Microchip electrophoresis bioanalytical applications

Microchip electrophoresis (MCE) is a novel analytical technique resulting from miniaturization of capillary electrophoresis (CE) to a planar microfabricated separation device. The consequences of the transfer of CE to MCE in terms of benefits and drawbacks have been identified and commented. The strategies developed to overcome the unfavourable features of the chip with respect to the capillary are briefly described. A method for simultaneous separation of catecholamines and their cationic metabolites has been developed on the microchip. The addition of three modifiers was required to resolve all analytes. The sensitivity of on-chip amperometric detection has been improved by employing an enzyme-catalyzed reaction on the amperometric electrode, as well as by using a carbon nanotube-modified electrode. The developed analytical methodology has been successfully applied for a direct on-chip determination of catecholamines and their metabolites in a mouse brain homogenate. The feasibility of performing affinity measurements as well as isoelectric focusing on the microchip has been demonstrated and available applications of these two electrophoretic modes on a chip have been reviewed. A commercial Shimadzu microchip station has for the first time been applied for high-throughput microchip isoelectric focusing of therapeutic proteins and obtained results have been compared to conventional capillary isoelectric focusing

Review on the development of truly portable and in-situ capillary electrophoresis systems

Capillary electrophoresis (CE) is a technique which uses an electric field to separate a mixed sample into its constituents. Portable CE systems enable this powerful analysis technique to be used in the field. Many of the challenges for portable systems are similar to those of autonomous in-situ analysis and therefore portable systems may be considered a stepping stone towards autonomous in-situ analysis. CE is widely used for biological and chemical analysis and example applications include: water quality analysis; drug development and quality control; proteomics and DNA analysis; counter-terrorism (explosive material identification) and corrosion monitoring. The technique is often limited to laboratory use, since it requires large electric fields, sensitive detection systems and fluidic control systems. All of these place restrictions in terms of: size, weight, cost, choice of operating solutions, choice of fabrication materials, electrical power and lifetime. In this review we bring together and critique the work by researchers addressing these issues. We emphasize the importance of a holistic approach for portable and in-situ CE systems and discuss all the aspects of the design. We identify gaps in the literature which require attention for the realization of both truly portable and in-situ CE systems

Spectrum of polysaccharides degradation products of ales and lager beers

The saccharide spectrum, as a distribution of fractions of different molecular mass, of sixteen beers was determined by ultracentrifugation using filters with cut-offs of 1, 5, 10 and 50 kDa. The saccharide concentrations in the filtrates were determined by density measurements. The saccharide composition was examined through HPAEC-PAD. The results were compared with the values of classic features of beers. The newly developed method provides additional information of the beers and is a simple and fast tool for exploring the effect of the saccharide spectrum on the industrial characteristics. The results revealed that similar top fermentation beers and similar lager beers have different saccharide spectra

Silicate determination in sea water: toward a reagentless electrochemical method

ilicate has been determined in sea water by four different electrochemical methods based on the detection of the silicomolybdic complex formed in acidic media by the reaction between silicate and molybdenum salts. The first two methods are based on the addition of molybdate and protons in a seawater sample in an electrochemical cell. Cyclic voltammetry presents two reduction and two oxidation peaks giving four values of the concentration and therefore increasing the precision. Then chronoamperometry is performed on an electrode held at a constant potential. A semi-autonomous method has been developed based on the electrochemical anodic oxidation of molybdenum, the complexation of the oxidation product with silicate and the detection of the complex by cyclic voltammetry. This method is tested and compared with the classical colorimetric one during ANT XXIII/3 cruise across Drake Passage (January–February 2006). The detection limit is 1 μM and the deviation between both methods is less than 3% for concentrations higher than 10 μM. Finally a complete reagentless method with a precision of 2.6% is described based on the simultaneous formation of the molybdenum salt and protons in a divided electrochemical cell. This latter method should be very useful for developing a reagentless sensor suitable for long term in situ deployments on oceanic biogeochemical observatories

Microelectrode arrays of diamond-insulated graphitic channels for real time detection of exocytotic events from cultured chromaffin cells and slices of adrenal glands

A microstructured graphitic 4x4 multielectrode array was embedded in a single crystal diamond substrate (4x4 {uG-SCD MEA) for real-time monitoring of exocytotic events from cultured chromaffin cells and adrenal slices. The current approach relies on the development of a parallel ion beam lithographic technique, which assures the time effective fabrication of extended arrays with reproducible electrode dimensions. The reported device is suitable for performing amperometric and voltammetric recordings with high sensitivity and temporal resolution, by simultaneously acquiring data from 16 rectangularly shaped microelectrodes (20x3.5 um^2) separated by 200 um gaps. Taking advantage of the array geometry we addressed the following specific issues: i) detect both the spontaneous and KCl-evoked secretion simultaneously from several chromaffin cells directly cultured on the device surface, ii) resolve the waveform of different subsets of exocytotic events, iii) monitoring quantal secretory events from thin slices of the adrenal gland. The frequency of spontaneous release was low (0.12 Hz and 0.3 Hz respectively for adrenal slices and cultured cells) and increased up to 0.9 Hz after stimulation with 30 mM KCl in cultured cells. The spike amplitude as well as rise and decay time were comparable with those measured by carbon fiber microelectrodes and allowed to identify three different subsets of secretory events associated to "full fusion" events, "kiss and-run" and "kiss-and-stay" exocytosis, confirming that the device has adequate sensitivity and time resolution for real-time recordings. The device offers the significant advantage of shortening the time to collect data by allowing simultaneous recordings from cell populations either in primary cell cultures or in intact tissues

All-carbon multi-electrode array for real-time in vitro measurements of oxidizable neurotransmitters

We report on the ion beam fabrication of all-carbon multi electrode arrays (MEAs) based on 16 graphitic micro-channels embedded in single-crystal diamond (SCD) substrates. The fabricated SCD-MEAs are systematically employed for the in vitro simultaneous amperometric detection of the secretory activity from populations of chromaffin cells, demonstrating a new sensing approach with respect to standard techniques. The biochemical stability and biocompatibility of the SCD-based device combined with the parallel recording of multi-electrodes array allow: i) a significant time saving in data collection during drug screening and/or pharmacological tests over a large number of cells, ii) the possibility of comparing altered cell functionality among cell populations, and iii) the repeatition of acquisition runs over many cycles with a fully non-toxic and chemically robust bio-sensitive substrate.Comment: 24 pages, 5 figure

Prospects of pulsed amperometric detection in flow-based analytical systems: a review

Electrochemical (EC) detection techniques in flow-based analytical systems such as flow injection analysis (FIA), capillary electrophoresis (CE), and liquid chromatography (LC) have attracted continuous interest over the last three decades, leading to significant advances in EC detection of a wide range of analytes in the liquid phase. In this context, the unique advantages of pulsed amperometric detection (PAD) in terms of high sensitivity and selectivity, and electrode cleaning through the application of pulsed potential for noble metal electrodes (e.g. Au, Pt), have established PAD as an important detection technique for a variety of electrochemically active compounds. PAD is especially valuable for analytes not detectable by ultraviolet (UV) photometric detection, such as organic aliphatic compounds and carbohydrates, especially when used with miniaturised capillary and chip-based separation methods. These applications have been accomplished through advances in PAD potential waveform design, as well as through the incorporation of nanomaterials (NMs) employed as microelectrodes in PAD. PAD allows on-line pulsed potential cleaning and coupling with capillary or standard separation techniques. The NMs are largely employed in microelectrodes to speed up mass and electron transfer between electrode surfaces and to perform as reactants in EC analysis. These advances in PAD have improved the sensitive and selective EC detection of analytes, especially in biological samples with complex sample matrices, and detection of electro-inactive compounds such as aliphatic organic compounds (i.e., formic acid, acetic acid, maleic acids, and β-cyclodextrin complexes). This review addresses the fundamentals of PAD, the role of pulsed sequences in AD, the utilization of different EC detectors for PAD, technological advancements in PAD waveforms, utilisation of microelectrodes in PAD techniques, advances in the use of NMs in PAD, the applications of PAD, and prospects for EC detection, with emphasis on PAD in flow-based systems

Microchip Capillary Electrophoresis

For the study of neuromodulation in Cancer borealis we have designed a microfluidic device to separate and detect bioamine concentrations with a high temporal resolution. Our goal is to use this device to measure the concentration of continuous bioamine microdialysis samples directly from the pericardial cavity (the area surrounding the heart) of Cancer borealis. The microfluidic device that we designed is made from polydimethylsiloxane (PDMS) and exhibits an off-channel configuration of capillary electrophoresis (CE) by incorporating micellar electrokinetic chromatography (MEKC). CE is used to separate bioamines based on charge and size due to the applied electrical potential. In the off-channel configuration, the potential is applied across the separation channel and grounded by the palladium decoupler, which lies just before the detector. Microchip CE is advantageous because it uses small amounts of analyte and completes fast run times. We will use MEKC to separate dopamine and octopamine, since they are structural isomers, by their difference in affinity to sodium dodecyl sulfate (SDS) micelles. This results in different elution times for dopamine and octopamine. We were able to drive the fluid in the correct direction. The creation of this device has valuable implications, allowing for baseline concentrations of neuromodulators with the Cancer borealis to be established. The effect of different stimuli on these crabs can then be more accurately determined

Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors

Mammals express the sialic acids ​N-acetylneuraminic acid (​Neu5Ac) and ​N-glycolylneuraminic acid (​Neu5Gc) on cell surfaces, where they act as receptors for pathogens, including influenza A virus (IAV). ​Neu5Gc is synthesized from ​Neu5Ac by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). In humans, this enzyme is inactive and only ​Neu5Ac is produced. Ferrets are susceptible to human-adapted IAV strains and have been the dominant animal model for IAV studies. Here we show that ferrets, like humans, do not synthesize ​Neu5Gc. Genomic analysis reveals an ancient, nine-exon deletion in the ferret CMAH gene that is shared by the Pinnipedia and Musteloidia members of the Carnivora. Interactions between two human strains of IAV with the sialyllactose receptor (sialic acid—α2,6Gal) confirm that the type of terminal sialic acid contributes significantly to IAV receptor specificity. Our results indicate that exclusive expression of ​Neu5Ac contributes to the susceptibility of ferrets to human-adapted IAV strains