Affinity capillary electrophoresis on microchips

Stettler, Alexandra R.; Schwarz, Maria A.

doi:10.1016/j.chroma.2004.11.079

Cited by 42 publications

(28 citation statements)

References 36 publications

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“…Pre-equilibrium mixture analysis [12,[15][16][17][18]: The complex is formed by pre-incubating the analyte and the binding probe, and then the resulting equilibrium mixture is analyzed under non-equilibrium separation conditions based on electrophoresis. Mobility-shift analysis [19][20][21][22]: In this approach, the separation buffer contains the binding ligand while the sample contains the analyte; following injection, the analyte-ligand complex continuously undergoes dynamic dissociation/association cycles during CE separation; the migration time of the analyte is shifting with the concentrations of the ligand in the separation buffer. Pre-equilibriummixture analysis is preferred for relatively strong binding systems in which the complex has a slow-off rate and can be detected before it dissociates to a significant extent.…”

Section: Introductionmentioning

confidence: 99%

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

et al. 2008

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The use of traditional capillary electrophoresis (CE) to detect weak binding complexes is problematic due to the fast-off rate resulting in the dissociation of the complex during the separation process. Additionally, proteins involved in binding interactions often nonspecifically stick to the bare-silica capillary walls, which further complicates the binding analysis. Microchip CE allows flexibly positioning the detector along the separation channel and conveniently adjusting the separation length. A short separation length plus a high electric field enables rapid separations thus reducing both the dissociation of the complex and the amount of protein loss due to nonspecific adsorption during the separation process. Thrombin and a selective thrombinbinding aptamer were used to demonstrate the capability of microchip CE for the study of relatively weak binding systems that have inherent limitations when using the migration shift method or other CE methods. The rapid separation of the thrombin-aptamer complex from the free aptamer was achieved in less than 10 s on a single-cross glass microchip with a relatively short detection length (1.0 cm) and a high electric field (670 V/cm). The dissociation constant was determined to be 43 nM, consistent with reported results. In addition, aptamer probes were used for the quantitation of standard thrombin samples by constructing a calibration curve, which showed good linearity over two orders of magnitude with a limit of detection for thrombin of 5 nM at a 3-fold signal-to-noise ratio.

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Section: Introductionmentioning

confidence: 99%

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

et al. 2008

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“…Derived from the principles of affinity gel electrophoresis, ACE measures and determines the physicochemical and thermodynamic parameters of drug compounds, offering strategies to explore and characterize molecular interactions [25,26]. Although various miniaturized CE systems have been reported, ACE on microchips was developed just recently [27]. This method describes the estimation of the strength of interactions between a ligand and its substrate using UV and electrochemical detection.…”

Section: Cementioning

confidence: 99%

Capillary electrophoresis at the omics level: Towards systems biology

et al. 2006

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Emerging systems biology aims at integrating the enormous amount of existing omics data in order to better understand their functional relationships at a whole systems level. These huge datasets can be obtained through advances in high-throughput, sensitive, precise, and accurate analytical instrumentation and technological innovation. Separation sciences play an important role in revealing biological processes at various omic levels. From the perspective of systems biology, CE is a strong candidate for high-throughput, sensitive data generation which is capable of tackling the challenges in acquiring qualitative and quantitative knowledge through a system-level study. This review focuses on the applicability of CE to systems-based analytical data at the genomic, transcriptomic, proteomic, and metabolomic levels.

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“…The development of a fast analysis, i.e., high-throughput screening together with binding data is attractive but still in the prototype phase. Stettler et al [118] compared chip-based systems with UV and electrochemical detection with traditional CE-UV using the interaction between the neurotransmitters epinephrine and norepinephrine and CD in solution as a model application. The affinity microchip-CE system showed proofof-principle ACE in qualitative terms, especially at lower CD concentrations, but was hampered by peak broadening.…”

Section: Technical Innovationmentioning

confidence: 99%

Recent applications of affinity interactions in capillary electrophoresis

2006

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Systems biology depends on a comprehensive assignment and characterization of the interactions of proteins and polypeptides (functional proteomics) and of other classes of biomolecules in a given organism. High-capacity screening methods are in place for ligand capture and interaction screening, but a detailed dynamic characterization of molecular interactions under physiological conditions in efficiently separated mixtures with minimal sample consumption is presently provided only by electrophoretic interaction analysis in capillaries, affinity CE (ACE). This has been realized in different fields of biology and analytical chemistry, and the resulting advances and uses of ACE during the last 2.5 years are covered in this review. Dealing with anything from small divalent metal ions to large supramolecular assemblies, the applications of ACE span from low-affinity binding of broad specificity being exploited in optimizing selectivity, e.g., in enantiomer analysis to miniaturized affinity technologies, e.g., for fast processing immunoassay. Also, approaches that provide detailed quantitative characterization of analyte-ligand interaction for drug, immunoassay, and aptamer development are increasingly important, but various approaches to ACE are more and more generally applied in biological research. In addition, the present overview emphasizes that distinct challenges regarding sensitivity, parallel processing, information-rich detection, interfacing with MS, analyte recovery, and preparative capabilities remain. This will be addressed by future technological improvements that will ensure continuing new applications of ACE in the years to come.

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Affinity capillary electrophoresis on microchips

Cited by 42 publications

References 36 publications

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

Capillary electrophoresis at the omics level: Towards systems biology

Recent applications of affinity interactions in capillary electrophoresis

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