Szilveszter Gáspár

Biosensors based on novel plant peroxidases: a comparative study

Abstract Amperometric biosensors for hydrogen peroxide detection have been constructed using horseradish peroxidase (HRP) and two newly purified peroxidases extracted from tobacco (TOP) and sweet potato (SPP). The peroxidases were cross-linked to a redox polymer [poly(vinylimidazole) complexed with Os(4,4′dimethylbipyridine) 2 Cl] using poly(ethylene glycol) diglycidyl ether as the cross-linker. A comparative study with regard to their bioelectrochemical characteristics showed that, irrespective of peroxidase, the biosensors sensitivity was strongly influenced by hydrogel composition, curing procedure, film thickness and applied potential. The electrostatic interaction between the cationic redox polymer and the negatively charged peroxidases (TOP and SPP) enhanced the hydrogen peroxide signal. When operated in a FI system, the optimized SPP biosensor (48% redox polymer, 23% cross-linker and 29% enzyme, w/w %) displayed the highest sensitivity for H 2 O 2 (3.2 A M −1 cm −2 ), a linear range up to 220 μM, a detection limit of 25 nM (calculated as 2S/N) and a response time of about 2 min.

Hydrogen peroxide sensitive biosensor based on plant peroxidases entrapped in Os-modified polypyrrole films

Abstract An amperometric hydrogen peroxide biosensor was designed based on horseradish and tobacco peroxidase entrapped into a conducting redox-polymer immobilised on either glassy-carbon or platinum electrodes. A versatile one-step immobilisation method was carried out based on the electrochemical polymerisation of a pyrrole monomer functionalised with an Os-complex. Cyclic voltammetry and constant potential amperometry performed with the different peroxidases in solution or entrapped within the conducting redox-polymer film suggests that the redox centre within the active site of horseradish peroxidase exhibits a better accessibility for the either free-diffusing or polymer-bound Os-complexes than that of tobacco peroxidase. Therefore, the obtained sensitivities for the reduction of H 2 O 2 are significantly higher for the HRP-based sensors as compared with the tobacco peroxidase-based ones. The direct reduction of H 2 O 2 on the polymer backbone was identified as a side reaction even though the bioelectroreduction through horseradish peroxidase is a much more efficient reaction pathway.

Hydrogen Peroxide Biosensors Based on Direct Electron Transfer from Plant Peroxidases Immobilized on Self-Assembled Thiol-Monolayer Modified Gold Electrodes

A recently characterized tobacco peroxidase (TOP) has been adsorbed on differently modified mixed alkylthiol monolayers, and the direct electron transfer between the enzyme and the monolayer-modified electrode has been investigated. The comparison of the electrocatalytic activities of adsorbed TOP and horseradish peroxidase (HRP) showed no efficient direct electron transfer for HRP, whereas a significant electrocatalytic current could be observed for TOP in the presence of hydrogen peroxide. The use of differently charged monolayers suggests that the electron-transfer rate of the electrocatalytic reduction of hydrogen peroxide is dependent on the orientation of the adsorbed tobacco peroxidase, probably due to electrostatic interactions between charged head groups at the monolayer and the protein shell.

Development of a label-free aptasensor for monitoring the self-association of lysozyme

A novel aptamer and surface plasmon resonance (SPR)-based sensor was developed for the label-free detection of lysozyme. The aptasensor is characterised by a detection limit of 1 μg mL(-1) and a linear range of 5-50 μg mL(-1). As an application, we examined the usefulness of the aptasensor for monitoring the early stages of the aggregation of lysozyme. It was surprisingly found that, despite a significant decrease in monomer content during aggregation, the response of the aptasensor for protein solutions aged for 12 hours was similar to that for the fresh protein. To correlate the results obtained with the aptasensor with the composition of lysozyme solutions at various time points, we examined them in detail by atomic force microscopy (AFM), thioflavin T fluorescence, size-exclusion chromatography (SEC) and Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF-MS). All methods together indicated that during the initial hours of aggregation, the protein solutions contained small lysozyme oligomers (mainly dimers) and decreasing amounts of monomers. Our results thus suggest that the aptamer also recognizes lysozyme dimers/oligomers. A higher non-specific binding was observed for the aggregated lysozyme at the surface of the aptasensor as compared to the native protein. This was attributed to the hydrophobic patches which are exposed by the unfolded lysozyme and/or oligomer species, allowing for different adsorption and organisation at the surface of the aptasensor. This hypothesis is supported by square wave voltammetry (SWV) studies using solutions of aggregated lysozyme. A higher electrochemical signal due to the direct oxidation of tyrosine/tryptophan residues was observed for aged protein solutions as compared to the fresh solution, indicative of an increased number of such exposed electroactive residues and of overall increased surface hydrophobicity of the protein. Our work presents a label-free lysozyme aptasensor that is useful not only for the detection of the protein monomer but also for observing the onset of aggregation. The approach can be extended to other proteins which are prone to aggregation.

PFeW11-doped polymer film modified electrodes and their electrocatalytic activity for H2O2 reduction

A study of electrochemical and electrocatalytic properties, toward H 2 O 2 reduction, of the [PFeW 11 O 39 ] -4 polyoxoanion in solution as well as immobilized in partially oxidized polypyrrole (pPy) and in polyvinyl alcohol bearing styrylpyridinium groups (PVA) films is reported. Using cyclic voltammetry at different pH values and scanning rates it was observed that: (i) the two bielectronic waves corresponding to the tungstate-oxo cage are pH-dependent ; (ii) the Fe center of the polyoxoanion has an excellent electrocatalytic effect on H 2 O 2 reduction, giving a catalytic efficiency and an electrode sensitivity, both higher when dissolved than when immobilized onto electrode surface; and (iii) the one-step method used to obtain the pPy-doped film assures a greater amount of immobilized polyoxoanion than the ionic-exchange method used for PVA films. From rotating-disk electrode (RDE) measurements, the polyoxoanion diffusion coefficient was estimated at 4. 1x10 -6 cm 2 s -1 , which agrees to that obtained by cyclic voltammetry, 3.9x10 -6 cm 2 s -1 , and the heterogeneous rate constant was found to vary between 3.5 x 10 -3 and 1.9 x10 -3 cm s -1 at a variation of the electrode potential from 30 to -30 mV vs. SCE. The best amperometric response observed for the PFeW 11 -doped pPy membrane, showed a linear response to H 2 O 2 additions up to 9 mM and a sensitivity of 4.8 mA M -1 H 2 O 2 in the first day of utilization.

Effect of calcium oxalate on renal cells as revealed by real-time measurement of extracellular oxidative burst

Calcium oxalate is one of the main constituents of kidney stones and has a proved deleterious effect on renal cells that is mediated by oxidative stress. However, the subcellular source of this oxidative stress, and whether it is extending to the extracellular space or not, is still disputed. Therefore, an electrochemical superoxide biosensor was constructed, positioned above A6 renal cells, and used to measure in real-time the extracellular oxidative burst following addition of calcium oxalate crystals. It was observed that A6 cells do secrete superoxide into their extracellular space in few minutes after encountering calcium oxalate crystals. The amount of released superoxide peaks at about 20 min. Superoxide is cleared away from the extracellular space after approximately 3h. Superoxide secretion depends on the presence of superoxide-scavenging enzyme superoxide dismutase, the age of the cells, the amount of calcium oxalate crystals, and the temperature. Moreover, the effect of calcium oxalate crystals was mimicked by phorbol 12-myristate 13-acetate. The developed sensing system can be a useful tool for biologists investigating nephrolithiasis at cellular level.

Surface Plasmon Resonance based sensing of lysozyme in serum on Micrococcus lysodeikticus-modified graphene oxide surfaces

Lysozyme is an enzyme found in biological fluids, which is upregulated in leukemia, renal diseases as well as in a number of inflammatory gastrointestinal diseases. We present here the development of a novel lysozyme sensing concept based on the use of Micrococcus lysodeikticus whole cells adsorbed on graphene oxide (GO)-coated Surface Plasmon Resonance (SPR) interfaces. M. lysodeikticus is a typical enzymatic substrate for lysozyme. Unlike previously reported sensors which are based on the detection of lysozyme through bioaffinity interactions, the bioactivity of lysozyme will be used here for sensing purposes. Upon exposure to lysozyme containing serum, the integrity of the bacterial cell wall is affected and the cells detach from the GO based interfaces, causing a characteristic decrease in the SPR signal. This allows sensing the presence of clinically relevant concentrations of lysozyme in undiluted serum samples.

Magneto-plasmonic biosensor with enhanced analytical response and stability.

We present novel solutions to surpass current analytic limitations of Magneto-Optical Surface Plasmon Resonance (MOSPR) assays, concerning both the chip structure and the method for data analysis. The structure of the chip is modified to contain a thin layer of Co-Au alloy instead of successive layers of homogeneous metals, as currently used. This alloy presents improved plasmonic and magnetic properties, yet a structural stability similar to Au-SPR chips, allowing for bioaffinity assays in saline solutions. Analyzing the whole reflectivity curve at multiple angles of incidence instead of the reflectivity value at a single incidence angle provides a high signal-to-noise ratio suitable for detection of minute analyte concentrations. Based on assessment of solutions with known refractive indices as well as of a model biomolecular interaction (i.e. IgG-AntiIgG) we demonstrate that the proposed structure of the MOSPR sensing chip and the procedure of data analysis allows for long-time assessment in liquid media with increased sensitivity over standard SPR analyses.

Modification with hemeproteins increases the diffusive movement of nanorods in dilute hydrogen peroxide solutions

Nanorods were decorated with different hemeproteins that are able to convert hydrogen peroxide. When dispersed into hydrogen peroxide solutions, most of these nanorods are characterized by diffusion coefficients which increase with the concentration of hydrogen peroxide. Such a behaviour does not characterize unmodified nanorods.

DESIGN, VISUALIZATION, AND UTILIZATION OF ENZYME MICROSTRUCTURES BUILT ON SOLID SURFACES

Abstract in Undetermined Immobilized bioelements offer additional sensitivity and/or selectivity in analytical techniques, in biotechnological devices, and in different medical applications. Great research interest has recently been focused in all these fields on miniaturization techniques, special attention being accorded to the patterning of biomolecules (e.g. enzymes) on solid surfaces with micrometer resolution. Novel microanalysis systems consider the use of enzyme microstructures as e.g. part of mu- Total Analysis System platforms as biochemical microreactors or detection units. This mini-review highlights recent advances in the creation of enzyme microstructures by indirect methods (photolithography, microcontact printing. etc.) and active placement methods (electrospraying, microdispensing, etc.). Some key visualization techniques of enzyme microstructures (fluorescence microscopy, scanning electrochemical microscopy, etc.) are also mentioned together with examples of their applications or application possibilities. (Less)