WO2020081342A1 - Smart toilet paper - Google Patents

Abstract

The technology described herein is directed to devices and methods for low-cost, field-deployable nucleic acid detection.

Description

SMART TOILET PAPER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos. 62/747,669 filed October 19, 2018 and 62/898,132 filed September 10, 2019, the contents of which are incorporated herein by reference in their entireties.

TECHNICAL FIELD

[0002] The technology described herein relates to devices for the detection of nucleic acid targets.

BACKGROUND

[0003] Detection of microbial DNA in biological samples (e.g., fecal samples) currently involves the use of high precision instruments, which come with high costs. Such

instrumentation is rarely suitable for field use, particularly in less-developed portions of the world. Providing low-cost devices which do not require advanced infrastructure for microbial DNA detection would be of great assistance in treating numerous diseases, on both an individual and population level.

SUMMARY

[0004] Described herein is the combination and simplification of the three necessary functions of DNA detection (extraction, amplification, and visualization) into one paper- based microbial screening device. The device can be used in health screening, field research, or in the most modem facilities to permit rapid, low-cost detection of various microbes.

[0005] In one aspect of any of the embodiments, provided herein is a device comprising layers in the following order:

a. optionally, a sample collection layer comprising at least one opening;

b. a first water-resistant layer comprising at least one opening;

c. a microfluidics layer comprising:

i. a collection chamber aligned with the at least one opening in the first water- resistant layer;

ii. at least a first buffer in a first buffer reservoir;

iii. at least one microfluidic channel comprising:

1. a nucleic acid binding element;

2. a pair of nucleic acid primers; 3. the proximal zone of the microfluidic channel having a junction with the collection chamber and;

4. the distal zone of the microfluidic channel having a junction with at least one waste collection chamber;

iv. a nucleic acid visualization area comprising a western blot or lateral flow assay which has a junction with the microfluidic channel;

d. a second water-resistant layer; and

e. an axial element positioned to pass through each of the layers.

[0006] In some embodiments of any of the aspects, the layers are circular or cylindrical in shape. In some embodiments of any of the aspects, the width, length, and/or diameter of each of the layers is substantially the same as that of the other layers. In some embodiments of any of the aspects, the width, length, and/or diameter of each of the layers is within 10% of the average for the layers. In some embodiments of any of the aspects, the width, length, and/or diameter of each of the layers is the same as that of the other layers.

[0007] In some embodiments of any of the aspects, the sample collection layer comprises toilet paper, tissue paper, baby wipe material, personal hygiene wipe material, sanitary napkin material, tampon material, or the like. In some embodiments of any of the aspects, the at least one opening in the sample collection layer is a slit or hole which traverses from the exterior of the layer to the side of the layer facing the first water-resistant layer. In some embodiments of any of the aspects, the at least one opening in the sample collection layer is closer to the center of the layer’s most-planar dimension than to the edge of the layer’s most-planar dimension. In some embodiments of any of the aspects, the sample collection layer is disposable or removable. In some embodiments of any of the aspects, the sample collection layer is adhered to the first water-resistant layer with a chemical or physical adhesive.

[0008] In some embodiments of any of the aspects, the at least one opening in the first water-resistant layer is a slit or hole which traverses from the side of the layer facing the sample collection layer to the side of the layer facing the microfluidics layer. In some embodiments of any of the aspects, the at least one opening in the first water-resistant layer is closer to the center of the layer’s most-planar dimension than to the edge of the layer’s most- planar dimension. In some embodiments of any of the aspects, the at least one opening in the first water-resistant layer is aligned with the at least one opening in the sample collection layer. In some embodiments of any of the aspects, the at least one opening which is aligned with the at least one opening in the sample collection layer is a slit. In some embodiments of any of the aspects, the first-water resistant layer comprises at least one opening which is not aligned with the at least one opening in the sample collection layer. In some embodiments of any of the aspects, the at least one opening which is not aligned with the at least one opening in the sample collection layer is a mesh, hole, or slit.

[0009] In some embodiments of any of the aspects, the one or more microfluidic channels are hydrophobic. In some embodiments of any of the aspects, the collection chamber is located substantially in the center of the microfluidic layer’s most-planar dimension. In some embodiments of any of the aspects, the collection chamber further comprises a sample agitation element. In some embodiments of any of the aspects, the sample agitation element is one or more beads. In some embodiments of any of the aspects, the one or more microfluidic channels and/or the collection chamber comprise a particulate filter designed to retain large sample particulate or sample agitation elements in the collection chamber or proximal portion of the one or more microfluidic channels. In some embodiments of any of the aspects, the particulate filter is a nylon filter.

[0010] In some embodiments of any of the aspects, the first buffer reservoir comprises a lysis buffer (e.g., the first buffer is a lysis buffer). In some embodiments of any of the aspects, the lysis buffer further comprises RNase and a binding agent.

[0011] In some embodiments of any of the aspects, the collection chamber comprises a second buffer in a second, separate buffer reservoir and the second bugger is a buffer that can release nucleic acids from the nucleic acid binding element. In some embodiments of any of the aspects, the buffer than can release nucleic acids is TE buffer.

[0012] In some embodiments of any of the aspects, each buffer reservoir is a rupturable capsule that can be ruptured by applying force to the region of the device comprising the buffer reservoir. In some embodiments of any of the aspects, the force is no more than half the force required to rupture the water-resistant layers or the microfluidics layer.

[0013] In some embodiments of any of the aspects, the microfluidic layer comprises a plurality of microfluidic channels and each microfluidic channel comprises a single pair of nucleic acid primers. In some embodiments of any of the aspects, the microfluidic layer comprises a plurality of microfluidic channels and each microfluidic channel comprises a single unique pair of nucleic acid primers. In some embodiments of any of the aspects, the at least one pair of nucleic acid primers is provided in a rupturable primer capsule. In some embodiments of any of the aspects, the capsule comprising the pair of nucleic acid primer further comprises one or more RPA reagents. In some embodiments of any of the aspects, at least one pair of nucleic acid primers is specific for a pathogenic microorganism, a microorganism found in the subject species’ microbiome, or a microbial gene that affects host drug metabolism.

[0014] In some embodiments of any of the aspects, the junction between the microfluidic channel and the waste chamber comprises a separable or sealable element.

[0015] In some embodiments of any of the aspects, the visualization area comprises an LFA assay. In some embodiments of any of the aspects, the LFA assay comprises i) a LFA- compatible pad or pads and iii) gold particles, latex, and/or carbon nanotubes. In some embodiments of any of the aspects, the junction of the microfluidic channel and the visualization area comprises a rupturable or degradable barrier.

[0016] In some embodiments of any of the aspects, the second water-resistant layer comprises viewing windows aligned with one or more of the visualization areas. In some embodiments of any of the aspects, the second water-resistant layer comprises openings, indentations, or thinner zones aligned with one or more of the rupturable capsules.

[0017] In some embodiments of any of the aspects, the axial element is located substantially at the center of each layer’s most-planar dimension. In some embodiments of any of the aspects, the axial element is located at the center of each layer’s most-planar dimension. In some embodiments of any of the aspects, the axial element is not located at the center of each layer’s most-planar dimension. In some embodiments of any of the aspects, each layer comprises a portal or opening with an aperture which is not contiguous with any other element of the layer, and the axial element passes through the apertures. In some embodiments of any of the aspects, the axial element is a tensile element forming a closed loop and extending beyond the exterior faces of the sample collection layer and the second water-resistant layer. In some embodiments of any of the aspects, each layer comprises two portals or openings, each with an aperture which is not contiguous with any other element of the layer, and the axial element passes through the apertures.

[0018] In one aspect of any of the embodiments, provided herein is a method of detecting a microorganism or microbial gene in a sample, the method comprising:

a. contacting the sample collection layer of a device as described herein with the sample; b. opening or rupturing the first buffer reservoir;

c. agitating the device;

d. causing the device to spin about the axial element;

e. closing or sealing the at least one waste collection chamber; f. optionally, opening or rupturing the second buffer reservoir;

g. optionally, opening or rupturing any primer capsules present;

h. warming the device to at least 35°C;

i. observing or detecting any visible signals in the visualization areas.

In some embodiments of any of the aspects, when step f is performed, the method comprises a further step after step f of causing the device to spin about the axial element.

[0019] In some embodiments of any of the aspects, step h comprises warming the device to 37°C-39°C. In some embodiments of any of the aspects, in step h, the device is maintained at the temperature of at least 35°C for about 20 minutes or until at least one signal is visible in at least one visualization area. In some embodiments of any of the aspects, step h comprises placing the device in a water bath, placing the device in a sealed container which is placed in a water bath, or placing the device in close proximity to the surface of a subject’s skin.

[0020] In some embodiments of any of the aspects, the method further comprises a step after a. and before b. of sealing all or a portion of the sample collection layer’s outer surface or removing the sample collection layer and sealing all or a portion of the first water-resistant layers’ outer surface. In some embodiments of any of the aspects, the sealing comprises application of an adhesive cover or sticker.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Fig. 1 depicts diagrams of an exemplary embodiment of the compositions described herein.

[0022] Fig. 2 depicts a diagram of an exemplary paperfuge. From“Hand-Powered Ultralow-Cost Paper Centrifuge | Nature Biomedical Engineering.” Accessed December 10, 2018 (available on the world wide web at nature.com/articles/s4l55l-0l6-0009).

[0023] Fig. 3 depicts one embodiment of the technology described herein.

[0024] Fig. 4 depicts PCR vs. RPA technologies

[0025] Figs. 5-7 depict an exemplary method of operating one of the embodiments of the devices described herein.

[0026] Fig. 8 depicts an exemplary embodiment of the paper layer (Layer 1).

[0027] Fig. 9 depicts an exemplary embodiment of the second water-resistant layer

(Layer 4).

[0028] Fig. 10 depicts an exemplary embodiment of the microfluidics layer (Layer 3). [0029] Figs. 11-13 depict various parts and elements of an exemplary embodiment of the devices described herein.

DETAILED DESCRIPTION

[0030] Described herein are devices, kits, and methods that permit quick, low-cost detection of nucleic acid targets (e.g., those specific to certain microorganisms) in field conditions, e.g., without access to refrigeration, laboratory equipment, or even on-scene medical professionals. The devices, kits, and methods described herein can be used with any biological sample type to detect any known microorganism, but applications utilizing fecal samples are used as a non-limiting illustrative example throughout. The devices described herein are multi-layered compositions that permit sample collection, sample preparation, and a detection assay all within or on the single device.

[0031] In one aspect of any of the embodiments, described herein is a device comprising four layers in the following order:

1. a sample collection layer 10 comprising at least one opening 11;

2. a first water-resistant layer 20 comprising at least one opening;

3. a microfluidics layer 30 (e.g., a water-resistant microfluidics layer); and

4. a second water-resistant layer 50.

In some embodiments, e.g, after sample collection and the removal of the sample collection layer 10, or if sample collection is performed with a separate element or material than a sample collection layer 10, a device described herein can comprise three layers in the following order:

a first water-resistant layer 20 comprising at least one opening;

a microfluidics layer 30 (e.g., a water-resistant microfluidics layer); and

a second water-resistant layer 50.

The layers can be of any shape or size, e.g., circular, cylindrical, rectilinear, triganular, or the like. In some embodiments of any of the aspects, the layers are circular or cylindrical in shape. In some embodiments of any of the apsects, the width, length, and/or diameter of each of the layers is substantially the same, e.g., such that the layers can be arrayed in the order specified in this paragraph and no layer extends substantially beyond the other layers. In some embodiments of any of the apsects, the width, length, and/or diameter of each of the layers is within 10% of the average for the layers as a group. In some embodiments of any of the apsects, the width, length, and/or diameter of each of the layers is the same. In some embodiments of any of the apsects, one or two of the layers can have a width, length, or diameter which is greater than the width, length, or diameter of the remaining layers, such that that layer’s ends can be wrapped or sealed over the ends of the remaining layers, thereby sealing the device. For example, the water-resistant layers 20 and 50 could extend beyond the microfluidics layer 30 and join together around the perimeter of the device, e.g., with a post- and-hole, or tongue-and-groove architecture, such that the microfluidics layer is encased between them.

[0032] The sample collection layer 10, can be of any material suitable for obtaining biological samples. In some embodiments of any of the aspects, the sample collection layer 10 can comprise an absorbent material. In some embodiments of any of the aspects, the sample collection layer 10 can comprise one or more materials known in the art for manipulation, disposal, collection, or containment of biological matter, particularly those materials designed for use by lay persons. By way of non-limiting example, a sample collection layer 10, can comprise one or more of toilet paper, tissue paper, baby wipe material, personal hygiene wipe material, sanitary napkin material, tampon material, wound dressing material, cotton swab material, or the like.

[0033] The sample collection layer 10, can be disposable, removable, or can be coverable by an adhesive cover or sticker. Removing or covering the sample collection layer 10 after sample collection can avoid the user(s) being unduly exposed to biological matter or spreading biological matter in the area of use. A cover or sticker can be adhered or attached to the sample collection layer 10 after sample collection, or the sample collection layer 10 can be removed after sample collection, or the sample collection layer 10 can be removed after sample collection and a cover or sticker adhered or attached to the first water-resistant layer 20. Layers can be adhered to each other, or a cover or sticker can be adhered to a layer, by a chemical or physical adhesive, e.g., a glue or a fiber-based connection system such as Velcro™ or variations thereof.

[0034] The sample collection layer 10, can comprise at least one opening 11, which permits the sample to transit the sample collection layer 10 and reach the first water-resistant layer 20. The opening 11 can be a slit or hole, or collection/multiplicity of slits or holes, which traverse or penetrate from the exterior face of the sample collection layer 10 to the side of the sample collection layer facing the first water-resistant layer 20. In some embodiments of any of the aspects, the at least one opening 11, can be a mesh. The size of the at least one opening 11 can be selected to regulate the type and/or amount of biological matter which traverses the sample collection layer 10. Such sizes will be dependent upon the type of the sample and the target(s) / microorganism(s) to be detected and can be determined on the basis of those factors by one of skill in the art. For example, if the biological matter is fecal matter, the size of the at least one opening 11 can be selected to exclude undigested material.

[0035] In some embodiments of any of the aspects, the at least one opening 11 in the sample collection layer 10 is closer to the center of the layer’s most-planar dimension than to the edge of the layer’s most-planar dimension.

[0036] The first water-resistant layer 20, can comprise at least one opening which permits the sample to transit the first water-resistant layer 20 and reach the microfluidics layer 30.

The opening can be a slit or hole, or collection of slits or holes, which traverse/penetrate from the outside face of the first water-resistant layer 20 (e.g,. the face positioned towards the sample collection layer 10), to the side of the first water-resistant layer 20 facing the microfluidics layer 30. In some embodiments of any of the aspects, the at least one opening in first water-resistant layer 20 can be a mesh. The size of the at least one opening in first water-resistant layer 20 can be selected to regulate the type and/or amount of biological matter which traverses the first water-resistant layer 20. Such sizes will be dependent upon the type of the sample and the target(s) / microorganism(s) to be detected and can be determined on the basis of those factors by one of skill in the art. For example, if the biological matter is fecal matter, the size of the at least one opening can be selected to exclude undigested material.

[0037] In some embodiments of any of the aspects, the at least one opening in the first water-resistant layer 20 is closer to the center of the layer’s most-planar dimension than to the edge of the layer’s most-planar dimension. In some embodiments of any of the aspects, the at least one opening in the first water-resistant layer 20 is aligned with the at least one opening 11 in the sample collection layer 10, e.g., at least one opening in each layer is directly above or below at least one opening in the other layer. In some embodiments of any of the aspects, the at least one opening in the first water-resistant layer 20 is a slit aligned with the at least one opening 11 in the sample collection layer 10. In some embodiments of any of the aspects, the first water-resistant layer 20 comprises at least one opening which is not aligned with an opening 11 in the sample collection layer 10. In some embodiments of any of the aspects, the at least one opening in the first water-resistant layer 20 is a slit or hole which is not aligned with the at least one opening 11 in the sample collection layer 10. [0038] After the sample is collected using the sample collection layer 10 and at least a portion of the sample traverses the first water-resistant layer 20, the sample will enter the microfluidics layer 30. The microfluidics layer 30, comprises a collection chamber 31, which is aligned with at least one opening in the first water-resistant layer 20. In some embodiments of any of the aspects, the collection chamber 31, is aligned with any openings present in the first water-resistant layer 20.

[0039] In some embodiments of any of the aspects, the collection chamber 31, comprises one or more sample agitation elements 32. A sample agitation element 32 is a fixed or mobile structure that, when the device is agitated (e.g. shaken either by hand or by a machine), will exert physical forces (e.g. shear forces) that disrupt the biological matter of the sample. The sample agitation elements 32 can be fixed, e.g., posts, baffles, lanes, or funnels, or mobile, e.g., beads. The sample agitation elements 32 can be sized to disrupt

macrostructures in the sample, e.g., to break down clumps of fecal matter, and/or can be sized to disrupt cells (either eukaryotic and/or prokaryotic) depending on the preference for the sample type, the target to be detected, and the required sensitivity of the particular application planned. Multiple types and sizes of sample agitation elements 32 can be combined in a single collection chamber 31. Such sample agitation elements are well known in the art and available commercially in a number of materials and configurations.

[0040] The collection chamber 31, comprises at least one buffer in a buffer reservoir 34. In some embodiments of any of the aspects, the collection chamber 31 can further comprise a second buffer in a separate buffer reservoir 35. In some embodiments of any of the aspects, each buffer reservoir 34, 35 present is a rupturable capsule that can be opened or ruptured by applying force to the region of the device comprising the buffer reservoir. The first panel of Fig. 9 depicts an illustrative example of such rupturable capsules, e.g.,“blisters”. Such “blister” packaging is well-known in the art and includes foils, plastics, and the like. In some embodiments of any of the aspects, the force required to rupture either buffer reservoir 34, 35 is not more than half the force that would cause the water-resistant layers 20, 50 or the microfluidics layer 30 material to rupture, rip, or crack. In some embodiments of any of the aspects, the force required to rupture either buffer reservoir 34, 35 is within the force a health adult human’s fingers can exert. In some embodiments of any of the aspects, each buffer reservoir 34, 35 is designed (e.g., with partial perforations or a weaker/thinner zone) to rupture on a face that will direct the buffer directly into the collection chamber 31, e.g.

instead of towards another layer or a wall of the microfluidics layer 30. [0041] The first buffer reservoir 34 can comprise a lysis buffer, e.g., a cell lysis buffer. Suitable lysis buffers for various cell types are known in the art and commercially available. As but one example, a suitable lysis buffer can comprise a detergent and a buffer molecule(s). For example, an exemplary cell lysis buffer can comprise SDS (e.g., 10% SDS), Tris-HCl (e.g., at 1 M, pH 8.1), and EDTA (e.g., at 0.5 M, pH 8). In some embodiments of any of the aspects, the cell lysis buffer can further comprise RNAse. In some embodiments of any of the aspects, the cell lysis buffer can further comprise one or more binding agents, e.g., agents that will cause a nucleic acid to bind to a nucleic acid binding element 37, e.g., a silica membrane. Such interactions are well-understood in the art and one of skill in the art can readily selected suitable binding agents or purchase mixtures designed for this use

commercially. As a non-limiting example, such binding agents can comprise chaotropic salts and/or ethanol.

[0042] The second buffer reservoir 35, in embodiments that comprise the second buffer and second buffer reservoir 35, can comprise a buffer that can release nucleic acids from a nucleic acid binding element 37, e.g., a silica membrane. Such buffers can be those which do not comprise a binding agent as described above and/or which raise the concentration of buffers and/or chelators relative to the cell lysis buffer. Such interactions are well-understood in the art and one of skill in the art can readily select suitable binding agents or purchase mixtures designed for this use commercially, see, e.g., TE buffer, which is widely available commercially. As a non-limiting example, a buffer that can release nucleic acids from a silica membrane, suitable for use with the exemplary cell lysis buffer described above, can comprise Tris (e.g., lOmM, pH 8.0), HC1, and EDTA (e.g., lmM, pH 8.0). As noted, the use of this buffer is optional, as the nucleic acids can be retained on the nucleic acid binding element 37 while still providing a functional device.

[0043] In some embodiments of any of the aspects, the collection chamber 31 is closer to the center of the microfluidics layer’s 30 most-planar dimension than to the edge of the layer’s most-planar dimension. In some embodiments of any of the aspects, the collection chamber 31 is located substantially in the center of the microfluidics layer’s 30 most-planar dimension. In some embodiments of any of the aspects, the collection chamber 31 is located near the center of the microfluidics layer’s 30 most-planar dimension, but to one side of or around the at least one portal or opening 52.

[0044] The collection chamber 31 has at least one junction with at least one microfluidic channel 36. Each microfluidic channel 36 comprises a proximal zone of the microfluidic channel having a junction with the collection chamber 31, a distal zone of the microfluidic channel having a junction 42 with at least one waste collection chamber 38, and a junction 43 with a nucleic acid visualization area 39.

[0045] In some embodiments of any of the aspects, the one or more microfluidic channels 36, and optionally, other elements of the microfluidic layer 30, can be made of water-resistant material as described elsewhere herein. In some embodiments of any of the aspects, the one or more microfluidic channels 36, and optionally, other elements of the microfluidic layer 30, can be made of hydrophobic material or comprise a layer of a hydrophobic material on any surface that will be contacted by the sample or buffers described herein. For example, a layer of paraffin, silicone, or other hydrophobic coating can be applied to paper or another material which is used as the substrate or base for the microfluidics layer. In some embodiments of any of the aspects, the layer can be made of plastics, or paper coated in latex, epoxy or foil. In some embodiments of any of the aspects, the layer can be resistant to alcohols, e.g., ethanol.

[0046] In some embodiments of any of the aspects, the one or more microfluidic channels 36 and/or the collection chamber 31 comprise a particulate filter designed to retain large sample particulate or sample agitation elements 32 in the collection chamber 31 or proximal portion of the one or more microfluidic channels. Preferably, the particulate filter is located proximal of the nucleic acid binding element 37. Particulate filters may be nylon, plastic, or the like, but should not be of a material that will bind nucleic acids in the context of the cell lysis buffer selected. The particulate filters should have a minimum size sufficient to permit the target nucleic acids in the sample to pass through. For example, if the device is designed to detect/amplify a bacterial gene, the filter should be sized to permit the passage of bacterial chromosomes. If the device is designed to detect/amplify a parasite or host gene, the filter should be sized to permit the passage of the relevant parasite or host chromosome. The particulate filters should have a maximum size to prevent passage of any sample agitation elements 32 which are not fixed structures.

[0047] Each microfluidic channel 36 comprises a nucleic acid binding element 37 and at least one pair of nucleic acid primers. The nucleic acid binding element 37 is a membrane, surface, support, or structure (e.g., bead(s), including titanium beads) which can bind nucleic acid molecules, e.g., the primers and/or some or all of the nucleic acids present in the sample. The nucleic acid binding element 37 can bind in a sequence-specific manner (e.g., beads with nucleic acid probes conjugated to them) or in a manner which is not sequence specific (e.g. a silica membrane). The RPA reaction can occur“on-element”, e.g., while the targets and/or primers are bound to the nucleic acid binding element 37 or the nucleic acid binding element 37 can be exposed to a buffer that can release nucleic acids from a nucleic acid binding element prior to the RPA reaction such that the RPA reaction occurs“off-element”

[0048] In some embodiments of any of the aspects, at least one pair of nucleic acid primers in the device (or each pair of nucleic acid primers in the device, or at least one pair in each microfluidic channel 36 of the device) is specific for (e.g., specifically amplifies a sequence which is specific to) a pathogenic microorganism, specific for a microorganism found in the subject species’ microbiome, or specific for a microbial gene that affects host drug metabolism. In some embodiments of any of the aspects, at least one pair of nucleic acid primers in the device (or each pair of nucleic acid primers in the device, or at at least one pair in each microfluidic channel 36 of the device) is specific for a genus, species, or strain of a pathogenic microorganism (e.g., one that is pathogenic for the subject’s species). In some embodiments of any of the aspects, at least one pair of nucleic acid primers in the device (or each pair of nucleic acid primers in the device, or at least one pair in each microfluidic channel 36 of the device) of the device is specific for a genus, species, or strain of a microorganism found in the subject species’ microbiome. In this context a sequence is “specific” to a given target or target class if the sequence is unique to that target or target class, e.g., organisms or genes other than the target (or target class) do not contain the same sequence. Such genes, and nucleic acid sequences specific for the stated populations of microorganisms are known in the art and readily identified by, e.g., comparing the known sequence of the desired population of microorganisms to sequences from related

microorganisms and/or other microorganism that might be found in a sample of the same type. Exemplary such genes are provided in the examples herein. The primers can also be specific for a nucleic acid found in, or potentially found in the subject’s own genome or cells, e.g., to determine the target’s genetic susceptibility for a condition or infection, or to make personalized medicine-based decisions.

[0049] In some embodiments of any of the aspects, the microfluidic layer comprises a plurality of microfluidic channels and each microfluidic channel comprises at least a single pair of nucleic acid primers. In such scenarios, each microfluidic channel might permit detection of a single different gene, strain, species, or genus. Alternatively, two or more microfluidic channels might permit detection of different nucleic acid sequences specific for the same gene, strain, species, or genus, e.g., providing independent verification of the other channel’s detection. [0050] In embodiments where a microfluidic channel 36 comprises more than one pair of nucleic acid primers, the multiple pairs can each permit detection of a different gene, strain, species, or genus. Alternatively, two or more of the pairs of nucleic acid primers in a single microfluidic channel can permit detection of a different nucleic acid sequence specific for the same strain, species, or genus, e.g., providing independent verification of the other pair’s detection.

[0051] In some embodiments of any of the aspects, the microfluidic layer 30 comprises a plurality of microfluidic channels 36 and each microfluidic channel 36 comprises a single unique pair of nucleic acid primers.

[0052] In some embodiments of any of the aspects, the at least one pair of nucleic acid primers can be provided in a buffer-soluble primer capsule 40 or a rupturable primer capsule 40.Rupturable capsules are described elsewhere herein. A buffer-soluble primer capsule can be soluble in the presence of 1) the lysis buffer and 2) the buffer that can release nucleic acids from a nucleic acid binding element (e.g., a silica membrane). In some embodiments of any of the aspects, the buffer-soluble primer capsule can be soluble in the presence of the buffer that can release nucleic acids from a nucleic acid binding element (e.g., a silica membrane) , e.g., a TE buffer.

[0053] In some embodiments of any of the aspects, the capsule 40 comprising at least one pair of nucleic acid primers can further comprise one or more recombinase polymerase amplification (RPA) reagents or mixtures. In addition to the aforementioned pairs of primers and a template nucleic acid found in the biological sample, RPA utilizes at least a

recombinase such at T4 UvsX, a recombinase loading factor such as T4 UvsY, a single- stranded binding protein such as T4 gp32m, a DNA polymerase such as Bsu or Sau, a mix of deoxynucleotide triphosphates, and ATP. RPA reagent mixtures can further include magnesium acetate, potassium acetate, Tris, ATP, creatine kinase, phosphocreatine, DTT, and/or a crowding reagent like Carbowax20M or PEG. RPA reagents and conditions are further described in, e.g., Li et al. Analyst 2019 144:31-67 (incorporated by reference herein in its entirety), and RPA reagents and mixtures are available commercially (see, e.g.,

TWISTAMP Liquid Basic (Cat. No. TALWBAS01; TwistDx Cambridge UK)).

[0054] In some embodiments of any of the aspects, the at least one pair of nucleic acid primers can be provided in the microfluidic channel 36 or on the nucleic acid binding element 37 and the rupturable capsule 40 can contain one or more RPA reagents or mixtures. [0055] The microfluidic channel 36 has a junction 42 with a waste chamber 41. The waste chamber 41 can be a single chamber which has junctions with each microfluidic channel 36 or the device can comprise multiple waste chambers 41, each connected to one or more microfluidic channels 36. In some embodiments of any of the aspects, the waste chamber can be the element of the microfluidic layer 30 which is most distal from the axial element. The waste chamber 41 can be an unmodified chamber or can comprise baffles, absorbent material, or similar elements designed to improve the uptake and retention of the buffer and sample elements which reach the waste chamber 41.

[0056] Each junction (e.g., 42, 43) can comprise a separable or sealable element.

Numerous configurations and materials for sealing or separating two fluid channel s/chambers are known in the art. For example, the separable or sealable element can comprise a one-way valve, an area or zone which is crushable, or an area or zone which is crushable and comprises adhesives, or an area or zone which is crushable and comprises parts which can mate to hold the area in a closed configuration, or an area or zone which is crushable and comprises a water-resistant gel or matrix.

[0057] The nucleic acid visualization area 39 comprises a western blot or lateral flow assay, e.g., the materials and/or reagents necessary for detection/visualization or one or more nucleic acid molecules by way of western blot or lateral flow assay. Such

assays/materials/reagents are well known in the art and readily obtained commercially.

Briefly, lateral flow assay test (LFA), also known as the immunochromatographic assay, or strip test, is an assay intended to detect the presence (or absence) of a target, e.g. a nucleic acid, in a fluid sample. There are currently many LFA tests used for medical diagnostics, either for home testing, point of care testing, or laboratory use. In LFA tests the test sample flows along a solid substrate via capillary action. After or before the sample is applied to the pad of the visualization area it encounters a colored reagent (e.g., gold particles, latex, and/or carbon nanotubes) which mixes with the sample and transits the pad of the visualization area. LFA tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of targets from fluid samples such as urine, blood, water, and/or homogenized tissue samples etc. There are a number of variations on lateral flow technology. It is also possible to apply multiple capture zones to create a multiplex test. In some embodiments of any of the aspects, the nucleic acid visualization area 39 comprises a lateral flow assay, e.g., the materials and/or reagents necessary for detection/visualization of one or more nucleic acid molecules by way of lateral flow assay. A lateral flow assay can comprise i) a LFA-compatible pad or pads and ii) gold particles, latex, and/or carbon nanotubes. LFA assays are described in more detail at, e.g., Koczula and Gallotta. Essays Biochem (2016)

60: 111-120; which is incorporated by reference herein in its entirety.

[0058] The junction 43 of the microfluidic channel 36 and the visualization area 39 can comprise a rupturable or degradable barrier. When junction 43 comprises a degradable barrier, the barrier comprises a material which degrades in the presence of a recombinase agent or RPA mix. In some embodiments of any of the aspects, the material is not degraded by the 1) the lysis buffer and/or 2) the buffer that can release nucleic acids from a nucleic acid binding element 37 (e.g., a silica membrane).

[0059] In some embodiments of any of the aspects, the second water-resistant layer 50 can be a solid layer not comprising any openings, holes, slits, or gaps. Alternatively, in some embodiments of any of the aspects, the second water-re stistant layer 50 can comprise openings, holes, or gaps aligned with one or more of the visualization areas 39 or any rupturable capsule, rupturable barrier, sepearable element, or sealable element of the microfluidics layer 30. Further, in some embodiments of any of the aspects, the second water-restistant layer 50 can comprise viewing windows aligned with one or more of the visualization areas 39. In some embodiments of any of the aspects, the second-water resistant layer 50 can comprise viewing windows, openings, gaps, holes, indentations, or markings aligned with any rupturable capsule, rupturable barrier, sepearable element, or sealable element of the microfluidics layer 30. Viewing windows 51 are distinguished from openings, holes, or gaps in that viewing windows are a transparent portion of the second water-resistant layer 50 which permit the user and/or a device to visually/optically observe a portion of the microfluidics layer 30 without permitting fluids to exit the microfluidics layer through the second water-resistant layer 50.

[0060] The layers may be sealed together at any point, particularly near any edges, holes, or gaps, e.g., using an adhesive as described elsewhere herein. In some embodiments of any of the aspects, when the second water-resistant layer 50 is sealed or attached to the microfluidics layer 30, no channel or path for fluids in the microfluidic layer 30 to transit the second water-resistant layer 50 exists.

[0061] The axial element passes through each of the layers. The axial element permits the entire device to be spun or rotated around or with the axial element, causing at least a portion of the sample to move from the sample collection chamber 31 through one or more microfluidic channels 36 and into the waste collection chamber 41. [0062] In some embodiments of any of the aspects, each layer comprises at least one portal or opening 52 with an aperture which is not contiguous with any other element of the layer (other than the substrate of the layer), and the axial element passes through the apertures. In some embodiments of any of the aspects, the axial element, and/or the at least one portal or opening 52 is located substantially at the center of each layer’s most-planar dimension. In some embodiments of any of the aspects, the axial element, and/or the at least one portal or opening 52 is located at the center of each layer’s most-planar dimension. In some embodiments of any of the aspects, the axial element, and/or the at least one portal or opening 52 is not located at the center of each layer’s most-planar dimension.

[0063] In some embodiments of any of the aspects, the axial element is a string, cord, rope, wire, and/or tensile element. In some embodiments of any of the aspects, the axial element is a string, cord, rope, wire, and/or tensile element forming a closed loop and extending beyond the exterior faces of the sample collection layer 10 (or first water resistant layer 20) and the second water-resistant layer 50. In some embodiments of any of the aspects, the axial element is a tensile element forming a closed loop and extending beyond the exterior faces of the sample collection layer 10 (or first water resistant layer 20) and the second water-resistant layer 50. In some embodiments of any of the aspects, the axial element is a string, cord, rope, wire, and/or tensile element forming a closed loop and extending beyond the exterior faces of the sample collection layer 10 (or first water resistant layer 20) and the second water-resistant layer 50, and each layer of the device comprises two portals or openings 52. In some embodiments of any of the aspects, the axial element is a tensile element forming a closed loop and extending beyond the exterior faces of the sample collection layer 10 (or first water resistant layer 20) and the second water-resistant layer 50, and each layer of the device comprises two portals or openings 52.

[0064] As explained elsewhere herein, the operation of the device described herein can comprise a step of heating the device. In some embodiments of any of the aspects, the microfluic layer 30 can further comprise a group of exothermic reactants located in an area of the microfluidic layer 30 not occupied by the chambers, channels, visualization area, or capsules described above herein. Such groups of exothermic reactants, often activated by air exposure, or the flexing of a metal disk, are well known in the art. As a non-limiting example, a group of exothermic reagents suitable for use in a device described herein can comprise iron, water, vermiculite, charcoal, polymer, and salt. [0065] The devices described herein can be used to detect a target nucleic acid in a sample, the method comprising:

a. contacting the sample collection layer of a device described herein with the

sample;

b. opening or rupturing the first buffer reservoir;

c. agitating the device;

d. causing the device to spin about the axial element;

e. closing or sealing the at least one waste collection chamber;

f. optionally, opening or rupturing the second buffer reservoir if present;

g. optionally, opening or rupturing any primer/RPA capsules present;

h. warming the device to at least 35°C;

i. observing or detecting any visible signals in the visualization areas.

[0066] Contacting the sample collection layer 10 of a device with a sample can comprise using the device to wipe a biological surface (e.g., the anus or a wound), or removing a biological sample from a subject or storage container and applying it to the sample collection layer 10 by means of another tool, gravity, fluid pressure, digital manipulation, or the like. The sample collection layer 10 can then be removed, covered with an adhesive cover or sticker, or removed and the first water resistant-layer 20 covered with an adhesive cover or sticker. The sample collection layer 10 can be covered in whole or in part, depending on, e.g., the nature of the sample material and how it is applied to the sample collection layer 10. In some embodiments of any of the aspects, the sample collection layer 10 can then be removed and the first water-resistant layer 20 covered with an adhesive cover or sticker. The first water-resistant layer 20 can be covered in whole or in part, depending on, e.g., the nature of the sample material, how it is applied to the sample collection layer 10, and the design of the first water-resistant layer 20. Such adhesive covers or stickers can be included in the kits described herein.

[0067] Opening or rupturing the first buffer reservoir 34, can comprise the user applying pressure digitally or by any other means to cause the first buffer reservoir to rupture. The device is then agitated, e.g., by causing the device to be shaken. The agitation permits the sample to be physically broken down or disrupted by the sample agitation element(s) 32 as well as permitting the cell lysis buffer to act upon the sample. In some embodiments of any of the aspects, the device is shaken or agitated, at least partially, along one or more lines or directions found in the most-planar dimension of the device. [0068] The device is next spun or rotated about or with the axial element. The spinning or rotation can be accomplished using any device or digitally which causes the axial element and therefore the device to rotate. One such approach, termed“paperfuge” is describe in the Examples herein, but is not limiting. As further non-limiting examples, the axial element could be a stick which is turned or rubbed between fingers, hands, or a hand and a second surface; the axial element could form a top (i.e., the toy known as a tope)-like element that can be spun; or the axial element could be tethered at one or both ends to a solid support and the layers spun while the axial element remains stationary. The rotation or spinning will cause the sample and lysis buffer to transit the one or more microfluidic channels 36 and enter the one or more waste chambers 41. Nucleic acids present in the sample will be captured by the nucleic acid binding element 37 and particulate matter or sample agitation elements 32 can be retained in the sample collection chamber 31 by the particulate filter.

[0069] After completing the rotation or spinning of the device, the waste collection chamber 41, can be closed or sealed, e.g., to prevent the material therein re-entering the one or more microfluidic channels 36. The closing or sealing can be accomplished by causing the seperable or sealable element at the junction 42 of the waste collection chamber 41 and a microfluidic channel 36 to be separated or sealed. Such elements are described elsewhere herein and the separation or sealing can be accomplished by, e.g., applying pressure digitally to the element, e.g., from both sides of the device to cause crushing, collapsing, or to bring multiple aspects of the junction 42 into direct contact to achieve a mating of parts or joining by an adhesive. Other means of accomplishing the closing or sealing will be apparent to those of skill in the art depending on the identity of the the seperable or sealable element, e.g., a crimper tool could be provided with the device, or the user can apply heat (e.g., body heat or an external source) to the junction 42.

[0070] Optionally, the second buffer reservoir 35, if present, is then opened or ruptured, which permits the buffer contained therein to reach the nucleic acid binding element 37 and release the nucleic acid molecules retained there. The nucleic acid molecules can then enter the one or more microfluidic channels 36. Rupturable reservoirs and methods of causing them to rupture are described elsewhere herein. Optionally, the device can be spun or rotated again to encourage the buffer and nucleic acids to enter the one or more microfluidic channels 36. Alternatively, the movement of the liquid may be accomplished by capillary pressure and/or the pressure exerted by the opening or rupturing of the second buffer reservoir 35. In a further alternative, the nucleic acids molecules are not released from the nucleic acid binding element 37, e.g., when the nucleic acid binding element 37 is located within the microfluidic channel 36 and the RPA reaction will occur“on-element.” In embodiments utilizing the second buffer reservoir 35, the method of use can further comprise a step of causing the device to spin or rotate around or with the axial element again, e.g., to cause the buffer from the second buffer reservoir 35 to flow past the nucleic acid binding element(s) 37 and release the nucleic acids.

[0071] If a primer capsule 40 is present, the primer capsule 40 is then ruptured or opened. Rupturable reservoirs and methods of causing them to rupture are described elsewhere herein, and similar approaches can be used for the primer capsule 40. This causes the RPA reagents and/or mixes to come into contact with the nucleic acids which were present in the sample, and RPA will proceed. In some embodiments of any of the aspects, the user can be instructed to refrain from shaking or spinning the device after the second buffer reservoir 35 and/or primer capsule 40 rupture/opening, either for a set period of time and/or until one or more signals are visible in the nucleic acid visualization areas 39.

[0072] In some embodiments of any of the aspects, the device is warmed to at least 35°C after the second buffer reservoir 35 and/or primer capsule 40 rupture/opening, either for a set period of time and/or until one or more signals are visible in the nucleic acid visualization areas 39. In some embodiments of any of the aspects, the device is warmed to at least 37°C after the second buffer reservoir 35 and/or primer capsule 40 rupture/opening, either for a set period of time and/or until one or more signals are visible in the nucleic acid visualization areas 39. In some embodiments of any of the aspects, the device is warmed to a temperature of 35°C to 42°C after the second buffer reservoir 35 and/or primer capsule 40

rupture/opening, either for a set period of time and/or until one or more signals are visible in the nucleic acid visualization areas 39. In some embodiments of any of the aspects, the device is warmed to a temperature of 37°C to 49°C after the second buffer reservoir 35 and/or primer capsule 40 rupture/opening, either for a set period of time and/or until one or more signals are visible in the nucleic acid visualization areas 39.

[0073] In some embodiments of any of the aspects, the set period of time is at least 5 minutes. In some embodiments of any of the aspects, the set period of time is at least 10 minutes. In some embodiments of any of the aspects, the set period of time is at least 20 minutes. In some embodiments of any of the aspects, the set period of time is about 20 minutes. In some embodiments of any of the aspects, the set period of time is from 10 to 30 minutes. [0074] The heating of the device can be accomplished by any method of providing an increased temperature to the device without damaging the device, e.g., the device can be placed in an incubator set to the appropriate temperature, the device can be placed in a room or environment which is at an appropriate temperature, a group of exothermic reactants in the device can be activated to provide the appropriate temperature, the device can be placed in a water bath which is at an appropriate temperature, the device can be placed in a sealed container which is placed in a water bath which is at an appropriate temperature, or the device can be placed in close proximity to the surface of a subject’s skin.

[0075] In some embodiments of any of the aspects, an exterior-visible portion of the device, or a sealable bag or container provided with the device (e.g., a Tyvek bag) comprises a temperature-dependent, visually detectable signal. For example, the bag (or a portion thereof), will change color when the desired temperature is reached, permitting the user to reach and hold the desired temperature for the desired amount of time without access to laboratory equipment. Such temperature-sensitive color-change materials are known in the art and commercially available.

[0076] After the expiration of the set period of time, or after a signal is visible in at least one of the visualization areas 39, the visible signals are observed and/or detected (e.g, as viewed through viewing windows 51 in the second water-resistant layer 50). Observation and/or detected can be done by the human eye. For example, the device can be provided with a key or instructions that provide the user with the information necessary to interpret results, e.g., as in the case of home pregnancy test kits. In such embodiments, the possible results and number of visualization areas can be minimal to avoid confusion or incorrect

interpretation by lay users, e.g, the device can be designed to detect a single target or target class, such that a result is categorized as“yes” or“no.” Observation and/or detection by the human eye can also be performed by a medical professional. In such cases, the device can be provided with a key or instructions that provide the medical professional with the information necessary to interpret results. In such embodiments, the possible results and number of visualization areas can be complex enough to provide multiple possible outputs, e.g, the device can be designed to detect multiple targets or target classes (e.g., a multiplexed device). Particularly when the device is configured for evaluation by a computer or medical professional, the information can be interpreted beyond a binary“yes” or“no”, as the relative abundance of different targets may be informative and diagnostic. [0077] Observation and/or detection can also be performed with a device which can discern the signals. For example, a photograph of the visualization areas 39 can be taken (e.g, as viewed through viewing windows 51 in the second water-resistant layer 50) using a smartphone, tablet, digital camera, or the like. The captured image can then be analysed by a computer program and/or medical professional in order to interpret the results.

[0078] Interpretation of the results will depend on the identity of the pairs of nucleic acid primers provided in the device. For example, a given device may be provided that will permit screening of a sample for a single pathogen, or a device may be provided that will permit screening of a sample to determine which (if any) of a group of different pathogens is present. In the later case, the device can be provided to detect whether any of a group of pathogens that cause similar symptoms/etiology, or which are found in the same types of samples are present. For example, devices could be distributed to an area suffering an outbreak of gastrointestinal illness and subjects screened to determine if they have

Salmonella enterica , Vibrio cholera , or Eschericia coli present in their feces or fecal matter. In such a scenario, a device with a 3-4 visualization areas could provide sufficient information for a user to determine if they are a carrier/presymptomatic; for local relief workers to implement early or preventative care, track effectiveness of prevention methods, or select optimtal treatment/support strategies or epidemiological controls; and for medical professionals (either remote or on-site) to further track the epidemic and direct individual treatment. The device is provided with markings or instructions that provide the user with the necessary information to interpret the presence/absence of a signal in any given visualization area 39 or combination of visualization areas 39.

[0079] In some embodiments of any of the aspects, one or more visualization areas 39 or a mock visualization area not actually having a junction with a microfluidics channel 36 is provided with a predetermined concentration of one or more target sequences, or RPA reaction products, e.g., to serve as a control or reference marker, particularly when using photography, computers or the like to record, transmit, or analyze the results.

[0080] In one aspect, described herein is a kit comprising a device as described herein. A kit is any manufacture (e.g., a package or container) comprising at least one article, e.g., the device described herein, the manufacture being promoted, distributed, or sold as a unit for performing the methods described herein. The kits described herein can optionally comprise additional components useful for performing the methods described herein. By way of example, the kit can comprise one or more adhesive stickers or covers, a sealable bag or other container for placing the device in a water bath, one or more bags or containers for dispoal of biowastes and/or devices, a bag or container for storage/shipment of a device after use, items for sample collection (e.g., wipes, needles, swabs, and the like), an instructional material which describes performance of a method as described herein, and the like. Additionally, the kit may comprise an instruction leaflet and/or may provide information as to the relevance of the obtained results. Instructions can be printed on a separate leaflet, on the device, or on the packaging of the kit itself. Instructions can further provide a means (e.g., URL or QR code) for accessing complete instructions, including videos and timers, by way of a smartphone, tablet, computer, or the like. Instructions can also further provide a means of identifying the specific user, e.g., a unique identifier can be incorporated into the URL or QR code, or accessing a computer system via the URL or QR code can prompt the user to provide identifying information or be assigned a unique identifier.

[0081] As used herein“junction” refers to the interface between two elements. A junction does not necessarily indicate whether the two elements are connected or in communication in an operable manner, e.g., whether a fluid can travel from the first to the second element, merely that there is a physical interface between the two elements. The physical interface can comprise a wall, membrane, port, or the like which can be opened, ruptured, degraded, etc to provide intercommunication between the elements or their interior spaces. As used herein,“connection” refers to a junction where the two elements are joined or in communication in an operable manner, e .g., a fluid can travel from the first to the second element.

[0082] For clarity, where the placement of devices is referred to as“proximal” or“distal” herein, reference is made to the most-planar dimension of a layer. The center of that dimension is most proximal and the outer edge of that layer is most distal.

[0083] As used herein, the term“channel” refers to any capillary, channel, tube, or groove that is deposed within or upon a substrate. A channel can be a microchannel; i.e. a channel that is sized for passing through microvolumes of liquid.

[0084] A“chamber” refers herein to a space in or on a substrate that can retain and/or allow passage of an aqueous solution (e.g., water or buffer) and optionally, sample biomaterial and/or other elements described herein (e.g. sample agitation elements). A chamber can be a well, a depression, a channel, or an area bounded by walls. A chamber can comprise a junction or connection with another element described herein. A“reservoir” is a type of chamber that is initially configured such that there is no communication of the reservoir’s interior with the interior of any other chamber, reservoir, or channel but that such communication can be provided (e.g., the reservoir has one or more rupturable walls or a valve that can be opened). Provision of such communication can be reversible (e.g. a valve that can be moved between open and closed positions) or irreversible (e.g., a rupturable capsule). A“capsule” refers to a type of reservoir having a shell or cap which encloses an interior space. The interior space may be empty or may be fully or partially filled with a solution (e.g. a buffer or reaction mixture).

[0085] It should be noted that in referring to“water-resistant” layers (e.g., 20, 50), reference is made to the material of which the layer is made (or the outer coating/surface thereof), and it is not implied that aqueous solutions (e.g., water) cannot pass the layer at all. Rather, water and aqueous solutions will pass through the layer only where holes, slits, or openings are provided; as described above herein. As used herein, the term "water-resistant" refers to a tendency to repel, block or not significantly absorb or transmit aqueous solutions (e.g., water) in normal use and connotes at least substantial aqueous solution (e.g., water or buffer) blocking properties as opposed requiring total or complete aqueous solution (e.g., water or buffer) blocking properties. The water-resistant layers can optionally be aqueous solution (e.g., water or buffer) -impermeable or aqueous solution (e.g., water or buffer)-proof layers.

[0086] The layers (and various elements and structures) of the device described herein can be formed, such as by molding, cutting, printing, stamping, etching, 3-D printing, machining, or micro-machining.

[0087] The layers described herein can be made of a biocompatible flexible material or a biocompatible non-flexible material according to the design and application requirements. It should be noted that the designs depicted in the Figures are exemplary and the device described herein is not limited to the configurations shown in the Figures. The water-resistant layers 20, 50 and microfluidics layer 30 and/or portions thereof can be made of a flexible material, including but not limited to, a biocompatible material such as polydimethyl siloxane (PDMS), polyurethane or polyimide. The water-resistant layers 20, 50 and microfluidics layer 30 and/or portions thereof can also be made of non-flexible materials like paper, glass, silicone, polysulfone, hard plastic, and the like, as well as combinations of these materials.

[0088] A biocompatible polymer refers to materials which do not have toxic or injurious effects on biological functions. Biocompatible polymers include natural or synthetic polymers. Examples of biocompatible polymers include, but are not limited to, collagen, poly(alpha esters) such as poly(lactate acid), poly(glycolic acid), polyorthoesters and polyanhydrides and their copolymers, polyglycolic acid and polyglactin, cellulose ether, cellulose, cellulosic ester, fluorinated polyethylene, phenolic, poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether, polyester, polyestercarbonate, polyether, polyetheretherketone, polyetherimide, polyetherketone, polyethersulfone, polyethylene, polyfluoroolefm, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea-formaldehyde, polyglactin, or copolymers or physical blends of these materials.

[0089] A biocompatible material can also be, for example, ceramic coatings on a metallic substrate. But any type of coating material and the coating can be made of different types of materials: metals, ceramics, polymers, hydrogels or a combination of any of these materials. Biocompatible materials include, but are not limited to an oxide, a phosphate, a carbonate, a nitride or a carbonitride. Among the oxide the following ones are preferred: tantalum oxide, aluminum oxide, iridium oxide, zirconium oxide or titanium oxide. Substrates are made of materials such as metals, ceramics, polymers or a combination of any of these. Metals such as stainless steel, Nitinol, titanium, titanium alloys, or aluminum and ceramics such as zirconia, alumina, or calcium phosphate are of particular interest.

[0090] The biocompatible polymer may be shaped using methods such as, for example, solvent casting, compression molding, filament drawing, meshing, leaching, weaving and coating. In solvent casting, a solution of one or more polymers in an appropriate solvent, such as methylene chloride, is cast as a branching pattern relief structure. After solvent

evaporation, a thin film is obtained. In compression molding, a polymer is pressed at pressures up to 30,000 pounds per square inch into an appropriate pattern. Filament drawing involves drawing from the molten polymer and meshing involves forming a mesh by compressing fibers into a felt-like material. In leaching, a solution containing two materials is spread into a shape close to the final form of the RUG. Next a solvent is used to dissolve away one of the components, resulting in pore formation. (See Mikos, U.S. Pat. No.

5,514,378, hereby incorporated by reference). In nucleation, thin films in the shape of a RUG are exposed to radioactive fission products that create tracks of radiation damaged material. Next the polycarbonate sheets are etched with acid or base, turning the tracks of radiation- damaged material into pores. Finally, a laser may be used to shape and burn individual holes through many materials to form a RUG structure with uniform pore sizes. Coating refers to coating or permeating a polymeric structure with a material such as, for example liquefied copolymers (poly-DL-lactide co-glycolide 50:50 80 mg/ml methylene chloride) to alter its mechanical properties. Coating may be performed in one layer, or multiple layers until the desired mechanical properties are achieved. These shaping techniques may be employed in combination, for example, a polymeric matrix may be weaved, compression molded and glued together. Furthermore different polymeric materials shaped by different processes may be joined together to form a composite shape. The composite shape may be a laminar structure. For example, a polymeric matrix may be attached to one or more polymeric matrixes to form a multilayer polymeric matrix structure. The attachment may be performed by gluing with a liquid polymer, by suturing, or by heat press (e.g., melting or fusing). In addition, the polymeric matrix may be formed as a solid block and shaped by laser or other standard machining techniques to its desired final form. Laser shaping refers to the process of removing materials using a laser.

[0091] The term“sample” or“test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a blood, stool, mucus, or plasma sample from a subject. In some embodiments of any of the aspects, the present invention encompasses several examples of a biological sample. In some embodiments of any of the aspects, the biological sample is cells, or tissue, or peripheral blood, bodily fluid, or bodily wastes, secretions, or excretions. Exemplary biological samples include, but are not limited to, a biopsy, a tumor sample, biofluid sample; blood; serum; plasma; urine; feces; sperm; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva; and/or tissue sample etc. The term also includes a mixture of the above-mentioned samples. The term“test sample” also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments of any of the aspects, a test sample can comprise cells from a subject. In some embodiments of any of the aspects, a test sample can comprise microorganisms from a subject.

[0092] The test sample can be obtained by removing a sample from a subject, but can also be accomplished by using a previously isolated sample (e.g. isolated at a prior timepoint and isolated by the same or another person).

[0093] In some embodiments of any of the aspects, the test sample can be an untreated test sample. As used herein, the phrase“untreated test sample” refers to a test sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments of any of the aspects, the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein. After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein. In some embodiments of any of the aspects, the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample. In some embodiments of any of the aspects, a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof. In some embodiments of any of the aspects, the test sample can be treated with a chemical and/or biological reagent. Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing. The skilled artisan is well aware of methods and processes appropriate for pre-processing of biological samples required for determination of the level of an expression product as described herein.

[0094] In some embodiments of any of the aspects, the methods, described herein can further comprise a step of obtaining or having obtained a test sample from a subject. In some embodiments of any of the aspects, the subject can be a human subject.

[0095] The samples described herein comprise biological matter or biological material. “Biological matter” or“biological material”, used interchangeable herein refer to any matter or material obtained from a subject which comprises at least one of, tissue, fluids, cells, bodily waste products, and/or microorganisms. The biological matter or material is manipulated and/or seperated in the methods and devices described herein, but the resulting products and portions retain their identity as“biological matter” or“biological material.” [0096] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0097] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[0098] The terms“decrease”,“reduced”,“reduction”, or“inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments,“reduce,” “reduction" or“decrease" or“inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level.“Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0099] The terms“increased”,“increase”,“enhance”, or“activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”,“increase”,“enhance”, or“activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a lO-fold increase, or any increase between 2-fold and lO-fold or greater as compared to a reference level. In the context of a marker or symptom, a“increase” is a statistically significant increase in such level.

[00100] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus.

Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms,“individual,”“patient” and“subject” are used interchangeably herein.

[00101] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. A subject can be male or female.

[00102] As used herein, the terms“protein" and“polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and“polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[00103] As used herein, the term“nucleic acid” or“nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA.

[00104] In various embodiments, the methods and compositions described herein relate to performing a RPA regimen with at least one pair of oligonucleotide primers. As used herein, “primer” refers to a DNA or RNA polynucleotide molecule or an analog thereof capable of sequence-specifically annealing to a polynucleotide template and providing a 3' end that serves as a substrate for a template-dependent polymerase to produce an extension product which is complementary to the polynucleotide template. A primer useful in the methods described herein is generally single-stranded, and a primer and its complement can anneal to form a double-stranded polynucleotide. Primers according to the methods and compositions described herein can be less than or equal to 300 nucleotides in length, e.g., less than or equal to 300, or 250, or 200, or 150, or 100, or 90, or 80, or 70, or 60, or 50, or 40, and preferably 30 or fewer, or 20 or fewer, or 15 or fewer, but at least 10 nucleotides in length.

[00105] A pair of primers comprises at least one forward primer and at least one reverse primer specific for a target sequence, one of which anneals to a first strand of a target nucleic acid sequence and the other of which anneals to a complement of the first strand. The orientation of the primers when annealed to the target and/or its complement can be such that nucleic acid synthesis proceeding from primer extension of a one primer of the primer pair subset would produce a nucleic acid sequence that is complementary to at least one region of the second primer of the primer pair subset. The“first strand” of a nucleic acid target and/or sequence can be either strand of a double-stranded nucleic acid comprising the sequence of the target nucleotide, but once chosen, defines its complement as the second strand. Thus, as used herein, a“forward primer” is a primer which anneals to a first strand of a nucleic acid target, while a“reverse primer” of the same set is a primer which anneals to the complement of the first strand of the nucleic acid target.

[00106] As used herein,“specific” when used in the context of a primer specific for a target nucleic acid refers to a level of complementarity between the primer and the target such that the primer will anneal to and mediate amplification of the target nucleic acid and will not anneal to or mediate amplification of non-target sequences present in a sample under the conditions described herein.

[00107] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable

accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and/or to the translation of mRNA into a polypeptide.

[00108] In some embodiments, the expression of a biomarker(s), target(s), or

gene/polypeptide described herein is/are tissue-specific. In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is/are global. In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is systemic.

[00109] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[00110] "Marker" in the context of the present invention refers to an expression product, e.g., nucleic acid or polypeptide which is differentially present in a sample taken from subjects having having a condition, as compared to a comparable sample taken from control subjects (e.g., a healthy subject). The term "biomarker" is used interchangeably with the term "marker."

[00111] In some embodiments of any of the aspects, the primer pairs described herein are specific for a target, e.g., a target nucleic acid found or expressed on a particular cell type. Detection of the target indicates the presence in the subject of the associated particular cell type. The target can also be a mRNA, wherein detection of the target indicates expression of the target.

[00112] In some embodiments, the methods and devices described herein relate to measuring, detecting, or determining the presence/level of at least one marker or target. As used herein, the term "detecting" or“measuring” refers to observing a signal from, e.g. a probe, label, or target molecule to indicate the presence of an analyte in a sample. In some embodiments of any of the aspects, measuring can be a quantitative observation.

[00113] As used herein,“contacting" refers to any suitable means for delivering, or exposing, the device with a sample. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.

[00114] The term“statistically significant" or“significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[00115] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term“about.” The term“about” when used in connection with percentages can mean ±1%.

[00116] As used herein, the term“comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[00117] The term "consisting of' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[00118] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[00119] As used herein, the term“specific binding” refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.

[00120] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[00121] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can 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 herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[00122] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978- 0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN

9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081- 569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's

Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewiris Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X,

9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[00123] Other terms are defined herein within the description of the various aspects of the invention. [00124] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[00125] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[00126] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[00127] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.

[00128] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs: A device comprising layers in the following order:

a. Optionally, a sample collection layer comprising at least one opening;

b. A first water-resistant layer comprising at least one opening;

c. A microfluidics layer comprising:

i. a collection chamber aligned with the at least one opening in the first

water-resistant layer;

ii. at least a first buffer in a first buffer reservoir;

iii. at least one microfluidic channel comprising:

1. a nucleic acid binding element;

2. a pair of nucleic acid primers;

3. the proximal zone of the microfluidic channel having a junction with the collection chamber and;

4. the distal zone of the microfluidic channel having a junction with at least one waste collection chamber;

iv. a nucleic acid visualization area comprising a western blot or lateral flow assay which has a junction with the microfluidic channel; d. A second water-resistant layer; and

e. An axial element positioned to pass through each of the layers. The device of paragraph 1, wherein the layers are circular or cylindrical in shape.

The device of any of paragraphs 1-2, wherein the width, length, and/or diameter of each of the layers is substantially the same as that of the other layers.

The device of any of paragraphs 1-2, wherein the width, length, and/or diameter of each of the layers is within 10% of the average for the layers.

The device of any of paragraphs 1-2, wherein the width, length, and/or diameter of each of the layers is the same as that of the other layers The device of any of paragraphs 1-5, wherein the sample collection layer comprises toilet paper, tissue paper, baby wipe material, personal hygiene wipe material, sanitary napkin material, tampon material, or the like.

The device of any of paragraphs 1-6, wherein the at least one opening in the sample collection layer is a slit or hole which traverses from the exterior of the layer to the side of the layer facing the first water-resistant layer. The device of any of paragraphs 1-7, wherein the at least one opening in the sample collection layer is closer to the center of the layer’s most-planar dimension than to the edge of the layer’s most-planar dimension.

The device of any of paragraphs 1-8, wherein the sample collection layer is disposable or removable.

The device of paragraph 9, wherein the sample collection layer is adhered to the first water-resistant layer with a chemical or physical adhesive. The device of any of paragraphs 1-10, wherein the at least one opening in the first water- resistant layer is a slit or hole which traverses from the side of the layer facing the sample collection layer to the side of the layer facing the microfluidics layer.

The device of any of paragraphs 1-11, wherein the at least one opening in the first water- resistant layer is closer to the center of the layer’s most-planar dimension than to the edge of the layer’s most-planar dimension.

The device of any of paragraphs 1-12, wherein the at least one opening in the first water- resistant layer is aligned with the at least one opening in the sample collection layer. The device of paragraph 13, wherein the at least one opening which is aligned with the at least one opening in the sample collection layer is a slit.

The device of any of paragraphs 1-14, wherein the first-water resistant layer comprises at least one opening which is not aligned with the at least one opening in the sample collection layer.

The device of paragraph 1-15, wherein the at least one opening which is not aligned with the at least one opening in the sample collection layer is a mesh, hole, or slit. The device of any of paragraphs 1-16, wherein the one or more microfluidic channels are hydrophobic.

The device of any of paragraphs 1-17, wherein the collection chamber is located substantially in the center of the microfluidic layer’s most-planar dimension.

The device of any of paragraphs 1-18, wherein the collection chamber further comprises a sample agitation element.

The device of paragraph 19, wherein the sample agitation element is one or more beads. The device of any of paragraphs 1-20, wherein the one or more microfluidic channels and/or the collection chamber comprise a particulate filter designed to retain large sample particulate or sample agitation elements in the collection chamber or proximal portion of the one or more microfluidic channels.

The device of paragraph 21, wherein the particulate filter is a nylon filter.

The device of any of paragraphs 1-22, wherein the first buffer reservoir comprises a lysis buffer.

The device of paragraph 23, wherein the lysis buffer further comprises RNase and a binding agent.

The device of any of paragraphs 1-24, wherein the collection chamber comprises a second buffer in a second, separate buffer reservoir and the second bugger is a buffer that can release nucleic acids from the nucleic acid binding element.

The device of paragraph 25, wherein the buffer than can release nucleic acids is TE buffer.

The device of any of paragraphs 1-26, wherein each buffer reservoir is a rupturable capsule that can be ruptured by applying force to the region of the device comprising the buffer reservoir.

The device of paragraph 27, wherein the force is no more than half the force required to rupture the water-resistant layers or the microfluidics layer.

The device of any of paragraphs 1-28, wherein the microfluidic layer comprises a plurality of microfluidic channels and each microfluidic channel comprises a single pair of nucleic acid primers.

The device of any of paragraphs 1-29, wherein the microfluidic layer comprises a plurality of microfluidic channels and each microfluidic channel comprises a single unique pair of nucleic acid primers.

The device of any of paragraphs 1-30, wherein the at least one pair of nucleic acid primers is provided in a rupturable primer capsule.

The device of paragraph 31, wherein the capsule comprising the pair of nucleic acid primer further comprises one or more RPA reagents.

The device of any of paragraphs 1-32, wherein at least one pair of nucleic acid primers is specific for a pathogenic microorganism, a microorganism found in the subject species’ microbiome, or a microbial gene that affects host drug metabolism.

The device of any of paragraphs 1-33, wherein the junction between the microfluidic channel and the waste chamber comprises a separable or sealable element. The device of any of paragraphs 1-34, wherein the visualization area comprises an LFA assay.

The device of paragraph 35, wherein the LFA assay comprises i) a LFA-compatible pad or pads and iii) gold particles, latex, and/or carbon nanotubes.

The device of any of paragraphs 1-36, wherein the junction of the microfluidic channel and the visualization area comprises a rupturable or degradable barrier. The device of any of paragraphs 1-37, wherein the second water-resistant layer comprises viewing windows aligned with one or more of the visualization areas.

The device of any of paragraphs 1-38, wherein the second water-resistant layer comprises openings, indentations, or thinner zones aligned with one or more of the rupturable capsules. The device of any of paragraphs 1-39, wherein the axial element is located substantially at the center of each layer’s most-planar dimension.

The device of any of paragraphs 1-39, wherein the axial element is located at the center of each layer’s most-planar dimension.

The device of any of paragraphs 1-39, wherein the axial element is not located at the center of each layer’s most-planar dimension.

The device of any of paragraphs 1-42, wherein each layer comprises a portal or opening with an aperture which is not contiguous with any other element of the layer, and the axial element passes through the apertures.

The device of any of paragraphs 1-43, wherein the axial element is a tensile element forming a closed loop and extending beyond the exterior faces of the sample collection layer and the second water-resistant layer.

The device of paragraph 44, wherein each layer comprises two portals or openings, each with an aperture which is not contiguous with any other element of the layer, and the axial element passes through the apertures. A method of detecting a microorganism or microbial gene in a sample, the method comprising:

a. contacting the sample collection layer of a device of any of paragraphs 1-45 with the sample; b. opening or rupturing the first buffer reservoir;

c. agitating the device;

d. causing the device to spin about the axial element;

e. closing or sealing the at least one waste collection chamber;

f. optionally, opening or rupturing the second buffer reservoir;

g. optionally, opening or rupturing any primer capsules present;

h. warming the device to at least 35°C;

i. observing or detecting any visible signals in the visualization areas.

47. The method of paragraph 46, wherein when step f is performed, the method comprises a further step after step f of causing the device to spin about the axial element.

48. The method of any of paragraphs 46-47, wherein step h comprises warming the device to 37°C-39°C.

49. The method of any of paragraphs 46-47, wherein in step h, the device is maintained at the temperature of at least 35°C for about 20 minutes or until at least one signal is visible in at least one visualization area.

50. The method of any of paragraphs 46-49, wherein step h comprises placing the device in a water bath, placing the device in a sealed container which is placed in a water bath, or placing the device in close proximity to the surface of a subject’s skin.

51. The method of any of paragraphs 46-50, wherein the method further comprises a step after a. and before b. of sealing all or a portion of the sample collection layer’s outer surface or removing the sample collection layer and sealing all or a portion of the first water-resistant layers’ outer surface.

52. The method of paragraph 52, wherein the sealing comprises application of an adhesive cover or sticker.

EXAMPLES

[00129] Example 1: Smart Toilet Paper - Paper-based microbial screening

[00130] There is an increasing amount of research within the intersection of the human microbiome and human health - finding correlations between certain types of microbial communities and diseases. About 100 trillion microbes live within the human body.

Currently, technologies (including kits) allow researchers to extract these microbial DNA sequences from fecal samples. With most of the microbes living within the human gut, studying fecal samples offers a generous window into the overall human microbiome. The instruments used today are highly advanced, as they offer high-precision measurements, but they are also very costly. Among them are high-speed centrifuge machines, polymerase chain reaction (PCR) thermocyclers, and electrophoresis chambers. These correlate with the three main steps to obtain DNA markers from fecal sample: (1) extraction of DNA from the sample through a series of lysis buffers (centrifuge is required to mix the sample with the lysis buffers and separate the DNA from the rest of the cells), (2) amplifying of the DNA with a set of given primers using PCR, and (3) visualizing the DNA against known DNA markers of interest.

[00131] The‘Smart Toilet Paper’ technology described herein combines and simplifies the three functions mentioned above (extraction, amplification, and visualization) into one paper- based microbial screening. It is meant to be used in field research as well as in the house for an easy sample and data collection and basic health screening. This product combines a few known simple technologies that have already been proven successful in their individual applications.

[00132] A recent invention,‘Paperfuge’, developed by researcher Manu Prakash (Stanford University) is able to utilize the physics behind the old string-spin toy and reproduce the effects of an ultracentrifuge. In his application, the‘Paperfuge’ was used to separate individual components from blood samples, which can help detect malaria. Speeds can reach up to 125,000 rpm. Rather than for blood samples, the‘Smart Toilet Paper’ utilizes this simple technology to extract DNA sequences.

[00133] Instead of performing a conventional PCR, there is another recent method, loop mediated isothermal amplification (LAMP), which uses a single temperature rather than alternating between multiple temperatures as seen in PCR. LAMP operates at a temperature of 60-65 °C. In order to achieve this temperature, the‘Smart Toilet Paper’ employs another existing technology commonly found in the supermarket. Single-use heat packets consist of iron, water, vermiculite, charcoal, polymer, and salt, which when exposed to air, produces the required heat. There are recent studies that have also shown the ability to replace the gel, typically used in gel electrophoresis to visualize incoming DNA, with paper. The required high voltage can be generated by a simple rotor/stator or permanent magnet combination.

[00134] Simple microfluidics devices, including those formed of paper, also serve main functions for the‘Smart Toilet Paper’ in delivering fluids from one step to the next. [00135] Product Components/Specifications: Refer to Fig. 1 for an exemplary

embodiment.

[00136] The‘Smart Toilet Paper’ consists of 4 main layers: (1) disposable paper layer, (2) a water resistant layer, (3) water resistant microfluidics layer, and (4) another water resistant layer. For optimized balance, these layers are in the shape of identical circles.

[00137] The disposable paper layer is similar to that of any existing toilet paper. It is meant to the primary wipe onto which most of the fecal material is smeared. There are slits near the center that allow fecal matter to penetrate through this layer and into the center areas of second and third layers. Upon wiping, this layer would be discarded.

[00138] The water-resistant layer serves as a top layer over the microfluidics layer. Within it are a plurality of small holes spaced slightly apart from each other and located around the center of the circular paper. This layer also has slits, aligned directly those on with layer 1.

[00139] The microfluidics layer is of a material that comprises of a series of hydrophobic channels formed into them. The center has a compartment to allow for the lysis buffer solutions. Around the junction between the compartment and channels are filters. Along the length of each channel there is a specific DNA primer encased in buffer soluble capsules. Along the distal end of each channel is a dedicated area on which to visualize the DNA. In the areas not utilized by the channels, exothermic reactants are embedded into the layer.

[00140] The second water-resistant layer is similar to layer two, except without the formed slits.

[00141] A tensile element (i.e. string) is looped through the central holes across layers 2-4 and formed in a closed loop. This element serves as a main mechanism for the spinning process.

[00142] Example 2

[00143] Described herein is a simple, paper-based device that can quantify specific elements of the gut microbiome from fecal samples.

[00144] HUMAN MICROBIOME

[00145] The human body hosts over 39 trillion microbes, most of which contribute towards good health - in terms of helping us digest foods, develop our immune system, and help the overall body systems function. A large majority of these microbes reside within the intestinal tract, therefore stool samples extracted from the body can often be used as a non- invasive means to link microbiome with human health. [00146] Current research within the field of microbiome analyzes: (1) which microbial species are present, (2) their functionality, and (3) their chemistries. These studies range from seeing how the microbiome impacts the overall human immune system development, to detecting microbial communities that may lead to both infectious and chronic diseases, and to identifying microbial characteristics that effect drug metabolism. As a proof of concept, the Smart Toilet Paper project uses the case of the cardiac glycoside drug, Digoxin, to demonstrate its potential applicability.

[00147] Atrial fibrillation affects about 2.7-6.1 million individuals in the U.S. and about 33.5 million individuals globally. The cardiac glycoside drug, Digoxin, used to treat patients with atrial fibrillation, has a narrow therapeutic window. Current research shows how certain microbial communities within the body can inhibit the drug and render the initial prescribed dosage to be inaccurate (Haiser HJ et al. (2013) Predicting and manipulating cardiac drug inactivation by the human gut bacterium Eggerthella lenta. Science 341 : 295-298).

[00148] Most individuals are host to the bacterial species known as Eggerthella lenta. A subset of E. lenta community within the body may contain a cgr operon within their genetic make-up, which have been shown to be high inhibitors of Digoxin. The Smart Toilet Paper aims to visualize the ratio of E. lenta with to those without the cgr operon to determine the level of drug inhibition within each patient. Other studies have repeatedly shown that the microbiome changes quite rapidly depending on diet and life style. The Smart Toilet Paper would serve as a quick point-of-care measuring device to verify whether or not the current dosage is adequate.

[00149] Although the procedures and instruments used to analyze and measure the microbiome exist, they are complex and expensive and therefore inaccessible for most individuals who need to frequently monitor their microbiome to keep their dosage (of Digoxin) in check. Provided herein is a simple and affordable device that can effectively track the human microbiome for these specific microbial communities.

[00150] In order to produce a device that can replicate and condense the steps required to produce meaningful data directly from fecal samples, it is necessary understand the science behind the protocols. In the most basic terms, the device would need to (1) break down the fecal matter, (2) extract the microbial DNA fragments, (3) amplify the targeted DNA sequences, and (4) visualize the concentrations of the targeted DNA sequences found in the fecal sample. The science experiments performed on the device have adapted and modified (for simplificity) DNA extraction and amplification kit protocols. [00151] Certain embodiments of the compositions can include elements from the

Paperfuge. The Paperfuge was developed as a simple and affordable tool for detecting malaria. It consists of a paper disc with a looped string threaded through the center.

Additionally, attached to the disc is a sealed small plastic straw to hold the blood sample. Based on the common spin toy, the Paperfuge is able to separate plasma from blood samples (in order to detect pathogens) by replicating the spinning mechanism of a micro centrifuge (spinning at up to 20,000 rpm, assuming a radius of 50mm) (Fig. 2).

[00152] One of the most common methods to extract DNA from its host cells is through the spin-column method. The cells are first lysed (where the cell walls and components are broken down to expose the DNA fragments) using a lysis buffer. The mixture is then pipetted into a spin column and spun down in a micro centrifuge. A spin column is a standard tube which has a silica membrane at its bottom tip and typically has an attached waste tube.

Because silica has a positively charged surface, DNA (negatively charged on its surface) tends to bind to the silica under certain pH levels and salt concentrations, while all other cell components flow through and collect in the waste container. An early prototype consists of a series of tubes (including the spin column) attached to a chipboard disc. The lysate and live E.Coli cells are inserted into the tubes and spun down by hand using the disc-and-string spinning method (Figs. 2,3).

[00153] ON DEVICE : DNA EXTRACTION

[00154] In one embodiment, the design of the device accommodates the steps (previously mentioned) required to extract the microbial DNA. After wiping a small amount of fecal matter into the device, the user would pop the first bubble which contains the lysis buffer, RNAse, and binding agent. A gentle shake would allow the fecal matter to mix with the lysate to break open the cells. The device would then be spun so that everything but the DNA would pass through the silica membranes and into the waste chamber. The collection chambers would need to be sealed by applying pressure onto the designated areas prior to popping the second bubble. TE buffer, contained in the second bubble, would then release the captured DNA from the silica membranes and allow for the DNA to be collected (Fig. 5 and 10).

1. fecal matter is introduced

2. bubble 1 is popped (lysis, RNAse, binding)

3. fecal matter is mixed with the solutions from bubble 1

4. bacterial cells in fecal matter are lysed 5. device is spun

6. all fluids migrate through the silica membranes

7. all fluids (except the DNA) are collected in the waste chamber

8. DNA fragments are bound to the silica membranes

9. collection chambers are sealed by pressure

10. bubble 2 is popped (TE buffer)

11. device is spun, TE buffer migrate through silica membranes

12. DNA fragments are released from silica membranes and collected

[00155] DNA AMPLIFICATION

[00156] In order to visualize whether certain DNA sequences are present there needs to be enough signal to read (in other words, an increased concentration of those targeted sequences). DNA amplification is a method used to multiply DNA fragments that contain specific sequences. The specificity is established using primers (forward and reverse) that bookend the targeted sequence. Typically, the targeted sequence is much longer than what is shown below.

[00157] The target sequence is pre-established based on known genome sequencing and its known effects. In the case of Digoxin, the cgr operon gene along with the identifying gene for EJenta are the two primary targeted sequences. For the current prototype, E.Coli is used as a proof of concept to evaluate the device’s capability to identify the presence of a given species.

The conventional method for DNA amplification is known as Polymerase Chain Reaction (PCR), which typically takes about 120 mins and requires a thermocycler (95°C, 50°C, 72°C) to repeatedly split the DNA into single strands and allow for new nucleotides to bind and produce new copies of double-stranded DNA. The amplification method proposed for the Smart Toilet Paper is called Recombinase Polymerase Amplification (RPA). This method uses enzymes, rather than the alternating temperatures, to aid in the splitting and binding processes, and therefore can be isothermal (Fig. 4).

[00158] The primers used for the experiment were selected from Bauer, Andreas Peter, Sarah Maria Dieckmann, Wolfgang Ludwig, and Karl-Heinz Schleifer. 2007.“Rapid

Identification of Escherichia Coli Safety and Laboratory Strain Lineages Based on

Multiplex-PCR.” FEMS Microbiology Letters 269 (1): 36-40. (Tn7f for E.Coli strain C).

[00159] ON DEVICE : DNA AMPLIFICATION [00160] After the DNA has been extracted and collected, a series of small bubbles are popped to release the RPA reagents. The device should be put into its pouch/packaging and then placed either in warm water (~ 37-39°C) or on the body (~ 37°C body temperature) for about 20 minutes while the DNA sequences are being amplified. The pouch for the Smart Toilet Paper also includes a thermosensitive indicator which changes color when the optimal temperature has been reached. Each of the bubbles contain a unique set of primers that correspond to general E.lenta, E.lenta with cgr operon, E.lenta without cgr operon, and general bacteria (for the DNA reserve) (Fig. 6).

1. DNA has been collected

2. series of four small bubbles are popped

3. RPA reagents are released into each collection area

4. 4-6 DNA sequences are amplified

[00161] THE SCIENCE : VISUALIZATION

[00162] The most standard way to visualize the presence of specific sequences post amplification is through a gel electrophoresis. In this process, fluorescent dye is introduced into the gel mixture. DNA is pipetted into preformed wells within the gel and a current is applied across the gel, producing positive and negative charges on either ends. The dye in the gel binds to the DNA strands and migrate across the gel toward the positive charge. Since the dye is fluorescent, the bands need to be seen under UV light.

[00163] The goal for the Smart Toilet Paper is to reduce the need for equipment in every aspect. There are currently two basic methods that can visualize the results under normal lighting or daylight: (1) Western blot and (2) lateral flow assay, both of which use nano gold particles (seen as red) rather than fluorescent dye. While the Western blot requires a wash buffer to remove additional gold particles that are not adhered to DNA, lateral flow assay simply relies on capillarity of the pads to“wash” away the excess. For this project, the lateral flow assay method is more ideal since it eliminates the need for additional washes. Although lateral flow strips are often used to detect binary conditions (either there is some presence or none at all), current research also show their capability to quantify the resulting

concentrations through varying values of red. See, e.g., Gasperino, David T, Daniel Leon, Barry Lutz, David M. Cate, Kevin P. Nichols, David Bell, and Bernhard H. Weigh 2018. “Threshold-Based Quantification in a Multiline Lateral Flow Assay via Computationally Designed Capture Efficiency.” Analytical Chemistry 90 (11): 6643-50 and “Streptavidin Gold Conjugates - Cytodiagnostics.” n.d. Accessed May 9, 2019, available on the world wide web at cytodiagnostics.com/store/pc/ Streptavidin-Gold-Conjugates-cl83.htm.

[00164] ON DEVICE : VISUALIZATION

[00165] After 20 minutes of amplification, the material separating the collection chamber begins to break away and allow the solution to seep through the lateral flow pads. After about 10 minutes, the results can be visualized from the bottom side of the device - in various forms of red, depending on the resulting concentrations per chamber. The results would need to be photographed (i.e. with a smart phone) and either uploaded to the Smart Toilet Paper app or sent directly to the designated health physician. The computer vision algorithm on the app (either on the phone or on the physician’s computer) would be able to quantify the resulting concentrations based on a reference red patch located on the device (Fig. 7).

1. separating material breaks away

2. DNA and gold particles migrate across the lateral flow pads

3. results can be seen from the bottom side (Fig. 9)

[00166] The current experiments have successfully shown a version of the device able to extract the DNA from E.Coli (strain C) cells and amplify the same DNA sample using two sets of primers (correct/positive and incorrect/negative). The results have been examined using a gel electrophoresis.

[00167] Additionally, given the increased use of smart phones in the bathroom setting, a set of interactive instructions (with set timers) can also be accessed through an app.

[00168] It is contemplated herein that embodiments of the technology can be used for, e.g_·,:

[00169] The direct link of digoxin efficacy to the human microbiome has already been established, therefore it is appropriate as a first example of how the Smart Toilet Paper can be applied. The current development of the Smart Toilet Paper is a proof of concept that can be extended to other applications beyond drug modulation.

[00170] The Smart Toilet Paper can also be used for infectious disease detection, especially in cases where it is time-critical (in which point of care is required) or where resources are limited.

[00171] In order for microbiome research to penetrate into the clinical practice, more robust analytical data will be required. Although the field is fairly young, there is a constant increase in the number of research, publications, clinical trials, as well as funding that goes into microbiome-related research/projects. One of this project’s goals is to be used as a non- invasive means of detecting chronic diseases that may also be asymptomatic, which also depends on the advancement in microbiome research. The design of the Smart Toilet Paper can be modified to accommodate the varying applications but also to continue to increase simplicity and efficacy where needed.

[00172] Many tests and screenings will extend into the house, which would not only add convenience for the users, alleviate the clinics resources for more urgent matters, and also increase data collection for the purposes of clinical research.

Example 3

[00173] The development of the Smart Toilet Paper comprised of multiple (and simultaneous) trajectories: (1) Design/ Architecture, (2) Physics, (3) Chemistry, (4)

Material/Fabrication, and (5) User Experience.

[00174] The DESIGN development was in parallel with an understanding of obtaining and processing DNA information from fecal matter. Starting with an initial basic understanding of how DNA amplification functions, the architecture of the device simply shows a multiplex “plate” with chambers for 8 to 24 different primers. Upon learning and conducting DNA extraction from a first fecal sample, the design was altered to better reflect the real added complexity. This new design became the basis for the later developments on the device. The basic outline of the design was of (1) a central/collection chamber guarded by a line of filter, (2) a secondary chamber circumscribing the central chambers, (3) a series of 3-8 tertiary chambers radially distributed around the secondary chamber, (4) a silica membrane at each junction between the secondary and tertiary chambers, (5) a dedicated chamber for the lysis/RNAse/binding agent with a l-way valve which opens directly into the central chamber, (6) a dedicated chamber for the wash buffer with a l-way valve which opens directly into the secondary chamber, (7) a dedicated chamber for TE buffer with a l-way valve which also opens directly into the secondary chamber, (8) a series of 3-8 dedicated chambers for the RP A/primer solutions with l-way valves which each opens directly into the respective tertiary chambers, and (9) an outer continuous waste chamber located beyond the tertiary chambers.

[00175] The PHYSICS component was simply (1) to test the spinning mechanism to see whether fluids would be able to migrate to the peripheral waste chamber as expected and (2) to measure the rotational speed of the device and evaluate it against that of a microcentrifuge.

[00176] The CHEMISTRY experiments were to test whether certain proposed alterations from given kit protocols still yielded similar results. These were done for DNA extraction, DNA amplification, and DNA visualization. Additional experiments were required to determine a workable bacterial strain along the proper primer can be used in conjunction with the RPA mix. The alterations were first tested outside of the device (i.e. in 1.5 mL tubes) and when they were successful, similar steps were incorporated into the physical prototype devices to be further tested. Some of the prototypes were simplified (in terms of their design) as a means to control the multiple variables that may affect the final outcome.

[00177] The current experiments have successfully shown a simplified version of the device able to extract the DNA from E.coli (strain C) cells and amplify the same DNA sample using two sets of primers (correct/positive and incorrect/negative). The results have been examined using a gel electrophoresis.

[00178] The MATERIAL/FABRICATION development involved iterating on using hydrophobic materials, such as paraffin and PETG, and hydrophobic coatings over paper- based materials, such as epoxy, nail polish (nitrocellulose w/ ethyl acetate), and silicone.

With each new material, the fabrication also differs to include laser cutting, thermoforming, and embossing.

[00179] The ETSER. EXPERIENCE study used dry prototypes (not including any of the fluids mentioned above) of the ideal design and asked a number of volunteers to follow a series of written/visual/verbal instructions and perform the method of operation for the device. Each study was followed by a short survey which asks which component of the device and/or the instruction set was easiest and hardest to perform/follow and suggestions for improvements.

Claims

What is claimed herein is:

1. A device comprising layers in the following order:

a. Optionally, a sample collection layer comprising at least one opening;

b. A first water-resistant layer comprising at least one opening;

c. A microfluidics layer comprising:

i. a collection chamber aligned with the at least one opening in the first

water-resistant layer;

ii. at least a first buffer in a first buffer reservoir;

iii. at least one microfluidic channel comprising:

1. a nucleic acid binding element;

2. a pair of nucleic acid primers;

3. the proximal zone of the microfluidic channel having a junction with the collection chamber and;

4. the distal zone of the microfluidic channel having a junction with at least one waste collection chamber;

iv. a nucleic acid visualization area comprising a western blot or lateral flow assay which has a junction with the microfluidic channel; d. A second water-resistant layer; and

e. An axial element positioned to pass through each of the layers.

2. The device of claim 1, wherein the layers are circular or cylindrical in shape.

3. The device of any of claims 1-2, wherein the width, length, and/or diameter of each of the layers is substantially the same as that of the other layers.

4. The device of any of claims 1-2, wherein the width, length, and/or diameter of each of the layers is within 10% of the average for the layers.

5. The device of any of claims 1-2, wherein the width, length, and/or diameter of each of the layers is the same as that of the other layers

6. The device of any of claims 1-5, wherein the sample collection layer comprises toilet paper, tissue paper, baby wipe material, personal hygiene wipe material, sanitary napkin material, tampon material, or the like.

7. The device of any of claims 1-6, wherein the at least one opening in the sample collection layer is a slit or hole which traverses from the exterior of the layer to the side of the layer facing the first water-resistant layer.

8. The device of any of claims 1-7, wherein the at least one opening in the sample collection layer is closer to the center of the layer’s most-planar dimension than to the edge of the layer’s most-planar dimension.

9. The device of any of claims 1-8, wherein the sample collection layer is disposable or removable.

10. The device of claim 9, wherein the sample collection layer is adhered to the first water- resistant layer with a chemical or physical adhesive.

11. The device of any of claims 1-10, wherein the at least one opening in the first water- resistant layer is a slit or hole which traverses from the side of the layer facing the sample collection layer to the side of the layer facing the microfluidics layer.

12. The device of any of claims 1-11, wherein the at least one opening in the first water- resistant layer is closer to the center of the layer’s most-planar dimension than to the edge of the layer’s most-planar dimension.

13. The device of any of claims 1-12, wherein the at least one opening in the first water- resistant layer is aligned with the at least one opening in the sample collection layer.

14. The device of claim 13, wherein the at least one opening which is aligned with the at least one opening in the sample collection layer is a slit.

15. The device of any of claims 1-14, wherein the first-water resistant layer comprises at least one opening which is not aligned with the at least one opening in the sample collection layer.

16. The device of claim 1-15, wherein the at least one opening which is not aligned with the at least one opening in the sample collection layer is a mesh, hole, or slit.

17. The device of any of claims 1-16, wherein the one or more microfluidic channels are hydrophobic.

18. The device of any of claims 1-17, wherein the collection chamber is located substantially in the center of the microfluidic layer’s most-planar dimension.

19. The device of any of claims 1-18, wherein the collection chamber further comprises a sample agitation element.

20. The device of claim 19, wherein the sample agitation element is one or more beads.

21. The device of any of claims 1-20, wherein the one or more microfluidic channels and/or the collection chamber comprise a particulate filter designed to retain large sample particulate or sample agitation elements in the collection chamber or proximal portion of the one or more microfluidic channels.

22. The device of claim 21, wherein the particulate filter is a nylon filter.

23. The device of any of claims 1-22, wherein the first buffer reservoir comprises a lysis buffer.

24. The device of claim 23, wherein the lysis buffer further comprises RNase and a binding agent.

25. The device of any of claims 1-24, wherein the collection chamber comprises a second buffer in a second, separate buffer reservoir and the second bugger is a buffer that can release nucleic acids from the nucleic acid binding element.

26. The device of claim 25, wherein the buffer than can release nucleic acids is TE buffer.

27. The device of any of claims 1-26, wherein each buffer reservoir is a rupturable capsule that can be ruptured by applying force to the region of the device comprising the buffer reservoir.

28. The device of claim 27, wherein the force is no more than half the force required to rupture the water-resistant layers or the microfluidics layer.

29. The device of any of claims 1-28, wherein the microfluidic layer comprises a plurality of microfluidic channels and each microfluidic channel comprises a single pair of nucleic acid primers.

30. The device of any of claims 1-29, wherein the microfluidic layer comprises a plurality of microfluidic channels and each microfluidic channel comprises a single unique pair of nucleic acid primers.

31. The device of any of claims 1-30, wherein the at least one pair of nucleic acid primers is provided in a rupturable primer capsule.

32. The device of claim 31, wherein the capsule comprising the pair of nucleic acid primer further comprises one or more RPA reagents.

33. The device of any of claims 1-32, wherein at least one pair of nucleic acid primers is specific for a pathogenic microorganism, a microorganism found in the subject species’ microbiome, or a microbial gene that affects host drug metabolism.

34. The device of any of claims 1-33, wherein the junction between the microfluidic channel and the waste chamber comprises a separable or sealable element.

35. The device of any of claims 1-34, wherein the visualization area comprises an LFA assay.

36. The device of claim 35, wherein the LFA assay comprises i) a LFA-compatible pad or pads and iii) gold particles, latex, and/or carbon nanotubes.

37. The device of any of claims 1-36, wherein the junction of the microfluidic channel and the visualization area comprises a rupturable or degradable barrier.

38. The device of any of claims 1-37, wherein the second water-resistant layer comprises viewing windows aligned with one or more of the visualization areas.

39. The device of any of claims 1-38, wherein the second water-resistant layer comprises openings, indentations, or thinner zones aligned with one or more of the rupturable capsules.

40. The device of any of claims 1-39, wherein the axial element is located substantially at the center of each layer’s most-planar dimension.

41. The device of any of claims 1-39, wherein the axial element is located at the center of each layer’s most-planar dimension.

42. The device of any of claims 1-39, wherein the axial element is not located at the center of each layer’s most-planar dimension.

43. The device of any of claims 1-42, wherein each layer comprises a portal or opening with an aperture which is not contiguous with any other element of the layer, and the axial element passes through the apertures.

44. The device of any of claims 1-43, wherein the axial element is a tensile element forming a closed loop and extending beyond the exterior faces of the sample collection layer and the second water-resistant layer.

45. The device of claim 44, wherein each layer comprises two portals or openings, each with an aperture which is not contiguous with any other element of the layer, and the axial element passes through the apertures.

46. A method of detecting a microorganism or microbial gene in a sample, the method

comprising: a. contacting the sample collection layer of a device of any of claims 1-45 with the sample;

b. opening or rupturing the first buffer reservoir;

c. agitating the device;

d. causing the device to spin about the axial element;

e. closing or sealing the at least one waste collection chamber;

f. optionally, opening or rupturing the second buffer reservoir;

g. optionally, opening or rupturing any primer capsules present;

h. warming the device to at least 35°C;

i. observing or detecting any visible signals in the visualization areas.

47. The method of claim 46, wherein when step f is performed, the method comprises a further step after step f of causing the device to spin about the axial element.

48. The method of any of claims 46-47, wherein step h comprises warming the device to 37°C-39°C.

49. The method of any of claims 46-47, wherein in step h, the device is maintained at the temperature of at least 35°C for about 20 minutes or until at least one signal is visible in at least one visualization area.

50. The method of any of claims 46-49, wherein step h comprises placing the device in a water bath, placing the device in a sealed container which is placed in a water bath, or placing the device in close proximity to the surface of a subject’s skin.

51. The method of any of claims 46-50, wherein the method further comprises a step after a. and before b. of sealing all or a portion of the sample collection layer’s outer surface or removing the sample collection layer and sealing all or a portion of the first water- resistant layers’ outer surface.

52. The method of claim 52, wherein the sealing comprises application of an adhesive cover or sticker.