A Miniature 3D Printed LED-Induced Fluorescence Detector for Capillary Electrophoresis and Dual-Detector Taylor Dispersion Analysis.

Abstract

Taylor dispersion analysis (TDA) provides absolute determination of diffusion coefficients for analytes ranging from small molecules to particulate matter. TDA has seen a resurgence in recent years, as modern commercial capillary electrophoresis (CE) instrumentation is well equipped to meet the precision flow requirements of TDA. Discontinuous flow velocities, which occur during sample injection, can lead to substantial inaccuracies in single-point detection TDA. Dual-point detection allows TDA to be carried out under continuous flow in the volume between the detection points, but dual-point fluorescence detection has not previously been feasible within the confines of commercial CE instrumentation. Here, we describe a compact light-emitting diode (LED)-induced fluorescence detector designed for online, dual-point capillary detection within a commercial CE system. The three-dimensional (3D) printed detector houses an inexpensive LED excitation source, a bandpass excitation filter, an integral 3D printed pinhole collimator, and a ball lens, which collects fluorescence emission. Multivariate optimization of operating conditions yielded a detection limit of 613 ± 13 pM for CE of fluorescein disodium salt solution in borate buffer. The miniature size of the device allowed integration of two detectors within a commercial CE system without modification to the instrument, thereby enabling dual-detector assays including TDA and CE-TDA. Monitoring of the bioconjugation reaction between fluorescein isothiocyanate (FITC) and a model protein illustrates the utility of direct, calibration-free size determination, which enabled the resolution of fluorescence originating from free FITC from that of protein-bound FITC. TDA detection coupled to CE enabled the determination of peak identities without the need for standard solutions.