Towards controlled and simple design of non-enzymatic amperometric sensor for glycerol determination in yeast fermentation medium.

Glycerol is a widely used signaling bioanalyte in biotechnology. Glycerol can serve as a substrate or product of many metabolic processes in cells. Therefore, quantification of glycerol in fermentation samples with inexpensive, reliable, and rapid sensing systems is of great importance. In this work, an amperometric assay based on one-step designed electroplated functional Pd layers with controlled design was proposed for a rapid and selective measurement of glycerol in yeast fermentation medium. A novel assay utilizing electroplated Pd-sensing layers allows the quantification of glycerol in yeast fermentation medium in the presence of interfering species with RSD below 3% and recoveries ranged from 99 to 103%. The assay requires minimal sample preparation, viz. adjusting of sample pH to 12. The time taken to complete the electrochemical analysis was 3 min. Remarkably, during investigations, it was revealed that sensitivity and selectivity of glycerol determination on Pd sensors were significantly affected by its adsorption and did not depend on the surface structure of sensing layers. This study is expected to contribute to both fundamental and practical research fields related to a preliminary choice of functional sensing layers for specific biotechnology and life science applications in the future.

First principles of hydrazine electrooxidation at oxide-free and oxide-based palladium electrodes in complex media.

In this study, fundamental aspects that have impact on the electroanalytical detection of hydrazine in phosphate, acetate and yeast fermentation medium in an analytically significant concentration range by several types of palladium (Pd)-modified electrodes, namely, Pd-ink, Pd-sputtered films and palladium nanoparticles (Pd-NPs) were systematically studied. The efficiency of hydrazine electrooxidation is not affected by the composition of multicomponent medium (i), presence of oxygen (ii), morphology or electroactive area (iii), but more likely depends on the purity degree of the electrode surface from residual palladium oxides (iv). In addition, using advanced methods of nanoanalytics and quantum chemistry, the crucial role of hydrazine surface adsorption (v) on oxide-free and oxide-based Pd-electrodes is highlighted. The obtained knowledge will provide future development strategies of electrodes based on nanoparticles of noble metals for tuned and efficient hydrazine electrooxidation in complex fermentation media.

A rapid in vitro electrochemical screening of extracellular matrix of Saccharomyces cerevisiae by palladium nanoparticles-modified electrodes.

Herein, a rapid electrochemical screening of yeasts (Saccharomyces cerevisiae) in vitro mode depending on their optical density, cultivation time and growth medium used was conducted in 3 min by palladium nanoparticles (Pd-NPs)-modified electrodes. Pd-NPs-modified electrodes operated in cyclic voltammetry mode at low scan rates, i.e. 5-20 mV/s supported a low oxidative process in the yeast extracellular matrix. The electrochemical screening relied on an efficient electrooxidation of secondary metabolites, i.e. organohydrazines formed in the extracellular medium as a result of microbial activity of yeast cells. More importantly, during the study the impact of fundamental parameters, viz. type of the matrix and pH on electroanalytical response of Pd-NPs-based electrodes in real fermentation medium was investigated in detail. The efficiency of the proposed in vitro electrochemical screening of yeast extracellular matrix was not affected by pH of the samples or composition of the multicomponent medium, but more likely exclusively depended on the presence of organohydrazines. The potential of this electroanalytical approach towards profiling of the extracellular matrix of Saccharomyces cerevisiae was compared with results obtained by gas chromatography mass-spectrometry (GC-MS) and genetically encoded biosensor (ro-GFP2) assays.

In situ modulation of enzyme activity via heterogeneous catalysis utilizing solid electroplated cofactors.

During product isolation the received bioreceptors often do not exhibit a sufficient biochemical activity due to multistep dissociation and loss of cofactors. However, for bioelectrochemical applications the presence of cofactors is necessary for a successful oxidative or reductive conversion of the substrates to the products. Herein, we show how the immobilization of the required electroplated cofactors in a design of amperometric electrodes can in situ assist the activity of apo-enzymes. Compared to conventional approaches used in enzyme engineering this tailored nanoengineering methodology is superior from economic point of view, labor and time costs, storage conditions, reduced amount of waste and can fill the gap in the development of tuned bioelectrocatalysts.

The development of alginate-based amperometric nanoreactors for biochemical profiling of living yeast cells.

This study describes the development of a one-pot electrochemical miniaturized system for simultaneous cultivation and monitoring of the oxidative status of living cells. This system consisted of screen-printed electrodes modified by electroplated Pd-NPs as an electrocatalyst (i) and living yeast cells (Saccharomyces cerevisiae) (ii) immobilized on the cytocompatible alginate layer (iii). Briefly, during the course of electrochemical investigations a novel electroactive compound methylhydrazine derivative as a secondary metabolite and result of microbial activity was found in yeast cells and used as a signaling molecule for their biochemical profiling. Under the optimized experimental conditions the signal corresponding to the found electroactive secondary metabolite formed in medium of living cells was measured without sample collecting, transport, storage or pre-treatment steps (i.e. extraction, pre-concentration, chemical derivatization or labeling). The electrochemical dependencies, which were derived by a miniaturized electroanalytical system, were fully validated in a conventional three-electrode system under inert atmosphere (Ar) and in the presence of oxygen (air, O2). It is believed that the proposed one-pot nanoreactors serving simultaneously as nanofermenters and amperometric detectors for the quantification of secondary metabolites formed in medium of living cells can significantly enhance the understanding of ongoing fermentation processes in the future and our knowledge on the biochemistry of yeasts.

Electrochemical operational principles and analytical performance of Pd-based amperometric nanobiosensors.

Palladium nanoparticles (Pd-NPs) have been approved as an effective catalyst for hydrogen peroxide decomposition which is released during specific enzymatic reactions. However, the general operational principles and electrochemical performance of Pd-NPs-based nanobiosensors have been poorly exploited. Here, the electrochemical behavior of oxidase-associated peroxide oxidation co-catalysis of the modelled microanalytical system based on screen-printed electrodes modified by electroplated Pd-NPs as an electrocatalyst, glucose oxidase (GOx) or alcohol oxidase (AOx) as a bioreceptor and the ionomer Nafion as a polymeric binding agent was studied in detail. The impact of palladium surface oxides and adsorbed oxygen on the activity and product selectivity in an oxidase type of nanobiosensor was ascertained. To avoid PdO and oxygen electroreduction affecting the entire amperometric response of Pd-NPs-based nanobiosensors, a special two-step polarization procedure was proposed. Under the established electrochemical conditions, Pd-NPs-based nanobiosensors with encapsulated oxidases showed a wide dynamic range towards selective bioanalyte detection, excellent basic line stability, accuracy and resistance to the presence of interfering electrochemical species. This work can serve as a guideline for the search and validation of operational principles of novel biosensors based on nanoparticles.

Mechanistic aspects of functional layer formation in hybrid one-step designed GOx/Nafion/Pd-NPs nanobiosensors.

Amperometric nanobiosensors are crucial time and cost effective analytical tools for the detection of a wide range of bioanalytes, viz. glucose present in complex environments at very low concentrations. Although the excellent analytical performance of nanobiosensors is undoubted, their exact molecular structure often remains unclear. Here, by combining advanced nanoanalytical approaches with theoretical modeling, we conducted a comprehensive study towards the investigation of the molecular structure of a hybrid GOx/Nafion/Pd-NPs layer deposited by electroplating from the multicomponent electrolyte solution on the surface of screen printed electrodes modified with graphene oxide. Specifically, we revealed that Pd2+ cations were adsorbed on GOx amino acid residues, forming the GOx·nPd2+ enzymatic complex. The highest adsorption energy of Pd2+ cations on GOx was found during their interaction with the side chains of basic amino acids and methionine. In addition, we showed and fully validated the end-structure of the one-step designed GOx/Nafion/Pd-NPs nanobiosensor as a structural model mainly composed of GOx and water molecules incorporated into the metal-polymer scaffold. Our approach will thus serve as a guideline for the study of molecular interactions occurring in complex systems and will contribute to the design of the next generation of hybrid nanobiosensors. The proposed mechanism, driving the self-assembly of the hybrid layer, will allow us to construct modular enzymatic nanoanalytical devices with tailored sequences in the future.

Data describing the cofactor additives effect on bioelectrocatalytic activity of «crude¼ extracts.

«Crude¼ extracts obtained via simple ultrasonic disintegration of microbial cell membrane are perspective bioelectrocatalysts. This extract contains all the necessary enzymes and cofactors required for oxidative or reductive conversion. The technology of synthesis of «crude extract¼ is simpler and less costly in comparison with technology of obtaining pure enzymes. Dialysis of the obtained extracts was performed with different molecular weight cut-off (3.5 kDa, 12-14 kDa, 25 kDa, 50 kDa). The obtained data show that after dialysis extracts lose their dehydrogenase and bioelectrocatalytic activity due to the loss of cofactors. However, the addition of NAD and NADP cofactors leads to a recovery of activity. The obtained data demonstrate that the concentration of the cofactor directly affects the rate of the bioelectrocatalytic reaction. Also, the obtained data indicate that the composition of the enzyme systems of the extract includes succinate dehydrogenase. Analyzing this data set can provide insight on increase of the electrocatalytic activity of a new type of bioelectrocatalyst.

[The impact of retraction cord design on marginal gingiva status in patients with fixed dentures].

The study examines the impact of weaved retraction cords on microcirculation in periodontal tissues and cytokines content in gingival fluid. The relationship between the strength of the traumatic agent during retraction of the gums and marginal periodontal phenotype was found out. Individual retraction method in each clinical case should be considered in order to avoid adverse aesthetic consequences of orthopedic treatment with non-removable dentures.