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.

LDI-MS scanner: Laser desorption ionization mass spectrometry-based biosensor standardization.

Amperometric biosensors have been widely utilized for the cost-effective and rapid analysis of various bioanalytes, for example glucose. However, a lack of standardization and validation procedures remains a major limitation in biosensor development. Therefore, despite rapid advances in material science driving the development of amperometric biosensors, to date only a few biosensors, detecting a limited range of analytes, are available on the market. It is believed, once this issue is addressed, it can significantly facilitate the next step in the overall concept "go to the market" production and implementation of amprerometric biosensors for a large industrial scale. Herein, we report on the use of laser desorption ionization mass spectrometry (LDI-MS) for the standardization of amperometric biosensors, based upon a complete and non-destructive characterization and validation of layer-by-layer (LbL) biosensors at each fabrication step. We reveal that specific ionization pathways of mediators, polymers and enzymes from the biosensor surface allows for robust quality control during LbL biosensor manufacture. Furthermore, this LDI-MS approach can also be used to monitor, and therefore ensure, the encapsulation of enzymes in one-step nanobiosensors. Specifically, we show that LDI-MS can be used for the rapid chemical profiling of LbL biosensors and one-step synthesized nanobiosensors, as well as to assess their synthesis quality and to monitor for batch-to-batch and intra- and inter-day changes in their function and behavior. Our novel approach will thus contribute to the future development, improved design and fine tuning of both conventional LbL-fabricated amperometric biosensors and one-step designed nanobiosensors.

Towards one-step design of tailored enzymatic nanobiosensors.

The manufacturing of conventional enzymatic biosensors produced via a layer-by-layer (LbL) approach requires expensive instrumentation, and in most cases involves a complex, resource and time-consuming fabrication process. Moreover, LbL assemblies are prone to mechanical instability that leads to irreversible changes in sensor architecture and morphology resulting in degradation of enzymatic activities and insufficient signal reproducibility. Hence, novel fabrication techniques for the production of enzymatic biosensors that are instrumentally controlled and allow reproducible, simultaneous multi-analyte detection with high specificity, temporal and spatial resolution are greatly required. Herein, we report on the development of a novel, fully instrumentally controlled, one-step synthesis approach for the production of nanoparticle-based enzymatic biosensors. The approach relies on a simultaneous encapsulation of the enzyme (glucose and alcohol oxidases), a fluoropolymer (Nafion) and noble metal nanoparticles via co-deposition from a phosphate multiple electrolyte on top of the sensor surface. Remarkably, electrochemical studies revealed that nanoparticle-based biosensors produced by this novel fabrication approach display a significantly enhanced mechanical stability (more than several orders of magnitude higher) without loss of biological activity or leakage of the enzyme or Nafion, and advanced synthesis reproducibility (40 times higher) in comparison to LbL analogues.

One-step encapsulation, storage and controlled release of low molecular weight organic compounds via electroplated nanoparticles.

Herein, we introduce an original strategy toward one-step encapsulation, storage and controlled release of low molecular weight organic compounds via electroplated nanoparticles. This concept is demonstrated on the basis of the encapsulation of several organic matrices typically used for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) as a case study via co-deposition with palladium nanoparticles (Pd-NPs). Remarkably, Pd-NPs act as a capsule for MALDI matrices and thus provide their controlled release depending on the external factors, viz. applied laser fluence or pH of the surrounding media. The proposed approach is considered as a simple, fast and inexpensive preparation method towards the formation of ultimate self-assembled hybrid MALDI matrices with a less pronounced "sweet spot" phenomenon and improved long-term stability.

Exploring the potential of electroless and electroplated noble metal-semiconductor hybrids within bio- and environmental sensing.

Over the last two decades, the rapid development and widespread application of nanomaterials has significantly influenced research in various fields, including analytical chemistry and biosensing technologies. In particular, the simple functionalization and tuning of noble metal nanoparticle (NP) surface chemistry resulted in the development of a series of novel biosensing platforms with quick read-out and enhanced capabilities towards specific analyte detection. Moreover, noble metal NPs possess a number of unique properties, viz. high surface-to-volume ratio and excellent spectral, optical, thermal, electrical and catalytic characteristics. This manuscript provides an elaborate review on galvanic noble metal NPs deposited onto semiconductor surfaces, from the preparation stage towards their application in biosensors and gas sensing. Two types of deposition approaches, viz. galvanic displacement/electroless and conventional electroplating, are introduced and compared. Furthermore, the analytical merit of hybrid nanomaterials towards the improvement of sensing abilities is highlighted. Finally, some limitations and challenges related to progress in the development and application of analytical devices based on electroless and electroplated noble metal NPs-semiconductor hybrids (NMNPsHs) in biochemical and environmental sensing are discussed.