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1. Separation of Cells and Microparticles in Insulator-Based Electrokinetic Systems

   https://doi.org/10.1021/acs.analchem.2c04366  

   Vaghef-Koodehi, Alaleh; Ernst, Olivia D.; Lapizco-Encinas, Blanca H. (January 2023, Analytical Chemistry)

   Presented here is the first continuous separation of microparticles and cells of similar characteristics employing linear and nonlinear electrokinetic phenomena in an insulator-based electrokinetic (iEK) system. By utilizing devices with insulating features, which distort the electric field distribution, it is possible to combine linear and nonlinear EK phenomena, resulting in highly effective separation schemes that leverage the new advancements in nonlinear electrophoresis. This work combines mathematical modeling and experimentation to separate four distinct binary mixtures of particles and cells. A computational model with COMSOL Multiphysics was used to predict the retention times (tR,p) of the particles and cells in iEK devices. Then, the experimental separations were carried out using the conditions identified with the model, where the experimental retention time (tR,e) of the particles and cells was measured. A total of four distinct separations of binary mixtures were performed by increasing the level of difficulty. For the first separation, two types of polystyrene microparticles, selected to mimic Escherichia coli and Saccharomyces cerevisiae cells, were separated. By leveraging the knowledge gathered from the first separation, a mixture of cells of distinct domains and significant size differences, E. coli and S. cerevisiae, was successfully separated. The third separation also featured cells of different domains but closer in size: Bacillus cereus versus S. cerevisiae. The last separation included cells in the same domain and genus, B. cereus versus Bacillus subtilis. Separation results were evaluated in terms of number of plates (N) and separation resolution (Rs), where Rs values for all separations were above 1.5, illustrating complete separations. Experimental results were in agreement with modeling results in terms of retention times, with deviations in the 6–27% range, while the variation between repetitions was between 2 and 18%, demonstrating good reproducibility. This report is the first prediction of the retention time of cells in iEK systems. 

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2. Assessing the Discriminatory Capabilities of iEK Devices under DC and DC-Biased AC Stimulation Potentials

   https://doi.org/10.3390/mi14122239  

   Nasir_Ahamed, Nuzhet Nihaar; Mendiola-Escobedo, Carlos A; Perez-Gonzalez, Victor H; Lapizco-Encinas, Blanca H (December 2023, Micromachines)

   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 (Rs), showed a complete separation (Rs = 1.51) with the application of a DC-biased low-frequency AC signal but an incomplete separation (Rs = 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. 

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3. Fine-Tuning Electrokinetic Injections Considering Nonlinear Electrokinetic Effects in Insulator-Based Devices

   https://doi.org/10.3390/mi12060628  

   Miller, Abbi; Hill, Nicole; Hakim, Kel; Lapizco-Encinas, Blanca H. (June 2021, Micromachines)

   null (Ed.)

   The manner of sample injection is critical in microscale electrokinetic (EK) separations, as the resolution of a separation greatly depends on sample quality and how the sample is introduced into the system. There is a significant wealth of knowledge on the development of EK injection methodologies that range from simple and straightforward approaches to sophisticated schemes. The present study focused on the development of optimized EK sample injection schemes for direct current insulator-based EK (DC-iEK) systems. These are microchannels that contain arrays of insulating structures; the presence of these structures creates a nonuniform electric field distribution when a potential is applied, resulting in enhanced nonlinear EK effects. Recently, it was reported that the nonlinear EK effect of electrophoresis of the second kind plays a major role in particle migration in DC-iEK systems. This study presents a methodology for designing EK sample injection schemes that consider the nonlinear EK effects exerted on the particles being injected. Mathematical modeling with COMSOL Multiphysics was employed to identify proper voltages to be used during the EK injection process. Then, a T-microchannel with insulating posts was employed to experimentally perform EK injection and separate a sample containing two types of similar polystyrene particles. The quality of the EK injections was assessed by comparing the resolution (Rs) and number of plates (N) of the experimental particle separations. The findings of this study establish the importance of considering nonlinear EK effects when planning for successful EK injection schemes. 

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4. Determination of the Empirical Electrokinetic Equilibrium Condition of Microorganisms in Microfluidic Devices

   https://doi.org/10.3390/bios10100148  

   Coll De Peña, Adriana; Hill, Nicole; Lapizco-Encinas, Blanca H. (October 2020, Biosensors)

   null (Ed.)

   The increased concern regarding emerging pathogens and antibiotic resistance has drawn interest in the development of rapid and robust microfluidic techniques to analyze microorganisms. The novel parameter known as the electrokinetic equilibrium condition (EEEC) was presented in recent studies, providing an approach to analyze microparticles in microchannels employing unique electrokinetic (EK) signatures. While the EEEC shows great promise, current estimation approaches can be time-consuming or heavily user-dependent for accurate values. The present contribution aims to analyze existing approaches for estimating this parameter and modify the process into an accurate yet simple technique for estimating the EK behavior of microorganisms in insulator-based microfluidic devices. The technique presented here yields the parameter called the empirical electrokinetic equilibrium condition (eEEEC) which works well as a value for initial approximations of trapping conditions in insulator-based EK (iEK) microfluidic systems. A total of six types of microorganisms were analyzed in this study (three bacteria and three bacteriophages). The proposed approach estimated eEEEC values employing images of trapped microorganisms, yielding high reproducibility (SD 5.0–8.8%). Furthermore, stable trapping voltages (sTVs) were estimated from eEEEC values for distinct channel designs to test that this parameter is system-independent and good agreement was obtained when comparing estimated sTVs vs. experimental values (SD 0.3–19.6%). The encouraging results from this work were used to generate an EK library of data, available on our laboratory website. The data in this library can be used to design tailored iEK microfluidic devices for the analysis of microorganisms. 

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5. Antagonism between killer yeast strains as an experimental model for biological nucleation dynamics

   https://doi.org/10.7554/eLife.62932  

   Giometto, Andrea; Nelson, David R; Murray, Andrew W (December 2021, eLife)

   Antagonistic interactions are widespread in the microbial world and affect microbial evolutionary dynamics. Natural microbial communities often display spatial structure, which affects biological interactions, but much of what we know about microbial antagonism comes from laboratory studies of well-mixed communities. To overcome this limitation, we manipulated two killer strains of the budding yeastSaccharomyces cerevisiae, expressing different toxins, to independently control the rate at which they released their toxins. We developed mathematical models that predict the experimental dynamics of competition between toxin-producing strains in both well-mixed and spatially structured populations. In both situations, we experimentally verified theory’s prediction that a stronger antagonist can invade a weaker one only if the initial invading population exceeds a critical frequency or size. Finally, we found that toxin-resistant cells and weaker killers arose in spatially structured competitions between toxin-producing strains, suggesting that adaptive evolution can affect the outcome of microbial antagonism in spatial settings. 

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