Studying the Behavior of T. Brucei Under Electric Field Gradients Implemented Using Optoelectronic Tweezers
Authors: Gullette Emily , Hanson Natalie , Kluttz Emily , Stuart Callie , Hammer Meredith , Pitman Anna , Wallace Kayla , Keck Devin Monroe , Martinez-Duarte Rodrigo 
Published: February 2020

Here we present a study of the response of Trypanosoma brucei (T. brucei) to electric field gradients. T. brucei is a parasitic protozoan that causes trypanosomiasis or African Sleeping Sickness. Unfortunately, accurate detection of the parasite is now costly, requiring considerable technical training as in the case of cerebrospinal fluid testing (1) and card agglutination tests (2), which hinders the diagnosis and treatment of the disease in patients living in developing countries. Further characterization of T.brucei is thus necessary to create more affordable and accessible diagnostic techniques. The long term objective of this research is to develop alternatives to detecting T. brucei by aggregating the parasites to a specific area which will facilitate their observation.
The work presented here is aimed at characterizing the response of T. brucei to electric field gradients towards assessing the potential of dielectrophoresis as a tool for parasite concentration. To do this, we implemented an Optoelectronic tweezer (OET) setup to create an electric field gradient by simple illumination of specific areas of a photoconductive layer (3). Briefly, an OET setup features two plates of transparent indium tin oxide (ITO) polarized by a function generator and spaced apart a specific distance to enclose a volume filled with media. A photoconductive layer must be deposited on one of the ITO plates such that electrical coupling between the plates only occurs on those areas that are illuminated by visible light. Fabrication of such devices only requires film coatings, which makes production scalable and relatively inexpensive. Here we generate the light pattern using a general purpose DLP-based projector fitted with reduction optics. Hence, by changing this light pattern we are able to efficiently introduce electric field gradients in the fluidic volume. We utilize this capability to expose T. brucei to gradients of varying magnitude and frequency. We seek those parameters under which T. brucei shows a clear migration towards the gradient that will enable its concentration.
To run OET experiments on T. brucei, the parasite was centrifuged twice and resuspended in a sugar solution with its conductivity adjusted to values from 500 μS to 1,500 μS using Phosphate Buffered Saline (PBS). 10 μL of such sample were then introduced in the OET setup. A powerpoint slide with a black background and white, square rings was used to illuminate the photoconductive layer. The ITO plates were polarized using a sinusoidal signal with varying voltage and frequency, in the range 3.5 - 10V and 100kHz- 20MHz respectively. The parasite behavior was monitored throughout the experiment to assess its response to the light-induced electric field gradient.
Initial results when probing the frequency range mentioned above showed attraction of the parasite to the illuminated square only at 18 and 19 MHz across the complete range of tested signal amplitudes (3.5 Vpp, 5 Vpp, 6 Vpp, 7.5 Vpp and 10 Vpp) with a conductivity of 500 μS/cm. When there was no attraction, the parasites did not move towards the light, or passed through the light, showing no restriction of movement from an attractive force. By increasing the voltage at a frequency that causes attraction, the speed of movement towards the light increases proportionally. Lowering the electrical conductivity of the media also causes a stronger attraction of the parasite. The use of conductivity values below 500 μS and amplitudes above 10 V renders the parasite non viable regardless of the frequency value.
The focus of ongoing work includes validating the frequencies where attraction was seen, 18-19 MHz, and further optimizing the voltage to maximize attraction of the parasite while keeping them viable. Future work includes experimentation with different shapes and colors of the incident light to assess their importance in the rapid concentration of the parasite working towards an inexpensive and accurate diagnostic test for T. brucei.

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.

Kremer, C., Neale, S., Menachery, A., Barrett, M., Cooper, M. (2012). Optoelectronic Tweezers for Medical Diagnostics. Frontiers in Biological Detection: From Nanosensors to Systems IV, 8(212).