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

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.