Studying the Behavior of T. Brucei Under Electric Field Gradients Implemented Using Optoelectronic Tweezers

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 IT