PROJECT SUMMARY/ABSTRACT The human gastrointestinal (GI) tract hosts the largest number of bacterial species found in the body, often referred to as the gut microbiota. This diverse and complex microbial community functionally expands the host genome, critically impacting metabolism, physiology, nutrition, and immune function. Current methods for assessing the human gut microbiota are based on fecal sampling, visualization, and various endoscopy- assisted biopsy techniques. Endoscopy-assisted techniques are invasive and usually incapable of retrieving samples from the small intestine. While stool samples provide noninvasive assessment, they are incapable of preserving spatial and temporal information of the bacteria through the GI tract. Therefore, we propose to develop an ingestible non-invasive smart sampling capsule (SSC)-based device that will provide a targeted sampling of viable gut microbes. The developed technology would allow unique insights into the impact that live microbes and other microbiota modulators have on the gut microbiota in hard-to-access GI regions. In the R21 phase of the project, we will design and develop two SSCs that provide targeted sampling in the small or large intestines, respectively. These SSCs are composed of acrylic casing that will house an absorbent hydrogel and a gas permeable membrane, as well as a nonmagnetic tracer to detect the capsule’s excretion in the feces. The hydrogel acts as a passive actuator such that upon swelling, it aspirates the intestinal fluid with its containing microbiota and seals the capsule. Single-layer pH-triggered and double pH- and microbial-triggered polymeric coatings will be utilized to delay the sampling until the capsule reaches the proximal small or large intestine, respectively. During this phase, in-depth capsule performance (e.g. sampling and retrievability) of the two SSCs will be assessed in vitro and in diet-controlled pigs. The R33 phase with two aims will clinically assess the effects of a U.S.-style Healthy Eating Pattern with low- versus high-dietary fiber content on segmental GI microbiota in humans using the two developed SSCs. We will first work to adapt the SSC methodology from the animal model to human subjects. This proof-of-principle study will be conducted to validate the effectiveness of SSC recovery from participants away from the testing facility. We will also assess the targeted sampling performance of the SSCs at appropriate locations along the GI tract using an FDA- approved large intestine-specific delivery drug (Balsalazide) as a testing control compound. For the clinical study, we will use the two SSCs to assess and compare the effects of high- versus low-fiber U.S.-style Healthy Eating Pattern on the gut microbiota in the proximal small and large intestine of 40 young, middle-aged, and older adults. The overall goal of this R21/R33 project is to produce highly reliable, practical, and cost-effective smart sampling capsules, suitable for use for re...