There is a rising need for rapid and reliable analytical methods for separating microorganisms in clinical and biomedical applications. Microscale-insulator-based electrokinetic (iEK) systems have proven to be robust platforms for assessing a wide variety of microorganisms. Traditionally, iEK systems are usually stimulated with direct-current (DC) potentials. This work presents a comparison between using DC potentials and using DC-biased alternating-current (AC) potentials in iEK systems for the separation of microorganisms. The present study, which includes mathematical modeling and experimentation, compares the separation of bacterial and yeast cells in two distinct modes by using DC and DC-biased AC potentials. The quality of both separations, assessed in terms of separation resolution ($R_s$), showed a complete separation ($R_s$ = 1.51) with the application of a DC-biased low-frequency AC signal but an incomplete separation ($R_s$ = 0.55) with the application of an RMS-equivalent DC signal. Good reproducibility between experimental repetitions (<10%) was obtained, and good agreement (~18% deviation) was observed between modeling and experimental retention times. The present study demonstrates the potential of extending the limits of iEK systems by employing DC-biased AC potentials to perform discriminatory separations of microorganisms that are difficult to separate with the application of DC potentials.

This study proposes a strategy for improving the separation resolution of binary microparticle mixtures by modifying the post array arrangement in insulator-based electrokinetic devices.

Insulator-based electrokinetic (iEK) platforms integrate insulating structures within microchannels to amplify nonlinear electrophoresis (EPNL) effects and improve separation efficiency, as such effects scale with the electric field magnitude and introduce more discriminatory characteristics. In contrast, free solution platforms, used in conventional capillary electrophoresis (CE) systems, traditionally neglect EPNL effects and rely on the electrical charge of target analytes alone to perform separations by employing linear electrophoresis (EPL). In this work, the effects of EPNL in free solution platforms are tested by separating a binary mixture of microparticles and a charge-identical binary mixture of microparticles and Bacillus cereus cells. While analytical techniques typically face a trade-off between being ″good″ (high resolution) or ″fast″ (short duration), the results show that EPNL can be exploited to deliver on both aspects simultaneously. Using EPNL increased separation resolution (Rs) by up to 50% and decreased separation duration by 56%, making separations with EPNL both faster and better compared to platforms relying on EPL alone. Furthermore, separations of a binary mixture of microparticles and cells achieved resolution values >2.0 when EPNL effects were accentuated, well above the gold standard of 1.5. To the best of our knowledge, this work represents the first of its kind where such a comprehensive advantage of EPNL in free solution is reported, effectively overcoming the traditional performance trade-offs of open channel electrokinetic separations.