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. Competitive immunoassay reagents are mixed online and streamed into the μFFE device, where bound and free labelled species are separated in real-time based on their electrophoretic mobility. In the current manuscript 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 9-seconds, a 2.7-fold improvement in temporal response. The response time of the assay worsened when immunoassay reagents were incorporated due to 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.