Project Summary This R21/R33 proposal is submitted in response to PAR-20-133. The proposal focuses on the development of an ingestible device termed “micro-pill”. The micro-pill will be designed to autonomously sample intestinal content from predetermined anatomical sites in the gastrointestinal (GI) tract, a process which currently requires invasive methods. The development of the micro-pill benefits from the applicants' experimentation with an earlier version of the pill that utilizes an osmotic pump for sampling. To ensure accurate sampling of GI luminal content from specific anatomical sites, the micro-pill will be fitted with a miniaturized motor controlled by pH and pO2 sensors. The concept takes advantage of the proximal-to-distal rising pH and falling oxygen gradient, such that the micro-pill will sample autonomously based on pre-programmed pH and O2 thresholds. This innovative control mechanism is optimal for this application because it dispenses with the need for tomography or built-in camera that may be needed to track the pill's location for spatially selective sampling. The new micro-pill will run on power from a coin cell battery and will also support wireless magnetic activation in individuals where pH/pO2 levels may be altered due to atypical GI tract conditions. The architecture of the micro-pill is optimized to enable sampling of viscous intestinal content and reduce leakage and contamination, while maximizing the volume of sample collected and maintaining low specific weight for improved buoyancy and motility through the GI tract. During the R21 phase micro-pill prototypes will be built and tested in vitro in conditions simulating the viscosity of the GI lumen and ex vivo in pig intestines. Optimized designs will be tested in live pigs. The R21 phase is organized in 3 specific aims: (1), To design, fabricate and validate in vitro a motorized micro-pill; (2) To design, integrate and test pH/pO2 sensors in vitro and ex vivo using electronics integrated in the micro-pill; (3) To test the ingestible micro-pill in live pigs in vivo. The aims of the R33 phase are to assess the efficacy of the micro- pill (4) and conducting a 2-phase randomized controlled cross-over diet trial in humans (5). In Specific aim 4 the feasibility of sampling human adults' luminal content from pre-determined GI tract regions will be tested in a clinical trial. The trial will not only assess the micro-pill's function, but also compare the effects of a plant-based and a meat-based diet on the GI tract microbiota collected from different anatomical GI sites. This proposal was conceived by three Tufts University researchers with complementary expertise. PI Dr. Sameer Sonkusale from the School of Engineering heads an interdisciplinary Nano Lab and specializes in micro- and nano-fabrication, and biomedical devices. Dr. Giovanni Widmer from the School of Veterinary Medicine has extensive experience with animal models, microbiota analysis and bioinformatics. Dr. Al...