Ingestible apparatus for collecting samples in the gastrointestinal tract

Abstract

A device may include a housing defining a sample compartment having a first portion and a second portion, the housing comprising one or more access ports formed therein, and the sample compartment being communicable with the one or more access ports. A device may include a functional substance arranged in the second portion of the sample compartment. A device may include a separation layer arranged between the first portion and the second portion of the sample compartment, the separation layer being configured to change at least one property in response to interaction with GI fluid.

Description

I NG ESTI BLE APPARATUS FOR COLLECTING SAMPLES IN THE GASTROINTESTINAL TRACT BACKGROUND

[0001] The gastrointestinal (Gl) tract is a series of joined, hollow organs substantively in the form of a long and twisting tube from the mouth to the anus. The contents in the Gl tract such as tissues, mucosal cells, microbiota, molecules, and the like contain information regarding the condition of the Gl tract. Samples of Gl contents may be collected via stool sampling. However, the spatial and temporal information of the Gl contents is poorly preserved in stool samples. Therefore, it is desirable in many situations to collect samples of Gl contents directly from the interior of the Gl tract by using a suitable sampling tool. Different organs of the Gl tract have varying degrees of accessibility. For example, the small intestine is a deep and long organ which is difficult to access using transoral or transanal catheters. Therefore, various sampling tools and methods, including, but not limited to, tubes with endoscopic-assisted aspiration or biopsy means and micro-electro- mechanical system (MEMS)-based capsules, have been developed for accessing the Gl tract and sampling therein.

[0002] Effective sampling within the Gl tract requires a device that is able to start and end sampling within a target region of the Gl tract, and that is able to effectively absorb fluid during the sampling period. This requirement becomes of the upmost significance when sampling in regions that are of relatively low biomass as residual sampling beyond the targeted region can cause significant contamination to collected sample and fluid levels can vary.

[0003] It is therefore desirable to have a device that closes the sampling chamber within a controlled and reproducible period of time, as well as provide for effective absorption and / or sample preservation while the device passes through the remainder of the Gl tract.SUMMARY

[0004] In some aspects, the techniques described herein relate to an ingestible apparatus for collecting a sample from a target area of a gastrointestinal (Gl) tract of a subject, the ingestible apparatus including: a housing defining a sample compartment having a first portion and a second portion, the housing including one or more access ports formed therein, and the sample compartment being communicable with the one or more access ports; a functional substance arranged in the second portion of the sample compartment; and a separation layer arranged between the first portion and the second portion of the sample compartment, the separation layer being configured to change at least one property in response to interaction with Gl fluid.

[0005] In some aspects, the separation layer is configured to change the at least one property by dissolving in response to interaction with Gl fluid. In some aspects, the separation layer is configured to change the at least one property by changing permeability in response to interaction with Gl fluid. Additionally, in response to the separation layer changing the at least one property, the functional substance is configured to move from the second portion of the sample compartment to the first portion of the sample compartment.

[0006] In some aspects, the separation layer is a polymer.

[0007] In some aspects, the functional substance is in a solid state.

[0008] In some aspects, the functional substance is a preserving substance.

[0009] In some aspects, the ingestible apparatus further includes an absorbent material arranged in the first portion of the sample compartment.

[0010] In some aspects, the separation layer is disposed between the absorbent material and the functional substance.

[0011] In some aspects, the absorbent material is sized and / or shaped such that the absorbent material cannot exit the housing via the one or more access ports.

[0012] In some aspects, the absorbent material is an organized presentation of fiber.

[0013] In some aspects, the organized presentation of fiber is configured to increase a surface area of the absorbent material in proximity to a sealing surface of the housing.

[0014] In some aspects, the organized presentation of fiber is an assembly of fiber. For example, the assembly of fiber optionally includes a plurality of loops. Optionally, the plurality of loops is between about 5 and 100 loops.

[0015] In some aspects, the assembly of fiber is a ball of fiber.

[0016] In some aspects, the organized presentation of fiber includes one or more loops, balls, knots, braids, or brushes.

[0017] In some aspects, the organized presentation of fiber is sized and / or shaped such that no portion of the organized presentation of fiber can exit the housing via the one or more access ports.

[0018] In some aspects, the absorbent material is spaced apart from the functional substance.

[0019] In some aspects, the separation layer is configured to prevent the absorbent material from contacting the functional substance before the separation layer changes the at least one property.

[0020] In some aspects, the absorbent material contacts the separation layer. Optionally, the absorbent material is attached to or partially embedded with the separation layer.

[0021] In some aspects, the absorbent material does not contact the separation layer.

[0022] In some aspects, the absorbent material defines a first surface adjacent to the separation layer and a second surface opposite to the first surface, the separation layer being arranged in the sample compartment such that the second surface is spaced about 1 millimeter (mm) from a sealing surface of the housing.

[0023] In some aspects, the one or more access ports are located in a port region of the housing, the housing further including a plurality of access ports and a plurality of struts in the port region of the housing, each of the plurality of struts being arranged between adjacent access ports of the plurality of access ports.

[0024] In some aspects, a surface area ratio of the plurality of access ports to the plurality of struts in the port region of the housing is greater than about 50%.

[0025] In some aspects, the housing further includes a sealing surface, and wherein the port region of the housing is spaced apart from the sealing surface.

[0026] In some aspects, the ingestible apparatus further includes an actuation assembly that is movable between a first working position where the sample compartment is open and a second working position where the sample compartment is closed.

[0027] In some aspects, the actuation assembly includes a compression spring, wherein the compression spring is in a first compressed state in the first working position and a second compressed state in the second working position. In some aspects, the actuation assembly includes an extension spring, wherein the extension spring is in a first extension state in the first working position and a second extension state in the second working position.

[0028] In some aspects, the actuation assembly further includes a piston.

[0029] In some aspects, the ingestible apparatus further includes a stopper attached to the actuation assembly, wherein the stopper is configured to seal the sample compartment in thesecond working position. In some aspects, the housing further includes a sealing surface, and wherein the stopper is configured to seal against the sealing surface.

[0030] In some aspects, the ingestible apparatus further includes a crosslinked block configured to prevent the actuation assembly from moving between the first working position and the second working position until the ingestible apparatus is within the target area of the Gl tract of the subject.

[0031] In some aspects, the crosslinked block has an undeformed state and a deformed state, wherein the crosslinked block is configured to hold the actuation assembly in the first working position in the undeformed state, and wherein the crosslinked block is configured to release the actuation assembly to the second working position in the deformed state.

[0032] In some aspects, the crosslinked block is a disc having a central hole, wherein the actuation assembly includes a piston and a spring, wherein a first diameter of the central hole in the undeformed state is configured to engage the piston to hold the actuation assembly in the first working position, and wherein a second diameter of the central hole in the deformed state is configured to disengage the piston to release the actuation assembly to the second working position.

[0033] In some aspects, the crosslinked block is configured to gradually transition from the undeformed state to the deformed state in response to interaction with Gl fluid.

[0034] In some aspects, the actuation assembly is configured to rapidly transition from the first working position to the second working position when released by the crosslinked block.

[0035] In some aspects, the undeformed state is an unexpanded state and the deformed state is an expanded state.

[0036] In some aspects, the crosslinked block is porous.

[0037] In some aspects, the ingestible apparatus further includes a groove disposed on an exterior surface of the housing. Optionally, the groove is positioned at about the same height along the exterior surface of the housing as the separation layer is positioned within the sample compartment.

[0038] In some aspects, the techniques described herein relate to an ingestible apparatus for collecting a sample from a target area of a gastrointestinal (Gl) tract of a subject, the ingestible apparatus including: a housing defining a sample compartment, the housing including one or more access ports formed therein, and the sample compartment being communicable with the one or more access ports; and an absorbent material arranged in the sample compartment, wherein the absorbent material is an organized presentation of fiber.

[0039] In some aspects, the assembly of fiber optionally includes a plurality of loops. Optionally, the plurality of loops is between about 5 and 100 loops.

[0040] In some aspects, the assembly of fiber is a ball of fiber.

[0041] In some aspects, the organized presentation of fiber includes one or more loops, balls, knots, braids, or brushes.

[0042] In some aspects, a method of intestinal analysis includes: administering an ingestible apparatus for collecting a sample from a target area of a gastrointestinal (Gl) tract of a subject, the ingestible apparatus comprising: a housing defining a sample compartment having a first portion and a second portion, the housing comprising one or more access ports formed therein, and the sample compartment being communicable with the one or more access ports; a functional substance arranged in the second portion of the sample compartment; and a separation layer arranged between the first portion and the second portion of the sample compartment, the separation layer being configured to change at least one property in response to interaction with Gl fluid; analyzing a Gl fluid collected from the target area of the Gl tract to create an intestinal profile, wherein analyzing the Gl fluid comprises sequencing a microbiome and wherein the intestinal profile comprises a report of a microbiome constituent of the Gl fluid.

[0043] In some aspects sequencing includes metagenomic, genomic, metabolomic, proteomic, lipodomic and the profile can include a representation of a microbial metagenome, genome, metabolome, proteome, lipodome or any combination thereof.

[0044] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The components in the drawings are not necessarily to scale relative to each other. Like reference numerals designate corresponding parts throughout the several views.

[0046] FIGURE 1 is a cross sectional view of an ingestible apparatus according to an implementation described herein.

[0047] FIGURE 2 is a perspective view of an ingestible apparatus according to an implementation described herein.

[0048] FIGURE 3A is a cross sectional view of an ingestible apparatus with an actuation assembly in a first working position according to an implementation described herein. FIGURE 3B is across sectional view of an ingestible apparatus with an actuation assembly in a second working position according to an implementation described herein.

[0049] FIGURES 4A-4D are cross sectional views of an ingestible apparatus according to an implementation described herein. Fig. 4A illustrates the ingestible apparatus with an actuation assembly in a first working position (e.g. sample compartment is open) and a separation layer fully intact. Fig. 4B illustrates the ingestible apparatus with the actuation assembly in a second working position (e.g. sample compartment is closed) and a permeable separation layer (e.g. having microchannels). Fig. 4C illustrates the ingestible apparatus with the actuation assembly in the second working position and a dissolved separation layer. Fig. 4D illustrates the ingestible apparatus with the actuation assembly in the first working position (e.g. sample compartment is open), the separation layer fully intact, and an absorbent material.

[0050] FIGURES 5A-5F are cross sectional views of an ingestible apparatus with an absorbent material according to implementations described herein.

[0051] FIG. 6 illustrates an example configuration of an implementation of the present disclosure traveling along a gastrointestinal tract.

[0052] FIGS. 7A and 7B illustrate bar plots of genus-level 16S microbiome sequencing profiles, according to a study of an example implementation of the present disclosure.

[0053] FIGS. 8A-8D illustrate in situ x-ray images of an example implementation of the present disclosure in sealed and released configurations, according to implementations of the present disclosure.

[0054] FIGS. 9A and 9B illustrate differences between experimental groups, according to a study of an example implementation of the present disclosure.

[0055] FIGS. 10A-10D illustrate a summary of pairwise group comparisons by machine learning classifiers, according to a study of an example implementation of the present disclosure.

[0056] FIG. 11 illustrates Pirate plots showing a selection of most abundant taxa, according to a study of an example implementation of the present disclosure.

[0057] FIG. 12 illustrates 16S V4 region taxonomic barplots summarized by top 5 bacterial genera by relative abundance and grouped by experimental condition, according to a study of an example implementation of the present disclosure.

[0058] FIG. 13 illustrates alpha diversity metrics of baseline (endoscopic aspirate reference sample at time 0) compared against device under simulated Gl transit conditions with and without stabilizer, according to a study of an example implementation of the present disclosure.

[0059] FIG. 14 illustrates a PCoA plot summary of Bray-Curtis distances, according to a study of an example implementation of the present disclosure.

[0060] FIG. 15 illustrates an example method of sampling showing that capsules according to implementations of the present disclosure can consistently pass through the stomach and enter the small intestine within one hour.

[0061] FIG. 16 illustrates an example method of sampling including configuring the capsule to be ingested 60 minutes before a liquid intervention.DETAILED DESCRIPTION

[0062] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. As used in the specification, and in the appended claims, the singular forms "a," "an," "the" include plural referents unless the context clearly dictates otherwise. The term "comprising" and variations thereof as used herein is used synonymously with the term "including" and variations thereof and are open, non-limiting terms. The terms "optional" or "optionally" used herein mean that the subsequently described feature, event or circumstance may or may not occur, and that the description includes instances where said feature, event or circumstance occurs and instances where it does not. Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, an aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0063] As used herein, the terms "about" or "approximately" when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, or ±1% from the measurable value.

[0064] The term "subject" is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In some embodiments, the subject is a human.

[0065] The term "gastrointestinal fluid" or "Gl fluid" is defined herein to include luminal fluid, mucosal fluid, or a combination of luminal and mucosal fluid from the gastrointestinal tract, as well as any semi-solid or solid substances contained in the fluid. The functionality of the gastrointestinal tract also leads to its Gl fluid carrying semi-solid or solid substances, which may originate from food, shedding cells from the body, or metabolic byproducts of intestinal microbes.These substances undergo continuous transformation within the dynamic environment of the gastrointestinal tract.

[0066] The terms "stool sampling" and "fecal sampling" are used interchangeably herein.

[0067] Referring now to Figs. 1 and 2, an ingestible apparatus 100 according to an implementation described herein is shown. This disclosure contemplates that the ingestible apparatus 100 can be used to collect a sample from and only from a target area of a gastrointestinal (Gl) tract of a subject. Ingestible apparatuses for collecting Gl fluid samples are described in U.S. 2023 / 0061826, which is incorporated herein by reference in its entirety. The ingestible apparatus 100 includes a housing 102 defining a sample compartment 104. As shown in Fig. 1, the housing 102 has exterior surfaces 102A and interior surfaces 102B. The sample compartment 104 is at least partially enclosed by interior surfaces 102B of the housing 102. Additionally, the sample compartment 104 has a first portion 104A and a second portion 104B.

[0068] The housing 102 also includes one or more access ports 106 formed therein. It should be understood that Gl fluid can flow into the sample compartment 104 through the one or more access ports 106. As described herein, Gl fluid may be luminal fluid, mucosal fluid, or a combination of luminal and mucosal fluid. In other words, the sample compartment 104 is communicable with the one or more access ports 106 such that Gl fluid can be collected in the sample compartment 104. The one or more access ports 106 are formed in a port region of the housing 102. Additionally, the housing 102 include a sealing surface 116, and optionally the port region of the housing 102 is spaced apart from the sealing surface 116. Optionally, the sealing surface 116 is centrally located within the sample compartment 104.

[0069] The ingestible apparatus 100 is configured with a relatively large opening to facilitate Gl fluid flowing into the sample compartment 104. Such a large opening makes it possible to collect mucosal fluid or combination of luminal and mucosal fluid, or the semi-solid and solid substances that Gl fluid may carry. For example, as shown in Fig. 2, the housing 102 can include a plurality of access ports 106 and a plurality of struts 114, where each of the plurality of struts 114 is arranged between adjacent access ports 106. The plurality of access ports 106 and the plurality of struts 114 are located in the port region of the housing 102. The struts 114 are narrow and the access ports 106 are wide. Thus, a surface area ratio of the plurality of access ports 106 to the plurality of struts 114 in the port region of the housing 102 is greater than 50%. Optionally, the surface area ratio of the plurality of access ports 106 to the plurality of struts 114 in the port region of the housing 102 is between about 50% and about 90%. For example, the surface area ratio may be 50%, 51%, 52%, 53%, 54%, 55%, ... 85%, 86%, 87%, 88%, 89%, or 90%.

[0070] In one implementation, each port 106 has a relatively rectangular shape, which is 3.7 mm width and 3.2 mm height. The piston (described below) inside the housing 102 is seated below the top of the openings, functionally reducing such openings by 54%. The larger surface area on the outside housing 102 functions to minimize surface tension and encourage inflow of Gl fluid into the sample compartment 104. There are four (4) ports 106 that are equidistantly distributed around the circumference of the housing 102. The struts 114 between ports 106 are about 1mm wide. Therefore, the total open perimeter is 4 x 3.7 mm = 14.6 mm and the total closed perimeter is 4 x 1 mm = 4 mm, and the ratio of open to closed is 14.6 / (14.6 +4) = 78.5%. The total port size area around the perimeter is 47.5 mm2; but functionally because of the piston positioning this is decreased by 54%, to 25 .7 mm2. It should be understood that the number, sizes, and / or arrangement of the ports and struts above are provided only as an example. This disclosure contemplates providing an apparatus having different numbers, sizes, and / or arrangement of the ports and struts.

[0071] The ingestible apparatus 100 also includes a functional substance 108 arranged in the sample compartment 104. In Figs. 1 and 2, the functional substance 108 is arranged in the second portion 104B of the sample compartment 104. In this arrangement, the second portion 104B is located at the furthest position from the one or more access ports 106 (e.g., the port opening). It should be understood that the arrangement shown in Figs. 1 and 2 is only provided as an example. This disclosure contemplates that the second portion 104B can be arranged differently, including closer or directly beside the port opening. As used herein, a "functional substance" refers to any material or compound designed or recognized for having specific functions that can be used in various applications beyond its basic structural use. For example, such functions can include, but are not limited to, chemical reactivity, physical properties, biological activity, or specific interactions with other substances (e.g. Gl fluid). Optionally the functional substance can be any substance that mixes with a fluid for a biological and / or chemical reaction. For example, the substance can be a gluten substance, that mixes with the fluid after collection and then the change within the fluid is measured to determine a patient outcome. Optionally, one specific type of material or compound can have multiple different types of such functions. In some implementations, the functional substance 108 is a preserving substance, which is configured to preserve the Gl fluid collected in the sample compartment 104. Optionally, the preserving substance is ammonium sulfate. When added to a medium or solution, ammonium sulfate increases the osmolarity, or concentration of solutes, outside the bacterial cell, impairing the cell growth. When concentration of ammonium ions further increases, it can be toxic to bacteria by disrupt various cellular processes, such as enzyme activity and protein synthesis, ultimately leading to cell death. When mixing with DNA or RNA, the high ionicstrength of the ammonium sulfate causes the nucleic acids to precipitate out of solution while leaving other contaminants behind. This process effectively concentrates and purifies the DNA or RNA, making it easier to isolate and work with. Additionally, the presence of ammonium sulfate can also help stabilize nucleic acids by shielding them from enzymatic degradation and other denaturing agents. It should be understood that ammonium sulfate is provided only as an example preserving substance. This disclosure contemplates using preserving substances other than ammonium sulfate. For example, the functional substance 108 can be made of different compositions to preserve various types of the bioinformation depending on the target application. For metabolomic / proteomic material preservation, both microbial and enzymatic reactions must be quenched. In this case, the preserving substance may be a blend (e.g. cocktail solution) of the ammonium sulfate (which quenches the microbial activities) and other preserving agents (which quenches the enzymatic activities). Additionally, for preserving the viability of microorganisms such as bacteria, ingredients that are nutritious to a wide-spectrum of the microorganisms and / or to a selected range of the microorganisms can be included in the substance.

[0072] In some implementations, the functional substance 108 is in a solid state. Optionally, the functional substance 108 is in a powdered form, which promotes mixing when in the presence of Gl fluid. For example, when in a powered form, the functional substance 108 can more easily mix with Gl fluid as compared to a bulk solid, which would be required to dissolve and / or break apart before mixing with Gl fluid. Additionally, when in a powdered form, the granules can be grinded to a small enough size so a relatively flat surface of the granules is formed, which allows a separation layer (described below) to be spread more evenly across the surface. It should be understood that the functional substance 108 in a powered form is provided only as an example. This disclosure contemplates that the functional substance 108 can be in another state or form including, but not limited to, bulk solids, liquids, gels, or combinations thereof.

[0073] The ingestible apparatus 100 also includes a separation layer 110 arranged in the sample compartment 104. In Figs. 1 and 2, the separation layer 110 is arranged between the first portion 104A and the second portion 104B of the sample compartment 104. Additionally, the separation layer 110 is configured to change at least one property in response to interaction with the Gl fluid. In some implementations, the separation layer 110 is configured to change the at least one property by dissolving in response to interaction with Gl fluid. The separation layer 110 partially dissolves in some implementations, while in other implementations dissolves completely. In other implementations, the separation layer 110 is configured to change the at least one property by changing permeability in response to interaction with Gl fluid. Changing permeability can be accomplished through the formation of microchannels in the separation layer 110. Additionally, inresponse to the separation layer 110 changing the at least one property, the functional substance 108 is configured to move from the second portion 104B of the sample compartment 104 to the first portion 104A of the sample compartment 104, which allows for mixing with the collected Gl fluid. The controlled release of the functional substance 108 is based on relatively slow release through the separation layer 110, which can be well-timed relative to closing of the sample compartment 104. Eventually the change of the property stops when the closed system reaches the state of equilibrium. A closed system means, for example, that the actuation assembly 118 is in a second working position, where the sample compartment is closed such that Gl fluid can no longer flow through the one or more access ports 106. In some implementations, the state of equilibrium is achieved in about in few hours after piston release so the preservation will soon become fully effective for the rest of the journey (e.g. Gl transit by the ingestible apparatus 100 and shipping transit to the lab).

[0074] In one implementation, the separation layer 110 is a polymer. Example polymers suitable for the separation layer 110 include Eudragit retardant soluble (Eudragit RS) and Eudragit retardant long-acting (Eudragit RL), both of which are used in pharmaceutical formulations. Each is part of the Eudragit family, which consists of various types of acrylic and methacrylic acid copolymers. These copolymers are used in pharmaceuticals due to their biocompatibility, filmforming properties, and ability to modify drug release characteristics. It should be understood that Eudragit RS and Eudragit RL are provided only as example polymers.

[0075] Optionally, the separation layer 110 is a combination of Eudragit RS and Eudragit RL (Eudragit RS / RL). Such a polymer does not dissolve to release the functional substance 108. Instead, it changes permeability by gradually swelling and creating microchannels in between molecular bindings to allow the functional substance 108 to permeate through the separation layer 110. For example, after 2 hours of exposure to Gl fluid, approximately 30% of the functional substance 108 has been found to permeate through the separation layer 110 in example bench tests. It should be understood that permeating speed is affected by factors including, but not limited to, the thickness of the separation layer 110, the mechanism (e.g. dissolving or permeating) of the separation layer 110, the density and / or the size of the microchannels (if the separation layer 110 is permeating), the gradient of the concentration of the functional substance 108 on the two sides of the separation layer 110.

[0076] In Figs. 1 and 2, the separation layer 110 provides a divider between the first portion 104A and second portion 104B, and the functional substance 108 is arranged in the second portion 104B. It should be understood that the functional substance 108 have a different arrangement. For example, the functional substance 108 may be embedded within the separationlayer 110, such that the functional substance 108 is released as the separation layer 110 changes a property. Alternatively, the functional substance 108 may be both arranged in the second portion 104B and also embedded in the separation layer 110.

[0077] Referring now to Figs. 4A-4D, an ingestible apparatus according to an implementation described herein is shown. This disclosure contemplates that the ingestible apparatus can be used to collect a sample from a target area of a Gl tract of a subject. The ingestible apparatus includes a housing 402 defining a sample compartment 404, which has a first portion 404A and a second portion 404B. The housing 402 also includes one or more access ports 406 formed therein. It should be understood that Gl fluid can flow into the sample compartment 404 through the one or more access ports 406. In other words, the sample compartment 404 is communicable with the one or more access ports 406 such that Gl fluid can be collected in the sample compartment 404. The ingestible apparatus also includes a functional substance 408 arranged in the sample compartment 404. In Figs. 4A-4D, the functional substance 408 is arranged in the second portion 404B of the sample compartment 104. The ingestible apparatus also includes a separation layer 410 arranged in the sample compartment 104. In Figs. 4A-4D, the separation layer 410 is arranged between the first portion 404A and the second portion 404B of the sample compartment 104.

[0078] As described above, the separation layer 410 is configured to change at least one property (e.g. dissolves or changes permeability) in response to interaction with the Gl fluid such as Gl fluid. For example, with reference to Fig. 4A, an actuation assembly 418 is in a first working position, where the sample compartment is open such that Gl fluid flows through the one or more access ports 406 into the first portion 404A of the sample compartment. The Gl fluid therefore comes into contact with the separation layer 410, and a result, the separation layer 410 changes at least one property such that the functional substance 408 can begin to move from the second portion 404B of the sample compartment 404 to the first portion 404A of the sample compartment 404. In Fig. 4B, the separation layer 410 developed microchannels (e.g. changed permeability) by interacting with the Gl fluid, allowing the functional substance 408 to move into the first portion 404A of the sample compartment 404, mixing with the Gl fluid. In Fig. 4C, the separation layer 410 dissolved in the Gl fluid, allowing the functional substance 408 to move into the first portion 404A of the sample compartment 404, mixing with the Gl fluid.

[0079] In Figs. 4B and 4C, the actuation assembly 418 is in a second working position, where the sample compartment is closed such that Gl fluid can no longer flow through the one or more access ports 406 into the first portion 404A of the sample compartment. As a result, the functional substance 408 is contained with the sample compartment 404 even after permeating ortraveling through the separation layer 410. Optionally, the separation layer 410 changes at least one property over a period of time when exposed to the Gl fluid. Optionally, the separation layer 410 is designed such that the change in at least one property does not occur until after the actuation assembly 418 is in the second working position (i.e. the sample compartment is closed). The separation layer 410 can be designed such that the change in property is well-timed with respect to sample compartment closing.

[0080] With reference to Fig. 4D, the ingestible apparatus optionally includes an absorbent material 412. Optionally, the separation layer 410 is configured to prevent the absorbent material 412 from contacting the functional substance 408 before the separation layer 410 changes the at least one property. Absorbent materials are described in detail below.

[0081] Referring again to Figs. 1 and 2, in some implementations, the functional substance 108 is a preserving substance such as ammonium sulfate as described above. The ingestible apparatus 100 can have a large port design to ensure a representative mix of the Gl material is collected and to ensure that the port stays open for an extended period of time as described herein. The preserving substance is placed in contact with the collected and sealed Gl fluid to mix and preserve it during transit. Since the ingestible apparatus 100 design is not a one-way valve, Gl fluid can enter and exit the sample compartment 104 during the sampling period until the sampling is completed. Therefore, there is the concern that adding a preserving substance to the sample compartment 104 could mix with the Gl fluid during collection period, which could then flow out of the sample compartment 104 and into the subject's body. This could have a deleterious impact to the subject as they may be exposed to large amounts of the preserving substance. In addition, this could result in insufficient or inconsistent quantities of the preserving substance being available to stabilize the collected sample. Additionally, if the functional substance is substance intended to mix with the intestinal fluid to gauge a biological or chemical reaction, it may endanger the user and / or affect the results (e.g., by changing the quantity of the sample) if it is released outside of the sampling chamber.

[0082] The separation layer 110 as described herein delays the mixing of the preserving substance with the Gl fluid until the sampling is completed. Thus, in one implementation, the preserving substance is placed in a solid form (e.g. a powder) and the separation layer 110 is a polymer. When the Gl fluid is collecting, the fluid is not in contact with the preserving substance. However, the collected fluid is in contact with the separation layer 110 and it begins to dissolve or change permeability. The timing of the dissolution or change of permeability can be timed with the closure of the sample compartment 104, such that substantive mixing of the preserving substance only takes place after closure. This is in contrast to other techniques for achieving delayed mixingsuch as mechanically poking a preserving-substance sealant to release it into the sample chamber, which can interfere with effective sealing, and cannot be timed to begin mixing a portion of the preserving agent during extended sampling durations. Additionally, this is in contrast to other techniques for preventing loss of a functional substance during sampling such as impregnating an absorbent material with the preserving substance, which detracts from the ability of the preserving substance to mix with the Gl fluid and / or bacteria and therefore negatively impacts the function. An impregnated absorbent material may also negatively impact the absorbent properties and decrease the amount of fluid collected.

[0083] Fluid collection in the Gl tract is made challenging by inconsistent fluid volumes. It is desirable to present the absorbent material close to the fluid interface, however having the absorbent material present at the surface of the port would interfere with the sealing action of the piston 122 against the sealing surface 116 in the closed position. To achieve a sealing surface that can mate with the piston 122 given the linear axis of the actuation assembly 118, and to enabling full circumferential opening to promote sample collection in all orientations of the ingestible apparatus 100 relative to the intestinal walls where pockets of the Gl fluid resides, the sealing surface 116 is not provided at the outer walls of the housing 102, rather the opening of the sample compartment 104 and the sealing surface 116 are located centrally, within the interior of the housing 102. The absorbent material can be contained within this central portion, and can therefore not be placed close to the exterior walls where it would be best exposed to the surrounding fluid. Further, the absorbent material is intended to collect fluid with Gl contents (bacteria, metabolites etc.) and therefore it is desirable to increase the surface area of the absorbent material and to increase the access to the absorbent material with a high portion of port opening. However having a frayed ends, or multiple wicks, can further challenge of obtaining adequate seal between the piston 122 and the sealing surface 116.

[0084] Accordingly, in some implementations, the ingestible apparatus 100 also includes an absorbent material 112 arranged in the sample compartment 104. In Figs. 1 and 2, the absorbent material 112 is arranged in the first portion 104A of the sample compartment 104. The separation layer 110 is therefore disposed between the absorbent material 112 and the functional substance 108. Optionally, the absorbent material 112 is sized and / or shaped such that the absorbent material 112 cannot exit the housing 102 via the one or more access ports 106. Alternatively or additionally, the absorbent material 112 is sized and / or shaped such that no portion of the absorbent material 112 can exit the housing 102 via the one or more access ports 106. Optionally, the absorbent material 112 is sized and / or shaped such that no portion of the absorbent material 112 extendsbeyond the sealing surface 116. Optionally, the absorbent material 112 is sized and / or shaped such that no portion of the absorbent material 112 interferes with the sealing surface 116.

[0085] Optionally, in some implementations, the surface of the absorbent material 112 adjacent to the sealing surface 116 is spaced about 1 millimeter (mm) from the sealing surface 116 of the housing 102. Additionally, the surface of the absorbent material 112 adjacent to the sealing surface 116 is spaced no more than about 5 mm from the sealing surface of the housing 102. In one example, at 1mm away, 60% of tested ingestible apparatuses collected sufficient (>20mg) of fluid, while at 5mm away, 100% of the tested ingestible apparatuses failed to collect a sufficient sample. Optionally, the surface of the absorbent material 112 adjacent to the sealing surface 116 is spaced between about 0 mm and 2 mm from the sealing surface 116 of the housing 102. For example, the second surface can be spaced about 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mm from the sealing surface 116 of the housing 102.

[0086] In some implementations, the absorbent material 112 is an organized presentation of fiber. Organized presentation of fibers provide a high surface area to volume ratio that is beneficial for increasing the volume of the collected Gl fluid, and also for increasing the concentration of DNA collected from the Gl fluid. This effect is particularly amplified when the ingestible apparatus 100 remains open for a substantial period of time to promote uniform sampling. As used herein, an "organized presentation of fiber" refers to a fiber or plurality of fibers prepared to maintain organization, increase ease of handling, and / or increase ease of insertion into the ingestible apparatus 100. The organized presentation is used to provide a specific shape and ensure consistency in the amount of fiber in any one portion of the sampling chamber. For example, near the sampling port. The organized presentation of fiber may include one or more loops, balls, knots, and / or brushes. This disclosure contemplates that the fiber or plurality of fibers can be prepared by looping, twirling, knotting, balling, winding, etc. a fiber or plurality of fibers. For example, an organized presentation of fiber can be prepared by winding a fiber or plurality of fibers around a spool. Optionally, an organized presentation of fiber can be prepared such that there is consistency across devices, i.e. multiple organized presentations of fiber share common features or characteristics (e.g. loops, balls, knots, braids and / or brushes). In some implementations, the organized presentation of fiber is not fastened, bound, or fixed together. In other implementations, the organized presentation of fiber is fastened, bound, or fixed together (e.g. tied, glued, etc.). When fastened, bound, or fixed together, an organized presentation of fiber is referred to herein as a bundle of fiber. Additionally, it should be understood that an organized presentation of fiber should be understood in contrast to a disorganized collection of fiber or fibers (see Fig. 5E).

[0087] In some implementations, the organized presentation of fiber is configured to increase a surface area of the absorbent material 112 in proximity to a sealing surface 116 of the housing 102. For example, the absorbent material 112 can define a first surface adjacent to the separation layer 110 and a second surface opposite to the first surface. The second surface is therefore adjacent to the sealing surface 116. The second surface of the absorbent material 112 can be arranged below the sealing surface 116. To ensure that the second surface of the absorbent material 112 is in close proximity to the fluid of the Gl tract, the housing 102 is designed with one or more access ports 106 having large openings, e.g. evenly-spaced ports around the perimeter of the housing 102, with only small struts 114 separating them. This allows for Gl fluid to flow about the sample compartment 104, which is centrally located within the housing 102, and over the second surface of the absorbent material 112.

[0088] In some implementations, the organized presentation of fiber is an assembly of fiber. For example, the assembly of fiber optionally includes a plurality of loops. Optionally, the plurality of loops is between about 5 and 100 loops. For example, the plurality of loops can be 5, 6, 7, 8, 9, 10, ... 95, 96, 97, 98, 99, 100 loops. Optionally, the plurality of loops is between about 50 and 90 loops. Optionally, the plurality of loops is about 80 loops. In other implementations, the assembly of fiber is a ball of fiber. In comparison to loops, which is a relatively less dense organization of fiber, a ball of fiber is more densely organized. In other implementations, the assembly of fiber is a bundle of fiber.

[0089] In one example implementation, the absorbent material 112 is an organized presentation of fiber including 80 loops. In particular, the organized presentation of fiber is prepared by winding a fiber around a spool. The fiber has a thickness of Metric Count: 54.237 m / g or 54.237NM (British Count: 325 or 32N). Additionally, the fiber has a length of 117 ± 17.5 cm, weight of 20 ± 3 mg, and the diameter of each loop is <1 cm. Forty (40) loops wound around the spool are removed and doubled over to create eighty (80) loops before placing the absorbent material 112 in the ingestible apparatus 100. Alternatively or additionally, the fiber has the following characteristics: 0.204 mg / cm and about 20 mg for the fiber length and weight. 20 mg of the fiber has been shown as effectively keeping about 130mg of the liquid samples, and adding weight of the fibers beyond 20 mg is not expected to increase the amount of the liquid retained in the sampling chamber (difference of approximately + / - 0.05mg between 20 and 25 mg). After 20mg of fiber weight, the sampling chamber becomes the limiting factor. It should be understood that the organized presentation of fiber described in this example is provided only as an example. This disclosure contemplates that an organized presentation of fiber can have different characteristics than those described in this example.

[0090] In some implementations, the absorbent material 112 contacts the separation layer 110. For example, the separation layer 110 can serve as a shelf, surface, strut, etc. on which the absorbent material 112 rests. Optionally, the absorbent material 112 is attached to and / or partially embedded with the separation layer 110. In other implementations, the absorbent material 112 does not contact the separation layer 110. Optionally, in some implementations, the absorbent material 112 defines a first surface adjacent to the separation layer 110 and a second surface opposite to the first surface, the separation layer 110 being arranged in the sample compartment 104 such that the second surface is spaced about 1 millimeter (mm) from a sealing surface 116 of the housing 102. Additionally, the second surface is spaced no more than about 5 mm from the sealing surface of the housing 102. In one example, at 1mm away, 60% of tested ingestible apparatuses collected sufficient (>20mg) of fluid, while at 5mm away, 100% of the tested ingestible apparatuses failed to collect a sufficient sample. Optionally, the second surface is spaced between about 0 mm and 2 mm from the sealing surface 116 of the housing 102. For example, the second surface can be spaced about 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mm from the sealing surface 116 of the housing 102.

[0091] Referring now to Figs. 5A-5E, an ingestible apparatus according to an implementation described herein is shown. This disclosure contemplates that the ingestible apparatus can be used to collect a sample from a target area of a Gl tract of a subject. The ingestible apparatus includes a housing 502 defining a sample compartment. The housing 502 also includes one or more access ports 506 formed therein. It should be understood that Gl fluid can flow into the sample compartment through the one or more access ports 506. In other words, the sample compartment is communicable with the one or more access ports 506 such that Gl fluid can be collected in the sample compartment. In Figs. 5A-5D, the ingestible apparatus also includes an absorbent material arranged in the sample compartment, where the absorbent material is an organized presentation of fiber, and specifically an assembly of fiber. Fig. 5A illustrates an ingestible apparatus including an assembly of fiber 512A that includes a plurality of loops. Fig. 5B illustrates an ingestible apparatus including an assembly of fiber 512B that is a ball of fiber. As described above, the assembly of fiber 512A or 512B can be formed from a single fiber or a plurality of fibers. Figs. 5C and 5D illustrates an arrangement of the assembly of fiber relative to a sealing surface 516 of the housing 502. In Fig. 5C, the assembly of fiber 512C is spaced too far from the sealing surface 516. As described above, a top surface of the fiber 512C may be spaced 5 mm or more from the sealing surface 516 in Fig. 5C. On the other hand, the assembly of fiber 512D is spaced appropriately from the sealing surface 516. As described above, a top surface of the fiber 512D may be spaced between about 1-2 mm from the sealing surface 516 in Fig. 5D. Additionally, Figs. 5E and 5F illustrates non-optimal arrangements for the absorbent material. In Fig. 5E, at least a portion of the assembly of fiber 512E is disposed such that it will interfere with the sealing surface 516. Fig. 5F illustrates an absorbent material 512F that is disorganized (i.e. not an organized presentation of fiber) and as a result is too distance from the fluid transfer interface.

[0092] Referring again to Figs. 1 and 2, the ingestible apparatus 100 optionally includes an actuation assembly 118 that is movable between a first working position where the sample compartment 104 is open (see Fig. 3A) and a second working position where the sample compartment 104 is closed (see Fig. 3B). As shown in Figs. 1-3B, the actuation assembly 118 includes a spring 120 and a piston 122. Additionally, the ingestible apparatus 100 includes a stopper 124 attached to the actuation assembly 118. In some implementations, the piston 122 and stopper 124 are a unitary component. In other implementations, the piston 122 and stopper 124 are different components fixed together. Optionally, a gasket 130 can be provided around at least a portion of the stopper 124, for example to assist in providing a seal. In these implementations, the gasket 130 provides a seal against the sealing surface 116.

[0093] In Figs. 1-3B, the spring 120 is a compression spring. As shown in Fig. 3A, the compression spring is in a first compressed state in the first working position. As shown in Fig. 3B, the compression spring is in a second compressed state in the second working position. A potential energy associated with the second compressed state is greater than zero but less than a potential energy associated with the first compressed state. Thus, the stopper 124 is configured to seal the sample compartment 104 in the second working position. For example, as shown in Fig. 3B, the stopper 124 and gasket 130 provide a seal against the sealing surface 116. It should be understood that the compression spring shown in Figs. 1-3B is provided only as an example. This disclosure contemplates that the actuation assembly 118 can include an extension spring in other implementations. For example, the extension spring can be in a first extension state in the first working position and a second extension state in the second working position, and a potential energy associated with the second extension state can be greater than zero but less than a potential energy associated with the first extension state. Thus, similarly to the compression spring implementation, the stopper 124 would be configured to seal the sample compartment 104 in the second working position.

[0094] Additionally, the ingestible apparatus 100 optionally includes a block 126 configured to prevent the actuation assembly 118 from moving between the first working position (see Fig. 3A) and the second working position (see Fig. 3B) until the ingestible apparatus is within the target area of the Gl tract of the subject and has completed the sampling. For example, the actuation assembly 118 can be configured to withstand the closing force (e.g. spring force) toeffectively hold the piston 122 in place during the sampling period and then effectively release the piston 122 at the end of the sampling period. The configuration of the block 126 can for example be cross linked (also referred to herein as a "crosslinked block"). The cross linking can be used to maintain certain structural aspects of the block needed to hold the piston 122 in place. For example, to maintain a complete ring shape, or maintain a height or both. During the sampling period the crosslinked block 126 is deforming with continued exposure to the Gl fluid, however the deformation should not change the position of the piston 122 during this period. For example, the piston 122 cannot slowly start to close during the sampling period because the block 126 is deforming and becoming thinner, as even small changes in the size of the port opening can impact the sampling efficacy. In addition, the piston 122 should not tilt to one side during the sampling period because the block 126 is preferentially deforming on one side versus the other as the titling may cause the piston 122 to become caught or jammed, or may result in an ineffective seal. Instead, the actuation assembly 118 can be configured to effectively hold the piston 122 during transport, storage, and first transit through the Gl tract and then to effectively move from the first working position (i.e. start position) to the second working position (i.e. end position). Effective movement from the first working position (i.e. start position) to the second working position (i.e. end position) occurs within a short period of time (e.g. sudden or over a few minutes). Additionally, the sampling period should be repeatable. This requires that the change in state of the actuation assembly 118 be controlled such that it occurs at a similar rate and in a similar manner each time across different devices. At times it is desirable to have the timing of the sampling period reduced, for example in individuals with a known rate of increased motility. Or where it is desirable to target a specific sampling region, for example the upper Gl tract or a portion of the upper Gl tract, for example the duodenum, jejunum or ileum. It is also desired for the actuation assembly 118 to stay open for sufficient time so as to sample a region of the small intestine, for example one that stays open with sufficient time to travel a portion of the distal small intestine.

[0095] In some implementations, the crosslinked block 126 has an undeformed state and a deformed state such that the crosslinked block 126 is configured to: (i) hold the actuation assembly 118 in the first working position in the undeformed state, and (ii) release the actuation assembly 118 to the second working position in the deformed state. The crosslinked block 126 is configured to gradually transition from the undeformed state to the deformed state in response to interaction with Gl fluid. Optionally, the gradual transition from undeformed to deformed states occurs over a period of time. The period of time relates to the sampling time. For example, in some implementations, the period of time may be relatively lengthy such as between about 15 and 150 minutes. Optionally, the period of time is about 110 minutes, e.g. the crosslinked block 126undergoes deformation and releases the piston 122 at about 110 minutes. It should be understood that the above range is provided only as an example and that the period of time may include values less or more than those in the example and dependent on the sampling time. This disclosure contemplates that the crosslinked block 126 can be designed accordingly. In contrast, the actuation assembly 118 is configured to rapidly transition from the first working position to the second working position when released by the crosslinked block 126. In other words, once the crosslinked block 126 transitions to the deformed state, the actuation assembly 118 rapidly moves from the first working position to the second working position, for example due to the spring force. As described herein, the crosslinked block 126 can be make of a crosslinked starch (e.g. heat cured and specially cooled), which ensures structural strength and repeatable deformation. Optionally, the crosslinked block 126 is porous and / or is formed to create small internal defects such as bubbles, which increases the rate of deformation change of the crosslinked block 126.

[0096] In some implementations, as shown in Figs. 3A-3B, the crosslinked block 126 is a disc having a central hole. The crosslinked block 126 is arranged externally with respect to the housing 102. In Figs. 3A-3B, the undeformed state is an unexpanded state and the deformed state is an expanded state. In Fig. 3A, which illustrates the crosslinked block 126 in the undeformed state, a first diameter of the central hole is configured to engage the piston 122 to hold the actuation assembly 118 in the first working position. In Fig. 3B, which illustrates the crosslinked block 126 in the deformed state, a second diameter of the central hole (i.e. larger than the first diameter) is configured to disengage the piston 122 to release the actuation assembly 118 to the second working position. In other words, the crosslinked block 126 expands between states shown in Figs. 3A-3B such that the central hole disengages the piston 122 to release the actuation assembly 118 to the second working position. It should be understood that the size, shape, and / or arrangement of the crosslinked block 126 shown in Figs. 3A-3B are provided only as examples. It should be understood that the crosslinked block 126 can have a different size, shape, and / or arrangement than shown in Figs. 3A-3B.

[0097] In one implementation, the crosslinked block 126 is a donut-shaped disc made of a deformable material. The piston 122 is positioned through the central hole of the crosslinked disc. The piston 122 has a cap that is in contact with the top surface of the crosslinked disc. The contact area is sufficiently large to ensure the piston 122 does not slip or tilt through the hole. The disc is made from a material that is deformable when in contact with the Gl fluid. The deformation includes a change in the diameter of the central hole. The contact area of the cap on the crosslinked disc is less than the diameter change of the central hole. The effect is that once the dimensional transformation has been achieved, the piston 122 is released in a manner that allows the actuationassembly 118 to instantly move from the first working position to second working position while maintaining an upright position. Additionally, the working height of the crosslinked disc is designed to ensure that the working length of the spring 120 remains in compression during transformation. This behavior is achieved by providing a disc that is made of a crosslinked starch (e.g. heat cured and specially cooled) to control the disc's change in shape and to ensure structural strength. By controlling the crosslinking, it is possible to get repeatable deformation behavior change profiles.

[0098] The crosslinked disc is not dissolving, rather its shape is substantially maintained. This prevents a solute from mixing with the Gl fluid which could have an impact on the sample. For example, releasing a substantial amount of starch to the bacteria of the small intestine could change their metabolic behavior. Instead, the crosslinked disc swells when in contact with the Gl fluid and expands. As a result, the crosslinked disc expands and the central hole becomes larger. The piston 122 eventually falls through the central hole, releasing the actuation assembly 118 to the second working position. Since the central hole expands uniformly, the piston 122 travels straight without tilting. The actuation assembly 118 that ensures an aligned piston change is particularly important in a multi-port design, where the ports are of sufficient size to allow for a tilted piston to be caught. Additionally, while crosslinking is important to ensure the structural integrity of the disc, introducing bubbles or pores within the disc increases the rate of change while maintaining the structural strength.

[0099] The ingestible apparatus 100 also includes a groove 128 disposed on an exterior surface of the housing 102. Optionally, the groove 128 is positioned at about the same height along the exterior surface of the housing 102 as the separation layer 110 is positioned within the sample compartment 104.

[0100] The sample collected with the ingestible apparatus 100 is from the upper gastrointestinal tract, where the concentration of bacteria is less than what is present in the lower tract (colon). Once the ingestible apparatus 100 opens and closes to collect the sample, and transit the remainder of the tract, the ingestible apparatus 100 is surrounded by the matter of higher bacterial concentration (e.g. feces). In addition to requiring a good seal to prevent any contamination during transit, it is important to ensure that the sample is removed without contamination. Even the smallest amount can impact the bacterial profile.

[0101] Opening an ingestible apparatus may introduce contamination to the collected fluid sample. For example, some ingestible apparatuses known in the art include threaded portions to facilitate opening of the device. The opening of threaded portions, however, is an easy way to introduce contamination. Other techniques to remove the collected sample from ingestible apparatuses include insertion of a needle through the housing, or through a special port in thehousing. One challenge to this approach for the present device is that the sample is collected with the absorbent material 112, e.g. an organized collection of fiber, which has collected a significant portion of the deoxyribonucleic acid (DNA). Attempts to use only the fluid portion extracted via needle for further analysis, without spinning the fiber have shown that the collected DNA is not sufficient in the fluid only. Thus, the collected fluid sample and the fiber should be removed but without introducing contamination. Use of the groove 128 as a cutting guide addresses problems with known techniques. In particular, the groove 128 is positioned at the height of the separation layer 110 inside the housing 102. Cutting the housing 102 open at the groove 128 enables ready access to the absorbent material 112.

[0102] Examples. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0103] First Example Apparatus

[0104] In one example, an ingestible apparatus for collecting a sample from a target area of a Gl tract of a subject includes: a housing (see Figs. 1-2, housing 102) defining a sample compartment (see Figs. 1-2, sample compartment 104) having a first portion (see Figs. 1-2, first portion 104A) and a second portion (see Figs. 1-2, second portion 104B), the housing including one or more access ports (see Figs. 1-2, one or more access ports 106) formed therein, and the sample compartment being communicable with the one or more access ports; a functional substance (see Figs. 1-2, functional substance 108) arranged in the second portion of the sample compartment; and a separation layer (see Figs. 1-2, separation layer 110) arranged between the first portion and the second portion of the sample compartment, the separation layer being configured to change at least one property in response to interaction with Gl fluid.

[0105] Second Example Apparatus

[0106] In another example, an ingestible apparatus for collecting a sample from a target area of a Gl tract of a subject includes: a housing (see Figs. 5A-5B, housing 502) defining a sample compartment, the housing including one or more access ports (see Figs. 5A-5B, ports 506) formed therein, and the sample compartment being communicable with the one or more access ports; and an absorbent material (see Figs. 5A-5B, assembly of fiber 512A, 512B) arranged in the sample compartment, wherein the absorbent material is an organized presentation of fiber.

[0107] Example 1

[0108] A study was performed using an example implementation of the present disclosure including a capsule and methods for collecting DNA samples using the capsule.

[0109] Methods. Individuals with previously diagnosed IBS (Rome 4 criteria, diagnosed by a gastroenterologist) and healthy / control volunteers were recruited from clinical practice, recruitment posters at the University of Calgary-affiliated gastroenterology clinics. Recruited participants were between 18 - 70 years of age, and selected based on the following criteria; 1) did not have prior gastrointestinal disease, surgery, or radiation treatment; 2) did not use any medications a week prior to the study that would affect Gl motility or acidity; 3) if female, were not pregnant, not breastfeeding, and practicing birth control; 4) did not take antibiotics, colon cleanses / colonics, or bowel preparation for colonoscopy within 2 weeks prior to recruitment; 5) if in the control group, did not have fewer than 2 bowel movements a week.

[0110] All participants ingested two capsules at two separate visits separated by 7 to 21 days. In the first visit, the two capsules were ingested with the participants in a fasted state (minimum 8 hour fast) with water. For this study a small radiopaque bead was inserted between the external coating and the capsule; when the bead became detached from the capsule on X-ray it indicated that the external coating had sloughed off and that the capsule had started collecting content. Capsules have a mechanical closure mechanism which can be assessed by X-ray to be open (i.e. collecting content) or approximately every 15 - 45 minutes until capsules completed sampling) to document sample collection start-, end-point locations, sampling durations and timing from ingestion to start- and end-point sampling. After completing their X-ray visit, participants returned home with instructions and materials for retrieving the capsules by screening each bowel movement until they were retrieved. When a capsule was found, subjects also collected a separate fecal sample from the same stool.

[0111] Between 7 and 21 days following the initial X-ray visit, fasting participants underwent an esophagogastroduodenoscopy (EGD) procedure to collect a duodenal aspirate, duodenal cytological brush, and saliva sample. Saliva was collected prior to the endoscopy. Standard conscious sedation with fentanyl and midazolam was used, and oral spray anesthetic was not used. The gastroscope was intubated as far as possible into the duodenum (typically to the fourth part; at least third part in all cases). Aspirate was taken first using sterile technique around scope handling and particularly regarding the scope biopsy channel. A sterile aspiration catheter was used to collect a fluid sample from the distal duodenum. Duodenal mucosal brushing was then taken. All EGD evaluations were visually normal.

[0112] The day after endoscopy, two further capsules were ingested by the participants. Identical with the first phase of the study, capsules were ingested with water on a fasted stomach and collected along with matched fecal samples at home. Collected capsules and fecal samples were returned via courier immediately and processed within 24 hours upon receipt using sterile technique.

[0113] Samples were received, extracted, and prepared for 16S sequencing and metabolomics analysis. A subset of the remaining DNA samples were prepared for shotgun metagenomic sequencing to further assess the DNA quality.

[0114] RESULTS. The ingestible capsules were evaluated for their ability to reliably and reproducibly collect small intestinal luminal samples both by physical assessment and comparison of 16S rRNA gene amplicon sequencing and metabolomics data to samples collected from patient matched feces and endoscopy aspirate. A total of 30 participants were recruited, comprising 10 reference healthy controls and 20 with impaired Gl motility (8 IBS-Constipation or IBS-C, 10 IBS- Diarrhea or IBS-D, and 2 IBS-Mixed or IBS-M), with a median age 43 years (min 23; max 67; IQR 32 to 53), and male / female ratio of 12 / 18.

[0115] Of the 120 total capsule ingestions performed, all capsules were confirmed to have passed spontaneously, with 93% (112 / 120) being successfully retrieved and returned (58 / 60 following X-Ray and 54 / 60 following Endoscopy). There was no participant dropout between visits, and no serious adverse events were reported that concern the safety of the capsule. Minimal adverse events were reported and were not device-related: 1 event of prolonged capsule retention (> 7 days) in an IBS patient likely associated with opioid used at endoscopy.

[0116] X-ray tracking of capsule sampling endpoints indicated a high targeting accuracy of the capsule to the SI. X-rays were read by an expert radiologist blinded to any other subject information other than time post-ingestion. With an X-ray sampling interval of 15-45 minutes, 41 of the 60 capsules ingested had confirmed sampling start locations (start was observed by the marker displacement floating away once the capsule shell dissolved) and 47 had confirmed sampling end locations (end being observation of spring deployment). Sampling location was deemed determinate if it could be confidently assigned by the radiologist and if capsule opening or closing events were seen at least one observation interval after capsule Gl-regional transit events. All 41 determinately tracked capsules for start-point initiated sampling in the SI, which also included 3 / 4 capsules that were observed to remain in the epigastric region for longer than 2 hours, and of these, one capsule remained in the epigastric region without observed shell dissolution, indicating no impact of gastric pH on outer shell integrity. Of the determinate tracked capsules for capsule closure, 44 / 49 (~90 %) were observed to complete sampling in the distal SI, with 15 sampling in the jejunum region and 29in the ileum region. No significant difference in sampling end location was observed across participant motility groups (Chi-squared goodness of fit test p-value ~ 0.41). The remaining 5 also sampled primarily in the distal SI but final sealing was observed in the proximal colon. The median capsule sampling duration from sampling start to end was ~1.5 hours (min = 1.04 hours; max = 2.02 hours) and was also not found to significantly differ by sampling end location (SI vs. Colon) or participant motility group (Kruskal-Wallis rank-sum test p-value ~0.78). The median capsule total Gl transit time was ~46.1 hours and was only found to significantly differ between control and IBS-C groups (median transit time control vs. IBS-C = 29.7 hours vs. 54.7 hours; two-sided Mann-Whitney test p-value ~ 0.006). Excluding one participant where dual-capsules remained in the gastric region for the duration of X-ray observation, X-ray tracking was able to definitively discern that the capsule successfully collected a SI sample, with either a determinate starting or end location in the SI, for 29 / 29 ~ 100% of study participants.

[0117] To evaluate the efficacy of the capsule in capturing and preserving an uncontaminated sample of the SI, microbiome and metabolomics profiles of key Gl metabolites (BAs and SCFAs) were compared between capsules and matched fecal samples. An examination of 16S microbiome taxa plots revealed distinct microbiome compositions between capsule and fecal samples, with the former dominated by bacteria of the genera Streptococcus, and the latter dominated a more diverse profile of Blautia, Bacteroidetes, and Fecalibacterium, consistent with previous findings [4]. Principal component analysis of microbiome beta-diversities (weighted Unifrac distance) confirmed a significant difference in microbiome composition between capsule and fecal samples (PERMANOVA P-value ~ 0.001, n = 66 samples).

[0118] To have a better assessment of the quality of the DNA extracted from the capsules, DNA samples from 16 participants (For each participant, 1 capsule, 1 matched endoscopic aspirate, 1 cytological brush, 1 saliva, and 1 feces sample) for a follow-up shotgun metagenomic sequencing. Five samples (1 / 16 saliva samples, 0 / 16 brush samples, 2 / 16 aspirate samples, and 1 / 16 capsule samples) failed library preparation and in total 76 samples were sequenced. Good sequencing data was obtained for all samples with an average of 38.5 M read pairs per sample and a minimum of 32.9 M read pairs. The majority of the reads (on average 99.6%) were of high quality. Cytological brush and aspirate samples contained high proportions of host (human) DNA (93.1 - 96.4 % of all reads) which decreases the proportion of microbial DNA available for microbiome analysis.Cytological brush samples had, on average, only 0.04 M read pairs per sample that could be mapped to the gene catalog. For capsule and fecal samples we obtained low proportions of host DNA and high numbers of read pairs mapping to the gene catalog. The small intestine microbiome profiles from the shotgun metagenomic testing look similar with what we observed from the 16s sequencingresults: Streptococcus (n=192) appears among the top genus in the capsule samples. The high quality non-human reads of the capsules (average 32.06M per sample, or 88.01% of 36.53 Million reads) is comparable with those of the fecal samples (average 37.35M per sample, or 85.25% of 43.74 Million reads) reflected the quality of the data collection with the fiber construct.

[0119] The differences between capsule and fecal sampling were also shown by the concentrations of key Gl metabolites. For example, the concentrations of all SCFAs examined were found to be significantly increased in fecal samples compared to capsules (two-sided Mann-Whitney test p-value range: 1.65 xlO14- 2.29 x 1012). Furthermore, a striking difference was observed between conjugated and deconjugated BAs, which were nearly all exclusively associated with capsule and fecal samples, respectively. The only exception was the presence of the primary cholic acid (CA) in feces, which was most likely the result of production via alternate colonic microbial deconjugation pathways. Together these results confirm that capsules capture a distinct microbiome and metabolomic profile from the SI that is effectively preserved against fecal contamination during Gl transit and performs robustly under different gut motility conditions.

[0120] As a final evaluation of capsule performance as an accurate and reliable SI sampling tool, microbiome composition (using 16S rRNA gene sequencing, for its level of completeness) and metabolomic profiles from a second round of capsule ingestions were compared against gold- standard endoscopic aspirate and cytology brush samples from the duodenum. In addition to fecal samples, which served to demonstrate the effective sealing performance of capsules against potential fecal contamination, saliva samples were also included to assess potential oral contamination of endoscopy sampling.

[0121] Principal co-ordinates analysis of 165 microbiome beta-diversities revealed a noticeable degree of overlap between capsule, endoscopic aspirate and cytological brush samples, particularly on the primary-axis of variation (~28.1% total variance explained). Interestingly, the second axis of variation (~9.9% of total variance explained) also indicated a separation between capsules from a subset of endoscopic and saliva samples, likely reflecting biologically relevant differences between duodenal and ileum microbiome communities resulting from underlying physiological pH gradients (see following section). Further PERMANOVA statistical testing (regressing weighted Unifrac beta diversity against sample type as an ordered factor with capsules as the intercept) did reveal a significant difference in 165 microbial composition between capsules and other sampling methods (p-value ~ 0.001, R2~ 0.41). However when broken down by sample type the majority of variation was contributed by fecal and saliva samples (feces PERMANOVA p-value ~ 0.001, R2~ 0.17; saliva PERMANOVA p-value ~ 0.001,5 R2~ 0.13), while endoscopic aspirate and cytology brush contributed a substantially lower effect-size in terms of proportion of varianceexplained (endoscopic aspirate PERMANOVA p-value ~ 0.001, R2~ 0.048; cytology brush PERMANOVA p-value ~ 0.001, R2~ 0.061). Overall, microbiome profiles captured by capsule and endoscopy were both substantially different to fecal samples and to similar degrees (capsule vs. feces median Unifrac distances ~ 0.538; endoscopic aspirate vs. feces ~ 0.554; cytology brush vs. feces ~ 0.541).

[0122] The analysis of targeted metabolomics profiles also demonstrated that both endoscopic aspirates and capsules recapitulated significant differences in BA and SCFA concentrations in comparison to fecal samples. The presence of conjugated BAs was also found to be exclusive to the SI, with capsules showing no significant differences in concentration compared to gold standard endoscopic aspirates (two-sided Mann-Whitney test FDR adjusted p-values: min = 0.194, max = 0.917, mean ~ 0.5). Deconjugated BAs were again exclusively found in feces, except for cholic acid (CA) which are also detected in the Capsule and Aspirate samples.

[0123] SCFA concentrations were also found to be significantly increased in feces in comparison to both capsule and endoscopic aspirates (two-sided Mann-Whitney test FDR-corrected p-values, endoscopic aspirate vs. feces = 2.72 x 10'9to 1.23 x 10'8), similar to previous results from the X-ray visit. Interestingly, SCFA concentrations were also found to be significantly increased in capsules relative to endoscopic aspirates (two-sided Mann-Whitney test FDR-corrected p-values, capsule vs. endoscopic aspirate = 2.72 x 10'9to 3.84 x 10'6). This result suggests an increasing gradient in SCFA production from proximal SI (endoscopy), distal SI (capsule), and colon (feces), and furthermore implies that SCFA production is not an exclusive metabolic function of the colonic microbiome.

[0124] Additional semi-targeted metabolomics analyses of a panel of 85 metabolites also revealed a strong differentiation between capsules and fecal sample and similarity to endoscopic aspirates, as indicated by hierarchical clustering of metabolite profiles. Given the substantial differences in total spectral abundance between fecal and SI samples (feces vs. capsule and endoscopic aspirate median loglO total spectral abundances: ~7.5 vs. 8.3), spectral counts were normalized using the following procedure to aid comparison normalization by median metabolite spectral count; loglO transformation; and sample-wise auto-correlation / unit scaling of metabolite profiles. Additional K-means clustering of loglO transformed metabolite intensities further revealed markedly different patterns of intensity for different sets of metabolites across sample types. Although the majority of metabolites (endoscopy visit) were generally of high intensity in fecal samples and low in saliva (63 / 85 significantly elevated metabolite concentrations in feces by two- sided Mann-Whitney test FDR adjusted p-value <= 0.05), several were identified (10 / 85) that were significantly increased in capsule samples (two-sided Mann-Whitney test FDR adjusted p-valuerange < 0.04 to 1.5 x 10'9), of which (8 / 10) were also significantly increased in endoscopic aspirates. As expected from the targeted metabolomics analyses, the primary bile-acid glycocholate was identified, as well as several amino-acids (L-Arginine, L-Histidine, and L-Cysteine). Taken together, these results demonstrate that the capsule performs on par with endoscopy in sampling the SI, capturing microbiome profiles and broad metabolic profile distributions that are significantly distinct from feces, and identifying metabolic markers associated with important physiological differences between the SI and colon.

[0125] To understand whether the differences in 16S microbiome composition between capsule and endoscopy samples were due to potential oral contamination, or a biologically meaningful difference in community composition due to differences in SI sampling location, a summary of all unique and shared amplicon sequence variants (ASVs) detected across the distinct sample types was generated, representing the biogeographic distribution of bacteria across the Gl tract.

[0126] Although the vast majority of ASVs detected were unique to fecal samples (256 / 446 ~ 57% of ASVs), followed by saliva samples (46 / 446 ~ 10% of ASVs detected), several intersections of interest were found that revealed insights into the distribution of bacterial genera across the Gl tract. The first intersection of particular interest represented ASVs that were only identified in endoscopic aspirate and cytology brush, and saliva samples (34 / 446 ~ 7% of ASVs detected).Notably, this intersection contained several ASVs annotated to the family Prevotellaceae, a known acid-tolerant bacterium present in the oral microbiome, which were frequently detected (>= 50%) across saliva and endoscopic samples (duodenum), but notably absent in capsules (jejunum + ileum). These results would indicate that the differences between capsule and endoscopy likely reflect biologically relevant regional differences in microbiome composition influenced by proximity of sampling location to the stomach. A second substantial intersection included ASVs identified in capsule and endoscopy and saliva samples (39 / 446 ~ 8%), which were relatively increased in relative abundance and prevalence in endoscopy and saliva compared to capsule. The last intersection of interest contained ASVs identified in all sample types (12 / 446 ~ 2%), of which Streptococcus, a keystone genus of the SI was found to be particularly dominant across capsule, endoscopic aspirate, and cytological brush samples (~30% relative abundance) and reduced in saliva (~10%) and feces (< 1%). Taken together, these results further support that the capsule is capturing a sample representative of the SI, specifically the distal region.

[0127] Example 2

[0128] Implementations of the present disclosure address the lack of tools and methods to study Gl regions that are difficult to access. An example implementation, describedherein, includes a fully autonomous and passive sampling method, , for convenient, high-quality, and reliable sampling to study the diet-microbiota interactions in the SI. The sealing efficacy and microbial DNA preservation capacity of the capsules was first validated through in vitro simulation assays. Then, a clinical study was conducted with 20 healthy participants to validate the in vivo use of the capsules to reliably capture samples for SI microbiome analysis before and after an intervention. Participants ingested the capsules at baseline and 7 days later, with a probiotic capsule containing a blend of L. rhamnosus R0011 and B. longum R0175. Following baseline capsule ingestion, multiple low-dosage x-ray scans were performed to track the sampling location. Fecal samples corresponding with the baseline and intervention capsule were analyzed for comparison. The capsules' performance in vitro demonstrated the potential for contamination-free sampling with preservation of the microbial communities. Within the clinical study, the capsules performed safely and reliably for collection of SI content. X-ray tracking confirmed that 97.2% of the capsules completed sample collection in the SI regions before reaching the colon. Importantly, our data showed that the capsules sampled in the right area of the intestines and that baseline microbiome profile from the capsule collected sample is significantly different from fecal microbiome profile. The capsule successfully detected a concurrent probiotic intervention in the small intestine, which was not detectable using stool samples. The high accuracy of sampling location and sealing efficacy of the capsules makes them potentially useful research tools in clinical trials for studying dietmicrobiota interactions in health and disease.

[0129] This is the first report of spatial and temporal accuracy, including use in humans with and without intervention. In this study, we developed a pH-based autonomous and passive Small Intestine MicroBiome Aspiration capsule. The S capsules have unique features of large sampling ports for reliable sampling volume, strong sample sealing performance and embedded microbial DNA preserving agents to ensure sample quality during the capsule transit time and capsule return process. The results of in vitro simulation assays and clinical study presented herein confirm that the capsules are well-tolerated, minimally invasive capsule that passively captures, seals, and preserves small intestine luminal fluid, providing samples that are suitable for downstream microbiome analysis.

[0130] Methods. The capsule of the example implementation has overall dimensions of 25.4 mm (L) x 8 mm (D), the size of a OOEL capsule. The functional device is contained within a pH sensitive small intestine targeting outer shell. The outer shell is designed to disintegrate at a nearly neutral pH, which is similar to the pH of the proximal small intestine region. The capsule comprises a main body which includes a sampling chamber with sufficient volume (~105 pL) to capture a representative sample of intestinal fluid. The intestinal fluid enters the chamber throughfour evenly spaced ports which are accessed by the fluid only upon disintegration of the outer shell (FIG. 6).

[0131] The ports are radially facing towards the mucosal layer of the small intestine to collect luminal samples near the mucosa. The ports are sufficiently wide to allow easy inflow of the liquid sample. Hydrophilic fibers are placed in the chamber to wick in and retain the liquid sample. On the top of the sampling chamber, a piston is held in the open position by a latch until it is exposed to small intestine fluid and dissolved in a time-controlled manner after the outer shell dissolution. Then, the sampling chamber is closed and sealed with a compressive spring forced by the piston on its top end.

[0132] A preserving agent is embedded within the sampling chamber to maintain the integrity of the collected sample during the capsule transit time in the gut and the capsule return process. The sampling chamber is closed on the lower end by a cap. After retrieval of the device, the cap is easily removed to access the collected sample for downstream processing and analysis.

[0133] In vitro sealing performance and preserving agent efficacy. To assess the sealing performance, 37 capsules without outer shells were submerged in sterile PBS for 4 hours, automatically triggering the sampling and sealing mechanism in the presence of an aqueous solution. 34 capsules were then transferred to a healthy donor fecal slurry spiked with L. rhamnosus R0011 (~109CFU / mL). Out of these 34 capsules, 3 were manually unsealed as a positive contamination control. The 3 remaining capsules were not exposed to the fecal slurry. All the control capsules and the treatment capsules, while being submerged in the fecal slurry, were anaerobically incubated at 37 °C for 72 hours (to simulate the gut transit environment), then at 4 °C for 72 hours (to simulate a cold shipping condition). Then, the capsules were opened, and samples were recovered by pipetting. Contamination was assessed using a strain-specific SYBR Green qPCR assay targeting R0011.

[0134] Another set of 20 capsules were subjected to the same sealing procedure in PBS followed by a 24 h incubation in ROOll-spiked healthy donor fecal slurry. While these capsules remained in the slurry, 8 capsules were frozen at -20 °C for 24 h or 168 h, and 2 capsules were incubated at 4 °C for the same time points (Table 1). After thawing, capsules were removed from the slurry and then samples were retrieved from the capsules by pipetting and contamination was assessed with a strain-specific SYBR Green qPCR assay targeting R0011.

[0135] For preserving agent efficacy testing, fresh SI endoscopic aspirates from 3 patients collected on the same day from the Intestinal Inflammation Tissue Bank (I ITB), University of Calgary were pooled, homogenized and inoculated into 30 capsules containing the embedded preserving agent and 4 capsules without the preserving agent. The filled capsules were then sealedand incubated at 37 °C for 144 hours (6 days) under anaerobic conditions before sample retrieval for DNA extraction.

[0136] DNA extraction and quantification. Once the capsules were received by the lab, the samples were removed from the capsules using a sterile pipette to avoid crosscontamination by the fecal matters attached to the outer surface of the capsule body. The samples from the capsules and their associated control samples collected at various timepoints were stored at -20 °C for up to 7 days until the scheduled DNA extraction. DNA Extraction of the capsule and fecal samples was performed with the Qiagen QIAamp PowerFecal Pro DNA Kit, following the manufacturer's protocol with modifications for the capsule samples. The Qubit dsDNA HS Assay Kit was used with the Qubit® 2.0 Fluorometer to measure the capsule aspirate and fecal sample DNA concentration.

[0137] 16S library preparation and sequencing for in-vitro preserving agent efficacy testing. For the preserving agent efficacy testing, the 16S rRNA gene V4 variable region was amplified using PCR primers with internal barcodes (primers: F: AATGATACGGCGACCACCGAGATCTACAC-barcode-TATGGTAATTGTGTGCCAGCMGCCGCGGTAA, R: CAAGCAGAAGACGGCATACGAGAT-barcode-AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT) in a 35 cycle PCR using the KAPA HiFi HotStart master mix (Roche Sequencing). The conditions for the thermocycler were as follows: 98 °C for 2 minutes, followed by 35 cycles of 98 °C for 30 seconds, 55 °C for 30 seconds and 72 °C for 20 seconds, after which a final elongation step at 72 °C for 7 minutes. Amplified PCR products were checked in a 1 % agarose gel. The PCR products were then purified using NucleoMag NGS Clean-up and Size Select (Macherey-Nagel) and concentrations normalized using SequalPrep Normalization Plate (Invitrogen). Amplicons were pooled and concentration and quality were determined using the Qubit HS DNA kit (Invitrogen) and the Tapestation D1000 assay (Agilent), respectively. Amplicon sequencing was done on a MiSeq Benchtop DNA sequencer (Illumina) using a V2-500 cycle kit (Illumina Inc). The pooled library was then denatured and prepared for loading on an Illumina MiSeq cartridge with a 5% PhiX Control.

[0138] On the morning of the first day (termed the Baseline day), fasted participants visited the X-ray facility and then ingested two capsules simultaneously. After ingesting the capsules, low-dose (70-80kVp), multiple X-ray imaging was used to confirm the timing and location of the capsules. The protocol specifies a scanning interval of approximately 30 minutes, and each volunteer is not permitted to undergo more than 12 scans on the same day. Once capsules were seen finished sampling on an X-ray image, participants were provided with capsule and stool collection kits and discharged. Participants were then allowed to leave the clinic and resume normal activities. Participants were asked to resume normal eating 4 hours after the capsule ingestion andmaintain a stable diet until the second visit. Participants were asked to monitor their stool for the passing of the capsule, and upon excretion they were asked to collect the capsules and a stool sample from the same bowel movement using the provided retrieval kit and return it promptly in an ice box for analysis.

[0139] After at least 5 days, but no more than 21 days following Baseline day, participants returned for a second visit (termed Intervention day) at the Investigators office or a clinic. Participants were required to fast overnight in advance of this visit. In the morning, they first ingested the probiotic capsule (40 billion CFUs, containing a blend of L. rhamnosus R0011 (71%) and B. longum R0175 (29%)) under instruction and then immediately ingested two capsules under supervision. Participants did not undergo X-ray monitoring on their second visit. The participants were allowed to resume normal activities and were allowed to eat 4 hours after the ingestion of the capsules. Participants were provided with stool and capsule collection kits and were instructed again on procedures for capsule return. Upon excretion of the capsule, patients collected the capsules and a stool sample from the same bowel movement and returned it promptly for analysis.

[0140] 16S library preparation and sequencing for Clinical Samples. The 16S targeted amplicon sequencing library was prepared by amplifying 10 pL of each capsule gDNA extracts (or 25 ng for the fecal extracts) with IX KAPA Hi Fi HotStart ReadyMix (Roche, cat # KK2802) and 200 nM universal 16S primers (forward 5'-CCTACGGGNGGCWGCAG-3' and reverse 5'- GACTACHVGGGTATCTAATCC-3') targeting V3-V4 regions in a 25 pL reaction volume . PCR products were visualized on a 2% agarose precast E-Gel stained with SYBR Safe dye (Invitrogen cat # G72080). Amplicons were purified with Agencourt AMPure beads (Beckman Coulter, cat # A63881) following Illumina 16S Metagenomic sequencing library preparation's protocol. A second round of amplification using 5 pL of the purified amplicon PCR reaction as template, 2.5 pL each of Nextera XT V2 primers sets A and D (Illumina, cat # FC-131-2001 and FC-131-2004) and IX KAPA Hi Fi ReadyMix was performed in 25 pL reactions with the same cycling conditions as the Amplicon PCR except that only 8 cycles were used. PCR reactions were again purified with AMPure beads before individual fluorescent quantification by Quant-iT PicoGreen dsDNA assay (Life Technologies, cat # P7589). Volumes corresponding to 100 ng of each purified Index PCR reaction were pooled using an EpMotion 5075 liquid handling robot (Eppendorf) and this pool was quantified with QuBit Broad Range assay (ThermoScientific, cat # Q32853) following manufacturer's instructions. This pool was also quality controlled for the presence of the desired amplicon (size obtained 630 bp) and the absence of secondary amplification by running a High Sensitivity D1000 TapeStation assay (Agilent, cat # 5067-5584 / 5585). Library was denatured with 0.2 N NaOH and loaded at 8 pM with 5 % PhiX(Illumina, cat # FC-110-3001) on an Illumina MiSeq instrument using MiSeq V3 Reagent Kit (Illumina, cat # MS- 102-3003) for 2x 301 cycles.

[0141] Microbiome analysis. The demultiplexed fastq sequences were imported into QIIME2 (Quantitative Insight Into Microbial Ecology-2) as artefacts and inspected for overall quality (visual inspection of the q-Scores per base plots). The reads were determined to be very high quality on the 40nt->280nt for the forward and 40nt->260nt for the reverse reads. These parameters were used to denoise the paired reads using the Dada2 denoiser (as a QIIME2 encapsulated version). Hence, the reads were clustered into amplicon sequence variants (ASVs). The feature classifier was used to attribute the ASVs to the closest known taxa using QIIME2's taxonomic classification module (linking ASV sequences to known bacterial groups). The taxonomy file was trained on a 99% clustered Silva_138 taxonomic database (V3-V4 subregion of the 16S). The ASV tables were exported as Level-6 (Genus Level) relative abundance tables for downstream analysis.

[0142] The 'core-metrics' module from QIIME2 was also used to generate the Alpha Diversity measures and the PCoA distance matrices. The alpha diversity algorithms include Pielou (Evenness), Faith (Phylogenetic Distance) and Shannon Entropy. For the PCoA, the Weighted UniFrac algorithm was used. The PCoA was viewed interactively with the Emperor module through the QI IM E 2 viewing server (https: / / view.qiime2.org / ) and a collection of images was captured for later reporting. The PCoA and diversity figures were calculated on ASV tables and rooted tree (phylogenetic relation between observed ASV sequences).

[0143] To determine group differences between the treatment groups, QIIME2's encapsulated Machine Learning Sample Classifier was used with the ExtraTreesClassifier algorithm. In order to assess the presence or absence of differences between treatment groups (or Capsules V. Stool samples), the algorithm trains on 2 / 3 of the sample's taxonomic tables at genus level (Training Set) and then test its predictive power on the remaining 1 / 3 of the samples from each group (Testing Set). If the algorithm is able to tell the samples from each other for the Label of interest (the variable used to make the groups, ex. Stool V. Capsule samples), then the groups are determined to be different. The accuracy results of the Test Set results are presented as confusion matrices, with the main classification indicator being the Final Accuracy.

[0144] The Capsule can effectively seal and protect collected samples. The goal of our in vitro validation experiments was to assess the possibility of contamination occurring after completion of the sample collection and sealing of the capsule, simulating the conditions of capsule transit through the Gl tract and potential shipping and storage conditions. For the first experimental design, there was no contamination in 29 out of the 31 test capsules as shown by the absence of detection of L. rhamnosus R0011 in the capsules' cargo. The 2 samples with positive detection ofR0011 are the result of one obstructed capsule preventing it from completely closing (which precludes from assessing sealing efficacy) and one capsule that incidentally touched the biosafety cabinet during the sample removal process, the cargo coming into contact with the outside of the capsule. Therefore, the detection of R0011 in these 2 samples was not due to failure of the seal and were excluded from the sealing efficacy evaluation (Table 1). Importantly, no target bacteria were detected in the negative controls, and R0011 was detected between 104and 105in the contamination positive controls (i.e., manually unsealed before immersion in the contaminating slurry).

[0145] In the second set of sealing efficacy testing (Table 1), all 16 frozen samples were negative for R0011 after thawing, regardless of the time spent at -20 °C. This temperature was tested to ensure that the spring-based mechanism and sealing capacity of the capsules were able to withstand freezing stress causing an expansion of the cargo. The 4 samples kept at 4 °C were also negative for R0011. Overall, the sealing efficacy of the capsule was conservatively evaluated at 2 positives / 51 tested capsules (96.1 % efficacy) (Table 1).

[0146] Table 1:

[0147] Most of the 16S microbiome sequencing profiles of the endoscopic aspirate samples stored in the capsules with the preserving agent (Group P, N=30) are similar to the TO control (FIG. 7A), while the 4 samples without the preserving agent (Group NP, N=4) are characterized by a notable dominance of Pasteurellaceae. The absence of preservative allows for theovergrowth of some species, which changes the relative abundance in the sample. Among samples with the preserving agent, 5 / 30 capsules showed a variable amount of staphylococcus contamination after 6 days at 37 °C. After removal of Staphylococci by bioinformatics filtering, the 5 samples displayed a 16S profile similar to the other samples and TO (FIG. 7B).

[0148] The Capsule Performs Safely and Reliably for Collection of Small Intestine Luminal Fluid in vivo. The clinical study results are summarized in Table 2. This single arm study enrolled 20 healthy volunteers (mean age (±SD): 38 ± 11, range 18-58 years; mean BMI 25.6 ± 4.7; 12 females and 8 males) recruited at the Foothills Hospital in Calgary Alberta. The capsules were well tolerated by participants (N = 20), with no adverse events or capsule retention, and only 5 out of the 40 incidences (2 capsule retrievals by each of the 20 participants) where participants judged the first-time retrieval of the capsules from stool in the baseline round was "difficult", 18 / 40 "Neutral", 13 / 40 "easy", 4 / 40 "very easy", and 1 / 40 "Lost". In the probiotic round, the difficulty of capsule retrieval was reported as follows: 5 / 40 "difficult", 11 / 40 "Neutral", 18 / 40 "easy", 3 / 40 "very easy", and 1 / 40 "Lost". Overall, 78 / 80 (97.5%) capsules (39 baseline + 39 probiotic intervention) were retrieved by participants, after a median number of 2 stools (range 1-7) and with total median transit time (ingestion to expulsion) of 30 hours (IQR 23-48). The two missing capsules were established as lost in feces in two participants through follow-up x-ray scans confirming capsule clearance.

[0149] Table 2:

[0150] Baseline capsules were monitored by X-ray (2 capsules per participant) following ingestion to determine the sampling time and region. FIG. 8A and 8B show examples of the full abdominal X-ray images taken from one participant at two different timepoints. FIG. 8C highlights the region of interest (ROI) in which the two capsules were still open (sampling); FIG. 8D shows the ROI in which the two capsules were closed (sampling completed). A radiopaque marker was attached on one of the two capsules ingested together in order to distinguish the two capsules in the X-ray images. In total, 35 / 40 (87.5%) capsules were confirmed as having completed sample collection in the targeted region of the small intestine. The only failed case was a capsule that was seen completing collection in the stomach before reaching the small intestine. Four capsules were classified as indeterminate due to the X-ray scanning frequency or the overall duration of the X-ray scanning period. Of these four capsules, two were still in the stomach at the end of the X-ray schedule, although sampling collection was not completed. Two other capsules were last seen open in the small intestine in one X-ray scan and were first seen closed on the next scan but had already reached the colon. If these four cases were excluded from the sampling location analysis, 35 / 36 (97.2 %) capsules were observed collecting samples in the small intestine before reaching the colon, with 1 / 36 (2.8 %) completing its collection while still in the stomach. Overall, 38 / 40 capsules completed sample collection within 210 minutes following ingestion (IQR 150-180 minutes).

[0151] The mean sample weight per capsule was 89 ± 1 mg (range 15-130 mg). In total, 87.2% (68 / 78) of the capsules collected more than 20 mg of sample, which is the threshold weight we set to assess the sample collection efficacy. However, the sample collection rate increased to 97.5% (39 / 40) by ingestion under the current dual-capsule ingestion protocol in this study. DNA extraction was performed on all 38 / 38 baseline capsule samples (1 baseline capsule and 1 post-intervention capsule samples were lost in feces), 28 / 40 post-intervention capsule samples and all fecal samples. 66 samples, including at least one from each ingestion time point, were allocated for 165 sequencing and a remaining 12 capsule samples were stored at -80°C for future analysis. 65 / 66 capsules allocated for 165 sequencing had sufficient DNA of suitable quality for downstream analyses. In the 100 pL of the DNA elution, the median DNA concentration of baseline small intestine sample collected from the capsules was 0.058 ng / pL (IQR 0.039 - 0.082 ng / pL). The median DNA amount of post-intervention small intestine sample collected from the capsules was 0.557 ng / pl (IQR 0.153 - 1.41 ng / pl), which represents the high concentration of probiotics in the capsules. In contrast, the fecal samples contain much more concentrated DNA: median 561.13 ng / pL (IQR 491.28-688.34 ng / pL) for the baseline fecal DNA concentration and 629.20 ng / pL (IQR 519.38- 698.11 ng / pL) for post-intervention fecal DNA concentration.

[0152] Microbiota composition is different between small intestinal capsule samples and stool samples. Samples were analysed using a PCoA with an unsupervised Weighted UniFrac algorithm revealing 3 main clusters based on spatial disposition (FIG. 9A). The orange (Baseline) and green (Probiotics) samples are concentrated on the left-center of the PCoA space and correspond to stool samples. The capsule samples are located on the right side and separated along the PC2 axis, suggesting group differences between stool samples and capsules, and between Capsules-Probiotics (red, bottom-right) and the Capsules-Baseline (blue, top-right). These differences are also visible on the grouped taxonomic bar plots (FIG. 9B), where the apparent difference between Capsules- Baseline and Capsules-Probiotics is caused by the high number of Lactobacilli and Bifidobacteria from the probiotic co-ingestion. Indeed, the removal of Lactobacilli and Bifidobacteria from the grouped bar plot analysis by bioinformatic filtering increased the similarity between the Capsules- Probiotics and the Capsules-Baseline in a manner similar to stool samples.

[0153] As expected for samples originating from the small intestine, alpha diversity measures were lower in the capsules compared to the stool samples, while the Capsules-Probiotics showed an even lower diversity than the Capsules-Baseline that is most likely due to the presence of high amounts of probiotic Lactobacilli and Bifidobacteria.

[0154] Comparisons between groups by machine learning confirms the difference between the capsule and stool samples

[0155] The overall differences between the stool and capsule samples were also assessed by machine learning group comparisons (FIG. 10A) with 3 comparisons. The first comparison (Comp 1) included all the stool vs. capsules samples without consideration for the probiotic intervention. The very high final accuracy at 100% means that from training on genus-level taxonomic tables, the algorithm was always able to distinguish between the stool and capsules samples (i.e. the group differences were clear and the capsules are consistently different from the stool samples) (FIG. 10B). In comparison 2 (Comp 2), a final accuracy of 45% (accuracy ratio of 0.83) means that the algorithm could not distinguish between stool-baseline or stool-probiotics very efficiently (FIG. 10C). In comparison 3 (Comp 3), a final accuracy of 100 % shows that the algorithm was able to clearly distinguish between the capsule-baseline and capsule-probiotics (FIG. 10D).

[0156] The main group classifiers are representative of their sampling region. Among the main classifiers identified by the machine learning algorithm (comparing Baseline stool samples versus Baseline capsule samples), several taxa previously associated with the SI microbiome were identified as enriched in the capsules while taxa known to be associated with the colon were enriched in the stool samples (FIG. 11). For example, Streptococcus, Veillonella, Actinomyces,Gemella and TM7x were enriched in the capsule baseline sample, while Bacteroides, Blautia, Faecalibacterium, Dorea and Anaerostipes were enriched in the stool baseline samples.

[0157] Discussion. The study introduced the Small Intestine MicroBiome Aspiration capsule, a minimally invasive device for collecting small intestine luminal fluid for microbiome analysis. The capsules will allow to address a critical research gap in gastrointestinal microbiome studies, where the upper gastrointestinal tract's unique characteristics are often overlooked due to the challenges of accessing and sampling this region.

[0158] In vitro, the capsules displayed an excellent sealing efficacy under conditions mimicking those encountered during a clinical trial (i.e., gut transit at 37 °C in fecal slurry and storage / shipping at 4 °C or -20 °C). For the preserving agent efficacy, our in vitro testing was very challenging, designed as a worst-case scenario with 6 days at 37 °C after manipulations for manual inoculations that are an unusual capsule usage. We observed that only 5 capsules out of 30 displayed a variable amount of staphylococcus contamination. However, it was clear with the negative controls that the preserving agent was successful at maintaining the community architecture of most samples in the absence of an external contamination. In a real-life setting, the capsule parts inside of their pH-sensitive outer shell are sterile and undergo DNA removal process during the manufacturing process. In the current experimental setting, it is likely that the capsules were contaminated with various amounts of Staphylococci during the inoculation or extraction of the cargo by pipetting, before the 16S amplification.

[0159] In the clinical study, the capsule showed a remarkable ability to collect samples from the small intestine using the multiple X-ray tracking method, 97.2% (35 / 36) completing sample collection in the targeted region. The companion X-ray tracking method provides a relatively affordable and minimally invasive approach to validate the timing and location of sampling with our passive sampling capsule technology.

[0160] Microbiome analysis revealed significant differences between the small intestine microbiome profiles obtained with the small intestinal and fecal microbiome profiles. This highlights the importance of directly sampling the small intestine for a more accurate understanding of diet-microbiota interactions. This technology possesses the potential to transform our comprehension of the gut microbiome and its implications in health and disease. Furthermore, it facilitates interventional studies for monitoring both the immediate and prolonged impacts on the small intestine microbiome resulting from various medications and nutraceutical products, including prebiotics, probiotics, and postbiotics. The capsule captured the co-ingested intervention, here a probiotic, which took a lot of space in the capsule and appeared clearly in the bar plots of the microbiome analysis. This is interesting because probiotic interventions are reputably difficult tomonitor after one dose using stool samples; this study proved that the probiotic bacteria reached the small intestine. In future probiotics studies, care should be given to allow enough time for washout of the probiotic bolus before ingesting the capsules in order to study the effects of repeated doses on the SI microbiome. We have succeeded in removing the probiotic signal by filtering out all the Bifidobacteria and Lactobacilli, but this is an artificial analysis shown here as a proof-of-concept and the validity of using this approach to further analyze microbiome composition after filtering the samples should be determined.

[0161] When analyzing the baseline capsule and stool samples, there was a clear difference in microbiome composition between the 2 sampling locations, and the main genera enriched in the capsule converge with the published microbiome composition of small intestine endoscopy samples. There is still a lack of direct comparison with small intestine samples collected by other means, which is a limitation of this study. As the next step, samples collected from the capsules will be compared against endoscopic aspirate samples.

[0162] The capsules may offer a minimally invasive alternative to endoscopic aspiration, making it more comfortable for study participants while providing comprehensive spatial representation along the gastrointestinal tract. Its embedded preservative agent ensures the retention of time-stamped microbiome snapshots, crucial for studying dynamic effect of a concurrent intervention to the small intestine microbiome, either pharmaceutical or dietary.

[0163] By addressing the need for minimally invasive devices to collect, seal and preserve the small intestine sample, the capsule opens new avenues for research into various digestive conditions, such as small intestine bacterial overgrowth, irritable bowel syndrome, obesity, metabolic diseases, and cancer.

[0164] Although the subject matter has been described in language specific to structural features and / or methodological acts, it is to be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts described above are disclosed as example forms of implementing the claims.

[0165] Example 3 - Sample Preservation

[0166] Implementations of the ingestible apparatus described herein enable direct microbiome sampling from the small intestine while preserving the microbiome composition during gastrointestinal (G I) transit with a functional substance. An example implementation's performance was assessed using a defined microbial consortium and human duodenal aspirate samples under simulated in vivo conditions (anaerobic, body-temperature environments) to evaluate the device's ability to preserve genomic profiles over six days at body temperature. Devices with a stabilizingagent effectively inhibited microbial growth, while those without stabilizer showed bacterial proliferation. DNA analysis via qPCR confirmed that the stabilizer-maintained DNA quantity and quality comparable to baseline, unlike non-stabilized samples, which exhibited increased microbial replication. Alpha diversity metrics (Chaol, Inverse Simpson, and Shannon indices) demonstrated stable within-sample diversity in stabilized capsules, with no significant loss over six days, whereas diversity declined significantly in non-stabilized samples. Beta diversity analysis (Bray-Curtis distances) showed that stabilized samples retained microbial compositions closely resembling baseline profiles, unlike non-stabilized samples, which experienced significant shifts. These findings confirm that capsules with stabilizer effectively maintain microbiome composition and DNA integrity during up to six days of Gl transit, offering a reliable tool for small intestine microbiome sampling and metagenomic analysis.

[0167] Methods and Results. The Altered Schaedler Flora (ASF), a model community of eight microorganisms representing G I microflora, was used to assess the stabilizer's effectiveness in inhibiting bacterial growth. The cultivated bacterial strains were used to create an ASF multi-strain consortium for inoculation and tested their stability using the devices.

[0168] The ASF strains were successfully cultivated, normalized, and inoculated into the capsules. Cell densities ranged from 1.33 x 105to 3.57 x 1011CFU / ml before normalization. No additional bacterial growth was observed in the capsules (4 capsules per each time point) with stabilizer during the simulated Gl transit condition across all time points (24, 72, and 144 hours). In contrast, the capsules (2 capsules per each time point) without stabilizer showed bacterial growth, confirming the stabilizer's inhibitory effect

[0169] Endoscopic aspirate sample, collected from the duodenum region of participants, were pooled and inoculated into the ingestible apparatus and microtubes, which were incubated at 37°C (body temperature) under anaerobic condition for up to 144 hours (N=30, 6 days). Sequencing of the 16S rRNA gene was performed using Illumina MiSeq at the International Microbiome Centre at the University of Calgary.

[0170] Taxonomic barplots illustrated a stark contrast in taxonomic composition between stabilized (N = 31) and unstabilized (N = 4) device samples after 6 days of simulated Gl transit (Figure 15). The taxonomic profiles of device samples with stabilizer were dominated by known SI genera (e.g. Streptoccus, Veillonella, Gamella and Rothia) and were highly correlated by relative abundance to the baseline reference (N = 1) endoscopic aspirate sample (median Spearman correlation to baseline ~ 0.71, p-value ~ 2.03 x 10'55), while unstabilized samples failed to preserve the composition of the baseline sample (average Spearman correlation to baseline ~ 0.29, p-value ~ 0.0029). Four capsule samples with stabilizer were also found to be dominated by Caminibacter (>25% by relative abundance) which was determined as a contaminant introduced during the PCR barcoding stage of 16S V4 sequencing and was not as the result of stabilizer failure. Removal of Caminibacter revealed that the taxonomic profiles of outliers were similar to uncontaminated samples, further confirming the effectiveness of the stabilizer at maintaining a representative taxonomic profile of the SI during Gl transit conditions. Caminibacter was removed from all subsequent analyses.

[0171] FIG. 12 illustrates 16S V4 region taxonomic barplots summarized by top 5 bacterial genera by relative abundance and grouped by experimental condition: Aspirate at Time 0 (Baseline) (N = 1); Treatments -Device With Stabilizer (N = 53), Device Without Stabilizer (N = 4) incubated at 37°C for 6 days simulated Gl transit; Negative Sequencing Controls (NC - water). Top Panel - Unfiltered samples with Caminibacter contamination (yellow bars) indicated with red asterisk; Bottom Panel - Taxa barplots with Caminibacter removed and sample relative abundances recalculated.

[0172] Device samples with stabilizer also showed comparable microbial alpha diversity to baseline, while unstabilized samples showed a significant loss in species diversity (Figure 16; Device stabilized vs. unstabilized Mann- Whitney test P-values 0.004, 0 .014 and 0.011, respectively).

[0173] FIG. 13 illustrates Alpha diversity metrics of baseline (endoscopic aspirate reference sample at time 0) compared against device under simulated Gl transit conditions (6 days incubation at 37°C) - with and without stabilizer. Brackets above boxplots indicate the respective significance of Mann-Whitney tests with *'s indicating exponential degree of the calculated test p- value against an alpha of 0.05. Note - Baseline represents a single sample and statistical testing was not applicable.

[0174] An examination of microbiome compositional differences between device samples with and without stabilizer treatment was performed. PCoA ordination of between-sample Bray-Curtis distances showed that capsule samples with stabilizer clustered with the baseline reference sample and showed a clear and significant separation in microbiome community composition to samples without stabilizer.

[0175] FIG. 14 illustrates a PCoA plot summary of Bray-Curtis distances (beta diversity) for baseline aspirate samples (red point) and aspirate incubated at 37°C for 6 days in device with (green points) and without stabilizer (blue points).

[0176] The example implementation with functional substance stabilizes microbial diversity and composition, maintaining profiles representative of the original sample during Gl transit, and limiting bacterial growth for up to 6 days at 37°C. Samples with stabilizer maintainedmicrobial diversity, while those without showed shifts in composition. The data underscore the importance of the capsule's stabilization performance for accurate microbiome profiling, with capsules without stabilizer exhibiting significant loss of fidelity to the original microbiome composition.

[0177] Example 4 - Fiber Placement

[0178] Fibers placed within the sampling chamber enable fluid to wick into the sampling chamber to overcome the fluid tension at the port opening. However, the fibers cannot be positioned within the port opening without impacting sealing. The fiber placement within the sampling chamber, placed below the level of the sealing surface remains sensitive to the relative distance from the opening and can be controlled.

[0179] Methods. A total of 10 ingestible apparatus were prepared with a preweighed fiber bundled positioned either at 1 mm from the sealing surface (n=5) or 5 mm from the sealing surface (n=5). The devices were placed in a container with fluid for 2 minutes and then removed. The fiber was removed from the device and weighed. The fiber weight and any fluid remaining in the container was combined for a total fluid collection volume (1 ug = 1 mL). A minimum threshold of 20 mg was considered to be a collection pass criteria.

[0180] Results. Table 1'. Capsules with Fibers positioned at 1 mm below the sealing surface

[0181] The positioning of the fiber bundled within the sampling chamber relative to the port opening was found to impact the amount of fluid collected by the ingestible apparatus.

[0182] Example 5 - Sample removal without contamination

[0183] The sample removal procedure for the capsule was validated to confirm that the process effectively minimizes contamination. The validation employed quantitative PCR (qPCR) to measure contamination levels and assess amplification significance. The amplification is considered significant when the quantification cycle (Cq) is less than 35 (Cq<35) and insignificant when the Cq is greater than 35 (Cq>35). Using Absolute qPCR of R0011 Lacticaseibacil I us rhamnosus which contains 5 copies of the gene of interest per cell, the validation determined a significant threshold at which there was at least 5 copies / reaction.

[0184] Methods. A total of 27 test articles were activated within a sterile PBS dissolution for 3 hours at 37C to fill the sampling chamber with sterile PBS. The articles were then incubated in contaminating agent formed of an overnight culture of R0011 Lacticaseibacillus rhamnosus mixed with feces in a 1:1 ratio at 37C for 72 hours and then an additional 4C for 24 hours. The test articles were then cleaned and disinfected and test articles opened by cutting the housing open at the groove via a set of clippers to enables ready access to the absorbent fibers. The fibers were removed via sterile tweezers to remove the fiber construct in addition to a rinse with 100 ul rinse with Milli-Q H20. DNA Extraction done using Qiagen Power Fecal Pro DNA Kit (PFP50 011) and qPCR performed using p201 / pl370 primer set. Multiple controls, including positive, negative, build, and no-template controls (NTCs), were implemented to monitor contamination throughout DNA extraction and qPCR analysis. Specifically, four positive controls were subjected to a PBS dissolution for 3 hours at 37C, incubation in contaminating agent at 37C for 72 hours, incubation in contaminating agent at 4C for 24 hours, capsules were rinsed with water (30seconds) and then the sample was removed. Four negative controls were subject to a PBS dissolution for 3 hours at 37C, incubation in sterile PBS at 37C for 72 hours, incubation in sterile PBS at 4C for 24 hours, capsules were rinsed with water (30seconds) and then the sample was removed. In addition, two build controls where the capsule was sealed using the screwdriver, capsule was opened and twist cap was washed with Milli-Q water and the fiber removed. Four DNA extraction Kit controls to control for contamination during the DNA extraction and four NTCs to control for contamination during qPCR.

[0185] Results. The validation results demonstrated that the sample removal procedure was effective and reproducible. The fibers could be easily removed via the method of cutting the test articles at the groove. There was no evidence of contamination in the kit controls, capsule build contamination controls, the negative controls, the no template controls via 16S relative quantification qPCR or the R0011 absolute quantification qPCF indicated by the absence of an amplification curve on the Cycle vs. ARn graph. There was clear evidence of contamination in the positive controls with mean Cqs of 23.3 ,26.0,22.7 and 25.6. All but one test articles passed the 16s relative quantification failure with no significant amplification detected. The one failure detected at 34.6 Cq, slightly less than the cut off of 35 Cq. Two of the three failures of the 1 test articles in the R011 absolute quantification were deemed to have been contaminated during extraction process as there was no associated contamination detected during the 16s amplification. The remaining sample failed only one of three replicates and was granted a conditional pass.

[0186] Expected amplification was observed in the positive control capsules, confirming the sensitivity and accuracy of the qPCR system. The consistent and reliable Cq values across the test samples provide strong evidence that the method of sample removal effectively limits contamination during sample removal. The controlled performance across all test conditions supports the robustness and reliability of the validated procedure.

[0187] Example 6: Sample timing Implementations of the present disclosure include methods of scheduling capsule delivery. X-ray tracking of the capsule (over 150 cases), shows that due to its small size, most of the capsules can consistently pass through the stomach and enter the small intestine within one hour (FIG. 15). After this, its sampling time, even if passive, can accurately complete sampling approximately three hours after ingestion.

[0188] Based on these observations, implementations of the present disclosure include methods of scheduling the capsule intake for different scenarios, such as ingestion in nonfasted states to detect different types of food interventions. For example, to detect intervention in the small intestine in a non-fasted state, as analyzed in FIG. 16, the method can include configuring the capsule to be ingested 60 minutes before a liquid intervention. This allows the capsule to pass through the stomach and enter the small intestine before the intervention, and then slowly move within the small intestine for about 120 to 150 minutes, waiting for the liquid intervention to enter the small intestine and sample it from the small intestine contents.

Claims

WHAT IS CLAIMED:

1. An ingestible apparatus for collecting a sample from a target area of a gastrointestinal (Gl) tract of a subject, the ingestible apparatus comprising: a housing defining a sample compartment having a first portion and a second portion, the housing comprising one or more access ports formed therein, and the sample compartment being communicable with the one or more access ports; a functional substance arranged in the second portion of the sample compartment; and a separation layer arranged between the first portion and the second portion of the sample compartment, the separation layer being configured to change at least one property in response to interaction with Gl fluid.

2. The ingestible apparatus of claim 1, wherein the separation layer is configured to change the at least one property by dissolving in response to interaction with Gl fluid.

3. The ingestible apparatus of claim 1, wherein the separation layer is configured to change the at least one property by changing permeability in response to interaction with Gl fluid.

4. The ingestible apparatus of claim 2 or claim 3, wherein, in response to the separation layer changing the at least one property, the functional substance is configured to move from the second portion of the sample compartment to the first portion of the sample compartment.

5. The ingestible apparatus of claim 1, wherein the separation layer is a polymer.

6. The ingestible apparatus of any one of claims 1-5, wherein the functional substance is in a solid state.

7. The ingestible apparatus of any one of claims 1-6, wherein the functional substance is a preserving substance.

8. The ingestible apparatus of any one of claims 1-7, further comprising an absorbent material arranged in the first portion of the sample compartment.

9. The ingestible apparatus of claim 8, wherein the separation layer is disposed between the absorbent material and the functional substance.

10. The ingestible apparatus of claim 8 or claim 9, wherein the absorbent material is sized and / or shaped such that the absorbent material cannot exit the housing via the one or more access ports.

11. The ingestible apparatus of any one of claims 8-10, wherein the absorbent material is an organized presentation of fiber.

12. The ingestible apparatus of claim 11, wherein the organized presentation of fiber is configured to increase a surface area of the absorbent material in proximity to a sealing surface of the housing.

13. The ingestible apparatus of claim 11 or claim 12, wherein the organized presentation of fiber is an assembly of fiber.

14. The ingestible apparatus of claim 13, wherein the assembly of fiber comprises a plurality of loops.

15. The ingestible apparatus of claim 14, wherein the plurality of loops is between about 5 and 100 loops.

16. The ingestible apparatus of claim 13, wherein the assembly of fiber is a ball of fiber.

17. The ingestible apparatus of claim 11 or claim 12, wherein the organized presentation of fiber comprises one or more loops, balls, knots, braids, or brushes.

18. The ingestible apparatus of any one of claims 11-17, wherein the organized presentation of fiber is sized and / or shaped such that no portion of the organized presentation of fiber can exit the housing via the one or more access ports.

19. The ingestible apparatus of any one of claims 8-18, wherein the absorbent material is spaced apart from the functional substance.

20. The ingestible apparatus of any one of claims 8-18, wherein the separation layer is configured to prevent the absorbent material from contacting the functional substance before the separation layer changes the at least one property.

21. The ingestible apparatus of claim 20, wherein the absorbent material contacts the separation layer.

22. The ingestible apparatus of claim 21, wherein the absorbent material is attached to or partially embedded with the separation layer.

23. The ingestible apparatus of claim 20, wherein the absorbent material does not contact the separation layer.

24. The ingestible apparatus of any one of claims 20-23, wherein the absorbent material defines a first surface adjacent to the separation layer and a second surface opposite to the first surface, the separation layer being arranged in the sample compartment such that the second surface is spaced about 1 millimeter (mm) from a sealing surface of the housing.

25. The ingestible apparatus of any one of claims 1-24, wherein the one or more access ports are located in a port region of the housing, the housing further comprising a plurality of access ports and a plurality of struts in the port region of the housing, each of the plurality of struts being arranged between adjacent access ports of the plurality of access ports.

26. The ingestible apparatus of claim 25, wherein a surface area ratio of the plurality of access ports to the plurality of struts in the port region of the housing is greater than about 50%.

27. The ingestible apparatus of claim 25 or claim 26, wherein the housing further comprises a sealing surface, and wherein the port region of the housing is spaced apart from the sealing surface.

28. The ingestible apparatus of any one of claims 1-27, further comprising an actuation assembly that is movable between a first working position where the sample compartment is open and a second working position where the sample compartment is closed.

29. The ingestible apparatus of claim 28, wherein the actuation assembly comprises a compression spring, wherein the compression spring is in a first compressed state in the first working position and a second compressed state in the second working position.

30. The ingestible apparatus of claim 28, wherein the actuation assembly comprises an extension spring, wherein the extension spring is in a first extension state in the first working position and a second extension state in the second working position.

31. The ingestible apparatus of claim 29 or claim 30, wherein the actuation assembly further comprises a piston.

32. The ingestible apparatus of any one of claims 28-31, further comprising a stopper attached to the actuation assembly, wherein the stopper is configured to seal the sample compartment in the second working position.

33. The ingestible apparatus of claim 32, wherein the housing further comprises a sealing surface, and wherein the stopper is configured to seal against the sealing surface.

34. The ingestible apparatus of claim 28, further comprising a crosslinked block configured to prevent the actuation assembly from moving between the first working position and the second working position until the ingestible apparatus is within the target area of the Gl tract of the subject.

35. The ingestible apparatus of claim 34, wherein the crosslinked block has an undeformed state and a deformed state, wherein the crosslinked block is configured to hold the actuation assembly in the first working position in the undeformed state, and wherein the crosslinked block is configured to release the actuation assembly to the second working position in the deformed state.

36. The ingestible apparatus of claim 35, wherein the crosslinked block is a disc having a central hole, wherein the actuation assembly comprises a piston and a spring, wherein a first diameter of the central hole in the undeformed state is configured to engage the piston to hold the actuation assembly in the first working position, and wherein a second diameter of the central hole in the deformed state is configured to disengage the piston to release the actuation assembly to the second working position.

37. The ingestible apparatus of claim 35 or claim 36, wherein the crosslinked block is configured to gradually transition from the undeformed state to the deformed state in response to interaction with Gl fluid.

38. The ingestible apparatus of claim 37, wherein the actuation assembly is configured to rapidly transition from the first working position to the second working position when released by the crosslinked block.

39. The ingestible apparatus of any one of claims 35-38, wherein the undeformed state is an unexpanded state and the deformed state is an expanded state.

40. The ingestible apparatus of any one of claims 34-39, wherein the crosslinked block is porous.

41. The ingestible apparatus of any one of claims 1-40, further comprising a groove disposed on an exterior surface of the housing.

42. The ingestible apparatus of claim 41, wherein the groove is positioned at about the same height along the exterior surface of the housing as the separation layer is positioned within the sample compartment.

43. An ingestible apparatus for collecting a sample from a target area of a gastrointestinal (Gl) tract of a subject, the ingestible apparatus comprising: a housing defining a sample compartment, the housing comprising one or more access ports formed therein, and the sample compartment being communicable with the one or more access ports; and an absorbent material arranged in the sample compartment, wherein the absorbent material is an organized presentation of fiber.

44. The ingestible apparatus of claim 43, wherein the organized presentation of fiber is configured to increase a surface area of the absorbent material in proximity to a sealing surface of the housing.

45. The ingestible apparatus of claim 43 or claim 44, wherein the organized presentation of fiber is an assembly of fiber.

46. The ingestible apparatus of claim 45, wherein the assembly of fiber comprises a plurality of loops.

47. The ingestible apparatus of claim 46, wherein the plurality of loops is between about 5 and 100 loops.

48. The ingestible apparatus of claim 45, wherein the assembly of fiber is a ball of fiber.

49. The ingestible apparatus of claim 43 or claim 44, wherein the organized presentation of fiber comprises one or more loops, balls, knots, braids, or brushes.

50. A method of intestinal analysis comprising: administering an ingestible apparatus for collecting a sample from a target area of a gastrointestinal (Gl) tract of a subject, the ingestible apparatus comprising: a housing defining a sample compartment having a first portion and a second portion, the housing comprising one or more access ports formed therein, and the sample compartment being communicable with the one or more access ports; a functional substance arranged in the second portion of the sample compartment; and a separation layer arranged between the first portion and the second portion of the sample compartment, the separation layer being configured to change at least one property in response to interaction with Gl fluid; analyzing a Gl fluid collected from the target area of the Gl tract to create an intestinal profile, wherein analyzing the Gl fluid comprises sequencing a microbiome and wherein the intestinal profile comprises a report of a microbiome constituent of the Gl fluid.

51. The method of claim 50, wherein sequencing includes metagenomic, genomic, metabolomic, proteomic, lipodomic and the profile can include a representation of a microbial metagenome, genome, metabolome, proteome, lipodome or any combination thereof.

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