Systematic Approach to Optimization of Experimental Conditions in Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures

Abstract

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is an efficient method for studying intermolecular interactions. Optimization of NECEEM experiments is not a trivial task, due to the complex interrelation between numerous experimental parameters and their combined effects on the accuracy and precision of measurements. Here we present an "algorithmic" approach for NECEEM optimization, which eliminates all of the guesswork out of this process and allows researchers to approach it in a systematic manner. We have fully tested our approach using comprehensive in silico analysis and have showed its utility within a real experimental study. The new approach makes NECEEM more robust, resilient to errors, and easily approachable for researchers with varying experience in CE.

Dual Detection High-Speed Capillary Electrophoresis for Simultaneous Serum Protein Analysis and Immunoassays

“…[27][28][29] To maintain the SPE signal, however, approaches like NECEEM that begin with pre-equilibrated samples are the most straightforward to implement. [30][31][32][33][34][35] The three buffer systems optimized for SPE in Fig. 2, therefore, were tested for their ability to support successful immunoassays.…”

“…The broad background trailing the peak is often referred to as the bridge region and arises from the continuous dissociation of antigen from the complex during the non-equilibrium separation. [30][31][32][33][34][35] As the antigen dissociates, its higher mobility opposes the EOF, which eventually sweeps it past the detection zone. Finally the additional peaks are observed in Fig.…”

“…Antigen-antibody complexes initially at equilibrium can be separated from unbound species in CZE, as long as the electrophoretic mobilities of the species are sufficiently different and the separation time is competitive with the equilibrium kinetics. 30 Affinity-based CZE techniques have been thoroughly discussed in the literature and identified as affinity probe capillary electrophoresis (APCE) and more recently as non-equilibrium capillary electrophoresis in equilibrium mixtures (NECEEM). [30][31][32][33][34][35] CZE analysis of pre-equilibrated solutions were first used to analyze human growth hormone, where bound and unbound species were successfully separated and quantified using UV detection.…”

“…30 Affinity-based CZE techniques have been thoroughly discussed in the literature and identified as affinity probe capillary electrophoresis (APCE) and more recently as non-equilibrium capillary electrophoresis in equilibrium mixtures (NECEEM). [30][31][32][33][34][35] CZE analysis of pre-equilibrated solutions were first used to analyze human growth hormone, where bound and unbound species were successfully separated and quantified using UV detection. 36,37 The integration of fluorescence labeling methods increased specificity and substantially improved detection limits, expanding the reach of the technique.…”

Non-SELEX isolation of DNA aptamers for the homogeneous-phase fluorescence anisotropy sensing of tau Proteins

“…The bulk affinity of DNA pools to the t protein can be estimated through a binding analysis relying on the determination of peak areas for the complex, the free DNA and the decay bridge. Due to the large portion of the complex-dissociating DNA, a systematic method developed by Krylov and colleagues was used to reliably determine the overlap between the bridge and the DNA peak [31]. A constant reduction in the apparent dissociation constant value was reported as the number of rounds increased (Fig.…”