Atomic force microscopy imaging and electrical recording of lipid bilayers supported over microfabricated silicon chip nanopores: lab-on-a-chip system for lipid membranes and ion channels

We describe a silicon chip-based supported bilayer system to detect the presence of ion channels and their electrical conductance in lipid bilayers. Nanopores were produced in microfabricated silicon membranes by electron beam lithography as well as by using a finely focused ion beam. Thermal oxide was used to shrink pore sizes, if necessary, and to create an insulating surface. The chips with well-defined pores were easily mounted on a double-chamber plastic cell recording system, allowing for controlling the buffer conditions both above and below the window. The double-chamber system allowed using an atomic force microscopy (AFM) tip as one electrode and inserting a platinum wire as the second electrode under the membrane window, to measure electrical current across lipid bilayers that are suspended over the pores. Atomic force imaging, stiffness measurement, and electrical capacitance measurement show the feasibility of supporting lipid bilayers over defined nanopores: a key requirement to use any such technique for structure-function study of ion channels. Online addition of gramicidin, an ion-channel-forming peptide, resulted in electrical current flow across the bilayer, and the I-V curve that was measured using the conducting AFM tip indicates the presence of many conducting gramicidin ion channels.     

On-line solid-phase extraction-thermal desorption-gas chromatography with ion trap detection tandem mass spectrometry for the analysis of microcontaminants in water

An improved set-up for solid-phase extraction with thermal desorption coupled on-line to gas chromatography (SPETD-GC) is presented. It includes a newly designed liner for a programmable temperature vaporizer (PTV) and an improved water elimination system. The SPETD procedure now includes a washing step with HPLC-grade water to prevent degradation of analytes due to interaction with remaining sample constituents. The system was used to analyze surface and tap water samples over a 4-month period. No decrease of chromatographic or trace-enrichment performance was observed, and a liner packed with Tenax GR could be used for at least 150 analyses. The SPETD module was coupled to GC with ion-trap detection for mass spectrometric (MS) and MS/MS detection. The linearity and repeatability of the procedure for several pesticides which were tested in the 0.5–10 μg/1 range were fully satisfactory (1 μg/1, RSD range 5–11%; n = 5). When using sample volumes of 0.1 ml only, detection limits were as low as 0. 1-0.2 μg/1. As an example, the confirmation and quantification of a suspected pesticide in a real-life sample using electron impact and positive chemical ionization in both the MS and MS/MS mode is shown.     

Laser spectroscopy in analytical chemistry:light on the next millennium

To date, lasers are widely accepted tools in analytical spectroscopy, involved in various stand-alone and hyphenated techniques. Furthermore, significant progress can be noted in this field. In this paper, first of all some laser characteristics are discussed. Subsequently, five selected topics are outlined to illustrate recent achievements and future developments:

1. Laser-induced fluorescence for detection in capillary electrophoresis, including the use of ultraviolet, continuous-wave lasers in combination with wavelength-resolved emission detection; the use of diode laser-induced fluorescence in the red region of the electromagnetic spectrum and the use of Ti:sapphire lasers for multiphoton-excited fluorescence detection.

2. Degenerate four-wave mixing for detection in liquid microseparation systems (based on the coherence of laser light).

3. Fluorescence line-narrowing spectroscopy for identification purposes, a cryogenic high-resolution molecular fluorescence technique with a high potential in environmental analysis.

4. Recent developments in Raman spectroscopy (including ultraviolet-Resonance Raman and hyphenation of liquid chromatography and Raman spectroscopy).

5. Use of lasers for sample introduction in inorganic analysis based on controlled material ablation.

Pd/Cu Dual-Catalyzed Asymmetric Synthesis of Highly Functional All-Carbon Quaternary Stereocenters from Vinyl Carbonates

The challenging metal-catalyzed asymmetric synthesis of highly functional quaternary carbon centers using decarboxylative C(sp³)−C(sp³) bond formation reactions is reported. The key substrate, a vinyl cyclic carbonate, is activated to provide concomitantly both the requisite nucleophile (by formal umpolung) and electrophile reaction partner preceding the asymmetric cross-coupling process. A wide screening of reaction conditions, additives and catalyst precursors afforded a protocol that gave access to a series of compounds featuring densely functionalized, elusive quaternary carbon stereocenters in appreciable yield and with enantiomeric ratios (er's) of up to 90 : 10.     

Quantitative profiling of oxylipins through comprehensive LC-MS/MS analysis: application in cardiac surgery

Oxylipins, including eicosanoids, affect a broad range of biological processes, such as the initiation and resolution of inflammation. These compounds, also referred to as lipid mediators, are (non-) enzymatically generated by oxidation of polyunsaturated fatty acids such as arachidonic acid (AA). A plethora of lipid mediators exist which makes the development of generic analytical methods challenging. Here we developed a robust and sensitive targeted analysis platform for oxylipins and applied it in a biological setting, using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) operated in dynamic multiple reaction monitoring (dMRM). Besides the well-described AA metabolites, oxylipins derived from linoleic acid, dihomo-γ-linolenic acid, α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid were included. Our comprehensive platform allows the quantitative evaluation of approximately 100 oxylipins down to low nanomolar levels. Applicability of the analytical platform was demonstrated by analyzing plasma samples of patients undergoing cardiac surgery. Altered levels of some of the oxylipins, especially in certain monohydroxy fatty acids such as 12-HETE and 12-HEPE, were observed in samples collected before and 24 h after cardiac surgery. These findings indicate that this generic oxylipin profiling platform can be applied broadly to study these highly bioactive compounds in relation to human disease.     

Application of an omonasteine ligation strategy for the total chemical synthesis of the BRD7 bromodomain

The use of omonasteine (Omo) in sequential peptide ligation strategies extends the scope of homocysteine (Hcy) ligation to longer, methionine-rich proteins. Hcy-to-Omo conversion can be performed on-resin, while the Omo-to-Hcy deprotection can be performed in situ after peptide ligation. This strategy was successfully applied in the synthesis of the BRD7 bromodomain.     

iLIF: illumination by Laser-Induced Fluorescence for single flash imaging on a nanoseconds timescale

The challenge in visualizing fast microscale fluid motion phenomena is to record high-quality images free of motion-blur. Here, we present an illumination technique based on laser-induced fluorescence which delivers high-intensity light pulses of 7 ns. The light source consists of a Q-switched Nd:YAG laser and a laser dye solution incorporated into a total internal reflection lens, resulting in a uni-directional light beam with a millimeter-sized circular aperture and 3° divergence. The laser coherence, considered undesirable for imaging purposes, is reduced while maintaining a nanoseconds pulse duration. The properties of the illumination by laser-induced fluorescence (iLIF) are quantified, and a comparison is made with other high-intensity pulsed and continuous light sources.     

Millifluidic droplet analyser for microbiology

We present a novel millifluidic droplet analyser (MDA) for precisely monitoring the dynamics of microbial populations over multiple generations in numerous (≥10(3)) aqueous emulsion droplets (~100 nL). As a first application, we measure the growth rate of a bacterial strain and determine the minimal inhibitory concentration (MIC) for the antibiotic cefotaxime by incubating bacteria in a fine gradient of antibiotic concentrations. The detection of cell activity is based on the automated detection of an epifluorescent signal that allows the monitoring of microbial populations up to a size of ~10(6) cells. We believe that this device is helpful for the study of population dynamic consequences of microbe-environment interactions and of individual cell differences. Moreover, the fluidic machine may improve clinical tests, as it simplifies, automates and miniaturizes the screening of numerous microbial populations that grow and evolve in compartments with a finely tuned composition.