(307b) Microfluidic Detection Tool for Anaplasma Spp. Infections Via Electrokinetics

Anaplasmosis, caused by Anaplasma phagocytophilum and transmitted by ticks, particularly the black-legged tick (Ixodes scapularis), has become a significant public health concern. In 2021, the U.S. reported 6,729 cases, surpassing pre-pandemic levels [1]. Current diagnostic methods, including PCR and blood smear microscopy, have turnaround times of 2 to 4 weeks [2]. This research introduces a novel diagnostic tool that is label-free, minimally invasive, low-cost, and rapid by harnessing the unique dielectric signatures of HL-60 cells affected by Anaplasma spp. using dielectrophoresis (DEP), an electrokinetic technique [3, 4]. HL-60 cells, both healthy and Anaplasma spp.-infected, were incubated at 37 °C in a 5% CO₂ environment using a medium of 0.8% sucrose and 0.3% dextrose, conductivity adjusted using 1x PBS (0.01-0.05 S/m conductivity) [5]. The cell suspension pipetted into the 3DEP analyzer [6] microchip, was exposed to a fixed peak-to-peak voltage of 20 V_pp, and cell behavior was observed. The dielectric crossover frequency, defined as the frequency at which cells remain stationary in non-uniform electric fields, was measured between 0.5 Hz and 45 MHz. This correlates to the observed physiological changes in the cells' cytoplasm and membrane. The results show unique dielectric characteristics of infected cells compared to healthy cells, as evidenced by the distinct crossover frequencies. Specifically, a first crossover frequency of 40 kHz was observed for infected cells, which is significantly lower than the 120 kHz observed for healthy cells. Additionally, a decrease in cytoplasmic conductivity was noted in cells with over 75% infection, while an increase in specific membrane capacitance was observed in cells with moderate infection levels (greater than 50%). These changes in the bioelectric signatures will be harnessed to obtain a rapid and cost-effective diagnostic tool for diagnosing Anaplasma spp. infections. COMSOL Multiphysics software is utilized to design the optimal diagnostic tool yielding superior efficiency and specificity.

Figure 1: Experimental workflow for creating a microfluidic sorting device to differentiate between healthy and Anaplasma spp. infected HL-60 cells. (A) Anaplasmosis is transmitted through a tick bite, (B) Healthy and Anaplasma spp. infected HL-60 cells are cultured in an incubator, (C) A buffer medium containing 0.8% sucrose and 0.3% dextrose is prepared, with a conductivity range of 0.01-0.05 S/m, adjusted using 1x PBS, (D) The crossover frequency, where cells neither move toward nor away from the electrodes, is determined using the dielectrophoresis technique, (E) Data from healthy and infected cells are compared, (F) The COMSOL platform is used to simulate the sorting device based on the experimental data and (G) The optimized sorting platform is 3D printed using a CADWorks 3D printer.

References

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[3] R. Oladokun, E. Adekanmbi, M. Ueti, and S. Srivastava, "Dielectric characterization of Babesia bovis using the dielectrophoretic crossover frequency," ELECTROPHORESIS, vol. 44, no. 11-12, pp. 988-1001, 2023/06/01 2023, doi: https://doi.org/10.1002/elps.202200263.

[4] N. F. Doost, S. D. R. Yaram, K. Wagner, H. Garg, and S. K. Srivastava, "Bioelectric profiling of Rickettsia montanensis in Vero cells utilizing dielectrophoresis," Journal of Biological Engineering, vol. 19, no. 1, p. 18, 2025/02/18 2025, doi: 10.1186/s13036-025-00487-y.

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[6] K. F. Hoettges et al., "Ten-Second Electrophysiology: Evaluation of the 3DEP Platform for high-speed, high-accuracy cell analysis," (in eng), no. 2045-2322 (Electronic).