Headspace mass spectrometry methodology: application to oil spill identification in soils

In the present work we report the results obtained with a methodology based on direct coupling of a headspace generator to a mass spectrometer for the identification of different types of petroleum crudes in polluted soils. With no prior treatment, the samples are subjected to the headspace generation process and the volatiles generated are introduced directly into the mass spectrometer, thereby obtaining a fingerprint of volatiles in the sample analysed. The mass spectrum corresponding to the mass/charge ratios (m/z) contains the information related to the composition of the headspace and is used as the analytical signal for the characterization of the samples. The signals obtained for the different samples were treated by chemometric techniques to obtain the desired information. The main advantage of the proposed methodology is that no prior chromatographic separation and no sample manipulation are required. The method is rapid, simple and, in view of the results, highly promising for the implementation of a new approach for oil spill identification in soils. Figure PCA score plots illustrate clear discrimination of types of crude oil in polluted soil samples (e.g. results are shown for vertisol)     

Fluorescence detection techniques for protein kinase assay

Traditional methods for protein kinase (PK) assay are mainly based on use of ³²P-labeled adenosine triphosphate (ATP); applications of such methods are, however, hampered by radioactive waste and short half-life of ³²P-labeled ATP. Therefore non-radioactive methods, such as fluorescence detection techniques are good alternative. In this review, we describe the principles of four fluorescence techniques (fluorescence intensity endpoint measurement, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), and fluorescence lifetime imaging) and provide an overview of applications of these fluorescence detection techniques in protein kinase assay, underlining their relative advantages and limitations. Research trends in this field are also highlighted. Figure Schematic representation of kinase assay based on direct fluorescence polarization measurements. The fluorescent peptide, on phosphorylation by kinase, binds to a phosphospecific antibody, which leads to a high FP value     

Disposable screen-printed chemiluminescent biochips for the simultaneous determination of four point-of-care relevant proteins

A screen-printed (SP) microarray is presented as a platform for the achievement of multiparametric biochips. The SP platform is composed of eight (0.28-mm²) working electrodes modified with electroaddressed protein A-aryl diazonium adducts. The electrode surfaces are then used as an affinity immobilisation support for the orientated binding of capture monoclonal antibodies, having specificity against four different point-of-care related proteins (myoglobin, cardiac troponin I, C-reactive protein and brain natriuretic peptide). The immobilised capture antibodies are involved in sandwich assays of the four proteins together with biotinylated detection antibodies and peroxidase-labelled streptavidin in order to permit a chemiluminescent imaging of the SP platform and a sensitive detection of the assayed proteins. The performances of the system in pure buffered solutions, using a 25-min assay duration, were characterised by dynamic ranges of 0.5–50, 0.1–120, 0.2–20 and 0.67–67 μg/L for C-reactive protein, myoglobin, cardiac troponin I and brain natriuretic peptide, respectively. The four different assays were also validated in spiked 40-times-diluted human sera, using LowCross buffer, and were shown to work simultaneously in this complex medium. Figure Principle of the screen-printed POC microarray and a schematic representation of the assay architecture. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hydrophilic interaction liquid chromatography (HILIC) in proteomics

In proteomics, nanoflow multidimensional chromatography is now the gold standard for the separation of complex mixtures of peptides as generated by in-solution digestion of whole-cell lysates. Ideally, the different stationary phases used in multidimensional chromatography should provide orthogonal separation characteristics. For this reason, the combination of strong cation exchange chromatography (SCX) and reversed-phase (RP) chromatography is the most widely used combination for the separation of peptides. Here, we review the potential of hydrophilic interaction liquid chromatography (HILIC) as a separation tool in the multidimensional separation of peptides in proteomics applications. Recent work has revealed that HILIC may provide an excellent alternative to SCX, possessing several advantages in the area of separation power and targeted analysis of protein post-translational modifications. Figure Artistic impression of the HILIC separation mechanism     

Redox cycling in nanofluidic channels using interdigitated electrodes

Amperometric detection is ideally suited for integration into micro- and nanofluidic systems as it directly yields an electrical signal and does not necessitate optical components. However, the range of systems to which it can be applied is constrained by the limited sensitivity and specificity of the method. These limitations can be partially alleviated through the use of redox cycling, in which multiple electrodes are employed to repeatedly reduce and oxidize analyte molecules and thereby amplify the detected signal. We have developed an interdigitated electrode device that is encased in a nanofluidic channel to provide a hundred-fold amplification of the amperometric signal from paracetamol. Due to the nanochannel design, the sensor is resistant to interference from molecules undergoing irreversible redox reactions. We demonstrate this selectivity by detecting paracetamol in the presence of excess ascorbic acid. Figure  

Monitoring surface-assisted biomolecular assembly by means of evanescent-field-coupled waveguide-mode nanobiosensors

Biological self-assembly is a natural process that involves various biomolecules, and finding the missing partner in these interactions is crucial for a specific biological function. Previously, we showed that evanescent-field-coupled waveguide-mode sensor in conjunction with a SiO₂ waveguide, the surfaces which contain cylindrical nanometric holes produced by atomic bombardment, allowed us to detect efficiently the biomolecular interactions. In the present studies, we showed that the assembly of biomolecules can be monitored using the evanescent-field-coupled waveguide-mode biosensor and thus provide a methodology in monitoring assembly process in macromolecular machines while they are assembling. Evanescent-field-coupled waveguide-mode sensor Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Two-dimensional electrophoresis on a microfluidic chip for quantitative amino acid analysis

Analysis of complex biological samples requires the use of high-throughput analytical tools. In this work, a microfluidic two-dimensional electrophoresis system was developed with mercury-lamp-induced fluorescence detection. Mixtures of 20 standard amino acids were used to evaluate the separation performance of the system. After fluorescent labeling with fluorescein isothiocyanate, mixtures of amino acids were separated by micellar electrokinetic chromatography in the first dimension and by capillary zone electrophoresis in the second. A double electrokinetic valve system was employed for the sample injection and the switching between separation channels. Under the optimized conditions, 20 standard amino acids were effectively separated within 20 min with high resolution and repeatability. Quantitative analysis revealed linear dynamic ranges of over three orders of magnitudes with detection limits at micromolar range. To further evaluate the reliability of the system, quantitative analysis of a commercial nutrition supplement liquid was successfully demonstrated. Figure       

Optical sensing in microfluidic lab-on-a-chip by femtosecond-laser-written waveguides

We use direct femtosecond laser writing to integrate optical waveguides into a commercial fused silica capillary electrophoresis chip. High-quality waveguides crossing the microfluidic channels are fabricated and used to optically address, with high spatial selectivity, their content. Fluorescence from the optically excited volume is efficiently collected at a 90° angle by a high numerical aperture fiber, resulting in a highly compact and portable device. To test the platform we performed electrophoresis and detection of a 23-mer oligonucleotide plug. Our approach is quite powerful because it allows the integration of photonic functionalities, by simple post-processing, into commercial LOCs fabricated with standard techniques. Figure Femtosecond laser written waveguides can selectively excite fluorescence in a microfluidic channel of a commercial lab-on-a-chip. A compact scheme for on-chip detection by laser induced fluorescence is applied to capillary electrophoresis of a 23-mer Cy3-labeled oligonucleotide     

Outer-membrane proteomic maps and surface-exposed proteins of <Emphasis Type="Italic">Legionella pneumophila</Emphasis> using cellular fractionation and fluorescent labelling

Bacterial surface-associated proteins play crucial roles in host–pathogen interactions and pathogenesis. The identification of these proteins represents an important goal of bacterial proteomics for vaccine development, but also for environmental concerns such as microbial biosensing. Here, we developed such an approach for Legionella pneumophila, a bacterium that causes severe pneumonia. We propose a complementary strategy consisting of (1) a fluorescent labelling of surface-exposed proteins in parallel with (2) a fractionation of the outer-membrane protein extract. These two distinct protein populations were subsequently separated using two-dimensional gel electrophoresis and characterised by mass spectrometry. Within these populations, we found proteins which were expected for the compartments studied, but also a great number of proteins never experimentally described, and also a non-negligible fraction of proteins never described in these fractions. These data provided new routes of inspection for transport and host recognition for Legionella pneumophila. In addition, these results on the membranome and surfaceome show that Legionella in the stationary phase of growth possesses the major determinants to infect host cells. Figure Electron micrograph of Legionella pneumophila in stationnary phase of growth Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Presented at the Annual French National Symposium on Mass Spectrometry, Electrophoresis and Proteomics, 20–23 September 2007 in Pau, France     

Electrochemical time of flight method for determination of diffusion coefficients of glucose in solutions and gels

The diffusion coefficient of glucose in different media is an important parameter in life sciences, as well as in biotechnology and microbiology. In this work a simple, fast method is proposed that is based on the electrochemical time of flight principle. In most of the earlier time of flight experiments performed, a constant flight distance was applied. In the present work a scanning electrochemical microscope (SECM) was applied as a measuring tool. With use of the SECM, the flying distance could be changed with high precision, making measurements with several flight distances more accurate and reliable values could be obtained for solutions as well as for gels. The conventional voltammetric methods are not applicable for glucose detection. In our work electrocatalytic copper oxide coated copper microelectrodes and micro-sized amperometric enzyme sensors were used as detectors, while microdroplet-ejecting pneumatically driven micropipettes were used as a source. Figure Experimental set up for SCEM-TOF diffusion coefficient measurements Presented at the 9th International Symposium on Instrumental Analysis, Pécs, Hungary, 29 July-2 August 2008     

Simultaneous use of multiple fluorescent reporter dyes for glucose sensing in aqueous solution

The simultaneous use of several fluorescent reporter dyes in a multicomponent boronic acid-based glucose sensing system is reported. In one application, two dyes with widely different emission wavelengths are used to report changes in glucose concentration. A third glucose-insensitive dye was then added to act as a reference dye and provide for a ratiometric correction to the two reporter dye signals. The inclusion of such a reference dye reduces errors arising from sources such as fluctuations in lamp intensity and sample dilution. The simultaneous use of multiple fluorescent reporter dyes     

Nanobioanalytical luminescence: Förster-type energy transfer methods

Förster resonance energy transfer-based analytical techniques represent a unique tool for bioanalysis because they allow one to detect protein–protein interactions and conformational changes of biomolecules at the nanometer scale, both “in vitro” and “in vivo” in cells, tissues and organisms. These techniques are applied in diverse fields, from the detection and quantification of ligands able to bind to proteins or receptors to the development of RET-based whole-cell biosensors, microscope imaging techniques and “in vivo” whole-body imaging for the monitoring of physiological and pathological processes. However, their quantitative performances need further improvements and, even though RET measurement principles and procedures have been continuously improved, in some cases only qualitative or semiquantitative information can be obtained. In this review we report recent applications of RET-based analytical techniques and discuss their advantages and limitations.

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Figure RET-based techniques allow analysis of protein–protein interactions and conformational changes of biomolecules at the nanometer scale

     

Investigation of sample-purification procedures for MALDI-based proteomic studies

Purification methods for proteomics samples are of crucial concern for improving the quality of the sample delivered to the mass spectrometer. They constitute the link between the mass spectrometer and protein processing and peptide isolation steps that usually require solvents, buffers, or detergents completely incompatible with MS-analysis conditions. This work describes three new clean-up procedures using synthetic membranes and polymer media and compares them with standard procedures. The efficiency of each of the purification procedures was studied via application to four standards and two membrane proteins. This work highlights the importance of versatility in sample preparation, especially for MS-based proteomic investigations. Figure PMF spectra obtained after MALDI-TOF measurements of bovine mitochondrial complex III (A) and complex IV (B) in-solution digests, with and without purification     

Biosensors based on carbon nanotubes

Carbon nanotubes (CNTs) exhibit a unique combination of excellent mechanical, electrical and electrochemical properties, which has stimulated increasing interest in the application of CNTs as components in (bio)sensors. This review highlights various design methodologies for CNT-based biosensors and their employment for the detection of a number of biomolecules. In addition, recent developments in the fields of CNT-based chemiresistors and chemically sensitive field-effect transistors are presented. After a critical discussion of the factors that currently limit the practical use of CNT-based biosensors, the review concludes with an outline of potential future applications for CNTs in biology and medicine.      

Phase-changing sacrificial layers in microfluidic devices: adding another dimension to separations

The use of polymers in microchip fabrication affords new opportunities for the development of powerful, miniaturized separation techniques. One method in particular, the use of phase-changing sacrificial layers, allows for simplified designs and many additional features to the now standard fabrication of microchips. With the possibility of adding a third dimension to the design of separation devices, various means of enhancing analysis now become possible. The application of phase-changing sacrificial layers in microchip analysis systems is discussed, both in terms of current uses and future possibilities. Figure Phase-changing sacrificial materials enable multilayer microfluidic device layouts     

Enzyme-functionalized mesoporous silica for bioanalytical applications

The unique properties of mesoporous silica materials (MPs) have attracted substantial interest for use as enzyme-immobilization matrices. These features include high surface area, chemical, thermal, and mechanical stability, highly uniform pore distribution and tunable pore size, high adsorption capacity, and an ordered porous network for free diffusion of substrates and reaction products. Research demonstrated that enzymes encapsulated or entrapped in MPs retain their biocatalytic activity and are more stable than enzymes in solution. This review discusses recent advances in the study and use of mesoporous silica for enzyme immobilization and application in biosensor technology. Different types of MPs, their morphological and structural characteristics, and strategies used for their functionalization with enzymes are discussed. Finally, prospective and potential benefits of these materials for bioanalytical applications and biosensor technology are also presented. Figure Enzyme-functionalized mesoporous silica fibers and their integration in a biosensor design. The immobilization process takes place essentially in the silica micropores.     

Site-specific binding of chelerythrine and sanguinarine to single pyrimidine bulges in hairpin DNA

Spectrofluorometric titration, electrospray ionization time-of-flight mass spectrometric and UV melting methods were employed to study the binding of chelerythrine and sanguinarine to bulged DNA. The results showed that both alkaloids bind specifically to single pyrimidine (C, T) bulge sites. The ability of sanguinarine to bind to both regular and bulged hairpins was found to be stronger than that of chelerythrine, but the binding selectivity of chelerythrine toward single-base bulges was much larger than that of sanguinarine. Figure Association constants for chelerythrine and sanguinarine toward regular and single-base bulged hairpins obtained from fluorometric analysis     

Microchannel chips for the multiplexed analysis of human immunoglobulin G–antibody interactions by surface plasmon resonance imaging

We report the multiplexed, simultaneous analysis of antigen–antibody interactions that involve human immunoglobulin G (IgG) on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved by simultaneously monitoring protein–protein interactions under identical conditions. Antibody–antigen binding affinities towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement of protein–protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant molecules. Figure Microchannel SPR imaging for protein–protein interactions