Conclusion
In this work, a robotic pill was evaluated as a proof-of-concept biomarker sampling platform. The robotic pill was fabricated by incorporating simple materials commonly used in medical applications (e.g., PMMA, porous membranes, sodium polyacrylate). Experiments were performed to evaluate the robotic pill's ability to entrap microparticles, proteins, and bacteria as model target collectable biomarkers. Moreover, the delayed opening of the collection chamber was achieved by coating the porous membrane with an enteric like coating which was shown to delay collection in acidic environments while opening in subsequent incubation in physiological pH environments. The robust robotic locomotion, guidance in complex environments and docking for a prolonged time in a porcine intestine model under constant flow was also demonstrated.
There are still aspects that can be enhanced within the current platform. For example, the continuous sampling provided by this design increases the possibility of cross contamination with other regions of the GI compared to other methods that collect fluid at one timepoint and keep their collection chamber sealed off. This device can be fabricated inexpensively when compared to other more robust robotic pills which integrate electronic or moving components. Therefore, our proposed model could be used to explore and sample low-abundance biomarkers in resource-limited environments, owing to its simple design and relatively inexpensive cost. For instance, the pill contents could be evaluated with downstream omics and colorimetric assays, moving this technology towards improved biomarker sampling.
In the next phase, the robotic pill control system will be integrated with automated guidance and imaging providing a higher degree of control and data collection. Moreover, we plan to study other relevant biomarkers, such as extracellular vesicles and the gut biome. We expect that the robotic pill collector would not present any risk to the user, as its small size and shape have been widely used in other ingestible imaging pills.\cite{Steiger2018} The principles outlined by this work could provide the foundation for a rapidly adaptable platform capable of isolating signatures for disease detection at early stages and therapy monitoring at later stages.
Experimental Section/Methods
Robotic Pill Fabrication: The robotic pill was fabricated by integrating several components, including: (1) a 1.5 mm laser cut optically clear cast acrylic sheet (Mcaster-carr) as a rigid backbone, (2) a 5 µm cylcopore polycarbonate membrane (Whatman) manually cut from 1.5 mm plastic sheet adhered to the rigid backbone using a double-sided adhesive medical (ARcare@90445, Adhesives Research) tape with the shape of the rigid backbone, (3) a cylindrical neodymium magnet 3 mm in diameter and 1.5 mm in thickness (K&J Magnetics, 6451 Gauss) and (4) sodium polyacrylate (Caroline biological supply) as the absorbent materials.
Quantifying the microparticle, protein, and bacteria uptake of the pill: The material's absorption was characterized by adding 500 µL of water, phosphate buffer saline (PBS), and gastric simulant to 10 mg of dry absorbent powder. The powder absorbed the solution and was weighed to measure the captured solution and compared to the original weight of the added solution (500 µg) for normalization. To evaluate the capture contribution of each of the pill’s components, the robotic pill was incubated in a 1 mL solution containing 2 µm microbeads (Spherotech, 465 ± 57 particles/µL). After incubation, the different parts of the robotic pill (rigid backbone, membrane, absorbent material) were placed in a 1 mL solution and gently rotated for 15 min. 1 µL of the supernatant was pipetted onto a glass slide, The collection capability was analyzed by measuring the number of particles in the 200 × 200 μm2 area located at the center of each droplet using brightfield microscopy, and quantified using the ImageJ count function.
For protein analysis, the robotic pill was incubated overnight in 1% BSA solution in PBS. After collection, the absorbent material was removed from the robotic pill structure and placed inside a container with either PBS or 2M CaCl2. The amount of protein released from the absorbent material was quantified using microBCA (Pierce) using the microplate standard protocol with the samples at a 1:10 dilution. Similarly, to quantify bacterial uptake, the robotic pill was incubated with E. coli (ATTC, 25922GFP) at a starting concentration of ~1.5 OD600 for an hour under shaking conditions. The three absorbent gel matrixes were removed from the pill collector, and each placed in separate 2 ml of growth media for evaluating growth. 200 µL was removed from each tube every hour for 8 hours to quantify bacterial growth as measured using OD600.
Evaluating the effects of coating the pill membrane: The porous membrane was coated with a EUDRAGIT® L 100-55 (Evonik) using manufacturers protocols and drop cast over a porous membrane containing a plastic adhesive in the shape of the robotic pill. The borders of the adhesive layer help to contain the enteric solution in place. The material was left to dry overnight before use. UV green dye (Bitspower) was used to provide contrast in the evaluation of the presence of enteric coating in the porous membrane after incubation and as an indicator captured solution. The membranes and pills were placed in either simulated gastric fluid simulant (Ricca) or buffer solution.
Demonstrating pill locomotion using an external magnetic field: A 1.5-inch square neodymium magnet (K&J Magnetics) was placed 20 mm below a table substrate supporting the robotic pill to induce locomotion. The mangetic filed strength was measured at different distances from the surface of the magnet by a handheld digital teslameter (TD8620,Tunkia) as shown in Figure S8. The magnet was rotated manually to cause the rotation of the robotic pill. The robotic pill was forced to walk across small stones (<10 mm) and an inch thick porcine belly strip (bought at the local supermarket). For fluidic experiments, either a one-inch diameter plastic tube or porcine intestine (bought at the local market) were connected to a fountain pump to generate a closed-loop fluidic system. The data that support the findings of this study are available from the corresponding author upon reasonable request.
Acknowledgements
This work was supported by the Stanford RISE COVID-19 Crisis Response Seed Grant Program, and the Canary Center at Stanford for Cancer Early Detection Seed Award. F.S was supported by the Stanford Molecular Imaging Scholars program, 5R25CA118681. (NIH T32 postdoctoral fellowship) and the Schmidt Science Fellows in partnership with the Rhodes Trust. P.D.S. was supported by the James D. Plummer Graduate Fellowship, EDGE Doctoral Fellowship Program, the Dean’s Office of the Stanford School of Engineering, and the Department of Chemical Engineering. The authors thank C. F. Guimarães for the helpful discussion.
Conflict of interest
Prof. Utkan Demirci (UD) is a founder of and has an equity interest in: (i) DxNow Inc., a company that is developing microfluidic IVF tools and imaging technologies, (ii) Koek Biotech, a company that is developing microfluidic technologies for clinical solutions, (iii) Levitas Inc., a company focusing on developing microfluidic sorters using magnetic levitation, (iv) Hillel Inc., a company bringing microfluidic cell phone tools to home settings, and (v) Mercury Biosciences, a company developing vesicle isolation technologies. UD's interests were viewed and managed in accordance with the conflict-of-interest policies.