Biomedical subjects

Joël S Rossier

Publications and source records attributed to Joël S Rossier.

16 recordsLinked to original sources

PubMed2006

Disposable microfluidic ELISA for the rapid determination of folic acid content in food products.

A micro-analytical system for rapid and quantitative analysis by inhibition immunoassay is presented and applied to the detection of folic acid. Eight polymer microchannels of 65-nL volume each and containing microelectrodes are embedded in a cartridge so that they can be operated simultaneously. All fluidic steps as well as the amperometric detection in the channels are operated by an instrument and software developed in-house. The fluidic steps of the immunoassay occur through hydrodynamic loading of the different solutions through the channels. The speed and duration of the flow and incubation parameters can thus be adapted to the biological and testing requirements. The effectiveness of the system was demonstrated by analysing folic acid concentrations in real infant formula samples within 5 min. In an effort to get a fully monitored assay, each fluidic step is monitored thanks to continuous amperometric detection of oxygen in the microchannel.

Alkaline Phosphatase↗

PubMed2006

Identification of brain cell death associated proteins in human post-mortem cerebrospinal fluid.

Following any form of brain insult, proteins are released from damaged tissues into the cerebrospinal fluid (CSF). This body fluid is therefore an ideal sample to use in the search for biomarkers of neurodegenerative disorders and brain damage. In this study, we used human post-mortem CSF as a model of massive brain injury and cell death for the identification of such protein markers. Pooled post-mortem CSF samples were analyzed using a protocol that combined immunoaffinity depletion of abundant CSF proteins, off-gel electrophoresis, SDS-PAGE and protein identification by LC-MS/MS. A total of 299 proteins were identified, of which 172 proteins were not previously described to be present in CSF. Of these 172 proteins, more than 75% have been described as intracellular proteins suggesting that they were released from damaged cells. Immunoblots of a number of proteins were performed on individual post-mortem CSF samples and confirmed elevated concentrations in post-mortem CSF compared to ante-mortem CSF. Interestingly, among the proteins specifically identified in the post-mortem CSF, several have been previously described as biochemical markers of brain damage.

Biomarkers↗

PubMed2004

Gravity-induced convective flow in microfluidic systems: electrochemical characterization and application to enzyme-linked immunosorbent assay tests.

A way of using gravity flow to induce a linear convection within a microfluidic system is presented. It is shown and mathematically supported that tilting a 1 cm long covered microchannel is enough to generate flow rates up to 1000 nL.min(-1), which represents a linear velocity of 2.4 mm.s(-1). This paper also presents a method to monitor the microfluidic events occurring in a covered microchannel when a difference of pressure is applied to force a solution to flow in said covered microchannel, thanks to electrodes inserted in the microfluidic device. Gravity-induced flow monitored electrochemically is applied to the performance of a parallel-microchannel enzyme-linked immunosorbent assay (ELISA) of the thyroid-stimulating hormone (TSH) with electrochemical detection. A simple method for generating and monitoring fluid flows is described, which can, for instance, be used for controlling parallel assays in microsystems.

Electricity↗

PubMed2004

A thin chip microsprayer system coupled to Fourier transform ion cyclotron resonance mass spectrometry for glycopeptide screening.

A thin polymer microchip was coupled with a Fourier transform ion cyclotron resonance (FTICR) 9.4 T mass spectrometer and the method was optimized in negative ion mode for glycopeptide screening. The interface between the polymer microchip and FTICR mass spectrometer consists of an in-laboratory conceived and designed mounting system that exhibits robust and controllable alignment of the chip toward the inlet of the mass spectrometer. The particular attribute of the polymer chip coupled to the FTICR mass spectrometer, to achieve an increase in ionization efficiency and sensitivity under the premise of high mass accuracy of detection, is highlighted by the large number of major and minor glycopeptide structures detected and identified in highly heterogeneous mixtures obtained from urine matrices. Glycoforms expressing various saccharide chain lengths ranging from tri- to dodecasaccharide, bearing up to three sialic acid moieties, could be detected and assigned based on the accuracy of the mass measurement (average mass deviation below 6 ppm) of their molecular ions. -Thin chipESI-FTICRMS is a potent novel system for glycomic screening of complex mixtures, as demonstrated for identification of singly sialylated O-glycosylated amino acids and peptides from urine matrices, and could be considered for general applicability in the glycoanalytical field.

Amino Acid Sequence↗

PubMed2004

Why the move to microfluidics for protein analysis?

There has been a recent trend towards the miniaturization of analytical tools, but what are the advantages of microfluidic devices and when is their use appropriate? Recent advances in the field of micro-analytical systems can be classified according to instrument performance (which refers here to the desired property of the analytical tool of interest) and two important features specifically related to miniaturisation, namely reduction of the sample volume and the time-to-result. Here we discuss the contribution of these different parameters and aim to highlight the factors of choice in the development and use of microfluidic devices dedicated to protein analysis.

Animals↗

PubMed2004

Generation of mass tags by the inherent electrochemistry of electrospray for protein mass spectrometry.

We present herein a review of our work on the on-line electrochemical generation of mass tags toward cysteine residues in peptides and proteins. Taking advantage of the inherent electrochemical nature of electrospray generated from a microfabricated microspray emitter, selective probes for cysteine were developed and tested for on-line nonquantitative mass tagging of peptides and proteins. The nonquantitative aspect of the covalent tagging thus allows direct counting of free cysteines in the mass spectrum of a biomolecule through additional adduct peaks. Several substituted hydroquinones were investigated in terms of electrochemical properties, and their usefulness for on-line mass tagging during microspray experiments were assessed with L-cysteine, peptides, and intact proteins. Complementarily, numerical simulations were performed to properly understand the respective roles of mass transport, kinetics of electrochemical-chemical reactions, and design of the microspray emitter in the mass tagging overall efficiency. Finally, the on-line electrochemical tagging of cysteine residues was applied to the analysis of tryptic peptides of purified model proteins for protein identification through peptide mass fingerprinting.

Cysteine↗

PubMed2003

Microfluidic systems in proteomics.

We present the state-of-the-art in miniaturized sample preparation, immunoassays, one-dimensional and multidimensional analyte separations, and coupling of microdevices with electrospray ionization-mass spectrometry. Hyphenation of these different techniques and their relevance to proteomics will be discussed. In particular, we will show that analytical performances of microfluidic analytical systems are already close to fulfill the requirements for proteomics, and that miniaturization results at the same time in a dramatic increase in analysis throughput. Throughout this review, some examples of analytical operations that cannot be achieved without microfluidics will be emphasized. Finally, conditions for the spreading of microanalytical systems in routine proteomic labs will be discussed.

Electrophoresis↗

PubMed2003

Prefractionation techniques in proteome analysis.

The present review deals with a number of prefractionation protocols in preparation for two-dimensional map analysis, both in the fields of chromatography and in the field of electrophoresis. In the first case, Fountoulaki's groups has reported just about any chromatographic procedure useful as a prefractionation step, including affinity, ion-exchange, and reversed-phase resins. As a result of the various enrichment steps, several hundred new species, previously undetected in unfractionated samples, could be revealed for the first time. Electrophoretic prefractionation protocols include all those electrokinetic methodologies which are performed in free solution, essentially all relying on isoelectric focusing steps. The devices here reviewed include multichamber apparatus, such as the multicompartment electrolyzer with Immobiline membranes, Off-Gel electrophoresis in a multicup device and the Rotofor, an instrument also based on a multichamber system but exploiting the conventional technique of carrier-ampholyte-focusing. Other instruments of interest are the Octopus, a continuous-flow device for isoelectric focusing in a upward flowing liquid curtain, and the Gradiflow, where different pI cuts are obtained by a multistep passage through two compartments buffered at different pH values. It is felt that this panoply of methods could offer a strong step forward in "mining below the tip of the iceberg" for detecting the "unseen proteome".

Chemical Fractionation↗

PubMed2002

Finite element simulation of pinched pressure-driven flow injection in microchannels.

A pinched pressure-driven flow injection on a microchip is numerically simulated in order to optimize the relative values of the operational parameters. The geometry studied is a two-dimensional rectangular channel featuring a cross-junction with a large depth-over-width ratio. The hydrodynamic and convection-diffusion equations are solved for the two steps of the process: first, the sample solution is pinched into the transversal channel (injection channel), and then it is injected into the longitudinal one (separation channel), where the time evolution of the concentration is analyzed for different types of the detectors. Electroosmotic flow calculations have also been performed and have shown a good agreement with literature. The results for pressure-driven flow point out that the shape of the detection signal is strongly dependent on the velocity in the separation channel and on the position of the detection probes. The so-called double-humped peak, caused by the parabolic flow profile at high driving flow rate is analyzed. A tight pinch greatly decreases the amount of injected sample and, consequently, the signal sensitivity without increasing its quality. A proper pullback of the sample during the separation process can decrease the tailing due to the sample leakage from the injection channel. Although a high sample pullback causes a considerable decrease in the signal sensitivity, it also greatly enhances the peak resolution. Finally, it is shown that a wider injection channel with high sample pullback ensures an improved signal sensitivity with good resolution.

Journal Article↗

PubMed2002

Plasma etched polymer microelectrochemical systems.

This paper presents a novel technique based on plasma etching for the mass production of polymer microchip devices. The method consists of the patterning of a photo-resist by a high resolution printer on a foil composed of three layers (5 microm copper/50 microm polyimide/5 microm copper). After this step, both copper layers are chemically etched in order to serve as a contact mask on the polyimide surface so as to produce the desired microstructure pattern. The foil is placed into a reactive plasma chamber in order to etch the exposed polyimide by means of an oxidizing plasma. The method enables holes, lines or larger areas to be etched, thereby generating either microholes, microchannels or electrodes in the plastic material. The copper can then be chemically removed or further patterned to produce conductive pads which are further electroplated with gold. The microchannel is then covered with a polyethylene terephthalate/polyethylene (PET/PE) lamination. The strength of this technology is that access holes for the fluid inlet and outlet, as well as gold coated electrodes can be fabricated without post-processing in a batch process. Demonstration of the application of such microelectrochemical systems is shown here by voltammetric detection inside a 60 nL microchannel, which presents the special feature of linear depletion of the analytes in the direction parallel to the microchannel.

Journal Article↗

PubMed2002

Polymer microchips bonded by O2-plasma activation.

This paper presents a fabrication of polymer microchips with homogeneous material technique due to surface treatment by plasma before sealing. UV laser photoablation was used for fast prototyping of microstructures, and oxygen plasma was used as a surface treatment for both the microfabricated substrate and the polymer cover. It was found that with an oxidative plasma treatment, successful bonding could be achieved without adhesive material between polymer sheets substantially below the glass transition temperature of the polymer. Homogeneous polyethylene terephthalate (PET) microstructures were characterized by scanning electron microscopy (SEM) and analyzed by X-ray photoelectron spectroscopy (XPS) surface analyses after different surface treatments. The electroosmotic flow characteristics including the velocity and the stability over 20 days have been tested and compared to composite channels, in which the cover presents a polyethylene (PE) adhesive layer. Capillary zone electrophoresis in both homogeneous and composite microanalytical devices were then performed and compared in order to evaluate the separation efficiency. In preliminary experiments, a plate height of 0.6 microm has been obtained with homogenous microchannels. The surface analysis pointed out that the surface chemistry is of prime importance for the performance of microfluidic separation.

Biotinylation↗

PubMed2002

Finite element simulation of Off-Gel trade mark buffering.

The protonation of an aqueous solution of two ampholytes AH and BH next to a gel buffered by immobilized acid moieties IH has been studied by finite element simulation in an iterative scheme. A ten species model has been formulated, taking into account transient diffusion and equilibrium kinetics of the two amphoteric species AH and BH, of water and of the immobilized species IH. This model has been developed to illustrate the pH evolution between an ampholyte solution and an Immobiline gel, and to study the influence of the Immobiline concentration on protons and ampholyte distributions. It has been demonstrated that a minimum initial Immobiline concentration of 10(-2) M is necessary to maintain the pH in the gel in contact with a closed chamber, when the difference between the isoelectric points of AH and BH is 4 and when the initial concentration of the ampholytes in solution is in the micromolar range. This approach provides a first theoretical framework for the recently developed Off-Gel trade mark electrophoresis.

Buffers↗

PubMed2002

Integration of a membrane-based desalting step in a microfabricated disposable polymer injector for mass spectrometric protein analysis.

A simple desalting procedure for the coupling of a polymer microchip injector to mass spectrometry is proposed. The overall process is based on the adsorption of proteins on a poly(vinylidene difluoride) (PVDF) membrane, which are then directly eluted in the spraying solution. This microchip-based approach has been successfully applied to small drugs, peptides and proteins originally diluted in phosphate-buffered saline (PBS). Moreover, when eluting the retained proteins in small volumes, a preconcentration is obtained. The combination of single-use, mass-produceable, low-sample-consumption, easy-to-automate, miniaturized polymer injectors with easy-to-handle solution-exchange membranes makes this system particularly amenable to screening applications.

Angiotensin II↗

PubMed2002

Protein purification by Off-Gel electrophoresis.

A novel free-flow protein purification technique based on isoelectric electrophoresis is presented, where the proteins are purified in solution without the need of carrier ampholytes. The gist of the method is to flow protein solutions under an immobilised pH gradient gel (IPG) through which an electric field is applied perpendicular to the direction of the flow. Due to the buffering capacity of the IPG gel, proteins with an isoelectric point (pI) close to pH of the gel in contact with the flow chamber stay in solution because they are neutral and therefore not extracted by the electric field. Other proteins will be charged when approaching the IPG gel and are extracted into the gel by the electric field. Both a demonstration experiment with pI markers and a simulation of the electric field distribution are presented to highlight the principle of the system. In addition, an isoelectric fractionation of an Escherichia coli extract is shown to illustrate the possible applications.

Bacterial Proteins↗

PubMed2002

Microfabricated polymer injector for direct mass spectrometry coupling.

This paper demonstrates the coupling of a plasma etched polymer microfluidic system with an electrospray mass spectrometer by generation of a nanospray. Taking advantage of the microtechnology processes and polymer properties, high volume production with good reproducibility of hydrophobic interfaces could be obtained. The nanospray was directly produced from the outlet of the plastic microfabricated chip positioned in front of the capillary entrance of the mass spectrometer. No chemical background due to the polymer has been observed under standard nanospray conditions. The performances of the spray as well as its efficiency have been demonstrated by flow measurements, stability establishment and tandem mass spectrometry experiment on angiotensin II. The spray was actuated without additional flow in methanol: water:acetic acid (50:49:1%) solution. A 40 fmol/microL detection limit could be reached.

Angiotensin II↗

PubMedDate not supplied

Added value for tandem mass spectrometry shotgun proteomics data validation through isoelectric focusing of peptides.

A very popular approach in proteomics is the so-called "shotgun LC-MS/MS" strategy. In its mostly used form, a total protein digest is separated by ion exchange fractionation in the first dimension followed by off- or on-line RP LC-MS/MS. We replaced the first dimension by isoelectric focusing in the liquid phase using the Off-Gel device producing 15 fractions. As peptides are separated by their isoelectric point in the first dimension and hydrophobicity in the second, those experimentally derived parameters (pI and R(T)) can be used for the validation of potentially identified peptides. We applied this strategy to a cellular extract of Drosophila Kc167 cells and identified peptides with two different database search engines, namely PHENYX and SEQUEST, with PeptideProphet validation of the SEQUEST results. PHENYX returned 7582 potential peptide identifications and SEQUEST 7629. The SEQUEST results were reduced to 2006 identifications by validation with PeptideProphet. Validation of the PeptideProphet, SEQUEST and PHENYX results by pI and R(T) parameters confirmed 1837 PeptideProphet identifications while in the remainder of the SEQUEST results another 1130 peptides were found to be likely hits. The validation on PHENYX resulted in the fixation of a solid p-value threshold of <1 x 10(-04) that sets by itself the correct identification confidence to >95%, and a final count of 2034 highly confident peptide identifications was achieved after pI and R(T) validation. Although the PeptideProphet and PHENYX datasets have a very high confidence the overlap of common identifications was only at 79.4%, to be explained by the fact that data interpretation was done searching different protein databases with two search engines of different algorithms. The approach used in this study allowed for an automated and improved data validation process for shotgun proteomics projects producing MS/MS peptide identification results of very high confidence.

Algorithms↗