WO2023091792A1

WO2023091792A1 - Robotic pill system for biomarker sampling in body cavities

Info

Publication number: WO2023091792A1
Application number: PCT/US2022/050746
Authority: WO
Inventors: Fernando Soto; Utkan Demirci; Demir Akin
Original assignee: The Board Of Trustees Of The Leland Stanford Junior University
Priority date: 2021-11-22
Filing date: 2022-11-22
Publication date: 2023-05-25

Abstract

An untethered robotic pill system for collecting biomarkers from human and animal body cavities includes (i) a robotic pill adapted to collect a biofluid containing biomarkers as the robotic pill travels through a human cavity and (ii) a motive system adapted to perform at least one of directing a position of the robotic pill and maintaining the position of the robotic pill within the human cavity. The robotic pill can collect biomarkers such as extracellular vesicles at a particular position or as it travels through the body cavity or cavities. In this way, the robotic pill and untethered robotic pill system can be used to collect useful spatially-specific biomarkers and be used to map a gastrointestinal or genitourinary or respiratory tract. Such a system may be used to map the secretome of these locations, such as gut, and may be used in targeted therapies, such as molecularly- or immunologically-targeted therapies.

Description

ROBOTIC PILL SYSTEM FOR BIOMARKER SAMPLING IN BODY CAVITIES

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/281,926 entitled "Robotic Pill System for Biomarker Sampling in Body Cavities" filed November 22, 2021, the contents of which are incorporated by reference herein in its entirety for all purposes.

STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] Not applicable.

TECHNICAL FIELD

[0003] This disclosure relates to the collection of biological samples, in liquid or solid or mixed form, from body cavities, such as gastrointestinal tract, genitourinary track, peritoneal and pleural cavities or respiratory track, and vasculature.

BACKGROUND

[0004] Health relevant information is coded in circulating signatures or biomarkers, such as exosomes, genetic materials, biome, proteome, secretome, and other markers. The collection and analysis of such biomarkers can serve as an early sign of disease initiation and its progression thereafter. Nevertheless, it remains challenging to isolate and/or sample biomarkers from regions of the body with limited accessibility, such as the gastrointestinal tract, peritoneal cavity, lungs, urogenital track and spine. In this direction, invasive medical procedures such as surgery, biopsies, and colonoscopies, are required to obtain biomarkers from hard-to-reach regions. These techniques involve trained practitioners, expensive instruments, and specialized facilities, making them hard to expand to large populations. Moreover, both endoscopy and colonoscopy can only reach so far into certain cavities, such as the gastrointestinal tract, which is more than 30 feet in length.

[0005] On the other hand, minimally-invasive approaches can also be used for sampling biomarkers. For instance, liquid biopsy is a standard approach to obtain biomarkers from biofluids (blood, urine, or stool samples). Although useful, it provides indirect information, averaged data of the sample, and is not specific to a particular body cavity. Smart pills have also been deployed to sample biomarkers inside the body. Nevertheless, they isolate fluid at one point, which might result in limited capture of low abundance biomarkers.

[0006] The ability to isolate biomarkers with high resolution, quantity and specificity while aiming for a minimally-invasive dynamic sampling system could help discover new potential signals of disease at early stages and monitor health status by allowing generation of comprehensive datasets or knowledge about parameters involved in the shift from health to disease states and vice versa.

SUMMARY

[0007] Techniques, devices, and systems are disclosed herein for collecting biological samples, including but not limited to fluids, tissues, chemicals, molecules, and other targets from the body cavities such as gastrointestinal tract. The devices / robotic pill can collect, isolate and/or enrich biomarkers (exosomes, genetic materials, biome, protein/peptide-based biomarkers, carbohydrates, lipids, secretome, and other markers of health and disease states) directly, near the source of generation, over prolonged periods of time, while being retained in the collection site by self-produced or externally generated mechanical force. The device includes a passive locomotion mechanism or a propulsion engine that enables the locomotion and retention of the device at a point of interest over the desired period, an enrichment and storage module that allows the collection of different biomarkers (including but not limited to small and large molecules, such as proteins, peptides, amino acids, DNA, RNA, exosomes, extracellular vesicles, bacteria, immune cells, viruses, circulating cancer cells, ions, pharmaceuticals) in a collection matrix while letting the biofluid pass through it, and an opening/actuation mechanism that controls the biofluid/biomarker access to the enrichment and storage module. The cargo collected by the pill can be further analyzed in detail by downstream processes such as comprehensive omics analysis methods for example proteomics, genomics, metabolomics and lipidomics.

[0008] The robotic pill device can be used as an individual unit or placed in a magnetic holder filled with multiple collection cartridges aimed at sampling at different locations serially or in parallel and either programmed or externally or internally controlled durations. This magnetic holder can be controlled by constant or changing magnetic fields enabling retention or locomotion of the cartridge in a specific location. After the device collects the biofluid, biomarkers can be enriched and isolated using diverse techniques (centrifugation, filtering, immunocapture), enabling identification of the changes in their concentration.

[0009] The system described here can serve for the discovery, and detection of diseases including but not limited to genetic, allergic, infectious, metabolic diseases or disruptions of the normal flora of the cavities, such as irritable bowel syndrome, pancreatic cancer, colon cancer, microbiome imbalance, nutrient malabsorption, among many others. Moreover, it could provide information regarding an individual metabolism, secretome, biome or composition of the relevant locations and cavities mentioned above.

[0010] The device can also be used to monitor the functional status of transplants, the immune response and rejection in transplant applications such as small bowel transplant and other Gl tract related surgeries.

[0011] According to one aspect, an untethered robotic pill system for collecting biomarkers and other targets of interest from human and animal body cavities and liquid resources is disclosed. The system includes a robotic pill and a motive system. The robotic pill is adapted to collect a biofluid containing biomarkers when the robotic pill is received or introduced or taken through a human orifice, other natural or surgical openings and cavity. The motive system is adapted to perform at least one of directing a position of the robotic pill within the human cavity and maintaining the position of the robotic pill within the human cavity or sample site. [0012] In some forms, the robotic pill may be adapted to collect and isolate a target of interest in a gastrointestinal tract and includes an opening adapted to receive a biofluid or a sample or a tissue specimen or cells from the human cavity and further includes a collection module configured to draw the biofluid across one or more membranes from the opening toward the collection module within the robotic pill in which the membranes divide an interior of the robotic pill into various chambers.

[0013] In some forms, the robotic pill may include a frame. The frame may support a magnet, the magnet being part of the motive system of the robotic pill. The frame may be composed of a deformable material and the magnet may be received within the deformable material of the frame with the elasticity of the deformable material maintaining the magnet within the frame.

[0014] In some forms, the frame may support a metasponge material for the collection of the biomarkers, in which the metasponge comprising an absorbent material and an elastomer support matrix. The absorbent material may be entangled within a mesh of the elastomer matrix. In one specific form, the absorbent material may be sodium polyacrylate. The metasponge may be tuned to selectively sample in the gastrointestinal tract as the metasponge provides limited absorption or growth in acidic environment and comparably higher absorption or growth in neutral environments.

[0015] In some forms, the robotic pill may include a collection chamber for collecting the biofluid containing biomarkers. The collection chamber may include a single large container or multiple enrichment sites inside a base, each containing their opening and venting independent of the others. The collection chamber may comprise a metasponge including absorbent material and an elastomer support matrix. In some forms, the system may be activated autonomously to provide access to the collection chamber in response to environmental changes or external signals. For example, the environmental changes may include changes in pH of the environment (as may occur, for example, while traversing a gastrointestinal tract). In some forms, an interior of the collection chamber may include a swelling component that absorbs liquid, swells up, and blocks the further fluid entrance. In some forms, the robotic pill includes a functionalized surface to collect biomarkers.

[0016] According to another aspect, a method is disclosed of using an untethered robotic pill system to analyze a human or animal cavity. This may be any of the pills and systems described above or herein involving any or all workable features or combination of features. The robotic pill is received in the human or animal cavity or in a tract leading to these cavities. The robotic pill is directed through the human or animal cavity, optionally to one or more specific positions. Biofluids, samples, tissues, cells, and other materials are collected from the human or animal cavity within the robotic pill (or assembly of several of them).

[0017] In some forms, the tract may be a gastrointestinal tract and the step of receiving the pill may involve administering the robotic pill through ingestion. The method may further include the step of recovering the robotic pill of the system from excretion. [0018] In some forms, after the collecting step, the biofluids, samples, tissues, cells, and other materials may be analyzed.

[0019] In some forms, during the directing step, the robotic pill may be maintained at a position of interest in the human or animal cavity to collect the biofluids, samples, tissues, cells, and other materials at the position of interest.

[0020] These and still other advantages of the invention will be apparent from the detailed description and drawings. What follows is merely a description of some preferred embodiments of the present invention. To assess the full scope of the invention, the claims should be looked to as these preferred embodiments are not intended to be the only embodiments within the scope of the claims.

BRIEF DESCRIPTION OF THE FIGURES

[0021] FIG. 1 illustrates an exemplary engine module in the form of a magnet for retention of the robotic pill in a point of interest for prolonged sampling, isolation and enrichment of biomarkers, signaling molecules, indicators of health and disease states.

[0022] FIG. 2A shows a photograph of a robotic pill for locomotion and retention.

[0023] FIG. 2B is a scheme and photographs of locomotion mechanism based on response to an external magnetic field.

[0024] FIG. 2C is a time-lapse image of directed locomotion of robotic pill.

[0025] FIGS. 2D, 2E, and 2F illustrate the retention of robotic pill under flow conditions illustrated by photographs (FIG. 2D), displacement (FIG. 2E), and speed quantification of pill placed under constant fluid flow in a small intestine tubing model (FIG. 2F) in the presence of no magnetic field (i) and magnetic field (ii).

[0026] FIG. 2G provides photographs showing some different possible orientations to align a robotic pill by rotating an external magnetic field in the presence of constant fluid flow.

[0027] FIG. 2H is a schematic illustrating the use of an external magnetic field to localize the robotic pill in a specific small intestine location.

[0028] FIG. 21 is a photograph depicting a robotic pill next to a pig's small intestine.

[0029] FIG. 2J is a photograph illustrating the robotic pill inside the pig's intestine. [0030] FIG. 2K is a photograph illustrating the robotic pill being retained in a specific location within a pig's small intestine with fluid flow.

[0031] FIG. 3 shows examples of different types of enrichment modules, including labeled capture and physical-based isolation.

[0032] FIG. 4A provides a schematic and images of a swelling-based enrichment module before and after capture the capture.

[0033] FIG. 4B illustrates micrographs showing 2 pm microparticles trapped by and recovered from the collector from different components of the pills, (i) control, (ii) plastic structure, (iii) porous membrane, and (iv) absorbent material.

[0034] FIG. 4C shows the quantification of particles retained at the stages described in FIG. 4F with reference to the i-iv designations in the previous sentence.

[0035] FIG. 4D shows colorimetric assay quantification of captured BSA Protein by robotic pill based on different release protocols, including under release by (a) NaCa2 solution and shaking and (b) only shaking.

[0036] FIG. 4E illustrates a scheme of robotic pill capture and retention of bacteria.

[0037] FIG. 4F shows the quantification E. coli growth after isolation (inset photograph of capture and transfer of E. coli bacteria using pill collector).

[0038] FIG. 5 illustrates three examples of opening / actuation modules including degradable material, shrinking, and a micropump controller.

[0039] FIG. 6A illustrates a scheme of opening / actuation modules based on porous membrane coated with pH sensitive degradable polymers.

[0040] FIG. 6B shows fluorescent micrographs of i) only porous membrane, ii) porous membrane pores filled with enteric coating containing fluorescent dye, iii) porous membrane pores filled with enteric coating containing fluorescent dye after 1 hour incubation in gastric simulant (pH 2), and iv) porous membrane pores filled with enteric coating containing fluorescent dye after 1 hour incubation in buffer (pH 7.4).

[0041] FIG. 6C shows the quantification described in the micrographs in FIG. 6B with reference to the i-iv designations in the previous figure description. [0042] FIG 6D shows a comparison of absorbent-loaded robotic pills with coating and without coating after sequential incubation in acidic pH and neutral pH solutions containing dye solution.

[0043] FIG. 6E shows the quantification of captured fluorescent dye described in FIG 6D.

[0044] FIG. 7 illustrates a stretchable and sellable material matrix (that is, a "metasponge" structure) for integration into the robotic pill design towards blocking the collection chamber and sampling and transporting liquid samples and, more specifically, shows the reversible size change of a material composed of sodium polyacrylate (absorbent) and stretchable matrix (elastomer), the material grows in volume (3x times) as it absorbs liquid sample.

[0045] FIG. 8A is a kinetics graph illustrating the growth of a metasponge over time after incubation in water in which the center solid line is an average and the grey line provides the bounds of three individual experiments.

[0046] FIG. 8B illustrates the reversible swelling and deswelling of metasponge structure during multiple dehydration cycle (n=5).

[0047] FIG. 8C shows the tunable swelling of metasponge by adjusting the elastomer to absorbent material ratio.

[0048] FIG. 8D shows the effect of salt concentration on metasponge swelling under distinct types of salt and molar concentrations after a 1-hour incubation.

[0049] FIG. 8E illustrates the effect of temperature on metasponge swelling after a time of 30 minutes.

[0050] FIG. 9A provides photographs of mechanical testing performed on dry and swollen metasponge films. The white scale bar is 20 mm.

[0051] FIG. 9B provides, at its top panel, a scheme illustrating an optical fiber input-output setup for light guiding using metasponge and, below that, photographs of experiments using [i] elastomer only, [ii] dry metasponge (original), and [iii] fully swollen metasponge fibers.

[0052] FIG. 9C is a plot showing the optical output power transmitted through metasponge fibers under different experimental conditions (n=3) .

[0053] FIG. 9D illustrates the transformation of a star-shaped metasponge actuator based on the original state (left panel) and swollen state (right panel). [0054] FIG. 9E provides a scheme of different components of robocar in which the metasponge material forms part of the wheels of the robocar. The car is actuated by an external rotating magnetic field.

[0055] FIG. 9F illustrates the use of metasponge wheels to sample solution (acid solution) in a collection chamber followed by transport to detection chamber where the solution changes color based on pH. The black scale bar is 5 mm.

[0056] FIG. 10A shows the applicability of this metasponge material as a time delayed growing plug illustrating i) y microfluidic chip moving blue-colored water through one entrance and two exits and ii) blocking a bottom exit thus encapsulating fluid in the collection chamber.

[0057] FIG. 10B is a schematic of a metasponge being used for selective sampling in the gut, with the inset graph in the upper right corner illustrating the change in weight of the metasponge after incubation in different pH (n=5) .

[0058] FIG. 10C shows the use of a metasponge for sampling bacteria, illustrated by a schematic and a confocal image projection showing bacteria internalization into the metasponge's interior.

[0059] FIG. 10D shows the use of a metasponge to isolate hemoglobin from a liquid environment, hemoglobin is a relevant marker for gastrointestinal (Gl) disease with the left panel being before incubation and the right panel being after incubation.

[0060] FIG. 10E illustrates that, after sampling, the metasponge can be integrated with typical sensing modalities, including as specifically illustrated lateral flow based fecal immunochemical tests.

[0061] FIG. 10F illustrates that, after sampling, the metasponge can be integrated with other typical sensing modalities, such as and as specifically illustrated, commercial electronic, optical sensors.

[0062] FIG. 10G illustrates that, after sampling, the metasponge can be integrated with still other typical sensing modalities, including colorimetric assays, such as Guaiac oxidation, as specifically illustrated. DETAILED DESCRIPTION

[0063] Disclosed herein is a robotic pill for sample collection as the device travels through the remote areas of the gastrointestinal tract and other body's cavities. The robotic pill can collect and investigate, for example, extracellular vesicles, among other biomarkers, as the robotic pill travels through the gastrointestinal tract. This collected information can be used to generate a map or atlas of the gastrointestinal tract secretome as a function of spatial and temporal locations. Once a healthy or normal gastrointestinal tract is characterized, this robotic pill could also be used as a diagnostics tool to look for signals that might reflect an unhealthy gastrointestinal tract (for example, from food poisoning, cancer nodules, a benign development, and so forth) or be used to monitoring a treatment or surgery (for example, monitoring the health of a transplanted small bowel without a full biopsy).

[0064] While much of the description herein describes the use of a robotic pill of this type as being used in the gastrointestinal tract, it is contemplated that this robotic pill might be adapted to navigate other body tracts and pathways such as airflow pathways or portions of the circulatory system and collect extracelluar vesicles or secretomes in other bodily contexts. In the more general context, the robotic pill might be thought of as a small-scale robotic device. This robotic pill can be integrated with downstream proteomic and genomic analysis to investigate and decipher the collected samples and signals.

[0065] "On-site" sampling and collection platforms aimed for novel extracellular vesicle markers may offer more precise detection of disease. In particular, this may be especially helpful for the early detection and diagnosis of gastrointestinal tract diseases that have been a challenge for clinicians and researchers, as most cases are asymptomatic until advanced stages. When diagnosing a gastrointestinal-related health problem, the first clinical analysis commonly relies on indirect monitoring of blood and stool samples. Anemia or blood in stool could indicate bacterial infection, micro bleeding in the walls of the gastrointestinal tract, or gastric cancer. However, fecal sampling does not provide complete information for the diagnosis due to sample intermixing through the downstream gastrointestinal environment, where environmental markers and microorganisms vary significantly. The ability to map and provide accurate identification of relevant biomarker populations and their relative abundance in the gut with higher resolution and specificity stands to expand our understanding of relevant gastrointestinal biomarkers and their relationship with a patient's health.

[0066] The use of prolonged sampling periods using the ingestible robotic pill with diverse built-in collection mechanism (e.g., mechanical pump, osmotic gradients, passive absorption) may enhance selective sampling and collection of gastrointestinal biomarkers, when compared to a single collection time point. The collection mechanism can be integrated into the system and controlled from outside, for example, in the case of a mechanical pump. A deployable, active sampling robotic pill collector platform can offer in vivo gastrointestinal sample collection assays. Comprehensive analysis of the gastrointestinal biofluid can serve to establish libraries of gut biomarkers, or bleeding indications. This new approach can provide a robust test for routine gastric measurements in the clinical practice towards early detection of disease.

[0067] The robotic pill can be fabricated in large-scale batches using diverse methods such as laser cutting, 3D stereolithography, 3D printing, micromachining, micromolding, stamping, laser cutting, directed assembly, and injection molding. The modular microstructure design method can employ distinct modules to target and isolate specific biomarkers. The collector module can contain a series of interconnected chambers with different sized porous membranes, thus enabling sequential sample collection with minimal cross-contamination between sampling pockets. The opening of the robotic pill can be controlled using a built-in delay activation mechanism based on FDA-approved polymeric sustained release or targeted delivery formulation such as enteric coatings composed of poly(meth)acrylate. For instance, the opening of the sampling robotic pill can be coated with the enteric coating, which will start dissolving under specific environmental pH conditions found in the different sections of gut, thus inducing the autonomous opening of the robotic pill in different target sections of the gastrointestinal tract (the duodenum, jejunum, and ileum). Moreover, the triggered opening of the robotic pill collection chamber can be induced by applying external fields (for example, ultrasound, electrostatic forces, electrical and electromagnetic fields).

[0068] A passive or active RFID tag with integrated circuits or a magnetically or elementally encoded barcode can serve to locate and relate the robot pill inside a patient's body and record other relevant parameters such as temperature and location, and its relation to human health. [0069] The foremost broader impact of this approach is the potential to reduce the barriers to access of analytical platforms into the gastrointestinal tract and address challenges related to the cost and discomfort associated with endoscopy and endoscopy preparative procedures. Although biomarker collection is a focus, the robotic pill sampler platform may translate to other application areas, such as the study of pharmaceutical concentrations in the body to evaluate and monitor treatment adherence and efficacy. It is envisioned that this new approach can provide a robust test for routine gastric and enteric measurements in the clinical practice towards early disease detection.

[0070] The platform offers an innovative approach to examine several fundamental cellular characteristics spanning numerous biomedical and biotechnology applications. The simplicity, small size-scale and versatility of the robotic pill design make the system compatible with mobile devices for telemedicine, for screening and diagnosis of infectious diseases in resourcelimited settings, and offers an easy to set-up and use system for biological, biomedical or clinical laboratories.

[0071] In one form, an untethered robotic pill system for collecting samples and/or biomarkers from human cavities is disclosed herein. The system can include an apparatus that places and retains the robotic pill position at a target point of interest over desired time periods as depicted for example in FIG. 1. External fields (for example, light, ultrasound, or magnetic fields) can induce a retention mechanism, or local chemical reaction (chemical gradient, bubble generation) that drives the device to convert those types of energy into mechanical motion. [0072] For example, an exemplary pill is shown in FIG. 2A, in which a polymeric frame has a magnet supported therein and which frame also provides a number of chambers that could support collection materials (some of which will be described below and herein) for collecting fluids during use. FIGS. 2B through 2C, in particular, show the use of the magnet in this pill structure to effectuate a "walking" movement of the pill-like structure. By manipulating a magnet external to the pill, such as by rotation of a magnet as stepwise depicted in FIG. 2B, the magnet in the frame of the pill can be caused to flip taking the frame of the pill with it and creating the walking-like action. When the magnetic field is repeatedly applied, it can provide continuing directional movement and, when the field is differentially applied with respect to direction, it can result in movement of the pill in a non-linear, but still directed fashion. See, for example, the time lapse results of FIG. 2C.

[0073] Although the application of a varying magnetic field can result in motive action as depicted, the magnet and an applied magnetic field could also be used to retain the robotic pill within a portion of the Gl tract for a period of time or at another static position. Such retention of a pill in place is demonstrated in FIGS. 2D through 2F (as well as schematically in FIG. 1). In FIG. 2D, a pill with a magnet in a frame is placed in tube under flow conditions. In the top panels of FIG. 2D [indicated by (i)], no external magnet is present and the pill simply flows left to right within the tube. In the bottom panels of FIG. 2D [indicated by (ii)], an external magnet is present outside the tube that positionally captures the pill at the position of the magnet, even as flow within the tube continues around the pill. The differential results are illustrated in FIGS. 2E and 2F respectively, which shows how, when the magnet is present, the pill is able to be positionally captured and no longer displaces over time (see FIG. 2E) and how the velocity stops (see FIG. 2F).

[0074] As a variation to this demonstration of pill capture, FIG. 2G shows that by rotating the external magnet (compare the N-S pole arrangement in the left and right panels of FIG. 2G), the pill may be orientated differently in place.

[0075] Those having skill in the art will appreciate that the sizing of magnets or strength of magnetic field may need to be varied to obtain the desired effect. Nonetheless, this demonstrates that the positioning of a pill by magnetic field is certainly possible.

[0076] With reference to the schematic of FIG. 2H, it is contemplated that this magnetic pill locating scheme can be used to locate the robotic pill in a specific gastrointestinal location by location of a magnet relative to Gl tract or intestine. This is further evidenced by the sequence of figures that follow - specifically, FIGS. 21, 2J and 2K - which illustrate the pill next to a small intestine of a pig for scale in FIG. 21 and then within the intestine in FIG. 2J. Under simulated flow as depicted in FIG. 2K, a magnet is illustrated as retaining the pill in position against that flow. See for example, the bulge in FIG. 2K with the dashed white line depicting the rough location of the robotic pill.

[0077] The system can further include a microfluidic and biomarker collection container or enrichment module (cartridge) in which the biomarkers can be enriched in a trapping matrix

- In while letting the biofluid pass through it. The designs can include, but are not limited, to diverse types of shapes and procedures, individual or multiple flat microfluidic chips and three- dimensional containers. The capture of biomarkers can result from swellable materials, filter structures and functionalized surfaces as depicted, for example, in FIGS. 3 and 4A-4F. FIG. 3 depicts swelling based, filter based and functionalized surfaced based capture of biomarkers. In the swelling based concept (top of FIG. 3), a swelling agent can expand or swell upon the entrance of biofluid to effectively trap the biomarkers within the collection space. In the filterbased design (center of FIG. 3), a biofluid may flow through the collection space and one or more filters or intermediate membranes may capture the biomarkers within the collection space or divisions thereof, with not all biomarkers able to pass through some or all of the filters. In a functional surface type design (bottom of FIG. 3), a functionalized surface may retain biomarkers of interest at binding sites. In all instances, after collection, the collected biomarkers may eventually be recoverable and/or analyzable in order to investigate the biomarkers collected.

[0078] The system can further include an opening/actuation module that controls the collection and retention of surrounding biofluid by actuation of device components, for instance, either by opening/closing of the chamber, mechanical pumps, pressure gradients, swelling materials or osmotic gradients. See FIGS. 5 and FIG. 6A. In the top panels of FIG. 5, for example, a degradable material may break down to provide initial access to the collection chamber. In the middle panels of FIG. 5, the collection chamber could be structured to shrink under certain conditions and expand in others. In such case, the mechanical adjustment of shrinking or expanding may result in opening or closing of an opening to the collection chamber. And as indicated in the lowermost panel of FIG. 5, the collection chamber could involve one or more micropumps to bring fluids into or expel fluids out of one or more collection chambers.

[0079] In the case of FIG. 6A, a schematic is provided illustrating porous membranes coated with pH sensitive degradable polymers. In the upper panels, in which the pill is received in an acid pH material, the membrane may not be readily dissolved and so access to the collection chamber can be limited. In contrast, in the lower panels, a neutral pH environment may provide the ability for biomarkers to enter the channel. [0080] FIG. 6B shows fluorescent micrographs of i) only porous membrane, ii) porous membrane pores filled with enteric coating containing fluorescent dye, iii) porous membrane pores filled with enteric coating containing fluorescent dye after 1 hour incubation in gastric simulant (pH 2), and iv) porous membrane pores filled with enteric coating containing fluorescent dye after 1 hour incubation in buffer (pH 7.4). This may also be considered in conjunction with FIG. 10B below, which shows that within a Gl environment, such pH may vary in this way. FIG. 6C shows the quantification described in the micrographs in FIG. 6B with reference to the i-iv designations in FIG. 6D. FIG 6D shows a comparison of absorbent-loaded robotic pills with coating and without coating after sequential incubation in acidic pH and neutral pH solutions containing dye solution and FIG. 6E shows the quantification of captured fluorescent dye described in FIG 6D.

[0081] Moreover, the robotic pill can integrate swelling and stretchable materials to serve as sampling reservoirs or as blocking agents. The device size or various components of the device can actively change size to be able to travel through various terrain.

[0082] For instance, a material based sampling method involving a metamaterial hydrogel or "metasponge" can be employed. As illustrated in FIG. 7A, which shows both the structure and expansion, such a "metasponge" material can operate by integrating the properties of an absorbent hydrogel (sodium polyacrylate) within a highly stretchable and robust elastomer (Ecoflex® obtainable from BASF of Ludwigshafen, Germany). Such a metasponge can be fabricated by mixing two main components: a dry sodium polyacrylate powder (which may be obtained from Carolina Scientific) and uncured liquid elastomer (Ecoflex® 00-30, Smooth). The Ecoflex liquid solution is prepared by mixing a base and a curing agent in equal volumes. The powder is added to the mixed Ecoflex solution and mixed to generate a paste. The resulting paste was poured over a mold and let in a vacuum pump for one minute, followed by incubation in an oven at 65 °C to dry for 10 minutes to cure. This step can be done in a sequential manner to add an extra metasponges or elastomer layer. The molds can be fabricated using a 3D printer (ANYCUBIC-Photon) or by engraving a clear cast acrylic sheet (McMaster-Carr) using a laser cuter (Versa Laser). In certain cases, color pigments can be included in the metasponge or elastomer matrix for contrast purposes. [0083] When the metasponge is submerged in an aqueous solution, the absorbent material swells within the elastomer matrix resulting in volumetric expansion. Such absorption leads to sampling liquid solution and storing it in the stable matrix of the metasponge. The elastomeric matrix enables the metasponge structure to withstand mechanical deformation while retaining hydrogel proprieties (for example, water retention and diffusivity). Furthermore, active regions composed of absorbent/elastomer can be spatially distributed within a passive support matrix (elastomer only, non-swelling), resulting in asymmetric swelling behaviors.

[0084] A paste-like material results from mixing the absorbent material (sodium polyacrylate, dry powder) and an elastomer support matrix (Ecoflex®, uncured liquid). The paste can be molded or extruded into the desired shape. The metasponge grows in volume as the sodium polyacrylate polymeric chains uncoil by absorbing water molecules, creating a wetting network that propagates through the core of the metasponge. The absorbent material is entangled within the mesh of the elastomer matrix; thus, there is non-significant leaking or release of the passive material from the metasponge. In addition, the elastomer matrix provides mechanical robustness to the metasponge compared to a pure hydrogel structure and enables it to expand compared to other stiff matrixes, such as Polydimethylsiloxane (PDMS). [0085] Metasponge growth under different environmental conditions was studied in detail. A metasponge rectangular cuboid (3.5mm*3.5mm*1.5mm) was used as a standard with equal absorbent and support matrix material ratio by volume unless otherwise stated. First, as illustrated in FIG. 8A, the swelling kinetics of the metasponge was evaluated by measuring the growth of individual structures (measuring ratio between final length L between Lo initial length) during overnight incubation. The metasponge presented a rapid growth profile during the first hour, almost doubling in length, followed by slower growth during its plateau for 12 hours, resulting in a 3.5-fold increase in size. The inner material surface wetted slower than the outer surface, causing the plateau. The metasponge swelling process is reversible when left dry overnight or in an oven to dehydrate the sample. The metasponge structure retained swelling capabilities - and therefore potential sampling capability - for multiple cycles measured as depicted in FIG. 8B which shows a progressive growth from dry to wet of 3.5-fold, followed by a comparable decrease. [0086] As illustrated by the data collected and presented in FIG. 8C, the metasponge liquid sampling capabilities can be engineered by tuning the ratio between the absorbent and matrix materials. Growth upon absorption might be tuned from just over 1 to a 4.5-fold increase in size or potentially more.

[0087] As illustrated in FIG. 8D, the metasponge had different sampling profiles (that is, exhibited different growth) in different ionic environments. This can lead to targeted sampling at biofluids with specific ionic environments. The metasponge grew differently under ionic environments (NaCI, CaCI₂, FeCI₃, NbCI₃) and molar concentrations. (10^-3, 10^-2, 10¹ and 10° M). Ions present in the solution can compete with water molecules to interact with the negatively charged carboxylic groups from the absorbent material. The presence of positively charged ions with a higher charge (Fe³⁺ and Nb³⁺) hindered the growth of the material more significantly when compared to lower charge cations (Na⁺ and Ca²⁺). Moreover, the molarity of the salt concentration played a significant role in the swelling of the metasponge structure. For instance, at low relative ionic strength (10^-3M), the metasponge structure presented similar growth to water. In contrast, in high ionic strength (IM), the metasponge structure did not present any significant growth compared to its original size.

[0088] Still further and as illustrated in FIG. 8E, the sampling behavior of the metasponge can be accelerated by the function of temperature. The metasponge was incubated under different fluid temperatures (4, 23, 40 °C) for 30 minutes. The metasponge grew at different rates based on the incubation temperate as follows: 2.7-fold at a high temperature (40 °C), 2- fold at room temperature (23 °C) and 1.4-fold at a cold temperature (4 °C). The combination of a higher temperature led to an increased diffusion rate of water molecules into the metasponge matrix driven by the faster movement of the solvent molecules and the change in mechanical proprieties of the polymer matrix resulting in enhanced growth

[0089] The mechanical and physical properties of metasponge can change depending on its original and swollen state. As depicted in the photographs of FIG. 9A, the metasponge can sustain constant mechanical strain and deformation, including stretching and torsion, in both original and swollen conditions indicating its resiliency for medical applications.

[0090] It is observed that, in its original form, the metasponge is opaque and does not allow light to travel through. Its swollen state is translucent and allows light to travel from the input to the output port. Thus, the metasponge can be used to sample biofluids and analyze them optically by effectively guiding light as an optical metasponge fiber, as shown in the photographs in FIG. 9B and quantification graph of FIG. 9C. From FIGS. 9B and 9C, fibers made of only elastomer (indicated by "i") or dry metasponge (indicated by "ii") did not present guidance, where the readout sensor only detected environmental noise. However, the swollen fiber (indicated by "iii") was able to transmit light, providing an output signal. The light-guiding capability of the metasponge optical fibers from these figures was quantified using a light source (0SL2 Fiber Illuminator, Thorlabs of Newton, NJ) and a power meter/spectrometer collection setup (Thorlabs). The light was illuminated through an objective (lOOx) and the illumination angle was controlled with an angular stage. The incident angle was changed to get a maximum light transfer through the fiber. The light was illuminated on one end of the metasponge fiber, and transmitted light was measured on the end of the metasponge fiber using a power meter (Thorlabs).

[0091] It is contemplated in some instances that the engineered growth of the metasponge can serve as an actuation mechanism for physical sampling method. For example and with reference being had to FIG. 9D, a star-shaped sampling device was designed using a bottom layer of passive material with active top material. The metasponge materials swelled, while the passive support served as a resistor, generating a spring-like muscle that bent the arms of the star while they dynamically swelled, resulting in a fully closed sphere-like structure that served to capture targets in its interior.

[0092] The use of metasponge collectors can be integrated with locomotion. For example, a robocar (see FIG. 9E) in which the wheels were composed of the metasponge material was, as depicted in FIG. 9F, guided to a reservoir containing an acidic solution (pH 2) and was left sampling for 1 minute. Next, the robocar was guided to a second reservoir containing a pH- responsive colorimetric solution. The absorbed acidic solution was transferred to the assay reservoirs changing its color from yellow to pink.

[0093] It is contemplated that while the example above illustrates the use of the metasponge in a robocar, that a metasponge might be integrated into the robotic pills as described above, in which those pills have other motive or position retaining means, such as magnets or magnetic fields, or can be driven at least in part by the environment itself (such as, for example, by the action of a digestive tract).

[0094] Metasponges can also serve as autonomous volumetric flow control valves that close after a specific desired volume passes through the chamber. For example, FIG. 10A shows the applicability of this material as a time delayed growing plug illustrating, in the left panel, y microfluidic chip moving blue-colored water through one entrance and two exits and, in the right panel blocking bottom exit thus encapsulating fluid in the collection chamber.

[0095] Another potential use of the metasponge relies on biomarker sampling in the body. With reference being made to FIG. 10B, it is also contemplated that the metasponge can selectively sample in the gastrointestinal tract as it has limited growth in an acid environment but not in physiological environments. Looking at the leftmost panel of FIG. 10B, the metasponge is schematically shown in relatively acidic and relatively normal pH regions of the intestine. In high acidity regions, the metasponge intakes a comparably low amount of fluid as compared to less acid and relatively neutral regions.

[0096] As proof of concept, metasponges were mixed with nutrients in a solution containing fluorescent E. coli and incubated. After one day of incubation, the metasponge was cross-sectioned and imaged using a confocal microscope. During incubation, metasponge squares were incubated in an E. coli (ATTC, 25922GFP) solution and left incubated overnight. The metasponge was sliced in half and imaged using a confocal microscope to study the internalization of the bacteria into the gel. Looking at FIG. 10C, this image illustrates that bacteria can migrate to the metasponge interior serving as a sampling collector, as shown by the cross-section of a metasponge using a confocal microscope interior. It was also found that metasponges produced containing nutrients (2:1:1, elastomer, sodium polyacrylate, LB broth powder), presented higher internalization when compared to bare metasponge.

[0097] As yet another proof of concept, and with reference being made to FIG. 10D, the metasponge was used to isolate hemoglobin from a liquid environment, with hemoglobin being a relevant marker for Gl disease. The metasponges were incubated in hemoglobin solution for 1 hour and a colorimetric solution used to detect the recovery of hemoglobin from the solution. The hemoglobin sampling was validated using via a commercial electronic sensor and lateral flow immunoassay. [0098] As generally indicated in FIGS. 10E, 10F, and 10G, after sampling, the metasponge can be integrated with typical sensing modalities, including lateral flow based fecal immunochemical tests (FIG. 10E), commercial electronic, optical sensors (FIG. 10F) and colorimetric assays, such as Guaiac oxidation (FIG. 10G). In the fecal immunochemical tests of FIG. 10D, the samples of metasponge plus hemoglobin (indicated by "i", lower left panel) provided positive tests while the samples of metasponge plus buffer (indicated by "ii", lower right panel) provided negative tests. In the optical sensor tests of FIG. 10F, it is demonstrated that the hemoglobin (Hgb) in solution corresponded closely with the hemoglobin recovered in the metasponge. Finally, FIG. 10G provides qualitative photographs illustrating the hemoglobin capture efficiency after incubation of a metasponge in Guaiac solution. For the guaiac procedure, samples were placed in a mixture of guaiac gum powder with 200 proof ethanol and 3% hydrogen peroxide. Germaine Laboratories' AimStrip® Hb Hemoglobin Meter was used to detect hemoglobin concentrations and controls were measured against Germaine Laboratories' AimStrip Hb Control Set.

[0099] It is contemplated that the pill can collect and deliver samples with active pumping inwards and outwards via auto actuation or actuation by an external trigger. The pill can be integrated with various valving mechanisms enabling collection from multiple locations in the gastrointestinal and genitourinary tract. This ability to collect sample in multiple locations can be integrated with imaging and spatial location of the pill as a function of its travel and location through the tract allowing the system to create an ATLAS and map of samples collected though the tract matching the collected secretome and samples with the specific anatomical location in the travelled tract. The travel of the device through the tract can be manipulated by external or internal magnetic or acoustic or electronic means allowing the device to travel on precise paths. Internal paths can include the device being integrated with acoustic sensors and actuators including piezoelectric transducers, capacitive micromachined ultrasonic transducers or piezoelectric micromachined ultrasonic transducers.

[00100] In some forms, the system can be administered through ingestion or implanted in the body or delivered through the natural orifices, openings or via the surgically created laparoscopic holes, cannulas, tubes and likes.

[00101] In some forms, the robotic pill of the system can be recovered from body excretion. [00102] In some forms, the system can include a sensing module to send information in realtime upon detection of desired analyte or biomarker.

[00103] In some forms, the system can collect biofluid dynamically in different locations based on mechanical actuation or time delay opening of the microfluidic container.

[00104] In some forms, the system can be activated autonomously to open the enrichment module in response to environmental changes or external signals.

[00105] In some forms, the system can be actuated to move the robotic pill to desired locations under external control or driven to that location by itself by following biological gradients within the body cavities.

[00106] In some forms, the system retains the robotic pill in a specific area using its mechanical force to overcome fluid force, gut motion, or other forces to collect for more extended periods in a particular location.

[00107] In some forms, a collection chamber can include, but is not limited to, a single large container or multiple enrichment sites inside a base, each containing their opening and venting independent of the others. These can be stacked to offer dynamic sampling.

[00108] In some forms, each cartridge of the system can contain responsive materials that respond to environmental triggers allowing access to the enrichment and storage module. For example, from acidic environment in the stomach to a higher pH in the gastrointestinal tract. This change leads to the opening of the porous gate or opening chamber, enabling the collection point to be selected by tailoring the material's properties of the time-delayed gate. [00109] In some forms, the cartridges can collect biomarkers inside the storage module using, but not limited to, biomarker diffusion, capillarity forces, negative pressure, or piezoelectric pumps integrated in the device and powered by an external field or attached battery.

[00110] In some forms, the base holder of the cartridges can be made of a deformable material and filled with magnetic materials to enable magnetic actuation.

[00111] In some forms, the base holder of the cartridges can be made of a deformable material and filled with reactive materials that enable chemical actuation. The combustion chamber can employ propellants such as magnesium beads, which react with body fluids generating hydrogen bubbles that displace the device. Other catalysts and enzymes can also convert the chemical fuel (body fluid) into motion.

[00112] In some forms, the interior of the collection chamber can contain a swelling component that absorbs liquid, swells up, and blocks the further fluid entrance.

[00113] In some forms, the collector chamber can contain a biological stabilizing agent that maintains viability of the collected sample.

[00114] In some forms, the system can trap biomarkers in the enrichment and storage module using swelling materials, microstructures or functionalized surfaces that bind to specific molecules at the surface of exosomes or containing complementary oligonucleotide base pairs. [00115] In some forms, the collector can be made of soft or rigid biocompatible materials including but not limited medical grade polymers, metals, and ceramic materials.

[00116] In some forms, the use of functionalized surface that bind to specific targets can be integrated into the enrichment module.

[00117] In some forms, the porosity of the enrichment module microstructure or swelling polymer can be tuned to target different sized biomarkers.

[00118] In some other forms, the robotic pill is capable sampling other types of samples such as liquid biopsy samples from patients or sampling for pollutants and nutrient from water resources.

[00119] It should be appreciated that various other modifications and variations to the preferred embodiments can be made within the spirit and scope of the invention. Therefore, the invention should not be limited to the described embodiments. To ascertain the full scope of the invention, the following claims should be referenced.

Claims

CLAIMS What is claimed is:

1. An untethered robotic pill system for collecting biomarkers and other targets of interest from human and animal body cavities and liquid resources, the system comprising: a robotic pill adapted to collect a biofluid containing biomarkers when the robotic pill is received or introduced or taken through a human orifice, other natural or surgical openings and cavity; a motive system adapted to perform at least one of directing a position of the robotic pill within the human cavity and maintaining the position of the robotic pill within the human cavity or sample site.

2. The untethered robotic pill system of claim 1, wherein the robotic pill is adapted to collect and isolate a target of interest in a gastrointestinal tract and includes an opening adapted to receive a biofluid or a sample or a tissue specimen or cells from the human cavity and a collection module configured to draw the biofluid across one or more membranes from the opening toward the collection module within the robotic pill in which the membranes divide an interior of the robotic pill into various chambers.

3. The untethered robotic pill system of claim 1, wherein the robotic pill includes a frame.

4. The untethered robotic pill system of claim 3, wherein the frame supports a magnet, the magnet being part of the motive system of the robotic pill.

5. The untethered robotic pill system of claim 4, wherein the frame is composed of a deformable material and the magnet is received within the deformable material of the frame and the elasticity of the deformable material maintains the magnet within the frame.

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6. The untethered robotic pill system of claim 3, wherein the frame supports a metasponge material for the collection of the biomarkers, the metasponge comprising an absorbent material and an elastomer support matrix.

7. The untethered robotic pill system of claim 6, wherein the absorbent material is sodium polyacrylate.

8. The untethered robotic pill system of claim 6, wherein the absorbent material is entangled within a mesh of the elastomer matrix.

9. The untethered robotic pill system of claim 6, wherein the metasponge is tuned to selectively sample in the gastrointestinal tract as the metasponge provides limited absorption or growth in acidic environment and comparably higher absorption or growth in neutral environments.

10. The untethered robotic pill system of claim 1, wherein the robotic pill includes a collection chamber for collecting the biofluid containing biomarkers.

11. The untethered robotic pill system of claim 10, wherein the collection chamber includes a single large container or multiple enrichment sites inside a base, each containing their opening and venting independent of the others.

12. The untethered robotic pill system of claim 10, wherein the collection chamber comprises a metasponge comprising absorbent material and an elastomer support matrix.

13. The untethered robotic pill system of claim 10, wherein the system is activated autonomously to provide access to the collection chamber in response to environmental changes or external signals.

14. The untethered robotic pill system of claim 13, wherein the environmental changes include changes in pH of the environment.

15. The untethered robotic pill system of claim 10, wherein an interior of the collection chamber includes a swelling component that absorbs liquid, swells up, and blocks the further fluid entrance.

16. The untethered robotic pill system of claim 10, wherein the robotic pill includes a functionalized surface to collect biomarkers.

17. A method of using the untethered robotic pill system of claim 1 to analyze a human or animal cavity, the method comprising: receiving the robotic pill in the human or animal cavity or in a tract leading to these cavitites; directing the robotic pill through the human or animal cavity, optionally to one or more specific positions, and collecting biofluids, samples, tissues, cells and other materials from the human or animal cavity within the robotic pill.

18. The method of claim 17, wherein the tract is a gastrointestinal tract and wherein the step of receiving involves administering the robotic pill through ingestion and further comprising the step of recovering the robotic pill of the system from excretion.

19. The method of claim 17, further comprising, after the collecting step, analyzing the biofluids, samples, tissues, cells, and other materials.

20. The method of claim 17, wherein during the directing step, the robotic pill is maintained at a position of interest in the human or animal cavity in order to collect the biofluids, samples, tissues, cells, and other materials at the position of interest.