Serum protein electrophoresis (SPE) separates serum proteins into bands whose shape and amplitude can alert clinicians to a range of disorders. This is followed by more specific immunoassays to quantify important antigens and confirm a diagnosis. Here we develop a high-speed capillary electrophoresis (HSCE) platform capable of simultaneous SPE and immunoassay measurements. A single laser excitation source is focused into the detection zone of the capillary to measure both refractive index (SPE) and fluorescence signals (immunoassays). The refractive index signal measures characteristic SPE profiles for human serum separated in 100 mM boric acid (pH 10), 100 mM arginine (pH 11), and 20 mM CHES (pH 10). For the immunoassay, the fluorescence electropherograms reveal that CHES provides the optimal buffer for measuring the immunocomplex and separating it from the free antigen. Immunoassays in CHES yield a LOD of 23 nM and a LOQ of 70 nM for the detection of fluorescein. The high pH reduces protein adsorption but reduces antibody affinity. Preliminary studies carried out in 50 mM barbital at pH 8 show improved stability of the immunocomplex and better separation for immunoassay quantification. Further optimization will open new capabilities for measuring orthogonal diagnostic signals in seconds with HSCE.
The appearance of unexpected peaks in capillary electrophoresis (CE) is common and can lengthen the time of method development as assay conditions and experimental parameters are varied to understand and mitigate the effects of the additional peaks. Additional peaks can arise when a single-analyte zone is split into multiple zones. Understanding the underlying mechanism of these phenomena, recognizing conditions that favor its presence, and knowing how to confirm and eliminate the effect are important for efficient method optimization. In this study, we examine how the overlap of analyte zones with the sample plug can lead to peak splitting. This is explored experimentally using dual detection CE, which enables both the sample plug and analyte zones to be independently and simultaneously measured from the same detection volume. Simulations performed via COMSOL Multiphysics confirm the origin of the splitting and help guide experiments to reduce and eliminate the effect. Our findings show that this peak splitting mechanism can arise in separations of both small and large molecules but is, especially, prevalent in separations of slowly migrating macromolecules. This effect is also more prevalent when using a short length-to-detector, as is commonly found in microfluidic applications. A simple diffusion-less model is introduced to develop strategies for reducing peak splitting that avoids modifying the apparatus, such as by lengthening the separation length, which can be difficult. Decreasing the sample plug length and slowing the electroosmotic flow can both reduce this effect, which is confirmed experimentally.
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