Here, we describe the development and testing of an innovative noninvasive technology to sample the intestinal lumen in vivo. Beyond the prospects and opportunities for ingestible GI sampling devices discussed in a prior article, 12 the proposed device is specifically targeted to study the gut microbiome. We created an ingestible, biocompatible, 3D‐printed microengineered pill with an integrated osmotic sampler that requires no battery for its operation. Stereo‐lithography (SLA)‐based 3D printing was used to fabricate the miniaturized ingestible device with sophisticated microfluidic functions for spatial sampling of the gut lumen. The pill was covered with a pH‐sensitive enteric coating to delay sampling until it entered the small intestine, where the coating dissolves in the higher pH environment. A magnetic holding mechanism was designed to enable the pill to sample more time from a targeted region of the gut. Natural peristaltic motion endows mobility to the pill through the GI tract without any active parts. The sampling function of the pill has been extensively validated in vitro and in vivo in pigs and primates. The magnetic hold for spatial targeting has been validated in vitro.

The gut microbiota represents trillions of bacteria belonging to around 1000 species. 1 Among the various microbial communities associated with the human body, the gut microbiome is noted for its diversity, elevated concentration, 2 , 3 and many beneficial functions. Dysbiosis (microbial imbalance) has been associated with conditions such as inflammation, recurrent infections, and increased susceptibility to enteric pathogens. 4 - 8 Most studies infer the condition of the gut microbiome from the analysis of fecal DNA and fecal metabolites. 1 Because the gut environment changes as the gut content moves down the gastrointestinal (GI) tract, 9 analyses of feces are inadequate to identify abnormal conditions upstream of the distal colon. Whereas the analysis of complex bacterial populations has benefitted from new DNA sequencing techniques, our capacity to precisely and noninvasively sample different organs has not improved. Consequently, medical research is often based on easily accessible samples, like feces, in spite of the inherent limitations of the conclusions that can be drawn from the analysis of such samples. 10 For instance, samples important for understanding the interaction between enteric pathogens and the host remain out of reach, unless invasive sampling techniques are used. 11 To gain new insights into the many beneficial functions of the gut microbiota, it is essential to sample in vivo different locations in the gut, particularly organs located upstream of the colon.

2 Results and Discussion

2.1 Osmotic Pill Sampler Design The overall design of the pill is shown in Figure 1 a. The osmotic pill sampler consists of three main parts—a top sampling head, a semipermeable membrane in the middle, and a bottom salt chamber. The head comprises four inlets, a stilling basin connected to four helical channels, all of them leading to one small chamber. The salt chamber consists of a cavity that contains dry calcium chloride salt powder, a second cavity to hold a small neodymium magnet, two tube‐like reservoirs to hold fluorescent dye, and a horn‐shaped exit nozzle at the bottom. The pill works on the principle of osmosis where a pressure differential is created across the semipermeable membrane, which creates a passive pumping action (see Figure 1a). This mechanism facilitates the flow of water across the membrane from the helical channels toward the salt chamber. The porosity of the membrane blocks the flow of larger particles (e.g., microorganisms), leaving them trapped in the helical channels (Figure 1a). Figure 1 Open in figure viewer PowerPoint Osmotic pill sampler design and working principle. a) A 3D schematic of the overall design of the pill, b) fabricated osmotic pill samplers, c) proof of concept, the pill samples of a water solution containing blue food dye (the sample did not penetrate the salt chamber), d) the pill surface and the fluorescent dye in the pill are detectable under UV light, e) scanning electron microscope (SEM) images of the osmotic membrane used in the pill, f) marked inlet at the top chamber is designed for initiating the pill (priming), and g) overall working principle of the osmotic pill sampler in an enteric capsule. The fabrication of this pill is explained in detail in the Experimental Section. Briefly, the pill sampling head and the bottom salt chamber are 3D printed using an SLA 3D printer. A semipermeable membrane is used to construct the osmotic pump. The membrane itself is made of woven 5 μm‐thick cellulose acetate fibers commonly used in reverse osmosis–based water filters (Figure 1e,f). A small neodymium magnet is placed and sealed inside the salt chamber. To facilitate locating the pill after it is excreted in the feces, two lines of a green fluorescent dye are painted and sealed in the salt chamber. The salt chamber is filled with calcium chloride salt. The top and bottom chambers are separated by the semipermeable membrane assembled and attached together using a UV curable adhesive. The fabricated osmotic pill sampler is shown in Figure 1b. Note that the proposed pill has no electrical components or moving parts. We have used a biocompatible photocurable polymer for the pill casing. Furthermore, the only chemical used in the pill is common salt. Considering the materials and chemicals used in the construction of the pill, it is safe to conclude that the pill is biocompatible.

2.2 Pill Operation Concept 2.2.1 Priming the Pill The pill is primed by injecting approximately 200 μL water into the salt chamber through the exit nozzle using a syringe with a 36 G needle. Similarly, the helical channels are filled with water from a marked inlet, which is connected to one of the helical channels (see Figure 1a, and Supplementary video 1). By aspirating water through the marked inlet, the water flows first through the corresponding channel and then fills the chamber at the bottom of the sampling head and finally fills all the other helical channels and the stilling chamber on the top of the pill in this sequence. The head can hold approximately 120 μL of sample. 2.2.2 Sampling Strategy After priming, the pill is ready for oral administration. To avoid sampling the stomach lumen and limit the sampling to the small and large intestine, we placed the pill in a commercially available size‐0 enteric coating capsule. The enteric coating resists the acidic environment of the stomach and only dissolves in the neutral/basic environment of the intestine. The dissolution profile for the enteric coating can be controlled by an appropriate choice of polymer(s). Testing of different enteric coatings was not the focus of this study. The osmotic pill sampler moves down the GI tract primarily due to natural peristalsis. The pill, however, can be held at a specific location inside the GI tract using an external magnet. This is made possible by a small neodymium magnet embedded in the pill (Figure 1a). Immobilizing the pill enables preferential sampling of specific regions of the gut, so that more of the collected sample originates from this region. Without this magnet, the pill would sample more or less uniformly along the entire length of the GI tract. We have added two fluorescent marks on the pill to facilitate detection following excretion (Figure 1d). The details of our sampling strategy are shown in Figure 1g.

2.3 Physical Characterization of the Pill Osmosis is a net movement of a solvent through a semipermeable membrane toward a region of high solute concentration. Once the pill is primed, the process of osmosis causes solvent (water) to flow across the membrane from the helical channels into the salt chamber (Figure 1f). The flow rate depends on the properties of this membrane (e.g., thickness, porosity, area) and the salt gradient across it. The fluid sampler consists of an osmotic pump that continuously pulls the fluid from the gut into the narrow microfluidic collection channels (<1 mm diameter) at a flow rate of 2–20 μL h−1. We characterized the flow created by osmosis across the semipermeable membrane, which drives the sampling function of the pill (see Section 1a, Supporting Information). The flow rate through the membrane assembled in the pill was also measured over time (Figure S1b, Supporting Information). Initially, the flow rate was ≈2.5 μL h−1 and remained approximately constant for almost 48 h. The flow rate decreased to around ≈0.5 μL h−1 after 4 days. The design of the exit nozzle in the salt chamber is important because it facilitates the discharge of water being accumulated in the salt chamber as a result of osmotic action. Numerical simulations were carried out to study flow through the discharge hole for two different hole diameters, 100 μm and 50 μm (see Figure S2, Supporting Information). The maximum fluid velocity for the 50 μm hole was 0.6 mm s−1, which was 4.28 times higher than that for the hole of 100 μm size. A higher continuous flow rate at the discharge point has two advantages: first, it reduces the diffusion from the environment into the salt chamber, essentially acting as a one‐way valve, and second, a high flow rate reduces the chance of blockage of the discharge hole by solid debris present in the gut.

2.4 In Vitro Sampling The gut microbiome comprises prokaryotic and eukaryotic cells that may have no active motility or may be able to swim propelled by flagella or other mechanisms. We validated whether the osmotic pump can sample microorganisms regardless of their motion by testing the pill in vitro with nonmotile solid particulates as well as with highly motile bacteria. The first environment included polystyrene microparticles of 10 μm diameter in a highly viscous liquid (160 mPa s−1) to mimic the gut environment. These particles exhibit negligible diffusion, especially in the highly viscous solution of 50% w/v polyethylene glycol (PEG) hydrogel. Because these particles are neutrally buoyant in this solution, the effect of gravity on their motion is insignificant. The second environment included highly motile bacteria, wild‐type Bacillus subtilis, in an aqueous solution. These bacteria perform a run‐and‐tumble motion,13 meaning that they swim in a certain direction for a short time (≈1 s) and then change their swimming direction at random. This results in a highly diffusive transport of the bacteria over a long time with effective diffusion coefficients comparable to small gas molecules in water.

2.5 In Vitro Sampling of Nonmotile Microparticles −1 at 25 °C (USS‐DVT4 Rotary Viscometer Viscosity Meter, U.S. Solid Inc., Cleveland, OH, USA). Next, polystyrene particles of 10 μm diameter were added to these solutions to mimic partially digested food particles in the GI tract. These microparticles are large enough so that Brownian motion can be ignored. Therefore, any microparticles captured by the pill would have entered the helical channels mainly due to osmotic flow. Initially, pills were primed with deionized water without PEG. Then, a group of three primed pills were placed in each of the pH 4 and pH 8 solutions described earlier. At different time points, a pill was taken out of the solution, and the sample was extracted from the pill using a pipette. On average 120 ± 6 μL of the sample was recovered from each pill. The extracted sample was weighed and imaged using an optical microscope and finally dried on a hotplate at 40 °C and weighed again. The samples were weighed before and after drying to evaluate the amount of hydrogel aspirated by the pill. The number of microparticles acquired by the pill at two different time steps was also quantified (see D, for microparticles in the viscous medium through the Stokes–Einstein relation D = k b T 3 π η d (1) where k b is Boltzmann's constant, T is the absolute temperature, d is the diameter of the particle and η is the dynamic viscosity of the medium. The diffusion coefficient of a 10 μm‐diameter spherical particle at 37 °C in a fluid with viscosity of 160 mPa s−1 is D = 2.8 × 10 − 4 μ m 2 s − 1 . To diffuse one particle diameter under such conditions would require a time t ≈ d 2 / 2 D = 2.1 days. Thus, on time scales relevant to our experiments, diffusion is ineffective in generating any appreciable uptake of particles into the pill. We tested the pill in highly viscous environments similar to the GI tract by using solutions of 50% w/v of PEG, with a molecular mass of 10 000, in two different aqueous buffers of pH 4 and pH 8. Different pH conditions help model the fact that pH varies along the GI tract. The measured viscosity of the solution was 160 ± 5 mPa sat 25 °C (USS‐DVT4 Rotary Viscometer Viscosity Meter, U.S. Solid Inc., Cleveland, OH, USA). Next, polystyrene particles of 10 μm diameter were added to these solutions to mimic partially digested food particles in the GI tract. These microparticles are large enough so that Brownian motion can be ignored. Therefore, any microparticles captured by the pill would have entered the helical channels mainly due to osmotic flow. Initially, pills were primed with deionized water without PEG. Then, a group of three primed pills were placed in each of the pH 4 and pH 8 solutions described earlier. At different time points, a pill was taken out of the solution, and the sample was extracted from the pill using a pipette. On average 120 ± 6 μL of the sample was recovered from each pill. The extracted sample was weighed and imaged using an optical microscope and finally dried on a hotplate at 40 °C and weighed again. The samples were weighed before and after drying to evaluate the amount of hydrogel aspirated by the pill. The number of microparticles acquired by the pill at two different time steps was also quantified (see 3 for details). To exclude the effect of background fluid motion on the sampling capability of the pill, the tests were performed in static conditions with no agitation. To justify exclusion of the uptake in the pill by diffusion, we calculate the diffusion coefficient,, for microparticles in the viscous medium through the Stokes–Einstein relationwhereis Boltzmann's constant,is the absolute temperature,is the diameter of the particle andis the dynamic viscosity of the medium. The diffusion coefficient of a 10 μm‐diameter spherical particle at 37 °C in a fluid with viscosity of 160 mPa sis. To diffuse one particle diameter under such conditions would require a timedays. Thus, on time scales relevant to our experiments, diffusion is ineffective in generating any appreciable uptake of particles into the pill. Figure 2 shows the number of particles recovered from the helical channels for both acidic (pH 4) and basic (pH 8) environments. The number of particles increases over time (Figure 2a), which validates that the osmotic pill can indeed sample nonmotile microparticles in a highly viscous environment. We also evaluated the amount of PEG aspirated into the pill's helical channels over time. Initially, the channels contain only pure water used for priming. Over time, the PEG from the surrounding environment is sampled by the pill along with microparticles, causing the concentration of the PEG in the pill to increase. Figure 2b shows a quantitative comparison of the hydrogel concentration in the pill over time in acidic (pH 4) and basic (pH 8) conditions. The hydrogel concentration increases almost linearly with time. However, in basic conditions, a higher sampling rate was noted, as was a higher hydrogel concentration inside the pill, exceeding concentration in the surrounding environment. We believe this is because the pH protonates and deprotonates the functional groups of the membrane itself and of the molecules in solution. This effect will change the effective charge on the membrane and alter the size of the pores, which will impact the membrane's nanofiltration properties and affect the flux of water.14, 15 The zeta potential (ζ) of a cellulose acetate membrane is negative for pH 3 or higher and becomes more negative as the pH increases.16 Therefore, the membrane is more negatively charged at pH 8 (ζ ≈ −35 mV) compared with pH 4 (ζ ≈ −10 mV), which can result in bigger pore sizes and higher flux at pH 8. We repeated these experiments for a total of 12 pills, six under acidic (pH 4) and the rest at basic (pH 8) conditions. The same 50% PEG w/v solution and black 10 μm polystyrene beads were used as discussed earlier. The results shown in Figure 2 validate the sampling capability of the pill in both acidic and basic conditions. The number of particles is larger under basic conditions as compared to acidic conditions for the reasons explained earlier and is approximately equal to the control after 24 h of operation. Here, the control is the liquid containing the particles in which pills are immersed. Figure 2 Open in figure viewer PowerPoint Osmotic sampling by the pill of solid particles in acidic and basic high‐viscosity environments (50% PEG hydrogel). a) microparticles (polystyrene beads) captured by the pill after 8 and 24 h in solutions of different pH. Control shows particle suspension in which pills were immersed. b) weight percentage of the dried sample extracted from the pill at different time points. c) number of particles captured by the pill in two different pH conditions and compared with control (control is considered the environment outside of the pill).

2.6 In Vitro Sampling of Motile Bacteria B. subtilis was chosen as the test bacterium, which is ubiquitous in the GI tract of ruminants and humans. We used a fluorescent strain to facilitate imaging. B. subtilis is a multiflagellated bacterium that swims with an average speed of 52 μm s−1 at room temperature (25 °C) when the flagella rotate in synchrony to form a bundle. 17 18 D ≈ U 2 τ (2) where U is the swimming speed of the cells and τ is the persistence time of cell orientation, which is approximately ≈1 s for B. subtilis. 19 2 s−1, which is 107 larger than the polystyrene beads used in the previous experiment. In the second in vitro test, we aimed to evaluate the sampling performance of the osmotic pills with motile bacteria. Wild‐typewas chosen as the test bacterium, which is ubiquitous in the GI tract of ruminants and humans. We used a fluorescent strain to facilitate imaging.is a multiflagellated bacterium that swims with an average speed of 52 μm sat room temperature (25 °C) when the flagella rotate in synchrony to form a bundle.Fluctuations in flagellar rotation can cause the flagella to unbundle and generate a random reorientation of the cells. Subsequently, the bundle reforms resulting in the canonical run‐and‐tumble motion of the bacteria, which is characterized by an effective diffusion coefficientwhereis the swimming speed of the cells andis the persistence time of cell orientation, which is approximately ≈1 s forTherefore, the effective diffusion coefficient is 2700 μ m, which is 10larger than the polystyrene beads used in the previous experiment. Figure 3 shows the motile bacteria sampled by the pill. Nine osmotic pills were placed in separate test tubes containing bacterial suspension, and three pills were removed at two time points. Their content was examined under a fluorescent microscope and compared with a sample of bacterial suspension (see 3 for details). The concentration of bacteria in the pill was almost equal to the concentration in the environment at 1.5 h but continued to increase thereafter. This outcome was in fact expected as these bacteria are very motile and their diffusion factor is orders of magnitude higher than passive particles of the same size. Significantly, after 3 h, the bacteria in the pill were almost three times more concentrated than in the surrounding environment, likely the effect of continuous osmotic sampling. The osmotic membrane is water permeable, which in essence is filtering the bacteria and accumulating them in the collection chamber. Moreover, the helical geometry of the channels reduces the probability that motile bacteria can leave the collection chambers. The slight increase of the cell concentration in the surrounding medium outside the pill (control) as shown in Figure 3b is consistent with slow bacterial multiplication at room temperature. Moreover, the randomness of the position where the cells were taken from can contribute to this effect because the cell distribution inside the control volume is not completely uniform. This variation is negligible compared with a threefold increase in the concentration of bacteria inside the pill after 3 h. The in vitro test was undertaken for 24 h. As expected, the bacteria captured by the pill remained motile. Figure 3 Open in figure viewer PowerPoint Osmotic sampling of motile bacteria by the pill in aqueous suspension (medium). Bacillus subtilis was used to model live microorganisms. a) a snapshot of bacteria concentration in the pill and surrounding environment outside of the pill (control) at different time points. b) number of bacteria sampled by the pill and outside of the pill (control). The error bars show the standard deviation of three replicate counts of the same sample. c) a snapshot of bacteria in the pill after 24 h.

2.7 Pill Mobility in the Gut (Ex Vivo Study) To study how the pill moves inside the GI tract, we conducted ex vivo experiments using intestines freshly dissected from pigs (Figure S3, Supporting Information). At a flow rate of 4 and 8 mL min−1, the pills moved with an average speed of ≈10 and ≈13 cm min−1, respectively. The pill, however, moves with much higher velocity (≈22 cm min−1) when the flow rate is increased to 10 mL min−1, demonstrating nonlinear behavior attributed to the viscoelastic nature of the intestine that expands under increased peristaltic pressure. The natural peristaltic flow rate of fluid through the proximal small intestine varies widely from an average of 2.5 mL min−1 in fasting subjects to as high as 20 mL min−1 after meals.20-23 The ex vivo observation indicates that the pill readily moves inside the GI tract under realistic flow conditions.