Bastien Doumèche

Paper electrodes for bioelectrochemistry: Biosensors and biofuel cells.

Paper-based analytical devices (PAD) emerge in the scientific community since 2007 as low-cost, wearable and disposable devices for point-of-care diagnostic due to the widespread availability, long-time knowledge and easy manufacturing of cellulose. Rapidly, electrodes were introduced in PAD for electrochemical measurements. Together with biological components, a new generation of electrochemical biosensors was born. This review aims to take an inventory of existing electrochemical paper-based biosensors and biofuel cells and to identify, at the light of newly acquired data, suitable methodologies and crucial parameters in this field. Paper selection, electrode material, hydrophobization of cellulose, dedicated electrochemical devices and electrode configuration in biosensors and biofuel cells will be discussed.

1-Ethyl-3-methylimidazolium ethylsulfate/copper catalyst for the enhancement of glucose chemiluminescent detection: effects on light emission and enzyme activity.

The effect of the ionic liquid 1-ethyl-3-methylimidazolium ethylsulfate ([Emim][EtSO(4)]) on the copper-catalyzed luminol chemiluminescence (CL) is reported. A drastic light emission enhancement is observed, related to a strong interaction between Cu(2+) and the imidazolium ring. In these conditions, the CL reaction was able to produce light efficiently at pH as low as 6.5 (amplification factor: Intensity(+IL)/Intensity(-IL) = 2900). Interesting effects of [Emim][EtSO(4)] on the enzyme glucose oxidase activity were also evidenced, and advantages were taken from this enhancement to perform sensitive chemiluminescent glucose detection (LOD = 4 microM) at pH 8.0.

Contribution of dynamic and static quenchers for the study of protein conformation in ionic liquids by steady-state fluorescence spectroscopy.

The study of protein conformation in ionic liquids (ILs) is crucial to understand enzymatic activity. Steady-state fluorescence is a proven, rapid and easy method to evaluate the protein structure in aqueous solutions, but it is discussed when used in ILs. In this work, the structure of the formate dehydrogenase from Candida boidinii (FDH, EC: 1.2.1.2) in three imidazolium-based ILs (dimethylimidazolium dimethylphosphate [MMIm][Me(2)PO(4)], 1-butyl-3-methylimidazolium acetate [BMIm][CH(3)COO], and dimethylimidazolium methylphosphonate [MMIm][CH(3)HPO(2)(OCH(3))]) is studied by fluorescence spectroscopy. The UV-vis spectroscopic analysis shows that the decrease of the FDH fluorescence is not only due to the high light absorption of these ILs. The Stern-Volmer analysis clearly shows that these ILs are quenchers of the indole fluorescence, while this quenching property is not found when imidazole is used. Fluorescence spectra of the FDH in the presence of the ILs show that a maximal ionic liquid concentration (MILc), which could be used for steady-state fluorescence study, should be defined. Therefore, FDH conformation could not be directly related to the decrease of its fluorescence in ILs. Nevertheless, the structure of the FDH could be evaluated with dynamic and static quenchers like iodide or acrylamide, used below the MILc, demonstrating the relevance of this parameter. The Stern-Volmer constants (K(SV)(Q)), calculated in the presence of the different ILs, demonstrate that these ILs are strong denaturing agents, each one acting with a different mechanism. This report provides a suitable and easy-to-apply method to study any enzyme structures in ILs by steady-state fluorescence.

Ionic Liquid-Inspired Cations Covalently Bound to Formate Dehydrogenase Improve its Stability and Activity in Ionic Liquids

Ionic liquids (ILs) are important new solvents for electrochemistry and biocatalysis, but dehydrogenases usually do not work in ionic liquids. Adding more than 40 % (v/v) of the water miscible ionic liquid [MMIm][Me2PO4] (MMIm: 1‐methyl‐3‐methyl imidazolium dimethylphosphate) inactivates the formate dehydrogenase (FDH) from Candida boidinii. The grafting of a variety of IL‐inspired hydroxylated cations (hydroxyalkyl imidazolium, hydroxylalkyl pyrrolydinium, and cholinium) on the enzyme through lysine coupling was performed to understand the relationship between grafted cation, enzyme activity, and protein structure. As a general trend, the more a cation was kosmotropic (e.g., presenting a high B coefficient), the larger the resulting modifications were. The ability of these enzymes to bind the substrates was studied by fluorescence quenching in the presence of nicotinamide adenine dinucleotide (NAD+) and azide. The dissociation constant for NAD+ was only slightly affected by the grafting of the cations, however, the quenching efficiency was reduced. Azide binding was more affected by the cations. In the presence of 30 % (v/v) [MMIm][Me2PO4], the catalytic efficiency of the wild‐type enzyme was reduced by 2.8 fold. In comparison, the catalytic efficiency of the modified FDH was preserved in these conditions and even improved after modification by hydroxypropyl imidazolium. The grafting of the chaotropic cations prevented the unfolding of the FDH due to [MMIm][Me2PO4].

Directed Evolution of a Formate Dehydrogenase for Increased Tolerance to Ionic Liquids Reveals a New Site for Increasing the Stability

The formate dehydrogenase (FDH) from Candida boidinii is a well‐known enzyme in biocatalysis for NADH regeneration. Nevertheless, it has low activity in a water‐miscible ionic liquid (1,3‐dimethylimidazolium dimethyl phosphate, [MMIm][Me2PO4]). In this work, this enzyme was subjected to directed evolution by using error‐prone PCR, and a mutant (N187S/T321S) displaying higher activity was obtained following selection based on the formazan‐based colorimetric assay. The mutation N187S is responsible for improved activity both in aqueous solution and in [MMIm][Me2PO4], through an enhancement of the kcat value by a factor of 5.8. Fluorescence experiments performed in the presence of a quenching agent revealed that the mutant does not unfold in the presence of 50 % (v/v) [MMIm][Me2PO4] whereas the wild‐type enzyme does. Molecular modelling revealed that the mutation is located at the monomer–monomer interface and causes an increase in the pKa of residue E163 from 4.8 to 5.5. Calculation of the pKa of this residue in other microbial FDHs showed that thermostable FDHs have a highly basic glutamate at this position (pKa up to 6.2). We have identified a new site for improving FDH thermostability and tolerance to ionic liquids, and it is linked to the local charge of the enzymes in this class.

Micro-contact printing of PEM thin films: effect of line tension and surface energies

Polyelectrolyte multilayer (PEM) thin films are popular candidates for surface coating due to their versatility, tunability and simple production method. Often these films are used in a 2D structured manner for creating defined cell scaffolds or electronic applications. Although these films were successfully printed in the past, the conditions and energies necessary for a successful printing were only investigated as isolated parameters or as a function of the substrate but not the PEM surface energy and therefore the dominating forces remained controversial. We hereby present a theory and method for microcontact printing of condensed polyelectrolyte multilayer thin films, based on surface energies and the line tension. The theory relies on the surface energy of the substrate, stamp and PEM as well as the PEM line tension ratios to create the desired pattern. The presented theory is able to predict the printability, quality and resolution limit of a chosen system and was evaluated with experiments. A reduction of the production time from the beginning of PEM assembly to the final pattern from several hours down to 30 minutes was achieved while increasing reproducibility and resolution of the printed patterns at the same time. We would like to point out that this approach can generally be used for any kind of adsorbed thin film on substrates.

A 96-well electrochemical method for the screening of enzymatic activities.

The rapid electrochemical screening of enzyme activities in bioelectronics is still a challenging issue. In order to solve this problem, we propose to use a 96-well electrochemical assay. This system is composed of 96 screen-printed electrodes on a printed circuit board adapted from a commercial system (carbon is used as the working electrode and silver chloride as the counter/reference electrode). The associated device allows for the measurements on the 96 electrodes to be performed within a few seconds. In this work, we demonstrate the validity of the screening method with the commercial laccase from the fungus Trametes versicolor. The signal-to-noise ratio (S/N) is found to be the best way to analyze the electrochemical signals. The S/N follows a saturation-like mechanism with a dynamic linear range of two decades ranging from 0.5 to 75 ng of laccase (corresponding to enzymatic activities from 62 × 10(-6) to 9.37 × 10(-3) μmol min(-1)) and a sensitivity of 3027 μg(-1) at +100 mV versus Ag/AgCl. Laccase inhibitors (azide and fluoride anions), pH optima, and interfering molecules could also be identified within a few minutes.

Thiamine biosensor based on oxidative trapping of enzyme-substrate intermediate.

In the present work, we describe a new thiamine amperometric biosensor based on thiamine pyrophosphate (ThDP)-dependent transketolase (TK)-catalyzed reaction, followed by the oxidative trapping of TK intermediate α,β-dihydroxyethylthiamine diphosphate (DHEThDP) within the enzymatic active site. For the biosensor design purpose, TK from Escherichia coli (TKec) was immobilized in Mg2Al-NO3 Layered Double Hydroxides (LDH) and the electrochemical detection was achieved with the TKec/LDH modified glassy carbon electrode (GCE). The transduction process was based on the ability of Fe(CN)63- to oxidize DHEThDP to glycolic acid along with ThDP regeneration. The released Fe(CN)64- was re-oxidized at +0.5V vs Ag-AgCl and the reaction was followed by chronoamperometry. The TKec/LDH/GCE biosensor was optimized using the best TK donor substrates, namely l-erythrulose and d-fructose-6-phosphate. ThDP was assayed with great sensitivity (3831mAM-1cm-2) over 20-400nM linear range.

Peroxide detected in imidazolium-based ionic liquids and approaches for reducing its presence in aqueous and non-aqueous environments

Imidazolium-based ionic liquids were discovered to contain micromolar (μM) quantities of a peroxide species. A general approach using catalase (aqueous solution) or a salen–manganese complex (neat IL) for reducing the presence of the peroxide species is described herein.

Hexokinase and glucokinases are essential for fitness and virulence in the pathogenic yeast Candida albicans

Metabolic flexibility promotes infection and commensal colonization by the opportunistic pathogen Candida albicans. Yeast cell survival depends upon assimilation of fermentable and non-fermentable locally available carbon sources. Physiologically relevant sugars like glucose and fructose are present at low level in host niches. However, because glucose is the preferred substrate for energy and biosynthesis of structural components, its efficient metabolization is fundamental for the metabolic adaptation of the pathogen. We explored and characterized the C. albicans hexose kinase system composed of one hexokinase (CaHxk2) and two glucokinases (CaGlk1 and CaGlk4). Using a set of mutant strains, we found that hexose phosphorylation is mostly assured by CaHxk2, which sustains growth on hexoses. Our data on hexokinase and glucokinase expression point out an absence of cross regulation mechanisms at the transcription level and different regulatory pathways. In the presence of glucose, CaHxk2 migrates in the nucleus and contributes to the glucose repression signaling pathway. In addition, CaHxk2 participates to oxidative, osmotic and cell wall stress responses, while glucokinases are overexpressed under hypoxia. Hexose phosphorylation is a key step necessary for filamentation, that is affected in the hexokinase mutant. Virulence of this mutant is clearly impacted in the Galleria mellonella and macrophage models. Filamentation, glucose phosphorylation and stress response defects of the hexokinase mutant prevent host killing by C. albicans. By contributing to metabolic flexibility, stress answer response and morphogenesis, hexose kinase enzymes play an essential role in the virulence of C. albicans. Author summary The pathogenic yeast C. albicans is both a powerful commensal and pathogen of humans that can infect wide range of organs and body sites. To grow in its host and establish an infection, the pathogen must assimilate carbon from these heterogenous environments. C. albicans regulates central carbon metabolism in a niche-specific manner, activating alternatively gluconeogenesis, glyoxylate cycle and the glycolytic metabolism. For yeast and other microorganisms, glucose is the preferred carbon and energy source and its accurate detection and metabolism is essential. However, the glycolytic hexose kinase system has not been investigated yet in C. albicans. In this report, we showed that hexokinase and glucokinases contribute to the fitness and virulence of C. albicans. We revealed the main metabolic role of the hexokinase CaHxk2 which impacts on growth, glucose signalling, morphological transition and virulence. However, glucokinases contribute to the anoxic response and their implication in regulation processes is suggested.