| 1. |
- Bollella, Paolo, et al.
(författare)
-
Highly sensitive, stable and selective hydrogen peroxide amperometric biosensors based on peroxidases from different sources wired by Os-polymer : A comparative study
- 2018
-
Ingår i: Solid State Ionics. - : Elsevier BV. - 0167-2738. ; 314, s. 178-186
-
Tidskriftsartikel (refereegranskat)abstract
- A comparison was made between two plant peroxidases, cationic horseradish peroxidase (HRP) and anionic tobacco peroxidase (TOP), combined with a highly cationic osmium polymer [Os(4,4'-dimethyl-2,2'-bipyridine)2poly(N-vinylimidazole)10Cl]+2/+ ([Os(dmp)PVI]+/2+) to develop highly sensitive, stable and selective hydrogen peroxide biosensors. The two different plant peroxidases were individually immobilized onto graphite rod (G) electrodes by a three steps drop-casting procedure consisting of the subsequent deposition of an aqueous solution of ([Os(dmp)PVI]+/2+), followed by a solution of poly(ethyleneglycol) diglycidyl ether (PEGDGE), used as a cross linking agent and finally an aliquot of a solution of cationic HRP or anionic TOP to make HRP/PEGDGE/[Os(dmp)PVI]+/2+/G and TOP/PEGDGE/[Os(dmp)PVI]+/2+/G based electrodes, respectively. Electrochemical experiments were carried out to investigate the influence of the surface charge of the enzyme and the charge of the polymer on the efficiency of the electron transfer (ET) between the enzyme and the wiring redox polymer and the efficiency for electrocatalytic reduction of H2O2. In the case of HRP a decrease in the ET rate was observed due to the repulsion between this enzyme and the polymer, both positively charged, whereas with TOP there was an enhanced ET rate due to the attraction between the anionic enzyme and the cationic polymer. The effects of enzyme loading and pH were investigated. Both peroxidase modified electrodes exhibited a wide dynamic response range (1-500μM H2O2) and a low detection limit (0.3μM H2O2). The TOP based electrode showed a higher sensitivity (470nAμM-1 cm-2) compared to that of the HRP based electrode (300nAμM-1 cm-2) and an improved long-term stability (decrease in 17.3% upon 30days compared with 50% for HRP). Both enzyme electrodes showed a response time of 3s. The HRP based sensor was more sensitive to the presence of phenolic compounds acting as alternative electron donors, whereas the TOP based sensor was virtually interference free. Both HRP and TOP based electrodes were successfully tested in contact lens cleaning samples and real "spiked" samples from different sources such as tap water, milk and dairy products.
|
|
| 2. |
- Ciogli, Leonardo, et al.
(författare)
-
Highly Sensitive Hydrogen Peroxide Biosensor Based on Tobacco Peroxidase Immobilized on p-Phenylenediamine Diazonium Cation Grafted Carbon Nanotubes : Preventing Fenton-like Inactivation at Negative Potential
- 2021
-
Ingår i: ChemElectroChem. - : Wiley. - 2196-0216. ; 8:13, s. 2495-2504
-
Tidskriftsartikel (refereegranskat)abstract
- Herein, we present a novel electrode platform for H2O2 detection based on the immobilization of recombinant Tobacco Peroxidase (r-TOP) onto graphite electrodes (G) modified with p-phenylenediamine (p-PD) diazonium cation grafted multi-walled carbon nanotubes (MWCNTs). The employment of both p-phenylenediamine moieties and covalent cross-linking by using glutaraldehyde allowed us to enhance the sensitivity, stability, and selectivity toward H2O2 detection, as well as preventing enzyme inactivation due to the electro-Fenton reaction. This reaction continuously produces hydroxyl radicals, whose high and unselective reactivity is likely to reduce drastically the operating life of the biosensor. The protection against the electro-Fenton reaction is mainly ascribed to a beneficial enzyme immobilization leading to a correct orientation achieved through cross-linking the enzyme in combination with interaction between the uncoupled -NH2 groups (mainly uncharged at pH 7, considering a pKa of 4.6) available on the electrode surface and the enzyme. In particular, the electrode based on the r-TOP/p-PD/MWCNTs/G platform showed a lower limit of detection of 1.8 μM H2O2, an extended linear range between 6 and 900 μM H2O2, as well as a significant increase in sensitivity (63.1±0.1 μA mM−1 cm−2) compared with previous work based on TOP. Finally, the r-TOP/p-PD/MWCNTs/G electrode was tested in several H2O2 spiked food samples as a screening analytical method for the detection of H2O2.
|
|
| 3. |
- Olloqui-Sariego, José Luis, et al.
(författare)
-
Influence of tryptophan mutation on the direct electron transfer of immobilized tobacco peroxidase
- 2020
-
Ingår i: Electrochimica Acta. - : Elsevier BV. - 0013-4686. ; 351
-
Tidskriftsartikel (refereegranskat)abstract
- A major challenge in the design of electrochemical biodevices is to achieve fast rates of electron exchange between proteins and electrodes. In this work, we show that a significant increase in the direct electron transfer rate between a graphite electrode and Tobacco Peroxidase takes place when a surface exposed leucine, located in the vicinity of the heme pocket, is replaced by tryptophan. The analysis of the Fe(III)/Fe(II) voltammetric responses of native and mutated proteins, as a function of solution pH and temperature, leads to similar values of the reduction entropy and reorganization energy, but to a higher electronic coupling in the case of the mutant. In addition, the mutated and native proteins are shown to display similar electrocatalytic activities to reduce hydrogen peroxide at positive potentials, indicating that the molecular structure of the heme pocket is largely unaffected by the mutation.
|
|
| 4. |
- Olloqui-Sariego, José Luis, et al.
(författare)
-
Interprotein Coupling Enhances the Electrocatalytic Efficiency of Tobacco Peroxidase Immobilized at a Graphite Electrode.
- 2015
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 87:21, s. 10807-10814
-
Tidskriftsartikel (refereegranskat)abstract
- Covalent immobilization of enzymes at electrodes via amide bond formation is usually carried out by a two-step protocol, in which surface carboxylic groups are first activated with the corresponding cross-coupling reagents and then reacted with protein amine groups. Herein, it is shown that a modification of the above protocol, involving the simultaneous incubation of tobacco peroxidase and the pyrolytic graphite electrode with the cross-coupling reagents produces higher and more stable electrocatalytic currents than those obtained with either physically adsorbed enzymes or covalently immobilized enzymes according to the usual immobilization protocol. The remarkably improved electrocatalytic properties of the present peroxidase biosensor that operates in the 0.3 V ≤ E ≤ 0.8 V (vs SHE) potential range can be attributed to both an efficient electronic coupling between tobacco peroxidase and graphite and to the formation of intra- and intermolecular amide bonds that stabilize the protein structure and improve the percentage of anchoring groups that provide an adequate orientation for electron exchange with the electrode. The optimized tobacco peroxidase sensor exhibits a working concentration range of 10-900 μM, a sensitivity of 0.08 A M(-1) cm(-2) (RSD 0.05), a detection limit of 2 μM (RSD 0.09), and a good long-term stability, as long as it operates at low temperature. These parameter values are among the best reported so far for a peroxidase biosensor operating under simple direct electron transfer conditions.
|
|
| 5. |
- Olloqui-Sariego, José Luis, et al.
(författare)
-
The Fe (III)/Fe(II) redox couple as a probe of immobilized tobacco peroxidase : Effect of the immobilization protocol
- 2019
-
Ingår i: Electrochimica Acta. - : Elsevier BV. - 0013-4686. ; 299, s. 55-61
-
Tidskriftsartikel (refereegranskat)abstract
- Non-turnover voltammetry is a sensitive tool to characterize the electrochemical properties of redox proteins. However, the catalytically competent oxidation states of most peroxidases do not display the required electrochemical reversibility. In this report, we circumvent this limitation and exploit the voltammetric response associated with the Fe(III)/Fe(II) redox couple of tobacco peroxidase to probe the energetics and electronic connectivity of the heme pocket. We have applied this approach to rationalize the previously reported influence of the immobilization protocol on the electrocatalytic activity of tobacco peroxidase. To decouple proton and electron transfer steps, measurements have been carried out over the 3 ≤ pH ≤ 9 range and a 1e−/2H+ ladder scheme has been adopted for their analysis. At each pH, thermodynamic and kinetic parameters associated with the Fe(III)/Fe(II) redox conversion were determined as a function of temperature in the 0-30 °C range. Reduction entropies and reorganization energies displayed different values for covalently immobilized and physisorbed enzymes, pointing to a larger involvement of the solvent in the last case. These findings, together with a larger electronic coupling between the prosthetic group and the electrode, are indicative of a partial denaturation of the physisorbed enzymes as the origin of their lower electrocatalytic activity.
|
|
