3D Printed Instrument for Taylor Dispersion Analysis with Two-Point Laser-Induced Fluorescence Detection.

Precisely controlling the size of engineered biomolecules and pharmaceutical compounds is often critical to their function. Standard methods for size characterization, such as dynamic light scattering or size exclusion chromatography, can be sample intensive and may not provide the sensitivity needed for mass- or concentration-limited biological systems. Taylor dispersion analysis (TDA) is a proven analytical method for direct, calibration-free size determination which utilizes only nL-pL sample volumes. In TDA, diffusion coefficients, which are mathematically transformed to hydrodynamic radii, are determined by characterizing band broadening of an analyte under well-controlled laminar flow conditions. Here, we describe the design and development of a 3D printed instrument for TDA, which is the first such instrument to offer dual-point laser-induced fluorescence (LIF) detection. The instrument utilized a fully 3D printed eductor as a vacuum source for precise and stable pressure-driven flow within a capillary, evidenced by a linear response in generated static pressure to applied gas pressure (R2 = 0.997) and a 30-fold improvement in stability of static pressure (0.05% RSD) as compared to a standard mechanical pump (1.53%). Design aspects of the LIF detection system were optimized to maximize S/N for excitation and emission optical axes, and high sensitivity was achieved as evidenced by an 80 pM limit of detection for the protein R-Phycoerythrin and low nM limits of detection for three additional fluorophores. The utility of the instrument was demonstrated via sizing of R-Phycoerythrin at pM concentrations.

Taylor dispersion analysis in fused silica capillaries: a tutorial review.

Biological and pharmaceutical analytes like liposomes, therapeutic proteins, nanoparticles, and drug-delivery systems are utilized in applications, such as pharmaceutical formulations or biomimetic models, in which controlling their size is often critical. Many of the common techniques for sizing these analytes require method development, significant sample preparation, large sample quantities, and lengthy analysis times. In other cases, such as DLS, sizing can be biased towards the largest constituents in a mixture. Therefore, there is a need for more rapid, sensitive, accurate, and straightforward analytical methods for sizing macromolecules, especially those of biological origin which may be sample-limited. Taylor dispersion analysis (TDA) is a sizing technique that requires no calibration and consumes only nL to pL sample volumes. In TDA, average diffusion coefficients are determined via the Taylor-Aris equation by characterizing band broadening of an analyte plug under well-controlled laminar flow conditions. Diffusion coefficient can then be interpreted as hydrodynamic radius (RH) via the Stokes-Einstein equation. Here, we offer a tutorial review of TDA, intended to make the method better understood and more widely accessible to a community of analytical chemists and separations scientists who may benefit from the unique advantages of this versatile sizing method. We first provide a tutorial on the fundamental principles that allow TDA to achieve calibration-free sizing of analytes across a wide range of RH, with an emphasis on the reduced sample consumption and analysis times that result from utilizing fused silica capillaries. We continue by highlighting relationships between operating parameters and critically important flow conditions. Our discussion continues by looking at methods for applying TDA to sample mixtures via algorithmic approaches and integration of capillary electrophoresis and TDA. Finally, we present a selection of reports that demonstrate TDA applied to complex challenges in bioanalysis and materials science.