Vratislav Kostal

Capillary Electrophoresis in Bioanalysis

For the period January 2006 to December 2007, Web of Science reports ∼4800 hits on capillary electrophoresis (CE) including 410 reviews. Because of the prominence of CE techniques in bioanalysis, we decided make them the focus of this review and selected 200 hundred papers representing advances in this field. The selected papers cover advances in CE theory, instrumentation, and methodologies that are specific to various analytes of biological origin or relevance. The group of analytes includes nucleic acids, proteins and peptides, carbohydrates, lipids, single cells, and bioparticles. In addition, we have included advances in the use of CE to define functional assays or to investigate biomolecular interactions. The use of microfabricated devices for CE analysis was not included because this is already covered in other review.

Recent advances in the analysis of biological particles by capillary electrophoresis

This review covers research papers published in the years 2005–2007 that describe the application of capillary electrophoresis to the analysis of biological particles such as whole cells, subcellular organelles, viruses and microorganisms.

Fast Determination of Mitochondria Electrophoretic Mobility Using Micro Free-Flow Electrophoresis

Fast, continuous separation of mitochondria from rat myoblasts using micro free-flow electrophoresis (muFFE) with online laser-induced fluorescence detection (LIF) is reported. Mitochondrial electrophoretic profiles were acquired in less than 30 s. In comparison to macroscale FFE instruments, muFFE devices consumed approximately 100-fold less sample, used 10-fold less buffer, and required a 15-fold lower electric field. Mitochondrial electrophoretic mobility distributions measured using muFFE were compared to those measured with a capillary electrophoresis instrument with laser-induced fluorescence detection (CE-LIF). There was high similarity between the two distributions with CE-LIF distribution being offset by 1.8 x 10(-4) cm(2) V(-1) s(-1) with respect to the microFFE distribution. We hypothesize that this offset results from the differences in electric field strength used in the techniques. In comparison to CE-LIF, analysis of mitochondria using muFFE greatly decreased separation time and required less separation voltage, while maintaining low sample (125 nL) and buffer (250 microL) volumes. These features together with the potential for collecting separated organelle fractions for further characterization make microFFE a very attractive tool for the high-throughput analysis of organelle subpopulations as well as investigating the fundamentals of the electrophoretic mobility of biological particles.

Review on recent advances in the analysis of isolated organelles.

The analysis of isolated organelles is one of the pillars of modern bioanalytical chemistry. This review describes recent developments on the isolation and characterization of isolated organelles both from living organisms and cell cultures. Salient reports on methods to release organelles focused on reproducibility and yield, membrane isolation, and integrated devices for organelle release. New developments on organelle fractionation after their isolation were on the topics of centrifugation, immunocapture, free flow electrophoresis, flow field-flow fractionation, fluorescence activated organelle sorting, laser capture microdissection, and dielectrophoresis. New concepts on characterization of isolated organelles included atomic force microscopy, optical tweezers combined with Raman spectroscopy, organelle sensors, flow cytometry, capillary electrophoresis, and microfluidic devices.

Capillary Isoelectric Focusing of Individual Mitochondria

Mitochondria are highly heterogeneous organelles that likely have unique isoelectric points (pI), which are related to their surface compositions and could be exploited in their purification and isolation. Previous methods to determine pI of mitochondria report an average pI. This article is the first report of the determination of the isoelectric points of individual mitochondria by capillary isoelectric focusing (cIEF). In this method, mitochondria labeled with the mitochondrial-specific probe 10-N-nonyl acridine orange (NAO) are injected into a fused-silica capillary in a solution of carrier ampholytes at physiological pH and osmolarity, where they are focused then chemically mobilized and detected by laser-induced fluorescence (LIF). Fluorescein-derived pI markers are used as internal standards to assign a pI value to each individually detected mitochondrial event, and a mitochondrial pI distribution is determined. This method provides reproducible distributions of individual mitochondrial pI, accurate determination of the pI of individual mitochondria by the use of internal standards, and resolution of 0.03 pH units between individual mitochondria. This method could also be applied to investigate or design separations of organelle subtypes (e.g., subsarcolemmal and interfibrillar skeletal muscle mitochondria) and to determine the pIs of other biological or nonbiological particles.

Individual organelle pH determinations of magnetically enriched endocytic organelles via laser-induced fluorescence detection.

The analysis of biotransformations that occur in lysosomes and other endocytic organelles is critical to studies on intracellular degradation, nutrient recycling, and lysosomal storage disorders. Such analyses require bioactive organelle preparations that are devoid of other contaminating organelles. Commonly used differential centrifugation techniques produce impure fractions and may not be compatible with microscale separation platforms. Density gradient centrifugation procedures reduce the level of impurities but may compromise bioactivity. Here we report on simple magnetic setup and a procedure that produce highly enriched bioactive organelles based on their magnetic capture as they traveled through open tubes. Following capture, in-line laser-induced fluorecence detection (LIF) determined for the first time the pH of each magnetically retained individual endocytic organelle. Unlike bulk measurements, this method was suitable to describe the distributions of pH values in endocytic organelles from L6 rat myoblasts treated with dextran-coated iron oxide nanoparticles (for magnetic retention) and fluorescein/TMRM-conjugated dextran (for pH measurements by LIF). Their individual pH values ranged from 4 to 6, which is typical of bioactive endocytic organelles. These analytical procedures are of high relevance to evaluate lysosomal-related degradation pathways in aging, storage disorders, and drug development.

Susan Müller, Thomas Bley (Eds.): High resolution microbial single cell analytics

Book’s topic High Resolution Microbial Single Cell Analytics focuses on recent advances in technology for characterizing the complexity of microbial populations and their effects on biotechnologically important processes. The book covers both the traditional methods of single-cell analysis (e.g. fluorescence microscopy) and specialized techniques, for example compression testing, microfluidic devices, and capillary electrophoresis. The objective of the book is to familiarize the reader with the techniques that are available and to demonstrate their advantages over bulk measurements in answering important questions in microbial biology and bioengineering.