| 1. |
- Alpeeva, IS, et al.
(author)
-
Bi-enzyme alcohol biosensors based on genetically engineered alcohol oxidase and different peroxidases
- 2005
-
In: Microchimica Acta. - : Springer Science and Business Media LLC. - 1436-5073 .- 0026-3672. ; 152:1-2, s. 21-27
-
Journal article (peer-reviewed)abstract
- We report on the development of a bi-layer bi-enzyme biosensor architecture using different peroxidases and alcohol oxidase from Hansenula polymorpha C-105 as biological recognition elements. The sensor architecture comprises a first layer containing either horseradish peroxidase or royal palm tree peroxidase crosslinked with an Osmium complex-modified redox hydrogel. On top, a second layer was formed by electrochemically induced precipitation of a cathodic electrodeposition paint simultaneously entrapping alcohol oxidase isolated from a genetically modified strain of Hansenula polymorpha C-105. The sensor architecture was optimized with respect to effective electron transfer and stability of the enzyme. The main characteristics of the biosensors are an apparent maximal current I-max(app) of 572-940 nA, an apparent Michaelis constant K-M(app) of 9.5 mM, a sensitivity of 60-98 nA mM(-1) and an improved operational stability represented by a deactivation constant of 1.5-2.0 x 10(-4) min(-1).
|
|
| 2. |
- Alpeeva, I, et al.
(author)
-
Cyclometalated Ruthenium(II) Complexes As Efficient Redox Mediators in Peroxidase Catalysis
- 2003
-
In: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 1432-1327 .- 0949-8257. ; 8:6, s. 683-688
-
Journal article (peer-reviewed)abstract
- Cyclometalated ruthenium(II) complexes, [Ru II(C~N)(N~N) 2]PF 6 [HC~N=2-phenylpyridine (Hphpy) or 2-(4'-tolyl)pyridine; N~N=2,2'-bipyridine, 1,10-phenanthroline, or 4,4'-dimethyl-2,2'-bipyridine], are rapidly oxidized by H 2O 2 catalyzed by plant peroxidases to the corresponding Ru III species. The commercial isoenzyme C of horseradish peroxidase (HRP-C) and two recently purified peroxidases from sweet potato (SPP) and royal palm tree (RPTP) have been used. The most favorable conditions for the oxidation have been evaluated by varying the pH, buffer, and H 2O 2 concentrations and the apparent second-order rate constants ( k app) have been measured. All the complexes studied are oxidized by HRP-C at similar rates and the rate constants k app are identical to those known for the best substrates of HRP-C (10 6–10 7 M -1 s -1). Both cationic (HRP-C) and anionic (SPP and RPTP) peroxidases show similar catalytic efficiency in the oxidation of the Ru II complexes. The mediating capacity of the complexes has been evaluated using the SPP-catalyzed co-oxidation of [Ru II(phpy)(bpy) 2]PF 6 and catechol as a poor peroxidase substrate as an example. The rate of enzyme-catalyzed oxidation of catechol increases more than 10,000-fold in the presence of the ruthenium complex. A simple routine for calculating the rate constant k c for the oxidation of catechol by the Ru III complex generated enzymatically from [Ru II(phpy)(bpy) 2] + is proposed. It is based on the accepted mechanism of peroxidase catalysis and involves spectrophotometric measurements of the limiting Ru II concentration at different concentrations of catechol. The calculated k c value of 0.75 M -1 s -1 shows that the cyclometalated Ru II complexes are efficient mediators in peroxidase catalysis.
|
|
| 3. |
- Alpeeva, IS, et al.
(author)
-
Palm tree peroxidase-based biosensor with unique characteristics for hydrogen peroxide monitoring
- 2005
-
In: Biosensors & Bioelectronics. - : Elsevier BV. - 1873-4235 .- 0956-5663. ; 21:5, s. 742-748
-
Journal article (peer-reviewed)abstract
- Three amperometric enzyme electrodes have been constructed by adsorbing anionic royal palm tree peroxidase (RPTP), anionic sweet potato peroxidase (SPP), or cationic horseradish peroxidase (HRP-C) on spectroscopic graphite electrodes. The resulting H2O2-sensitive biosensors were characterized both in a flow injection system and in batch mode to evaluate their main bioelectrochemical parameters, such as pH dependency,I-max, K-M(app), detection limit, linear range, operational and storage stability. The obtained results showed a distinctly different behavior for the plant peroxidase electrodes, demonstrating uniquely superior characteristics of the RPTP-based sensors. The broader linear range observed for the RPTP-based biosensor is explained by a high stability of this enzyme in presence of H2O2. The higher storage and operational stability of RPTP-based biosensor as well as its capability to measure hydrogen peroxide under acidic conditions connect with an extremely high thermal and pH-stability of RPTP.
|
|
| 4. |
- Bontidean, Ibolya, et al.
(author)
-
Biosensors for detection of mercury in contaminated soils
- 2004
-
In: Environmental Pollution. - : Elsevier BV. - 0269-7491. ; 131:2, s. 255-262
-
Journal article (peer-reviewed)abstract
- Biosensors based on whole bacterial cells and on bacterial heavy metal binding protein were used to determine the mercury concentration in soil. The soil samples were collected in a vegetable garden accidentally contaminated with elemental mercury 25 years earlier. Bioavailable mercury was measured using different sensors: a protein-based biosensor, a whole bacterial cell based biosensor, and a plant sensor, i.e. morphological and biochemical responses in primary leaves and roots of bean seedlings grown in the mercury-contaminated soil. For comparison the total mercury concentration of the soil samples was determined by AAS. Whole bacterial cell and protein-based biosensors gave accurate responses proportional to the total amount of mercury in the soil samples. On the contrary, plant sensors were found to be less useful indicators of soil mercury contamination, as determined by plant biomass, mercury content of primary leaves and enzyme activities. (C) 2004 Elsevier Ltd. All rights reserved.
|
|
| 5. |
- Bontidean, Ibolya, et al.
(author)
-
Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection
- 2003
-
In: Biosensors & Bioelectronics. - 1873-4235. ; 18:5-6, s. 547-553
-
Journal article (peer-reviewed)abstract
- A novel capacitance biosensor based on synthetic phytochelatins for sensitive detection of heavy metals is described. Synthetic phytochelatin (Glu-Cys)20Gly (EC20) fused to the maltose binding domain protein was expressed in Escherichia coli and purified for construction of the biosensor. The new biosensor was able to detect Hg2+, Cd2+, Pb2+, Cu2+ and Zn2+ ions in concentration range of 100 fM–10 mM, and the order of sensitivity was SZn>SCu>SHg>>SCdSPb. The biological sensing element of the sensor could be regenerated using EDTA and the storage stability of the biosensor was 15 days.
|
|
| 6. |
- Castillo Leon, Jaime, et al.
(author)
-
Bienzyme biosensors for glucose, ethanol and putrescine built on oxidase and sweet potato peroxidase
- 2003
-
In: Biosensors & Bioelectronics. - 1873-4235. ; 18:5-6, s. 705-714
-
Journal article (peer-reviewed)abstract
- Amperometric biosensors for glucose, ethanol, and biogenic amines (putrescine) were constructed using oxidase/peroxidase bienzyme systems. The H2O2 produced by the oxidase in reaction with its substrate is converted into a measurable signal via a novel peroxidase purified from sweet potato peels. All developed biosensors are based on redox hydrogels formed of oxidases (glucose oxidase, alcohol oxidase, or amine oxidase) and the newly purified sweet potato peroxidase (SPP) cross-linked to a redox polymer. The developed electrodes were characterized (sensitivity, stability, and performances in organic medium) and compared with similarly built ones using the 'classical' horseradish peroxidase (HRP). The SPP-based electrodes displayed higher sensitivity and better detection limit for putrescine than those using HRP and were also shown to retain their activity in organic phase much better than the HPR based ones. The importance of attractive or repulsive electrostatic interactions between the peroxidases and oxidases (determined by their isoelectric points) were found to play an important role in the sensitivity of the obtained sensors
|
|
| 7. |
- Castillo Leon, Jaime, et al.
(author)
-
Biosensors for life quality - Design, development and applications
- 2004
-
In: Sensors and Actuators B: Chemical. - : Elsevier BV. - 0925-4005. ; 102:2, s. 179-194
-
Journal article (peer-reviewed)abstract
- Biosensors, combining a selective biological recognition element and a sensitive transducer, are versatile analytical tools applied more and more in different fields, such as medicine, food quality and safety control, and environment pollution monitoring. They are expected to play an increasingly important role in the improvement of life quality. In this context, the present work covers recent approaches in design and development of biosensors applied for analysis of real samples of medical, environmental or industrial relevance. The described sensors meet the sensitivity, selectivity, and response time required by their applications. Moreover, they are designed to avoid contamination of the monitored systems with undesirable components and to minimise the damage of living organisms (when it comes to in vivo monitoring). (C) 2004 Elsevier B.V. All rights reserved.
|
|
| 8. |
- Castillo Leon, Jaime, et al.
(author)
-
Glutamate detection from nerve cells using a planar electrodes array integrated in a microtiter plate
- 2005
-
In: Biosensors & Bioelectronics. - : Elsevier BV. - 1873-4235 .- 0956-5663. ; 20:10, s. 2116-2119
-
Journal article (peer-reviewed)abstract
- There is an increasing interest in new strategies for replacing animal tests in research. The use of cell cultures and integrated electrodes is seen as a promising alternative that could potentially solve this problem. In this work, we present a L-glutamate sensor based on a bienzyme redox hydrogel, capable of detecting the release of this excitatory neurotransmitter from adherently growing cells upon stimulation. The low working potential required for the operation of the sensor decreases the possibility of interference by easily oxidizable compounds always present in complex biological samples. A low detection limit of 0.5 mu M L-glutamate, a response time of about 35 s, and a linear range of up to 60 mu M are the main characteristics of the sensor. The system has been successfully employed to monitor the release of L-glutamate from HN10 and C6 cells upon stimulation with K+-ions. The developed integrated electrochemical platform will be used in future for drug screening and potentially for replacing animal models in neurological experiments.
|
|
| 9. |
- Castillo Leon, Jaime, et al.
(author)
-
Purification and substrate specificity of peroxidase from sweet potato tubers
- 2002
-
In: Plant Science. - 0168-9452. ; 163:5, s. 1011-1019
-
Journal article (peer-reviewed)abstract
- Previously the screening of tropical plants demonstrated a high peroxidase activity in sweet potato (Ipomoea batatas) tubers. The major peroxidase pool is localized in peel. Using peel of sweet potato as a source, the sweet potato peroxidase (SPP) has been isolated and purified to homogeneity. The enzyme purification included homogenization, extraction of colored compounds and consecutive chromatographies on Phenyl-Sepharose and DEAE-Toyopearl. The purified SPP had specific activity of 4900 U mg(-1) protein, RZ (ratio of absorbances at 403 and 280 nm, respectively) 3.4, molecular mass of 37 kDa and isoelectric point of 3.5. The spectrum of peroxidase from sweet potato is typical for plant peroxidases with a Soret maximum at 401 nm and the maxima in the visible region at 497 and 638 nm, respectively. The substrate specificity of SPP is distinct from the specificity of other plant peroxidases, ferulic acid being the best substrate for SPP.
|
|
| 10. |
- Castillo Leon, Jaime, et al.
(author)
-
Simultaneous detection of the release of glutamate and nitric oxide from adherently growing cells using an array of glutamate and nitric oxide selective electrodes
- 2005
-
In: Biosensors & Bioelectronics. - : Elsevier BV. - 1873-4235 .- 0956-5663. ; 20:8, s. 1559-1565
-
Journal article (peer-reviewed)abstract
- The simultaneous detection of nitric oxide and glutamate using an array of individually addressable electrodes, in which the individual electrodes in the array were suitably modified with a highly sensitive nitric oxide sensing chemistry or a glutamate oxidase/redox hydrogel-based glutamate biosensor is presented. In a sequence of modification steps one of the electrodes was covered first with a positively charged Ni porphyrin entrapped into a negatively charged electrodeposition paint followed by the manual modification of the second working electrode by a bienzyme sensor architecture based on crosslinked redox hydrogels with entrapped peroxidase and glutamate oxidase. Adherently growing C6-glioma cells were grown on membrane inserts and placed in close distance to the modified sensor surfaces. The current responses recorded at each electrode after stimulation of glutamate and NO release by means of K+ and bradykinin clearly demonstrate the ability of the individual electrode in the array to detect the analyte towards which its sensitivity and selectivity was targeted without interference from the neighbouring electrode or other analytes present in the test mixture.
|
|
