospinels with transition metals.
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| 4. |
- Gligor, Delia, et al.
(författare)
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Amperometric Biosensor For Ethanol Based On A Phenothiazine Derivative Modified Carbon Paste Electrode
- 2008
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Ingår i: Studia Universitatis Babes-Bolyai, Chemia. - 2065-9520. ; 53:1, s. 55-62
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Tidskriftsartikel (refereegranskat)abstract
- A new amperometric biosensor for ethanol, based on carbon paste electrode modified with alcohol dehydrogenase (ADH), polyethylenimine (PEI) and using a phenothiazine derivative (DDDP; 16H,18H-dibenzo[c,1]7,9-dithia-16,18-diazapentacene) as redox mediator for NADH recycling, was developed. The biosensor response is the result of mediated oxidation of NADH, generated in the enzymatic reaction between ADH and ethanol (in the presence of NAD(+)). The biosensor sensitivity (calculated as the ratio I-max/K-M(app)) was 0.035 mA M-1 and the detection limit was 0.26 mM, while the linear response range was from 0.1 to 20 mM ethanol.
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| 5. |
- Gligor, Delia, et al.
(författare)
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Influence of the Electrode Material on the Electrochemical Behavior of Carbon Paste Electrodes Modified with Meldola Blue and Methylene Green Adsorbed on a Synthetic Zeolite
- 2010
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Ingår i: Electroanalysis. - : Wiley. - 1040-0397 .- 1521-4109. ; 22:5, s. 509-512
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Tidskriftsartikel (refereegranskat)abstract
- Carbon paste electrodes modified with a phenoxazine derivative, Meldola blue, and a phenothiazine derivative, methylene green, both strongly adsorbed on a synthetic zeolite were investigated using either glassy carbon powder (Sigradur K, SK) or single-walled carbon nanotubes (SWCNT) as conductive electrode material. In the case of SWCNT based electrodes, the formal potential of both mediators was pH dependent, as expected for a redox process involving proton transfer. In contrast, the formal potential of both mediators of SK based modified electrodes was practically insensitive to pH. This behavior is discussed in terms of interactions existing in the heterogeneous system mediator-zeolite-electrode material.
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| 6. |
- Gligor, Delia, et al.
(författare)
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Photoelectrocatalytic Oxidation of NADH at a Graphite Electrode Modified with a New Polymeric Phenothiazine
- 2009
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Ingår i: Electroanalysis. - : Wiley. - 1040-0397 .- 1521-4109. ; 21:3-5, s. 360-367
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Konferensbidrag (refereegranskat)abstract
- A new approach for photoelectrocatalytic oxidation of NADH is described, based on a graphite electrode (G) modified with a new phenothiazine derivative, polyphenothiazine formaldehyde (PPF). Electrochemical measurements reveal that G/PPF presents a quasireversible voltammetric response, corresponding to a 2e(-)/2H(+) transfer and that G/PPF modified electrodes show electrocatalytic activity for NADH oxidation. When the surface of the G/PPF modified electrode was irradiated with a light source (250 W Halogen lamp), a significant increase in the electrocatalytic current was observed. The second order electrocatalytic rate constant (k(obs,[NADH]=0) = 3.9 x 10(3) M-1 s(-1), pH 7.0) was calculated from rotating disk electrode experiments, performed at various NADH concentrations. From amperometric measurements, it was noticed that, in the presence of light irradiation, the sensitivity of NADH detection (calculated as I-max/K-M ratio) increased ca. 3 times.
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| 7. |
- Gligor, Delia, et al.
(författare)
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Poly-phenothiazine derivative-modified glassy carbon electrode for NADH electrocatalytic oxidation
- 2009
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Ingår i: Electrochimica Acta. - : Elsevier BV. - 0013-4686. ; 54:11, s. 3124-3128
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Tidskriftsartikel (refereegranskat)abstract
- Electropolymerization of a new phenothiazine derivative (bis-phenothiazin-3-yl methane; BPhM) on glassy carbon (GC) electrode generates a conducting film of poly-BPhM. in stable contact with the electrode surface. The heterogeneous electron-transfer process corresponding to the modified electrode is characterized by a high rate constant (50.4 s(-1), pH 7). The CC/poly-BPhM electrode shows excellent electrocatalytic activity toward NADH oxidation. The rate constant for catalytic NADH oxidation, estimated from rotating disk electrode (RDE) measurements and extrapolated to zero concentration of NADH, was found to be 9.4 x 10(4) M-1 s(-1) (pH 7). The amperometric detection of NADH, at +200 mV vs. SCE, is described by the following electroanalytical parameters: a sensitivity of 1.82 mA M-1, a detection limit of 2 mu M and a linear domain up to 0.1 mM NADH. (c) 2008 Elsevier Ltd. All rights reserved.
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| 8. |
- Killyeni, Aniko, et al.
(författare)
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Effect of deglycosylation on the mediated electrocatalytic activity of recombinantly expressed Agaricus meleagris pyranose dehydrogenase wired by osmium redox polymer
- 2014
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Ingår i: Electrochimica Acta. - : Elsevier BV. - 0013-4686. ; 126, s. 61-67
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Tidskriftsartikel (refereegranskat)abstract
- The effect of deglycosylation of pyranose dehydrogenase (PDH) obtained from Agaricus meleagris (Am) and recombinantly expressed in Pichia pastoris on its electrocatalytic activity was investigated. Glycosylated (gAmPDH) and deglycosylated PDH (dgAmPDH) were immobilised on spectrographic graphite (G) simultaneously with an osmium redox polymer (Os-RP) using poly(ethylene glycol)(400) diglycidyl ether (PEGDGE) as cross-linking agent. The amperometric response to glucose, recorded at G/(Os-RP)-gAmPDH and G/(Os-RP)-dgAmPDH bioelectrodes, was optimised under flow injection conditions concerning the applied potential, enzyme loading, working pH and flow rate. The G/(Os-RP)dgAmPDH bioelectrode is characterised by better kinetic and electroanalytical parameters compared with the G/(Os-RP)-gAmPDH bioelectrode: (i) a higher value of the maximum catalytic current density, j(max) = (146.6 +/- 2.6) mu A cm(-2) vs. j(max) = (80.9 +/- 1.9) mu A cm(-2); (ii) a lower value of the apparent Michaelis-Menten constant, K-M(app) = (2.4 +/- 0.1) mu M vs. K-M(app) = (7.5 +/- 0.3) mM; (iii) a higher slope of the linear domain, (43.6 +/- 1.1) mu A cm(-2) mM(-1) vs. (9.74 +/- 0.16) mu A cm(-2) mM(-1). Additionally, the time dependent decay of the amperometric response to glucose shows a slightly better operational stability for the G/(Os-RP)-dgAmPDH bioelectrode than that for the G/(Os-RP)-gAmPDH. The enzyme deglycosylation induces significant changes in the order of substrate selectivity for gAmPDH and dgAmPDH. (C) 2013 Elsevier Ltd. All rights reserved.
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| 9. |
- Killyeni, Aniko, et al.
(författare)
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EFFECT OF ENZYME DEGLYCOSYLATION ON THE AMPEROMETRIC DETECTION OF GLUCOSE AT PDH-MODIFIED ELECTRODE
- 2012
-
Ingår i: Studia Universitatis Babes-Bolyai, Chemia. - 2065-9520. ; 57:4, s. 87-99
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Tidskriftsartikel (refereegranskat)abstract
- The effect of deglycosylation of pyranose dehydrogenase (PDH), obtained from Agaricus meleagris and recombinantly expressed in Pichia pastoris, on the amperometric detection of glucose was investigated. Glycosylated (gPDH) and deglycosylated (dgPDH) PDH were immobilized on spectrographic graphite (G) simultaneously with an Os redox polymer (Os-RP). The amperometric response of G/Os-RP/gPDH and G/Os-RP/dgPDH to glucose was recorded using flow injection measurements and cyclic voltammetry. A significant increase in the maximum catalytic current density was observed for G/Os-RP/dgPDH [(148.7 +/- 0.14) mu A/cm(2)) compared with G/Os-RP/gPDH [(81.4 +/- 1.4) mu A/cm(2)]. Additionally, the deglycosylation of the enzyme resulted in a higher substrate-enzyme affinity (K-M (app) = 2.44 +/- 0.10 mM), compared with glycosylated PDH (K-M(app) = 7.52 +/- 0.34 mM).
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| 10. |
- Kovacs, Gabor, et al.
(författare)
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Graphite electrodes modified with Neurospora crassa cellobiose dehydrogenase: Comparative electrochemical characterization under direct and mediated electron transfer
- 2012
-
Ingår i: Bioelectrochemistry. - : Elsevier BV. - 1878-562X .- 1567-5394. ; 88, s. 84-91
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Tidskriftsartikel (refereegranskat)abstract
- The electrochemical characterization of a class II cellobiose dehydrogenase (CDH), from the ascomycete fungus Neurospora crassa. adsorbed on graphite (G), was performed in regard to direct (DET) and mediated electron transfer (MET). The effects of the applied potential, mediator (1,4 benzoquinone) concentration and flow carrier pH on the amperometric response of the G/CDH modified electrodes were investigated under flow conditions. From the calibration curves, recorded at two pH values (5.2 and 7.0) for nine different sugars, the kinetic and the analytical parameters were evaluated under DET and MET operation modes. These results together with those obtained from long term operational stability measurements showed that: (i) for all nine investigated sugars the sensitivity was higher for MET than for DET and for pH 5.2 compared to pH 7.0; (ii) irrespective of DET or MET operation mode, the sensitivity of the new enzyme towards the investigated sugars decreased in the following sequence: cellobiose > lactose > (cellotriose approximate to cellopentaose) > > (maltotriose approximate to maltotetraose approximate to maltopentaose) > (maltose approximate to glucose); (iii) for all tested substrates, the apparent CDH affinity was roughly higher in DET than in MET operation mode. (C) 2012 Elsevier B.V. All rights reserved.
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| 11. |
- Ladiu, Carmen Ioana, et al.
(författare)
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NADH electrocatalytic oxidation at glassy carbon paste electrodes modified with meldola blue adsorbed on acidic alpha-zirconium phosphate
- 2007
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Ingår i: Revue Roumaine de Chimie. - 0035-3930. ; 52:1-2, s. 67-74
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Tidskriftsartikel (refereegranskat)abstract
- The electrochemical behavior and the electrocatalytic activity for the NADH oxidation at glassy carbon paste electrodes (GCPEs) modified with Meldola Blue (MB) adsorbed onto acidic cc-zirconium phosphate (ZP*) have been investigated. Cyclic voltammetry and rotating disc electrode measurements, performed in Tris buffer solution (pH 7) in absence and in presence of different NADH concentrations, showed that: (i) the formal standard potential (E degrees') of NIB was found pH dependent, according to the equation E = E degrees (c)-0.059pH + 0.029log(1 + 10(pH-pKa)), where the best fitting value for pK(a) Was estimated to be 4.3 +/- 0.4; (ii) the best electrocatalytic response was obtained for MB-ZP*-modified GCPEs having a ratio between the glassy carbon powder (Sigradur K) and MB-ZP* of 19:1; (iii) the presence of polyethyleneimine in the paste has a significant beneficial effect on the electrocatalytic activity of the investigated modified electrodes. As a general conclusion, it was stated that the particular features of the MB-ZP*-GCPEs are related to the ZP* crystallinity.
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| 12. |
- Ladiu, Carmen Ioana, et al.
(författare)
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NADH electrocatalytic oxidation at glassy carbon paste electrodes modified with Meldola blue adsorbed on alpha-titanium phosphate
- 2007
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Ingår i: Revista de Chimie. - 0034-7752. ; 58:5, s. 465-469
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Tidskriftsartikel (refereegranskat)abstract
- Basic electrochemistry of glassy carbon paste electrodes (GCPEs) modified with Meldola Blue adsorbed on crystalline a-titanium phosphate (MB-a-TP) electrodes and their ability to catalyze the NADH oxidation have been investigated. Various ratios of glassy carbon (Sigradur K) to MB-alpha-TP, were used to obtain GCPEs. Cyclic voltammetry (CV) and rotating disc electrode (RDE) measurements, performed in Tris buffer solution, showed that: (1) the MB formal potential vs. pH dependence is linear; (2) the addition of polyethyleneimine (PEI) to the paste electrode has a significant beneficial effect on the electrooxidation of NADH. As a general conclusion, it can be stated that GCPEs modified with MB-alpha-TP and incorporating 2.44% (w/w) PEI exhibits the best behavior for NADH electrocatalytic oxidation.
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| 13. |
- Munro, Keith, et al.
(författare)
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The high-pressure, high-temperature phase diagram of cerium
- 2020
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Ingår i: Journal of Physics. - : Institute of Physics (IOP). - 0953-8984 .- 1361-648X. ; 32:33
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Tidskriftsartikel (refereegranskat)abstract
- We present an experimental study of the high-pressure, high-temperature behaviour of cerium up to ~22 GPa and 820 K using angle-dispersive x-ray diffraction and external resistive heating. Studies above 820 K were prevented by chemical reactions between the samples and the diamond anvils of the pressure cells. We unambiguously measure the stability region of the orthorhombic oC4 phase and find it reaches its apex at 7.1 GPa and 650 K. We locate the α-cF4–oC4–tI2 triple point at 6.1 GPa and 640 K, 1 GPa below the location of the apex of the oC4 phase, and 1–2 GPa lower than previously reported. We find the α-cF4 → tI2 phase boundary to have a positive gradient of 280 K (GPa)−1, less steep than the 670 K (GPa)−1 reported previously, and find the oC4 → tI2 phase boundary to lie at higher temperatures than previously found. We also find variations as large as 2–3 GPa in the transition pressures at which the oC4 → tI2 transition takes place at a given temperature, the reasons for which remain unclear. Finally, we find no evidence that the α-cF4 → tI2 is not second order at all temperatures up to 820 K.
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| 14. |
- Muresan, Laura, et al.
(författare)
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Amine oxidase amperometric biosensor coupled to liquid chromatography for biogenic amines determination
- 2008
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Ingår i: MICROCHIMICA ACTA. - : Springer Science and Business Media LLC. - 0026-3672 .- 1436-5073. ; 163:3-4, s. 219-225
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Konferensbidrag (refereegranskat)abstract
- A selective and sensitive method is presented for biogenic amines (BA) determination. The novelty consists in coupling a highly selective electrochemical biosensor to a weak acid cation-exchange column for online detection of amines. A bienzyme design, based on a recently isolated amine oxidase from grass pea and commercial horseradish peroxidase, was used for the biosensor construction. The enzymes were co-immobilized on the surface of a graphite electrode together with the electrochemical mediator (Os-redox polymer). The electrochemical detection was performed at a low applied potential (-50 mV vs. Ag/AgCl, KCl0.1 M), where biases from interferences are minimal. The separation and determination of six BA, with relevance in food analysis (tyramine, putrescine, cadaverine, histamine, agmatine and spermidine), were investigated. Irrespective of the BA nature, the amine oxidase-based biosensor showed a linear response up to 5 mM, and its sensitivity decreases in the following order: cadaverine, putrescine, spermidine, agmatine, histamine and tyramine. The approach was used to estimate the BA content in fish samples, after their extraction with methanesulfonic acid.
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| 15. |
- Muresan, Laura, et al.
(författare)
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AMPEROMETRIC BIOSENSORS FOR GLUCOSE AND ETHANOL DETERMINATION IN WINE USING FLOW INJECTION ANALYSIS
- 2008
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Ingår i: Studia Universitatis Babes-Bolyai, Chemia. - 2065-9520. ; 53:1, s. 71-79
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Tidskriftsartikel (refereegranskat)abstract
- Reagentless amperometric biosensors for glucose and ethanol were developed and successfully applied for monitoring glucose and ethanol concentrations in wine during the fermentation process. The glucose biosensor was based on commercially available glucose oxidase and horseradish peroxidase co-immobilized on solid graphite using Os(II)-redox hydrogel (RH) [1]. In the case of ethanol biosensor, the quinohemoprotein dependent alcohol dehydrogenase was immobilized on the graphite electrode surface using the same RH [2]. Both biosensors were operated at low applied potentials (-50 mV vs. Ag/AgCl, KCl0.1M for glucose biosensor, and +250 mV vs. Ag/AgCl, KCl0.1M for ethanol biosensor), where biases from interferences are minimal. The bioelectroanalytical parameters, estimated from flow injection analysis measurements, were found as follows: sensitivity, 0.73 +/- 0.01 mu A mM(-1) for glucose and 0.45 +/- 0.01 mu A mM(-1) for ethanol; linear range up to 1 mM in both cases; detection limit, 7.0 mu M for glucose and 8.9 mu M for ethanol. The results for real samples were found in good agreement with those reported by Barsan et al. [3].
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| 16. |
- Muresan, Laura, et al.
(författare)
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Monitoring of glucose and glutamate using enzyme microstructures and scanning electrochemical microscopy.
- 2009
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Ingår i: Bioelectrochemistry. - : Elsevier BV. - 1878-562X .- 1567-5394. ; 76, s. 81-86
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Tidskriftsartikel (refereegranskat)abstract
- Glucose oxidase and glutamate oxidase lines, with typical width of 100 microm, were patterned on gold surfaces using a micro-dispensing system, by shooting 100 pl droplets of the corresponding enzyme solutions. The probe of a scanning electrochemical microscope (SECM) was then carefully positioned in the close proximity of the enzyme microstructure and poised to +600 mV vs. Ag/AgCl, KCl 0.1 M. The H(2)O(2), generated by the enzyme lines at different concentrations of glucose and glutamate in the surrounding solution, was sequentially monitored. Reproducible calibration curves for glucose and glutamate were obtained in one single experiment, proving that the combination of enzyme microstructures with SECM can provide a new way of achieving multianalyte detection.
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| 17. |
- Muresan, Laura, et al.
(författare)
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Reagentless Amperometric Biosensor For Nadh Detection
- 2009
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Ingår i: Revue Roumaine de Chimie. - 0035-3930. ; 54:9, s. 755-760
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Tidskriftsartikel (refereegranskat)abstract
- A sensitive and selective amperometric biosensor, based on NADH-oxidase (NADOx) working in tandem with horseradish peroxidase (HRP), which is electrically connected to graphite electrode via Os-redox polymer (RP), was developed. Two designs were considered for the biosensor optimization: (i) the first one, [G/HRP-NADOx], exploited the direct electron transfer between HRP and the graphite electrode (G); (ii) the second one, [G/RP-HR-P-NADOx], used R-P as mediator. In both cases the enzyme matrix was cross-linked using poly(ethylenglycol) diglycidyl ether. The biosensors sensitivities, estimated as the slopes of linear ranges from the calibration curves, recorded in a single line flow injection setup, showed that G/RP-HRP-NADOx electrode (1.62 +/- 0.05 mA M-1) presents a higher efficiency than G/HRP-NADOx electrode (0.30 +/- 0.03 mA M-1).
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| 18. |
- Yakovleva, Maria, et al.
(författare)
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Further Insights into the Catalytical Properties of Deglycosylated Pyranose Dehydrogenase from Agaricus meleagris Recombinantly Expressed in Pichia pastoris
- 2013
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 85:20, s. 9852-9858
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Tidskriftsartikel (refereegranskat)abstract
- The present study focuses on fragmented deglycosylated pyranose dehydrogenase (fdgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris. Fragmented deglycosylated PDH is formed from the deglycosylated enzyme (dgPDH) when it spontaneously loses a C-terminal fragment when stored in a buffer solution at 4 degrees C. The remaining larger fragment has a molecular weight of similar to 46 kDa and exhibits higher volumetric activity for glucose oxidation compared with the deglycosylated and glycosylated (gPDH) forms of PDH. Flow injection amperometry and cyclic voltammetry were used to assess and compare the catalytic activity of the three investigated forms of PDH, "wired" to graphite electrodes with two different osmium redox polymers: [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(poly(vinylimidazole))(10)Cl](+) [Os(dmbpy)PVI] and [Os(4,4'-dimethoxy-2,2'-bipyridine)(2)(poly-(vinylimidazole))(10)Cl](+) [Os(dmobpy)PVI]. When "wired" with Os(dmbpy)PVI, the graphite electrodes modified with fdgPDH showed a pronounced increase in the current density with J(max). 13- and 6-fold higher than that observed for gPDH- and dgPDH-modified electrodes, making the fragmented enzyme extraordinarily attractive for further biotechnological applications. An easier access of the substrate to the active site and improved communication between the enzyme and mediator matrix are suggested as the two main reasons for the excellent performance of the fdgPDH when compared with that of gPDH and dgPDH. Three of the four glycosites in PDH: N-75, N-175, and N-252 were assigned using mass spectrometry in conjunction with endoglycosidase treatment and tryptic digestion. Determination of the asparagine residues carrying carbohydrate moieties in PDH can serve as a solid background for production of recombinant enzyme lacking glycosylation.
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| 19. |
- Yakovleva, Maria, et al.
(författare)
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Recombinant pyranose dehydrogenase-A versatile enzyme possessing both mediated and direct electron transfer
- 2012
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Ingår i: Electrochemistry Communications. - : Elsevier BV. - 1388-2481. ; 24, s. 120-122
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Tidskriftsartikel (refereegranskat)abstract
- The catalytical properties of glycosylated pyranose dehydrogenase (gPDH) and deglycosylated PDH (dgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris were studied. Both gPDH and dgPDH were "wired" to an osmium redox polymer on graphite electrodes mounted in a flow-injection system. The current from oxidation of glucose by immobilised gPDH and dgPDH was compared using flow injection amperometry and cyclic voltammetry. An increase in the current density was observed for dgPDH (190 mu A cm(-2)) compared with that for gPDH (90 mu A cm(-2)) due to the improved electron transfer between the active site and the electrode. Additionally, the ability of dgPDH for direct electron transfer (DET) was discovered, which is rather unique among FAD-containing enzymes. The ability to oxidise a variety of sugars at a rather low potential makes dgPDH attractive for construction of biofuel cells with high power output. (C) 2012 Elsevier B.V. All rights reserved.
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