Electronic‐Free Traceable Smart Capsule for Gastrointestinal Microbiome Sampling

Non‐invasive smart electronic‐free sampling capsules have revolutionized the exploration of microbiome‐disease interactions in inaccessible regions of the gastrointestinal (GI) tract. However, a significant impediment to the broader use of electronic‐free capsules is the challenge of reliably tracking and determining their in vivo location. Variability in patient motility introduces uncertainties in capsule position. Thus, there is a critical need for effective solutions that ensure traceability in microbiome studies employing such capsules. While tracking methods are explored in previous smart ingestible capsule designs, most have relied on RF, imaging, and radiation‐based techniques, limiting sampling volume, increasing costs, complicating design, and raising health concerns due to ionizing radiation exposure. To address these challenges, the design of an electronic‐free smart capsule is introduced that integrates a metal tracer for easy metal detection, serving as a reliable tracking mechanism. The capsule is housed in a 3D‐printed casing and includes a superabsorbent hydrogel serving as both a sampling medium and an actuator within the capsule. The capsule's targeted sampling of the GI tract is accomplished by covering the capsule's sampling port with a pH‐responsive coating. Optimal dimensions and material for the cylindrical shaped metal tracer on the capsule are determined through extensive optimizations, considering factors such as gastric flotation, corrosion resistance, read distance, and omnidirectional detectability. The results of these investigations reveal that a 12 mm stainless steel (SS 316L) cylinder offers the necessary detection and tracing capabilities with minimal toxicity and excellent corrosion resistance under relevant physiological conditions in the GI tract. Validation studies, both in vitro and in vivo, confirmed the capsule's trackability using a handheld metal detector. These findings are further validated by X‐ray imaging and CT scans, demonstrating the metal detector's ability to distinguish approximate GI tract regions and determine the time point of excretion. This innovative approach provides a reliable and cost‐effective solution for tracking electronic‐free smart capsules, enhancing their applicability in microbiome research for both human and animal studies.


Introduction
9][10][11][12] One common method used to study the gut microbiome involves the analysis of a collected fecal sample.This non-invasive approach is a relatively cheap and quick method to evaluate the population and diversity of microbes that reside in the gastrointestinal (GI) tract. [13,14]Despite the simplicity of this method, it has limitations in preserving spatial and temporal information about the bacteria community throughout the GI tract. [15,16][23] Consequently, relying solely on fecal sampling as an analysis technique poses a challenge in comprehensively assessing the exact effects of these microbiomes on overall gut health and physiology throughout the GI tract.The limitations of fecal analysis and the need for more region-specific sampling have led to the development of different methods over the last decade, mainly categorized into probe-based and ingestible sampling capsule-based approaches.Probe-based methods include endoscopy with biopsy tools, allowing for more targeted sampling ensuring that the sample accurately represents the area of interest. [24,25]However, this procedure is invasive, expensive, time-consuming, and requires skilled technicians. [26]dditionally, the collected sample is susceptible to contamination during extraction, as procedure often requires the probe to transition back through the GI tract during the retrieval phase, resulting in unwanted cross-contamination between regions.29][30] To address such issues, in recent years, there has been a significant focus on more non-invasive targeted sampling methods, leading to the development of smart ingestible capsules for localized sampling throughout the length of the GI tract. [31,32]In general, these capsules can be categorized as either electronic-based (active) or electronic-free (passive) modes of sampling mechanisms.][35][36][37] The advantage of such a design alleviates the concerns of device localization, while also providing a mechanism for sample collection and effective sealing.However, there are drawbacks associated with the inclusion of such components within this design, as the inclusion of electronics increases cost and design complexity, and occupies a significant proportion of the capsule size, reducing the available volume for sample collection.Further, the possibility of malfunctions within the capsule electronics or battery leakage increases the risk associated with the usage of active sampling capsules for health concerns.
On the other hand, smart passive ingestible capsules exhibit advantages over active capsules by eliminating onboard electronics and demonstrating a safer design. [38]These platforms often utilize stimuli-responsive polymers to take advantage of the physiological conditions within the GI tract, such as pH and microbiome [39][40][41][42][43][44][45][46][47][48] to trigger the sampling process and use electronic-free methods to perform the mechanical actuation required for sampling.As a direct comparison between the active and passive capsules, the onboard electronics and sensors are replaced with the use of stimuli-responsive polymers for targeted sampling, and the mechanical actuators are replaced with passive methods such as hydrogels, microfluidics etc.One such approach that takes advantage of these elements is shown by Nejad et al, [49] wherein a passive sampling capsule design is based on the principle of osmotic pressure to facilitate the intake and sampling of intestinal contents. [49]The capsule contains helical channels to transport and hold the intestinal contents, and a salt chamber below a semipermeable membrane to facilitate the osmotic gradient.Additionally, the designed capsule is placed within an enteric coating capsule such that the sampling occurs at the small intestine.Another example of passive capsule design is a model previously developed by our lab.[52] As the capsule enters the small intestine, the elevated pH levels trigger the dissolution of the polymer coating, allowing gastrointestinal luminal fluid to enter the capsule.The hydrogel inside the capsule absorbs the fluid, swells, and seals the sampling aperture.This mechanism effectively prevents contamination of the collected sample as the capsule progresses through the remaining sections of the GI tract.However, despite the numerous advantages and enhancements observed in passive capsule designs, a persistent challenge remains in precisely ascertaining the capsule's location throughout the GI tract and its time of excretion.The predominantly polymeric structure of most passive capsules makes them translucent, posing a challenge for visualization using common in vivo imaging modalities like Xray and CT scans.Additionally, in clinical trials or animal studies where there can be significant variations in motility between subjects, determining when a capsule has successfully traversed the GI tract and been excreted becomes a considerable challenge.This uncertainty impedes timely sample collection and subsequent analysis.
To mitigate these concerns, a straightforward method is needed to ascertain the capsule's presence within the GI tract.Over the years, various methods have been employed to track different types of ingestible smart devices.These methods can be broadly categorized into electromagnetic (EM) and magnetic tracking-based approaches.[55][56] Examples of such methods include the utilization of radioisotopes like In111 and Tc99m to label the capsule, enabling tracking through gamma scintigraphy, as demonstrated by Parker et al. [57] This approach renders the capsule visible in different regions of the GI tract.Another method involves the use of radiopaque markers for tracking through X-ray radiography, as illustrated by Klausner et al. [58] While these methods simplify the capsule design compared to active capsules, concerns arise due to the frequent exposure of the patient to ionizing radiation.Additionally, the complexity and cost of the required equipment pose challenges to the frequent imaging and tracking of the capsule.
An alternative and more practical approach with lower health concerns involves the utilization of magnetic tracing methods, employing magnetic sensors and magnets in tandem.In one method, a magnetic sensor is embedded within the capsule, and external magnets are employed to estimate the capsule's location.Another approach integrates a magnet within the capsule, relying on external magnetic sensors to track changes in the magnetic field strength and detect the capsule's position in the GI tract.Examples of this approach are demonstrated by Weitschies et al. [59,60] wherein a set of 83 external magnetic sensors was utilized to monitor the movement of an ingested capsule containing a permanent magnet.Although this method was reported to offer higher spatial resolution compared to gamma scintigraphy, challenges related to the cost of intricate sensors and readout equipment as well as potential sampling volume capability of such approaches impedes their practical usage.
To overcome current limitations, we explored the possibility of using external metal tracing modalities instead of imaging and magnetic-based methods to determine the in vivo position of the capsule.This serves as a simple, rapid, and practical tracking method for the capsule's position in the GI tract.The use of metal detectors for diagnostic purposes dates back to Alexander Graham Bell in 1879, who developed the technology for identifying embedded bullets in tissue.More recently, portable handheld metal detectors have been widely reported in localizing swallowed metallic objects, particularly in pediatric coin ingestion cases. [61,62]The diagnostic use of metal detectors for localizing ingested foreign metal objects was systematically studied by Arena and Baker in 1990, demonstrating 100% sensitivity and specificity compared to X-ray radiography. [63]This approach was considered cheaper, faster, and less harmful to the patient.More recently, metal detectors have been employed to study gastric emptying time.For instance, K. Ewe et al. showcased the capability of metal detection in determining gastric emptying using plasticcoated metal beads (11 × 6 mm) with a simple metal detector. [64]his method has been utilized to study variations in gastric emptying time with different foods, the impact of laxatives on colonic transit time, and the effects of various diseases such as diabetes and scleroderma on GI transit times.Inspired by these successful applications, we adapted this metal detection method to enable the straightforward tracking of a passive sampling capsule.This involved integrating a metal tracer into the body structure of the capsule to minimize any uptake in real estate volume within the capsule's structure while ensuring easy detectability through an external handheld reader.
To leverage the in vivo metal detection capability demonstrated in previous works for use in a capsule design, we have modified the existing passive sampling capsule by incorporating a metal cylinder on its outer walls, illustrated in Figure 1a.The capsule consists of a 3D printed housing with a specially designed compartment that accommodates the cylindrical metal tracing element within the body structure of the capsule.Comprising two separate compartments connected through a simple screw clamp mechanism, the capsule contains a superabsorbent hydrogel and a silicone elastomer plunger (PMDS).The sampling aperture on the capsule is shielded with a pH-responsive coating, which delays the activation and sampling of the targeted fluid in the GI tract until it reaches the designated area. [65,66]This coating takes advantage of the pH profile of the GI tract for targeted sampling, dissolving only at a specific pH level and designated region in the GI tract, allowing GI fluid to enter the capsule, either in the small intestine or large intestine. [40,48]pon initiation of sampling, the superabsorbent hydrogel within the capsule swells and captures the fluid, pushing the attached flexible silicone disc toward the sampling aperture.This action effectively seals the capsule, preserving the sampled contents to prevent contamination as the capsule traverses through the remaining sections of the GI tract.While the capsule is in transit, the metal detector, as shown in Figure 1b, can be used to provide audiovisual feedback, enabling the determination of capsule localization.This new capsule design, coupled with metal detection, significantly reduces the cost and time needed to confirm the presence of the ingested passive capsule as it passes through the GI tract.It ensures the motility of the capsule and determines the time point of extraction for timely sample collection.Once the capsule is expelled and detected by the metal detector, it is disassembled by unscrewing the two compartments, allowing the extraction of collected microbes from the hydrogel inside the capsule.
To validate the performance of the capsule design, systematic in vitro tests were conducted to determine the optimal material and dimensions for corrosion resistance, capsule density, bioinert properties, and omnidirectional detectability through a portable metal detector and X-ray imaging.Optimized capsule designs were tested in pig models for in vivo passage and tracking efficacy assessment.The in vivo study highlighted the handheld metal detector's high accuracy and reliability in distinguishing the capsule's location between approximate regions of the GI tract, further validated with 100% comparable accuracy to standard X-ray and CT scan imaging.
These findings suggest that physicians can be assisted in making medical decisions regarding the status of an administered passive sampling capsule.Therefore, the implementation of this design element can address concerns regarding motility variation among individuals and directly tackle a longstanding issue of passive capsules' traceability within the GI tract.This can have wide-ranging applications in microbiome analysis in humans, livestock, and veterinary research.

Materials and Methods
The capsule housing was fabricated using a stereolithography 3D printer with clear resin (Formlabs Inc., Somerville, MA).The Dow Sylgard 184 Silicone Elastomer Kit, used to fabricate the polydimethylsiloxane (PDMS) disks, was obtained from Dow Corning Corporation.Chemicals and reagents, including acrylamide, ammonium persulfate (APS), N,N′-methylene bisacrylamide (MBA), and phosphate-buffered saline, were purchased from Sigma-Aldrich (USA).Eudragit L100-55 (methacrylic acidethyl acrylate copolymer [1:1]), exhibiting dissolution characteristics at pH levels above 5.5, was procured from Evonik (USA) and applied as the pH-responsive coating on the capsule aperture.This coating delayed the sampling activity of the capsule until it reached the small intestinal region of the GI tract.Hollow brass and stainless steel (SS 316L) cylinders, with a thickness of 0.35 mm and an outer diameter of 9.5 mm, were acquired from McMaster-Carr (USA) and resized to fit the 3D printed capsule designs.Corn starch and gelatin, used in tissuemimicking phantoms, were procured from Walmart (USA).Finally, a portable MD-300 handheld metal detector from Walfront was used for all experimental capsule tracking procedures in both in vitro and in vivo studies with pig models (USA).

Capsule Design and Fabrication
The two-part capsule housing was designed using Autodesk Inventor software, featuring an assembly with overall dimensions (inner diameter, outer diameter, and height) of 7, 9.5, and 20 mm, respectively, and a sampling aperture of 2 mm in diameter.This design, incorporating a screw-on assembly, was executed using a Form 3 3D printer (Formlabs, USA).Following the 3D printing process, the capsules were immersed in pure isopropyl alcohol (IPA) for 1 h and UV photocured for 15 min at 60 °C.This step ensured full polymerization and the removal of any unbound polymer resin from the printed parts before assembly.For the PDMS disk within the capsule, a 1 mm thick sheet was prepared, and cylindrical PDMS disks with a 6 mm diameter were cut using a disposable punch biopsy.PDMS films were created by mixing a 1:10 ratio of curing agent to PDMS base, followed by degassing and curing at 70 °C for 4 h.Hydrogel synthesis involved creating a pre-gel stock solution by dissolving 334.5 mg of acrylamide and 16.35 mg of MBA (crosslinker) in 1.2 mL of DI water.After bubbling the solution with nitrogen gas for 30 min, a separate solution of 80 mg mL −1 APS in DI water was added to the pre-gel solution at a 5:1 ratio. [67]The mixture was poured into cylindrical molds with a diameter of 5 mm and height of 10 cm, allowing the hydrogels to polymerize overnight at room temperature.Subsequently, the hydrogels were dehydrated at 70 °C for 6 h.
The fully dried hydrogel cylinders were affixed to the bottom compartment of the capsule, bonded to the PDMS disks using GelBond film.The upper compartment's sampling aperture was coated with a viscous pH-responsive enteric coating to delay the sampling process until the capsule reached the designated region in the GI tract.The coating solution, comprising Eudragit L100-55 powder dissolved in an organic solvent (acetone 57%, isopropanol 38%, water 5% w/w), was drop-casted onto the capsule surface and allowed to dry overnight.Before full assembly, a cylindrical metal tracer, cut to appropriate dimensions, was fitted onto the outer wall of the capsule shell between the two halves.The design was optimized to provide a flush fit, resulting in a smooth surface after full assembly, accomplished by attaching and rotating the two compartments to screw onto each other.Figure 2 illustrates the separate components of the sampling capsule, including the 3D-printed capsule housing, hydrogel, pH-responsive PDMS disk, and metal cylinder tracer, both before and after full assembly, emphasizing the smooth capsule surface achieved by assembling the components.

Corrosion Test
Given the exposure of the capsule to varying pH levels during its journey through the GI tract, selecting an appropriate metal tracer design is crucial.The chosen design needs to not only ensure effective detectability but also exhibit stability against corrosion and potential degradation in these challenging environments.To evaluate this, capsules with optimized metal tracer designs, providing effective density, underwent standard corrosion characterization through electrochemical procedures.In this assessment, open circuit potential (E oc ) and potentiodynamic polarization scans were conducted to evaluate the corrosion rate of the metal used in the capsule's construction.These tests were performed using a Gamry instrument (Reference 3000 Potentiostat/Galvanostat/ZRA, USA) controlled by framework data acquisition software (version 6.23).A 3-electrode setup was employed, where the test metal surface served as the working electrode, and a silver-silver chloride (Ag/AgCl) (saturated KCl) electrode and a platinum rod functioned as the reference electrode and counter electrode, respectively.Corrosion rates and characteristics of each metal were analyzed under both acidic conditions (pH 1.2 to mimic the gastric environment) and basic conditions (pH 7.5 to simulate the intestinal environment), replicating the extreme conditions observed throughout the GI tract.The solutions were prepared using 12 m HCL (Sigma-Aldrich, USA) and pH 8 buffer solution (Fisher Scientific, USA) mixed with DI water until a pH of 1.2 and 7.5 were reached, all measurements were performed with a digital pH meter (HQ440D, Hach, USA).Potentiodynamic polarization scans were acquired within the voltage range of −0.5 to +0.5 V (vs Eoc) at a sweep rate of 1 mV s −1 .This comprehensive electrochemical assessment provided valuable insights into the corrosion behavior of the metals under conditions that simulate the harsh environment of the GI tract.

Simulation of Capsules Detectability
To simulate the detectability of the capsule design within the human body, an analysis was conducted to evaluate how well the capsule could be detected at varying depths and angular orientations.This simulation aimed to model the sensitivity/readability performance of the portable handheld metal detector and its capacity to detect the capsule under different conditions within the body.The simulation was performed using Ansys HFSS, employing electromagnetic simulations based on geometric models.The simulations helped us understand how factors such as the length of the metallic cylinder of the capsule (L), the orientation of the capsule (), and the read distance between the capsule and the reader coil (r) influenced the detectability of the capsule in different conditions.In developing the electromagnetic simulations, the size of the capsule was modeled as 20 mm × 9.5 mm.The reader was modeled as a metallic coil with an inner diameter of 8 cm and an outer diameter of 12 cm, similar to the dimensions of the commercially available metal detector.During the simulation, a fixed AC current of 1 A was applied to the reader coil at 25 kHz.The induced voltage on the reader coil, resulting from inductive coupling with the metal tracer on the capsule at various positions and orientations, was estimated.The level of induced voltage served as a direct indicator of the capsule's detectability by the portable metal detector.This simulation provided valuable insights into how the capsule design interacts with the metal detector under different scenarios, aiding in the optimization of capsule detectability in real-world conditions within the body.

Biocompatibility and Bioinert Assessment
Ensuring the capsule's materials do not adversely impact the gastrointestinal microbial community and avoiding leaching of chemicals or toxins during traversal through the GI tract is vital.Two in vitro experimental procedures were conducted to validate these aspects before commencing in vivo tests.

Biocompatibility Assessment
To evaluate the safety of all capsule materials, a cell-based assay utilized the human ileocecal HCT-8 (CCL-244, ATCC, Manassas, VA, USA) cell line.HCT-8 cells were cultivated in Dulbecco's modified Eagle medium (DMEM) supplemented with lglutamine, sodium pyruvate, and fetal bovine serum (FBS) at 37 °C with 5% CO 2 and 95% humidity. [68]In brief, HCT-8 cells were seeded at 1.0 × 10 5 cells per well in 12-well tissue culture plates and allowed to form monolayers. Next, the materials were placed in the center of each well-containing cell monolayer and incubated.After incubation (1, 2, and 3 days), cell viability was assessed using the MTT assay and live/dead staining. [69,70]In this process, the old media in each well was replaced with fresh DMEM media containing 0.5 mg/mL of MTT and incubated for another 4 h under the same conditions.Finally, the MTT was removed, and the formazan crystals were dissolved in 200 μL of DMSO and incubated for 30 min.The samples were then removed from the well plate, and the SpectraMax Paradigm Multi-Mode Detection Platform (Molecular Devices, USA) was used to measure the optical density (OD) values of the colored solutions.Cell viability was calculated as the percentage ratio of the sample absorbance to the control absorbance. [71,72]In addition, Live/dead staining (ThermoFisher Scientific) was performed using ethidium homodimer-1 and calcein-AM according to the manufacturer's instructions, and imaging was done using an epifluorescence microscope.

Microbial Interaction Experiment
In the initial set of experiments, capsules with different metal tracers were exposed to bacterial cultures commonly found in the GI tract-Gram-positive Enterococcus faecalis ATCC 29 212 (E.faecalis) and Gram-negative Escherichia coli ATCC 25 922 (E.coli).Metal cylinders within the capsules were immersed in 10 mL Tryptic Soy Broth (TSB) solutions with bacteria cultures (10 9 CFU/mL, OD 600nm = 0.4).Samples were withdrawn at 2, 4, 8, and 24 h, plated onto TSB agar after serial dilution, and compared to control conditions. [73,74]The experiments were conducted in triplicate, and averages were reported.

Capsule Detectability Assessment
Before transitioning to in vivo testing, a series of in vitro assessments were conducted to validate simulation results, ensuring the detectability of the capsules through both portable metal detection and standard X-ray imaging.

Metal Detection Performance
The first part of these in vitro tests aimed to validate simulation results concerning metal detection performance and accuracy with various metal tracer geometries integrated into the capsule structure.Capsules with different lengths of metal tracer were paired and placed in containers with water and agarose gel to simulate physiological conditions.The capsules, affixed at the container's bottom in different orientations using double-sided adhesive tape, were assessed for detectability using a portable handheld metal detector at varying distances.A 1% w/w agarose gel, prepared by dissolving agarose in Phosphate buffer saline (PBS), was used to mimic tissue conditions.

X-Ray Imaging Visibility
For X-ray imaging visibility assessments, tissue-mimicking phantoms were created by introducing capsules with metal cylinder tracers of varying lengths into the phantoms.These phantoms comprised DI water and agarose gel with different concentrations of corn starch (0.2%, 0.4%, and 0.8% w/w) to enhance X-ray interference and gray contrasts.Radiographs were captured using a Del Medical X-ray universal system.The visibility of metallic cylinders in radiograph images was analyzed and quantified by comparing the cylinder's gray-scale intensity to the background medium's intensity using ImageJ software.

In Vivo Capsule Tracking
To validate the effective traceability of the capsules with the optimized integrated metal tracer, an in vivo animal study was conducted using three pig models.This study received approval from the Purdue University Animal Care and Use Committee (protocol no. 1 911 001 975).The pigs, with weights between 45 and 55 kg, were fed three times the daily maintenance energy requirement (4% of body weight per day) composed mainly of corn and soybeans.Before the trial, the pigs underwent a 5-day acclimation period in their pens (2 m × 1.3 m) with ad libitum access to water.On the sixth day, after a 6-h fasting period, a single capsule was administered to each pig.Daily rapid capsule localization using a handheld metal detector was performed.Ground truth measurements and confirmation of capsule position were conducted at various time points during the study using X-ray imaging (Del Medical X-ray universal system) and CT-Scan (GE Healthcare GmbH, Germany).For all imaging procedures, the pigs were anesthetized via a face mask with isoflurane (3-5%) mixed with 2L min −1 of oxygen.After 15 minutes, the pigs were transported to the imaging facility and maintained under isoflurane inhalation (1-3% mixed with 2 L min −1 oxygen, using a face mask).Post-imaging, the pigs were allowed to recover in their pens.All X-ray radiographs were analyzed using VetRocket software.

Capsule Density Characterization
In previous reported in vivo pig studies, it has been observed that ingested objects, like capsules, with a density lower than gastric fluids could float in the stomach, potentially causing prolonged gastric retention.On the other hand, non-floating objects with higher densities are more likely to be propelled by peristaltic waves in both animals and humans.Therefore, ensuring the capsule's density surpassed that of gastric fluid was crucial.Moreover, for effective locomotion through peristaltic action, it was necessary to distribute weight uniformly along the capsule's long axis to prevent uneven sinking that might hinder passage through the pyloric sphincter.
[77] The study, depicted in Figure 3a, revealed that the capsule without the metal tracer had a density of 0.72 g cm −3 .The integration of a titanium metal tracer, even up to 12 mm, did not significantly alter the capsule's density (< 1 g cm −3 ), rendering titanium unsuitable for capsule design despite its common use in biomedical applications.
In contrast, theoretical analyses showed that capsules with cylindrical brass and SS 316L tracers, with densities of 8.47 and 7.98 g cm −3 , respectively, at a minimum length of 9 mm provided a substantial density (>1.004 g cm −3 ) which can enable the full capsules submersion.Figure 3b,c depicts the experimental tests on fully assembled capsules with brass and SS 316L cylindrical tracers at various lengths, placed in vials containing PBS.The metal cylinders, centrally located in the capsule shell, aided in better weight distribution and horizontal settling in the test media.Experimental results showed that capsules with brass and SS 316L traces of length 6 mm or less exhibited flotation, while those with lengths of 9 mm or more sank, confirming the theoretical density analysis results.The sinking of the capsules (with 12 mm cylindrical metal tracers) was also validated in freshly collected gastric fluid from pig models.

Corrosion Test
As previously mentioned, both SS 316L and brass, when of sufficient length, demonstrated the appropriate density for aiding the device's submersion in fluid.However, owing to the significant pH variability throughout the gut, a potentiodynamic corrosion test was conducted to assess the corrosion resistance of these two materials, crucial for identifying the optimal tracer metal for the final capsule construction.In Figure 3d, the experimental setup for the accelerated potentiodynamic corrosion test is illustrated, where the rate of corrosion on the capsule's metal tracer surface was analyzed by connecting it to a potentiostat through a wire connection and partially immersing it in a test solution with varying pH levels.The partial immersion allowed for a visual inspection of the corroded surface before and after the potentiodynamic test on the same metal tracer surface.
Figure 3e compares the potentiodynamic measurements of SS 316L and brass in acidic (pH 1.2) and slightly alkaline (pH 7.5) solutions.In the acidic environment, both metals exhibit similar ca-thodic behavior below their corrosion potential regions.However, as the potential increases above their corrosion potentials, a notable difference in their anodic region becomes apparent.Brass shows a steep increase in the anodic current with a relatively small increase in potential, indicating a significant rise in the corrosion rate on the metal surface.Through Tafel slope analysis, the corrosion current for brass was measured at 9.52 μA at the corrosion potential, corresponding to a corrosion rate of 0.112 mm per year for the acidic environment.Conversely, SS 316L showed clear passivation on the surface as the anodic current remains constant with a substantial increase in potential, demonstrating high corrosion resistance in the low pH conditions simulating the stomach's environment.The corrosion current and rate of SS 316L in the acidic environment were measured at 1.6 μA and 0.017 mm per year, significantly lower than brass.In both SS 316L and brass, a passive layer formed on their surfaces in the alkaline environment, with low corrosion currents of 0.5 and 3.5 μA with an estimated corrosion rate of 0.006 and 0.039 mm per year for the SS 316L and brass respectively.Figure 3f,g presents images of the metal surfaces after the potentiodynamic tests in pH 1.2 for gastric conditions and pH 7.5 for intestinal conditions.For better visualization of corrosion on the sample surface, only half of the cylinders were immersed inside the solutions.While no noticeable corrosion was observed on the metal surfaces tested in pH 7.5, visible corrosion and discoloration were observed on the brass surfaces tested in pH 1.2 conditions.In contrast, neither of the tested SS 316L samples in pH 1.2 and 7.5 exhibited a visible difference compared to the pristine sample, indicating high corrosion resistance in conditions simulating the GI tract.Based on these observations, it was concluded that while both metals had efficient density for effective submersion, SS 316L was a preferable choice due to its low risk of corrosion during its brief usage in the GI tract.

Capsule Bioinert Assessment
Figure 4a presents the results of the in vitro MTT assay, assessing the viability of HCT-8 cells when exposed to all materials utilized in the construction of the capsule.A cell viability percentage >85% was observed for all materials over the 3-day period.Figure 4b displays representative microscope images of cells interfacing with different materials.The results indicate high cell viability, as evidenced by the predominantly green appearance, signifying live cells. [78]Additionally, <10% of cells were found to be dead, a common observation in normal cell cultures.Importantly, none of the tested materials used in capsule construction exhibited immediate toxicity, as the recorded signals for all compounds and the control group were comparable.Moreover, the materials did not interfere with cellular growth, as no statistically significant difference was noted between the values obtained for samples and controls.
To investigate whether the fully assembled capsule, during its passage through the GI tract, exerts any adverse effects on the surrounding microbial population, a surface bioinert characterization of the capsule using representative gut microbes was per-formed.Figure 4c illustrates four vials filled with 10 9 CFU mL −1 bacterial solutions of E. coli and E. faecalis, both with and without capsules containing the SS 316L metal tracer.E. coli and E. faecalis were chosen as common strains of bacteria present in the human GI tract.As depicted in Figure 4d, the viable concentration of E. coli in the vicinity of the capsule remained at 9.84 ± 0.18 Log CFU mL −1 throughout the 24-h study, with no significant difference compared to the population observed in the control condition (9.46 ± 0.35 Log CFU mL −1 ) without the capsule.Similarly, the viable concentration of E. faecalis surrounding the capsule exhibited a bacterial population of 9.70 ± 0.60 Log CFU mL −1 , similar to the control conditions (9.74 ± 0.67 Log CFU mL −1 ) over the 24 h (Figure 4e).This analysis demonstrates that the capsule with the SS 316L metal tracer can effectively maintain bioinert properties, without altering the microbial population in its surroundings.

Capsule Traceability Using Metal Detection
Figure 5a shows a representative image of the assembled capsule with the SS 316L metal tracer and the portable metal detector that was utilized for its detection.Before experimental analysis, a systematic simulation-based study was conducted in Ansys HFSS to understand the effect of the angular orientation and length of the metal tracer on its detectability using the portable metal detector (Figure 5b).In the simulations, reader probe was modeled as a circular coil with a diameter of 10 cm.For simulating the angular orientation effect on the capsules detectability, the read distance between the capsule and reader coil was fixed at 10 cm, and the angular orientation of the capsule () was varied from 0 0 (vertical) to 90 0 (horizontal) for various values the metal tracer's lengths (L).The simulations showed that the induced voltage (V induced ) decreased as  increased from 0 0 to 90 0 for all values of L (Figure 5c).According to Faraday's law of induction, the induced voltage is proportional to the rate of change of the magnetic flux ( B ): Here, the magnetic flux comprises two components:  z , the magnetic flux through the hollow area of the metal cylinder, and  sw , the magnetic flux through the sidewalls of the cylinder.
In this formulation, the components of the magnetic flux can be expressed as: Where B z is the magnitude of the magnetic field, A z is the hollow area of the metal cylinder, dA is the differential area on the sidewall of the metal tracer, r 0 is the distance from the coil center to the hollow area of the cylindrical-shaped metal tracer, and r is the distance from the reader coil center to the differential area of the cylindrical-shaped metal tracer.
Based on the simulations, a cylinder length of 12 mm was identified as the ideal length to achieve orientation-independent readability.To study the effect of the read distance of the metallic cylinder from the reader coil on its detectability, V induced was simulated for various values of read distance in the air from 1 to 10 cm (Figure 5d).The simulations indicated a steep decrease in V induced as the read distance increased from 1 to 10 cm.Similar simulations were conducted in water and agarose gel to mimic tissuelike environments.The results demonstrated negligible impact on the read distance by the environments.This was attributed to the fact that the media found in tissues do not have noticeable diamagnetic properties, maintaining the intensity of inductive coupling and not influencing the read distance.After simulations, experiments were conducted using the commercial metal detector to determine the maximum read distance and the effect of various media on the detectability of the capsule with different lengths of SS 316L metal tracers.The capsule was aligned vertically and horizontally along the line of sight of the reader coil, and the maximum read distance was recorded.In both orientations, the media surrounding the capsule showed a negligible effect on the read distance (Figure 5e,f).In the vertical orientation (Figure 5e), the length of the capsules metal tracer had a negligible effect on the maximum read distance, consistent with simulations, with a maximum read distance of ≈10 cm for all values of L. In the horizontal configuration, the maximum read distance was <10 cm when L ≤ 9 mm (Figure 5f).However, the maximum read distance surpassed the 10 cm threshold when L = 12 mm.Given the necessity for the capsule to be readable in all angular orientations with the maximum possible read distance, the optimal length of the metallic tracer on the capsule was conclusively determined to be 12 mm.

Radiography Detectability Assessment
To ascertain the traceability of the capsule using standard Xray imaging procedures, a series of X-ray tests with capsules in tissue-mimicking phantoms were conducted.The gray intensity difference between the metal tracer element on the capsules and the tissue-mimicking phantoms was employed as a quantitative marker for the visibility/detectability of the capsules with X-ray imaging.Figure 6a-d shows the X-ray images of the capsules with different metal tracer lengths in the phantoms (DI water and agarose gel with varying concentrations of corn starch (0.2%, 0.4%, and 0.8% w/w)).As observed in the images, the metal tracer on all capsules was distinctly visible, presenting a lighter area compared to the background tissue phantom and the 3D-printed plastic body of the capsules, irrespective of the background and metal tracer length.This clear visibility is attributed to the higher density of the metal, resulting in high levels of X-ray radiation absorption and manifesting as brighter regions on the X-ray image.The level of gray-scale intensity measurements across all tissue phantoms exhibited a relatively similar intensity distribution of 142-158, with a peak at ≈149 ± 3 (Figure 6e-h).However, the metal tracers on the capsules displayed a notably higher grayscale intensity distribution of 179-207, and the grayscale intensity was independent of the length of the metal tracer.To determine the overall visibility of the capsules in X-ray images, the effective difference between the background intensity peak and the center peak point of the grayscale intensity of the capsules with their varied metal tracer lengths was measured.As observed, the grayscale intensity differences were 38, 36, 32, and 41 units between the capsules and the background tissue phantoms, including DI water and media with varying concentrations of corn starch (0.2%, 0.4%, and 0.8%).This underscores their high visibility and ease of detection through standard X-ray imaging, either via automated image processing or with the assistance of a radiograph technician.

In Vivo Capsule Tracking
As a proof-of-concept test to validate the traceability of the capsules (with an optimized metal tracer length of 12 mm) under in vivo conditions, the capsules were administered to pig models.The capsule's passage through the GI tract and excretion points were tracked using a portable metal detector, and the accuracy of the measurements was compared against standard X-ray and CT scan imaging.and d) detection of the capsule in the stomach on day 1, e) in the colon on day 4, and f) confirmation of excretion on day 6, g) image capturing the CT-scan imaging procedure of Anesthetized pig 1, and h) detection of the capsule in the stomach on day 1, i) in the colon on day 4. j) Individual transit time of the capsules in three pigs, and their position determined throughout the GI tract using metal detector, X-ray, and CT scan imaging.
Due to the ambulatory nature and rapid screening capability of the portable metal detector, the pigs were scanned throughout the ventral side and in the excreted fecal matter.Regions across the GI tract where auditory and light feedback signals given by the metal detector were recorded signified the area/region at which the capsule was detected.To collect ground truth measurements and confirm the metal detector's accuracy, the capsule's location was validated with X-ray and CT scan imaging after anesthetizing the pigs throughout the study.
Figure 7a-i shows a representative example of the collected data for pig 1 in this study.During the first 3 days, the metal detector emitted an auditory signal in the upper body area, signifying the presence of the capsule in the gastric environment, Figure 7a.To validate this finding on pig 1, the animal was anesthetized, and X-ray and CT-scan imaging were performed, revealing the capsule in the stomach, as displayed in Figure 7d,h.However, on day 4, the auditory signal was emitted by the metal detector while scanning the lower section and around the colonic region of pig 1′s anatomy, as shown in Figure 7b.
The collected X-ray and CT-scan images performed on the anesthetized pig within the same day further confirmed that the capsule had moved to the colonic region, Figure 7e,i.On day 6, no auditory signal was detected with scanning the pig, yet it showed a clear auditory signal with scanning the fecal matter in the pig's pen, confirming the exclusion of the capsule from the pig.X-ray and CT scan images also confirmed no detectable trace in the pig's body, confirming the successful excretion of the capsule, Figure 7f.A similar procedure was followed for capsule localization in pigs 2 and 3 throughout the entire in vivo study duration.Figure 7j highlights the results of tracking the capsule in different sections of the GI tract obtained by metal detection and X-ray and CT-scan imaging during the 17 days of the in vivo study.
The results showed that the gastric emptying time for the capsules was 3, 8, and 7 days for pigs 1, 2, and 3, respectively.The capsules' prolonged residence time in the stomach was associated with the pig's gastric anatomy, including a moderately stenotic pylorus, which restricts the passage of non-disintegrated matter. [79,80]Unlike humans, gravity is unable to assist with the passage of food through the Torus pyloricus, located on top of the pig's stomach.In addition, the "U-shaped" swine stomach, compared to the "J-shaped" human stomach, can obstruct the emptying of large non-disintegrated dose forms.In other words, the dosage form would need to be propelled towards the pylorus during stomach contractions, resulting in extended retention.82][83] During the in vivo study, once the capsule entered the small intestine, the narrow diameter of this section and the intense peristaltic contractions facilitated the rapid transit of the capsule through the small intestine, leading to no capsule detection during capsule tracking studies with both metal detector and imaging approaches.In contrast, the capsule device exhibited a prolonged residence time within the colon.It was observed that the duration of capsule residence in the colon was 2, 8, and 5 days in pigs 1, 2, and 3, respectively.The prolonged transit times in the colon can be attributed to the wider diameter of this segment leading to a damped peristaltic wave specifically for non-digested matter such as the capsule device.
Finally, despite being of identical age and kept under similar dietary and environmental conditions, the overall passage rate of the capsules was 6, 17, and 13 days for pigs 1, 2, and 3, respectively, and there were no observable indications of bowel obstruction during the study.These results further confirmed the necessity of having simple tracking capabilities for the capsule to ensure its safe passage and understand the time points where the capsule is excreted from the body.
The overall in vivo results demonstrate the metal detection method's ability to accurately track the location of a capsule throughout the digestive tract of the pigs with comparable accuracy to X-ray and CT scan imaging.Furthermore, it can be inferred that metal detection offers a fast and cost-effective approach for quick screening and determining if the capsule is present within the GI tract or has been excreted.X-ray and CTscan can be used as methods to identify the capsule's exact location more precisely in different organs throughout the GI tract.It should also be pointed out that the metal detector may face limitations in scenarios involving more obese patients, where the capsule might be deeply situated, potentially affecting its detectability.In such cases, it is conceivable that the capsule may not be promptly detected with the metal detector probe.To address this, a practical approach would involve multiple scans over the course of several hours.The GI tract is a dynamic and three-dimensional structure, and the capsule's position within it can vary, coming closer or moving away from the outer abdominal wall.Multiple scans with the metal detector can help overcome the limitation of limited detection depth and ensure tracking of the capsule's movement.

Conclusion
The GI tract's microbiome is integral to maintaining host health, contributing to protection against harmful microorganisms, digestion facilitation, and metabolic support.Imbalances in this microbial community are associated with various diseases and may impact the bioavailability and efficacy of therapeutics.Monitoring the microbiome's conditions and population along the GI tract is crucial for understanding disease dynamics.While smart ingestible passive sampling capsules have emerged as economically viable tools for targeted and non-invasive sampling throughout the GI tract, their lack of traceability and the inability to determine the moment of excretion pose challenges.To address this limitation, we have enhanced a previously developed sampling capsule technology with an optimized metal tracer, enabling straightforward traceability using a portable handheld metal detector.To identify the optimal tracer geometry, maximizing detection range and omnidirectional detectability, we conducted systematic studies and simulations with various materials and geometries.Based on simulation and experimental results, cylindrical metal tracers made with SS 316L and a length of 12 mm were identified as optimal.Additionally, the biocompatibility and bioinert characteristics of the capsule with the metal tracer ensure the capsule's safety, preserving the integrity of the collected sample's representation of the local environment without altering the GI tract microbiome.The successful administration and tracking performance of the metal detection method were confirmed in in vivo studies with pig models, validating its accuracy through comparison with X-ray and CT scan imaging.This capsule tracking method holds promise in aiding healthcare professionals in accurately determining the capsule's location, and identifying excretion or slow motility.This information can inform decisions regarding further treatment interventions in patients, livestock, and research animals.Overall, our modified capsule technology with enhanced traceability offers a valuable tool for advancing understanding and interventions in GI health.

Figure 1 .
Figure 1.Electronic-Free Traceable Smart GI Tract Sampling Capsule: a) 3D schematic depicting the comprehensive design and assembly process of the capsule components.b) Illustration outlining the working principle and tracking process of the capsule: (i) The sampling process is initiated in the stomach, where the pH-responsive enteric coating delays aperture exposure.(ii) Upon entering the target region in the GI tract, the enteric coating dissolves, allowing luminal content to enter the capsule and be absorbed by the hydrogel.(iii) Hydrogel swelling leads to self-sealing, preserving the capsule from contamination beyond the targeted sampling area.The capsule's position, traversal through the GI tract, and excretion into the fecal matter are detected via a metal detector, providing both auditory and visual feedback.

Figure 2 .
Figure 2. Capsule Components Assembly: a) Photograph showcasing all components utilized in capsule assembly, featuring a two-part 3D printed capsule housing, superabsorbent hydrogel, silicone elastomer, cylindrical-shaped metal tracer, and a pH-sensitive polymer coating on the sampling aperture.b) Image of the final screw-on assembly of the capsule.

Figure 3 .
Figure 3. Assessment of Capsule Density and Corrosion Reliability with Different Metal Tracers: a) Theoretical analysis of capsule density with integrated metal tracers of varying length and material (brass, stainless steel (SS), and titanium).Experimental analysis of capsules' sinking ability with different integrated b) brass and c) SS metal tracer lengths.d) Potentiodynamic test setup for characterizing the corrosion rate of the metal tracers on the capsules.e) Potentiodynamic polarization curves obtained from the brass and SS in acidic (pH 1.2) and slightly alkaline (pH 7.5) solutions.f) Image of Brass and g) SS cylinder surface before and after the potentiodynamic corrosion test in pH 1.2 and 7.5.

Figure 4 .
Figure 4. Biocompatibility and Bioinert Assessment of the Smart Capsule: a) Graph showing the percentage of viable HCT-8 cells over 3 days of direct exposure to different materials used in the construction of the capsule.b) Live (green)/Dead (red) staining of cells after 3 days.c) Test setup exposing cultures of E. coli and E. faecalis to the fully assembled capsule.Viable bacterial population of d) E. coli and e) E. faecalis in the culture vials after different durations of direct exposure to the capsule.

Figure 5 .
Figure 5. Capsule Detectability at Different Distances and Orientations: a) Photograph of the metal detector (with reader coil) and capsule with integrated SS 316L metal tracer with 12mm length.b) Simulation setup showing the reader coil and a capsule demonstrating the magnetic field distribution and test conducted to assess the detectability of the capsule at different distances and orientations.c) Simulated induced voltage in the reader coil as a function of the angular orientation of the capsule with different metal tracer lengths.d) Simulated induced voltage in the reader coil as a function of the distance with the capsule placed in different media conditions (air, water, and gel).Experimental results showing the maximum distance that the capsules with different SS tracer lengths can be detected with the metal detector in different media conditions (air, water, and gel) positioned at an angular orientation of e) 0°and f) 90°with respect to the reader coil.

Figure 6 .
Figure 6.Capsules Visibility Assessment through X-ray Imaging: Comparison of X-ray images collected from capsules with SS metal tracer of different lengths (6mm, 9mm, and 12 mm) placed in tissue-mimicking phantoms including a) DI, agarose gel containing b) 0.2%, c) 0.4%, and d) 0.8% cornstarch filler.Histogram of gray-scale intensity of the capsules with SS metal tracer of different lengths with respect to the background tissue-mimicking phantoms including (a) DI, agarose gel containing (b) 0.2%, (c) 0.4%, and (d) 0.8% cornstarch filler.

Figure 7 .
Figure 7. Capsule Tracking in Pigs: a) Representative photo of capsule detection in pig 1, in the stomach on day 1, and b) in the colon on day 4 using the portable metal detector.c) An image capturing the X-ray procedure of Anesthetized pig 1,and d) detection of the capsule in the stomach on day 1, e) in the colon on day 4, and f) confirmation of excretion on day 6, g) image capturing the CT-scan imaging procedure of Anesthetized pig 1, and h) detection of the capsule in the stomach on day 1, i) in the colon on day 4. j) Individual transit time of the capsules in three pigs, and their position determined throughout the GI tract using metal detector, X-ray, and CT scan imaging.