Chromatography

Role of streaming potential on pulsating mass flow rate control in combined electroosmotic and pressure-driven microfluidic devices

In the present study, we investigate the implications of streaming potential on the mass flow rate control in a microfluidic device actuated by the combined application of a pulsating pressure gradient and a pulsating, externally applied, electric field. We demonstrate that the temporal dynamics due to streaming potential effects may lead to interesting non-trivial aspects of the resultant transport characteristics. Our results highlight the importance of an adequate accounting of the streaming potential effects for temporally tunable mass flow rate control strategies, which may act as a useful design artifice to augment mass flow rates in practical scenarios.

Human complement factor 3 polymorphism determination by capillary electrophoresis of serum

Variability of complement factor 3 (C3) mobility in serum protein electrophoresis was investigated. We found that the migration time of C3 can be reproducibly determined (beween-run CV=0.76%) using clinical capillary electrophoresis (CE) equipment (the Capillarys™ 2 system, Sebia). Moreover, we found a significant difference (p<0.001) in migration times of the major C3 phenotypes FF (fast-fast), FS (fast-slow) and SS (slow-slow). Glycosylation did not significantly affect test results. This is the first report on the migration time of C3 phenotypes on a clinical CE instrument. The presented method allows faster data than agarose-electrophoresis or genotyping. Moreover, reference ranges for serum C3 concentration depend on C3 phenotype, which allows a better tailored clinical interpretation of C3 concentrations.

A homogeneous assay for protein analysis in droplets by fluorescence polarization

We present a novel homogeneous (“mix-incubate-read”) droplet microfluidic assay for specific protein detection in picoliter volumes by fluorescence polarization (FP), for the first time demonstrating the use of FP in a droplet microfluidic assay. Using an FP-based assay we detect streptavidin concentrations as low as 500 nM and demonstrate that an FP assay allows us to distinguish droplets containing 5 μM rabbit IgG from droplets without IgG with an accuracy of 95%, levels relevant for hybridoma screening. This adds to the repertoire of droplet assay techniques a direct protein detection method which can be performed entirely inside droplets without the need for labeling of the analyte molecules.

Mutation scanning-based analysis of anisakid larvae from Sillago flindersi from Bass Strait, Australia

Anisakidosis is an important fish-borne disease caused by the larvae of anisakid nematodes, which affects humans and a range of other animals. The accurate identification of members of this nematode group is central to investigating the epidemiology of the parasites and in the surveillance and control of anisakidosis. It is now well known that morphological identification alone does not allow specific identification, particularly of larval stages. To better understand the epidemiology of anisakid nematodes in southern Australian fishes and the potential risks posed to human health, a survey of 50 specimens of the commercially important fish, Sillago flindersi, from Bass Strait, Australia was conducted. We characterised anisakid larvae by PCR-coupled mutation scanning, sequencing and phylogenetic analyses of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA. This study revealed that 92% of the S. flindersi examined were infected with anisakids (n=194), which were represented by seven genotypes. Phylogenetic analyses of the genotypes defined herein, together with reference sequence for Anisakis pegreffii and Hysterothylacium sp. from public databases (i.e. GenBank), revealed the presence of A. pegreffii (n=24), Hysterothylacium larval type IV (n=90) and Hysterothylacium larval type VIII (n=80) in S. flindersi. Thus, the PCR-coupled mutation scanning approach employed herein is an effective tool for the genetic characterisation of anisakid nematodes for diagnostic and analytical purposes (nucleotide sequences reported in this paper are available in the GenBank database under accession nos. JN631796-809).

Cover Picture: Electrophoresis 3’2012

Issue no. 3 is a regular issue consisting of 17 contributions distributed over 5 separate parts. The issue starts with 4 research articles on micro- and nanofluidics making up Part I. This is followed by Part II that has 5 research articles involving studies on proteins, peptides and proteomics. Part III has 3 contributions dealing with studies on nucleic acids. Part IV has 3 research articles on enantioseparation methodologies. The last part (Part V) consists of 2 contributions on various aspects of preconcentration in CE of drug of abuse and their metabolites and the sensitive monitoring for ribonucleotide reductase activity. This issue, with as few as 17 contributions, is rich in very important ideas in the field for further investigations.Featured articles include:Modeling of droplet traffic in interconnected microfluidic ladder devices ((10.1002/elps.201100320))Role of streaming potential on pulsating mass flow rate control in combined electroosmotic and pressure-driven microfluidic devices ((10.1002/elps.201100414))Carotid atherosclerotic plaques: Proteomics study after a low-abundance protein enrichment step ((10.1002/elps.201100395))Quantitative experimental determination of primer-dimer formation risk by free-solution conjugate electrophoresis ((10.1002/elps.201100452))Electromembrane extraction combined with cyclodextrin-modified capillary electrophoresis for the quantification of trimipramine enantiomers ((10.1002/elps.201100426))

Editorial Board: Electrophoresis 3’2012

Investigation of in-line solid-phase extraction capillary electrophoresis for the analysis of drugs of abuse and their metabolites in water samples

In this study, in-line solid-phase extraction (SPE) was used as an enrichment technique in combination with CE for the preconcentration and separation of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), cocaine (COC), codeine (COD) and 6-acetylmorphine (6AM). The separation buffer (BGE) used was 80 mM disodium phosphate anhydrous and 6 mM of HCl (final BGE pH of 3). The SPE extractor consists of a small segment of capillary filled with Oasis HLB sorbent and inserted into the inlet section of the electrophoretic capillary. Different parameters affecting preconcentration were evaluated, such as sample pH, the volume of the elution plug and sample injection time. The detection limits (LODs) reached for standard samples by in-line SPE-CE-UV ranged between 50 and 200 ng/L, with sensitivity enhancement factors ranging from 2300 to 5300. Reproducibility values (expressed in terms of relative standard deviation) were below 7.6% for standard samples. This is a simple and an effective method for the determination of the studied drugs of abuse and their metabolites. The applicability of the developed method was demonstrated in tap and river water samples which were directly analyzed without any off-line pretreatment. Analytical parameters were evaluated and LODs were between 70 and 270 ng/L with relative recoveries between 85 and 97%.

Contents – Electrophoresis 3’2012

Call for Papers

Analytical solutions and validation of electric field and dielectrophoretic force in a bio-microfluidic channel

In a microbiological device, cell or particle manipulation and characterization require the use of electric field on different electrodes in several configurations and shapes. To efficiently design microelectrodes within a microfluidic channel for dielectrophoresis focusing, manipulation and characterization of cells, the designer will seek the exact distribution of the electric potential, electric field and hence dielectrophoresis force exerted on the cell within the microdevice. In this paper we describe the approach attaining the analytical solution of the dielectrophoretic force expression within a microchannel with parallel facing same size electrodes present on the two faces of channel substrates, with opposite voltages on the pair electrodes. Simple Fourier series mathematical expressions are derived for electric potential, electric field and dielectric force between two distant finite-size electrodes. Excellent agreement is found by comparing the analytical results calculated using MATLAB™ with numerical ones obtained by Comsol. This analytical result can help the designer to perform simple design parametric analysis. Bio-microdevices are also designed and fabricated to illustrate the theoretical solution results with the experimental data. Experiments with red blood cells show the dielectrophoretic force contour plots of the analytical data matched to the experimental results.

Carotid atherosclerotic plaques: Proteomics study after a low-abundance protein enrichment step

Atherosclerosis is one of the most important causes of cardiovascular and cerebrovascular events. Although phenotypic differentiation between stable and unstable plaques is currently possible, proteomic analysis of the atherosclerotic plaque could offer a global view of the atherosclerosis pathology. With the objective to highlight the detection of low-abundance proteins, we reduced the dynamic range of proteins by combinatorial peptide ligand library treatment of human carotid artery atherosclerotic plaques. After enrichment step, abundance of major proteins was decreased, revealing different protein profiles as assessed by both SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis comparative analyses. Identification of proteins that were contained in a spot allowed finding large differences between noncomplicated and complicated plaques from carotid atherosclerotic lesions. Novel low-abundance proteins were detected correlating very well with biological alterations related to atherosclerosis (heat shock protein 27 (HSP27) isoforms, aldehyde dehydrogenase, moesin, Protein kinase C delta-binding protein, and inter-α trypsin inhibitor family heavy chain-related protein (ITIH4)). At the same time, the differential expression of known proteins of interest such as hemoglobin β-chain and heat shock protein 27 between noncomplicated and hemorrhagic complicated plaques was maintained after enrichment step. The detection of different isoforms of a low-abundance protein such as heat shock protein 27 species was actually improved after enrichment of tissue protein extracts. All of these findings clearly support further investigations in view to confirm the role of these proteins as possible biomarkers.