Fluorescence polarization detection for affinity capillary electrophoresis

Abstract

Affinity capillary electrophoresis (ACE) with laser-induced fluorescence polarization (LIFP) detection is described, with examples of affinity interaction studies. Because fluorescence polarization is sensitive to changes in the rotational motion arising from molecular association or dissociation, ACE-LIFP is capable of providing information on the formation of affinity complexes prior to or during CE separation. Unbound, small fluorescent probes generally have little fluorescence polarization because of rapid rotation of the molecule in solution. When the small fluorescent probe is bound to a larger affinity agent, such as an antibody, the fluorescence polarization (and anisotropy) increases due to slower motion of the much larger complex molecule in the solution. Fluorescence polarization results are obtained by simultaneously measuring fluorescence intensities of vertical and horizontal polarization planes. Applications of CE-LIFP to both strong and weak binding systems are discussed with antibody-antigen and DNA-protein binding as examples. For strong affinity binding, such as between cyclosporine and its antibody, complexes are formed prior to CE-LIFP analysis. For weaker binding, such as between single-stranded DNA and its binding protein, the single-stranded DNA binding protein is added to the CE separation buffer to enhance dynamic formation of affinity complexes. Both fluorescence polarization (and anisotropy) and mobility shift results are complementary and are useful for immunoassays and binding studies.

Highly sensitive detection of human thrombin in serum by affinity capillary electrophoresis/laser-induced fluorescence polarization using aptamers as probes

“…In fact, CE/LIFP analysis not only provides the information on electrophoretic mobility (derived from migration), but also offers the information on the diffusional rotation of the analytes (fluorescence polarization). It has been demonstrated that the FP values can be used to differentiate between the unbound aptamers and the protein bound aptamer complexes even when they are not well separated [35,36]. Therefore, here we used the CE/LIFP approach to identify the thrombin-aptamer complex in CE separation of complex matrix.…”

Aptamer fluorescence anisotropy sensors for adenosine triphosphate by comprehensive screening tetramethylrhodamine labeled nucleotides

“…Since the discovery of aptamers in 1990s, aptamer-based assays for small molecules have drawn increasing attentions in environmental sensing, food safety, and clinical analysis due to the unique features of aptamers, such as easy generation, facile labeling, good thermal stability, small size, and target-binding induced structure change (Citartan et al, 2012;Feng et al, 2014;Juskowiak, 2011;Kim and Gu 2014;Li et al, 2015;Liu et al, 2009;McKeague and Derosa, 2012). Taking advantage of fluorescence anisotropy (FA) or fluorescence polarization (FP) in sensitivity, reproducibility, and simplicity (Lea and Simeonov, 2011;Gradinaru et al, 2010;Le et al, 2002;Smith and Eremin, 2008), the aptamer-based FA/FP sensors are attractive Liu et al, 2009;Ruta et al, 2009;Zhang et al, 2011).…”

Recent advances in the study of biomolecular interactions by capillary electrophoresis

“…1). A similar approach has been used to study oder interactions, such as antibodydigoxin, antibody-gentamicin, and antibody-vancomycin [77][78][79][80][81]. When in a CE system at a low concentration of protein as additive a steady state is reached, the additive entering the analyte plug is unaffected by the mobility of the additive.…”