Direct electrochemistry of recombinant tobacco peroxidase on gold

Direct electron transfer (DET) reactions of recombinant tobacco peroxidase (rTOP), namely direct electroreduction of Compound I/Compound II and heme Fe^3+/2+ conversion, were studied on gold electrodes. rTOP of wild type, non-glycosylated, was produced using an Escherichia coli expression system. At pH 5.0, the redox potential for direct electrochemical transformation of the Fe^3+/2+ of the peroxidase heme was −143 mV vs. AgAgCl, and 0.26 ± 0.07 pmol of the adsorbed rTOP were in DET contact with the gold electrode. The total amount of the adsorbed rTOP estimated from QCM data was 53 ± 5 pmol/cm² or 1.67 pmol when referred to the surface area of the electrodes used for electrochemical measurements. Of 1.67 pmol of adsorbed rTOP, only 0.76 pmol were catalytically active. DET between Au and the enzyme was also studied in the reaction of the bioelectrocatalytic reduction of H₂O₂ by cyclic voltammetry and amperometric detection of H₂O₂ at +50 mV with rTOP-modified Au electrodes placed in a wall-jet flow-through electrochemical cell. Maximal bioelectrocatalytic current response of the rTOP-modified gold electrodes to H₂O₂ was observed at pH 5.0 and stemmed from its bioelectrocatalytic reduction based on DET between Au and the active site of rTOP. Kinetic analysis of the DET reactions gave 52% of the adsorbed rTOP molecules active in DET reactions (0.4 pmol of adsorbed catalytically active rTOP, correspondingly), which correlated well with the non-catalytic-voltammetry data. DET was characterised by a heterogeneous ET rate constant of 13.2 s⁻¹, if one takes into account the QCM data, and 19.6 s⁻¹, if the amount of rTOP estimated from the data on DET transformation of Fe^3+/2+ couple of rTOP is considered. The sensitivity for H₂O₂ obtained for the rTOP-modified Au electrodes was 0.7 ± 0.1 A M⁻¹ cm⁻². These are the first ever-reported data on DET reactions of anionic plant peroxidases on bare gold electrodes.     

Heat resistance of composite sorption-active materials based on zeolite and fluoroethylene polymers

Relationship was found between the temperature of thermal degradation of composite sorption-active materials based on fluorinated ethylene polymers and the content of NaX zeolite in these materials.     

Electroanalysis of single-nucleotide polymorphism by hairpin DNA architectures

Genetic analysis of infectious and genetic diseases and cancer diagnostics require the development of efficient tools for fast and reliable analysis of single-nucleotide polymorphism (SNP) in targeted DNA and RNA sequences often responsible for signalling disease onset. Here, we highlight the main trends in the development of electrochemical genosensors for sensitive and selective detection of SNP that are based on hairpin DNA architectures exhibiting better SNP recognition properties compared with linear DNA probes. SNP detection by electrochemical hairpin DNA beacons is discussed, and comparative analysis of the existing SNP sensing strategies based on enzymatic and nanoparticle signal amplification schemes is presented.     

Physicochemical properties of composite sorption-active materials based on zeolite and fluorinated ethylene derivatives

A procedure was suggested for preparing composite sorption-active materials based on zeolite and fluorinated ethylene polymers. The morphology of these materials, their resistance to mechanical action, and their ability to sorb water vapor under static and dynamic conditions were examined.     

Direct electrochemistry of heme multicofactor-containing enzymes on alkanethiol-modified gold electrodes

Direct electrochemistry of heme multicofactor-containing enzymes, e.g., microbial theophylline oxidase (ThOx) and D-fructose dehydrogenase (FDH) from Gluconobacter industrius was studied on alkanethiol-modified gold electrodes and was compared with that of some previously studied complex heme enzymes, specifically, cellobiose dehydrogenase (CDH) and sulphite oxidase (SOx). The formal redox potentials for enzymes in direct electronic communication varied for ThOx from -112 to -101 mV (vs. Ag|AgCl), at pH 7.0, and for FDH from -158 to -89 mV, at pH 5.0 and pH 4.0, respectively, on differently charged alkanethiol layers. Direct and mediated by cytochrome c electrochemistry of FDH correlated with the existence of two active centres in the protein structure, i.e., the heme and the pyrroloquinoline quinone (PQQ) prosthetic groups. The effect of the alkanethiols of different polarity and charge on the surface properties of the gold electrodes necessary for adsorption and orientation of ThOx, FDH, CDH and SOx, favourable for the efficient electrode-enzyme electron transfer reaction, is discussed.     

Bioelectrochemical analysis of neurodegeneration: Refocusing efforts

Since 1970s, electrochemistry is enthusiastically used for studies of severe neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, or prion-associated transmissible spongiform encephalopathies, associated with the neuronal death in the brain. The existing electrochemical sensors can be used both for direct neurotransmitter analysis in the brain and for detection of both proteins/amyloid peptides and the extent of their aggregation/oligomerisation. However, these sensors' application in body fluids or certain brain areas of interest may be restricted by the presence of structurally or electrochemically related species interfering with electroanalysis. Thus, recent efforts are refocusing on bioelectroanalysis with the apatmer- and antibody-modified electrodes, enabling obtaining more specific, interference-free results that allow better correlations with the disease state. In this opinion, I consider these recent efforts aimed at deeper studies and better understanding of neurotransmitter and protein/peptide patterns linked to neurodegenerative disorders.     

Adsorption of differently charged forms of horseradish peroxidase on metal electrodes of different nature: effect of surface charges

The adsorption and bioelectrocatalytical activity in the reaction of H(2)O(2) reduction of two forms of horseradish peroxidase (HRP) offering different surface charges at pH 6.0 were studied on gold and silver electrodes. Positively charged HRP was assessed at pH 6.0 for the case of native HRP (isoenzyme C, pI=8.8), and negatively charged HRP for the case of native HRP exposed to previous oxidation of carbohydrate residues and further introduction of sulfonate groups (pI=5.0). Under oxidative pretreatment, the gold electrode surface was considered to be negatively charged. Data on the direct immobilisation of HRPs on the bare gold surfaces were estimated with quartz crystal microbalance and data on bioelectrocatalytical activity of peroxidases on gold and silver electrodes were obtained in the course of direct and mediated amperometric detection of H(2)O(2). The presented results demonstrate that the surface charges of both the enzyme and the electrode play a dominant role in the immobilisation and, thereby, in the efficiency of the bioelectrocatalytical processes.     

Effect of proton donors on direct electron transfer in the system gold electrode–horseradish peroxidase

The effect of proton donors (PD) on the direct electron transfer (ET) reaction between polycrystalline Au electrodes and horseradish peroxidase (HRP) was investigated. HRP was immobilised directly on the bare Au surface. The pH of the contacting solution was varied at a constant ionic strength and the following different PDs were used as additives: H₃O⁺, NH₄⁺, [La(H₂O)]³⁺, [Y(H₂O)]³⁺, [Lu(H₂O)]³⁺. The kinetics of the bioelectrocatalytic reduction of H₂O₂ catalysed by HRP was studied with linear sweep voltammetry (LSV) in the potential range from 700 to −100 mV vs. SCE as well as amperometrically at −50 mV vs. Ag|AgCl with the HRP-modified Au electrodes placed in a wall-jet flow through electrochemical cell. An increase of [H₃O⁺] results in an enhancement of the current of the bioelectroreduction of H₂O₂ due to a more facilitated direct ET between Au and the enzyme over the potential range involved. It is shown that at high overvoltages (E<0.4 V) the PDs do not affect the rate of the enzymatic reduction of H₂O₂ but rather increase significantly the rate of direct ET between Au and HRP and the efficiency of acting as a PD is strongly correlated with their PD properties. The dependence of the apparent heterogeneous rate constant of direct ET, k_s, on [H₃O⁺] makes it possible to suggest that the reaction mechanism involves the participation of a proton in the elementary step of the charge transfer.     

Electrochemical assays for microbial analysis: how far they are from solving microbiota and microbiome challenges

Bacterial sensors are indispensable in environmental monitoring, analysis of food and drink safety, prevention and treatment of pathogenic infections, antibiotic resistance screening, in combatting biocorrosion, and in biodefense. Recent discoveries within Human Microbiome project disclosed vital bacteria's role in human health and disease prognosis and treatment; they also placed in focus new analytical tools for bacterial analysis. Here, I discuss several basic concepts underlying the electrochemical bacterial biosensors: metabolic sensors, biosensors for DNA and RNA extracted from bacterial cells, and whole bacterial cell sensors, and their contribution to practically sought solutions for bacterial analysis. Current analytical issues and perspectives are outlined.     

Quantifying protein adsorption and function at nanostructured materials: enzymatic activity of glucose oxidase at GLAD structured electrodes

Nanostructured materials strongly modulate the behavior of adsorbed proteins; however, the characterization of such interactions is challenging. Here we present a novel method combining protein adsorption studies at nanostructured quartz crystal microbalance sensor surfaces (QCM-D) with optical (surface plasmon resonance SPR) and electrochemical methods (cyclic voltammetry CV) allowing quantification of both bound protein amount and activity. The redox enzyme glucose oxidase is studied as a model system to explore alterations in protein functional behavior caused by adsorption onto flat and nanostructured surfaces. This enzyme and such materials interactions are relevant for biosensor applications. Novel nanostructured gold electrode surfaces with controlled curvature were fabricated using colloidal lithography and glancing angle deposition (GLAD). The adsorption of enzyme to nanostructured interfaces was found to be significantly larger compared to flat interfaces even after normalization for the increased surface area, and no substantial desorption was observed within 24 h. A decreased enzymatic activity was observed over the same period of time, which indicates a slow conformational change of the adsorbed enzyme induced by the materials interface. Additionally, we make use of inherent localized surface plasmon resonances in these nanostructured materials to directly quantify the protein binding. We hereby demonstrate a QCM-D-based methodology to quantify protein binding at complex nanostructured materials. Our approach allows label free quantification of protein binding at nanostructured interfaces.