Microfluidic device for sample elution

Abstract

Devices and systems for extraction and detection of an analyte from a sampling device, as well as methods of making and using such devices and systems, are described herein. An example device includes a collection panel configured to receive the sampling device, a reagent panel configured to hold sample preparation reagents and / or detection reagents, and a detection panel that includes a capture reagent configured to bind to the analyte. In some examples, devices described herein are configured to extract the analyte from a channel disposed in the sampling device.

Description

MICROFLUIDIC DEVICE FOR SAMPLE ELUTIONCROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the priority of 63 / 683,571 , filed on August 15, 2024, which is incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. R35 GM 128648, awarded by the National Institutes of Health (NIH). The government has certain rights in the inventionTECHNICAL FIELD

[0003] This application relates to microfluidic devices and techniques for automated sample elution from sample collection devices.BACKGROUND

[0004] Diagnostics play an essential role in detection, identification, and monitoring of a wide variety of pathological conditions. Diagnostic tools can enable early detection, which may significantly increase the chances for successful treatment. Rapid tests, including at-home tests that became widespread during the COVID-19 pandemic, can provide timely results, often within minutes, allowing for prompt intervention. In the case of infectious diseases, early detection helps reduce transmission and improve outcomes. In emergency settings, rapid diagnostics support faster clinical decisions for patients in critical condition. Further, rapid diagnostics can support point-of-care testing, making healthcare more accessible in resource-limited or remote settings. Improving the accessibility and ease of use of rapid tests can enhance both patient care and public health responses.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] FIG. 1 illustrates an example fluidic device for automated sample elution from a sampling device.

[0006] FIG. 2 illustrates an example process for eluting a sample from a sampling device using a fluidic device

[0007] FIGs. 3A-3C illustrate renderings of a fluidic device disclosed herein.

[0008] FIGs. 4A-4D illustrate computer-aided design (CAD) drawings of a fluidic device disclosed herein.

[0009] FIGs. 5A and 5B illustrate example workflows of a fluidic device disclosed herein.

[0010] FIG. 6 illustrates an example user workflow of a fluidic device disclosed herein.

[0011] FIG. 7 illustrates example results of analyte detection using a fluidic device disclosed herein.

[0012] FIG. 8 illustrates example results of Wilcoxon matched pairs tests comparing the manual and microfluidic methods for the prepared elution volumes.

[0013] FIG. 9 illustrates example results of analyte detection using 100 L samples with a device disclosed herein.

[0014] FIGs. 10A-10L illustrate lateral flow strip images and corresponding image analysis.DETAILED DESCRIPTION

[0015] Various implementations described herein relate to automated sample elution within a fluidic device. In various implementations, devices described herein are configured to couple to a sample collection device and elute a sample captured in the sample collection device. In various examples, a fluidic device enables extraction of analytes from a channel of a microfluidic sampling device. However, implementations of the present disclosure are not so limited. Various examples of the fluidic device, as described herein, can be used with conventional sampling devices (e.g., nasal swabs, nasopharyngeal swabs, etc.). Various implementations described herein include a detection mechanism configured to capture an analyte in the sample.

[0016] Samples can be extracted from sample collection devices using manual manipulation, which can suffer from sample contamination, human error, and low throughput. Various implementations of the present disclosure provide devices and techniques for automated sample extraction and, in some examples, analyte detection directly from a sample collection device. Accordingly, various implementations can improve sample processing efficiency and reduce the risks of sample contamination and sample degradation.

[0017] Systems including examples of the fluidic device are also described. For instance, a system may include the fluidic device and a sample collection device. Methods of making and using implementations of the fluidic device are also described. An example method includes receiving the sampling device within a receptacle disposed in the collection panel of a fluidic device, causing the solution to flow from the channel disposed in the reagent panel of the fluidic device and into the receptacle, and receiving the solution within the detection panel of fluidic the device. In some examples, methods described herein include detecting, at the detection panel, a signal based on the analyte binding to the capture reagent.

[0018] Implementations of the present disclosure will now be described with reference to the accompanying figures.

[0019] FIG. 1 illustrates an example fluidic device 100 for automated sample elution from a sampling device 102. In some implementations, the fluidic device 100 can be utilized for sample processing and / or analyte detection.

[0020] In various cases, the fluidic device 100 is configured to receive a sampling device 102. The sampling device 102 is configured to retain a sample. The sample may be a fluid sample. In some instances, the sampling device 102 is configured to receive the sample. In various cases, the sample is obtained from a subject. For instance, the sample may include a saliva sample, a nasal sample, an oral sample, a throat sample, a vaginal sample, a urethral sample, a wound sample, an ear canal sample, a dermatological sample a blood sample, a urine sample, a fecal sample, a tissue sample, or any other sample. The subject may be a human, a non-human primate, a mammal, a rodent, or the like. In some instances, the sample includes an environmental sample (e.g., a water sample, soil sample, air sample,or the like), an agricultural sample (e.g., a plant sample), a chemical sample (e.g., a reagent sample), a food sample, or the like.

[0021] In some cases, the sampling device 102 is a device that is configured to collect the sample within a channel. The channel may be an open channel. An “open channel,” as used herein, may refer to a channel that is recessed into an outer surface of a panel or device. For instance, at least one sidewall of the channel is exposed to an external environment. The depth of the recess is defined as the distance from the outer surface of the panel or device to the surface of the recess. The depth of the panel or device is defined herein as the dimension of the panel or device along the axis perpendicular to the outer surface of the panel or device. In some examples, the depth of the recess is less than the depth of the panel or device. In some examples, the open channel runs through the depth of the panel or device (e.g., the depth of the recess is equal to the depth of the panel or device. Exemplary sampling devices that include an open channel are described in International Application No. PCT / US2022 / 017042. In various instances, the channel is a closed channel (e.g., walls connected by a base and a floor). A “closed channel,” as used herein, may refer to a channel with a closed shape as a cross-section perpendicular to the direction of fluid flow. In some cases, the sampling device 102 includes a swab, a brush, a capillary tube, a collection tube, a syringe, a scraper, a spatula, an absorbent material (e.g., a sponge, a cloth), or the like. In various implementations, a hole is disposed through the sampling device. In some cases, the hole is fluidically connected to the channel of the sampling device 102.

[0022] In some cases, the sample is obtained, using the sampling device 102, and stored prior to use with the fluidic device 100. For instance, the sampling device 102 may be stored to allow the sample to dry. In some cases, the sampling device 102 may be cooled (e.g., in a refrigerator or a freezer), for instance, to preserve the sample (e.g., prevent degradation of analytes in the sample).

[0023] In various implementations, the sample includes one or more analytes. Analytes include proteins, nucleic acids, enzymes, cells, pathogens (e.g., viruses, bacteria, parasites, etc.), or the like. In various examples, the analyte(s) are associated with a pathogenic condition of the subject, such as an infection, a chronic disease, progression of a condition, or the like. Examples of infections include viral infections, bacterial infections, fungal infections, or yeast infections. Examples of chronic conditions include diabetes, chronic kidney disease, chronic liver disease, chronic obstructive pulmonary disease (COPD), chronic inflammatory diseases, cancer, cardiovascular disease, or the like. In some examples, the analyte(s) are associated with a non-pathogenic condition of the subject. For instance, the analyte may be associated with pregnancy, menopause, hormonal fluctuations, or the like. In some cases, the analyte is indicative of a deficiency (e.g., a nutritional, vitamin, mineral, or other deficiency). Detecting the analyte(s) may enable, for instance, diagnosis of the pathogenic condition of the subject. In various instances, the analyte(s) include elements (e.g., lead, mercury, arsenic, etc.), chemicals (e.g., per- and polyfluoroalkyl substances (PFAs), polychlorinated biphenyls (PCBs), pesticides, toxins (e.g., aflatoxins, mycotoxins, etc.), ions (e.g., to detect pH), or the like. In order to detect the analyte(s), the sample may be extracted from the sampling device 102.

[0024] Sample collection devices generally undergo manual manipulation to elute the sample from the collection device. For instance, a sample collection device may be immersed in a sample preparation buffer and agitated to cause mixing of the sample and the sample preparation buffer. In some examples, microfluidic sample collection devices, such as blood capillary tubes, undergo centrifugation or manual extraction (e.g., using a pipette, or by tapping the device against an absorbent material). Manual sample processing carries the risk of exposure to biohazards and sample contamination, as well as sample loss or degradation. The workflow of some rapid tests, such as COVID at-home tests, involve sample collection and elution steps performed by a lay person (e.g., an untrained user). However, these tests can be affected by failure to follow instructions, as well as the risks associated with manual sample processing described above.

[0025] In various implementations, these and other issues can be addressed by using the fluidic device 100 that is configured to facilitate sample elution and, in some examples, reagent delivery using fluid flow. The fluidic device 100 is configured to couple to the sampling device 102. For instance, the sampling device 102 may be directly coupled to the fluidic device 100 (e.g., without undergoing any manual manipulation, or after undergoing minimal manual manipulation). Accordingly, using the fluidic device 100 can reduce the risks associated with manual sample processing and improve the efficiency of sample processing and analyte detection.

[0026] According to some examples, the fluidic device 100 includes a top panel 104 that is configured to be disposed on the sampling device 102. The top panel 104, in various cases, includes a channel 106. For instance, the channel 106 may be disposed within the top panel 104. In some instances, the channel 106 is configured to hold microfluidic volumes (e.g., volumes less than 1 milliliter (mL)). In some instances, the channel 106 has a length in a range of 1 to 1000 micrometers (pm). In some examples, the channel 106 may be configured to hold mesofluidic volumes (e.g., volumes less than 1 centiliter). The surface of the channel 106 may include, in various cases, a hydrophilic material. Examples of hydrophilic materials include glass, silicone, poly(methyl methacrylate) (PMMA), polyvinyl alcohol (PVA), or the like. In various implementations, the surface of the channel 106 includes a surface treatment. For instance, the surface treatment may increase the hydrophilicity of the surface of the channel 106. Examples of surface treatments include plasma treatment, polyethylene glycol (PEG) treatment, ultraviolet (UV) light treatment, UV-ozone treatment, silanization, or the like.

[0027] When the top panel 104 is coupled to the sampling device 102, the channel 106 is fluidically connected between a pump 108 and the sampling device 102. The pump 108, in various cases, is configured to cause a fluid to flow through the channel 106 towards the sampling device 102. The pump 108 may include a finger-actuated pump, a bulb pump, a squeeze pump, a syringe pump, a peristaltic pump, a pipette, a piezoelectric pump, a diaphragm pump, a button, a compressed gas, or the like. The pump 108 may be configured to generate a pressure-driven flow, a vacuum-driven flow, a gravity-driven flow, or an electroosmotic flow of the fluid through the channel 106.

[0028] In some cases, a reservoir is fluidically connected to the channel 106. The reservoir may include a seal that is configured to retain a fluid within the reservoir. For instance, the reservoir may be disposed between the pump 108and the channel 106. In some cases, the reservoir is disposed along the channel 106. According to various implementations, a cross-sectional area of the reservoir is larger than a cross-sectional area of the channel 106. The cross-sectional area, as used herein, refer to the area perpendicular to the flow of fluid within the fluidic device 100 when the fluidic device 100 is in use.

[0029] In various examples, the seal includes a material that can be removed or punctured in order to release the fluid from the reservoir. The material may include aluminum foil, metalized plastic film, a polymer film (e.g., a film that include polyolefin, polyethylene, polypropylene, polytetrafluoroethylene, or the like), silicone, a thermoplastic elastomer, latex, nitrile, paper (e.g., wax-coated paper), or the like. In some examples, the seal includes a peel-off seal, a tear-away seal, or the like. In some cases, the seal includes a mechanism configured to control the flow of a fluid stored in the reservoir. For instance, the seal may include a valve (e.g., a rotary valve, a diaphragm valve, a pinch valve, a capillary stop valve, a hydrophobic barrier, a wax valve, a solenoid valve, a ball valve, or the like).

[0030] The sampling device 102 is disposed between the top panel 104 and a base panel 110. In some cases, a receptacle disposed into a top surface on the base panel 110 is configured to receive the sampling device 102.

[0031] While FIG. 1 illustrates the sampling device 102 disposed between the top panel 104 and the base panel 110, implementations of the present disclosure are not so limited. In various examples, the top panel 104, the sampling device 102, and the base panel 110 may be disposed laterally next to each other. In some examples, both the top panel 104 and the base panel 110 are disposed on top of or beneath the sampling device 102. In some examples, the base panel 110 is disposed on the sampling device 102 and / or the sampling device 102 is disposed on the top panel 104. For instance, the pump 108 may be configured to cause the fluid in the top panel 104 to flow, against gravity, towards the sampling device 102.

[0032] The base panel 110 is, in various examples, coupled to a detection mechanism 112. The detection mechanism 112 may include a membrane configured to receive a fluid from the sampling device 102. For instance, the membrane include cellulose, nitrocellulose, polyethersulfone (PES), polycarbonate (PC), polyvinylidene fluoride (PVDF), polypropylene (PP), nylon, chitosan, or the like. In some cases, a hole 118 may be disposed through the base panel 110, such that the detection mechanism 112 1s fl uid ically connected to the sampling device 102. As illustrated in FIG. 1 , in some implementations, the detection mechanism 112 may be disposed (e.g., physically connected) on a bottom surface of the base panel. In other implementations, the detection mechanism 112 is disposed within or on a top surface of the base panel 110.

[0033] In various cases, the detection mechanism 112 may include a lateral flow assay, a flow-through immunoassay, a microfluidic immunoassay, a dipstick test, or the like. In some cases, the detection mechanism 112 includes a container configured to receive a fluid from the sampling device 102. For instance, the container may include a reagent configured to bind to an analyte in the sample. The container, in some cases, may enable a fluid to be transported (e.g., to a polymerase chain reaction (PCR) machine, an optical detector (e.g., a spectrophotometer), or the like) for further analysis.

[0034] In various examples, a gasket is disposed between the top of the sampling device 102 and the top panel 104. The gasket, in some cases, is configured to control fluid flow from the channel 106 to the sampling device 102. For instance, a hole disposed through the gasket may facilitate the flow of fluid from the channel 106 to a particular location of the sampling device 102. In some examples, the gasket is configured to seal an open channel of the sampling device 102. In various cases, the gasket includes a flexible material. The gasket may include silicone, a thermoplastic elastomer, ethylene propylene diene monomer, polyurethane, polyethylene, polyvinyl chloride, ethylene vinyl acetate, or the like. In some cases, the gasket may be attached to the top panel 104 and / or the base panel 110.

[0035] In various examples, the fluidic device 100 may include an attachment mechanism configured to securely couple (e.g. , removably or permanently attach) two or more of: the top panel 104, the sampling device 102, or the base panel 110. For instance, the attachment mechanism may be configured to securely couple the top panel 104 and the base panel 110 together after the sampling device is positioned in the receptacle of the base panel. In some cases, the attachment mechanism is configured to securely couple the top panel 104 and the base panel 110 while the sampling device 102 is inserted into the fluidic device 100. The attachment mechanism may include a clip, a clamp, a latch, a hook-and-loop fastener, a hook-and-eye fastener, a press stud, a threaded fastener, an adhesive, or the like.

[0036] In some cases, the fluidic device 100 includes an alignment mechanism configured to align the top panel 104 and the bottom panel 110. For instance, the fluidic device 100 may orient the top panel 104 relative to the bottom panel 110. The alignment mechanism, in some cases, aligns the sampling device 102 to the top panel 104 and / or the bottom panel 110. The alignment mechanism may include posts that are configured to fit within holes disposed through the top panel 104 or the bottom panel 110. In some cases, the alignment mechanism includes a guide pin, a tab, a slot, a recess, a rib, a channel, a ball, a socket, a magnet, or the like.

[0037] FIG. 2 illustrates an example process 200 for eluting a sample from a sampling device (e.g., the sampling device 102) using a fluidic device (e.g., the fluidic device 100). The process 200 may be performed by a fluidic device, another device (e.g., a robotic device, a specialized device, or the like), an entity (e.g., a technician, a researcher, a trained user, or the like), or a combination thereof. In some implementations, one or more of the steps of process 200 may be omitted

[0038] At 202, a sampling device is received at a collection panel (e.g., the base panel 110) of a fluidic device. In some examples, the sampling device is received in a receptacle recessed into the collection panel and configured to secure the sampling device in place. According to various cases, a sample is disposed on the sampling device, and the sample includes one or more analytes. In some examples, a gasket may be placed on the sampling device, for instance, in order to seal an open channel of the sampling device.

[0039] At 204, the entity causes a solution to flow from a reagent panel (e.g., the top panel 104) of the fluidic device to the sampling device. In various cases, the reagent panel is coupled to the sampling device and / or the collection panel. For instance, the reagent panel may be disposed on the sampling device. The solution flows from a channel(e.g., the channel 106) disposed in the reagent panel to the sampling device. In some cases, the solution is disposed in the channel of the reagent panel. In some cases, the solution is disposed in a reservoir of the reagent panel For instance, a seal may be configured to retain the solution in the reservoir. The solution may be released from the reservoir by removing or breaching (e.g., piercing) the seal. In some examples, the entity applies the solution into the channel of the reagent panel. For instance, the solution may include an unstable reagent, such as an oxygensensitive reagent. According to various implementations, an actuation mechanism (e.g., the pump 108) is actuated in order to cause the flow of the solution. For instance, the entity may squeeze the actuation mechanism to cause pressure-driven flow of the solution through the channel and onto the sampling device In particular implementations, the actuation mechanism may be configured to release the solution from the reservoir. For instance, actuating the actuation mechanism may cause one or more needles to pierce the seal of the reservoir.

[0040] The solution, in various examples, includes at least one sample preparation reagent and / or at least one detection reagent. Sample preparation reagents include extraction reagents configured to extract and / or stabilize the analyte(s) in the sample. Examples of extraction reagents include sodium nitrite, acetic acid, ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), a chaotropic salt (e.g., guanidinium thiocyanate (GuSCN), guanidinium hydrochloride (GuHCI), etc.), proteinase K, tris buffer, an extraction solvent (e.g., isopropanol, ethanol, methanol, acetone, etc.), a lysis buffer, a detergent (e.g., Triton X-100), urea, a protease, dithiothreitol (DTT), p-mercaptoethanol, an RNase inhibitor, cetyltrimethylammonium bromide (CTAB) buffer, polyvinylpolypyrrolidone (PVPP), or the like. In some examples, sample preparation reagents include amplification reagents, such as primers, loop primers, DNA polymerases, strand-displacing polymerases, deoxynucleotide triphosphates (dNTPs), amplification buffers, magnesium chloride (MgCh), reverse transcriptase, RNase inhibitors, template nucleic acid molecules, probes (e.g., molecular beacons), fluorescent dyes, or the like.

[0041] In various cases, detection reagents include at least one reagent configured to bind to the analyte(s). For example, the detection reagents may include an antibody, an antigen binding fragment, an aptamer, a nucleic acid molecule, a small molecule, a protein, a peptide, or the like. In some examples, detection reagents include one or more detectable labels. In some cases, the reagent configured to bind to the analyte is conjugated to a detectable label. Examples of detectable labels include a nanoparticle, a bead, a probe (e.g., a fluorescent probe, a molecular beacon, etc.), a dye (e.g., a colorimetric dye, a fluorescent dye, etc.), a quantum dot, streptavidin, biotin, avidin, an enzyme, an enzyme substrate, a chemilumiscent label (e.g., luminol), a bioluminescent label (e.g., luciferase), a radioactive label (e.g., an isotope), a DNA barcode, an electrochemical label (e.g., a metal ion, a conductive polymer, a redox-active molecule), a magnetic label (e.g., a magnetic particle), or the like. For instance, the solution may include an enzyme and an enzyme substrate that are configured to produce a colorimetric signal, wherein the enzyme or the enzyme substrate is conjugated to a detectable label. Examples of enzymes include horseradish peroxidase, alkaline phosphatase, P-Galactosidase, urease. Examples of enzyme substrates include 3,3'- diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)(ABTS), p-nitrophenyl phosphate (pNPP), ortho-nitrophenyl-p-galactoside (ONPG), o-phenylenediamine dihydrochloride (OPD), or the like.

[0042] At 206, the solution is received at a detection panel (e.g., the detection mechanism 112) of the fluidic device. For instance, based on the entity actuating the pump, the solution may flow from the top panel, through the sampling device, and to the detection panel of the fluidic device. In some implementations, based on coupling the reagent panel and the collection panel to the sampling device, a channel of the sampling device is fluidically connected between the channel of the reagent panel and a hole disposed through the collection panel. The hole disposed through the collection panel is, in various examples, fluidically coupled to the detection panel. In various cases, the detection panel is disposed on or within the collection panel of the fluidic device. According to some implementations, the detection panel includes one or more capture reagents configured to bind to the analyte(s). Examples of capture reagents include an antibody, an antigen binding fragment, an aptamer, a nucleic acid molecule, a small molecule, a protein, a peptide, or the like. For example, the capture reagent may include a nucleic acid molecule configured to hybridize to the analyte. In some examples, the capture reagent is disposed on a solid substrate, such as a membrane, a surface of a container configured to hold the solution, a channel disposed in the collection panel, or the like. For example, the solution may flow, by capillary flow, along the membrane towards the capture reagent. In various implementations, the detection panel is configured to capture more than one analyte in the sample. For instance, the detection panel may include a first capture reagent configured to bind to a first analyte in the sample and a second capture reagent configured to bind to a second analyte in the sample.

[0043] At 208, in some examples, the entity detects a signal corresponding to the detectable label immobilized by the capture reagent. Examples of the signals include a colorimetric signal, a fluorescent signal, a refractive index, an absorbance, an electrochemical signal, a chemiluminescent signal, an electrochemiluminescent signal, radiation, or the like. For instance, a signal corresponding to the detectable label may be detected visually or by imaging the detection panel. In various cases, the signal is detected by emitting photons towards the detection panel and detecting a reflection of the photons. For instance, the photons may have a wavelength in a range of 300 to 1000 nm. In some examples, detecting the signal includes detecting an electrical signal, for instance by using an electrode disposed on the detection panel.EXPERIMENTAL EXAMPLE

[0044] This Experimental Example describes an implementation of the disclosure. In particular, this Experimental Example describes a microfluidic device, and methods of making and using the microfluidic device, configured to extract and detect an analyte from a sampling device The microfluidic device (also referred to herein as the “fluidic device,” the “fluidic system,” or the “Open to Closed (O2C) system”) can enable early detection of various illnesses, such as respiratory illness, expand healthcare access, and improve patient experience.

[0045] This Example utilized a microfluidic sampling platform that effectively captures bacteria from saliva using open microchannels (Lee, U. N. et al, Lab. Chip 2022, 22 (18), 3555-3564). Multiple user steps are often involved in eluting bacteria from sampling devices, such as the microfluidic sampling platform, limiting usability and decentralized testing. This Experimental Example describes use of an example fluidic device for integrated bacteria capture, inline elution, and user-friendly detection.

[0046] The fluidic system is a lay-user operable device that consists of a sampling device along with a fluidic device that includes top and bottom layers with closed microchannels (FIGs. 3A-3C and 4A-4D). FIGs. 3A-3C illustrate renderings of a fluidic device disclosed herein FIG. 3A illustrates the three-dimensional (3D) printed system and corresponding fabricated components of an example fluidic device. FIG. 3B illustrates an exploded computer-aided design (CAD) rendering of the fluidic device components. FIG. 3C illustrates an image of the 3D-pri nted fluidic device with the components assembled together. The inset to the right highlights the closed microchannel connection between the top layer of the fluidic device and a sampling device. FIGs. 4A-4D illustrate CAD drawings of a fluidic device disclosed herein. The two-dimensional (2D) engineering drawings of the components of an example fluidic device are shown.

[0047] In this Example, the microfluidic design of the top and bottom layers of the fluidic device can transform the open channels of the sampling device into a closed microfluidic system that integrates into high-performance microfluidic network. In this Example, with the push of a button, the user will initiate a cascade of autonomous capillary microfluidic steps that are “coded” into the hardware, increasing useability and reducing error in comparison to standard home tests where the user is asked to perform multiple steps. RADT elution reagents in the top layer flow through the sampling device, interfacing with bacteria retained in the sampling device, and through the outlet of the bottom layer upon activation of a finger pump retrofitted from a silicone e-cigarette cap (Ehtashamul Haque, M. et al, Lab. Chip 2023, 23 (1), 62-71) (FIGs. 5A, 5B). FIGs. 5A and 5B illustrate operation and fluid pathway of an example fluidic device. FIG. 5A illustrates procedural steps for the fluidic device: Step i) collect sample (in this Example, a sampling device was inoculated with bacteria for 10 minutes; red food coloring was used for visualization to represent the sample); Step ii) pipette elution buffer and / or rapid antigen detection test (RADT) reagents into closed microchannel of top layer (blue flood coloring used for visualization); Step iii) stack components and clamp together to ensure a tight seal between all layers; Step iv) attached finger pump and press to elute; Step v) elute sample from the sampling device into reservoir. FIG. 5B illustrates a side view and top view of the device with fluid pathway highlighted in blue (elution buffer) and red (saliva with bacteria). When the finger pump is pressed, the two liquids will mix together (depicted in purple) and output through the bottom layer outlet into, in this Example, a petri dish for analysis with a lateral flow strip.

[0048] This device enables precise elution of a pathogen (or antigen) from a sampling device, automated sample preparation, and rapid pathogen detection readout. The device can be used with a variety of salient readouttechnologies used in rapid tests, such as a lateral flow assay technology for Group A Streptococcus (GAS) testing and a next generation flow-based immunoassay technology developed out of Colorado State University.

[0049] Integration of the fluidic device with a commercially available GAS RADT.

[0050] Methods. This Example describes methods for integrating a fluidic device with a sampling device and a commercially available rapid antigen detection test (RADT) for GAS, the Areta Strep A Swab Test™ kit (Easy Healthcare Corporation, Darien, IL). Using spiked saliva samples applied to the sampling device, the instructions of the Areta kit were modified for integration.

[0051] In this Example, sampling devices were micromilled and modified to have a thru-hole in the center of the spiral for liquid to flow through to the 3D-printed layers. The top layer and the sampling devices were plasma treated for 5 minutes at 70W for hydrophilicity. A silicone gasket was placed on top of the sampling device, prior to assembling the fluidic device, to prevent liquid leakage. Once assembled, clamps held the top and bottom layers together to create a tight seal between the two parts. GAS spiked in saliva at concentrations ranging from 1 x105- 1x107CFU / mL, including 1.5x105CFU / mL (the Areta kit’s reported limit of detection), a positive control at 1x109CFU / mL, and a negative control of pooled saliva were tested with the manual elution procedure and the fluidic system procedure. Spiked samples at each concentration (N=3) were tested at the previously optimized elution volume of 200 piL (Sanchez, J., et al., Anal. Chem. 2025, 97(14): 7692-7701) and a lower volume of 100 piL. Images of the lateral flow strips were captured with a flatbed scanner and analyzed in Python to quantify the test line and baseline signal from two non-reporting regions of the strip, resulting in a signal-to-baseline measurement (SBR) (Smith, C., et al., Lab. Chip 2023, 23, 451-465). The positivity threshold was defined as the mean SBR + 3 standard deviations of the negative control.

[0052] The positivity threshold is calculated as such:Positivity Threshold ^SBR + 3OSBR (Equation 1 ) where / ZSBR is the average signal-to-baseline and CTSBR is the standard deviation signal-to-baseline of the three negative controls. A python script finds the negative signal by analyzing a segment of the profile located a specific distance from the negative control line peak, a distance which is informed by the test line peak locations in positive test strips.

[0053] Results. The standard Areta elution reagents were found to be sufficient for removal of the antigen used to detect GAS. A prototype of the fluidic system, which flows Areta RADT elution reagents through the sampling device with the press of a finger pump, was developed, eliminating the need for multiple manual steps in the Areta workflow (FIGs. 3-6). FIG. 6 illustrates an example workflow of preparing and using the O2C system . At Step (i), the blue food coloring pipetted into the top closed channel represents the rapid antigen detection test (RADT) reagents. At Step (ii), a sample is collected, using a microfluidic device in this Example, from a subject. Red food coloring was used for visualization to represent saliva. At Step (iii), the fluidic device is assembled with the ceiling layer and align layer clamped together. At Step (iv), after assembly, the finger pump is pressed, and the blue food coloring flows throughthe channel of the fluidic device, the channel of the microfluidic sampling device, and out through the outlet of the align layer; entirely eluting the red food coloring from the channels. The resulting solution is collected in a reservoir in this Example. At Step (v), lateral Flow strips are inserted into the solution collected in the reservoir. At Step (vi), the lateral flow strips are analyzed in order to determine results of analyte detection.

[0054] Using a standard elution volume of 200 pL, both the manual and microfluidic procedures provided signal above the positivity threshold at GAS concentrations > 5x105CFU / mL, with the microfluidic method (e.g., using the fluidic system) generally providing higher signals (FIG. 7). FIG. 7 illustrates example results of analyte detection using a device disclosed herein. 200 pL elution of saliva spiked with a range of S. pyogenes concentrations were prepared. Quantification and corresponding scanned images of lateral flow strips is shown. Positivity threshold is the mean signal-to-baseline ratio (SBR) + 3 standard deviations of the negative controls. Concentrations > 5x105CFU / mL were above the positivity threshold.

[0055] The fluidic system provided a higher SBR than the manual method at higher concentrations. At the published LOD (1 .5x105CFU / mL), both methods produced a faint positive by eye, where the median values of the SBR of the manual and microfluidic methods were 1 .03 (interquartile range [IQR]: 1 .03-1 .04) and 1 .03 ([IQR]: 1 .02- 1.05), respectively. A Wilcoxon matched pairs signed rank test, used to compare the median SBRs of the manual and microfluidic method, showed there was no statistical difference (p > 0.999, n=4) between the SBRs (FIG. 8). FIG. 8 illustrates example results of Wilcoxon matched pairs tests comparing the manual and microfluidic methods for the prepared elution volumes. Quantification between the SBRs obtained with both methods is shown. There was no statistical significance between the SBRs

[0056] A smaller elution volume (100 pL) was also tested to increase bacteria concentration in the eluted sample and could distinguish GAS concentrations > 1x105CFU / mL from the positivity threshold (FIG. 9). FIG. 9 illustrates example results of analyte detection using a device disclosed herein. 100 pL elution of saliva spiked with a range of S. pyogenes concentrations were prepared. Quantification and corresponding scanned images of lateral flow strips are shown. All tested concentrations were above the positivity threshold for both protocols. The median values of the SBR of the manual and microfluidic methods were 1.04 ([IQR]: 1.03-1.05) and 1.05 ([IQR]: 1.04-1.13), respectively.

[0057] FIGs. 10A-10L illustrate lateral flow strip images and corresponding image analysis of samples eluted using an example fluidic device. FIGs. 10A-10D illustrate lateral flow strip images taken from GAS concentrations ranging from 0 CFU / mL to 109CFU / mL, with the level of detection at 1.5x105CFU / mL. The concentration range were tested at an elution buffer volume of 200 pL. FIGs. 10E-10H illustrate lateral flow strip images taken from the same GAS concentrations as FIGs. 10A-10D with an elution buffer volume of 100 pL. FIG. 101 illustrate lateral flow strip images taken with a default setting of the scanner set at a DPI of 300 and a darkness setting of 5. In FIG. 10J, the lateral flow strip images were optimized by adjusting the settings to a DPI of 600 and a darkness of 7. FIGs. 10K and 10L illustrate image analysis of the 200 pL and 100 pL datasets. Positivity threshold is the mean signal to baseline ratio + 3 standard deviations of the negative controls. ‘Dip’ refers to the method of using the kit-provided swab and dippingthe swab into the saliva sample prior to performing the elution steps. Generally, the 100 piL elution with the O2C device produced the highest signal to baseline ratio at the different concentration levels with a signal to baseline ratio above the positivity threshold for all values except 0.

[0058] Conclusion. As demonstrated in this Example, the fluidic system provided comparable or better results than the manual procedure, while automating multiple user steps, minimizing user interaction with potentially infectious agents, and enabling elution into smaller sample volumes. This integrated platform has the potential to be used for rapid and user-friendly bacteria detection in point-of-care settings.EXAMPLE CLAUSES1 . A device including: a collection panel including a receptacle configured to receive a sampling device; a reagent panel including a channel, the channel being connected to the receptacle; a solution disposed in the channel; and a detection mechanism physically connected to the collection panel and configured to receive the solution flowing out of the receptacle, the detection mechanism including a capture reagent configured to bind to an analyte.2. The device of clause 1 , wherein the solution includes detection reagents configured to bind to the analyte.3. The device of clause 2, wherein the detection reagents include an antibody, an antigen binding fragment, or an aptamer.4. The device of clause 2 or 3, wherein the detection reagents are conjugated to a nanoparticle, a bead, a fluorescent probe, a colorimetric dye, a quantum dot, streptavidin, biotin, or avidin.5. The device of any of clauses 2-4, wherein the solution includes an enzyme and an enzyme substrate configured to produce a colorimetric signal, and wherein the detection reagents are conjugated to the enzyme or the enzyme substrate.6. The device of any of clauses 1 -5, wherein the solution includes amplification reagents.7. The device of any of clauses 1 -6, wherein the solution includes at least one extraction reagent configured to release the analyte from the sampling device.8. The device of clause 7, wherein the at least one extraction reagent includes sodium nitrite and / or acetic acid.9. The device of any of clauses 1 -8, wherein the capture reagent includes a nucleic acid that hybridizes to the analyte.10. The device of any of clauses 1 -9, wherein the capture reagent includes an antibody, an antigen binding fragment, or an aptamer.11. The device of any of clauses 1-10, wherein the capture reagent is disposed on a membrane.12. The device of any of clauses 1-11 , wherein the channel is a first channel, and the capture reagent is disposed on a surface of a second channel disposed in the detection mechanism.13. The device of any of clauses 1-12, wherein the analyte is a first analyte, the capture reagent is a first capture reagent, and the detection mechanism includes a second capture reagent configured to bind to a second analyte.14. The device of any of clauses 1-13, wherein the analyte includes a protein or a nucleic acid associated with a disease.15. The device of clause 14, wherein the disease includes an infection or a chronic disease.16. The device of clause 15, wherein the infection is a viral infection, a bacterial infection, a fungal infection, or a yeast infection.17. The device of any of clauses 1-16, wherein the reagent panel is disposed on top of the collection panel.18. The device of any of clauses 1-17, wherein the collection panel is disposed on top of the detection mechanism.19. The device of any of clauses 1-18, further including an actuation mechanism disposed on the reagent panel, connected to the channel, and configured to cause the solution to flow from the channel into the receptacle.20. The device of clause 19, wherein the actuation mechanism includes a pump, a button, or a compressed gas.21 . The device of clause 19 or 20, wherein the actuation mechanism is configured to cause pressure-driven flow of the solution through the channel towards the receptacle.22. The device of any of clauses 1-21 , further including a gasket configured to be disposed on the sampling device.23. The device of clause 22, wherein the gasket is configured to seal an open channel of the sampling device.24. The device of any of clauses 1 -23, wherein a surface of the channel includes a surface treatment or a hydrophilic material.25. The device of clause 24, wherein the surface treatment includes a hydrophilic treatment.26. A system including the device of any of clauses 1 -25, the system further including the sampling device configured to capture a sample.27. The system of clause 26, wherein the sample includes a saliva sample, a nasal sample, an oral sample, a throat sample, a vaginal sample, a urethral sample, a wound sample, an ear canal sample, or a dermatological sample.28. The system of clause 26 or 27, wherein the sample includes an environmental sample, a food sample, or an agricultural sample.29. The system of any of clauses 26-28, the channel being a first channel, wherein the sampling device includes a second channel configured to be fluidically coupled between the first channel and the detection mechanism.30. The system of clause 29, wherein a hole is disposed through the sampling device.31 . The system of clause 30, wherein the second channel is configured to connect to the first channel at a first end of the second channel and is connected to the hole at a second end of the second channel.32. A method including: receiving a sampling device within a receptacle disposed in a collection panel of a device; causing a solution to flow from a channel disposed in a reagent panel of the device and into the receptacle, the sampling device being disposed in the receptacle; and receiving the solution within a detection mechanism of the device, the detection mechanism including a capture reagent configured to bind to an analyte.33. The method of clause 32, wherein causing the solution to flow includes generating, by a pump disposed on the reagent panel, a pressure-driven flow of the solution through the channel towards the receptacle.34. The method of clause 32 or 33, wherein causing the solution to flow includes generating a vacuum-driven flow, a gravity-driven flow, or an electroosmotic flow of the solution through the channel towards the receptacle.35. The method of any of clauses 32-34, wherein receiving the sampling device includes sealing an open channel of the sampling device, and wherein the solution flows from the channel to the sealed open channel of the sampling device.36. The method of any of clauses 32-35, wherein a hole is disposed through the sampling device, and receiving the solution within the detection mechanism includes causing the solution to flow through the hole into the detection mechanism.37. The method of any of clauses 32-36, wherein the detection mechanism includes the capture reagent disposed on a membrane.38. The method of clause 37, wherein the solution is configured to flow, by capillary flow, along the membrane.39. The method of any of clauses 32-38, wherein the channel is a first channel and wherein the detection mechanism includes the capture reagent disposed on a surface of a second channel disposed in the detection mechanism, the second channel being connected to the receptacle.40. The method of any of clauses 32-39, further including: detecting, at the detection mechanism, a signal based on the analyte binding to the capture reagent.41 . The method of clause 40, wherein the signal includes a colorimetric signal, a fluorescent signal, a refractive index, an absorbance, an electrochemical signal, a chemiluminescent signal, or an electrochemiluminescent signal.42. The method of clause 41 , wherein the solution includes a detection reagent configured to bind to the analyte, the detection reagent being configured to provide the colorimetric signal or the fluorescent signal.43. The method of any of clauses 40-42, wherein detecting the signal includes imaging the detection mechanism.44. The method of any of clauses 40-43, wherein detecting the signal includes emitting, toward the detection mechanism, a beam of light; and detecting a reflection of the beam of light.

[0059] The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or processfor attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be used for realizing implementations of the disclosure in diverse forms thereof.

[0060] As will be understood by one of ordinary skill in the art, each implementation disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of’ excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of’ limits the scope of the implementation to the specified elements, steps, ingredients or components and to those that do not materially affect the implementation. As used herein, the term “based on” is equivalent to “based at least partly on,” unless otherwise specified.

[0061] Unless otherwise indicated, all numbers expressing quantities, properties, conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11 % of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1 % of the stated value.

[0062] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0063] The terms “a,” “an,” “the” and similar referents used in the context of describing implementations (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. Allmethods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as’’) provided herein is intended merely to better illuminate implementations of the disclosure and does not pose a limitation on the scope of the disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of implementations of the disclosure.

[0064] Groupings of alternative elements or implementations disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0065] "Specifically binds" refers to an association of a binding domain (of, for example, a CAR binding domain or a nanoparticle selected cell targeting ligand) to its cognate binding molecule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M’1, while not significantly associating with any other molecules or components in a relevant environment sample. “Specifically binds” is also referred to as “binds” herein. Binding domains may be classified as "high affinity" or "low affinity". In particular embodiments, "high affinity" binding domains refer to those binding domains with a Ka of at least 107M1, at least 108M’1, at least 109M-1, at least 1010M'1, at least 1011M’1, at least 1012M-1, or at least 1013M-1. In particular embodiments, "low affinity" binding domains refer to those binding domains with a Ka of up to 107M’1, up to 106M’1, up to 105M'1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10’6M to 10'13M). In certain embodiments, a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka (equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd (dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off-rate (Koff) for the cognate binding molecule that is less than that of the reference binding domain. A variety of assays are known for detecting binding domains that specifically bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE® analysis (see also, e.g., Scatchard, et al., 1949, Ann. N.Y. Acad. Sci. 51 :660; and US 5,283,173, US 5,468,614, or the equivalent).

[0066] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4nd Edition (2012); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (2003); the series Methods In Enzymology (Academic Press, Inc.); Behlke, et al., Polymerase Chain Reaction: Theory and Technology (2019); Greenfield, ed.Antibodies, A Laboratory Manual, Second Edition (2014); and Capes-Davis and R. I. Freshney, eds. Freshney's Culture of Animal Cells 8th Edition (2021 ).

[0067] Certain implementations are described herein, including the best mode known to the inventors for carrying out implementations of the disclosure. Of course, variations on these described implementations will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for implementations to be practiced otherwise than specifically described herein. Accordingly, the scope of this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by implementations of the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMS1. A device comprising: a collection panel comprising a receptacle configured to receive a sampling device; a reagent panel comprising a channel, the channel being connected to the receptacle; a solution disposed in the channel; and a detection mechanism physically connected to the collection panel and configured to receive the solution flowing out of the receptacle, the detection mechanism comprising a capture reagent configured to bind to an analyte.

2. The device of claim 1 , wherein the solution comprises detection reagents configured to bind to the analyte.

3. The device of claim 2, wherein the detection reagents comprise an antibody, an antigen binding fragment, or an aptamer; and / or wherein the detection reagents are conjugated to a nanoparticle, a bead, a fluorescent probe, a colorimetric dye, a quantum dot, streptavidin, biotin, or avidin.

4. The device of claim 1 , wherein the solution comprises amplification reagents and / or at least one extraction reagent configured to release the analyte from the sampling device.

5. The device of claim 4, wherein the at least one extraction reagent comprises sodium nitrite and / or acetic acid.

6. The device of claim 1 , wherein the capture reagent comprises a nucleic acid that hybridizes to the analyte, an antibody, an antigen binding fragment, or an aptamer.

7. The device of claim 1 , wherein the capture reagent is disposed on a membrane.

8. The device of claim 1 , wherein the analyte comprises a protein or a nucleic acid associated with a disease, the disease comprising an infection or a chronic disease.

9. The device of claim 1 , wherein the reagent panel is disposed on top of the collection panel; and / or wherein the collection panel is disposed on top of the detection mechanism.

10. The device of claim 1 , further comprising an actuation mechanism disposed on the reagent panel, connected to the channel, and configured to cause the solution to flow from the channel into the receptacle.11 The device of claim 1 , further comprising a gasket configured to be disposed on the sampling device and to seal an open channel of the sampling device.

12. A system comprising the device of claim 1, the system further comprising the sampling device configured to capture a sample.

13. The system of claim 12, wherein the sample comprises a saliva sample, a nasal sample, an oral sample, a throat sample, a vaginal sample, a urethral sample, a wound sample, an ear canal sample, a dermatological sample, an environmental sample, a food sample, or an agricultural sample.

14. The system of claim 12, the channel being a first channel, wherein the sampling device comprises a second channel configured to be fluidically coupled between the first channel and the detection mechanism.

15. A method comprising: receiving a sampling device within a receptacle disposed in a collection panel of a device; causing a solution to flow from a channel disposed in a reagent panel of the device and into the receptacle, the sampling device being disposed in the receptacle; and receiving the solution within a detection mechanism of the device, the detection mechanism comprising a capture reagent configured to bind to an analyte.

16. The method of claim 15, wherein causing the solution to flow comprises generating, by a pump disposed on the reagent panel, a pressure-driven flow of the solution through the channel towards the receptacle.

17. The method of claim 15, wherein causing the solution to flow comprises generating a vacuum-driven flow, a gravity-driven flow, or an electroosmotic flow of the solution through the channel towards the receptacle.

18. The method of claim 15, wherein receiving the sampling device comprises sealing an open channel of the sampling device, and wherein the solution flows from the channel to the sealed open channel of the sampling device.

19. The method of claim 15, wherein a hole is disposed through the sampling device, and receiving the solution within the detection mechanism comprises causing the solution to flow through the hole into the detection mechanism.

20. The method of claim 15, further comprising: detecting, at the detection mechanism, a signal based on the analyte binding to the capture reagent.

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