Carbon-Electrode Dielectrophoresis for Concentrating Trypanosoma Brucei

Abstract

Trypanosoma brucei (T. brucei) is a parasite that causes African Trypanosomiasis, or African sleeping sickness. This disease is spread through bite from a Tsetse fly or congenitally. Current tests used to properly diagnose the disease, such as cerebrospinal fluid testing (1) and card agglutination tests (2), are unaffordable in developing countries. For example, impoverished areas in Africa suffering from the disease do not have proper resources to house and maintain equipment needed for these tests. Hence, the goal of this research is to find a more cost-efficient way to identify the disease in rural settings. Dielectrophoresis (DEP) is a separation technique that utilizes the characteristic response of different microorganisms to a non-uniform electric field to segregate a specimen of interest from its background (3). In particular, positive DEP enables the concentration of the targeted organism in regions of high electric field gradient. Here, we postulate the use of inexpensive carbon electrodes to implement positive DEP of T. brucei. We aim at attracting and concentrating T. brucei parasites around the electrodes to facilitate and ease Trypanosomiasis detection by untrained personnel, thus reducing costs and enabling faster treatment of African Trypanosomiasis. The experimental setup used here included a microfluidics chip featuring multiple rows of intercalated carbon microelectrodes as detailed elsewhere (4). The experimental sample was prepared by re-suspending T. brucei parasites in a sugar solution with a conductivity of 500 μS/cm. The sample was flowed through the polarized electrode array at a flow rate of 0.2 μL/min for 2 minutes. After this time the field was turned off and the parasites that may had been trapped were released for elution. The electrode array was polarized with a sinusoidal signal at different frequencies, ranging from 100 kHz to 10 MHz at 100 kHz intervals, but at an optimized constant amplitude of 2 Vpp. The array was monitored to characterize the movement of the parasite around the electrodes and sequential images were taken at the end of the array to characterize the parasite elution pattern once the the field was turned off. These images were analyzed for each experiment to assess the difference in parasite attachment before and after turning the field off. Preliminary results indicate that the use of a voltage value higher than 2 Vpp leads to parasite death. At such polarizing voltage, attraction of the parasite around the electrodes was observed in the frequency range 500-900 kHz. However, trapping is predominantly around the electrically-insulating SU-8 structure around the electrodes. The attraction to this part of the chip, rather than to the carbon electrodes where it was expected, indicates a possible affinity to topography but most likely the possibility of a distorted electric field that surrounds the insulator and generates a high field gradient. Such distortion is the primary hypothesis for the attraction witnessed because we do not see this behavior at other frequencies outside the range 500-900 kHz. While some trapping was observed at experiments run at 10 Vpp, many parasites died when exposed to a high voltage. Shear stress was also examined as a possible cause of parasite death and the flow rate used lead to continued parasite viability. Ongoing work includes investigating the attraction of the parasites to the SU-8 layer, and whether this is caused by the non-uniform electric field around the curvature of the insulator layer. To this end, we are implementing numerical modeling of the electric field in the device to further identify the magnitude of the gradient that causes trapping. Once this is identified, the effect of sample conductivity on the trapping will be addressed to further understand the DEP behavior of T. brucei and facilitate its detection. References Chappuis, F., Loutan, L., Simarro, P., Lejon, V., & Büscher, P. (2005). Options for field diagnosis of human African trypanosomiasis. Clinical Microbiology Reviews, 18(1), 133-146. Chappuis, F., Stivanello, E., Adams, K., Kidane, S., Pittet, A., & Bovier, P. A. (2004). Card agglutination test for trypanosomiasis (CATT) end-dilution titer and cerebrospinal fluid cell count as predictors of human African Trypanosomiasis (Trypanosoma brucei gambiense) among serologically suspected individuals in southern Sudan. The American Journal of Tropical Medicine and Hygiene, 71(3), 313-317. Menachery, M., Kremer, C., Wong, P., Carlsson, A., Neale, S., Barrett M., Cooper, J. (2012). Counterflow Dielectrophoresis for Trypanosome Enrichment and Detection in Blood. Scientific Reports, 775(2), 1-5. Martinez-Duarte, R., Renaud, P., Madou, M. (2011). A Novel Approach to Dielectrophoresis Using Carbon Electrodes. Electrophoresis, 32(17), 2385-2392. Figure 1