Simple, rapid, and reproducible capillary gel electrophoresis separation and laser-induced fluorescence detection of DNA topoisomers with unmodified fused silica separation capillaries.

The topology of DNA is a critical quality attribute for plasmid-based pharmaceuticals, making quantification of trace levels of plasmid topoisomers an important analytical priority. An automated and cost-effective method based on capillary gel electrophoresis laser-induced fluorescence detection is described. The method outlined in this report is significant because it is easily implemented by any laboratory for which routine analyses of plasmid topology are critical for the development of new plasmid-based therapies as well as for quality control of gene therapies utilizing supercoiled DNA.

Protein Sieving with Capillary Nanogel Electrophoresis.

Protein sieving, which is a fundamental tool in the biotechnology field, can be automated using capillary gel electrophoresis. The high-viscosity and biocompatible linear gels required for capillary sieving must be replaced for each run using high pressures. Thermally responsive gels are easier to renew in the capillary as they can be repetitively switched between low- and high-viscosity solutions.

Quantification of the α2-6 Sialic Acid Linkage in Branched N-Glycan Structures with Capillary Nanogel Electrophoresis.

Sialylation and sialic acid linkage in N-glycans are markers of disease but are analytically challenging to quantify. A capillary electrophoresis method is reported that integrates a unique combination of enzymes and lectins to modify sialylated N-glycans in real time in the capillary so that N-glycan structures containing α2-6-linked sialic acid are easily separated, detected, and quantified. In this study, N-glycans were sequentially cleaved by enzymes at the head of the separation capillary so that the presence of α2-6-linked sialic acids corresponded to a shift in the analyte migration time in a manner that enabled interpretation of the N-glycan structure.

Capillary Electrophoresis Separations of Glycans.

Capillary electrophoresis has emerged as a powerful approach for carbohydrate analyses since 2014. The method provides high resolution capable of separating carbohydrates by charge-to-size ratio. Principle applications are heavily focused on N-glycans, which are highly relevant to biological therapeutics and biomarker research.

Advances in enzyme substrate analysis with capillary electrophoresis.

Capillary electrophoresis provides a rapid, cost-effective platform for enzyme and substrate characterization. The high resolution achievable by capillary electrophoresis enables the analysis of substrates and products that are indistinguishable by spectroscopic techniques alone, while the small volume requirement enables analysis of enzymes or substrates in limited supply. Furthermore, the compatibility of capillary electrophoresis with various detectors makes it suitable for K determinations ranging from nanomolar to millimolar concentrations.

Capillary electrophoresis with stationary nanogel zones of galactosidase and Erythrina cristagalli lectin for the determination of β(1-3)-linked galactose in glycans.

A thermally responsive nanogel is used to create stationary zones of enzyme and lectin in a separation capillary. Once patterned in the capillary, analyte is driven through the zone, where it is converted to a specific product if an enzyme is used or captured if a lectin is used. These stationary zones are easily expelled after the analysis and then re-patterned in the capillary.

Microscale Measurements of Michaelis-Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage.

Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enables rapid quantification of Michaelis-Menten constants (K) for different substrates and ultimately provides a means to quantify the linkage (i.

Capillary electrophoresis applied to DNA: determining and harnessing sequence and structure to advance bioanalyses (2009-2014).

This review of capillary electrophoresis methods for DNA analyses covers critical advances from 2009 to 2014, referencing 184 citations. Separation mechanisms based on free-zone capillary electrophoresis, Ogston sieving, and reptation are described. Two prevalent gel matrices for gel-facilitated sieving, which are linear polyacrylamide and polydimethylacrylamide, are compared in terms of performance, cost, viscosity, and passivation of electroosmotic flow.

In vitro selection of a single-stranded DNA molecular recognition element against atrazine.

Widespread use of the chlorotriazine herbicide, atrazine, has led to serious environmental and human health consequences. Current methods of detecting atrazine contamination are neither rapid nor cost-effective. In this work, atrazine-specific single-stranded DNA (ssDNA) molecular recognition elements (MRE) were isolated.

Online enzymatic sequencing of glycans from Trastuzumab by phospholipid-assisted capillary electrophoresis.

CE separations of glycans taken from the cancer drug, Trastuzumab (Herceptin(®)), were accomplished using phospholipid additives. Glycans were labeled with 1-aminopyrene-3,6,8-trisulfonic acid and were separated with efficiencies as high as 510000 theoretical plates in a 60.2 cm 25 μm id fused-silica capillary.