Data in Support of Identifying and Minimizing Primary Sources of Temporal Broadening in Online Affinity Micro Free-Flow Electrophoresis

Affinity micro free-flow electrophoresis (µFFE) takes advantage of continuous separations to enable real-time measurements in biological systems. Here, competitive immunoassay reagents are mixed online and streamed into the µFFE device, where bound and free labeled species are separated in real-time based on their electrophoretic mobility. In the manuscript corresponding to this dataset, we optimized the affinity µFFE platform to minimize the response time of the assay. Analyte response time was improved by 1) mitigating temporal broadening as analyte transited the fluidic system and 2) reducing surface adsorption. Fast iteration of the microfluidic parameters enabled optimization of temporal broadening due to longitudinal diffusion and parabolic flow. Minimizing incubation capillary length while ensuring adequate equilibration time for the competitive immunoassay improved rise times from 25-seconds to 10-seconds, a 2.5-fold improvement in temporal response. Incorporation of immunoassay reagents hindered response time as a result of peptide surface adsorption. Dynamic coating with 0.005% w/v bovine serum albumin was shown to mitigate surface adsorption without interfering with the binding and separation of the immunoassay components, resulting in 2.8-fold improvement in overall signal and 2-fold improvement in temporal response, which improved from 61 seconds to 30 seconds. This data set includes raw and processed data supporting this publication, as well as MATLAB code used for data processing and figure generation.