Abstract
Hydrodynamic radius (R(H)) is a descriptive metric of protein structure with the potential to impact drug development, disease diagnosis, and other important research areas of molecular biology. Common instrumental methods for molecular size characterization are disadvantageous due to high sample consumption, measurements made in non-physiological conditions, and/or inaccurate size determinations. Capillary Taylor dispersion analysis (TDA) is a molecular sizing method that utilizes nL sample volumes and achieves absolute size determination without calibration or comparison to standards. One key drawback of TDA is that it reports the concentration-weighted average R(H), which may be limiting in the analysis of complex sample mixtures. Here, we describe the development of a 3D printed instrument to integrate capillary electrophoresis (CE) separations online with TDA size characterization. Dual laser-induced fluorescence detectors were developed to enable two-channel detection using a single PMT and fluorescence filter set, achieving detection limits for AlexaFluor 532 of 0.6 ± 0.4 nM and 1.1 ± 0.2 nM for detectors 1 and 2, respectively. Joule heating during CE separations was observed to introduce bias in subsequent TDA measurements. The effects of Joule heating were mitigated by integrating a water circulating sheath flow on the portion of the capillary used for CE. The utility of CE-TDA in bioanalysis was demonstrated by standard-free peak identification in the ficin digestion of IgG1. CE-TDA was further applied to characterizing denaturation dynamics of the Group II heat resistant protein apolipoprotein A-1 (ApoA), in which R(H) was observed to increase from 2.3 ± 0.2 nm at 20 °C to 5.2 ± 0.5 nm while heated at at 90 °C, then returned to a quasi-native state with R(H) = 2.9 ± 0.5 nm after cooling to 20 °C. CE-TDA is a powerful analysis mode with potential to impact various domains of bioanalysis. The instrument developed in this work offers a low barrier to entry for researchers interested in adopting CE-TDA.