Substrate Injection Research Articles

Combination of on-line CE assay with MS detection for the study of drug metabolism by cytochromes P450.

A new CE-MS method with enzymatic reaction inside the capillary was developed for the study of drug metabolism by cytochromes P450. This automated method, based on the transverse diffusion of laminar flow profiles methodology, is comprised of the injection of substrates and enzyme, their mixing, incubation, and separation of the reaction products, all performed by CE, and their detection, identification, and quantification by MS. The developed and validated method was finally used to conduct a kinetic study of cytochrome P450 isoform 2C9 or human liver microsomes with diclofenac in order to demonstrate its practical functionality. All the estimated kinetic values--apparent Michaelis constants and apparent maximum reaction velocities were in agreement with literature data obtained using other techniques. In addition, the consumption of reactants was in the tens of nL per analysis. The method's usability was further demonstrated on tolbutamide, the other probe substrate of cytochrome P450 isoform 2C9. As a result, the method is conceptually applicable for the screening of any other cytochrome P450 isoform and its substrates and inhibitors after adapting the incubation and separation conditions.

Dynamic hysteresis in the velocity and dissipation of stochastic Michaelis–Menten kinetics

In this work, we have studied a single molecule enzyme catalysis reaction in presence of oscillatory substrate concentration. The stochastic kinetics is modelled in terms of a chemical master equation. Depending on the oscillation frequency, hysteresis can occur in the system which is dynamic in nature. The time-dependent driving keeps the system out-of-equilibrium associated with dissipation. The interplay between the timescales of the system kinetics and the external driving necessitates the splitting of the total entropy production rate into adiabatic and nonadiabatic contributions. Analyses of these quantities give insights into the various balance conditions of the reaction fluxes and their roles in governing the nonequilibrium thermodynamics of the system. Interestingly, the net velocity of catalysis and the dissipation along with its various parts are found to exhibit hysteresis that vanish in the low and high-frequency ranges of substrate oscillation. However, the average (over a period) velocity as well as the average dissipation show hyperbolic increase with frequency to saturation. We have proposed an experimental protocol to realize such features using periodic stepwise injection of substrate at specified rates.

Improved Performance of OLEDs on Cellulose/Epoxy Substrate Using Adenine as a Hole Injection Layer

Organic light-emitting diodes (OLED) fabricated from all “natural” materials will lead to renewable, sustainable, and potentially inexpensive organic optoelectronics. Achieving this goal will require reimagining all the various aspects of the OLED with natural-based renewable materials. Here, we replace the common substrate, electrode, and hole injection layer in the OLED structure with cellulose, gold, and the DNA nucleobase adenine. Gold films are used as semitransparent electrodes on plant-based cellulose substrates, providing flexible anodes that are highly conductive without annealing. A lift-off fabrication method (template stripping) employed UV curable epoxy to transfer patterned gold from silicon to cellulose. The gold has excellent adhesion to the epoxy (is not damaged when wiped), and the two serve as a smoothing layer for the cellulose substrate, resulting in very uniform OLED emission. DNA and DNA nucleobases are demonstrating to be versatile materials in natural electronics due to their wide...

Evaluation of the Antithrombin Potential in a Thrombin Generation Assay Performed at the Surface of Endothelial Cells

Thrombin generation assay (TGA) was recently evaluated on a living endothelial-derived cell line (Coll et al. J. Thromb. Haemost. 2013; 11, 1916). This innovative assay brought into an hemostasis assay the cellular components of the anticoagulation pathway (APC and TFPI pathways) as well as a activated cell surface. It might help elucidate the relationship between hemostasis and inflammation in a more complex system.In the aim of evaluating the potential of antithrombin (AT) connecting both processes we set-up a similar assay on human vein endothelial cells (HUVEC). We first demonstrated that thrombin generation can be measured in flat-bottom 96-wells in factor IX-or factor VIII-deficient plasma substituted by either 0.1 or 1 U/ml of FIX or FVIII, respectively. Next, HUVEC were grown and expanded in a complete commercial medium (EndoGRO-LS, Millipore) for no more than 6 passages. Wells were then coated with gelatin 1% and cells seeded at 10,000 cells/well. The binding of plasma-derived AT (Aclotine ®, LFB; France dialyzed in cell culture medium) to HUVEC was demonstrated as being dose- (0.5; 1; 2.5 and 5 U/ml) and time- (0-6 hours) dependent. Saturating conditions were found using 2.5 U/ml AT for a 2h incubation. We also showed that the binding was moderately affected in the presence of heparin at concentrations up to 50 U/ml (loss of 19% of the signal) and not at all following an heparanase I+II+III treatment suggesting that another receptor(s) than cellular heparan sulfates being responsible for this interaction.The effect of AT on coagulation was then compared in the presence of cells or not. To do this cells were grown to confluence, washed with non-supplemented medium and incubated in the presence of the TGA mix (plasma containing AT or not, 0.5 pM Tissue Factor, 4 µM Phospholipids). The reaction was initiated by injection of the FluCa kit thrombin substrate (Stago). In the presence of HUVEC, the efficiency of thrombin generation from a control plasma (Unicalibrator, Stago) was decreased with a lag time increased (from 5.67 min to 6.83 min), the peak height diminished from 204.4 nM thrombin to 150.4 nM and the velocity from 55.8 nM/min to 33.4 nM/min. However, the overall amount of thrombin generated was less affected, diminishing from 1515.5 nM to 1482 nM. These data confirms that the presence of the HUVEC anticoagulants pathways can effectively diminish the thrombin generation.Without cells, the presence of 0.5, 1 or 2 U/ml AT dose-dependently decreased the generation of thrombin from the control plasma. The velocity was decreased by 23.2%, 57.6% and 75.5% and the peak height by 33.5%, 61.5% and 78.8%, respectively. When the same experiment was performed in the presence of HUVEC cells, the concentrations of AT similarly decreased the velocity by 34.2%, 54% and 70 % and the peak height by 39%, 59.1% 74.3%, respectively. There was no difference in the TGA parameters if AT was pre-incubated at the surface of the cells for up to 2h prior the TGA or if it was added extemporaneously. These results indicate that the presence of HUVEC did not modulate the in vitro effect of AT during coagulation. The effect of AT on the cell response during this process are in the process of being investigated with a particular focus on the anti-inflammatory properties of AT. DisclosuresCatieau:LFB Biotechnologies: Employment. Chtourou:LFB Biotechnologies: Employment. Plantier:LFB Biotechnologies: Employment.

Evaluation of imaging techniques for the assessment of tumour progression in an orthotopic rat model of malignant pleural mesothelioma†.

An orthotopic rat tumour recurrence model for malignant pleural mesothelioma (MPM) provides clinical similarity to patients and is useful for drug testing combined with surgical intervention. Importantly, a reliable imaging method is required allowing for noninvasive and repetitive evaluation of the tumour load. We compared the tumour load assessed by bioluminescence and magnetic resonance imaging (MRI) to the macroscopic tumour volume as a reference standard. A total of 500,000 syngeneic rat MPM cells transfected with luciferase were implanted underneath the parietal pleura of immunocompetent rats (n=13). From the second day after implantation, bioluminescence measurements of the tumour load expressed as the maximum bioluminescent intensity (photon/second) were performed daily after intraperitoneal injection of the luciferase substrate, d-luciferin, to observe the first occurrence of tumour. Six days after the first detection of tumour, bioluminescence, MRI and macroscopic tumour volume measurement were conducted. For MRI, a 4.7-Tesla small animal imager equipped with a 1H whole-body rat coil was employed using T2-weighted fast spin-echo sequences. Tumour burden (mm3) was quantified from magnetic resonance transverse images by two independent readers by manual segmentation. Finally, the tumour burden assessed by bioluminescence and MRI was correlated (Pearson's correlation) with the macroscopic measurement of tumour (ellipsoid) volume. In all rats, a single tumour nodule was found at the inoculation site with a median macroscopic volume of 46 mm3 (18-377 mm3). For tumour burden quantification of MRIs, we observed good interobserver correlation (R2=0.81, P<0.0001) as well as significant association with the macroscopic tumour volume (R2=0.59, P=0.002). However, the signal intensity of bioluminescence did not correspond to the macroscopic tumour volume (R2=0.01, P=0.76). MRI is a reliable and reproducible noninvasive in vivo imaging method for MPM tumour burden assessment for the present MPM model.

Simulations of Microbial-Enhanced Oil Recovery: Adsorption and Filtration

In the context of microbial-enhanced oil recovery (MEOR) with injection of surfactant-producing bacteria into the reservoir, different types of bacteria attachment and growth scenarios are studied using a 1D simulator. The irreversible bacteria attachment due to filtration similar to the deep bed filtration (DBF) is examined along with the commonly used reversible equilibrium adsorption (REA). The characteristics of the two models are highlighted. The options for bacteria growth are the uniform growth in both phases and growth of attached bacteria only. It is found that uniform growth scenario applied to filtration model provides formation of two oil banks during recovery. This feature is not reproduced by application of REA model or DBF with growth in attached phase. This makes it possible to select a right model based on the qualitative analysis of the experimental data. A criterion is introduced to study the process efficiency: the dimensionless time at which average recovery between pure water injection and maximum surfactant effect is reached. This characteristic recovery period (CRP) was studied as a function of the different MEOR parameters such as bacterial activity, filtration coefficients, and substrate injection concentrations. For both growth scenarios, there is a zone of optimal activity at which the CRP is minimal. Dependence of the CRP on substrate concentration for uniform growth scenario has also an optimal zone. Therefore, growth rate and the substrate concentration should be above a certain threshold value and still not be too high to obtain the minimum CRP. On the other hand, no such zone was found if the bacteria could grow only in the attached phase. Dependencies on both the injected concentration and filtration coefficient are monotonous in this case.

Lipid and 13C signatures of submicron and suspended particulate organic matter in the Eastern Tropical North Pacific: Implications for the contribution of Bacteria

The contribution of bacterial biomass to total particulate organic matter (POM) in the ocean, including exported material, is poorly constrained. To examine potential signatures for the presence and export of bacterioplankton and their detrital remains, here we provide a detailed compound-specific 13C characterization of fatty acids from membrane polar lipids obtained from a water column profile in the Eastern Tropical North Pacific. POM of submicron size (0.2–0.7μm; “X-POM”) was sampled and analyzed separately from the size class typically collected as “suspended” POM (0.7–53μm; “L-POM”). The distributions of polar head group classes, specific fatty acid side chains, and natural 13C contents all vary, both between particle size classes and with depth in the water column. In general, the polar lipids in submicron material – and by inference, lipids of bacterial origin – have higher 13C content than polar lipids from larger POM and are equally abundant. Lipid signatures from the photic zone appear to be partially conserved in the suspended pool during transit down the water column. However, bacterial heterotrophy and possibly chemoautotrophy partially overprint these surface signatures. In addition, active metabolisms in the oxygen minimum zone (OMZ) appear to mediate the disaggregation of POM transported from the surface, thus adding complexity to the pathways of mid-water carbon flux and providing additional organic substrates to the OMZ and below. This “substrate injection” may provide important fuel for the denitrification and anammox reactions. Finally, examination of 13C content in polar lipids provides a basis for new interpretation of depth-related variations in δ13C values of bulk suspended POM.

Factors Affecting the Performance of Single Chamber Microbial Fuel Cell Using a Novel Configuration

In this study, the single chamber microbial fuel cell (MFC) with annular configuration and spiral anode geometry is proposed for future large-scale applications. Dairy wastewater containing complex organic matter was used as substrate. Factors affecting power generation and internal resistance of this MFC such as substrate injection, electrode surface area, and substrate concentration were investigated to elucidate the effects of the MFC configuration and operating conditions on its performance. The results show that with decreasing anode surface area output current is reduced. With decreasing anode surface area from 2×63 to 2×38 cm, output current is reduced about 34%. In measurement of open circuit voltage, replacement of old wastewater by fresh ones the open circuit voltage significantly increased. Also, enhancement of wastewater concentration shows an increase in the MFC power density. The MFC which was fed with wastewater with lower COD had a shorter batch time than that of the higher wastewater COD.

A system for accurate and automated injection of hyperpolarized substrate with minimal dead time and scalable volumes over a large range

Over recent years hyperpolarization by dissolution dynamic nuclear polarization has become an established technique for studying metabolism in vivo in animal models. Temporal signal plots obtained from the injected metabolite and daughter products, e.g. pyruvate and lactate, can be fitted to compartmental models to estimate kinetic rate constants. Modeling and physiological parameter estimation can be made more robust by consistent and reproducible injections through automation. An injection system previously developed by us was limited in the injectable volume to between 0.6 and 2.4ml and injection was delayed due to a required syringe filling step. An improved MR-compatible injector system has been developed that measures the pH of injected substrate, uses flow control to reduce dead volume within the injection cannula and can be operated over a larger volume range. The delay time to injection has been minimized by removing the syringe filling step by use of a peristaltic pump. For 100μl to 10.000ml, the volume range typically used for mice to rabbits, the average delivered volume was 97.8% of the demand volume. The standard deviation of delivered volumes was 7μl for 100μl and 20μl for 10.000ml demand volumes (mean S.D. was 9 ul in this range). In three repeat injections through a fixed 0.96mm O.D. tube the coefficient of variation for the area under the curve was 2%. For in vivo injections of hyperpolarized pyruvate in tumor-bearing rats, signal was first detected in the input femoral vein cannula at 3–4s post-injection trigger signal and at 9–12s in tumor tissue. The pH of the injected pyruvate was 7.1±0.3 (mean±S.D., n=10). For small injection volumes, e.g. less than 100μl, the internal diameter of the tubing contained within the peristaltic pump could be reduced to improve accuracy. Larger injection volumes are limited only by the size of the receiving vessel connected to the pump.

Simultaneous Degradation of Trichloroethylene and Toluene by Burkholderia cepacia G4 and the Effect of Biotransformation on Bacterial Density

The effect of biomass density, effect of different ratios of trichloroethylene (TCE) and toluene on biomass, and strategies to cope with problems using acclimated Burkholderia cepacia G4 were investigated. Complete degradation of TCE was achieved when B. cepacia G4 was exposed to 0.5 mg/L TCE and 10 mg/L toluene after starvation of 100 h. Overall results of this study show that degradation rates of toluene decreased as TCE concentration increased. On the contrary, degradation rates of TCE increased up to 10 mg/L TCE and then decreased. Transformation capacity of B. cepacia G4 increased as toluene concentration increased but transformation yield decreased showing that excessive substrate supply may not help to improve specific performance parameters. Results of these experiments bear that instead of simultaneous injection of TCE and substrate, the supply of substrate in pulses can increase the efficiency of B. cepacia G4 or similar bacterial strains.

IRFP is a sensitive marker for cell number and tumor growth in high-throughput systems

GFP and luciferase are used extensively as markers both in vitro and in vivo although both have limitations. The utility of GFP fluorescence is restricted by high background signal and poor tissue penetrance. Luciferase throughput is limited in vitro by the requirement for cell lysis, while in vivo, luciferase readout is complicated by the need for substrate injection and the dependence on endogenous ATP. Here we show that near-infrared fluorescent protein in combination with widely available near-infrared scanners overcomes these obstacles and allows for the accurate determination of cell number in vitro and tumor growth in vivo in a high-throughput manner and at negligible per-well costs. This system represents a significant advance in tracking cell proliferation in tissue culture as well as in animals, with widespread applications in cell biology.

Impact of electrical stress on the electrical characteristics of 2 MeV electron irradiated metal-oxide-silicon capacitors with atomic layer deposited Al2O3, HfO2 and nanolaminated dielectrics

In this work, the impact of electrical stress on the electrical characteristics of 2MeV electron irradiated metal-oxide-silicon capacitors with atomic layer deposited (ALD) high permittivity (high-k) dielectric layers of Al2O3, HfO2 and a nanolaminate of them is evaluated. The aim is to investigate the susceptibility to electrical stress of the radiation effects created in irradiated MOS structures, paying especial attention to any possible interaction between the radiation-induced damage and the subsequent electrical stress degradation. For this study, MOS capacitors with a nominal dielectric physical thickness of 10nm (equivalent oxide thickness (EOT) between 3nm and 7nm) on different p-type and n-type silicon substrates were investigated. An exponentially increasing stress current was forced to flow in accumulation through the different dielectric layers, registering the evolution of gate voltage versus stress time until dielectric breakdown occurred. Capacitance–voltage characteristics of the different irradiated and non-irradiated structures are analyzed as a function of electrical stress. Different charge trapping behaviors and significant polarity dependence in interface state generation are observed for the dielectric layers subjected to substrate and gate injections. No clear interaction between radiation and electrical stress damages is noticed under substrate injection (n-type samples). However, higher negative charge trapping near the metal/dielectric interface is registered for the irradiated Al2O3 and nanolaminate layers subjected to gate injection and, in the case of the most irradiated samples, the radiation-induced interface states damage is found to dominate against the damage generated in early stages of the electrical stress.

MR Signal Amplification for Imaging of the Mutant EGF Receptor in Orthotopic Human Glioma Model

To investigate the potential of targeted MR signal amplification strategy for imaging of EGF receptor variant III (EGFRvIII) overexpression associated with the infiltrating margin of aggressive orthotopic brain tumors. F(ab')2 fragments of humanized anti-EGFRvIII monoclonal antibody (EMD72000) were linked to deglycosylated horseradish peroxidase (HRP) and glucose oxidase (GOX). Detection of the F(ab')2 conjugate pair colocalization in vivo was enabled by a subsequent IV injection of a low molecular weight paramagnetic substrate of HRP, diTyr-GdDTPA. The delivery of the targeted fragments to the tumor was validated using SPECT/CT imaging of radiolabeled anti-EGFRvIII F(ab')2 conjugates. Further, by using 3T MRI, we observed time-dependent differences in tumor signal intensity and signal retention at the endpoint depending on whether or not the animals were pre-injected with the anti-EGFRvIII F(ab')2 conjugates. Imaging of EGFRvIII expression in vivo was enabled by consecutive administration of targeted F(ab')2 conjugates and a paramagnetic substrate resulting in a tumor-specific receptor detection with high specificity and resolution.

Advanced high-k gate dielectric amorphous LaGdO3 gated metal-oxide-semiconductor devices with sub-nanometer equivalent oxide thickness

Careful selection of pulsed laser deposition conditions was executed to achieve sub-nanometer EOT (equivalent oxide thickness) in amorphous LaGdO3 based high-k/metal gate stacks. The lowest EOTs attained were ∼5.4 Å and 8.4 Å with and without quantum mechanical correction, respectively. The electrical measurements yielded a high permittivity of 20.5 ± 2.4, a thin bottom interfacial layer of thickness 4.5 ± 1 Å, and interface (cm−2 eV−1) and fixed (cm−2) charge densities of ∼1012. Analysis of temperature dependent leakage currents revealed that gate injection current was dominated by Schottky emission below 1.2 MV/cm and quantum mechanical tunneling above this field. The physical origin of substrate injection was found to be a combination of Schottky emission and trap assisted tunneling.

Comparative Study of Atomic-Layer-Deposited Stacked (HfO2/Al2O3) and Nanolaminated (HfAlOx) Dielectrics on In0.53Ga0.47As

The high-k gate dielectric structures in stacked (HfO2/Al2O3) and nanolaminated (HfAlOx) forms with a similar apparent accumulation capacitance were atomic-layer-deposited on n-type In0.53Ga0.47As substrates, and their electrical properties were investigated in comparison with a single-layered HfO2 film. Al-oxide interface passivation in both forms proved to be effective in preventing a significant In incorporation in the high-k film and reducing the interface state density. The measured valence band spectra in combination with the reflection electron energy loss spectra were used to extract the energy band parameters of various dielectric structures on In0.53Ga0.47As. A further decrease in the interface state density was achieved in the stacked structure than in the nanolaminated structure. However, in terms of the other electrical properties, the nanolaminated sample exhibited better characteristics than the stacked sample, with a smaller border trap density and lower leakage current under substrate injection conditions with and without voltage stressing.

In-situ zinc bioprecipitation by organic substrate injection in a high-flow, poorly reduced aquifer

We investigated if in-situ metal bioprecipitation (ISMP) is applicable to remediate a highly permeable zinc-contaminated aquifer at a metal-processing factory in Maasmechelen, Belgium. A large (more than 200m long and 70m wide) groundwater contamination plume has developed, with zinc concentrations in the range of 1-100mg/L, whereas the legal Flemish clean-up standard is 0.5mg/L. The estimated groundwater flow velocity is in the range 0.2-1m/d. The groundwater is relatively oxidized, naturally low in DOC (<1mg/L) and relatively low in sulfate (40-50mg/L). We conducted both laboratory feasibility tests as well as a long-term field pilot test in two sections of the plume. In the laboratory microcosm tests, zinc bioprecipitation (following addition of organic substrate and sulfate) removed more than 99% of the zinc from the water phase. Lactate, glycerol and vegetable oil were equally effective as substrates. 28-day anaerobic leaching tests indicated that the metal precipitates that were formed are stable, but they also suggested that substrate addition increases the solubility (leachability) of arsenic and manganese. In the field test, Zn concentrations were reduced by 2 to 3 orders of magnitude within the 232 day testing period and stayed low for the following 6 months in both pilot zones. In the field, no mobilization of arsenic occurred but manganese groundwater concentrations increased from 0.01-0.6mg/L to 0.4-6.5mg/L. Dissolved iron concentrations also increased markedly from below detection limits to concentrations as high as 67mg/L. Zinc concentrations in groundwater were closely correlated to pH and redox potential (Eh): plotting y=[Zn] against x=pH/log(Eh), an exponential relationship was found:

Neural network processing of microbial fuel cell signals for the identification of chemicals present in water

Biosensing is emerging as an important element of water quality monitoring. This research demonstrated that microbial fuel cell (MFC)-based biosensing can be integrated with artificial neural networks (ANNs) to identify specific chemicals present in water samples. The non-fermentable substrates, acetate and butyrate, induced peak areas (PA) and peak heights (PH) that were generally larger than those caused by the injection of fermentable substrates, glucose and corn starch. The ANN successfully identified peaks associated with these four chemicals under a variety of experimental conditions and for two MFCs that had different levels of sensitivity. ANNs that employ the hyperbolic tangent sigmoid transfer function performed better than those using non-continuous transfer functions. ANNs should be integrated into water quality monitoring efforts for smart biosensing.

Charge trapping and electrical degradation in atomic layer deposited Al2O3 films

In this work, we focus on the effects of electrical stress on the charge trapping behavior and electrical degradation of Al2O3 layers deposited by ALD. For this purpose, current–voltage (I–V) and capacitance–voltage (C–V) characteristics of Al/Al2O3/n+Si capacitors are analyzed before and after constant voltage stress (CVS). The results show that electron trapping and hard breakdown (HBD) phenomena are the main degradation mechanisms observed under positive bias stress (or substrate injection). While, the generation of positive charge and new defects as well as the occurrence of soft breakdown (SBD) and progressive breakdown (PBD) failure modes, are identified as the dominant contributions when a highly enough negative bias (or gate injection) stress is applied.

Boosting Bioluminescence Neuroimaging: An Optimized Protocol for Brain Studies

Bioluminescence imaging is widely used for optical cell tracking approaches. However, reliable and quantitative bioluminescence of transplanted cells in the brain is highly challenging. In this study we established a new bioluminescence imaging protocol dedicated for neuroimaging, which increases sensitivity especially for noninvasive tracking of brain cell grafts. Different D-Luciferin concentrations (15, 150, 300 and 750 mg/kg), injection routes (iv, ip, sc), types of anesthesia (Isoflurane, Ketamine/Xylazine, Pentobarbital) and timing of injection were compared using DCX-Luc transgenic mice for brain specific bioluminescence. Luciferase kinetics was quantitatively evaluated for maximal photon emission, total photon emission and time-to-peak. Photon emission followed a D-Luciferin dose-dependent relation without saturation, but with delay in time-to-peak increasing for increasing concentrations. The comparison of intravenous, subcutaneous and intraperitoneal substrate injection reflects expected pharmacokinetics with fastest and highest photon emission for intravenous administration. Ketamine/Xylazine and Pentobarbital anesthesia showed no significant beneficial effect on maximal photon emission. However, a strong difference in outcome was observed by injecting the substrate pre Isoflurane anesthesia. This protocol optimization for brain specific bioluminescence imaging comprises injection of 300 mg/kg D-Luciferin pre Isoflurane anesthesia as an efficient and stable method with a signal gain of approx. 200% (compared to 150 mg/kg post Isoflurane). Gain in sensitivity by the novel imaging protocol was quantitatively assessed by signal-to-noise calculations of luciferase-expressing neural stem cells grafted into mouse brains (transplantation of 3,000–300,000 cells). The optimized imaging protocol lowered the detection limit from 6,000 to 3,000 cells by a gain in signal-to-noise ratio.

Oxygen Plasma Treatment of Platinized Ultramicroelectrodes Increases Sensitivity for Hydrogen Peroxide Detection on Mitochondria

AbstractHydrogen peroxide is a major bio‐active molecule produced by mitochondria during aerobic metabolism. In order to monitor that production, platinized ultramicroelectrodes with high surface‐area were used. Their sensitivity was significantly improved following an oxygen plasma treatment. Cyclic voltammetry and chronoamperometry analyses of ferricyanide reduction and hydrogen peroxide oxidation showed that ultramicroelectrodes’ capacitive current was decreased 3‐fold, while faradaic currents remained constant. Then, plasma‐treated ultramicroelectrodes allowed the chronoamperometric detection of 100 nM H2O2 injections. This eventually enabled monitoring the mitochondria release of H2O2 following injection of substrates and a respiratory inhibitor; a flux of few hundreds picomoles H2O2/mg protein/min was detected.