The antioxidant capacity of erythrocyte concentrates is increased during the first week of storage and correlated with the uric acid level.

BACKGROUND AND OBJECTIVES: Red blood cells (RBCs) suffer from lesions during cold storage, depending in part on their ability to counterbalance oxidative stress by activating their antioxidant defence. The aim of this study was to monitor the antioxidant power (AOP) in erythrocyte concentrates (ECs) during cold storage. MATERIALS AND METHODS: Six ECs were prepared in saline-adenine-glucose-mannitol (SAGM) additive solution and followed during 43 days. The AOP was quantified electrochemically using disposable electrode strips and compared with results obtained from a colorimetric assay. Haematological data, data on haemolysis and the extracellular concentration of uric acid were also recorded. Additionally, a kinetic model was developed to extract quantitative kinetic data on the AOP behaviour. RESULTS: The AOP of total ECs and their extracellular samples attained a maximum after 1 week of storage prior to decaying and reaching a plateau, as shown by the electrochemical measurements. The observed trend was confirmed with a colorimetric assay. Uric acid had a major contribution to the extracellular AOP. Interestingly, the AOP and uric acid levels were linked to the sex of the donors. CONCLUSION: The marked increase in AOP during the first week of storage suggests that RBCs are impacted early by the modification of their environment. The AOP behaviour reflects the changes in metabolism activity following the adjustment of the extracellular uric acid level. Knowing the origin, interdonor variability and the effects of the AOP on the RBCs could be beneficial for the storage quality, which will have to be further studied.

Aluminium foil as a single-use substrate for MALDI-MS fingerprinting of different melanoma cell lines.

Herein, we present the intact cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the fingerprinting of human melanoma cancer cell lines grown on aluminium foil. To perform the MALDI-MS assay, melanoma cells were cultured on a flat and thin foil, which was directly transferred to the target plate of MALDI-MS for analysis. The influence of a wide range of cell fixation protocols (i.e. formalin-based and alcohol-based methods) and MALDI matrices on the obtained characteristic spectra was investigated. For the optimization of the MALDI-MS protocol, the MS fingerprints of the melanoma WM-239 cell line with and without an overexpressed enhanced green fluorescent protein were employed. The fingerprints obtained from WM-239 cells grown on aluminium foil were compared with the intact cell MALDI-MS of the cell pellet and presented higher sensitivity in a high m/z range. The optimized protocol was subsequently applied to characterise melanoma cell lines derived from different cancer stages and allowed identification of unique MS signals that could be used for differentiation between the studied cell lines (i.e. molecular weight equal to 10.0 kDa and 26.1 kDa).

Modeling the isoelectric focusing of peptides in an OFFGEL multicompartment cell.

In proteomic analysis of complex samples at the peptide level (termed shotgun proteomics), an effective prefractionation is crucial to decrease the complexity of the peptide mixture for further analysis. In this perspective, the high-resolving power of the IEF fractionation step is a determining parameter, in order to obtain well-defined fractions and correct information on peptide isoelectric points, to provide an additional filter for protein identification. Here, we explore the resolving power of OFFGEL IEF as a prefractionation tool to separate peptides. By modeling the peak width evolution versus the peptide charge gradient at pI, we demonstrate that for the three proteomes considered in silico (Deinococcus radiodurans, Saccharomyces cerevisiae, and Homo sapiens), 90% of the peptides should be correctly focused and recovered in two wells at most. This result strongly suggests OFFGEL to be used as a powerful fractionation tool in shotgun proteomics. The influence of the height and shape of the compartments is also investigated, to give the optimal cell dimensions for an enhanced peptide recovery and fast focusing time.

Nanowires network for biomolecular detection using contactless impedance tomoscopy technique.

Here we present the detection of ultralow concentrations of biomolecules in a device made from a polycarbonate membrane containing a network of gold nanowires and using a "contactless" impedance tomoscopy technique. The sensor comprises a thin dielectric layer with two parallel band electrodes on the one side and a microchannel containing gold nanowires onto which the adsorption of antibodies occurs. Upon applying a high-frequency ac voltage between the two electrodes, the adsorption process occurring at the surface of the gold nanowires can be followed through contactless impedance measurements. The configuration allows the real-time detection of biomolecules with a bulk concentration in the picomolar range.

Electrochemical multi-tagging of cysteinyl peptides during microspray mass spectrometry: numerical simulation of consecutive reactions in a microchannel.

On-line electrogeneration of mass tags in a microspray emitter is used to quantify the number of cysteine groups in a given peptide. A finite-element simulation of the multi-step process yields the relative distribution and concentration of tags, untagged and tagged species in the microchannel before the spray event. The work focuses on the tagging of cysteine moieties in peptides or proteins by electrogenerated quinone mass probes. The main chemical parameters determining the kinetics of the labelling are assessed and discussed considering the microfluidic aspects of the process. The control of the tagging extent allows the simultaneous MS analysis of both the unmodified and modified peptide(s). The number of cysteine groups corresponds to the number of characteristic mass shifts observed from the unmodified peptide. The present theoretical work establishes the range of optimum conditions for the determination of the number of cysteine groups in peptides containing up to five cysteine groups.

Preparation of silver nanoparticles in solution from a silver salt by laser irradiation.

A new method is proposed for the fabrication of a well-defined size and shape distribution of silver nanoparticles in solution; the method employs direct laser irradiation of an aqueous solution containing a silver salt and a surfactant in the absence of reducing agents.

Generalization of ionic partition diagrams to lipophilic compounds and to biphasic systems with variable phase volume ratios.

The ionic partition diagram methodology has been generalized to address both hydrophilic and lipophilic compounds and to consider biphasic systems with variable phase volume ratios. With this generalized approach electrochemical measurements of ion transfer potentials afford the determination of the standard partition coefficients of all forms of ionizable molecules, including the neutral form, as well as the evaluation of the dissociation constant of monoprotic substances. An interesting consequence of this approach is the definition of an extraction pK(a,ext) which is the apparent pK(a) of neutral acids and bases when dissolved in the organic phase.

Photomodification of polymer microchannels induced by static and dynamic excimer ablation: effect on the electroosmotic flow.

This paper presents a study of polymer surfaces modified by laser ablation using poly(ethylene terephthalate) (PET) as a model system. The surface properties induced by static and dynamic ablation with the 193-nm pulsed radiation of an ArF excimer laser (4 x 10(7) W/cm2) in air have been successfully used to control the electroosmotic flow (EOF) in photoablated PET microchannels. Through the creation of well-defined static ablation patterns onto the walls of a trapezoidal channel, it was found that the resulting reduction in the EOF could be controlled. For example, a reduction of 25% in the EOF was observed in 42-microm-deep microchannels when using a static ablation pattern treating 50% of the total wall surface area. A numerical study describing the fluidic behavior induced by a static pattern is also presented. Moreover, X-ray photoelectron spectroscopy has been used to point out surface changes between static and dynamic ablation, thereby demonstrating an ability to create new functionalities in microchannels by laser treatment.

The apparent lipophilicity of quaternary ammonium ions is influenced by galvani potential difference, not ion-pairing: a cyclic voltammetry study.

PURPOSE: This work examines whether ion-pairing contributes to the apparent lipophilicity of cations, which is seen by a shake-flask or titrimetic method to be influenced by the nature and concentration of counter-ions. METHODS: To solve this problem, the lipophilicity of several quaternary ammonium drugs was measured by cyclic voltammetry in the 1,2-dichloroethane/water system. The standard ionic partition coefficient values so obtained (log Pdce(o,C)) were correlated with log Poct values calculated by the CLOGP algorithm for the respective neutral molecules. RESULTS: The standard (i.e., intrinsic) lipophilicity values are shown to depend on a, the structure of the ion (nature, volume, charge), and b, on the Galvani potential difference at the ITIES (interface between two immiscible electrolyte solutions). CONCLUSIONS: The standard lipophilicity values were not influenced by counter-ions. In contrast, simulations showed that the increased apparent lipophilicity of cations, as measured by the shake-flask method in the presence of lipophilic anions, is fully accounted for by the resulting increase in the Galvani potential difference.

Electroosmotic flow in composite microchannels and implications in microcapillary electrophoresis systems.

The electroosmotic flow in laminated excimer laser-ablated microchannels has been studied as a function of the depth of the rectangular channels, and particular emphasis has been given to the difference in the zeta-potentials between the lamination layer and the ablated substrate. Experimental electroosmotic flow follows the tendency predicted by a recently published model. The zeta-potentials of lamination and ablated surfaces were determined for poly(ethylene terephthalate) and poly(carbonate) substrates by fitting the experimental data with a numerical implementation of this model. In the experimentally investigated range of channel cross sections, a linear fit to the data gives a good approximation of the zeta-potentials for both materials. Moreover, a flow injection analysis of fluorescein dye has been performed to show the severe loss in numbers of theoretical plates, caused by Taylor dispersion, when such microchannels, dedicated to microcapillary electrophoresis, are used.

MicroITIES detection of nitrate by facilitated ion transfer.

A microITIES array, created by laser photoablation of a 12-microm polyester film, was used to investigate electroassisted anion transfer between two immiscible electrolyte solutions. Besides measuring directly the transfer of nitrate to the organic phase, the enhancement of transfer of the cation (K+) by facilitated anion (counterion) transfer was measured as well. In the presence of a triamide derived from tris(2-aminoethyl)amine (tren), which is known to function as a host for nitrate, the current responses for both nitrate and potassium transfer were monitored. The linear relationships between the current responses and nitrate concentration formed the basis of an anion sensor with a dynamic range from 0.1 to 5 mM. A dual facilitated transfer mechanism is proposed to explain the enhancement phenomenon.

Polymer microspray with an integrated thick-film microelectrode.

A microfabrication process leading to a sheathless electrospray interface for mass spectrometry analysis is described. Photoablation is performed on a polymer substrate, allowing the integration of a thick-film conductive track in a sealed microchannel. High voltage is supplied close to the outlet, through an embedded microelectrode. The microspray is generated directly from the edge of the substrate without any tip addition. The flexibility of this technology provides a wide range of dimensions for the probe and the microelectrode design, including location, shape, and conductive material used. Thanks to the thick-film microelectrode and the hydrophobicity of the polymer, which avoids solution spreading at the outlet, the device has been found to be an efficient ionization source providing a stable MS signal through time. Moreover, the same device can be used several times without failure. The performance of the microspray has been studied in simple infusion mode for proteins and reserpine MS analyses. The detection limit of reserpine was found to be at the picomolar level in full-scan MS mode. It implies also that approximately 500 zmol was read consumed during 3 min of infusion. A dynamic range from pico- to millimolar level is also underlined.

Enzyme linked immunosorbent assay on a microchip with electrochemical detection.

This paper presents the development of a sandwich immunoassay in disposable plastic microchips. Photoablated microchannels with integrated electrodes have been used for the development of enzyme-linked-immunosorbent-assay (ELISA). The presence of the electrode inside the 40 nL microchannel enables the detection of the redox active enzyme substrate directly inside the reaction channel. Furthermore, due to the small diffusion distances, each incubation time can be reduced to five minutes instead of a few hours in standard microtiterplates. The initial characterisation of this immunoassay has been performed with a large protein complex D-Dimer-alkaline phosphatase. This system was used for the detection of immobilised antibodies on the surface of the photoablated microchannel. In a second step, a sandwich immunoassay with a horseradish peroxidase-secondary antibody conjugate (HRP-conjugate) was used to detect D-Dimer between 0.1 and 100 nM, which is the relevant concentration range of the clinical tests.

Pulse amperometric detection of salt concentrations by flow injection analysis using ionodes.

A sensitive novel approach of using an amperometric ion detector for the flow injection analysis of salts has been developed. The detection methodology is based on measuring the current associated with the transfer of ions across polarized microinterfaces between the aqueous sample solution and a 2-nitrophenyloctyl ether-poly(vinyl chloride) gel phase, referred to as ionodes. Different sodium salts of fluoride, chloride, bromide, nitrate, and sulfate were investigated. It was found that by employing an amperometric pulse detection mode and pure water as eluent, the detection limit of the ionode detector could be lowered to ppt level of salt concentrations under flowing conditions.

Second harmonic generation of glucose oxidase at the air/water interface.

We present a study of the adsorption of the glucose oxidase enzyme (GOx) at the air/water interface, using the nonlinear optical technique of surface second harmonic generation (SSHG). Resonant SSHG experiments were achieved by probing the pi-pi* transition of the flavin adenine dinucleotide (FAD) chromophores embedded in the GOx protein. Because of the subsequent resonance enhancement of the signal, the second harmonic (SH) wave arising from the GOx entities adsorbed at the interface was detectable for protein bulk aqueous concentrations as low as 70 nM. The protein adsorption was followed, and, at high GOx coverage, a change in the orientation of the FAD chromophore was observed, indicating either a rearrangement or a reorientation of the protein at the interface. Inasmuch as GOx is negatively charged at the biological pH of 7, its interactions with charged surfactants were also investigated. As expected, spreading positively charged surfactants onto a partial protein monolayer was found to increase the GOx surface concentration, whereas in the case of negatively charged surfactants, the GOx surface concentration decreased until the SH signal went back to the pure buffer solution response level. With the increasing GOx surface concentration, the rearrangement or reorientation of the protein was also observed.

The pH-partition profile of the anti-ischemic drug trimetazidine may explain its reduction of intracellular acidosis.

PURPOSE: The anti-ischemic drug trimetazidine (TMZ) acts by a combination of molecular mechanisms which begin to be understood. Thus, it acts in the micromolar range to significantly reduce intracellular acidification during ischemia. To search for a possible physicochemical explanation of this phenomenon, we investigated the transfer mechanisms of the various electrical forms of this dibasic drug. METHODS: The transfer characteristics of TMZ were studied by electrochemistry at the water/1,2-dichloroethane interface. Cyclic voltammetry was used to measure the formal transfer potentials of singly and doubly protonated forms of TMZ (noted TH+ and TH(2)2+, respectively) as a function of aqueous pH, and the partition coefficient of neutral TMZ (log P(T)) was measured by two-phase titration. RESULTS: log P(T) was measured to be 1.04 +/- 0.06, and the acid-base dissociation constants in water were deduced to be pK(w)a1 = 4.54 +/- .02 and pK(w)a2 = 9.14 +/- 0.02. The partition coefficients of TH+ and TH(2)2+ were found to be respectively log P0'TH+ = -3.78 +/- 0.16 and log P0'TH(2)2+ = -9.84 +/- 0.30, which agrees well with the charge being delocalized on two nitrogen atoms in TH+. The pH-partition profile of TMZ was then established in the form of its ionic partition diagram, which showed that the affinity of the ions for the organic phase is pH-dependent and strongly increased by the interfacial potential. CONCLUSIONS: This behavior suggests a physicochemical mechanism whereby efflux of protonated TMZ out of an acidified cell is facilitated, in effect exporting protons to extracellular space.

Microchannel networks for electrophoretic separations.

UV excimer laser photoablation was used to micro-machine polymer substrates not only to drill microchannel structures but also to change the surface physical properties of the substrates. We first describe how UV laser photoablation can be used for the patterning of biomolecules on a polymer and discuss parameters such as surface coverage of active antibodies and equilibration time. Secondly, we show how to design a single-use capillary electrophoresis system comprising an on-chip injector, column and electrochemical detector. The potential of this disposable plastic device is discussed and briefly compared to classical systems. Finally, preliminary results on protein separation by isoelectric focusing on a disposable microchip are presented.

Electrochemical detection in polymer microchannels.

A method, using UV laser photoablation, is presented for the fabrication and the integration of an electrochemical detector in a microchannel device, where carbon microband electrodes are placed either in the bottom or in the side walls of the rectangular microchannel. The different electrochemical cell geometries are tested with a model compound (ferrocenecarboxylic acid) in 40- and 100-µm-wide capillaries fabricated in planar polymer substrates. The experimental results are compared to numerical simulations for stagnant stream conditions. Depending on the scan rate and on the microchannel depth, the system behaves as a microband electrode until a linear diffusion field develops within the channel. The limit of detection for a one electron redox species within the 120-pL detection volume is ∼1 fmol with both cyclic voltammetry and chronoamperometric detection.

Charge effects on phospholipid monolayers in relation to cell motility.

It is shown that the interfacial free energy of adsorbed phospholipid layers is dependent on the interfacial inner potential difference and on the local pH at the surface. Gradients of potential and pH result in the onset of Marangoni hydrodynamic convective fluxes at the phase adjoining a phospholipid monolayer. It is proposed that the coupling between potential, pH and interfacial Gibbs energy can provide an elementary driving force for cell and organelle motility, phagocytosis and streaming effects. It is shown that the order of magnitude of the surface shear stress generated by surface tension gradients is sufficient to account for cytoplasmic streaming in cells.