A simple model of cardiac mitochondrial respiration with experimental validation.

Cardiac mitochondria are intracellular organelles that play an important role in energy metabolism and cellular calcium regulation. In particular, they influence the excitation-contraction cycle of the heart cell. A large number of mathematical models have been proposed to better understand the mitochondrial dynamics, but they generally show a high level of complexity, and their parameters are very hard to fit to experimental data. We derived a model based on historical free energy-transduction principles, and results from the literature. We proposed simple expressions that allow to reduce the number of parameters to a minimum with respect to the mitochondrial behavior of interest for us. The resulting model has thirty-two parameters, which are reduced to twenty-three after a global sensitivity analysis of its expressions based on Sobol indices. We calibrated our model to experimental data that consists of measurements of mitochondrial respiration rates controlled by external ADP additions. A sensitivity analysis of the respiration rates showed that only seven parameters can be identified using these observations. We calibrated them using a genetic algorithm, with five experimental data sets. At last, we used the calibration results to verify the ability of the model to accurately predict the values of a sixth dataset. Results show that our model is able to reproduce both respiration rates of mitochondria and transitions between those states, with very low variability of the parameters between each experiment. The same methodology may apply to recover all the parameters of the model, if corresponding experimental data were available.

Single-Particle Tracking Method in Fluorescence Microscopy to Monitor Bioenergetic Responses of Individual Mitochondria.

The spectroscopic methods commonly used to study mitochondria bioenergetics do not show the diversity of responses within a population of mitochondria (isolated or in a cell), and/or cannot measure individual dynamics. New methodological developments are necessary in order to improve quantitative and kinetic resolutions and eventually gain further insights on individual mitochondrial responses, such as studying activities of the mitochondrial permeability transition pore (mPTP ). The work reported herein is devoted to study responses of single mitochondria within a large population after isolation from cardiomyocytes. Mitochondria were preloaded with a commonly used membrane potential sensitive dye (TMRM), they are then deposited on a plasma-treated glass coverslip and subsequently energized or inhibited by additions of usual bioenergetics effectors. Responses were analyzed by fluorescence microscopy over few thousands of mitochondria simultaneously with a single organelle resolution. We report an automatic method to analyze each image of time-lapse stacks based on the TrackMate-ImageJ plug-in and specially made Python scripts. Images are processed to eliminate defects of illumination inhomogeneity, improving by at least two orders of magnitude the signal/noise ratio. This method enables us to follow the track of each mitochondrion within the observed field and monitor its fluorescence changes, with a time resolution of 400 ms, uninterrupted over the course of the experiment. Such methodological improvement is a prerequisite to further study the role of mPTP in single mitochondria during calcium transient loading.

Microwell Array Based Opto-Electrochemical Detections Revealing Co-Adaptation of Rheological Properties and Oxygen Metabolism in Budding Yeast.

Microdevices composed of microwell arrays integrating nanoelectrodes (OptoElecWell) are developed to achieve dual high-resolution optical and electrochemical detections on single Saccharomyces cerevisiae yeast cells. Each array consists of 1.6 × 105 microwells measuring 8 µm in diameter and 5 µm height, with a platinum nanoring electrode for in situ electrochemistry, all integrated on a transparent thin wafer for further high-resolution live-cell imaging. After optimizing the filling rate, 32% of cells are effectively trapped within microwells. This allows to analyse S. cerevisiae metabolism associated with basal respiration while simultaneously measuring optically other cellular parameters. In this study, the impact of glucose concentration on respiration and intracellular rheology is focused. It is found that while the oxygen uptake rate decreases with increasing glucose concentration, diffusion of tracer nanoparticles increases. The OptoElecWell-based respiration methodology provides similar results compared to the commercial gold-standard Seahorse XF analyzer, while using 20 times fewer biological samples, paving the way to achieve single cell metabolomics. In addition, it facilitates an optical route to monitor the contents within single cells. The proposed device, in combination with the dual detection analysis, opens up new avenues for measuring cellular metabolism, and relating it to cellular physiological indicators at single cell level.

Uphill production of dihydrogen by enzymatic oxidation of glucose without an external energy source.

Chemical systems do not allow the coupling of energy from several simple reactions to drive a subsequent reaction, which takes place in the same medium and leads to a product with a higher energy than the one released during the first reaction. Gibbs energy considerations thus are not favorable to drive e.g., water splitting by the direct oxidation of glucose as a model reaction. Here, we show that it is nevertheless possible to carry out such an energetically uphill reaction, if the electrons released in the oxidation reaction are temporarily stored in an electromagnetic system, which is then used to raise the electrons' potential energy so that they can power the electrolysis of water in a second step. We thereby demonstrate the general concept that lower energy delivering chemical reactions can be used to enable the formation of higher energy consuming reaction products in a closed system.

On-chip enzymatic microbiofuel cell-powered integrated circuits.

A variety of diagnostic and therapeutic medical technologies rely on long term implantation of an electronic device to monitor or regulate a patient's condition. One proposed approach to powering these devices is to use a biofuel cell to convert the chemical energy from blood nutrients into electrical current to supply the electronics. We present here an enzymatic microbiofuel cell whose electrodes are directly integrated into a digital electronic circuit. Glucose oxidizing and oxygen reducing enzymes are immobilized on microelectrodes of an application specific integrated circuit (ASIC) using redox hydrogels to produce an enzymatic biofuel cell, capable of harvesting electrical power from just a single droplet of 5 mM glucose solution. Optimisation of the fuel cell voltage and power to match the requirements of the electronics allow self-powered operation of the on-board digital circuitry. This study represents a step towards implantable self-powered electronic devices that gather their energy from physiological fluids.

PDMS microwells for multi-parametric monitoring of single mitochondria on a large scale: a study of their individual membrane potential and endogenous NADH.

Microwell arrays have been developed to monitor simultaneously, and on a large scale, multiple metabolic responses of single mitochondria. Wells of 50 to 1000 µm-diameter were prepared based on easy structuration of thin polydimethylsiloxane layers (PDMS; 100 µm thickness). Their surface treatment with oxygen plasma allowed the immobilization in situ and observation with time of populations of single isolated mitochondria. Their metabolic activities could be monitored individually by fluorescence microscopy under several activation/inhibition conditions. We measured the concomitant variations of two main metabolic parameters - the endogenous NADH level and the internal membrane potential difference Δψ owing to a cationic fluorescent probe (TMRM) - at energized, uncoupled and inhibited stages of the mitochondrial respiratory chain. Microwell arrays allowed analyses on large populations, and consequently statistical studies with a single organelle resolution. Thus, we observed rapid individual polarizations and depolarizations of mitochondria following their supply with the energetic substrate, while an averaged global polarization (increase of TMRM fluorescence within mitochondria) and NADH increase were detected for the whole population. In addition, statistical correlation studies show that the NADH content of all mitochondria tends toward a metabolic limit and that their polarization-depolarization ability is ubiquitous. These results demonstrate that PDMS microwell platforms provide an innovative approach to better characterize the individual metabolic status of isolated mitochondria, possibly as a function of their cell or organ origin or in different physio-pathological situations.

Optical microwell arrays for large-scale studies of single mitochondria metabolic responses.

Most of the methods dedicated to the monitoring of metabolic responses from isolated mitochondria are based on whole-population analyses. They rarely offer an individual resolution though fluorescence microscopy allows it, as demonstrated by numerous studies on single mitochondria activities in cells. Herein, we report on the preparation and use of microwell arrays for the entrapment and fluorescence microscopy of single isolated mitochondria. Highly dense arrays of 3 µm mean diameter wells were obtained by the chemical etching of optical fiber bundles (850 µm whole diameter). They were manipulated by a micro-positioner and placed in a chamber made of a biocompatible elastomer (polydimethylsiloxane or PDMS) and a glass coverslip, on the platform of an inverted microscope. The stable entrapment of individual mitochondria (extracted from Saccharomyces cerevisiae yeast strains, inter alia, expressing a green fluorescent protein) within the microwells was obtained by pretreating the optical bundles with an oxygen plasma and dipping the hydrophilic surface of the array in a concentrated solution of mitochondria. Based on the measurement of variations of the intrinsic NADH fluorescence of each mitochondrion in the array, their metabolic status was analyzed at different energetic respiratory stages: under resting state, following the addition of an energetic substrate to stimulate respiration (ethanol herein) and the addition of a respiratory inhibitor (antimycin A). Statistical analyses of mean variations of mitochondrial NADH in the population were subsequently achieved with a single organelle resolution.

Electrochemical monitoring of the early events of hydrogen peroxide production by mitochondria.

Mitochondria consume oxygen in the respiratory chain and convert redox energy into ATP. As a side process, they produce reactive oxygen species (ROS), whose physiological activities are still not understood. However, current analytical methods cannot be used to monitor mitochondrial ROS quantitatively and unambiguously. We have developed electrochemical biosensors based on peroxidase-redox polymer-modified electrodes, providing selective detection of H2O2 with nanomolar sensitivity, linear response over five concentration decades, and fast response time. The release of H2O2 by mitochondria was then monitored under phosphorylating or inhibited respiration conditions. We report the detection of two concomitant regimes of H2O2 release: large fluxes (hundreds of nM) under complex III inhibition, and bursts of a few nM immediately following mitochondria activation. These unprecedented bursts of H2O2 are assigned to the role of mitochondria as the hub of redox signaling in cells.

Switching an O2 sensitive glucose oxidase bioelectrode into an almost insensitive one by cofactor redesign.

In the 5-8 mM glucose concentration range, of particular interest for diabetes management, glucose oxidase bioelectrodes are O2 dependent, which decrease their efficiencies. By replacing the natural cofactor of glucose oxidase, we succeeded in turning an O2 sensitive bioelectrode into an almost insensitive one.

Optical microwell array for large scale studies of single mitochondria metabolic responses.

Microsystems based on microwell arrays have been widely used for studies on single living cells. In this work, we focused on the subcellular level in order to monitor biological responses directly on individual organelles. Consequently, we developed microwell arrays for the entrapment and fluorescence microscopy of single isolated organelles, mitochondria herein. Highly dense arrays of 3-µm mean diameter wells were obtained by wet chemical etching of optical fiber bundles. Favorable conditions for the stable entrapment of individual mitochondria within a majority of microwells were found. Owing to NADH auto-fluorescence, the metabolic status of each mitochondrion was analyzed at resting state (Stage 1), then following the addition of a respiratory substrate (Stage 2), ethanol herein, and of a respiratory inhibitor (Stage 3), antimycin A. Mean levels of mitochondrial NADH were increased by 29% and 35% under Stages 2 and 3, respectively. We showed that mitochondrial ability to generate higher levels of NADH (i.e., its metabolic performance) is not correlated either to the initial energetic state or to the respective size of each mitochondrion. This study demonstrates that microwell arrays allow metabolic studies on populations of isolated mitochondria with a single organelle resolution.

Electrochemical detection of single microbeads manipulated by optical tweezers in the vicinity of ultramicroelectrodes.

Latex micrometric beads are manipulated by optical tweezers in the vicinity of an ultramicroelectrode (UME). They are optically trapped in solution and approached the electrode surface. After the electrochemical measurement, they are optically removed from the surface. The residence time of the particle on the electrode is thus controlled by the optical tweezers. The detection is based on diffusional hindrance by the insulating objects which alters the fluxes of the redox Ru(NH3)6(3+) species toward the UME and thus its mass-transfer limited current. We have optically deposited successively 1, 2, and 3 beads of 3-µm radius on the UME surface, and we have recorded the variations of the current depending on their landing locations that were optically controlled. Finally we decreased the current by partially blocking the electroactive surface with a six-bead assembly. The variation of the steady-state current and the approach curves allow for the indirect electrochemical localization of the bead in the vicinity of the UME, not only when the bead is in contact but also when it is levitated at distances lower than the UME radius. These experiments show that single particles or more complex structures may be manipulated in situ in a contactless mode near the UME surface. From comparison with simulations, the electrochemical detection affords an indirect localization of the object in the UME environment. The developed approach offers a potential application for interrogating the electrochemical activity of single cells and nanoparticles.

Monitoring metabolic responses of single mitochondria within poly(dimethylsiloxane) wells: study of their endogenous reduced nicotinamide adenine dinucleotide evolution.

It is now demonstrated that mitochondria individually function differently because of specific energetic needs in cell compartments but also because of the genetic heterogeneity within the mitochondrial pool-network of a cell. Consequently, understanding mitochondrial functioning at the single organelle level is of high interest for biomedical research, therefore being a target for analyticians. In this context, we developed easy-to-build platforms of milli- to microwells for fluorescence microscopy of single isolated mitochondria. Poly(dimethylsiloxane) (PDMS) was determined to be an excellent material for mitochondrial deposition and observation of their NADH content. Because of NADH autofluorescence, the metabolic status of each mitochondrion was analyzed following addition of a respiratory substrate (stage 2), ethanol herein, and a respiratory inhibitor (stage 3), Antimycin A. Mean levels of mitochondrial NADH were increased by 32% and 62% under stages 2 and 3, respectively. Statistical studies of NADH value distributions evidenced different types of responses, at least three, to ethanol and Antimycin A within the mitochondrial population. In addition, we showed that mitochondrial ability to generate high levels of NADH, that is its metabolic performance, is not correlated either to the initial energetic state or to the respective size of each mitochondrion.

Heat and drying time modulate the O2 reduction current of modified glassy carbon electrodes with bilirubin oxidases.

Here we show that the magnitude of the O(2) reduction current of cathodes based on Bilirubin oxidases (BOD) immobilized into a redox hydrogel strongly depends on the drying conditions such as the curing time and temperature of drying as well as the thermostability of the BOD. To illustrate this effect, we performed experiments with two different BODs: one labile BOD from Trachyderma tsunodae and one highly thermostable BOD from Bacillus pumilus with different preparation protocols. The balance between the kinetics of formation of the hydrogel and the enzyme stability leads to optimal drying conditions of 2h at 25°C for both types of BODs when the most widespread protocol uses 18 hours at ambient temperature. For drying times longer than two hours, the catalytic current decreases because of the instability of T. tsunodae. Finally the optimal conditions for BOD from T. tsunodae lead to a faster preparation of electrodes than with the protocol currently in use (2h vs. 18h) and catalytic currents for oxygen reduction 100% higher (1040µA/cm(2) vs. 517µA/cm(2)).

Bilirubin oxidase from Bacillus pumilus: a promising enzyme for the elaboration of efficient cathodes in biofuel cells.

A CotA multicopper oxidase (MCO) from Bacillus pumilus, previously identified as a laccase, has been studied and characterized as a new bacterial bilirubin oxidase (BOD). The 59 kDa protein containing four coppers, was successfully over-expressed in Escherichia coli and purified to homogeneity in one step. This 509 amino-acid enzyme, having 67% and 26% sequence identity with CotA from Bacillus subtilis and BOD from Myrothecium verrucaria, respectively, shows higher turnover activity towards bilirubin compared to other bacterial MCOs. The current density for O(2) reduction, when immobilized in a redox hydrogel, is only 12% smaller than the current obtained with Trachyderma tsunodae BOD. Under continuous electrocatalysis, an electrode modified with the new BOD is more stable, and has a higher tolerance towards NaCl, than a T. tsunodae BOD modified electrode. This makes BOD from B. pumilus an attractive new candidate for application in biofuel cells (BFCs) and biosensors.

Spectroscopic and crystallographic characterization of "alternative resting" and "resting oxidized" enzyme forms of bilirubin oxidase: implications for activity and electrochemical behavior of multicopper oxidases.

While there is broad agreement on the catalytic mechanism of multicopper oxidases (MCOs), the geometric and electronic structures of the resting trinuclear Cu cluster have been variable, and their relevance to catalysis has been debated. Here, we present a spectroscopic characterization, complemented by crystallographic data, of two resting forms occurring in the same enzyme and define their interconversion. The resting oxidized form shows similar features to the resting form in Rhus vernicifera and Trametes versicolor laccase, characterized by "normal" type 2 Cu electron paramagnetic resonance (EPR) features, 330 nm absorption shoulder, and a short type 3 (T3) Cu-Cu distance, while the alternative resting form shows unusually small A(||) and high g(||) EPR features, lack of 330 nm absorption intensity, and a long T3 Cu-Cu distance. These different forms are evaluated with respect to activation for catalysis, and it is shown that the alternative resting form can only be activated by low-potential reduction, in contrast to the resting oxidized form which is activated via type 1 Cu at high potential. This difference in activity is correlated to differences in redox states of the two forms and highlights the requirement for efficient sequential reduction of resting MCOs for their involvement in catalysis.

Photopatterning of ultrathin electrochemiluminescent redox hydrogel films.

Photoinitiated polymerisation is efficiently and rapidly carried out to immobilise ultrathin electrochemiluminescent redox hydrogel films. Microscale patterns are fabricated on an electrode surface by a simple photolithographic procedure and revealed by ECL imaging.

Fast and easy enzyme immobilization by photoinitiated polymerization for efficient bioelectrochemical devices.

Immobilization and electrical wiring of enzymes is of particular importance for the elaboration of efficient biosensors and can be cumbersome. Here, we report a fast and easy protocol for enzyme immobilization, and as a proof of concept, we applied it to the immobilization of bilirubin oxidase, a labile enzyme. In the first step, bilirubin oxidase is mixed with a redox hydrogel "wiring" the enzyme reaction centers to electrodes. Then, this adduct is covered by an outer layer of PEGDA made by photoinitiated polymerization of poly(ethylene-glycol) diacrylate (PEGDA) and a photoclivable precursor, DAROCUR. This two-step protocol is 18 times faster than the current state-of-the-art protocol and leads to currents 25% higher. In addition, the outer layer of PEGDA acts as a protective layer increasing the lifetime of the electrode by 100% when operating continuously for 2000 s and by 60% when kept in dry state for 24 h. This new protocol is particularly appropriate for labile enzymes that quickly denaturate. In addition, by tuning the ratio PEGDA/DAROCUR, it is possible to make the enzyme electrodes even more active or more stable.

SPR imaging for label-free multiplexed analyses of DNA N-glycosylase interactions with damaged DNA duplexes.

Base excision repair (BER) is the major mechanism for the correction of damaged nucleobases resulting from the alkylation and oxidation of DNA. The first step in the BER pathway consists of excision of the abnormal base by several specific DNA N-glycosylases. A decrease in BER activity was found to be related to an increased risk of carcinogenesis and aging. To investigate BER activities we set up a new device for DNA repair analysis based on surface plasmon resonance imaging (SPRi). Oligonucleotides bearing an abnormal nucleoside, namely 8-oxo-7,8-dihydro-2'-deoxyguanosine and (5'S)-5',8-cyclopurine-2'-deoxynucleoside, were grafted by a pyrrole electro-copolymerization process on a glass prism coated with a gold layer. The latter label-free DNA sensor chip permits the detection of N-glycosylase/AP-lyase activity as well as the binding of repair proteins to DNA damage without cleavage activity. Thus, the Fapy DNA N-glycosylase (Fpg) protein is shown as expected to bind and then cleave its natural substrate, namely 8-oxo-7,8-dihydro-guanine, together with the resulting abasic site. Using the current SPR imaging-based DNA array we observed an original binding activity of Fpg towards the (5'S)-5',8-cyclodAdenosine residue. These results altogether show that SPR imaging may be used to simultaneously and specifically detect recognition and excision of several damaged DNA nucleobases, and constitutes an interesting technique to screen inhibitors of DNA repair proteins.

Real-time detection of lymphocytes binding on an antibody chip using SPR imaging.

We demonstrate the use of SPR imaging for the detection of site-specific binding of either B or T lymphocyte populations on an electrochemically-grafted antibody array.