Application of a Microfluidic Lab-on-a-Chip Platform Coupled with Automated Motility Analysis for Phenotypic Drug Screening of Trypanosoma cruzi Trypomastigotes

Chagas disease remains a major neglected tropical disease, highlighting the need for new anti-Trypanosoma cruzi compounds and improved approaches for their phenotypic evaluation. Here, we extended a previously developed Lab-on-a-Chip (LOC) platform integrating a serial dilution generator and automated label-free motility analysis (TR_KF) to the direct screening of extracellular T. cruzi trypomastigotes. The platform was validated in two steps using benznidazole as the reference drug. First, automated motility quantification in conventional 96-well plates was compared with manual Neubauer counting, yielding comparable concentration–response curves and IC50 values. Second, the complete LOC–TR_KF platform was compared with the conventional assay, producing an IC50 that was not significantly different from the reference method. The validated platform was subsequently applied to screen ten candidate samples, identifying combinatorial libraries 2161-X and 2160-X as the most active against trypomastigotes. Comparison with previously reported intracellular amastigote data revealed marked stage-dependent differences in compound activity, while pre-treatment with both libraries reduced parasite infectivity by approximately 50%. Overall, the integrated LOC–TR_KF platform provides pharmacological responses comparable to conventional assays while reducing assay-solution requirements, manual handling and enabling automated, label-free phenotypic screening of T. cruzi trypomastigotes.