Power-free fluidic device

Abstract

Fluidic devices that can be operated without the use of electrical power for controlling timing, fluid flow and transfer, chemical and / or biological reactions, and producing readout have performance and cost benefits. A power-free fluidic device may include a device body configured for substantially gravity-driven flow, one or more reaction chambers containing reagents, fluidic timing elements, and fluidic valves. The reaction chambers may contain reagents for conducting nucleic acid amplification reactions and other chemical and / or biological reactions under ambient conditions.

Description

Atty. Docket No.: 2013065-0954 POWER-FREE FLUIDIC DEVICE CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application Serial No.63 / 669,050, filed July 9, 2024 and entitled “POWER-FREE FLUIDIC DEVICE,” the disclosure of which is incorporated herein by reference in its entirety. BACKGROUND

[0002] Fluidic devices can be used to conduct chemical and / or biological assays using small quantities of liquid samples, and are useful for point of need (PON) applications such as diagnostic assays. Practical application and wider adoption of fluidic devices, particularly for diagnostic assays in locations with limited infrastructure and resources, would benefit from devices that can provide full functionality without the need for heating, electronics, motorized actuation, or other powered steps. In some locations where electrical power is not available, fluidic devices that can be operated in a power-free manner for operating assays, controlling timing, and producing readout, may be beneficial and more cost-effective. SUMMARY

[0003] Described herein are devices, systems, methods, compositions, and processes for fluidic devices that employ power-free actuation mechanisms such as water-soluble membranes, gravity flow, dead air space, expanding hygroscopic materials, serpentine channels, and slow and controlled air diffusion membranes. Fluidic actuation steps can be implemented without the use of any electronic or powered components.

[0004] In some aspects, provided embodiments are directed to a system including: a device body including: a sample inlet port for receiving a sample; a first flow channel (i.e., timer delay channel) fluidly coupled to the sample inlet port at an upstream end of the first flow channel, the first flow channel extending to a downstream end; at least one reaction chamber fluidly coupled to the first flow channel; a passively actuated valve disposed at the downstream end of the first flow channel; and a second flow channel (i.e., lateral flow chamber) disposed downstream of the first flow channel, wherein the passively actuated Page 1 of 164 12865804v1Atty. Docket No.: 2013065-0954 valve, when opened, is configured to allow flow of the sample from the first flow channel to the second flow channel.

[0005] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one lyophilized reaction bead contained within a reaction chamber.

[0006] In some embodiments, a reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure is coupled to a first flow channel between the upstream end and the downstream end via at least one side channel.

[0007] In some embodiments, a second flow channel provided by and / or utilized in accordance with embodiments of the present disclosure is disposed downstream of a passively actuated valve, and the passively actuated valve fluidly couples a first flow channel to the second flow channel.

[0008] In some embodiments, a passively actuated valve provided by and / or utilized in accordance with embodiments of the present disclosure is disposed downstream of a second flow channel, and the second flow channel fluidly couples a first flow channel to a passively actuated valve.

[0009] In some embodiments, a reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure is fluidly coupled to a first flow channel downstream of a sample inlet port via at least one side channel.

[0010] In some embodiments, a passively actuated valve provided by and / or utilized in accordance with embodiments of the present disclosure includes a soluble membrane.

[0011] In some embodiments, a soluble membrane provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one of polyvinyl alcohol (PVA) and a hydrogel.

[0012] In some embodiments, a soluble membrane provided by and / or utilized in accordance with embodiments of the present disclosure dissolves upon contact with a fluid. Page 2 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0013] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes air contained within a second flow channel; and sample contained within a reaction chamber.

[0014] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes air contained within a first flow channel downstream of a reaction chamber.

[0015] In some embodiments, air in at least one of a first flow channel and a second flow channel provided by and / or utilized in accordance with embodiments of the present disclosure prevents a biological solution containing a sample from flowing into the second flow channel.

[0016] In some embodiments, upon dissolution of a soluble membrane provided by and / or utilized in accordance with embodiments of the present disclosure, a passively actuated valve opens and at least a portion of the air in a second flow channel exits the second flow channel, thereby allowing a biological solution to flow into the second flow channel.

[0017] In some embodiments, a biological solution provided by and / or utilized in accordance with embodiments of the present disclosure flows into a second flow channel via gravity feed.

[0018] In some embodiments, at least a portion of a reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure is disposed at a lower vertical location than that of at least a portion of a sample inlet port when a device body is in an upright position, thereby allowing a biological solution to flow from the sample inlet port into the reaction chamber.

[0019] In some embodiments, a biological solution provided by and / or utilized in accordance with embodiments of the present disclosure flows from a sample inlet port to a reaction chamber via gravity and via a first flow channel.

[0020] In some embodiments, a biological solution provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one active nucleotide. Page 3 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0021] In some embodiments, a reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure includes multiple reaction chambers.

[0022] In some embodiments, multiple reaction chambers provided by and / or utilized in accordance with embodiments of the present disclosure includes two reaction chambers comprising a first reaction chamber and a second reaction chamber.

[0023] In some embodiments, a first reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure is located on a first side of a first flow channel and a second reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure is disposed on a second side of the first flow channel.

[0024] In some embodiments, a soluble membrane provided by and / or utilized in accordance with embodiments of the present disclosure dissolves upon contact with water molecules in an aqueous solution. For example, an aqueous solution may include a salt(s), a detergent(s) / surfactant(s), or other chemical component(s).

[0025] In some embodiments, hydroxyl groups of PVA provided by and / or utilized in accordance with embodiments of the present disclosure interact with water molecules, thereby forming hydrogen bonds.

[0026] In some embodiments, PVA provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one of a degree of hydrolysis in a range from about 60% mol hydrolysis to about 99% mol hydrolysis and a molecular weight in a range from about 26,000 to about 200,000, thereby producing enhanced solubility of the PVA with the water molecules.

[0027] In some embodiments, a PVA provided by and / or utilized in accordance with embodiments of the present disclosure includes a degree of hydrolysis in a range from about 65% mol hydrolysis to about 85% mol hydrolysis.

[0028] In some embodiments, a PVA provided by and / or utilized in accordance with embodiments of the present disclosure includes a degree of hydrolysis in a range from about 72% mol hydrolysis to about 78% mol hydrolysis. Page 4 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0029] In some embodiments, a PVA provided by and / or utilized in accordance with embodiments of the present disclosure includes a viscosity in a range from about 4 mPas to about 12 mPas.

[0030] In some embodiments, a PVA provided by and / or utilized in accordance with embodiments of the present disclosure includes a molecular weight in a range from about 5,000 to about 200,000.

[0031] In some embodiments, lyophilized reaction beads provided by and / or utilized in accordance with embodiments of the present disclosure, upon contact with a biological solution containing a sample cause amplification to occur on at least one active component of the sample.

[0032] In some embodiments, an amplification provided by and / or utilized in accordance with embodiments of the present disclosure occurs for a predetermined period of time.

[0033] In some embodiments, a predetermined period of time provided by and / or utilized in accordance with embodiments of the present disclosure concludes when a passively actuated valve opens; and the predetermined period of time comprises a period from about 5 minutes to about 30 minutes.

[0034] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes: a third flow channel fluidly coupled to a passively actuated valve; and an air diffusion component fluidly coupled to the third flow channel downstream of the passively actuated valve.

[0035] In some embodiments, an air diffusion component provided by and / or utilized in accordance with embodiments of the present disclosure allows air to flow therethrough, from a third flow channel, at a controlled rate.

[0036] In some embodiments, an air diffusion component provided by and / or utilized in accordance with embodiments of the present disclosure is composed of a material with an average pore diameter of about 0.025 microns (i.e., for example, in a range from about 0.01 microns to about 0.05, or from about 0.015 microns to about 0.04, from about 0.015 microns to about 0.035, or from about 0.02 microns to about 0.03 microns). Page 5 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0037] In some embodiments, an air diffusion component provided by and / or utilized in accordance with embodiments of the present disclosure includes a bulk density in a range from about 62% to about 82% (i.e., from about 67% to about 77%, i.e., from about 70% to about 75%).

[0038] In some embodiments, an air diffusion component provided by and / or utilized in accordance with embodiments of the present disclosure is composed of nitrocellulose (i.e., pyroxylin, i.e., cellulose nitrate, i.e., a mixture of nitric esters of cellulose).

[0039] In some embodiments, flow of air through an air diffusion component provided by and / or utilized in accordance with embodiments of the present disclosure enables air to be evacuated from a first flow channel, thereby enabling flow of a biological solution containing a sample into and through at least one of a passively actuated valve and a second flow channel.

[0040] In some embodiments, an average pore diameter provided by and / or utilized in accordance with embodiments of the present disclosure restricts the flow of air through an air diffusion component in order to slow down the flow of a biological solution containing a sample into and through a passively actuated valve and / or a second flow channel, thereby allowing the biological solution containing a sample to remain in a reaction chamber for a predetermined period of time.

[0041] In some embodiments, an air diffusion component provided by and / or utilized in accordance with embodiments of the present disclosure includes a thin layer (or sheet) of a material adhered to a surface of a device such that air diffuses from a third flow channel through the thin layer of the material and out at external edges of the thin layer of the material.

[0042] In some embodiments, an air diffusion component provided by and / or utilized in accordance with embodiments of the present disclosure includes a porous membrane sandwiched between a doubled-sided adhesive on one side and a single-sided adhesive on the other side.

[0043] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes a continuous flow path fluidly coupled to a passively actuated valve. Page 6 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0044] In some embodiments, a continuous flow path provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one of a sufficient distance and a sufficient resistance to flow to enable fluid (i.e., timing fluid) flowing within a continuous flow path to remain flowing therethrough for at least a pre-determined amount of time.

[0045] In some embodiments, a pre-determined amount of time provided by and / or utilized in accordance with embodiments of the present disclosure includes from about 5 to about 20 minutes.

[0046] In some embodiments, a continuous flow path provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one serpentine flow path including at least one turn.

[0047] In some embodiments, a turn provided by and / or utilized in accordance with embodiments of the present disclosure includes a 180-degree turn.

[0048] In some embodiments, a serpentine flow path provided by and / or utilized in accordance with embodiments of the present disclosure includes multiple turns, at least one turn of the multiple turns comprising a 180-degree turn, thereby resulting in multiple, substantially parallel passes (or portions, or channels) of the serpentine flow path.

[0049] In some embodiments, a continuous fluid path provided by and / or utilized in accordance with embodiments of the present disclosure includes: a first serpentine flow path; and a second serpentine flow path downstream of the first serpentine flow path.

[0050] In some embodiments, a first serpentine flow path provided by and / or utilized in accordance with embodiments of the present disclosure includes a first flow path nominal diameter, wherein a second serpentine flow path provided by and / or utilized in accordance with embodiments of the present disclosure includes a second flow path nominal diameter, and wherein the second nominal diameter is larger than the first nominal diameter.

[0051] In some embodiments, a first flow path nominal diameter (or channel height / width) provided by and / or utilized in accordance with embodiments of the present disclosure is in a range from about 2 microns to about 500 microns. Page 7 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0052] In some embodiments, a second flow path nominal diameter (or channel height / width) provided by and / or utilized in accordance with embodiments of the present disclosure is in a range from about 1 mm to about 10 mm.

[0053] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes a timing fluid inlet port.

[0054] In some embodiments, a timing fluid inlet port provided by and / or utilized in accordance with embodiments of the present disclosure is fluidly coupled upstream of a continuous flow path and configured to allow a fluid to gravity flow into the continuous flow path.

[0055] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes a lateral flow strip (LFS) fluidly coupled downstream or upstream of a passively actuated valve.

[0056] In some embodiments, a second flow channel provided by and / or utilized in accordance with embodiments of the present disclosure includes a lateral flow strip.

[0057] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes a timing fluid contained within a timing fluid inlet port.

[0058] In some embodiments, a timing fluid provided by and / or utilized in accordance with embodiments of the present disclosure does not include a sample.

[0059] In some embodiments, a timing fluid provided by and / or utilized in accordance with embodiments of the present disclosure is different than a sample.

[0060] In some embodiments, a timing fluid inlet port provided by and / or utilized in accordance with embodiments of the present disclosure is fluidly uncoupled from a second flow channel when a passively actuated valve is closed, and the timing fluid inlet port is fluidly coupled to the second flow channel when the passively actuated valve is opened.

[0061] In some embodiments, when a timing fluid provided by and / or utilized in accordance with embodiments of the present disclosure contacts the passively actuated valve, it causes the passively actuated valve to open. Page 8 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0062] In some embodiments, a timing fluid provided by and / or utilized in accordance with embodiments of the present disclosure causes a passively actuated valve to open by dissolving the passively actuated valve.

[0063] In some embodiments, a timing fluid provided by and / or utilized in accordance with embodiments of the present disclosure causes a passively actuated valve to open by being absorbed into a wicking layer that expands and forces a soluble membrane against a piercing feature (e.g., a pointed feature / protrusion, a puncturing feature / protrusion, a knife-like feature, a needle), thereby causing the soluble membrane to be punctured.

[0064] In some embodiments, a second flow channel provided by and / or utilized in accordance with embodiments of the present disclosure includes or connects to at least one lateral flow strip (LFS) used for testing a sample for the presence of one or more target nucleic acids.

[0065] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes a waste shunt coupled to a sample inlet port upstream of a first flow channel, the waste shunt including: a fourth flow channel connecting to the sample inlet port; a U-bend connecting to a downstream end of the fourth flow channel; and a vent disposed at the downstream end of the fourth flow channel.

[0066] In some embodiments, a waste shunt provided by and / or utilized in accordance with embodiments of the present disclosure prevents overfilling of a system.

[0067] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure further includes: a bulb disposed adjacent to a reaction chamber; a vertical fill line fluidly connected to a bottom portion of the reaction chamber.

[0068] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes a side channel, wherein an upstream end of a vertical fill line is fluidly coupled to a downstream end of the side channel, and wherein a downstream end of the vertical fill line is fluidly coupled to a bottom of portion of a reaction chamber. Page 9 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0069] In some embodiments, a vertical fill line provided by and / or utilized in accordance with embodiments of the present disclosure is at least partially oriented in a vertical direction.

[0070] In some embodiments, a vertical fill line provided by and / or utilized in accordance with embodiments of the present disclosure is oriented vertically.

[0071] In some embodiments, a vertical fill line provided by and / or utilized in accordance with embodiments of the present disclosure is oriented at an angle from a vertical direction (or at an angle from a longitudinal dimension of a device body).

[0072] In some embodiments, a bulb provided by and / or utilized in accordance with embodiments of the present disclosure includes a substantially spherical chamber.

[0073] In some embodiments, a bulb provided by and / or utilized in accordance with embodiments of the present disclosure is fluidly connected to a reaction chamber via a ridge feature and a permeable membrane disposed in a top portion of the reaction chamber.

[0074] In some embodiments, a bottom portion of a reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure includes a bottom 50% of the reaction chamber, and wherein the top portion of the reaction chamber comprises the top 50% of the reaction chamber.

[0075] In some embodiments, a ridge feature (in connection with surface tension) and / or a permeable membrane provided by and / or utilized in accordance with embodiments of the present disclosure allows air to pass through, but prevents or restricts liquid from passing through.

[0076] In some embodiments, a vertical fill line provided by and / or utilized in accordance with embodiments of the present disclosure enables a reaction chamber to be filled with a biological solution (i.e., a solution containing a sample) from the bottom up.

[0077] In some embodiments, a vertical fill line provided by and / or utilized in accordance with embodiments of the present disclosure enables biological solution to flow vertically in order to flow into a reaction chamber. Page 10 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0078] In some embodiments, a bulb provided by and / or utilized in accordance with embodiments of the present disclosure enables gas (i.e., air) disposed within a reaction chamber to exit a system.

[0079] In some embodiments, each of a bulb and a reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure is substantially spherical; wherein an interface between the bulb and the reaction chamber includes a substantially circular opening; and wherein a permeable membrane spans the substantially circular opening, or wherein a ridge feature extends around the substantially circular opening.

[0080] In some embodiments, each of a bulb and a reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure is substantially cylindrical; wherein each of the cylinders defining the respective bulb and the reaction chamber including a larger diameter than height; wherein an interface between the bulb and reaction chamber includes a substantially rectangular opening; and wherein a permeable membrane spans the substantially rectangular opening, or wherein a ridge feature extends around the substantially rectangular opening.

[0081] In some embodiments, a soluble membrane provided by and / or utilized in accordance with embodiments of the present disclosure covers (or spans, or extends across) a downstream end of a first flow channel.

[0082] In some embodiments, a passively actuated valve provided by and / or utilized in accordance with embodiments of the present disclosure includes: a hub defining a downstream end of a first flow channel, a soluble membrane attached (or adhered) to the hub; and a wicking layer disposed between the hub and the soluble membrane.

[0083] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes a needle for puncturing a soluble membrane.

[0084] In some embodiments, a wicking layer provided by and / or utilized in accordance with embodiments of the present disclosure includes a hole disposed therethrough. Page 11 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0085] In some embodiments, a hole provided by and / or utilized in accordance with embodiments of the present disclosure is disposed concentrically within the center of a wicking layer, with, in a closed position of a passively actuated valve, a soluble membrane disposed (or laid) thereover.

[0086] In some embodiments, a needle provided by and / or utilized in accordance with embodiments of the present disclosure is positioned such that, in a closed position of a passively actuated valve, a tip of the needle is centered approximately over the center of the hole a distance from a soluble membrane on an opposite side of the soluble membrane from a wicking layer.

[0087] In some embodiments, a wicking layer provided by and / or utilized in accordance with embodiments of the present disclosure expands as it absorbs a biological solution, thereby pushing a soluble membrane into a needle allowing the needle to puncture the soluble membrane and opening a passively actuated valve.

[0088] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure is power-free.

[0089] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes no external or internal power source.

[0090] In some embodiments, a device body is provided by and / or utilized in accordance with embodiments of the present disclosure configured such that each of a first channel and a second channel are oriented in a substantially vertical direction, gravity causes a biological solution to flow vertically downward through the first flow channel, and capillary action causes the biological solution to flow vertically upward through the second flow channel.

[0091] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes no moving parts.

[0092] In some embodiments, a device body provided by and / or utilized in accordance with embodiments of the present disclosure is 3D printed of a transparent material. Page 12 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0093] In some embodiments, a transparent material provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one of PLA (polylactic acid), PLA+ (i.e., PLA plus at least one of an impact modifier (i.e., methacrylate butadiene styrene (MBS) terpolymer, acrylate polymethacrylate copolymer (acrylic), chlorinated polyethylene (CPE), ethylene vinyl acetate copolymer (EVA), acrylonitrile butadiene styrene terpolymer (ABS), etc., a flexibilizing agent (i.e., a curing agent), and a plasticizer (i.e., phthalate esters, ethanolamine, caproic acid, lauric acid and glycerol triacetate (triacetin)), and PETG (polyethylene terephthalate glycol).

[0094] In some embodiments, a device body provided by and / or utilized in accordance with embodiments of the present disclosure is injection molded, compression molded, or machined from a transparent material.

[0095] In some embodiments, a transparent material provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one of polycarbonate, acrylic, nylon, poly(methyl methacrylate) (PMMA), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polycarbonate (PC), polypropylene (PP), acrylonitrile butadiene styrene (ABS), and polystyrene (PS).

[0096] In some embodiments, a biological solution provided by and / or utilized in accordance with embodiments of the present disclosure includes a surfactant.

[0097] In some embodiments, a biological solution provided by and / or utilized in accordance with embodiments of the present disclosure includes surfactant on a weight percent basis in a range from about 0.01% to about 1.0%.

[0098] In some embodiments, a surfactant provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one of alcohol ethoxylate, polysorbate 20, sodium lauryl sulfate, sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS) and sodium laureth sulfate.

[0099] In some embodiments, a reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure includes a first reaction chamber and a second reaction chamber, a second flow channel includes or connects to a lateral flow strip (LFS) used for testing a sample for the presence of one or more analytes or conditions, a lyophilized reaction bead includes a first bead contained within the first reaction chamber and Page 13 of 164 12865804v1Atty. Docket No.: 2013065-0954 a second bead contained within the second reaction chamber, and the first bead is configured for a first analyte or condition of the one or more analytes or conditions and the second bead is configured for a second analyte or condition of the one or more analytes or conditions. In some embodiments according to the present disclosure, the device includes a single reaction chamber (i.e., only a single reaction chamber). In some embodiments, the device according to the present disclosure may include one, two, three, or more than three reaction chambers, the device further including a corresponding channel connecting each reaction chamber to a lateral flow stip. In some embodiments, each reaction chamber includes a single lyophilized bead. In some embodiments, each reaction chamber includes multiple lyophilized beads.

[0100] In some embodiments, a lateral flow strip (LFS) provided by and / or utilized in accordance with embodiments of the present disclosure includes multiple capture regions configured to provide indication of the presence of each of a first analyte or condition and a second analyte or condition in a biological solution.

[0101] In some embodiments, a first condition provided by and / or utilized in accordance with embodiments of the present disclosure is gonorrhea and a second condition provided by and / or utilized in accordance with embodiments of the present disclosure is chlamydia. For example, the device and / or system of the present disclosure amplifies and detects nucleic acid(s) from one or more microorganisms in the biological sample that are known to cause gonorrhea and chlamydia.

[0102] In some embodiments, a device body provided by and / or utilized in accordance with embodiments of the present disclosure is composed of a transparent material, a lateral flow strip is disposed internally between a front surface of the device body and a rear surface of the device body, and the lateral flow strip is configured to provide at least one indication that is visible from a perspective external to the device body, the indication indicating the result of at least one of a biological assay and a chemical assay.

[0103] In some embodiments, a device body provided by and / or utilized in accordance with embodiments of the present disclosure includes a continuous, 3-dimensional body with voids disposed therein, the voids composing at least a first void defining a first flow channel, a second void or voids defining a reaction chamber into which a lyophilized bead can be inserted, and a third void or voids into which a lateral flow strip (LFS) can be inserted. Page 14 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0104] In certain aspects, provided embodiments are directed to a system including a device body including: a sample inlet port for receiving a fluid sample; a first flow channel fluidly coupled to the sample inlet port at an upstream end of the first flow channel, the first flow channel extending to a downstream end; at least one reaction chamber fluidly coupled to the first flow channel; a passively actuated valve disposed at the downstream end of the first flow channel; and a second flow channel disposed downstream of the first flow channel, wherein the passively actuated valve, when opened, is configured to allow flow of the fluid sample from the first flow channel to the second flow channel.

[0105] In some embodiments, a passively actuated valve provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one of a soluble membrane and a puncturable or pierceable membrane.

[0106] In some embodiments, a second flow channel provided by and / or utilized in accordance with embodiments of the present disclosure includes a lateral flow strip.

[0107] In some aspects, provided embodiments are directed to a method of tuning a fluidic timer including: providing a device body of the fluidic timer, the device body including: a fluid inlet port; a first fluid channel fluidly connected downstream of the fluid inlet port, the first fluid channel comprising an upstream end and a downstream end; and a passively actuated valve disposed downstream of the first fluid channel and coupled (directly or indirectly) thereto; providing a fluid sample to be disposed within the fluid inlet port; and adjusting at least one parameter of the device body and / or the fluid sample such that the fluid sample flows from the fluid inlet port to the downstream end of the first fluid channel for a duration of time to match or exceed a pre-determined amount of time.

[0108] In some embodiments, adjusting at least one parameter of a device body and / or a fluid sample provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one of: adjusting a length of a first fluid channel; adjusting an internal dimension (i.e., a diameter) of the first fluid channel; adjusting a weight percent of surfactant in the fluid sample in a range from about 0.01% to about 10%; adjusting a length of an air diffusion component disposed downstream of a downstream end of the first flow channel; and adjusting at least one of a length and an internal dimension of a continuous flow path fluidly connected to a passively actuated valve. Page 15 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0109] In some embodiments, a pre-determined amount of time provided by and / or utilized in accordance with embodiments of the present disclosure includes a period from about 1 minute to about 30 minutes (i.e., from about 5 minutes to about 25 minutes, i.e., from about 5 minutes to about 20 minutes, i.e., from about 7 minutes to about 20 minutes, i.e., from about 10 minutes to about 20 minutes).

[0110] In some aspects, provided embodiments are directed to a fluidic device, including: a device body arranged substantially vertically; a sample fluid input reservoir disposed at a top end of the device body; a sample flow channel fluidly connected to the sample fluid input reservoir; at least one reaction chamber fluidly connected to the sample flow channel; an outlet fluidly connected to the sample flow channel; a fluidic valve fluidly connected to and controlling flow through the outlet; and a fluidic timing element configured to control a duration of time for the sample fluid to remain in the at least one reaction chamber and in the sample flow channel before opening the fluidic valve and causing the sample fluid to flow out of the outlet, wherein operation of the fluidic device to perform a reaction in the at least one reaction chamber does not use electrical power.

[0111] In some embodiments, each reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure is fluidly connected to a sample flow channel via a side channel.

[0112] In some embodiments, each reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure is fluidly connected to an air vent.

[0113] In some embodiments, an air vent provided by and / or utilized in accordance with embodiments of the present disclosure includes an air pinning chamber adjacent to a reaction chamber.

[0114] In some embodiments, each reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure is configured to contain reagents for performing a reaction.

[0115] In some embodiments, a device provided by and / or utilized in accordance with embodiments of the present disclosure is configured to contain reagents in a lyophilized reaction bead configured to be rehydrated by a sample fluid. Page 16 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0116] In some embodiments, a fluidic timing element provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one of a slow air diffusion timer and a serpentine fluidic timer.

[0117] In some embodiments, a slow air diffusion timer provided by and / or utilized in accordance with embodiments of the present disclosure includes: an air channel fluidly connected to a sample flow channel and open to an external surface of a device body at an air channel opening; an air diffusion pad including: a porous membrane including a thin sheet of a porous material; an opening in a center of the thin sheet (or layer) that is at least as large as the air channel opening; and a coating on an exterior planar surface of a porous membrane that is not permeable to air, wherein the air diffusion pad is adhered on an uncoated planar surface of the thin sheet to the surface of the device body exterior surrounding the air channel opening, such that air can diffuse from the air channel through an interior of the thin sheet to an edge of the thin sheet.

[0118] In some embodiments, an area of a thin sheet of porous material provided by and / or utilized in accordance with embodiments of the present disclosure controls venting of air from an air channel and a sample flow channel.

[0119] In some embodiments, a serpentine fluidic timer provided by and / or utilized in accordance with embodiments of the present disclosure includes: a timer fluid input reservoir disposed at the top of a fluidic device; a timer fluid flow channel fluidly connected to the timer fluid input reservoir; a fluidic resistance element fluidly connected to the timer fluid flow channel; a serpentine fluid channel fluidly connected to the fluidic resistance element; and a timer exit channel fluidly connected between the serpentine fluid channel and a fluidic valve.

[0120] In some embodiments, a fluidic resistance element provided by and / or utilized in accordance with embodiments of the present disclosure includes a thin channel arranged with a plurality of turns.

[0121] In some embodiments, a serpentine fluid channel provided by and / or utilized in accordance with embodiments of the present disclosure includes a channel arranged with a plurality of turns. Page 17 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0122] In some embodiments, a fluidic valve provided by and / or utilized in accordance with embodiments of the present disclosure includes a valve membrane including a material dissolvable by a sample fluid, wherein the valve membrane is adhered to an exterior surface of a device body covering an opening of a sample flow channel, and wherein the valve membrane prevents flow of air through the sample flow channel.

[0123] In some embodiments, a fluidic valve provided by and / or utilized in accordance with embodiments of the present disclosure further includes a thin sheet of absorbent material disposed between a valve membrane and an exterior surface of a device body, and wherein the absorbent material covers an opening of a sample flow channel, and wicks sample fluid from the sample flow channel to the valve membrane.

[0124] In some embodiments, a fluidic valve provided by and / or utilized in accordance with embodiments of the present disclosure further includes a hygroscopic sponge disposed between a valve membrane and an exterior surface of a device body, wherein the hygroscopic sponge covers an opening of a sample flow channel, and expands when absorbing sample fluid, the hygroscopic sponge pushing outward against the valve membrane, and wherein the device body includes a counterbore recess to fit the hygroscopic sponge.

[0125] In some embodiments, a hygroscopic sponge provided by and / or utilized in accordance with embodiments of the present disclosure further includes a hole through an interior of a hygroscopic sponge, wherein the hole axis is perpendicular to an exterior surface of a device body, and wherein a fluidic valve provided by and / or utilized in accordance with embodiments of the present disclosure further includes a piercing feature (e.g., a pointed feature / protrusion, a puncturing feature / protrusion, a knife-like feature, a needle) disposed at an exterior surface of a valve membrane to puncture the valve membrane when the hygroscopic sponge pushes against the valve membrane.

[0126] In some embodiments, a needle provided by and / or utilized in accordance with embodiments of the present disclosure is attached to a device base configured to accept a fluidic device in a vertical orientation, and wherein the needle is positioned with a pointed end adjacent to an exterior surface of a valve membrane when the fluidic device is accepted in the device base. Page 18 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0127] In some embodiments, a sample fluid provided by and / or utilized in accordance with embodiments of the present disclosure flows substantially by gravity flow.

[0128] In some embodiments, a sample fluid provided by and / or utilized in accordance with embodiments of the present disclosure flows out of an outlet into at least one of a downstream reaction chamber, a readout mechanism, and a lateral flow chamber including a lateral flow strip.

[0129] In some embodiments, a sample provided by and / or utilized in accordance with embodiments of the present disclosure includes a target nucleic acid.

[0130] In some embodiments, a lyophilized reaction bead provided by and / or utilized in accordance with embodiments of the present disclosure includes a composition configured for carrying out an amplification process.

[0131] In some embodiments, an amplification process provided by and / or utilized in accordance with embodiments of the present disclosure includes strand displacement amplification (SDA).

[0132] In some embodiments, a composition provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one of an oligonucleotide binder, a ligase, a reverse transcriptase, a cleavage enzyme, a restriction enzyme, a single-strand binding protein, a nickase, a strand displacing polymerase, and a dNTP.

[0133] In some embodiments, a composition provided by and / or utilized in accordance with embodiments of the present disclosure includes an oligonucleotide binder including at least one of an SDA primer binding sequence, a first and / or second nucleic acid sensor part, modified nucleotides, a reverse primer, and a forward primer.

[0134] In some embodiments, a lyophilized reaction bead provided by and / or utilized in accordance with embodiments of the present disclosure includes a composition configured for lysing and preparing a sample.

[0135] In some embodiments, a composition configured for lysing a sample provided by and / or utilized in accordance with embodiments of the present disclosure includes at least Page 19 of 164 12865804v1Atty. Docket No.: 2013065-0954 one of sodium hydroxide (NaOH), at least one enzyme, a low pH level, and potassium hydroxide (KOH).

[0136] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes a base for supporting a device body, wherein a second flow channel includes a lateral flow chamber sized and shaped to allow insertion of a lateral flow strip therein. In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes multiple lateral flow chambers and multiple lateral flow strips.

[0137] In some embodiments, a base provided by and / or utilized in accordance with embodiments of the present disclosure includes: a reservoir disposed therein for containing a fluid sample; and a plenum disposed therein for collecting the fluid sample and delivering it to the reservoir; wherein the reservoir and the plenum fluidly connect a first flow channel to a second flow channel when a passively actuated valve is opened.

[0138] In some embodiments, a base provided by and / or utilized in accordance with embodiments of the present disclosure is integral with the device body.

[0139] In some embodiments, a device body provided by and / or utilized in accordance with embodiments of the present disclosure is configured to be inserted into (i.e., seated within) the base.

[0140] In some aspects, provided embodiments are directed to a device including a device body including a top surface and a bottom surface, the device including: a first fluid inlet port [i.e., a sample inlet port] disposed in the top surface, the first fluid inlet port for receiving a fluid sample; a second fluid inlet port [i.e., a timer fluid reservoir] disposed in the top surface, the second fluid inlet port for receiving a timer fluid; at least one reaction chamber disposed in the top surface and coupled fluidly downstream of the first fluid inlet port for receiving the first fluid sample; a fluidic timing circuit disposed within the device body, the fluidic timing circuit coupled downstream of the second fluid inlet and for receiving the timer fluid from the second fluid inlet; and a release valve disposed fluidly downstream of the fluidic timing circuit for receiving the timer fluid from the fluidic timing circuit. Page 20 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0141] In some embodiments, a release valve provided by and / or utilized in accordance with embodiments of the present disclosure is coupled fluidly downstream of the at least one reaction chamber.

[0142] In some embodiments, a device provided by and / or utilized in accordance with embodiments of the present disclosure included a diagnostic area fluidly coupled downstream of a reaction chamber and upstream of a release valve.

[0143] In some embodiments, a release valve provided by and / or utilized in accordance with embodiments of the present disclosure includes a dissolvable membrane including polyvinyl acid (PVA) fluidly sealing a diagnostic area from atmosphere.

[0144] In some embodiments, a dissolvable membrane valve provided by and / or utilized in accordance with embodiments of the present disclosure, when contacted by a timer fluid, dissolves and fluidly couples a diagnostic area to atmosphere, thereby allowing a fluid sample to flow into the diagnostic area.

[0145] In some embodiments, a device body provided by and / or utilized in accordance with embodiments of the present disclosure is substantially planar and oriented in a horizontal plane when in use.

[0146] In some embodiments, a sample fluid provided by and / or utilized in accordance with embodiments of the present disclosure is a biological sample, and wherein a diagnostic area includes a lateral flow chamber sized to contain at least one visible readout strip therewithin, the visible readout strip including at least one of a lateral flow strip (LFS) and a colorimetric reaction test strip.

[0147] In some embodiments, a device body provided by and / or utilized in accordance with embodiments of the present disclosure includes a continuous, single-piece body formed via 3-D printing, compression molding, or injection molding.

[0148] In some embodiments, a device provided by and / or utilized in accordance with embodiments of the present disclosure includes multiple features disposed within a bottom surface of the device body such that the multiple features are recessed and do not protrude beyond a plane defining the bottom surface of the device body. Page 21 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0149] In some embodiments, multiple features provided by and / or utilized in accordance with embodiments of the present disclosure include two or more of: a fluidic resistance element, a serpentine timer delay channel, a through hole, a vent channel, a lateral flow chamber, opposing side channels, a valve manifold or recess, a dead space or lacuna, and a fluid outlet channel.

[0150] In some embodiments, a lateral flow strip (LFS) provided by and / or utilized in accordance with embodiments of the present disclosure includes a carbon black conjugate and biotin on a first side and an oligo pulldown sequence on a second side.

[0151] In some embodiments, a fluidic timing circuit provided by and / or utilized in accordance with embodiments of the present disclosure includes a first plurality of serpentine channels and a second plurality of serpentine channels, wherein the first plurality of serpentine channels is fluidly coupled upstream of the second plurality of serpentine channels, wherein the first plurality of channels acts as a fluidic resistance element, and wherein the second plurality of channels acts as a serpentine timer delay channel.

[0152] In some embodiments, each channel of a first plurality of channels provided by and / or utilized in accordance with embodiments of the present disclosure includes a smaller internal diameter than each channel of a second plurality of channels.

[0153] In some embodiments, each channel of a first plurality of channels and a second plurality of channels provided by and / or utilized in accordance with embodiments of the present disclosure includes multiple 90-degree turns and multiple 180-degree turns.

[0154] In some embodiments, a device provided by and / or utilized in accordance with embodiments of the present disclosure includes a bottom layer adhered to a bottom surface of the device body, wherein the bottom layer fluidly seals multiple features disposed in the bottom surface of the device body.

[0155] In some embodiments, a bottom layer of a device provided by and / or utilized in accordance with embodiments of the present disclosure layer forms a bottom planar surface of the device devoid of any protrusions or recesses. Page 22 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0156] In some embodiments, each of a first inlet port, a second inlet port, and a reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure protrudes from a top surface of a device body.

[0157] In some embodiments, a device provided by and / or utilized in accordance with embodiments of the present disclosure includes a manifold housing a release valve, wherein the manifold housing the release valve protrudes from a top surface of the device body.

[0158] In some embodiments, a second plurality of channels provided by and / or utilized in accordance with embodiments of the present disclosure protrudes from a top surface of a device body, and a first plurality of channels provided by and / or utilized in accordance with embodiments of the present disclosure is contained within a thickness of the device body such that it does not protrude from the top surface of the device body.

[0159] In some embodiments, a lateral flow chamber provided by and / or utilized in accordance with embodiments of the present disclosure protrudes from a top surface of a device body.

[0160] In some embodiments, a device provided by and / or utilized in accordance with embodiments of the present disclosure includes a lid sized and shaped to be placed over a top surface of the device body.

[0161] In some embodiments, a lid provided by and / or utilized in accordance with embodiments of the present disclosure includes a puncturable, at-least partially flexible container or reservoir containing timer fluid located on an underside of the lid at a location corresponding to a second sample inlet when the lid is placed over a top surface of a device body.

[0162] In some embodiments, a device provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one puncture feature disposed within, and protruding from, a second inlet port such that the puncture feature punctures a puncturable, flexible container or reservoir when a lid is placed over a top surface of a device body, thereby causing a timer fluid to flow into the second inlet port.

[0163] In some embodiments, a puncture feature provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one thread. Page 23 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0164] In some embodiments, a device provided by and / or utilized in accordance with embodiments of the present disclosure includes: an O-ring disposed within a second inlet port for sealing a flexible container or reservoir to a second inlet port when a lid is placed on top of a device body, and / or a flexible sealing element integrated into the lid, wherein the flexible sealing element includes an elastomer material over-molded onto an internal substrate or surface of the lid.

[0165] In some embodiments, a reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure includes two reaction chambers.

[0166] In some embodiments, a device provided by and / or utilized in accordance with embodiments of the present disclosure includes separate inlets coupling a first inlet port to each of two reaction chambers, and wherein the device includes a separate fluid outlet coupled to each of the two reaction chambers.

[0167] In some embodiments, a device provided by and / or utilized in accordance with embodiments of the present disclosure includes a common inlet from a first inlet port that splits into two separate lines each coupled to one of two reaction chambers, and wherein the device includes a separate fluid outlet from each of the two reaction chambers that merge together downstream of the reaction chambers and upstream of a diagnostic area.

[0168] In some embodiments, a device provided by and / or utilized in accordance with embodiments of the present disclosure a pre-treatment chamber disposed fluidly upstream of a reaction chamber and downstream of a first inlet port, the pre-treatment chamber including at least one bead.

[0169] In some embodiments, a device provided by and / or utilized in accordance with embodiments of the present disclosure includes a valve manifold including at least one recess in which a release valve is disposed, the release valve including a PVA membrane; and at least one sponge disposed within the valve manifold positioned in the vicinity of the release valve to hydrate the PVA membrane with timer fluid.

[0170] In some embodiments, a device provided by and / or utilized in accordance with embodiments of the present disclosure includes a common outlet line coupling a reaction chamber to a diagnostic area, and wherein the common outlet line includes at least one flow feature. Page 24 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0171] In some embodiments, a flow feature provided by and / or utilized in accordance with embodiments of the present disclosure includes one or more diagonal hatch lines to help promote flow mixing of sample fluid from a reaction chamber before reaching a diagnostic area.

[0172] In some embodiments, an inlet portion of a diagnostic area provided by and / or utilized in accordance with embodiments of the present disclosure includes one or more pins protruding into a flow path of the diagnostic area to promote capillary action.

[0173] In some embodiments, a reaction chamber provided by and / or utilized in accordance with embodiments of the present disclosure is coupled to a second flow channel via at least one outlet channel.

[0174] In some embodiments, an outlet channel provided by and / or utilized in accordance with embodiments of the present disclosure includes a straight channel.

[0175] In some embodiments, an outlet channel provided by and / or utilized in accordance with embodiments of the present disclosure includes a serpentine channel.

[0176] In some embodiments, an outlet channel provided by and / or utilized in accordance with embodiments of the present disclosure includes a serpentine channel including a plurality of kite-line expansions.

[0177] In some embodiments, an outlet channel provided by and / or utilized in accordance with embodiments of the present disclosure includes a serpentine channel including a plurality of diagonal hatch lines.

[0178] In some embodiments, a second flow channel provided by and / or utilized in accordance with embodiments of the present disclosure includes a plurality of fluid pins.

[0179] In some aspects, provided embodiments are directed to a device including: a sample inlet port; at least one reaction chamber disposed fluidly downstream of the sample inlet port; a diagnostic area disposed downstream of the reaction chamber, the diagnostic area including a vent hole at a downstream end; and a manual venting feature for opening the vent hole to atmosphere, wherein manipulating the manual venting feature such that the vent hole is open to atmosphere causes sample fluid contained in the reaction chamber to flow into the diagnostic area. Page 25 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0180] In some embodiments, a manual venting feature provided by and / or utilized in accordance with embodiments of the present disclosure includes an adhesive pull tab.

[0181] In some embodiments, a manual venting feature provided by and / or utilized in accordance with embodiments of the present disclosure includes two adhesive pull tabs including: a first adhesive pull tab, removal of which vents a reaction chamber to atmosphere and causes sample fluid to flow from a sample inlet port into the reaction chamber; and a second adhesive pull tab covering the vent hole, removal of which vents a diagnostic area to atmosphere and causes sample fluid to flow from the reaction chamber into the diagnostic area.

[0182] In some aspects, provided embodiments are directed to a method of using a device including: providing a device, the device including: a sample inlet port; at least one reaction chamber disposed fluidly downstream of the sample inlet port; the reaction chamber including a first vent hole on a side; a diagnostic area disposed downstream of the reaction chamber, the diagnostic area including a second vent hole at a downstream end; a first adhesive pull tab covering the first vent hole; and a second adhesive pull tab covering the vent hole; providing a fluid sample to be disposed within the sample inlet port; removing the first adhesive pull tab, venting the reaction chamber to atmosphere and causing the sample fluid to flow from the sample inlet port into the reaction chamber; and removing the second adhesive pull tab, venting the diagnostic area to atmosphere and causing sample fluid to flow from the reaction chamber into the diagnostic area.

[0183] In some embodiments, removing a first adhesive pull tab provided by and / or utilized in accordance with embodiments of the present disclosure includes allowing the lapse of a pre-determined amount of time.

[0184] In some aspects, provided embodiments are directed to a method of using a device including: providing a device body, the device body including: a first fluid inlet port disposed in a top surface; a second fluid inlet port disposed in the top surface; at least one reaction chamber disposed in the top surface and coupled fluidly downstream of the first fluid inlet port; a fluidic timing circuit disposed within the device body, the fluidic timing circuit coupled downstream of the second fluid inlet; and a release valve disposed fluidly downstream of the fluidic timing circuit; a diagnostic area fluidly coupled downstream of the reaction chamber and upstream of the release valve; providing a fluid sample to be disposed Page 26 of 164 12865804v1Atty. Docket No.: 2013065-0954 within the first fluid inlet port; and providing a fluid timer to be disposed within the second fluid inlet port; wherein the release valve, when contacted by the timer fluid, dissolves and fluidly couples the reaction chamber to atmosphere, thereby allowing the fluid sample to flow into the diagnostic area.

[0185] In some embodiments, providing a fluid provided by and / or utilized in accordance with embodiments of the present disclosure includes providing a lid including a puncturable reservoir containing a timer fluid located on an underside of the lid at a location corresponding to a second fluid inlet port when the lid is placed over a top surface of a device body, and wherein the device body further includes at least one puncture feature disposed within the second inlet port such that the puncture feature punctures the puncturable reservoir when the lid is placed over the top surface of the device body, thereby causing the timer fluid to flow into the second inlet port.

[0186] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes a reagent bottle for collecting a biological sample and eluting the biological sample therein thereby creating a fluid sample prior to introduction of the fluid sample to a sample inlet port.

[0187] In some embodiments, a system provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one filter for filtering a biological sample.

[0188] In some embodiments, a filter provided by and / or utilized in accordance with embodiments of the present disclosure is disposed within a sample inlet port and / or a reagent bottle.

[0189] In some embodiments, a first fluid inlet port provided by and / or utilized in accordance with embodiments of the present disclosure is positioned at a higher vertical height than a reaction chamber when a device is in use.

[0190] In some embodiments, a puncturable, at-least partially flexible container or reservoir provided by and / or utilized in accordance with embodiments of the present disclosure includes a pierceable membrane or film. Page 27 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0191] In some embodiments, a piercing feature provided by and / or utilized in accordance with embodiments of the present disclosure includes at least one of a needle, pointed protrusion, and a knife-like protrusion.

[0192] In some aspects, provided embodiments are directed to a diagnostic method including: collecting a biological sample; eluting the biological sample with at least one reagent and / or solution, thereby creating a sample fluid; providing a device as described herein; adding one or more drops of the sample fluid to a sample inlet port of the device; adding timer fluid to a timer fluid inlet port of the device; waiting for a pre-specified period of time; and reading results of the diagnostic method via a diagnostic area of the device.

[0193] In some embodiments, adding timer fluid to a timer fluid inlet port provided by and / or utilized in accordance with embodiments of the present disclosure includes placing a lid containing the timer fluid on top of a device.

[0194] In some embodiments, a method provided by and / or utilized in accordance with embodiments of the present disclosure includes filtering a sample fluid via a filter prior to adding one or more drops of the sample fluid to a sample inlet port of a device, wherein the filter is disposed within the sample inlet port and / or a reagent bottle.

[0195] In some embodiments, a pre-specified period of time utilized in accordance with embodiments of the present disclosure includes a time period in a range from about 5 minutes to about 30 minutes.

[0196] In some embodiments, a method provided by and / or utilized in accordance with embodiments of the present disclosure includes a separate eluting step performed on the biological sample prior to introduction of the biological sample into the device. BRIEF DESCRIPTION OF THE DRAWING

[0197] The present teachings described herein will be more fully understood from the following description of various illustrative embodiments, when read together with the accompanying drawing. It should be understood that the drawing described below is for illustration purposes only and is not intended to limit the scope of the present teachings in any way. Page 28 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0198] FIG.1A is a schematic diagram illustrating a front view of a fluidic device featuring an air diffusion timer, according to aspects of the present embodiments.

[0199] FIG.1B is a schematic diagram illustrating a cross-sectional side view of a fluidic device featuring an air diffusion timer, focusing on a porous membrane, according to aspects of the present embodiments.

[0200] FIG.1C is a schematic diagram illustrating a front view of another embodiment of a fluidic device featuring an air diffusion timer, according to aspects of the present embodiments.

[0201] FIG.1D is a schematic diagram illustrating a front view of a lower portion of a fluidic device focusing on a passively actuated valve, according to aspects of the present embodiments.

[0202] FIG.1E is a schematic diagram illustrating a front view of a lower portion of a fluidic device focusing on a passively actuated valve, according to aspects of the present embodiments.

[0203] FIG.2A is a schematic diagram illustrating a front view of a fluidic device featuring an air diffusion timer, according to aspects of the present embodiments.

[0204] FIG.2B is a schematic diagram illustrating a front view of a fluidic device featuring an air diffusion timer, according to aspects of the present embodiments.

[0205] FIG.2C is a schematic diagram illustrating a front view of a fluidic device featuring an air diffusion timer, according to aspects of the present embodiments.

[0206] FIG.2D is a schematic diagram illustrating a front view of a fluidic device featuring an air diffusion timer, according to aspects of the present embodiments.

[0207] FIG.2E is a schematic diagram illustrating a front view of a fluidic device featuring an air diffusion timer, according to aspects of the present embodiments.

[0208] FIG.2F is a schematic diagram illustrating a front view of a fluidic device featuring an air diffusion timer, according to aspects of the present embodiments.

[0209] FIG.2G is a schematic diagram illustrating a front view of a fluidic device featuring an air diffusion timer, according to aspects of the present embodiments.

[0210] FIG.2H is a schematic diagram illustrating a front view of a fluidic device featuring an air diffusion timer, according to aspects of the present embodiments. Page 29 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0211] FIG.3 is a schematic diagram illustrating a front view of a fluidic device featuring a fluidic serpentine timing circuit, according to aspects of the present embodiments.

[0212] FIG.4A is a schematic diagram illustrating a front view of a fluidic device featuring a fluidic serpentine timer, according to aspects of the present embodiments.

[0213] FIG.4B is a schematic diagram illustrating a front view of a fluidic device featuring a fluidic serpentine timer, according to aspects of the present embodiments.

[0214] FIG.4C is a schematic diagram illustrating a front view of a fluidic device featuring a fluidic serpentine timer, according to aspects of the present embodiments.

[0215] FIG.4D is a schematic diagram illustrating a front view of a fluidic device featuring a fluidic serpentine timer, according to aspects of the present embodiments.

[0216] FIG.4E is a schematic diagram illustrating a front view of a fluidic device featuring a fluidic serpentine timer, according to aspects of the present embodiments.

[0217] FIG.4F is a schematic diagram illustrating a front view of a fluidic device featuring a fluidic serpentine timer, according to aspects of the present embodiments.

[0218] FIG.5A is a schematic diagram illustrating a side view of a passively actuated valve with a dissolvable membrane on a flat chip surface design, according to aspects of the present embodiments.

[0219] FIG.5B is a schematic diagram illustrating a side view of a passively actuated valve with a dissolvable membrane on a flat chip surface design, according to aspects of the present embodiments.

[0220] FIG.5C is a schematic diagram illustrating a side view of a passively actuated valve with a dissolvable membrane on a flat chip surface design, according to aspects of the present embodiments.

[0221] FIG.5D is a schematic diagram illustrating a side view of a passively actuated valve with a dissolvable membrane on a flat chip surface design, according to aspects of the present embodiments.

[0222] FIG.5E is a schematic diagram illustrating a side view of a passively actuated valve with a dissolvable membrane on a flat chip surface design, according to aspects of the present embodiments. Page 30 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0223] FIG.5F is a schematic diagram illustrating a side view of a passively actuated valve with a dissolvable membrane on a flat chip surface design, according to aspects of the present embodiments.

[0224] FIG.5G is a schematic diagram illustrating a side view of a passively actuated valve with a dissolvable membrane on a flat chip surface design, according to aspects of the present embodiments.

[0225] FIG.6A is a schematic diagram illustrating a side view of a wicking-assisted passively actuated valve design, according to aspects of the present embodiments.

[0226] FIG.6B is a schematic diagram illustrating a side view of a wicking-assisted passively actuated valve design, according to aspects of the present embodiments.

[0227] FIG.6C is a schematic diagram illustrating a side view of a wicking-assisted passively actuated valve design, according to aspects of the present embodiments.

[0228] FIG.6D is a schematic diagram illustrating a side view of a wicking-assisted passively actuated valve design, according to aspects of the present embodiments.

[0229] FIG.6E is a schematic diagram illustrating a side view of a wicking-assisted passively actuated valve design, according to aspects of the present embodiments.

[0230] FIG.7A is a schematic diagram illustrating a side view of a passively actuated valve design featuring a sponge to assist in membrane puncturing, according to aspects of the present embodiments.

[0231] FIG.7B is a schematic diagram illustrating a side view of a passively actuated valve design featuring a sponge to assist in membrane puncturing, according to aspects of the present embodiments.

[0232] FIG.7C is a schematic diagram illustrating a side view of a passively actuated valve design featuring a sponge to assist in membrane puncturing, according to aspects of the present embodiments.

[0233] FIG.7D is a schematic diagram illustrating a side view of a passively actuated valve design featuring a sponge to assist in membrane puncturing, according to aspects of the present embodiments. Page 31 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0234] FIG.8A is a schematic diagram illustrating a side view of a passively actuated valve design featuring a sponge and needle to assistant in membrane puncturing, according to aspects of the present embodiments.

[0235] FIG.8B is a schematic diagram illustrating a side view of a passively actuated valve design featuring a sponge and needle to assistant in membrane puncturing, according to aspects of the present embodiments.

[0236] FIG.8C is a schematic diagram illustrating a side view of a passively actuated valve design featuring a sponge and needle to assistant in membrane puncturing, according to aspects of the present embodiments.

[0237] FIG.8D is a schematic diagram illustrating a side view of a passively actuated valve design featuring a sponge and needle to assistant in membrane puncturing, according to aspects of the present embodiments.

[0238] FIG.8E is a schematic diagram illustrating a side view of a passively actuated valve design featuring a sponge and needle to assistant in membrane puncturing, according to aspects of the present embodiments.

[0239] FIG.8F is a schematic diagram illustrating a side view of a passively actuated valve design featuring a sponge and needle to assistant in membrane puncturing, according to aspects of the present embodiments.

[0240] FIG.9 shows a series of photographs of a fluidic device from 0 min to 20 min reaction time, showing the addition of sample fluid, flow of fluid, and lateral flow strip readout, according to aspects of the present embodiments.

[0241] FIG.10A shows a photograph of four fluidic devices showing empty devices, according to aspects of the present embodiments.

[0242] FIG.10B shows a photograph of four fluidic devices showing the addition of sample fluid, according to aspects of the present embodiments.

[0243] FIG.10C shows a photograph of four fluidic devices flow of fluid, according to aspects of the present embodiments.

[0244] FIG.10D shows a photograph of four fluidic devices showing lateral flow strip readouts, according to aspects of the present embodiments. Page 32 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0245] FIG.10E shows an enlarged view of the leftmost device from FIGS.10A- 10D, according to aspects of the present embodiments.

[0246] FIG.10F shows an enlarged view of the leftmost device from FIGS.10A- 10D, according to aspects of the present embodiments.

[0247] FIG.10G shows an enlarged view of the leftmost device from FIGS.10A- 10D, according to aspects of the present embodiments.

[0248] FIG.10H shows an enlarged view of the leftmost device from FIGS.10A- 10D, according to aspects of the present embodiments.

[0249] FIG.11A shows a photograph of fluidic devices and lateral flow strip readouts, according to aspects of the present embodiments.

[0250] FIG.11B shows a photograph of fluidic devices and lateral flow strip readouts, according to aspects of the present embodiments.

[0251] FIG.11C shows a photograph of fluidic devices and lateral flow strip readouts, according to aspects of the present embodiments.

[0252] FIG.12A shows a perspective view of a combined timer and air valve chip, according to aspects of the present embodiments.

[0253] FIG.12B shows a perspective view of a combined timer and air valve chip, according to aspects of the present embodiments.

[0254] FIG.12C shows a perspective view of a combined timer and air valve chip, according to aspects of the present embodiments.

[0255] FIG.12D shows a perspective view of a combined timer and air valve chip, according to aspects of the present embodiments.

[0256] FIG.12E shows a perspective view of a combined timer and air valve chip, according to aspects of the present embodiments.

[0257] FIG.12F shows a perspective view of a combined timer and air valve chip, according to aspects of the present embodiments.

[0258] FIG.12G shows a perspective view of a combined timer and air valve chip, according to aspects of the present embodiments. Page 33 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0259] FIG.12H shows a perspective view of a combined timer and air valve chip, according to aspects of the present embodiments.

[0260] FIG.12I shows a perspective view of a combined timer and air valve chip, according to aspects of the present embodiments.

[0261] FIG.12J shows a perspective view of a combined timer and air valve chip, according to aspects of the present embodiments.

[0262] FIG.13A shows a perspective view of a slow air diffusion chip, according to aspects of the present embodiments.

[0263] FIG.13B shows a perspective view of a slow air diffusion chip, according to aspects of the present embodiments.

[0264] FIG.13C shows a perspective view of a slow air diffusion chip, according to aspects of the present embodiments.

[0265] FIG.13D shows a perspective view of a slow air diffusion chip, according to aspects of the present embodiments.

[0266] FIG.13E shows a perspective view of a slow air diffusion chip, according to aspects of the present embodiments.

[0267] FIG.14A shows a perspective view of a base to support a slow air diffusion chip, according to aspects of the present embodiments.

[0268] FIG.14B shows a perspective view of a base to support a slow air diffusion chip, according to aspects of the present embodiments.

[0269] FIG.14C shows a perspective view of a base to support a slow air diffusion chip, according to aspects of the present embodiments.

[0270] FIG.14D shows a perspective view of a base to support a slow air diffusion chip, according to aspects of the present embodiments.

[0271] FIG.14E shows a perspective view of a base to support a slow air diffusion chip, according to aspects of the present embodiments.

[0272] FIG.14F shows a perspective view of a base to support a slow air diffusion chip, according to aspects of the present embodiments. Page 34 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0273] FIG.14G shows a perspective view of a base to support a slow air diffusion chip, according to aspects of the present embodiments.

[0274] FIG.14H shows a perspective view of a base to support a slow air diffusion chip, according to aspects of the present embodiments.

[0275] FIG.14I shows a perspective view of a base to support a slow air diffusion chip, according to aspects of the present embodiments.

[0276] FIG.14J shows a perspective view of a base to support a slow air diffusion chip, according to aspects of the present embodiments.

[0277] FIG.15A shows a photograph of a front side of a combined timer and air valve chip, according to aspects of the present embodiments.

[0278] FIG.15B shows a photograph of a front side of a combined timer and air valve chip, according to aspects of the present embodiments.

[0279] FIG.16A shows a photograph of a reverse side and front side of a combined timer and air valve chip, according to aspects of the present embodiments.

[0280] FIG.16B shows a photograph of a reverse side and front side of a combined timer and air valve chip, according to aspects of the present embodiments.

[0281] FIG.17A shows a plot of time required for fluid to reach a lateral flow strip in minutes as a function of the size in millimeters of a square air diffusion pad in air diffusion timer devices, according to aspects of the present embodiments.

[0282] FIG.17B shows a series of photographs from time t = 0 min to t = 33 min showing devices with different air diffusion pad sizes with different flow times to reach the lateral flow strip corresponding to the data in FIG.17A, according to aspects of the present embodiments.

[0283] FIG.18 is a flow chart illustrating a method of using a power-free fluidic device, according to aspects of the present embodiments.

[0284] FIG.19 is a flow chart illustrating a method of using a power-free fluidic device with a timer fluid, according to aspects of the present embodiments.

[0285] FIG.20A is a CAD drawing illustrating a top view of a horizontal fluidic device featuring a fluidic serpentine timer, according to aspects of the present embodiments. Page 35 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0286] FIG.20B is a CAD drawing illustrating a bottom view of a horizontal fluidic device featuring a fluidic serpentine timer, according to aspects of the present embodiments.

[0287] FIG.20C shows an enlarged top view of two reactions chambers, according to aspects of the present embodiments.

[0288] FIG.20D shows an enlarged top view of an outlet channel and a lateral flow chamber, according to aspects of the present embodiments.

[0289] FIG.21A is a CAD drawing illustrating a top view of a horizontal fluidic device, according to aspects of the present embodiments.

[0290] FIG.21B is a CAD drawing illustrating a top view of a horizontal fluidic device, according to aspects of the present embodiments.

[0291] FIG.21C is a CAD drawing illustrating a top view of a horizontal fluidic device, according to aspects of the present embodiments.

[0292] FIG.22A is a schematic diagram illustrating a side view of a fluidic device including a sponge, according to aspects of the present embodiments.

[0293] FIG.22B is a schematic diagram illustrating a side view of a fluidic device including a sponge, according to aspects of the present embodiments.

[0294] FIG.22C is a schematic diagram illustrating a side view of a fluidic device including a sponge, according to aspects of the present embodiments.

[0295] FIG.23A is a schematic diagram illustrating a side view of a fluidic device including a sponge, according to aspects of the present embodiments.

[0296] FIG.23B is a schematic diagram illustrating a side view of a fluidic device including two sponges, according to aspects of the present embodiments.

[0297] FIG.24A shows an exemplary fluidic device including a fluidic serpentine timer, according to aspects of the present embodiments.

[0298] FIG.24B shows an exemplary fluidic device including a fluidic serpentine timer, according to aspects of the present embodiments. Page 36 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0299] FIG.25A shows an exemplary fluidic device including a fluidic serpentine timer, according to aspects of the present embodiments.

[0300] FIG.25B shows an exemplary fluidic device including a fluidic serpentine timer, according to aspects of the present embodiments.

[0301] FIG.26A shows two fluidic devices positioned at a 15 ° tilt, according to aspects of the present embodiments.

[0302] FIG.26B shows two fluidic devices positioned at a 15 ° tilt, according to aspects of the present embodiments.

[0303] FIG.26C shows two fluidic devices positioned at a 15 ° tilt, according to aspects of the present embodiments.

[0304] FIG.27A illustrates a reaction chamber design including a splitting inlet / separate outlet arrangement, according to aspects of the present embodiments.

[0305] FIG.27B illustrates a reaction chamber design including a fully joined arrangement, according to aspects of the present embodiments.

[0306] FIG.27C illustrates a reaction chambers design including a fully separate arrangement, according to aspects of the present embodiments.

[0307] FIG.27D illustrates a reaction chambers design including a splitting inlet / merging outlet arrangement, according to aspects of the present embodiments.

[0308] FIG.28A illustrates a reaction chamber design including a mixing chamber, according to aspects of the present embodiments.

[0309] FIG.28B illustrates a reaction chamber design including unpinning air vents, according to aspects of the present embodiments.

[0310] FIG.28C illustrates a reaction chamber design including features that enable siphoning of excess sample, according to aspects of the present embodiments.

[0311] FIG.29A illustrates an air vent design including an outlet channel, according to aspects of the present embodiments. Page 37 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0312] FIG.29B illustrates an air vent design including a vent chamber, according to aspects of the present embodiments.

[0313] FIG.29C illustrates an air vent design including a membrane, according to aspects of the present embodiments.

[0314] FIG.29D illustrates an air vent design including an inlet channel, according to aspects of the present embodiments.

[0315] FIG.30 illustrates a mixer chamber featuring a serpentine design, according to aspects of the present embodiments.

[0316] FIG.31 illustrates a mixer chamber featuring an X-shape design, according to aspects of the present embodiments.

[0317] FIG.32A illustrates a mixer chamber featuring a four-turn design, according to aspects of the present embodiments.

[0318] FIG.32B illustrates a mixer chamber featuring a four-turn design, according to aspects of the present embodiments.

[0319] FIG.32C illustrates a mixer chamber featuring a four-turn design, according to aspects of the present embodiments.

[0320] FIG.32D illustrates a mixer chamber featuring a four-turn design, according to aspects of the present embodiments.

[0321] FIG.32E illustrates a mixer chamber featuring a four-turn design, according to aspects of the present embodiments.

[0322] FIG.32F illustrates a mixer chamber featuring a four-turn design, according to aspects of the present embodiments.

[0323] FIG.33A illustrates a mixer chamber featuring a merging burst-valve design, according to aspects of the present embodiments.

[0324] FIG.33B illustrates a mixer chamber featuring a merging burst-valve design, according to aspects of the present embodiments. Page 38 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0325] FIG.34A shows schematic diagrams of a U-bend reaction chamber design, according to the aspects of the present embodiments.

[0326] FIG.34B shows schematic diagrams of a U-bend reaction chamber design, according to the aspects of the present embodiments.

[0327] FIG.34C shows schematic diagrams of a U-bend reaction chamber design, according to the aspects of the present embodiments.

[0328] FIG.35 shows schematic diagrams of a reaction chamber design including a splitting outlet arrangement, according to the aspects of the present embodiments.

[0329] FIG.36A illustrates a reaction chamber inlet design, according to aspects of the present embodiments.

[0330] FIG.36B illustrates a reaction chamber inlet design, according to aspects of the present embodiments.

[0331] FIG.36C illustrates a reaction chamber inlet design, according to aspects of the present embodiments.

[0332] FIG.37A shows a straight channel connected to a lateral flow chamber, according to aspects of the present embodiments.

[0333] FIG.37B shows a basic serpentine channel connected to a lateral flow chamber, according to aspects of the present embodiments.

[0334] FIG.37C shows a serpentine channel with kite-line expansions connected to a lateral flow chamber, according to aspects of the present embodiments.

[0335] FIG.37D shows a serpentine channel with diagonal hatch-lines connected to a lateral flow chamber, according to aspects of the present embodiments.

[0336] FIG.37E shows a lateral flow strip indicating no mixing of reaction fluids, according to aspects of the present embodiments.

[0337] FIG.37F shows a lateral flow strip indicating full mixing of reaction fluids, according to aspects of the present embodiments. Page 39 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0338] FIG.38 shows a microfluidic device including a low ceiling and a microfluidic device including a high ceiling, according to aspects of the present embodiments.

[0339] FIG.39 shows a microfluidic device including a low ceiling and a microfluidic device including an insert, according to aspects of the present embodiments.

[0340] FIG.40 illustrates schematic diagrams of a standard lateral flow chamber and a high ceiling lateral flow chamber, according to aspects of the present embodiments.

[0341] FIG.41 shows an exemplary lateral flow chamber including a lateral flow strip and an insert, according to aspects of the present embodiments.

[0342] FIG.42 is a schematic diagram of a fluidic device featuring a metering chamber, according to aspects of the present embodiments.

[0343] FIG.43 is a schematic diagram of a fluidic device including a lid, according to aspects of the present embodiments.

[0344] FIG.44A is a schematic diagram of a fluidic device including a lid, according to aspects of the present embodiments.

[0345] FIG.44B shows an exemplary top view of a base chip, according to aspects of the present embodiments.

[0346] FIG.44C shows an exemplary top view of a base chip, according to aspects of the present embodiments.

[0347] FIG.45A is a schematic diagram of a fluidic device including a lid, according to aspects of the present embodiments.

[0348] FIG.45B shows an exemplary puncture, according to aspects of the present embodiments.

[0349] FIG.46 is an example of a fluidic device including a lid, according to aspects of the present embodiments.

[0350] FIG.47A is a schematic diagram of a fluidic device including a lid, according to aspects of the present embodiments. Page 40 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0351] FIG.47B shows an exemplary CAD drawing of the fluidic device, according to aspects of the present embodiments.

[0352] FIG.48A is a schematic diagram of a fluidic device including a lid, according to aspects of the present embodiments.

[0353] FIG.48B shows an exemplary CAD drawing of the fluidic device, according to aspects of the present embodiments.

[0354] FIG.48C shows an exemplary CAD drawing of the fluidic device, according to aspects of the present embodiments.

[0355] FIG.49 shows individual components of a fluidic device assembly / kit, according to aspects of the present embodiments.

[0356] FIG.50 shows a main chip, according to aspects of the present embodiments.

[0357] FIG.51 shows a fluidic device, according to aspects of the present embodiments.

[0358] FIG.52 is a flowchart of an assembly method of a microfluidic device, according to aspects of the present embodiments.

[0359] FIG.53A shows a front view of a manual activation fluidic device, according to aspects of the present embodiments.

[0360] FIG.53B shows a back view of a manual activation fluidic device, according to aspects of the present embodiments.

[0361] FIG.53C shows a top view of main components of a manual activation fluidic device, according to aspects of the present embodiments.

[0362] FIG.54A illustrates exemplary operational details of a manual activation fluidic device, according to aspects of the present embodiments.

[0363] FIG.54B illustrates exemplary operational details of a manual activation fluidic device, according to aspects of the present embodiments. Page 41 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0364] FIG.54C illustrates exemplary operational details of a manual activation fluidic device, according to aspects of the present embodiments.

[0365] FIG.54D illustrates exemplary operational details of a manual activation fluidic device, according to aspects of the present embodiments.

[0366] FIG.54E illustrates exemplary operational details of a manual activation fluidic device, according to aspects of the present embodiments.

[0367] FIG.54F illustrates exemplary operational details of a manual activation fluidic device, according to aspects of the present embodiments.

[0368] FIG.55A illustrates an exemplary view of a lateral flow chamber, according to aspects of the present embodiments.

[0369] FIG.55B illustrates an exemplary view of a lateral flow chamber, according to aspects of the present embodiments.

[0370] FIG.55C illustrates an exemplary view of a lateral flow chamber, according to aspects of the present embodiments.

[0371] FIG.55D illustrates an exemplary view of a lateral flow chamber, according to aspects of the present embodiments. DEFINITIONS

[0372] About, Approximately: As used herein, the terms “about” and “approximately” as used in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” and “approximately” in that context. For example, in some embodiments, the terms “about” and “approximately” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0373] Ambient temperature: As used herein, the term “ambient temperature” is the temperature of surroundings. In general, the term ambient temperature is to be understood as the temperature of any object or environment surrounding an item. Measuring an ambient Page 42 of 164 12865804v1Atty. Docket No.: 2013065-0954 temperature can be accomplished by using e.g., a thermometer or sensor. The ambient temperature of an item is dependent on the temperature of the surrounding of the item. The surroundings can have any temperature, such as a temperature below 95°C, such as below 90°C, such as below 85°C, such as below 80°C, such as below 75°C, such as below 70°C, such as below 65°C, such as below 60°C, such as below 55°C, such as below 50°C, such as below 45°C, such as below 40°C, such as below 35°C, such as below 30°C, such as below 25°C, such as below 24°C, such as below 23°C, such as below 22°C, such as below 21°C, such as below 20°C. Exemplary ambient temperature ranges include 5°C to 50°C, such as 10°C to 40°C, such as 15°C to 35°C, such as 20°C to 30°C, such as 20°C to 25°C, such as 20°C to 22°C. In some embodiments, ambient temperature may refer to room temperature or the temperature for ease of operation of a power-free fluidic device by a user. In connection with reactions that occur at ambient temperatures and do not need to be heated, the term ambient temperatures may refer to temperatures in a range from about 60°F to about 75°F.

[0374] Associated with: As used herein, the term, “associated with” refers to two events or entities when presence, level and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of and / or susceptibility to a disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0375] Biological Sample: As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a fluid or a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a source of interest is or comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; Page 43 of 164 12865804v1Atty. Docket No.: 2013065-0954 saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; tongue swabs, nasal swabs; washings or lavages such as a ductal lavages or bronchoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and / or excretions; and / or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, saliva, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components, etc.

[0376] Block or Cap: As used herein, the term “block” or “cap” or “capped” refers to an action of preventing an extension of a nucleic acid by an enzyme, e.g., a polymerase. In some embodiments, a nucleic acid is blocked by one or more modification to its 3’ end. In some embodiments, a nucleic acid is blocked by one or more modification to its 5’ end. In some embodiments, a blocked oligonucleotide is an oligonucleotide primer. In some embodiments, a blocked oligonucleotide is a primer. In some embodiments, a blocked oligonucleotide is a probe. In some embodiments, the term “block” and the term “cap” or ‘capped” are used interchangeably to indicate the same action. In some embodiments, an oligonucleotide binder comprises a 3’ blocking molecule. In some embodiments, a 3’ blocking molecule is a 3’ddNTP, 3’ Inverted dT, a 3’ carbon chain spacer, a 3’ hexanediol, a 3’ amino spacer, or a 3’ phosphorylation.

[0377] Cellular lysate: As used herein, the term “cellular lysate” or “cell lysate” refers to a fluid containing contents of one or more disrupted cells (i.e., cells whose membrane has been disrupted). In some embodiments, a cellular lysate includes both Page 44 of 164 12865804v1Atty. Docket No.: 2013065-0954 hydrophilic and hydrophobic cellular components. In some embodiments, a cellular lysate includes predominantly hydrophilic components; in some embodiments, a cellular lysate includes predominantly hydrophobic components. In some embodiments, a cellular lysate is a lysate of one or more cells selected from the group consisting of plant cells, microbes (e.g., viruses, bacteria, archaea, fungi and protist), animal cells (e.g., mammalian cells), human cells, and combinations thereof. In some embodiments, a cellular lysate is a lysate of one or more abnormal cells, such as cancer cells. In some embodiments, a cellular lysate is a crude lysate in that little or no purification is performed after disruption of the cells; in some embodiments, such a lysate is referred to as a “primary” lysate. In some embodiments, one or more isolation or purification steps is performed on a primary lysate; however, the term “lysate” refers to a preparation that includes multiple cellular components and not to pure preparations of any individual component.

[0378] Detectable entity: The term “detectable entity” as used herein refers to any element, molecule, functional group, compound, fragment or moiety that is detectable. In some embodiments, a detectable entity is provided or utilized alone. In some embodiments, a detectable entity is provided and / or utilized in association with (e.g., joined to) another agent. Examples of detectable entities include, but are not limited to: various ligands, radionuclides (e.g.,3H,14C,18F,19F,32P,35S,135I,125I,123I,64Cu,187Re,111In,90Y,99mTc,177Lu,89Zr etc.), fluorescent dyes (for specific exemplary fluorescent dyes, see below), chemiluminescent agents (such as, for example, acridinum esters, stabilized dioxetanes, and the like), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, carbon nanoparticles, enzymes (for specific examples of enzymes, see below), colorimetric labels (such as, for example, dyes, colloidal gold, and the like), biotin, dioxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available.

[0379] Determine: Many methodologies described herein include a step of “determining”. Those of ordinary skill in the art, reading the present specification, will appreciate that such “determining” can utilize or be accomplished through use of any of a variety of techniques available to those skilled in the art, including for example specific techniques explicitly referred to herein. In some embodiments, determining involves manipulation of a physical sample. In some embodiments, determining involves Page 45 of 164 12865804v1Atty. Docket No.: 2013065-0954 consideration and / or manipulation of data or information, for example utilizing a computer or other processing unit adapted to perform a relevant analysis. In some embodiments, determining involves receiving relevant information and / or materials from a source. In some embodiments, determining involves comparing one or more features of a sample or entity to a comparable reference.

[0380] Power Free: As used herein, in the context of the disclosed devices and systems, the terms “power free” and “power-free” mean that no external force or power sources other than fluid movement (including the movement of air) caused by gravity and / or capillary action (or chemical activity to dissolve the dissolvable membrane) are used to move fluids from one location to another within preselected periods of time. In addition, no external (or internal) power sources are used to initiate reactions and / or to heat fluids used in connection with the various disclosed reactions, as described herein.

[0381] Sample: As used herein, the term “sample” typically refers to an aliquot of material obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest is a biological or environmental source. In some embodiments, a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human). In some embodiments, a source of interest is or comprises biological tissue or fluid. In some embodiments, a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or component(s) thereof. In some embodiments, a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and / or a transcellular fluid. In some embodiments, a biological fluid may be or comprise a plant exudate. In some embodiments, a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, tongue, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., bronchoalvealar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of Page 46 of 164 12865804v1Atty. Docket No.: 2013065-0954 interest by any appropriate means. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and / or purification of certain components, etc. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0382] It is contemplated that methods, systems, compositions, and processes described herein encompass variations and adaptations developed using information from the embodiments described herein. Adaptation and / or modification of the methods, systems, compositions, and processes described herein may be performed, as contemplated by this description.

[0383] Throughout the description, where methods, systems, compositions, and / or processes are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are embodiments of the systems and / or compositions that consist essentially of, or consist of, the recited components, and that there are embodiments of the processes and methods that consist essentially of, or consist of, the recited steps.

[0384] It should be understood that the order of steps or order for performing certain action is immaterial so long as the method and / or processes remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0385] The mention herein of any publication, for example, in the Background section, is not an admission that the publication serves as prior art with respect to any of the claims presented herein. The Background section is presented for purposes of clarity and is not meant as a description of prior art with respect to any claim.

[0386] Documents are incorporated herein by reference as noted. Where there is any discrepancy in the meaning of a particular term, the meaning provided in the Definition section above is controlling. Page 47 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0387] Headers are provided for the convenience of the reader; the presence and / or placement of a header is not intended to limit the scope of the subject matter described herein.

[0388] Provided herein are improved technologies for performing assays such as diagnostic tests in portable devices without the use of electrical power. Applications of Fluidic Diagnostic Devices

[0389] The detection of various targets (e.g., target nucleic acids, pathogenic microbes, microorganisms, etc.) at the point of need (PON) would benefit from power-free fluidic devices that can be easily operated without the need for any electricity for heating, fluid movement, timing, and readout. A non-limiting example of a process that can be implemented in a power-free fluidic device is an ambient temperature nucleic acid amplification reaction known as strand displacement amplification (SDA), such as capped SDA (cSDA), that can amplify nucleic acid sequences without the need for any external heat source. Exemplary SDA, such as cSDA, is described in WO2024 / 006552 the entirety of which is herein incorporated by reference. Such a power-free fluidic device would perform the following steps as summarized in FIG.18: accept a fluid sample (e.g., a patient sample, e.g., a biological sample, e.g., a pre-filtered or otherwise treated sample, e.g., a processed sample such as a sample including a sample buffer; step 902); direct the sample fluid to rehydrate a lyophilized cSDA reaction bead with a metered volume (step 904); hold the fluid in a reaction chamber for a specific period of time for the reaction to take place (step 906); and transfer the fluid from the reaction chamber to a readout such as a lateral flow strip (908). Typical PON nucleic acid-based diagnostics employ heating elements and mechanically actuated fluidic components that require the use of a circuit board to control the timing of fluid transfer steps, as well as a power source (for example, at least one battery; see e.g., the Visby sexual health test: https: / / www.visbymedical.com / sexual-health-test / ).

[0390] In the present disclosures, there are three main design elements: 1. At least one reaction chamber that can be filled with a precise volume of sample fluid. 2. A mechanism whereby dead airspace is used to hold the sample fluid in place inside certain volumes (e.g., reaction chambers so that reactions can proceed), and where a dissolvable membrane (e.g., a PVA membrane) forms part of the containment of said dead airspace. Page 48 of 164 12865804v1Atty. Docket No.: 2013065-0954 3. A timing delay mechanism whereby a fluid traverses from one location to another at a controlled rate, ultimately coming into contact with the dissolvable membrane after a programmable amount of time, thus opening up the dead airspace and allowing fluid to exit from the certain volumes (e.g., reaction chambers). Fluidic Timers

[0391] In the present disclosures, several embodiments of fluidic timers are described. Fluidic timers are timing mechanisms in which a flow of fluid takes a prescribed duration of time to complete. By controlling various design parameters (e.g., materials properties, flow channel dimensions, etc.) the amount of time it takes for a certain flow of liquid or gas can be controlled. The fluidic timer embodiments described herein operate without the need for electrical power, circuits, or other control. Once a fluid is introduced to the fluidic timer, the timing mechanism is triggered to begin. Air Diffusion Timer

[0392] Air diffusion timers as described herein make use of the diffusion of air through porous membranes with very small pores, which controls the amount of time that a dead volume of air can be used to block the flow of a liquid.

[0393] FIG.1A is a schematic diagram illustrating a front view of a fluidic device 100 featuring an air diffusion timer, according to aspects of the present embodiments. In this embodiment, the device 100 is a vertical standing chip with gravity-driven flow (i.e., liquid flows from top to bottom). The slow, controlled diffusion of air through a porous membrane allows for the timed flow of a small volume of reaction fluid down a channel. The tunable timing of the flow creates a power-free timer that controls the time that the reactions proceed before being moved to a readout such as a lateral flow (LF) strip, or to another step (e.g., biochemical manipulation, a downstream reaction, electrical readout, etc.).

[0394] Referring still to FIG.1A, the device 100 includes a main chip surface 112 or chip body 112. In some embodiments, the main chip body 112 is made of a polymeric material (e.g., 3D printer resin plastic, e.g., Formlabs Clear Resin, Polymethylmethacrylate (PMMA), Cyclo-olefin-copolymer (COC), Cyclo-olefinpolymer (COP), Polycarbonate (PC), Polystyrene (PS), or other polymers for microfluidics). A sample inlet port and reservoir 101 Page 49 of 164 12865804v1Atty. Docket No.: 2013065-0954 at the top of the device 100 is where a sample fluid is input. In some embodiments, the reservoir 101 may hold a volume of approximately 100 μL to 5 mL (e.g., a volume of approximately 100 μL to about 2 mL, or from about 100 μL to about 1 mL, or from about 100 μL to about 500 μL, or from about 100 μL to about 200 μL, or from about 200 μL to about 5 mL, or from about 200 μL to about 2 mL, or from about 200 μL to about 1 mL, or from about 200 μL to about 500 μL, or from about 500 μL to about 5 mL, or from about 800 μL to about 2 mL, or from about 1.5 mL to about 2 mL, and / or other subranges therebetween). In some embodiments, the reservoir 101 may include dimensions (i.e., height, width, and / or length) in a range from approximately 5 mm to about 100 mm each. The sample flows down a first fluid channel 116, and then fills up two side channels 109 (e.g., one, two, or more side channels) to reach reaction chambers 103 that each contain a lyophilized reaction bead 104 comprising reagents needed for a particular reaction or assay. In some embodiments, the first fluid channel 116 may have dimensions (i.e., height, width, or diameter) in a range from about 0.1mm to about 2mm, with a length from about 2 mm to about 100mm. In some embodiments, the first fluid channel 116 may include a cross section that is substantially circular, oval, elliptical, square, rectangular, trapezoidal, triangular, and / or other shaped. In some embodiments, the side channels 109 may have dimensions (i.e., height, width, or diameter) in a range from about 0.1mm to about 2mm, with a length from about 2 mm to about 100mm. Reaction chamber air vents 105 allow air to escape so that the fluid can fully fill the reaction chambers 103. The side channels 109 may include a vertical portion directly fluidly connected to the reaction chambers 103 so that filling of the reaction chambers 103 occurs from the bottom up to minimize possible interactions between the fluid and the side walls of the reaction chambers 103. A waste shunt chamber 102 allows excess sample fluid to be held. The waste shunt chamber 102 may include a side channel from the input reservoir 101, a U-bend, and an air vent. In some embodiments, the waste shunt chamber 102 may have a volume in a range from about 100 μL to about 5 mL, and a total length in a range from about 5 mm to about 100 mm. In some embodiments, there may be one, two, three, four, or more reaction chambers each fluidly connected to a side channel that is fluidly connected to the sample reservoir. In some embodiments, each of these reaction chambers may contain a reaction bead containing dried components needed for conducting a different chemical, biological, or biochemical reaction or assay. In some embodiments, each of these reaction chambers may contain a reaction bead containing dried components needed for conducting the same chemical, biological, or biochemical reaction or assay. In some embodiments, each reaction chamber may contain same or different components, and / or may be empty. Page 50 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0395] Referring still to FIG.1A, the first flow channel 116 continues on to the timer delay channel 106. In some embodiments, the timer delay channel 106 may include dimensions (i.e., height, width, or diameter) in a range from about 0.1mm to about 2mm, with a length from about 10 mm to about 100mm, and various sub-ranges therebetween. At the bottom of the timer delay channel 106, the device 100 includes a dissolvable passively actuated valve 107 (e.g., a polyvinyl acid (PVA) comprising a membrane, e.g., FADE brand PVA tape) covering another opening at the side of the timer delay channel 106. Further details of the connection between the timer delay channel 106, the third flow channel 111, and the membrane 107 are described in relation to FIGS.1D-1E. The timer delay channel 106 is connected to an air channel 111 (or third flow channel 111) for controlled air diffusion via valve chamber 127 (not shown here), but visible in FIG.1D. The air channel 111 (or third flow channel 111) turns and moves up toward an air outlet port 110 disposed within a controlled air diffusion element 108. Air diffuses laterally through the controlled air diffusion element 108 (i.e., porous membrane 108), which may be in the shape of a square with side length L. In some embodiments, the diffusion element 108 may be other shapes (e.g., a circle, a rectangle, etc.). In some embodiments, the diffusion element 108 includes a porous membrane material (e.g., porous nitrocellulose membrane, e.g., Millipore MF 0.025 μm nitrocellulose filter). The air diffusion element is described in more detail below in relation to FIG.1B. As air gradually diffuses out of the controlled air diffusion element 108, air gradually empties from the third flow channel 111, and reaction fluid gradually flows down the timer delay channel 106 to eventually reach the membrane 107, and dissolve the membrane 107. Once the membrane 107 is dissolved, the fluid can exit out through the surface of the device (i.e., via a channel, plenum, or passageway at least partially aligned in the out of plane direction in the image, i.e., in the z-direction in the image) then reach a readout element such as a lateral flow strip. Various mechanisms for dissolving and / or breaking the membrane 107 are shown in the valve designs in FIGS.5-8. Further description of the channel / plenum through which liquid / fluid exits the valve chamber 107 is included herein in connection with FIGS.14A-14J.

[0396] FIG.1B is a schematic diagram illustrating a cross-sectional side view of a fluidic device 100 featuring an air diffusion timer, focusing on a porous membrane 108, according to aspects of the present embodiments. The controlled air diffusion element 108 is a porous membrane adhered to the chip surface 112 by double-sided adhesive 114 (or some other adhesive layer or connection means). There is a single-sided adhesive 113 on the outer Page 51 of 164 12865804v1Atty. Docket No.: 2013065-0954 surface of the porous membrane 108. In some embodiments, instead of a single-sided adhesive there is a non-porous coating. The porous membrane 108 is sandwiched between the single-sided adhesive 113 on one side and the double-sided adhesive 114 on the other side, both of which prevent air from diffusing out of the plane of the porous membrane 108, so that the only direction of air flow is by diffusion through the interior of the porous membrane 108 parallel to the surface of the device. That is, the airflow through the porous membrane 108 is constrained to the x-y dimension, and blocked in the z dimension. Since the only movement of air is through the interior of the porous membrane 108, the duration of time needed for a given volume of air to escape fully depends on the size of the porous membrane 108. The diffusion time of air through the porous membrane 108 is limited by the smallest lateral dimension of the porous membrane 108, as that is the shortest diffusion path for air to escape.

[0397] FIG.1C is a schematic diagram illustrating a front view of another embodiment of a fluidic device 120 featuring an air diffusion timer, according to aspects of the present embodiments. The device 120 is the same as device 100, except rather than having small air vent ports 105 above each of the reaction chambers 103, there are two larger air pinning chambers 125 above each of the reaction chambers 103. The air pinning chambers 125 may be any shape, but are shown as circles or spheres in FIG. 1C. Where the air pinning chambers 125 meet the reaction chambers 103 is a narrowing or neck 126, where the fluid in the reaction chamber 103 will be pinned by surface tension. In this manner, the volume within each reaction chamber 103 is effectively controlled. In some embodiments where the reaction chamber 103 and the air pinning chamber 125 are each substantially spherical, the combination of the reaction chamber 103 and air pinning chamber 125 resembles a two- sphere snowman as shown in FIG.1C. In some embodiments, the pinning chambers 125 facilitate rapid and even draining of the reaction chambers 103 because they have a larger radius of pinned surface tension at the junction of the air vent channel, compared with the wide radius of pinning in the pinning chamber 125. In some embodiments, air vent 128 allows air to escape from the pinning chamber 125.

[0398] FIG.1D-1E are schematic diagrams illustrating front views of a lower portion of a fluidic device 120 focusing on a passively actuated valve, according to aspects of the present embodiments. In FIG.1D, the membrane 107 is shown off to one side and not attached to the device surface 112. When the membrane 107 is not attached, the underlying flow pathways are more clearly visible. The timer delay channel 106 is fluidly connected to a valve chamber 127, which may include the shallow space 312 and a portion of a fluid flow Page 52 of 164 12865804v1Atty. Docket No.: 2013065-0954 path 308 that turns toward the exterior of the device in FIG.5A, which is then fluidly connected to the air channel for controlled air diffusion element (or third flow channel) 111. In FIG.1E, the valve membrane 107 is attached to the device surface 112 to cover over the valve chamber 127.

[0399] FIGS.2A-2H are a series of schematic diagrams illustrating front views of a fluidic device 100 featuring an air diffusion timer showing the addition of sample and progress of fluid flow through the device, according to aspects of the present embodiments. In FIG.2A, sample fluid 117 is added to the input reservoir 101. In FIG.2B, the sample fluid has filled the reaction chambers 103, and is rehydrating the lyophilized reaction beads 104. The reaction between the reagents in the lyophilized reaction bead and the sample begins. For example, in a nucleic acid-based detection assay, amplification of the target nucleic acid begins. In FIG.2C, excess waste sample is passed to the waste shunt. Flow of liquid down the timer delay channel 106 proceeds as air is displaced out through the air diffusion element 108.

[0400] In FIG.2D, the chemical reaction (e.g., the amplification reaction), proceeds for as long as it takes the sample fluid to travel down the timer delay channel 106. In some embodiments, the duration of time is tunable from about 15 min to 60 min (e.g., from 10 min to 60 min, from 5 min to 30 min, from 5 min to 120 min). In FIG.2E, the sample fluid is seen traveling partially down the timer delay channel 106. The air that is contained in the timer delay channel 106 is simultaneously pushed down the timer delay channel 106 and into the air channel 111 of the controlled air diffusion element 108, and exits the device by diffusion through the controlled air diffusion element 108. As the air gradually exits the device 100, the sample fluid continues further down the timer delay channel 106. In FIG.2F, the sample fluid has reached the soluble membrane 107 (e.g., PVA membrane), where it begins to dissolve and / or break through the membrane 107. Different mechanisms of breaking through the membrane are shown in FIGS.5-8.

[0401] In FIG.2G, the membrane 107 is partially dissolved. In FIG.2H, the membrane 107 is fully dissolved, so the entire reaction fluid and remaining sample fluid flow down the timer delay channel 106 and out through the now-open valve to a readout element such as a lateral flow strip. The flow is shown moving down from the device 100, but in some embodiments the flow is out of the plane of the page to another adjacent fluidic element such as a lateral flow strip, a container holding a lateral flow strip, or another readout element or Page 53 of 164 12865804v1Atty. Docket No.: 2013065-0954 device, or another chamber or volume for undergoing subsequent chemical, biological, or biochemical reaction step(s). Fluidic Serpentine Timer

[0402] Fluidic serpentine timers as described herein make use of the flow of fluid through a long path to control the duration of time needed for the flow to be completed. By changing the length, shape, and dimensions of the flow path, the duration of time can be controlled and engineered.

[0403] FIG.3 is a schematic diagram illustrating a front view of a fluidic device 200 featuring a fluidic serpentine timing circuit 210, 211, according to aspects of the present embodiments. In this embodiment, the device 200 is a vertical standing chip with gravity- driven flow (i.e., liquid flows from top to bottom), with a separate fluidic timing circuit composed of a fluidic resistance element 210 and a serpentine timer delay channel 211. A separate timer fluid 222 (shown in FIGS.4A-4F) flows through the fluidic timing circuit to control the duration of time for a sample fluid 220 to undergo a reaction. A sealed, airtight lateral flow chamber 207 holds the reaction fluid in place until the fluidic timer flow is completed after a programmable delay, which causes a PVA valve 208 to be opened to allow reaction fluid to enter the lateral flow chamber 207.

[0404] In some embodiments, the fluidic resistance element is any component that can produce a fluidic resistivity. In this case, the fluidic resistance element is itself also a serpentine channel with narrow channel dimensions. Other examples of fluidic components that can create fluidic resistivity include a narrow aperture (e.g., 10 μm cross-section), a porous material, or a Tesla valve flowing in the resistive direction.

[0405] In some embodiments, the serpentine timer delay channel, by contrast, is simply a reservoir for the accumulation of fluid coming out of the flow resistivity element. The reservoir should be designed so that the PVA valve is opened once the reservoir is filled. This reservoir does not have to be a serpentine, but could be an open chamber with any other geometry. In these embodiments, a serpentine shape was chosen for the reservoir (with relatively wider dimensions to allow for a significant volume) to make the design more robust to being knocked over. With an open chamber, if it is knocked over, fluid would wash up and to the top of the reservoir, thereby prematurely opening the PVA valve. If it is instead a Page 54 of 164 12865804v1Atty. Docket No.: 2013065-0954 serpentine, the liquid will remain trapped within the serpentine if it is knocked over, and the PVA valve is less likely to be prematurely opened.

[0406] Referring still to FIG.3, the device 200 includes a main chip surface 212 or chip body 212. A sample inlet port and reservoir 201 at the top of the device 200 is where a sample fluid is input. In some embodiments, the reservoir 201 may include dimensions (and may hold a fluid volume) that are approximately the same as the reservoir 101 of Fig.1A. The reservoir 201 is fluidly connected at its bottom to a first flow channel 214, which is then fluidly connected to two (e.g., one, two, or more) side channels 213 that flow up into reaction chambers 203. Each reaction chamber 203 may contain a lyophilized reaction bead 204 comprising reagents needed for a particular reaction or assay. In some embodiments, the first fluid channel 214 and the side channels 213 may have dimensions that are approximately the same as corresponding features of Fig.1A. Reaction chamber air vents 205 allow air to escape so that the fluid can fully fill the reaction chambers 203. The side channels 213 may include a vertical portion directly fluidly connected to the reaction chambers 203 so that filling of the reaction chambers 203 occurs from the bottom up to avoid possible interactions between the fluid and the side walls of the reaction chambers 203. A waste shunt chamber 202 allows excess sample fluid to be held. The waste shunt chamber 202 may include a side channel from the input reservoir 201, a U-bend, and an air vent. In some embodiments, the waste shunt chamber 202 may have a volume and dimensions that match those of the waste shunt chamber 102 shown in Fig.1A, and as described herein. In some embodiments, there may be one, two, three, four, or more reaction chambers each fluidly connected to a side channel that is fluidly connected to the sample reservoir. In some embodiments, each of these reaction chambers may contain one or more reaction beads containing dried components needed for conducting a different chemical, biological, or biochemical reaction or assay. In some embodiments, each of these reaction chambers may contain a reaction bead containing dried components needed for conducting the same chemical, biological, or biochemical reaction or assay. In some embodiments, each reaction chamber may contain same or different components, or may be empty.

[0407] Referring still to FIG.3, the first flow channel 214 continues on to the reaction chamber fluid outlet channel 206. In some embodiments, the reaction chamber fluid outlet channel 206 is configured to be substantially vertical to allow gravity flow of sample fluid. In some embodiments, there may be a partially diagonal or horizontal component of the reaction chamber fluid outlet 206 before it fluidly connects to the bottom of an airtight lateral Page 55 of 164 12865804v1Atty. Docket No.: 2013065-0954 flow chamber 207. The airtight lateral flow chamber 207 is configured to accommodate a lateral flow strip (for example, one or more lateral flow strips to accommodate embodiments in which multiple targets are amplified) arranged substantially vertical, with fluid reaching the bottom of the lateral flow chamber 207 to contact a lateral flow strip (LFS) 1102 (shown in Fig.15B) at a bottom portion 1104 (shown in Fig.15B) of the LFS 1102. At the top of the lateral flow chamber 207, a seal 208 (e.g., a water-soluble PVA valve, or other water-soluble valve, or water-soluble membrane) prevents air from escaping from the lateral flow chamber 207. The dead airspace within the reaction chamber fluid outlet channel 206 and the lateral flow chamber 207 hold in place the sample fluid within the reaction chambers, so that they do not flow out. The lateral flow chamber 207 may be sized to accommodate lateral flow strips 1102 of various sizes including those with a thickness in a range from about 2mm to about 15mm, a width in a range from about 4mm to about 15mm, and a length in a range from about 15mm to about 80mm. Accordingly, the lateral flow chamber 207 itself may have similar dimensions, albeit slightly larger (for example, 1-3% larger in each case) to allow for a tolerance to enable insertion and removal of the lateral flow strip 1102 into and out of the lateral flow chamber 207.

[0408] Referring further to FIG.3, the fluidic device 200 has a timer fluid inlet port and reservoir 209 disposed at the top of the device 200. The timer fluid inlet port and reservoir 209 is open to air at the top of the device 200 so that timer fluid can be added. In some embodiments, timer fluid may be or may include water, PEG, glycerol, and surfactants such as Ecosurf and Tween. In some embodiments, the reservoir 209 may have a volume in a range from about 0.1 mL to about 5 mL and various combinations of dimensions, each in a range from about 2 mm to about 100 mm. The reservoir 209 is fluidly connected at its bottom to a fluidic resistance element 210. In some embodiments, the fluidic resistance element 210 may include a narrow channel arranged in a series of serpentine turns connected to a vertical channel that leads to the lower end of the chip, where the channel fluidly connects to a serpentine timer delay channel 211. In some embodiments, the fluidic resistance element 210 may have dimensions (i.e., an inner diameter, or channel width) in a range from about 2 microns to about 500 microns. In some embodiments, the fluidic resistance element 210 may include a single aperture resistance element (i.e., without bends or turns) with an inner diameter or channel height / width in a range from about 2 microns to about 30 microns, (i.e., from about 2 microns to about 20 microns, or from about 2 microns to about 15 microns, or from about 2 microns to about 10 microns, or from about 20 microns to about 30 microns, or Page 56 of 164 12865804v1Atty. Docket No.: 2013065-0954 from about 15 microns to about 30 microns, or from about 10 microns to about 30 microns, or from about 25 microns to about 30 microns, or from about 5 microns to about 10 microns, or from about 10 microns to about 20 microns. In some embodiments, the fluidic resistance element 210 may include a serpentine configuration and may include an inner diameter or channel height / width in a range from about 50 microns to about 500 microns, or from about 50 microns to about 200 microns, or from about 50 microns to about 100 microns, or from about 200 microns to about 500 microns, or from about 300 microns to about 500 microns, or from about 400 microns to about 500 microns, or from about 100 microns to about 400 microns, or from about 150 microns to about 350 microns, or from about 200 microns to about 300 microns. In some embodiments, the serpentine timer delay channel 211 may include a series of serpentine turns, where timing fluid fills the channel 211 from the bottom up. In some embodiments, the serpentine timer delay channel 211 may include an inner diameter and / or channel height / width in a range of about 1 mm to about 10 mm. In some embodiments, both the fluidic resistance element 210 and the serpentine timer delay channel 211 may have serpentine turns with sharp corners as shown in FIG.3, but in some embodiments, they may have rounded corners as shown in FIGS.12A and 12E to ensure smoother fluid flow with minimal pinning of fluid in corners.

[0409] Referring still to FIG.3, the top of the serpentine timer delay channel 211 is sealed by the water-soluble seal 208 (e.g., PVA seal), the same seal 208 that holds the dead airspace in the airtight lateral flow chamber 207. Air exits the serpentine timer delay channel 211 and fluidic resistance element 210 via air vent 215. When the timer fluid reaches the water-soluble seal 208, the seal 208 is broken and air that was contained in the lateral flow chamber 207 is able to escape (also via air vent 215), thus allowing fluid in the reaction chamber fluid outlet 206 to flow into the lateral flow chamber 207. The valve 208 may have any of the designs illustrated in FIGS.5-8.

[0410] FIGS.4A-4F are a series of schematic diagrams illustrating front views of a fluidic device 200 featuring a fluidic serpentine timer showing the addition of sample and progress of fluid flow through the device, according to aspects of the present embodiments.

[0411] In FIG.4A, a fluidic device 200 similar to the device 200 of FIG.3 is shown. A quantity of sample fluid 220 is added to the sample inlet port and reservoir 201, and a quantity of timer fluid 222 is added to the timer fluid inlet port and reservoir 209. In some embodiments, the sample fluid 220 and the timer fluid 222 are added at the same time. In some embodiments, the sample fluid 220 may be added before or after the timer fluid 222 is Page 57 of 164 12865804v1Atty. Docket No.: 2013065-0954 added. In some embodiments, a timer fluid can be pre-loaded in the timer fluid reservoir 209, and then dispensed after an initiating step by the user (e.g., pressing a start button, opening a valve, piercing a membrane, etc.).

[0412] In FIG.4B, the sample fluid 220 travels down from the sample fluid inlet and reservoir 201 down the first flow channel 214, up the side channels 213, to fill the reaction chamber 203. In the reaction chambers 203, the sample fluid rehydrates reaction beads 204 initially contained in the reaction chambers 203. Excess sample fluid 220 is metered off into the waste shunt 202. As sample fluid 220 transfers out of its inlet 201, the timer fluid 222 also transfers out of its inlet 209, perfusing the fluidic resistance element 210.

[0413] In FIG.4C, the sample fluid 220 is held in place by the dead airspace in the downstream channel 206 and the lateral flow chamber 207, which is kept airtight by the water-soluble valve 208. The timer fluid has reached the serpentine timer delay channel 211 and is filling the serpentine timer delay channel 211 from the bottom up. The filling of the serpentine timer delay channel 211 occurs at a controlled flow rate programmable by tuning the fluidic resistance element 210 (e.g., cross-section size, overall length, number of turns, surfactant and other additives in the fluid (e.g., PEG, glycerol, etc.), and any fluidic obstacle- like features (e.g., Tesla valve)) and the height of the timer fluid reservoir 209. In some embodiments, factors such as the total volume of the channel 211, can also be tuned to control the flow rate and the total amount of time needed for the timer fluid 222 to reach the valve 208. One or more chemical reactions (e.g., nucleic acid amplification, or other reactions) proceed in the reaction chambers 203 for as long as it takes for the serpentine timer delay channel 211 to fill with timer fluid 222. In some embodiments, the time may be tunable from about 15 min to about 60 min (e.g., from 10 min to 50 min, from 10 min to 90 min, from 5 min to 30 min, from 5 min to 20 min, from 5 min to 15 min, from 10 min to 25 min, from 10 min to 20 min, and / or from 15 min to 45 min). The total time for the timer fluid to reach the valve 208 is determined by a combination of the length and volume of the serpentine timer delay channel 211 and the resistance of the fluidic resistance element 210.

[0414] In FIG.4D, the timer fluid 222 continues filling the serpentine timer delay channel 211. In FIG.4E, the timer fluid 222 reaches the valve 208 after the total delay time programmed by the chip design (i.e., the resistance of the fluidic resistance element 210 and the length and volume of the serpentine timer delay channel 211). Upon reaching the valve 208, the fluid comes into contact and hydrates the valve 208 (e.g., water-soluble membrane, PVA membrane), causing the valve 208 to stop being airtight. In some embodiments, fluid Page 58 of 164 12865804v1Atty. Docket No.: 2013065-0954 reaching the valve 208 may result in a combination of dissolution and / or rupturing of the valve membrane.

[0415] In FIG.4F, once the valve 208 is hydrated, air leaks out of the lateral flow chamber via an air vent (not shown) so that the sample fluid 220 held in the reaction chamber 203 is able to displace downstream air that had been keeping it in place. The air vent 215 also allows air to escape after the PVA seal is broken. As a result, the fluid in the reaction chambers 203 flows down the rest of the downstream channel 206 to enter the lateral flow chamber 207 near the bottom of the lateral flow chamber 207. The lateral flow chamber 207 may contain a lateral strip arranged substantially vertically within the lateral flow chamber 207. Fluidic Valves

[0416] In the present disclosures, fluidic valves are elements that control the flow of liquid between two channels in a fluidic device. Specifically, the present disclosures have fluidic valves that form a barrier preventing the flow of air or gas, which allows a volume of dead airspace to prevent the flow of a fluid. In some embodiments, a fluidic valve is a thin sheet or membrane of liquid-soluble (e.g., water-soluble) material (e.g., polyvinyl alcohol, PVA) that is adhered over an opening in a fluidic device or chip so that when liquid (e.g., water, sample fluid, timer fluid) comes into contact on one side of the thin sheet, the sheet is dissolved, thus opening the valve and allowing fluid to pass through the opening. In some embodiments, an additional element may be used in conjunction with the membrane and dissolving action of the fluid to mechanically disrupt the membrane. The additional element may be mechanically actuated by the presence of the fluid to simultaneously disrupt the membrane while it is also being dissolved by the fluid. FIGS.5-8 illustrate the operation of four embodiments of fluidic valves (e.g., PVA valves). In some embodiments, aqueous fluids are flowed so that water-soluble membranes are used as the membrane material in fluidic valves. Membrane Valve on Flat Chip Surface

[0417] In some embodiments, the present disclosure includes a membrane valve on a flat chip surface. FIGS.5A-5E are a series of schematic diagrams illustrating side views of a membrane valve (e.g., PVA valve) on a flat chip surface design, showing the flow of fluid and Page 59 of 164 12865804v1Atty. Docket No.: 2013065-0954 breaking of the PVA membrane to release fluid, according to aspects of the present embodiments.

[0418] In FIG.5A, a fluidic device 300 has a chip surface 302 shown in a side view cross section, with an empty fluid flow path 308. Fluid may enter the fluid flow path 308 and fill the volume by gravity or other pressure. In this image, the fluid flow path 308 is through the interior plane of the chip 302, and turns to exit at the top surface of the chip 302 via an opening 315. To construct the valve, a portion of the membrane 304 (e.g., PVA membrane) is cut to be larger than the size of the opening 315 that opens from the fluid path 308. There is a shallow space 312 (e.g., less than 0.5 mm thick) between the chip 302 and the membrane 304. In some embodiments, the membrane 304 is adhered to the chip 302 by a piece of double-sided adhesive 306 with a hole cut out in its center, so that fluid can pass through. The double-sided adhesive 306 is adhered around the fluid flow path 308 opening, and the membrane 304 is sealed over the double-sided adhesive 306. In some embodiments, another form of adhesive, glue, or sealant may be used to attach the membrane 304 to the chip 302 by forming a thin adhesive layer between the membrane 304 and the chip 302. In some embodiments, the shallow space 312 is determined by the thickness of the adhesive and any flexing of the membrane 304.

[0419] In FIG.5B, fluid 310 is flowing through the fluid flow path 308 of the device 300. In some embodiments, the fluid 310 may be water, aqueous solution, sample fluid, or other fluid that can dissolve the membrane 304. In FIG.5C, the fluid 310 has turned at a curve in the fluid path 308 to start filling the opening 315 adjacent to the membrane 304 and shallow space 312. In FIG.5D, the fluid 310 has reached the membrane 304, and capillary action wicks fluid between the membrane 304 and chip surface 302, so that the fluid completely fills the shallow space 312. As the membrane 304 comes into contact with the fluid 310, the membrane 304 dissolves and weakens. In FIG.5E, the membrane 304 is sufficiently dissolved so that there is a break in the membrane 304, and fluid bursts from the valve. The air seal is then broken, and fluid 310 can freely flow from the opening of the valve.

[0420] FIGS.5F-5G show schematic diagrams illustrating side views of a membrane valve (e.g., PVA valve) on a flat chip surface design 300, according to aspects of the present embodiments, where a continuation of the fluid flow path 308 leads to an air flow path 314 via passages 315a and 315b, which connect via shallow space 312. Air that is trapped in both the fluid flow path 308 and air flow path 314 ahead of the fluid 310 is gradually released via Page 60 of 164 12865804v1Atty. Docket No.: 2013065-0954 an air flow control element such as a slow air diffusion pad similar to the slow air diffusion pad 108 (i.e., porous membrane) illustrated in FIGS.1-2. In FIG.5G, as the fluid 310 enters passage 315a, the shallow space 312, and eventually passage 315b, the fluid 310 comes into contact with and wets the membrane 304, while the rest of the fluid 310 continues along the air flow path 314. The configuration shown in FIGS.5F and 5G is also applicable to the embodiments of the PVA valve / flat chip surface design 300 shown in FIGS.6A-6E, 7A-7D, and 8A-8F. Wicking-Assisted Membrane Valve

[0421] In some embodiments, the present disclosure includes a wicking-assisted membrane valve, which includes an additional wicking agent or absorbent material added to the otherwise flat chip surface. FIGS.6A-6E are a series of schematic diagrams illustrating side views of a wicking-assisted membrane valve (e.g., PVA valve) design, showing the flow of fluid and breaking of the membrane to release fluid, according to aspects of the present embodiments.

[0422] In FIG.6A, a fluidic device 400 has a chip surface 402 shown in a side view cross section, with an empty fluid flow path 408. Fluid may enter the fluid flow path 408 and fill the volume by gravity or other pressure. In this image, the fluid flow path 408 is through the interior plane of the chip 402, and returns to exit at the top surface of the chip 402 via an opening 415. To construct the valve, a portion of membrane 404 (e.g., PVA membrane) is cut to be larger than the size of the opening 415 of the fluid path 408, along with a portion of wicking material 412 to fill a shallow space 414 is similar to the shallow space 312 of the flat chip design 300 in FIG.5. The wicking material 412 is a thin layer of absorbent material (e.g., paper tissue, nitrocellulose, dried hydrogel, etc.) that is shaped larger than the opening of the fluid path 408 but smaller than the membrane 404, and is similar to the shape of the shallow space 414 (which is similar to shallow space 312). A portion of double-sided adhesive 406 is cut with an outer outline that is similar to the outline of the membrane 404 and with an inner hole similar to the outline of the wicking material 412. The valve is assembled by placing the wicking material 412 into the shallow space 414 covering the opening of the fluid path 408, the double-sided adhesive 406 is placed on the chip surface 402 around the wicking material 412, and the membrane 404 is placed on top of the wicking material 412 and adhesive 406 to form a seal. In some embodiments, another form of Page 61 of 164 12865804v1Atty. Docket No.: 2013065-0954 adhesive, glue, or sealant may be used to attach the membrane 404 to the chip 402 by forming a thin adhesive layer between the membrane 404 and the chip 402.

[0423] In FIG.6B, fluid 410 is flowing through the fluid flow path 408 of the device 400. In some embodiments, the fluid 410 may be water, aqueous solution, sample fluid, or other fluid that can dissolve the membrane 404. In FIG.6C, the fluid 410 has turned at a curve in the fluid path 408 to start filling the opening 415 adjacent to the wicking material 412. In FIG.6D, the fluid 410 has reached the wicking material 412, which rapidly draws fluid in, and helps to more quickly and reliably wet the membrane 404, rather than relying on capillary action alone. As the membrane 404 is in contact with the fluid 410 due to both capillary action and the wicking material 412, the membrane 404 dissolves. In FIG.6E, the membrane 404 is sufficiently dissolved and / or weakened so that there is a break in the membrane 404, and fluid bursts from the valve. The air seal is then broken, and fluid 410 can freely flow from the opening of the valve. The wicking material 412, once fully saturated with fluid 410, does not impede flow of the fluid 410 out of the valve. Membrane-Puncturing Sponge

[0424] In some embodiments, the present disclosure includes a membrane valve on a chip with the addition of a membrane-puncturing sponge. FIGS.7A-7D are a series of schematic diagrams illustrating side views of a membrane valve (e.g., PVA valve) design featuring a sponge to assist in membrane puncturing, showing the flow of fluid and breaking of the membrane to release fluid, according to aspects of the present embodiments.

[0425] In FIG.7A, a fluidic device 500 has a chip surface 502 shown in a side view cross section, with an empty fluid flow path 508. Fluid may enter the fluid flow path 508 and fill the volume by gravity or other pressure. In this image, the fluid flow path 508 is through the interior plane of the chip 502, and turns to exit at the top surface of the chip 502 via an opening 517. In this device 500, there is a further counterbore or recess 514 to accommodate a sponge 512 fitted over the opening 517 of the flow path 508. The area of the sponge 512 is larger than the area of the opening 517 of the fluid path 508. The sponge 512 is a portion of hygroscopic expanding material that expands in volume when it absorbs fluid. The sponge is cut to fit the recess 514 laterally, and extend outward slightly from the surface of the chip 502. A portion of double-sided adhesive 506 is cut to fit around the recess 514, but be smaller than the membrane 504 (e.g., PVA membrane). The double-sided adhesive 506 is placed on Page 62 of 164 12865804v1Atty. Docket No.: 2013065-0954 the chip 502 surface outside of the sponge 512 and recess 514, without contacting the sponge 512. The membrane 504 is then adhered to the adhesive 506 while covering over the entire outer surface of the sponge 512. In some embodiments, the sponge 512 material was Suge Cellulose Absorbent Material from QuestAlpha, 600g / m2compressed. Other possible materials for the sponge 512 include paper, cotton, nylon, hydrogels, or other hygroscopic materials.

[0426] In FIG.7B, fluid 510 is flowing through the fluid flow path 508 of the device 500. In some embodiments, the fluid 510 may be water, aqueous solution, sample fluid, or other fluid that can dissolve the membrane 504. In FIG.7C, the fluid 510 has reached the sponge 512, which starts to expand from fluid intake. As fluid travels through the expanding sponge 512, it comes into contact with the membrane 504 and begins to dissolve the membrane 504. The hygroscopic sponge 512 simultaneously pulls fluid 510 into contact with the membrane 504 in a rapid and robust manner, while also physically weakening and / or disrupting the membrane 504 and encouraging the membrane 504 to rupture. In FIG.7D, the membrane 504 is broken by a combination of dissolution in the fluid 510 and disruption by the expanding sponge 512. Fluid then bursts from the valve, and the air seal is broken. Membrane-Puncturing Sponge with Needle

[0427] In some embodiments, the present disclosure includes a membrane valve with a membrane-puncturing sponge with piercing feature (e.g., a pointed feature / protrusion, a puncturing feature / protrusion, a knife-like feature / protrusion or a needle). This membrane valve design is similar to the membrane-puncturing sponge design shown in FIG.7, but the sponge has an opening in it so that it is shaped like an O-ring or donut, to accommodate a needle to further puncture the membrane. FIGS.8A-8F are a series of schematic diagrams illustrating side views of a PVA valve design featuring a sponge and needle to assist in membrane puncturing, showing the flow of fluid and breaking of the membrane to release fluid, according to aspects of the present embodiments.

[0428] In FIG.8A, a fluidic device 600 has a chip surface 602 shown in a side view cross section, with an empty fluid flow path 608. Fluid may enter the fluid flow path 608 and fill the volume by gravity or other pressure. In this image, the fluid flow path 608 is through the interior plane of the chip 602, and returns to exit at the top surface of the chip 602. In this device 600, there is a further counterbore or recess 616 to accommodate a ring-shaped sponge Page 63 of 164 12865804v1Atty. Docket No.: 2013065-0954 612 fitted around the opening 619 of the flow path 608. There is a hole cut in the center of the sponge 612 that is around the same size or slightly larger than the opening of the fluid flow path 608. The outer dimensions of the sponge 612 fit within the recess 616. The ring-shaped sponge 612 is a portion of hygroscopic expanding material that expands in volume when it absorbs fluid. Possible materials for the sponge 612 include Suge Cellulose Absorbent Material from QuestAlpha, 600g / m2compressed, paper, cotton, nylon, hydrogels, or other hygroscopic materials. The sponge is cut to fit the recess 616 laterally, and extend outward slightly from the surface of the chip 602. A portion of double-sided adhesive 606 is cut to fit around the recess 616, but be smaller than the membrane 604 (e.g., PVA membrane). The double-sided adhesive 606 is placed on the chip 602 surface outside of the sponge 612 and recess 616, without contacting the sponge 612. The membrane 604 is then adhered to the adhesive 606 while covering over the entire outer surface of the sponge 612. A needle 614 is positioned adjacent to the outer surface of the membrane 604, but not in contact with the membrane 604. In some embodiments, the needle 614 may be a conical structure with its pointed end positioned adjacent to the membrane 604. The needle 614 is positioned so that its point is centered over the hole in the ring-shaped sponge 612 and over the opening 619 of the fluid flow path 608. In some embodiments, the needle 614 may be a 3D printed cone on the device body. In some embodiments, the needle 614 may be metal, other plastic, or any hard material. In some embodiments, the needle 614 may be a thin rod, a needle-like shape, or any other component with a ridge or point with which to rupture the membrane.

[0429] In FIG.8B, fluid 610 is flowing through the fluid flow path 608 of the device 600. In some embodiments, the fluid 610 may be water, aqueous solution, sample fluid, or other fluid that can dissolve the membrane 604. In FIG.8C, the fluid 610 has reached the sponge 612, which starts to absorb fluid 610. In FIG.8D, the sponge 612 has expanded outward from absorbing water, so that it pushes outward against the membrane 604 so that the membrane 604 is pushed against and pierced by the needle 614. In FIG.8E, the sponge 612 continues expanding outward and pushing the membrane 604 further against the needle 614. At the same time, the membrane 604 is rapidly and robustly wetted by being pressed against the sponge 612. In FIG.8F, the membrane 604 is broken by a combination of puncturing by the needle 614 and dissolution and / or weakening by contact with fluid 610 in the sponge 612. Fluid then bursts from the valve, and the needle puncture breaks the air seal. Materials and Components Page 64 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0430] Examples of materials and components used in the present disclosures are described throughout, and are summarized here. In some embodiments, these materials may be used. In some embodiments, other similar or equivalent materials with comparable properties and performance may be used. The present disclosed materials are not meant to be limiting as to the specific materials and components that may be used in connection with device 100.

[0431] The fluid-soluble membrane for valves (e.g., water-soluble membrane) were primarily made of polyvinyl alcohol (PVA), implemented using a One More Cast brand FADE PVA tape, 8 mm x 20 m roll. The air diffusion pads or membranes were porous nitrocellulose filters, implemented using a Millipore brand MF 0.025 μm pore size nitrocellulose filter, product code VSWP09025. The double-sided adhesive was 3M brand 300LSE pressure sensitive adhesive. The hygroscopic sponge material was QuestAlpha brand Suge Cellulose Absorbent Material with 600 g / m2density compressed. The 3D printer resin for the fluidic devices was Formlabs Clear Resin, product code RS-C2-GPCL-05. The pore size of the pores in the air diffusion pads or porous membranes as well as the dimensions (i.e., thickness) of the air diffusion pads or porous membranes (both of which affect flow resistance) have an impact on how quickly air may diffuse therethrough, and therefore the timeframe for how long the biological sample remains in the reaction chambers. Combinations of Features

[0432] The devices, components, and features described herein may be combined in different arrangements. For example, sample fluid from an initial input reservoir may fill one or more reaction chambers by side channels that may enter the reaction chambers at the bottoms, sides, or tops of the reaction chambers, or by any combination of entry points within a single fluidic device. Reaction chambers may be vented by any combination of venting channels, air pinning chambers, shunt channels, etc. Fluidic devices may contain multiple arrangements of reaction chambers, and different shapes and volumes of reaction chambers. Fluidic devices may include combinations of timing elements, such as slow air diffusion pads, fluidic resistance elements, serpentine timer channels, fluidic timer flow channels of different geometries, and fluidic holding chambers. Fluidic devices may contain one or more such timing elements in combination. Fluidic chambers may include more than one sample fluid input reservoir and more than one timing fluid input reservoir. In some embodiments Page 65 of 164 12865804v1Atty. Docket No.: 2013065-0954 according to the present disclosure, disclosed devices may include from about 1 to about 8 reaction chambers. Fluidic devices may contain user-actuated mechanisms (e.g., buttons, knobs, sliders, switches, valves, pull tabs, etc.) in addition to fluidic flow timing elements to control the timing and flow of fluids. Fluidic devices may include additional outputs after a specific timed flow portion is completed, including flow into a readout mechanism, flow into a lateral flow chamber with lateral flow strip for readout, flow into a subsequent channel and / or chamber for undergoing subsequent chemical and / or biological reactions, etc. Additional readout mechanisms may include colorimetric, electronic, optical, electrochemical, chemical, mechanical, and other combinations of readouts. Combined Fluidic Device Designs

[0433] The device designs of the present disclosures can be combined into single devices. For example, different reaction chamber and input reservoir designs, slow air diffusion filter pads, and serpentine timing circuits can be combined together. Combined Timer / Air Valve Chip

[0434] In some embodiments, the present disclosure includes a combined timer / air valve chip, in which a single monolithic fluidic device or chip contains both a serpentine fluidic timer and an air valve (i.e., dead air downstream of the reaction, in which the PVA membrane is keeping air sealed in, i.e., the air behaves as a valve by keeping the reaction liquid pinned in place by virtue of the air not being displaceable, so that when air is allowed to escape as the membrane dissolves, the fluid is able to flow downstream). FIGS.12A-12J show different perspective views of a combined timer and air valve chip 700, according to aspects of the present embodiments. FIG.12A shows a back view of the device 700, where there is a timer fluid input and reservoir 702 at the top of the device 700, which connects fluidly to a fluidic resistance element 704, and a serpentine timer channel 706. The device 700 is similar to the device 200 illustrated in FIG.3, except rather than having the sample flow portion and fluidic timer portion arranged side by side, they are arranged on the front and back sides of the device 700. The serpentine timer channel 706 is then connected to a fluid-soluble membrane valve 708. The device 700 has a device support base 710, which may include multiple horizontal supports or feet to help the device 700 stand vertically. FIG.12B shows a side view of the device 700. Page 66 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0435] FIG.12C shows a front view of the device 700. That is, it shows the device 700 rotated 180 degrees about the y-axis from FIG.12A. In this view, there is visible a sample input and reservoir 712 at the top of the device, which is fluidly connected at its bottom to two reaction chamber filling channels 714, which allows a sample fluid to fill two reaction chambers 718 from the top. Each reaction chamber 718 has an air vent channel 716 at the top, which ends in air outputs 720 to allow air to escape from the reaction chambers 718 when fluid fills into the reaction chambers 718. The reaction chambers 718 are fluidly connected from their bottoms to reaction chamber output channels 722, which end at outputs 724 that are connected to a lateral flow chamber 728 (or slot 728) (see FIGS.12G-12I). The lateral flow chamber 728 can accommodate a lateral flow strip, and is airtight due to the membrane valve 708. In some embodiments, each of the outputs 724 is connected to a separate lateral flow channel (each containing a separate lateral flow strip). Sample fluid will be held in the reaction chambers by the dead airspace in the output channels 722 and lateral flow chamber 728 until timer fluid reaches the membrane valve 708, which will allow air to escape and thus the reaction fluid to flow into the lateral flow chamber 728. The lateral flow chamber entrance 726, which may also be used as a positioning feature 726 to keep the lateral flow strip positioned against an opposing side of the lateral flow chamber (i.e., where the sample fluid enters) is also visible in the views of Figs.12C and 12F.

[0436] FIG.12D shows another side view of the device 700. FIG.12E shows a perspective view of the back of the device 700 showing the serpentine timer 706 design. FIG. 12F shows a perspective view of the front of the device 700 showing the reaction chambers.

[0437] FIGS.12G, 12H, and 12I show slightly different top views of the device 700. The sample input 712 and timer fluid input 702 are visible as open at the top. The lateral flow chamber 728 is also visible. A lateral flow strip may be inserted into the lateral flow chamber 728, and then inserted in an air-tight compartment. FIG.12J shows a bottom view of the device 700, including the device support base 710. Slow Air Diffusion Chip with Base

[0438] In some embodiments, the present disclosure includes a slow air diffusion chip. FIGS.13A-13E show different perspective views of a slow air diffusion chip 800, according to aspects of the present embodiments. The device 800 shown here is very similar to the device 120 illustrated in FIG. 1C. The device 800 has a sample fluid input and Page 67 of 164 12865804v1Atty. Docket No.: 2013065-0954 reservoir 802 at the top of the device, which is fluidly connected to a first flow channel 804, which is then connected to two side channels 806 to allow two reaction chambers 808 to be filled with fluid from the bottom up. The reaction chambers 808 are topped by air pinning chambers 810 to maintain the liquid volume in the reaction chambers 808. The air pinning chambers are vented to the top of the device 800 by air vents 812. Sample fluid in the reaction chambers 808 can flow down the timer delay channel 814, but are held in place by dead air space in the timer delay channel 814. A fluid-soluble membrane valve can be installed at valve chamber 816, which is connected to both the timer delay channel 814 and the air diffusion channel 818. The air vent 820 is to be covered by a slow air diffusion membrane (not shown), through which air can diffuse out. Once the air trapped in the channels 814 and 818 have diffused out, the sample fluid will be able to travel fully down the channel 814 to reach the fluid-soluble membrane valve. Once the valve is opened, the fluid can reach a lateral flow chamber 822 (see FIG.13C) on the reverse side of the device 800. In another embodiment, the fluid reservoir 853 may be used to collect the sample, with a lateral flow strip 1102 (shown in FIG.15B) inserted into the slot 858 (shown in FIG.14E).

[0439] FIG.13C shows a back view of the device 800, showing the air vent 820 for the slow air diffusion membrane, and the lateral flow chamber 822. FIG.13D shows a bottom view of the device 800, and FIG.13E shows a top view of the device 800.

[0440] FIGS.14A-14E show different perspective views of a base 850 to support the slow air diffusion chip 800 of FIGS.13A-13E, according to aspects of the present embodiments. FIG.14A shows a front view of the base 850, which features an upper compartment 854 to hold a device 800 vertically. The base 850 also features multiple horizontal support pieces 852 or feet 852 to hold the base 850 vertical. The base 850 also includes a needle 856, which in some embodiments may be configured as a conical protrusion with a surrounding ring, as illustrated in additional views in FIGS.14F-14J. As described earlier in relation to FIG.8, the needle 856 may be used in conjunction with a hygroscopic sponge and dissolvable membrane valve to facilitate breaking of the membrane (i.e., PVA membrane 304, which is part of the water-soluble PVA valve 107). In some embodiments, the base 850 is integral with the slow air diffusion chip 800 (i.e., device 800). In some embodiments, the slow air diffusion chip 800 (i.e., device 800) is inserted into the base 850.

[0441] FIG.14B shows a back view of the base 850, FIG.14C shows a bottom view of the base 850, and FIG.14D shows a side view of the base 850. FIG.14E shows a top Page 68 of 164 12865804v1Atty. Docket No.: 2013065-0954 view of the base 850, in which an opening to a narrow lateral flow chamber 858 for inserting a lateral flow strip is visible. The device 840 also includes a reservoir 853 for holding fluid that flows out of a membrane valve once the needle 856 facilitates the breaking of a membrane valve. In some embodiments, when a device 800 is installed in a base 850, a valve chamber 816 is aligned with a needle 856 so that the needle 856 may facilitate the breaking of a membrane installed over the valve chamber 816. As shown in FIGS.14A and 14E-14J, the base includes a plenum 855 positioned adjacent to the needle 856 providing an opening for fluid to be collected therethrough once the PVA membrane 304 has dissolved and / or been punctured. The plenum serves to fluidly connect the needle 856 area (i.e., after the PVA membrane has been punctured or dissolved) to the reservoir 853, which itself is fluidly coupled to the lateral flow channel (when the device 800 is inserted into the base 850). Therefore, the plenum 855 and the reservoir 853 (i.e., a fluid reservoir) collectively form a continuous fluid path from the water-soluble PVA valve 107 to the lateral flow strip once the PVA membrane 304 has dissolved and / or been punctured. Stated otherwise, the timer flow channel 666 (shown in FIG.10E) connects to the lateral flow chamber 676 (shown in FIG. 10E) via the base 850 (i.e., via the plenum 855 and the reservoir 853) once the PVA membrane 304 has dissolved and / or been punctured.

[0442] FIGS.14F-14J show additional views of the base 850, and show that there is a fluid path from the position of the needle 856 into the reservoir 853. The view in FIG.14J shows that there is an additional opening 860 at the bottom of the reservoir 853 that is in fluid communication with the bottom of the lateral flow chamber 858, so that once fluid escapes from the valve chamber 816, and flows into the reservoir 853, the fluid will be in contact with a lateral flow strip installed or inserted within the lateral flow chamber 858. Methods of Using Power-Free Fluidic Devices

[0443] FIG.18 is a flow chart illustrating a method 900 of using a power-free fluidic device, according to aspects of the present embodiments. In step 902, a sample fluid (e.g., a patient sample, a biological sample, or a chemical sample) is accepted into the device. In some embodiments, step 902 may include a user depositing a volume of sample fluid into an input reservoir of the device. In step 904, the sample fluid is directed to rehydrate at least one lyophilized reaction bead with a metered volume. In some embodiments, the directing of sample fluid may be under gravity flow and / or capillary flow. In step 906, the sample fluid is Page 69 of 164 12865804v1Atty. Docket No.: 2013065-0954 held in a reaction chamber for a specific period of time for a reaction to take place. In some embodiments, the holding time of the sample fluid is set by a fluidic timer circuit such as a slow air diffusion timer or a serpentine fluid timer. In step 908, the sample fluid is transferred from the reaction chamber to a next stage (e.g., a readout, a lateral flow strip, a subsequent chemical or biological reaction step, etc.).

[0444] FIG.19 is a flow chart illustrating a method 1000 of using a power-free fluidic device with a timer fluid, according to aspects of the present embodiments. In step 1002, a sample fluid (e.g., a patient sample, a biological sample, or a chemical sample) and a timer fluid are accepted into the power-free fluidic device. In some embodiments, step 1002 may include a user depositing a volume of sample fluid into an input reservoir of the device and depositing a volume of timer fluid into another input reservoir of the device. In some embodiments, the timer fluid is accepted into the device ahead of the sample fluid (e.g., pre- loaded). In step 1004, the sample fluid is directed to rehydrate at least one lyophilized reaction bead with a metered volume, and the timer fluid is directed to travel through a fluidic timer circuit. In some embodiments, the directing of sample fluid may be under gravity flow and / or capillary flow, while the directing of timer fluid may also be under gravity flow and / or capillary flow. In some embodiments where the timer fluid is pre-loaded into the device, the directing of timer fluid may include the user pressing a start button or operating some other actuation mechanism to start the flow of timer fluid. In step 1006, the sample fluid is held in a reaction chamber for a specific period of time for a reaction to take place. In some embodiments, the holding time of the sample fluid is by the time needed for the timer fluid to travel through the fluidic timer circuit (e.g., a serpentine fluidic circuit). In step 1008, the sample fluid is transferred from the reaction chamber to a next stage (e.g., a readout, a lateral flow strip, a subsequent chemical or biological reaction step, etc.). Compositions

[0445] In some embodiments, methods, systems, and processes provided herein comprises or utilizes a composition as provided herein. In some embodiments, compositions and components provided herein are useful in amplification of a target nucleic acid.

[0446] In some embodiments, the present disclosure provides compositions, components, and methods for amplification of a target nucleic acid e.g., wherein the target nucleic acid is from a pathogenic microorganism. In some embodiments, amplification is Page 70 of 164 12865804v1Atty. Docket No.: 2013065-0954 followed by detection of a target nucleic acid amplicon. Compositions and components provided herein are useful in devices, systems, methods, and processes provided herein, e.g., fluidic devices, such as power-free fluidic devices.

[0447] In some embodiments, amplification is strand displacement amplification (SDA). In some embodiments, strand displacement amplification is capped strand displacement amplification (cSDA).

[0448] In some embodiments, the present disclosure provides compositions useful in producing a target amplicon, preparing multiple copies of nucleic acid identical to the target amplicon and detection of at least one copy of nucleic acid identical or complementary to the target amplicon. In some embodiments, compositions of the present disclosure may be used to produce a single stranded DNA (ssDNA) or multiple copies of nucleic acids identical or complementary to the ssDNA, for example, at ambient temperature. In some embodiments, such compositions are used to produce ssDNA or multiple copies of nucleic acids identical or complementary to the ssDNA, for example, at isothermal conditions (e.g., without the need for temperature cycling). In some embodiments, compositions provided herein detect a ssDNA, for example, at ambient temperature. In some embodiments, compositions provided herein detect a ssDNA, for example, at isothermal conditions (e.g., without the need for temperature cycling).

[0449] In some embodiments, compositions provided herein comprise a target nucleic acid.

[0450] In some embodiments, compositions provided herein comprise an oligonucleotide binder (e.g., a primer and / or a probe). In some embodiments, compositions provided herein comprise one or more oligonucleotide binders (e.g., primers and / or probes). In some embodiments, oligonucleotide binders of the present disclosure are designed to bind specifically to a target nucleic acid.

[0451] In some embodiments, a composition comprises a ligase.

[0452] In some embodiments, a composition comprises a reverse transcriptase.

[0453] In some embodiments, a composition comprises a cleavage enzyme. In some embodiments, a composition comprises a restriction enzyme. In some embodiments, a composition comprises a nickase. Page 71 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0454] In some embodiments, a composition comprises a single-strand binding protein.

[0455] In some embodiments, a composition comprises a strand displacing polymerase.

[0456] In some embodiments, a composition comprises dNTPs. In some embodiments, a composition comprises one or more modified dNTPs.

[0457] In some embodiments, compositions for detecting a ssDNA sequence or multiple copies of nucleic acids identical or complementary to the ssDNA sequence are also included in compositions of the present disclosure. In some embodiments, a composition according to the present disclosure comprises a detectably labeled nucleic acid probe, a guide nucleic acid and a Cas enzyme (e.g., a Cas enzyme having collateral cleavage activity). Samples

[0458] In some embodiments, methods, systems, compositions, and / or processes described herein comprises or utilizes a sample. In some embodiments, a sample comprises a target nucleic acid. In some embodiments a sample is an environmental sample. In some embodiments a sample is a biological sample. In some embodiments, a sample is from a subject. In some embodiments, a sample is from a human subject. In some embodiments, a sample is blood, saliva, sputum, mucus, urine, or stool. In some embodiments, a sample is a swab of a surface in or on a human body. In some embodiments, a sample is a swab of a mucosal surface or membrane. In some embodiments, a sample is nasal swab, a cheek swab, an endocervical swab, a vulvovaginal swab, tongue swab or scrapping, or a throat swab.

[0459] In some embodiments, a sample is processed. In some embodiments, a sample is processed to isolate components of the sample. In some embodiments, a sample is processed to isolate nucleic acids (e.g., RNA and / or DNA). In some embodiments, a sample is processed to isolate RNA. In some embodiments, a sample is processed to isolate DNA. In some embodiments, a sample is processed to separate double stranded nucleic acids into single stranded nucleic acids. Page 72 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0460] In some embodiments, a sample is prepared or processed to provide a nucleic acid preparation. In some embodiments, a sample is prepared or processed using lysis buffers. In some embodiments, a lysis buffer comprises at least one surfactant or detergent. In some embodiments, samples (e.g., viral particles and / or cells) are lysed, (e.g., processed) using a zwitterionic detergent, a nonionic detergent or an anionic detergent.

[0461] In some embodiments, samples (e.g., viral particles and / or cells) are lysed, (e.g., processed) using a zwitterionic detergent as described in WO2024006548, which is incorporated herein by reference in its entirety. In some embodiments, such a zwitterionic detergent is selected from the group consisting of LAPAO, LDAO, and DDAO. Certain detergents demonstrate surprising effectiveness (e.g., certain zwitterionic detergents, such as LAPAO, LDAO, and DDAO) for use in lysing viral particles and / or releasing nucleic acids from viral particles, so that a nucleic acid preparation is obtained. Advantages of certain embodiments of provided lysis technologies may include, among other things, that a useful nucleic acid preparation is provided without use of one or more traditional processing steps – such as purification, isolation or extraction steps that are commonly required or utilized to remove detergents.

[0462] In some embodiments, the concentration of a zwitterionic detergent is within the range of 0.01% and 10% (w / v). In some embodiments, a lysis buffer comprises a zwitterionic detergent and HCl. In some embodiments, the concentration of HCl is within the range of 4 mM and 4M. In some embodiments, the pH of the lysis buffer is within the range of 0 and 6. In some embodiments, a zwitterionic detergent is selected from the group consisting of LAPAO, LDAO, and DDAO. In some embodiments, the concentration of LAPAO is within the range of 0.01% and 10% (w / v). In some embodiments, the concentration of LDAO is within the range of 0.02% and 4% (w / v). In some embodiments, the lysis buffer further comprises sodium decanoate.

[0463] In some embodiments, the nonionic detergent is selected from the group consisting of at least one of a primary alcohol ethoxylate (PAE), Polysorbate 20, ECOSURFTMsurfactant (e.g. ECOSURFTMEH-9, CAS Number 64366-70-7), Triton X, Tween, and Brij. In some embodiments, the concentration of nonionic detergent is within the range of 0.001% and 4% (w / v). In some embodiments, the concentration of nonionic detergent is within the range of 0.01% and 1% (w / v). Page 73 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0464] In some embodiments, the anionic detergent is selected from the group consisting of sodium lauryl sulfate (SLS), sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS), and sodium laureth sulfate or sodium lauryl ether sulfate (SLES). In some embodiments, the concentration of anionic detergent is within the range of 0.001% and 4%(w / v). In some embodiments, the concentration of anionic detergent is within the range of 0.01% and 1% (w / v).

[0465] In some embodiments, samples (e.g., viral particles and / or cells) are lysed (e.g., processed) using sodium hydroxide (NaOH). In some embodiments, samples are lysed with NaOH at ambient temperature (e.g., room temperature). In some embodiments, the concentration of NaOH is about 1 mM to about 200 mM. In some embodiments, the concentration of NaOH is about 10 mM to about 100 mM. In some embodiments, samples are lysed with NaOH for about 1 second to about 10 min, such as about 10 seconds to about 8 min, such as about 1 min to about 5 min, such as about 2 min to about 4 min. In some embodiments, samples are treated with NaOH to inhibit or reduce RNase activity. In some embodiments, NaOH releases viral nucleic acids from a viral sample. In some embodiments, NaOH denatures double stranded DNA or RNA (e.g., separates strands).

[0466] In some embodiments, samples (e.g., viral particles and / or cells) are lysed (e.g., processed) using potassium hydroxide (KOH). In some embodiments, samples comprising DNA (e.g., dsDNA) are treated with KOH. In some embodiments, samples are lysed with KOH at ambient temperature (e.g., room temperature). In some embodiments, the concentration of KOH is about 1 mM to about 200 mM. In some embodiments, the concentration of KOH is about 10 mM to about 100 mM. In some embodiments, samples are lysed with KOH for about 1 second to about 10 min, such as about 10 seconds to about 8 min, such as about 1 min to about 5 min, such as about 2 min to about 4 min. In some embodiments, samples are treated with KOH to inhibit or reduce RNase activity. In some embodiments, KOH releases viral nucleic acids from a viral sample. In some embodiments, KOH denatures double stranded DNA or RNA (e.g., separates strands). In some such embodiments, KOH denaturation separates dsDNA and produces ssDNA.

[0467] In some embodiments, a sample may be a “crude” sample in that it has been subjected to relatively little processing and / or is complex in that it includes components of relatively varied chemical classes. Page 74 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0468] In some embodiments, a cell free extract is a crude extract. In some embodiments, a cell free extract is generated by a cell-free protein expression system (such as, but not limited to PURExpress). Target Nucleic Acids

[0469] In some embodiments, technologies (e.g., methods or compositions) provided herein amplify and / or detect one or more target nucleic acid(s). In some embodiments, a target nucleic acid is a deoxyribonucleic acid (DNA). In some embodiments, a target nucleic acid is a ribonucleic acid (RNA). In some embodiments, a target nucleic acid is single stranded. In some embodiments, a target nucleic acid is double stranded. In some embodiments, a target nucleic acid is a DNA and an RNA hybrid.

[0470] A person skilled in the art is aware of methods to generate ssDNA from RNA (e.g., reverse transcriptase) or dsDNA (e.g., heat denaturation, NaOH denaturation, or KOH denaturation). In some embodiments, target RNA is converted to ssDNA. In some embodiments, target dsDNA is converted to ssDNA.

[0471] In some embodiments, a target nucleic acid is present in a sample. In some embodiments, a sample comprises one or more target nucleic acid(s). In some embodiments, a sample comprises one or more target nucleic acid(s) and nucleic acids other than the one or more target nucleic acid(s). In some embodiments, a target nucleic acid is from a eukaryote. In some embodiments, a target nucleic acid is from a prokaryote. In some embodiments, a target nucleic acid is parasitic (e.g., protozoan), bacterial, viral, or fungal. In some embodiments, a target nucleic acid is human.

[0472] In some embodiments, a target nucleic acid comprises a target nucleic acid region. In some embodiments, a target nucleic acid region is a nucleotide sequence to be amplified and / or detected by methods and compositions provided herein.

[0473] In some embodiments, a target nucleic acid comprises one or more restriction enzyme recognition sequences. In some embodiments, a target nucleic acid comprises two or more restriction enzyme recognition sequences. In some embodiments, one or more restriction enzyme recognition sequences are native restriction enzyme recognition sequences, i.e., such restriction enzyme recognition sequences are naturally occurring in a Page 75 of 164 12865804v1Atty. Docket No.: 2013065-0954 target nucleic acid and not added by any manipulation or amplification of a target nucleic acid. In some embodiments, a target nucleic acid comprises a restriction enzyme recognition sequence upstream or 5’ of a target nucleic acid region and a restriction enzyme recognition sequence downstream or 3’ of a target nucleic acid region.

[0474] In some embodiments, a restriction enzyme recognition sequence is a nickase recognition sequence. In some embodiments, a target nucleic acid comprises a nickase recognition sequence (e.g., a native nickase recognition sequence). In some embodiments, a target nucleic acid comprises one or more nickase recognition sequences (e.g., a native nickase recognition sequences). In some embodiments, a target nucleic acid comprises one or more nickase recognition sequences (e.g., a native nickase recognition sequences). In some embodiments, a target nucleic acid comprises a nickase recognition sequence upstream or 5’ of a target nucleic acid region and a nickase recognition sequence downstream or 3’ of a target nucleic acid region.

[0475] In some embodiments, a target nucleic acid does not comprise a restriction enzyme recognition sequence (e.g., a nickase recognition sequence) or a portion thereof. In some embodiments, a complete restriction enzyme recognition sequence (e.g., nickase recognition sequence) or partial restriction enzyme recognition sequence (e.g., nickase recognition sequence), or complements thereof, is added to a target nucleic acid sequence as provided herein below (e.g., by primers and / or probes).

[0476] In some embodiments, a target nucleic acid comprises one or more sequences that are capable of hybridizing to one or more oligonucleotide binders. In some embodiments, a target nucleic acid comprises at least one oligonucleotide binding sequence (i.e., a sequence capable of hybridizing to an oligonucleotide binder). In some embodiments, a target nucleic acid comprises two oligonucleotide binding sequences. In some embodiments, a target nucleic acid comprises a first oligonucleotide binding sequence and a second oligonucleotide binding sequence. For example, a target nucleic acid may comprise a first oligonucleotide binding sequence and a reverse complement of a second oligonucleotide binding sequence, wherein the first oligonucleotide binding sequence and the reverse complement of the second oligonucleotide binding sequences flank a target nucleic acid region. A target nucleic acid region refers to a sequence within the target nucleic acid that is specifically amplified. In some embodiments, an oligonucleotide binding sequence is a Page 76 of 164 12865804v1Atty. Docket No.: 2013065-0954 primer binding sequence. In some embodiments, an oligonucleotide binding sequence is a probe binding sequence.

[0477] In some embodiments, an oligonucleotide binding sequence comprises about 10 to about 16 nucleotides. In some embodiments, nucleotides in an oligonucleotide binding sequence are consecutive nucleotides in the primary sequence of the target nucleic acid (i.e., no additional intervening nucleotides or other molecules between the consecutive nucleotides). In some embodiments, an oligonucleotide binding sequence comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16 nucleotides. In some embodiments, an oligonucleotide binding sequence comprises at most 16, at most 15, at most 14, at most 13, at most 12, at most 11, at most 10 nucleotides. Oligonucleotide binders

[0478] In some embodiments, compositions and methods of the present disclosure comprise one or more oligonucleotide binders (e.g., probes or primers). In some embodiments, compositions and methods of the present disclosure comprise one or more primers (e.g., forward primers, reverse primers, strand displacement amplification (SDA) primers, or combinations thereof). In some embodiments, compositions and methods of present disclosure comprise one or more probes (e.g., a first probe, a second probe, or combinations thereof). In some embodiments, compositions and methods of present disclosure comprise one or more probes and one or more primers.

[0479] In some embodiments, compositions and methods provided herein utilize one or more oligonucleotide binders to produce a target amplicon, such as an ssDNA sequence.

[0480] In some embodiments, compositions and methods provided herein utilize one or more oligonucleotide binders to amplify a target nucleic acid sequence and / or an ssDNA sequence.

[0481] In some embodiments, compositions and methods provided herein utilize one or more oligonucleotide binders to detect a target nucleic acid, or copies thereof.

[0482] In some embodiments, an oligonucleotide binder comprises a sequence that is complementary to a target nucleic acid. In some embodiments, an oligonucleotide binder Page 77 of 164 12865804v1Atty. Docket No.: 2013065-0954 comprises a sequence that is complementary to an SDA primer or a portion thereof (e.g., an SDA primer binding sequence). In some embodiments, an oligonucleotide binder comprises an SDA primer binding sequence. In some embodiments, an oligonucleotide binder comprises a stabilization sequence. In some embodiments, an oligonucleotide binder comprises a blocking molecule. In some embodiments, oligonucleotide binders (e.g., probes) comprise one or more parts that encode reporting element components.

[0483] In some embodiments, oligonucleotide binders that are primers comprise a complete nickase recognition sequence or a partial nickase recognition sequence, or complements thereof. In some embodiments, complete nickase recognition sequences can be cleaved by a cleaving enzyme, e.g., a nickase.

[0484] In some embodiments, oligonucleotide binders comprise a complete restriction enzyme recognition sequence or a partial restriction enzyme recognition sequence, or complements thereof. In some embodiments, complete restriction enzyme recognition sequences can be cleaved by a cleaving enzyme, e.g., a restriction enzyme.

[0485] In some embodiments, oligonucleotide binders that are primers can be blocked with one or more modifications. In some embodiments, oligonucleotide binders that are primers may be blocked with one or more modifications at the 3’ end. In some embodiments, blocked oligonucleotide binders that are primers cannot be extended by a polymerase (e.g., a DNA polymerase, an RNA polymerase, a reverse transcriptase, etc.) from the 3’ end. Target Complementary Sequences

[0486] In some embodiments, an oligonucleotide binder (e.g., a primer or a probe) comprises a sequence that is complementary to a target nucleic acid sequence.

[0487] In some embodiments, an oligonucleotide binder comprises a sequence complementary to a target nucleic acid sequence that comprises a native restriction enzyme recognition sequence or portion thereof. In some embodiments, an oligonucleotide binder comprises a sequence complementary to a native restriction enzyme recognition sequence (e.g., a first native restriction enzyme recognition sequence and / or a second native restriction enzyme recognition sequence) or portion thereof. Page 78 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0488] In some embodiments, a portion of an oligonucleotide binder sequence that is complementary to a target nucleic acid sequence is at the 3’ end of the oligonucleotide binder.

[0489] In some embodiments, a sequence (e.g., an oligonucleotide binder sequence) that is complementary to a target nucleic acid sequence is at least 85%, 90%, 91%, 92%, 93%, 94%, 9596%, 97%, 98%, 99% complementary to a target nucleic acid sequence. In some embodiments, a sequence that is complementary to a target nucleic acid sequence is 100% complementary to the target nucleic acid.

[0490] In some embodiments, a sequence that is complementary to a target nucleic acid sequence (e.g., a portion of an oligonucleotide binder sequence that is complementary to a target nucleic acid sequence) comprises about 5 to about 30 nucleotides, e.g., 9 nucleotides and / or 16 nucleotides.

[0491] In some embodiments, a sequence that is complementary to a target nucleic acid sequence (e.g., a portion of an oligonucleotide binder sequence that is complementary to a target nucleic acid sequence) comprises about 5 to about 16 nucleotides. In some embodiments, a sequence that is complementary to a target nucleic acid sequence comprises at least 5 nucleotides, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16 nucleotides. In some embodiments, a nucleic acid sequence that is complementary to a target nucleic acid comprises at the most 16 nucleotides, at the most 15, at the most 14, at the most 13, at the most 12, at the most 11, at the most 10, at the most 9, at the most 8, at the most 7, at the most 6, at the most 5 nucleotides.

[0492] In some embodiments, a gap between the first and the second hybridization sequences in the target nucleic acid is 0 to 15 bases, 0 to 6 bases, 3 to 15 bases, 3 to 6 bases, e.g., 5, 7, or 11 bases. In some embodiments, hybridization sequences are overlapping by 1 to 2 bases.

[0493] In some embodiments, separating a first and a second hybridization sequence in the target nucleic acid provides target-based sequence specificity when using oligonucleotide probes. In some embodiments, the gap between the first and the second hybridization sequences in the target nucleic acid is at least 3 bases. In some embodiments, the gap between the first and the second hybridization sequences in the target nucleic acid is 3 to 15 bases or 3 to 6 bases. Page 79 of 164 12865804v1Atty. Docket No.: 2013065-0954 Restriction Enzyme Recognition Sequences

[0494] In some embodiments, oligonucleotide binders comprise a complete restriction enzyme recognition sequence, or a sequence complementary thereto. In some embodiments, oligonucleotide binders comprise a partial restriction enzyme recognition sequence, or a sequence complementary thereto.

[0495] In some embodiments, oligonucleotide binders comprise a complete nickase recognition sequence, or a sequence complementary thereto. In some embodiments, oligonucleotide binders comprise a partial nickase recognition sequence, or a sequence complementary thereto.

[0496] In some embodiments, a nickase recognition sequence is or comprises a nucleotide sequence listed in Table 1 or a sequence that is complementary thereto. In some embodiments, a partial nickase recognition sequence comprises a portion of a nickase recognition sequence listed in Table 1 or a sequence that is complementary thereto. A nickase recognition sequence and a nickase recognition site are used interchangeably herein.

[0497] In some embodiments, a nickase recognition sequence is about 3 to about 7 nucleotides. In some embodiments, a nickase recognition sequence is 3 nucleotides. In some embodiments, a nickase recognition sequence is 4 nucleotides. In some embodiments, a nickase recognition sequence is 5 nucleotides. In some embodiments, a nickase recognition sequence is 6 nucleotides. In some embodiments, a nickase recognition sequence is 7 nucleotides.

[0498] In some embodiments, a nickase recognition sequence comprises two cytosine nucleotides followed by a thymine, guanine or adenine nucleotide. SDA Primer Binding Sequences

[0499] In some embodiments, an oligonucleotide binder (e.g., primer or probe) comprises an SDA primer binding sequence. In some embodiments, an SDA primer binding sequence is located at the 5’ end of an oligonucleotide binder. In some embodiments, an SDA primer binding sequence comprises or consists of about 8 to about 12 nucleotides. In some embodiments, an SDA primer binding sequence comprises at least 8 nucleotides, at least 9, at least 10, at least 11, at least 12 nucleotides. In some embodiments, an SDA primer binding Page 80 of 164 12865804v1Atty. Docket No.: 2013065-0954 sequence comprises at the most 12 nucleotides, at the most 11, at the most 10, at the most 9, at the most 8 nucleotides.

[0500] In some embodiments, an SDA primer binding sequence comprises a complete restriction enzyme recognition sequence (e.g., nickase recognition sequence) or a complement thereof. In some embodiments, an SDA primer binding sequence comprises a partial restriction enzyme recognition sequence (e.g., a portion of a nickase recognition sequence) or a complement thereof. In some embodiments, a nickase recognition sequence is or comprises a nucleotide sequence listed in Table 1 or a complement thereof. In some embodiments, a partial nickase recognition sequence comprises portion of a nickase recognition sequence listed in Table 1 or a sequence that is complementary hereto.

[0501] In some embodiments, an SDA primer binding sequence comprises (i) a complete restriction enzyme recognition sequence (e.g., nickase recognition sequence), a partial restriction enzyme recognition sequence (e.g., nickase recognition sequence), or complements thereof and (ii) a further SDA primer binding sequence. In some embodiments, a further SDA primer binding sequence comprises about 2 to about 6 nucleotides. In some embodiments, a further SDA primer binding sequence is complementary to a target nucleic acid sequence (e.g., complementary to a nucleotide sequence within a target nucleic acid that is adjacent to an oligonucleotide binding sequence). Stabilization Sequences

[0502] In some embodiments, an oligonucleotide binder comprises a stabilization sequence. In some embodiments, a stabilization sequence extends from the 5’ end of an oligonucleotide binder. In some embodiments, a stabilization sequence comprises about 8 to about 20 nucleotides. In some embodiments, a stabilization sequence comprises at least 8 nucleotides, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 nucleotides. In some embodiments, a stabilization sequence comprises at the most 20 nucleotides, at the most 19, at the most 18, at the most 17, at the most 16, at the most 15, at the most 14, at the most 13, at the most 12, at the most 11, at the most 10, at the most 9, at the most 8 nucleotides.

[0503] In some embodiments, a stabilization sequence comprises a partial restriction enzyme recognition sequence (e.g., nickase recognition sequence). In some embodiments, a Page 81 of 164 12865804v1Atty. Docket No.: 2013065-0954 stabilization sequence comprises a partial restriction enzyme recognition sequence (e.g., nickase recognition sequence) and an additional nucleotide sequence. In some embodiments, such additional sequence is about 2 to about 20 nucleotides long. In some embodiments, nucleotides of an additional sequence can be any nucleotides that do not alone or together with the restriction enzyme recognition sequence (e.g., partial nickase recognition sequence) form a complete restriction enzyme recognition sequence (e.g., nickase recognition sequence). In some embodiments, a stabilization sequence does not comprise a restriction enzyme recognition sequence (e.g., partial nickase recognition sequence) or a complete restriction enzyme recognition sequence (e.g., nickase recognition sequence).

[0504] In some embodiments, a stabilization sequence comprises an RNA polymerase binding sequence. In some embodiments, a stabilization sequence is or comprises a T7 RNA polymerase promoter. In some embodiments, an RNA polymerase binding sequence is a eukaryotic RNA polymerase binding sequence. In some embodiments, an RNA polymerase binding sequence is a bacteriophage RNA polymerase binding sequence. In some embodiments, an RNA polymerase binding sequence is an RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, RNA polymerase V, Nr virion RNA polymerase, or T7 RNA polymerase binding sequence. In some embodiments, an RNA polymerase binding sequence is a T7 RNA polymerase binding sequence. In some embodiments, an RNA polymerase binding sequence is a bacterial RNA polymerase binding sequence.

[0505] In some embodiments, a stabilization sequence is a linear single stranded sequence.

[0506] In some embodiments, a stabilization sequence is a hairpin stabilizer. In some embodiments, a hairpin stabilizer includes a hairpin-loop structure, i.e., a stabilization sequence adapts a folded form where the 5’end of the stabilization sequence, or a portion thereof, hybridizes to the 3’end of the hybridization sequence, or a portion thereof, (e.g., a stem stabilizer). In some embodiments, a folded form includes a stabilizer stem portion and stabilizer loop portion. In some embodiments, a stabilizer loop is single stranded. In some embodiments, a stabilizer stem is double stranded. Blocking Molecules Page 82 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0507] In some embodiments, an oligonucleotide binder (e.g., a primer such as an SDA primer) comprises a 3’ blocking molecule.

[0508] In some embodiments, a blocking molecule blocks elongation of an SDA primer (e.g., stops elongation of a nucleotide sequence from proceeding) in the 3’ direction by chemically modifying the 3’ OH group of the 3’ terminal nucleotide of the SDA primer. In some embodiments, the 3’ OH chemical modification blocks the strand displacing polymerase from adding an additional nucleotide to the 3’ terminal nucleotide of the primer.

[0509] In some embodiments, a 3’ blocking molecule may also inhibit exponential amplification of primer dimers.

[0510] In some embodiments, a 3’ blocking molecule, when bound to the 3’ terminal of a primer, blocks elongation of the SDA primer in the 3’ direction. In some embodiments, a blocking molecule binds to the 3’ OH group of the 3’ terminal nucleotide of the primer.

[0511] In some embodiments, a blocking molecule is selected from the group consisting of 3’ddNTP, 3’ Inverted dT, a 3’ carbon chain spacer, a 3’ hexanediol, a 3’ amino spacer, and a 3’ phosphorylation.

[0512] In some embodiments, a 3’ddNTP is a dideoxynucleotide triphosphate that does not have a 3’ OH group required for elongation. In some embodiments, a deoxynucleotide triphosphate is selected from the group consisting of ddTTP, ddATP, ddGTP, and ddCTP.

[0513] In some embodiments, a 3’ Inverted dT has a 3’-3’ linkage that inhibits elongation.

[0514] In some embodiments, a 3’ carbon chain spacer is a carbon chain bound to the 3’ OH group blocking elongation. In some embodiments, a carbon chain spacer may be 3C, 4C, 5C, 6C, 7C, 8C, 9C, 10C, 11C, 12C or more in length.

[0515] In some embodiments, a 3’ hexanediol is a C6 glycol chain bound to the 3’ OH group blocking elongation.

[0516] In some embodiments, a 3’ amino spacer binds to the 3’OH group, of the oligonucleotide binder, required for elongation. In some embodiments, a 3’ amino spacer is a Page 83 of 164 12865804v1Atty. Docket No.: 2013065-0954 4C, 5C, 6C, 7C, 8C, 9C, 10C, 11C, 12C or more carbon chain with a methoxy group on C1, and an NH2 group bound to the last carbon of the spacer.

[0517] In some embodiments, a 3’ phosphorylation is a phosphate group bound to the 3’ OH required for elongation.

[0518] In some embodiments, a blocking molecule is hexanediol (3C6). In some embodiments, a blocking molecule is a 3SpC3. Sensor Parts

[0519] In some embodiments, an oligonucleotide binder (e.g., a probe) comprises a first and / or second nucleic acid sensor part. In some embodiments a first probe comprises a first sensory part. In some embodiments, a second probe comprises a second sensory part. A probe pair used herein may comprise a nucleic acid sensor set. A nucleic acid sensor set comprises at least a first nucleic acid sensor part and a second nucleic acid sensor part.

[0520] In some embodiments, a first nucleic acid sensor part comprises a sequence that is, encodes, or templates at least one reporting element. In some embodiments, a second nucleic acid sensor part comprises a sequence that is, encodes, or templates at least one reporting element. The first and second nucleic acid sensor parts are related to one another in that, when the system is in contact with a sample comprising a target nucleic acid, hybridization of the target nucleic acid with both of the first and second nucleic acid sensor parts juxtaposes the first and second nucleic acid sensor parts with one another so that the juxtaposed parts are susceptible to linkage by one or more of (i) ligation to generate a ligation product and / or (ii) templated copying to generate a linked template product (e.g., they form a “nicked arrangement”).

[0521] In some embodiments, one or both nucleic acid sensor parts may comprise a templating element that directs synthesis of a single, intact strand complementary to the nicked arrangement. For example, where a templating element is or comprises a promoter and / or one or more transcriptional regulatory elements, the system may be or comprise an RNA polymerase; where a templating element is or comprises an origin of replication and / or a binding site for an extendible primer, the system may be or comprise a DNA polymerase (which, in some embodiments, may be a thermostable DNA polymerase, particularly if the Page 84 of 164 12865804v1Atty. Docket No.: 2013065-0954 juxtaposed strand includes a sequence element corresponding to a second extendible primer and the system includes an appropriate pair of primers to amplify a duplex of the juxtaposed strand and its complement).

[0522] In some embodiments, linkage of first and second nucleic acid sensor parts generates a nucleic acid strand (i.e., a linked strand) that includes both of the first and second reporting elements (or their complements), which nucleic acid strand is a reporter in that it, or its complement (e.g., generated by transcription or extension [e.g., primed extension]), or an expression product (e.g., generated by transcription and / or translation) of either, is detectable or otherwise generates or participates in generation of a detectable signal indicative of presence and / or amount of the target nucleic acid in the sample.

[0523] In some embodiments, a linked strand may be transcribed and / or translated (e.g., via cell-free components such as a cell free protein synthesis expression system (CFPS)).

[0524] In some embodiments, linkage as provided herein generates a detectable output. In some embodiments, such detectable output is or is generated by a polypeptide. In some embodiments, a detectable output may be or comprise a catalytic output; in some embodiments, a detectable output may be or comprise a non-catalytic output.

[0525] In some embodiments, a catalytic output is or is generated by an enzyme that catalyzes a reaction, e.g., converting one or more substrates to one or more detectable outputs. In some embodiments, a non-catalytic output is or generates a detectable nucleic acid or polypeptide (e.g., that act as an antigen or other specific binding ligand).

[0526] Among other things, the present disclosure provides technology formats in which a detectable output is amenable to lateral flow analysis (e.g., is, comprises, or generates a product that is detectable by lateral flow). In some embodiments, the present disclosure provides an insight that coupling linkage mediated detection technologies with lateral flow assessment technologies may particularly facilitate multiplexed analyses (e.g., simultaneous detection of a plurality of products amenable to lateral flow). Furthermore, the present disclosure teaches that such coupling may have particular advantages that permit effective multiplexed analysis of products of different chemical class (e.g., two or more of nucleic acids, metals, polypeptides, small molecules, antibodies or fragments thereof etc.). Page 85 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0527] In some embodiments, a sensory part is positioned adjacent to the binder hybridization sequence. n some embodiments, technologies provided herein may include one or more bridging oligonucleotides (e.g., which may be referred to as “gap filling oligonucleotides (“GFO”) that hybridize to the target site between other, e.g., the first and second nucleic acid sensor parts. In some embodiments, a probe set, e.g., a first and second probe, comprises nucleic acid sensors that include only two (i.e., first and second) nucleic acid sensor parts. In some embodiments, technologies provided herein may include one or more bridging oligonucleotides that hybridize to the target site between the first and second nucleic acid sensors.

[0529] In some embodiments, a GFO can reduce background or off-target signal. In some embodiments, no detectable output is generated by ligation of a first nucleic acid sensor part and a second nucleic acid sensor part in the absence of a GFO. In some embodiments, an output generated by ligation of a first nucleic acid sensor part and a second nucleic acid sensor part in the absence of a GFO is not a reporting element. In some embodiments, an output generated by ligation of a first nucleic acid sensor part and a GFO or a second nucleic acid sensor part and a GFO is not a reporting element.

[0530] In some embodiments, a GFO comprises a primer element. In some embodiments, linkage of a first nucleic acid sensor part, a second nucleic acid sensor part, and a GFO generates a nucleic acid strand (i.e., a linked strand) that includes both of the first and second reporting elements (or their complements), which nucleic acid strand is a reporter in that it, or its complement (e.g., generated by transcription or extension [e.g., primed extension]), or an expression product (e.g., generated by transcription and / or translation) of either, is detectable or otherwise generates or participates in generation of a detectable signal indicative of presence and / or amount of the target nucleic acid in the sample. In some embodiments, a linked strand is amplified (e.g., by a polymerase chain reaction e.g., isothermal rolling circle amplification). In some embodiments, amplification utilizes at least a primer element in a GFO.

[0531] In some embodiments, a GFO comprises one or more nucleic acid sensor parts comprising one or more sequences that is / are, encodes, or templates at least one reporting Page 86 of 164 12865804v1Atty. Docket No.: 2013065-0954 element. In some embodiments, a GFO comprising one or more sequences that is / are, encodes, or templates at least one reporting element. Modifications

[0532] In some embodiments, an oligonucleotide binder provided herein comprises one or more modified nucleotides (e.g., modified ribonucleotides, modified deoxyribonucleotides, or a combination hereof).

[0533] In some embodiments, oligonucleotide binders are modified such that the phosphodiester bond of the restriction enzyme recognition sequence on one of the strands is protected using a nuclease resistant modification. In some embodiments, a nuclease resistant modification comprises phosphorothioate (PTO), boranophosphate, methylphosphate or a peptide internucleotide linkage. In some embodiments, modified internucleotide linkages, e.g., PTO linkages, can be chemically synthesized within oligonucleotide probes and primers or integrated into a double stranded nucleic acid by a polymerase, such as by using one or more alpha thiol modified deoxynucleotide. In some embodiments, an oligonucleotide is a modified oligonucleotide, wherein the internucleotide linkages are PTO linkages.

[0534] In some embodiments, dNTPs provided herein comprise dATP, or dTTP, or dCTP, or dGTP. In some embodiments, dNTPs provided herein comprise one or more modified nucleotides.

[0535] In some embodiments, a modified nucleotide is selected from the group consisting of an alpha thiol nucleotide, Borano derivatives, 2’-O-Methyl (2’OMe) modified bases and 2’-Fluoro bases. In some embodiments, a nucleotide is a modified stereoisomer dNTP. In some embodiments, an oligonucleotide binder comprises one or more alpha nucleotide binders. One of skill will understand a restriction enzyme is able to cleavage both strands in a double stranded DNA. In some embodiments, incorporation of modified nucleotides into one strand of a double stranded DNA prevents cutting of both strands by a restriction enzyme. In some embodiments, incorporation of modified nucleotides into one strand of a double stranded DNA allows a restriction enzyme to only cleave the unmodified strand and leaves the modified strand intact. Page 87 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0536] In some embodiments, a modified nucleotide is a peptide nucleic acid (PNA). In some embodiments, a modified nucleotide is a locked nucleic acid (LNA). Peptide nucleic acids, locked nucleic acids, or a combination hereof may be used to alter primer Tm and / or specificity. Primers comprising peptide nucleotides, locked nucleotides, or a combination may be particularly useful in methods of detecting a target nucleotide sequence having one or more SNP sites in order to increase specificity.

[0537] In some embodiments, a modified nucleotide is a 2’-Fluoro-nucleic acid or a 2’-O-methyl-nucleic acid. Primers comprising 2’-Fluoro-nucleic acid modifications, 2’-O- methyl-nucleic acid modifications or a combination have increased nuclease resistance compared to non-modified primers, as well as increased Tm of the 2’-Fluoro-nucleic acid modifications and / or 2’-O-methyl-nucleotide modified domain(s).

[0538] In some embodiments, a modified deoxyribonucleotide is a phosphorothioated deoxyribonucleotide. In some embodiments, a modified deoxyribonucleotide is a phosphodiester deoxyribonucleotide. In some embodiments, a modified deoxyribonucleotide as provided herein destabilizes helices. In some embodiments, a nucleic acid comprising a modified deoxyribonucleotide melts at lower temperatures relative to a control without modified deoxyribonucleotide. In some embodiments, a nucleic acid comprising a modified deoxyribonucleotide can be amplified at lower temperatures relative to a control without modified deoxyribonucleotide.

[0539] In some embodiments, a primer comprises a modified nucleotide in place of at least one guanine or adenine. In some embodiments, a modified nucleotide is a 2- Aminopurine (e.g., a purine analog of guanine and adenine). In some embodiments, a primer comprising a 2-Aminopurine is useful in fluorescence readouts.

[0540] In some embodiments, one of more of the modifications listed herein provides primers that are more resistant to nucleases and / or proteases compared to primers or other nucleotides without any modifications. Exemplary Oligonucleotides Reverse Primers Page 88 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0541] In some embodiments, compositions and methods of the present disclosure comprise one or more reverse primers. In some embodiments, an oligonucleotide binder is a reverse primer. In some embodiments, a reverse primer comprises a nucleic acid sequence complementary to a target nucleic acid (e.g., an RNA target polynucleotide). In some embodiments, a reverse primer comprises a sequence complementary to a target nucleic acid at the 3’ end of the reverse primer. In some embodiments, a reverse primer comprises an SDA primer binding sequence. In some embodiments, a reverse primer comprises a stabilization sequence.

[0542] In some embodiments, a reverse primer comprises from the 5’end to the 3’ end a stabilization sequence, an SDA primer binding sequence and a sequence that is complementary to a target nucleic acid. In some embodiments, a stabilization sequence and an SDA primer binding sequence are separated by one or more nucleotides. In some embodiments, an SDA primer binding sequence and a nucleic acid sequence that is complementary to a target nucleic acid are separated by one or more nucleotides. In some embodiments, a stabilization sequence comprises a partial nickase recognition sequence or complement thereof. In some embodiments, a stabilization sequence and an SDA primer binding sequence together form a complete restriction enzyme recognition sequence (e.g., a nickase recognition sequence). In some embodiments, a stabilization sequence comprises a restriction enzyme recognition sequence (e.g., a complete nickase recognition sequence).

[0543] In some embodiments, a reverse primer comprises a complete restriction enzyme recognition sequence (e.g., a complete nickase recognition sequence) or complement thereof. In some embodiments, a reverse primer comprises a partial restriction enzyme recognition sequence (e.g., a nickase recognition sequence) or complement thereof. Forward Primers

[0544] In some embodiments, compositions and methods of the present disclosure comprise one or more forward primers. In some embodiments, an oligonucleotide binder is a forward primer. In some embodiments, a forward primer comprises a nucleic acid sequence complementary a target nucleic acid (e.g., an RNA and / or DNA target polynucleotide). In some embodiments, a sequence complementary to a target nucleic acid is at the 3’ end of the forward primer. In some embodiments, a forward primer comprises an SDA primer binding sequence. Page 89 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0545] In some embodiments, a forward primer comprises from 5’end to 3’ end an SDA primer binding sequence and a nucleic acid sequence that is complementary to a target nucleic acid. In some embodiments, an SDA primer binding sequence and a nucleic acid sequence that is complementary to a target nucleic acid are separated by one or more nucleotides. In some embodiments, an SDA primer binding sequence comprises a partial restriction enzyme recognition sequence (e.g., a partial nickase recognition sequence).

[0546] In some embodiments, a forward primer comprises a partial restriction enzyme recognition sequence (e.g., a partial nickase recognition sequence). Bump primers

[0547] In some embodiments, compositions and methods of the present disclosure comprise one or more bump primers. In some embodiments, a bump primer is complementary to a target nucleic acid sequence and binds upstream of a primer (i.e., at the 5’end of the target nucleic acid relative to the binding to the primer). In some embodiments, a bump primer is useful when separating a newly synthesized strand of DNA from its template. In some embodiments, a bump primer binds to a DNA template upstream of a forward primer and thereby separates the synthesized strand ssDNA generated by the forward primer.

[0548] In some embodiments, one or more bump primers are not used when the sample is lysed using KOH. Probes

[0549] In some embodiments, compositions and methods of the present disclosure comprise one or more probes. In some embodiments, an oligonucleotide is a probe. In some embodiments, a probe comprises a nucleic acid sequence complementary to a target nucleic acid. In some embodiments, a probe comprises an SDA primer binding sequence. In some embodiments, a probe comprises a first and / or second nucleic acid sensor part, as described herein above. In some embodiments, when the first and second nucleic acid sensor parts are ligated together, they generate an ssDNA sequence that encodes at least one reporter and comprises one or more SDA primer binding sequences. Page 90 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0550] In some embodiments, a probe is a probe as described in PCT Publication, WO 2020 / 037038, entitled “In vitro detection of nucleic acid” and published 20 February 2020; PCT Publication WO 2020 / 191376, entitled “System” and published 24 September 2020; PCT publication WO 2021 / 050560, entitled “System” and published 18 March 2021, the contents of each of which is herein incorporated by reference in their entirety.

[0551] In some embodiments, a probe comprises a restriction enzyme recognition sequence (e.g., nickase recognition sequence). In some embodiments, a probe comprises a partial restriction enzyme recognition sequence (e.g., a partial nickase recognition sequence).

[0552] In some embodiments, a probe is a probe as described in PCT Publication WO / 2020 / 021272, entitled "Nucleic Acid Detection Method”.

[0553] In some embodiments, a probe does not comprise a partial or full restriction enzyme recognition sequence (e.g., nickase recognition sequence).

[0554] In some embodiments, methods and compositions provided herein comprises a first probe and a second probe. In some embodiments, a first probe is capable of hybridizing to a target amplification product. In some embodiments, a first probe is attached to a moiety that permits its detection. In some embodiments, a second probe is capable of hybridizing to a target amplification product sequence upstream or downstream of the first probe. In some embodiments, a second probe is attached to a solid material or to a moiety that permits its attachment to a solid material. In some embodiments, one or both of the first probe and second probe is blocked at the 3’ end from extension by a DNA polymerase. In some embodiments, one or both of the first probe and second probe is not capable of being cleaved by a restriction enzyme. In some embodiments, a probe that is blocked at the 3’ end from extension by a DNA polymerase and not capable of being cleaved by a restriction enzyme can be contacted with the sample simultaneously with amplification. In some embodiments, hybridization of the first and the second probes to at least one target amplification product produces a detectable entity. In some embodiments, the presence of the detectable entity indicates the presence of the target nucleic acid in the sample.

[0555] In some embodiments, a capture probe is a biotinylated capture probe. In some embodiments, a capture probe has a 5’ biotin modification. In some embodiments, a capture probe comprises a C-3 spacer modification to block the 3’ end of the probe. In some embodiments, a capture probe is about 10 to about 20 nucleotides. In some embodiments, a Page 91 of 164 12865804v1Atty. Docket No.: 2013065-0954 capture probe comprises a 3’ blocking molecule. In some embodiments, a capture probe comprises a stabilization sequence (e.g., a linear single stranded sequence, a hairpin stabilizer or a combination thereof). In some embodiments, a capture probe comprises a restriction enzyme recognition sequence, such as a nickase recognition sequence. In some embodiments, a restriction enzyme recognition sequence and / or nickase recognition sequence comprises one or more modifications (e.g., PTO bonds). In some embodiments, a capture probe comprises a target nucleic acid sequence or complement thereof. In some embodiments, a capture probe comprises a repeat strip pull down sequence.

[0556] In some embodiments, a probe is a conjugate probe. In some embodiments, a conjugate capture probe is about 9 to about 20 nucleotides. In some embodiments, a conjugate capture probe comprises a target nucleic acid sequence or complement thereof. In some embodiments, a conjugate capture probe comprises a repeat strip pull down sequence.

[0557] In some embodiments, a moiety that permits detection of a detectable entity and is a dye. In some embodiments, a dye attaches to a first probe. In some embodiments, a dye is a streptavidin attached carbon. In some embodiments, a dye is a gold particle. In some embodiments, a dye is a polystyrene particle. In some embodiments, a dye may be present in the conjugate pad of the nucleic acid lateral flow strip or during the amplification reaction. In some embodiments, a dye provides a rapid color-based visualization of the presence of the detectable entity in the presence of a target nucleic acid.

[0558] In some embodiments, a second probe may be attached to a solid material. In some embodiments, a second probe may be attached to a nitrocellulose surface of a nucleic acid lateral flow strip such that when the pre-detectable entity flows over it, sequence specific hybridization readily occurs, and the detector species becomes located at a defined location on the strip. In some embodiments, a second probe may be attached to a surface of an electrochemical probe, 96-well plate beads, or array surface, or may be attached to a moiety that permits its attachment to a solid material. In some embodiments, a second probe may be covalently attached to a solid material, such as but not limited to glass slides, or glass beads, or ferrite core polymer-coated magnetic microbeads, or silica micro-particles, or magnetic silica micro-particles, or silica-based capillary microtubes, or 3D-reactive polymer slides, microplate wells, or polystyrene beads, or poly(lactic) acid (PLA) particles, or poly(methyl methacrylate) (PMMA) micro-particles, or controlled pore glass resins, or graphene oxide surfaces or functionalized agarose or polyacrylamide surfaces. Page 92 of 164 12865804v1Atty. Docket No.: 2013065-0954 Strand Displacement Amplification (SDA) Primers

[0559] In some embodiments, compositions and methods provided herein comprise one or more strand displacement amplification (SDA) primers. In some embodiments, using short-capped SDA primer(s) during SDA amplification prevents nonspecific dimerization at low temperatures where DNA hybridization is less specific.

[0560] In some embodiments, an oligonucleotide binder is an SDA primer. In some embodiments, an SDA primer comprises a nucleotide sequence that is complementary to a target nucleic acid sequence. In some embodiments, an SDA primer comprises a sequence complementary to a native restriction enzyme recognition sequence (e.g., a native nickase recognition sequence). In some embodiments, an SDA primer comprises a nucleotide sequence that is complementary to an SDA primer binding sequence. In some embodiments, an SDA primer comprises a complete restriction enzyme recognition sequence (e.g., nickase recognition sequence) or a complement thereof. In some embodiments, an SDA primer comprises a blocking molecule. In some embodiments, an SDA primer comprises a 3’ blocking molecule. In some embodiments, a blocking molecule blocks elongation of an SDA primer (e.g., stops elongation of a nucleotide sequence from proceeding) in the 3’ direction. In some embodiments, an SDA primer comprises a stabilization sequence.

[0561] In some embodiments, an SDA primer hybridizes to a target nucleic acid sequence or complement thereof. In some embodiments, an SDA primer binds to an SDA primer binding sequence introduced to the ssDNA sequence by an oligonucleotide binder (e.g., primer and / or probes) or complement thereof.

[0562] In some embodiments, following binding of a SDA primer to a complementary target nucleic acid sequence comprising a native restriction enzyme recognition sequence (e.g., native nickase recognition sequence) or to an SDA primer binding sequence in an ssDNA sequence and subsequent polymerase-based elongation, a double stranded restriction enzyme recognition sequence (e.g., nickase recognition sequence) is produced (e.g., the amplicon generated comprises a double stranded restriction enzyme recognition sequence (e.g., nickase recognition sequence). When nicked and subsequent polymerase-based elongated, a reverse complement of the target nucleic acid sequence or ssDNA is produced. Nicking can occur by using a nickase or a restriction enzyme in combination with Page 93 of 164 12865804v1Atty. Docket No.: 2013065-0954 incorporation of one or more modified dNTP into one of the strands of the double stranded restriction enzyme recognition sequence, ensuring that only one strand is cleaved (e.g., the polymerase elongation or within the SDA primer).

[0563] In some embodiments, compositions and methods of the present disclosure comprise an SDA primer useful in amplifying a target nucleic acid sequence (e.g., comprising a native restriction enzyme recognition sequence, such as a native nickase recognition sequence). In some embodiments, compositions and methods of the present disclosure comprise an SDA primer useful in amplifying an ssDNA sequence. In some embodiments, compositions and methods provided herein produce multiple copies of nucleic acids identical to the ssDNA sequence and / or target nucleic acid sequence having one or more native restriction enzyme recognition sequences (e.g., native nickase recognition sequences). In some embodiments, an ssDNA sequence is amplified using an SDA primer as provided herein generating a plurality of amplified ssDNA sequence.

[0564] In some embodiments, an SDA primer comprises a 3’ blocking molecule. In some embodiments, an SDA primer having a 3’ blocking molecule prevents nonspecific dimerization at low temperatures (e.g., ambient temperatures) where DNA hybridization is less specific.

[0565] In some embodiments, an SDA primer is about 16 to about 33 nucleotides. In some embodiments, a SDA primer is at the most 33 nucleotides, such as at the most 32, such as at the most 31, such as at the most 30, such as at the most 29, such as at the most 28, such as at the most 27, such as at the most 26, such as at the most 25, such as at the most 24, such as at the most 23, such as at the most 22, such as at the most 21, such as at the most 20, such as at the most 19, such as at the most 18, such as at the most 17, such as at the most 16 nucleotides.

[0566] In some embodiments, an SDA primer comprises an RNA polymerase binding sequence.

[0567] In some embodiments, an SDA primer comprises a hairpin stabilizer (e.g., wherein the stabilization sequence forms a double stranded hairpin) and a sequence that is complementary to a target nucleic acid sequence comprising a complete nickase recognition sequence. Page 94 of 164 12865804v1Atty. Docket No.: 2013065-0954 Enzymes Cleavage enzymes

[0568] In some embodiments, compositions and methods provided herein utilize a cleavage enzyme aiding in amplification of a target nucleic acid sequence. In some embodiments, a cleavage enzyme may cleave one strand of a double stranded target nucleic acid, such as a double-stranded DNA, allowing a polymerase (e.g., a DNA polymerase having strand displacement activity) to extend the target nucleic acid sequence.

[0569] In some embodiments, a cleavage enzyme is a restriction enzyme. One of skill in the art is aware of restriction enzymes useful for methods and compositions described herein. For example, one of skill is aware of enzymes provided by commercial sources for example those listed at www.neb.com / products / restriction-endonucleases.

[0570] Restrictions enzymes are proteins isolated from bacteria that cleave DNA sequences at sequence-specific sites, producing DNA fragments with a known sequence at each end. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. Some restriction enzymes cut DNA by making two incisions, once through each sugar-phosphate backbone (i.e., each strand) of the DNA double helix.

[0571] In some embodiments, a restriction enzyme is used in combination with one or more modified dNTP. In some embodiments, following hybridization of an oligonucleotide binder to a target nucleic acid sequence, a strand displacement DNA polymerase extends the 3' end of the oligonucleotide binder using dNTPs and one or more modified dNTP. In some embodiments, a restriction enzyme recognition sequence for a restriction enzyme is formed with one or more modified dNTP base(s) incorporated into the reverse complementary strand acting to block the cleavage of said strand by cleavage of a restriction enzyme. In some embodiments, when a restriction enzyme recognizes its recognition sequence it cleaves only the primer strand that does not include a modified dNTP at the cleavage site thus keeping the other modified strand intact (i.e., a nick). In some embodiments, a nick can be extended by the strand displacement DNA polymerase using the dNTPs and the one or more modified dNTP and displacing the first primer strand. Page 95 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0572] A number of advantages of using a restriction enzyme exist compared to using e.g., nickases. One example thereof is that a much greater number of restriction enzymes that are not nickases are available than those that are nickases, which means that the restriction enzyme(s) for use in the method or composition of the disclosure can be selected from a large number of potential enzymes to identify those with superior properties for a given application, e.g., reaction temperature, buffer compatibility, stability and reaction rate (sensitivity).

[0573] In some embodiments, a cleavage enzyme is a nickase. Nickases (or nicking endonucleases) are a subgroup of restriction enzymes that only cleaves one strand of a dsDNA.

[0574] When restriction enzymes bind to their recognition sequences in a DNA sequence, they hydrolyze both strands of a double stranded target nucleic acid (i.e., a duplex) at the same time. Two independent hydrolytic reactions proceed in parallel, most often driven by the presence of two catalytic sites within the restriction enzymes, one for hydrolyzing each strand hereby cleaving the DNA strand. However, nickases are altered restriction enzymes that hydrolyze only one strand of the duplex, to produce DNA molecules that are “nicked” (e.g., one strand cut) rather than cleaved. Three naturally occurring nickases Nt.BstNBI, Nb.BtsI, and Nb.BsrDI exist. They consist of the large subunits of heterodimeric restriction endonucleases. As such, the catalytic site present in the small subunit that catalyzes cleavage of the other strand is entirely missing. In some embodiments, nickases display no double- strand cleavage activity. In some embodiments, a nickase recognizes a specific nickase recognition sequence within a nucleic acid sequence (e.g., a target polynucleotide).

[0575] In some embodiments, compositions and methods of the present disclosure comprise a nickase. In some embodiments, compositions and methods provided herein use nickases to introduce a nick in a double stranded DNA complex comprising a primer (e.g., a capped SDA primer) to allow removal of the 3’ blocking group during elongation of the primer. In some embodiments, compositions and methods provided herein use nickases to introduce a nick in double stranded nucleic acid to allow for strand displacement during elongation. Nick, nicking, and nicked all refer to cleaving one strand of a dsDNA molecule (e.g., a target polynucleotide) by a nickase. The nickases used herein may nick specific nickase recognition sequences. The term, cognate nickase is used to describe the pairing of Page 96 of 164 12865804v1Atty. Docket No.: 2013065-0954 nickase with its corresponding nickase recognition sequence. Cognate pairs are exemplified in Table 1.

[0576] In some embodiments, a nickase is cognate to a nickase recognition sequence on an oligonucleotide binder (e.g., a primer or a probe). In some embodiments, a nickase is cognate to a nickase recognition sequence within a target nucleic acid and hence within a target amplicon.

[0577] In some embodiments, a nickase binds to a newly formed ssDNA sequence or a target nucleic acid comprising one or more complete nickase recognition sites and nicks one strand (e.g., SDA primer), which enable the strand displacing polymerase to remove the 3’ blocking molecule and elongate the SDA primer.

[0578] In some embodiments, a nickase binds to a newly formed or already existing double stranded target amplicon and nicks the strand that elongates from the SDA primer. In some embodiments, the nickase nicks the double stranded target amplicon, which contributes to ambient temperature displacement of the strand by the polymerase (e.g., DNA polymerase).

[0579] In some embodiments, compositions and methods of present disclosure comprise a nickase. In some embodiments, a nickase is selected from the group consisting of Nt.CviPII, Nb.BbvCI, Nb.Bpu10I, Nb.Bsal, Nb.BsmI, Nb.BsrDI, Nb.BstNBIP, Nb.BstSEIP, Nb.BtsI, Nb.SapI, Nt.AlwI, Nt.BbvCI, Nt.BhaIIIP, Nt.BpulOI, Nt.BpulOIB, Nt.Bsal, Nt.BsmAI, Nt.BsmBI, Nt.BspD6I, Nt.BspQI, Nt.Bst9I, Nt.BstSEI, Nt.CviARORFMP, Nt.CviFRORFAP, Nt.BstNBI, Nt.CviQII, Nt.CviQXI, Nt.EsaSS1198P, Nt.MlyI and Nt.SapI. Nickases are associated to one or more nickase recognition sequences, see Table 1 herein below. A complete nickase recognition sequence is a sequence that would be recognized and nicked by a nickase. A partial nickase recognition site or a part of a nickase recognition site is a sequence that has a portion of a complete recognition sequence. Table 1 Nickase Nickase sitePage 97 of 164 12865804v1Atty. Docket No.: 2013065-0954 Nb.BssSI 5’-CACGAG-3’ Nb.Bpu10I 5’-CCTNAGC-3’

[0580] Those of ordinary skill in the art are aware that various nickases other than those listed in the present disclosure may be used in the present compositions and / or Page 98 of 164 12865804v1Atty. Docket No.: 2013065-0954 methods. In some embodiments, a nickase is an Nt.CviPII. In some embodiments, a nickase is an Nb.BbvCI.

[0581] In some embodiments, oligonucleotide binders and / or target nucleic acid sequences according to the present disclosure comprise a complete or partial nickase recognition site. In some embodiments, an oligonucleotide binder comprises a partial nickase recognition sequence. In some embodiments, a partial nickase recognition sequence from an SDA primer and a partial nickase recognition sequence from a primer binding sequence form a complete nickase recognition sequence.

[0582] Nickases as provided herein may be, for example, active at ambient temperature. In some embodiments, a nickase is stable and or active at temperatures ranging from about 14 °C to about 45 °C, such as about 15 °C to about 35 °C.

[0583] In some embodiments, a restriction enzyme is not a nickase. In some embodiments, a restriction enzyme that is not a nickase is capable of recognizing a restriction recognition sequence present within an oligonucleotide binder (e.g., primer) and cleave only the oligonucleotide binder (e.g., primer) of the cleavage site when said recognition sequence and cleavage site are double stranded (e.g., due to incorporation of modified dNTPs). Polymerases

[0584] Compositions and methods provided herein utilize, in some embodiments, polymerases having strand displacement activity to amplify a target nucleic acid sequence and / or a ssDNA sequence. In some embodiments, compositions and methods of present disclosure comprise a polymerase. In some embodiments, a polymerase is a polymerase comprising strand displacement activity. A polymerase comprising strand displacement activity is able to displace downstream DNA during elongation. In some embodiments, a polymerase is a DNA polymerase. In some embodiments, a strand displacing polymerase comprises elongation activity at ambient temperature. In some embodiments, a strand displacing polymerase comprises elongation activity at temperatures ranging from about 14 °C to about 45 °C, such as about 15 °C to about 35 °C. In some embodiments, a strand displacing polymerase has elongation activity at room temperature. Page 99 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0585] In some embodiments, a DNA polymerase having strand displacement activity is selected from the group consisting of Bsu DNA Polymerase I (Bsu DNAP), phi29, Bst 20 DNA Polymerase (Bst DNAP), Klenow Large Fragment (LF), Klenow Exo-, Bsu Large Fragment, Isopol, and Isopol SD+, or variants thereof. In some embodiments, a DNA polymerase having strand displacement activity is a Bsu or a variant thereof. In some embodiments, a DNA polymerase having strand displacement activity is selected from the group consisting of Bsu DNAP, Klenow LF, Klenow Exo-, and Isopol, and Bst DNAP. In some embodiments, a DNA polymerase having strand displacement activity is a Klenow or a variant thereof.

[0586] In some embodiments, a DNA polymerase having strand displacement activity is active at low temperature. In some embodiments, a DNA polymerase having strand displacement activity is active at 15 °C, at 14 °C, at 13 °C, at 12 °C. In some embodiments, a DNA polymerase having strand displacement activity is active at 14 °C to about 45 °C, such as about 15 °C to about 35 °C.

[0587] In some embodiments, a DNA polymerase having strand displacement activity extends an SDA primer from a nick after cutting and displacing the 3’ blocking molecule.

[0588] In some embodiments, one skilled in the art may enhance the polymerase- based amplification method by adding one or more additives, such as but not limited to 1- oxide, L-lysine free base, L-arginine, glycine, histidine, 5-aminovaleric acid, 1,5-diamino-2- methylpentane, N,N′-diisopropylethylenediamine, tetramethylenediamine (TEMED), tetramethylammonium chloride, tetramethylammonium oxylate, methyl sulfone acetamide, hexadecyltrimethylammonium bromide, betaine aldehyde, tetraethylammoniumchloride, (3- carboxypropyl)trimethylammoniumchloride, tetrabutylammoniumchloride, tetrapropylammoniumchloride, formamide, dimethylformamide (DMF), N-methylformamide, N-methylacetamide, N,N-dimethylacetamide, L-threonine, N,N-dimethylethylenediamine, 2- pyrrolidone, HEP (N-hydroxyethylpyrrolidone), NMP (N-methylpyrrolidone) and 1-methyl, 1-cyclohexyl-2-pyrrolidone (pyrrolidinones), δ-valerolactam, N-methylsuccinimide, 1- formylpyrrolidine, 4-formylmorpholine, DMSO, sulfolane, trehalose, glycerol, Tween-20, DMSO, betaine and BSA. Single stranded binding proteins Page 100 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0589] Compositions and methods provided herein, in some embodiments, utilize single strand binding proteins (SSBP). SSBP may stabilize a displaced strand during strand displacing polymerase elongation. In some embodiments, a composition or method provided herein comprises a SSBP. In some embodiments, a SSBP binds to the DNA strand that is displaced by the strand displacing polymerase. In some embodiments, a SSBP binds an oligonucleotide binder (e.g., a primer and / or a probe). In some embodiments, binding of a SSBP to an oligonucleotide binder prevents or reduces non-specific binding. In some embodiments, a SSB protein facilitates polymerase (e.g., DNA polymerase) nick extension. In some embodiments, the SSBP is selected from the group consisting of RpA, T7 gp2.5, T4 Gene 32 Protein (T4gp32), EcoSSB, TaqSSB, and TthSSB. In some embodiments, a SSBP is a T4gp32.

[0590] In some embodiments, the concentration of a single stranded binding protein in a composition according to the present disclosure is at least 100 ng / μl, at least 200 ng / μl, at least 300 ng / μl, at least 400 ng / μl.

[0591] In some embodiments, T4gp32 is present in the composition within the range of 100 ng / μl and 500 ng / μl, such as 200 ng / μl to 500 ng / μl, such as 300 ng / μl to 400 ng / μl. Reverse Transcriptase

[0592] In some embodiments, technologies provided herein comprises a reverse transcriptase. In some embodiments a reverse transcriptase has RNASEh activity. In some embodiments, a reverse transcriptase is selected from the group consisting of MMLV (alternatively, M-MuLV), AMV, Protoscript II, Superscript I and II and II and IV, RTx, GOScript, Sensiscript, Primescript, and Maxima. Ligases

[0593] embodiments, compositions and methods of present disclosure comprise a ligase. In some embodiments, ligase is selected from the group consisting of SplintR, T4 Ligase, T3 Ligase, and T7 Ligase. In some embodiments, a ligase is a SplintR ligase. In some embodiments, a ligase is a T4 DNA ligase. In some embodiments, the concentration of a ligase is within the range of 10 nM to 5 μM (e.g., 500 nM). Cas enzymes Page 101 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0594] Those skilled in the art, reading the present application, will appreciate that provided technologies can utilize, in various embodiments, any Cas enzyme (or variant, e.g., engineered variant, thereof) that has cleavage activity which is appropriate to the read-out to be utilized and which is activated by guide nucleic acid binding. Moreover, those skilled in the art, reading the present disclosure, will be well familiar with design choices etc. appropriate to match, for example, a particular type of Cas with a particular Cas-activating nucleic acid and / or cleavage substrate (e.g., nucleic acid reporter probe).

[0595] Certain Cas enzymes, specifically including certain Type V and Type VI Cas enzymes, such as Cas12, Cas13, and Cas14 (e.g., Cpf1 / Cas12a, C2c2 / Cas13a, Cas13b, Cas13c, Cas14a, etc.) have been demonstrated to have non-specific nuclease activity that is activated when their guide nucleic acid binds to its target. This non-specific cleavage activity is often referred to as “collateral cleavage”.

[0596] Nucleic acid detection systems have recently been developed that utilize the collateral cleavage activity of a Cas protein to detect presence of a target nucleic acid (or, more accurately, a nucleic acid whose nucleotide sequence includes a target site) of interest. In many embodiments, present compositions and methods utilize a Cas enzyme with collateral activity, and detects activation of that activity / cleavage of a nucleic acid reporter probe that is susceptible to Cas enzyme collateral cleavage activity.

[0597] In some embodiments, a Cas enzyme is a Cas12 enzyme. In some embodiments, a Cas12 enzyme is an LbaCas12 enzyme. In some embodiments, a Cas enzyme is a Cas13 enzyme. In some embodiments, a Cas13 enzyme is a Cas13a enzyme. In some embodiments, a Cas enzyme comprises multiple types of Cas enzymes used in a single reaction.

[0598] In some embodiments, a Cas enzyme is a thermostable Cas enzyme. In some embodiments, a Cas enzyme is thermostable within the range of about 4 °C to about 65 °C.

[0599] When an appropriate Cas for the type of nucleic acid (i.e., RNA, ssDNA, or dsDNA) present in the Cas activating nucleic acid is contacted with the Cas target nucleic acid, its cleavage (e.g., collateral cleavage) activity is activated, and an appropriate nucleic acid reporter probe is cleaved, resulting in a detectable signal. Page 102 of 164 12865804v1Atty. Docket No.: 2013065-0954 Guide polynucleotide

[0600] In some embodiments, a guide nucleic acid hybridizes to a target nucleic acid region within a target nucleic acid. In some embodiments, a guide nucleic acid is complementary to a target nucleic acid region within a target nucleic acid.

[0601] Cas enzymes are activated to cleave (whether specifically or non-specifically) nucleic acids when their guide nucleic acids hybridize with a complementary sequence (a target nucleic acid region or portion thereof). It is well established that guide nucleic acids can be engineered by researchers to hybridize with any target nucleic acid region. Additionally, it is well established that guide nucleic acids may include natural nucleotides, nucleotide analogs, and / or combinations thereof. All of that established knowledge is relevant to, and may be employed in the practice of, the present disclosure.

[0602] For example, those skilled in the art will appreciate that a guide nucleic acid may, in some embodiments, have a length (and / or a portion that hybridizes to a Cas recognition element) that is within a range of about 16-28 nucleotides (e.g., about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, or about 28 nucleotides).

[0603] One skilled in the art will also appreciate that, in certain embodiments, a guide nucleic acid may have less than 100% perfect complementarity with a relevant Cas recognition element (e.g., may be about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary). Buffers

[0604] The methods and compositions provided herein include buffers that provide reaction conditions suitable for amplification, reverse transcription, and / or detection of a target nucleic acid. In some embodiments, a buffer includes components that a skilled person would understand to be in a buffer for DNA amplification. In some embodiments, a composition further includes a buffer in which ambient amplification of the target nucleic acid can occur. In some embodiments, a buffer includes deoxynucleotide triphosphates (dNTPs). In some embodiments, a buffer includes ribonucleotide triphosphates (rNTPs). In some embodiments, a buffer includes Tris or acetate. In some embodiments, a buffer includes potassium ions (K+). In some embodiments, a buffer includes potassium acetate or potassium chloride. In some embodiments, a buffer includes magnesium ions (Mg2+). In some embodiments, a buffer includes magnesium chloride. In some embodiments, a buffer includes a polymerase chain reaction enhancer (e.g., Dimethyl sulfoxide (DMSO), Glycerol, Page 103 of 164 12865804v1Atty. Docket No.: 2013065-0954 Formamide, Bovine Serum Albumin, Ammonium sulfate, polyethylene glycol, gelatin, tween 20, triton X-100, or N,N,N- trimethylglycine (betaine)). In some embodiments, T7 RNA polymerase is active in the buffer. In some embodiments, Cas13 enzyme is active in the buffer. In some embodiments, Cas12 enzyme is active in the buffer.

[0605] In some embodiments, a buffer is selected from the group consisting of Tris, Phosphate, HEPES, DIPSO, MOBS, HEPPSO, TAPSO, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TEA, TAPS, AMPD, TABS, AMPSO, CHES, and CAPSO.

[0606] In some embodiments, a buffer has a buffering capacity in the range of about pH 7 to about pH 8.

[0607] In some embodiments, compositions and methods of the present disclosure include PEG. In some embodiments, a composition or a method includes 1% - 20% PEG, such as 5%- 15% PEG. In some embodiments, a composition includes 10% PEG. In some embodiments, a PEG is a PEG having a molecular weight ranging from 200 to PEG 3350; from 200 to 1000. In some embodiments, a PEG is a PEG 3350. Amplification

[0608] In some embodiments, the present disclosure provides methods of amplifying a target nucleic acid.

[0609] Amplification may be performed over a wide range of temperatures. The optimal temperature for amplification may be determined by the temperature optimum of the relevant polymerase and restriction enzymes and the melting temperature of the hybridizing regions of the oligonucleotide primers.

[0610] In some embodiments, methods provided herein do not use temperature cycling. Furthermore, the amplification step does not require any controlled oscillation of temperature, nor any hot or warm start, pre-heating or a controlled temperature decrease. In some embodiments, methods according to the present disclosure allow for amplification over a wide temperature range e.g., 15 °C to 60 °C, such as 20 °C to 60 °C, such as 15 °C to 45 °C or 15 °C to 35 °C.

[0611] In some embodiments, amplification is performed at ambient temperature. In some embodiments, amplification is performed without temperature cycling. In some Page 104 of 164 12865804v1Atty. Docket No.: 2013065-0954 embodiments, amplification is performed under isothermal conditions. In some embodiments, amplification is performed at most 50 °C, at most 45 °C, at most 40 °C, at most 35 °C, at most 30 °C, at most 25 °C, at most 20 °C, at most 15 °C. Reverse Transcription

[0612] In some embodiments, a target RNA is converted to DNA (e.g., ssDNA or dsDNA). In some embodiments, a target RNA is converted to DNA by a reverse transcriptase as described herein. In some embodiments, an RNA target nucleic acid sequence is converted to RNA by a method as described in WO2024 / 006552, the entirety of which is incorporated herein by reference.

[0613] In some embodiments, conversion of a target RNA to DNA may be performed over a wide range of temperatures. The optimal temperature for reverse transcription may be determined by the temperature optimum of the relevant polymerase and restriction enzymes and the melting temperature of the hybridizing regions of the oligonucleotide primers.

[0614] In some embodiments, methods provided herein do not use temperature cycling. Furthermore, the reverse transcription step does not require any controlled oscillation of temperature, nor any hot or warm start, pre-heating or a controlled temperature decrease. In some embodiments, methods according to the present disclosure allow for reverse transcription over a wide temperature range e.g., 15 °C to 60 °C, such as 20 °C to 60 °C, such as 15 °C to 45 °C or 15 °C to 35 °C.

[0615] In some embodiments, reverse transcription is performed at ambient temperature. In some embodiments, reverse transcription is performed without temperature cycling. In some embodiments, reverse transcription is performed under isothermal conditions. In some embodiments, reverse transcription is performed at most 50 °C, at most 45 °C, at most 40 °C, at most 35 °C, at most 30 °C, at most 25 °C, at most 20 °C, at most 15 °C. EXAMPLES Example 1: Fluid Flow in Two-Chamber Air Diffusion Device with Lateral Flow Strip Page 105 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0616] The present Example demonstrates the flow of fluid through power-free devices with two reaction chambers, air diffusion timers, and lateral flow strips. In each experimental result shown in this Example, colored liquid (i.e., water with 0.2% EcosurfTMsurfactant and red food dye) is used as a sample fluid so that the flow is clearly visible through the transparent device body in order to illustrate the flow behavior. The device used in this Example (see FIG.9) is similar to the device illustrated in FIG.1C, with two air pinning chambers above each of the two reaction chambers. Rather than a straight vertical timer delay channel like in FIG.1C, the timer delay channel in this device has a vertical portion leading into a diagonal portion. The air channel for the slow diffusion filter pad is also diagonal. These differences in channel geometry may, in some embodiments, enhance fluid flow, and allow the device to be made to be more compact. The slow air diffusion filter pad (i.e., the controlled air diffusion element, made of a porous membrane material) is visible as a white square with the air outlet visible as a small circle in the center of the slow air diffusion filter pad. The PVA valve at the bottom of the timer delay channel opens up to a chamber containing a lateral flow strip (not shown).

[0617] FIG.9 shows a series of photographs of a fluidic device from 0 min to 20 min reaction time, showing the addition of a red colored sample fluid, flow of fluid, and lateral flow strip readout, according to aspects of the present embodiments. In panel (i), at time t = 0 min, the empty device has two reaction beads (white spheres), one in each reaction chamber. In panel (ii), at time t = 2 min, the sample has been added to the sample input port and reservoir, and has filled up the two reaction chambers. The air present in the air pinning chambers pins the surface of the liquid in each reaction chamber so that the volume of liquid in each reaction chamber remains constant. In panel (iii), at time t = 10 min, the red liquid has visibly travelled down all of the vertical portion and about half of the diagonal portion of the timer delay channel. In panel (iv), at time = 18 min, the liquid has reached the bottom of the timer delay channel and has begun to dissolve the PVA valve. Throughout panels (i) to (iv), the air in the bottom portion of the timer delay channel and in the air channel for the controlled air diffusion element are slowly diffusing out through the slow air diffusion filter pad. Finally, in panel (v), at time t = 20 min, the PVA valve is completely opened (by dissolution and / or rupture of the PVA membrane), so that the sample liquid can reach the bottom of the lateral flow strip and begin to move upwards along the vertical length of the lateral flow strip. The liquid contained in the reaction chambers has completely emptied, with a small amount of liquid remaining in the timer delay channel. Page 106 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0618] FIGS.10A-10D show a series of photographs of four fluidic devices showing empty devices, with the addition of sample fluid, flow of fluid, and lateral flow strip readout, according to aspects of the present embodiments. In FIG.10A, four devices are shown side by side prior to addition of sample fluid. Each device has two reaction chambers, each reaction chamber having an air pinning chamber on top, and a slow air diffusion pad. The devices used in FIG.10 are very similar to the ones used in FIG.9. In FIG.10A, the lyophilized reaction beads are visible as white spheres in each of the reaction chambers, and the slow air diffusion pads are visible as off-white squares below the reaction chambers. There is also a lateral flow strip (visible as the narrow, tall rectangles) positioned vertically at the right side of each device.

[0619] In FIG.10B, sample fluid in the form of a red fluid (i.e., water with red dye and 0.2% EcosurfTMsurfactant) has been added to the devices so that the flow of fluid is clearly visible through the transparent device. The red fluid has flowed down the first fluid channel and up into each of the reaction chambers, where the lyophilized reaction beads are dissolved in the fluid.

[0620] In FIG.10C, the sample fluid has travelled down the timer delay channel, with the red color of the sample fluid allowing the timer delay channel to be clearly visible. In these devices, the timer delay channel has a vertical portion connecting down from the two channels leading to the reaction chambers, and a diagonal portion toward the PVA valve near the bottom of the device. Faintly visible is also a diagonal channel leading from the PVA valve up toward the center of the slow air diffusion pad. The amount of time it takes for the air to diffuse out from the slow air diffusion pad and for the sample fluid to reach the PVA valve is the needed time for the reaction to occur within the reaction chambers. Also visible in FIG.10C is the PVA valve, which is visible as a red circle due to the red sample fluid filling the space between the PVA membrane and the channel, so that the PVA membrane can be disrupted to allow the sample fluid to flow. The PVA membrane begins dissolving once the sample fluid has reached it.

[0621] In FIG.10D, the devices are shown after the PVA membranes are fully disrupted, so that the sample fluid can reach the lateral flow chamber and start wicking up the lateral flow strips. The lateral flow strips have visible red and pink color due to the red sample fluid being pulled up into them. There is some red sample fluid remaining visible within the timer delay channel. Page 107 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0622] FIGS.10E-10H show enlarged views of the leftmost device from FIGS.10A- 10D, according to aspects of the present embodiments. In FIG.10E, an empty device 650 is shown with sample fluid input reservoir 652, reaction chambers 656 topped by air pinning chambers 654 and containing reaction beads 658, and lateral flow strip 674. There is a dissolvable membrane covering the valve chamber 668 and preventing flow into the lateral flow chamber 676 where the lateral flow strip 674 is positioned. In FIG.10F, sample fluid 660 is added to the reservoir 652. The sample fluid 660 flows down the first flow channel 664, up the side channels 662, and into the reaction chambers 656 where the fluid 660 dissolves the reaction beads 658. The timer flow channel 666 is still filled with air, so the fluid 660 has not flowed into it. The timer flow channel 666 is fluidly connected to the valve chamber 668 and air flow channel 670, which is vented to a slow air diffusion pad 672 which allows air to escape slowly by diffusion through its porous interior. In FIG.10G, as the air has escaped, a portion of the fluid 660 has flowed down the timer flow channel 660 and reached the valve chamber 660. Part of the fluid 660 has pushed up into the air flow channel 670 as well. In FIG.10H, the dissolvable membrane covering the valve chamber 668 has been fully dissolved by the fluid 660, so that fluid can pass through into the chamber holding the lateral flow strip 674. The fluid 660 is visibly pooled at the bottom of the lateral flow chamber 676, and the fluid has wicked up the lateral flow strip 674. Example 2: On-Device Assay Testing for Neisseria Gonorrhoeae

[0623] The present example describes a clean system test of a Ng gDNA (Neisseria Gonorrhoeae genomic DNA) assay at high copy number. The purpose of the assay is testing for the presence of gonorrhoeae. FIGS.11A-11C show photographs of fluidic devices and lateral flow strip readouts, according to aspects of the present embodiments. FIG.11A shows a photograph of four devices prior to sample loading. The devices in this example each have two reaction chambers, an air diffusion pad to control the time that the reaction takes place, a PVA valve between the time delay channel and output port to a lateral flow strip as readout. In the present embodiment, the strips use carbon black as the conjugate, being pulled down by a set of dual probes that use biotin on one side to bind the carbon black, and an oligo pulldown sequence on the other side to bind the lateral flow strip surface. Lyophilized reaction beads containing reaction components for an ambient temperature SDA reaction are visible as white spheres inside each reaction chamber. In some embodiments, the reaction components may include 50 mM Tris-Acetate buffer pH 8.7, 50 mM KOAc, 0.5 mM dNTP mix, 0.5 U / μL Bsu DNAP, 0.05 U / μL Nt.CviPII, 0.2 ug / μL T4gp32, excipients including Page 108 of 164 12865804v1Atty. Docket No.: 2013065-0954 Raffinose, Dextran, and PEG, primers and probes for amplifying a Gonorrhoeae gDNA target (250 nM each of F and R primer, and 250 nM each of the two probe components).

[0624] The devices are labelled 1, 2, 3, and 4 from left to right. Devices 1 and 2 each contain gDNA, while devices 3 and 4 are no template controls (NTC). Liquid samples (i.e., water with 0.2% EcosurfTMsurfactant, 15 mM MgOAc, and the indicated concentration of target gDNA) are loaded into each device, and the user waits for the assay to complete. The flow of liquid is similar to what is schematically illustrated in FIGS.2A-2H.

[0625] FIG.11B shows a photograph of the four devices after assay run completion. The reaction beads are visibly gone, as they have dissolved upon introduction of the liquid sample into the reaction chambers. The regions of the lateral flow strips marked by the dashed rectangle are shown as enlarged photographs in FIG.11C. The lateral flow strips from devices 1 and 2, which contained samples of 1000 cp gDNA, show clear dark lines. The lateral flow strips from devices 2 and 3, which were the NTCs, show no such lines.

[0626] The results of the experiment in this Example clearly show that the power-free fluidic device is able to detect Neisseria gonorrhoeae in a sample, without the use of any electrical power or active input from the user. Example 3: Fluid flow in Combined Timer / Air Valve Chip with Serpentine Timer

[0627] The present example describes the flow of fluid in a combined timer / air valve chip 1100 with a serpentine timer built in. FIGS.15A-B show photographs of a front side of a combined timer and air valve chip 1100, according to aspects of the present embodiments. In FIG.15A, the empty device has a sample inlet, waste metering chamber, and reaction chambers visible. In this example, the two reaction chambers each has its own outlet channel toward the lateral flow strip, so that two separate reaction streams may reach the lateral flow strip. The lateral flow strip uses carbon black as the conjugate, being pulled down by a set of dual probes that use biotin on one side to bind the carbon black, and an oligo pulldown sequence on the other side to bind the lateral flow strip surface. In FIG.15B, sample fluid (i.e., a red aqueous fluid solution) has been added to the sample inlet and flowed into the two reaction chambers which are now filled. Excess sample fluid exits through channels at the top of the reaction chambers and flows into the waste metering chamber. The sample fluid has also started to exit the bottom of their respective channels. However, the flow through the exit channels is stopped by the dead airspace in the rest of the exit channel and in the lateral flow chamber. Page 109 of 164 12865804v1Atty. Docket No.: 2013065-0954

[0628] FIGS.16A-B show photographs of a reverse side of a combined timer and air valve chip 1100, according to aspects of the present embodiments. In FIG.16A, a timer fluid (i.e., a green liquid) has been added to the timer fluid reservoir, and has started to flow down the fluidic resistance element and up into the serpentine chamber. In FIG.16B, the timer fluid has reached the water-soluble PVA valve (i.e., fluid-soluble membrane), which is then dissolved or ruptured. Once the valve is opened, air can escape, and the sample fluid held in the exit channels can flow down toward the lateral flow strip. In FIG.16B, the red fluid is visible now passing upward into the lateral flow strip. In this example, the two reaction chambers did not contain lyophilized reaction beads. However, in some embodiments, each reaction chamber may contain a different lyophilized bead(s) for conducting a different chemical or biological reactions, which will act on a common sample fluid flowing into the reaction chambers. Upon flowing out of the reaction chambers, the sample fluid after undergoing a chemical and / or biological reaction, and after waiting the time delay for the serpentine timer to complete its flow, reaches the lateral flow strip and immediately starts moving up the lateral flow strip. If each reaction chamber contains a different type of lyophilized bead, each bead containing agents needed for conducting a different reaction, the lateral flow strip may also include different capture regions or chemicals to capture each of the products expected to be formed by each reaction. For example, each reaction chamber may contain a lyophilized bead containing reagents for reverse transcribing, amplifying and / or detecting a different virus from a single sample fluid from a patient. Example 4: Control of Fluid Timing by Air Diffusion Pad Size

[0629] The present example describes control of fluid timing by changing the size of an air diffusion pad in a slow air diffusion device. Being able to control the exact time when a sample fluid is held in place in reaction chambers for reactions to take place is very important. In a serpentine fluid timer circuit, the length, total volume, dimensions, and surface properties of a serpentine channel can be tuned to control the total time for a fluid to pass through. In a slow air diffusion device, the time it takes for air to diffuse through an air- diffusive volume can be tuned by the size of the porous membrane of the slow air diffusion timer. The total diffusion time is largely controlled by the shortest path from a central air vent connected to the rest of the device’s fluidic channels.

[0630] FIG.17A shows a plot of time required for fluid to reach lateral flow strip in minutes as a function of the size of the square air diffusion pad in air diffusion timer devices, according to aspects of the present embodiments. The data shown in the plot are from the Page 110 of 164 12865804v1Atty. Docket No.: 2013065-0954 devices in the photographs of FIG.17B. In this plot, since the diffusion pads were all squares, the x-axis shows the size of the diffusion pad in millimeters (mm). Three sizes of air diffusion pads were tested: 5 mm x 5 mm, 8 mm x 8 mm, and 12 mm x 12 mm. The y-axis shows the duration of time it takes for air to diffuse fully through the air diffusion pad so that the sample fluid is able to flow down to dissolve the water-soluble membrane to reach a lateral flow strip.

[0631] FIG.17B shows a series of photographs from time t = 0 min to t = 33 min showing devices with different air diffusion pad sizes with different flow times to reach the lateral flow strip corresponding to the data in FIG.17A, according to aspects of the present embodiments. The devices in FIG.17B are similar to the device design shown in FIG.13 fixed on top of the base design shown in FIG.14, and uses the needle-puncturing membrane valve design with an air diffusion pad. At the initial stage at t = 0 min, sample fluid (i.e., the red liquid) is loaded into each of the devices, and has filled the reaction chambers. At t = 23 min, the fluid has reached the lateral flow strips in the devices with 5 mm air diffusion pads. At t = 28 min, the fluid has also reached the lateral flow strips in the devices with 8 mm air diffusion pads, while the devices with 12 mm still have their fluids held back. Finally, at t = 33 min, the fluid in the remaining three devices with 12 mm air diffusion pads has reached the lateral flow devices as well. Thus, this experiment shows that the timer delay can be actively controlled by changing the size of the air diffusion pad. A larger air diffusion pad (as well as smaller pores) will result in a longer delay time before the sample fluid reaches the lateral flow strip. Horizontal Fluidic Device Featuring Serpentine Fluid Timer

[0632] FIGs.20A and 20B show an exemplary horizontal fluidic device 1100 featuring a fluidic serpentine timer 1111 (shown in FIG.20B), according to aspects of the present embodiments. FIG.20A is a CAD drawing illustrating a top view of the fluidic device 1100, and FIG.20B is a CAD drawing illustrating a bottom view of the fluidic device 1100. In this example, the device 1100 includes a main chip 1101 or chip body 1101 or device body 1101, a sample inlet port and reservoir 1114, two reaction chambers 1118, a timer fluid inlet port and reservoir 1108, a fluidic serpentine timer 1111 and a lateral flow chamber 1124. A timer fluid flows through the fluidic timer 1111 to control the duration of time for a sample fluid to undergo a reaction. The sealed, airtight lateral flow chamber 1124 holds the reaction fluid in place until the fluidic timer flow is completed after a programmable delay, which Page 111 of 164 12865804v1Atty. Docket No.: 2013065-0954 causes a PVA valve 1104 to be opened to allow reaction fluid to enter the lateral flow chamber 1124. The fluidic serpentine timer 1111 includes a fluidic resistance element 1110 and a serpentine timer delay channel 1112. In this case, the fluidic resistance element 1110 is itself a serpentine channel with narrow channel dimensions to produce a fluidic resistivity. The serpentine timer delay channel 1112 is a reservoir for the accumulation of timer fluid coming out of the fluidic resistance element 1110. In some embodiments, the serpentine timer delay channel 1112 and the fluidic resistance element 1110 may be coated with a hydrophobic plasma treatment and / or a superhydrophobic coating to help facility fluid movement therethrough. In some embodiments, the main chip 1101 comprises a single continuous body composed of a polymer material.

[0633] Still referring to FIGs.20A and 20B, in some embodiments, the main chip body 1101 is made of a polymeric material (e.g., 3D printer resin plastic, e.g., Formlabs Clear Resin, Polymethylmethacrylate (PMMA), Cyclo-olefin-copolymer (COC), Cyclo- olefinpolymer (COP), Polycarbonate (PC), Polystyrene (PS), polypropylene, or other polymers for microfluidics). In some embodiments, the main chip body 1101 is made of an injection molded plastic, such as Cyclo-olefinpolymer (COP), Polycarbonate (PC) and Polystyrene (PS). In some embodiments, the main chip body 1101 is 3D printed. The reservoir 1114 is disposed on the top of the device 1100 and is used to receive a sample fluid. In some embodiments, the reservoir 1114 may hold a volume of approximately 10 μL to 5 mL. In some embodiments, the main chip body 1101 may include more than one reaction bead (i.e., lyophilized reaction bead) per reaction chamber 1118. In some embodiments, the reservoir 1114 may include dimensions (i.e., height, width, and / or length) of in a range from approximately 0.5 mm to about 100 mm each. A bottom portion of the reservoir 1114 is fluidly connected to a first flow channel 1116, which is fluidly connected to two side channels 1117 that flow up into the reaction chambers 1118. Each reaction chamber 1118 may contain one or more lyophilized reaction beads comprising reagents needed for a particular reaction or assay. In some embodiments, the flow channel 1116 may have dimensions (i.e., height, width, or diameter) in a range from about 0.1 mm to about 5 mm, with a length from about 2 mm to about 100 mm. In some embodiments, the flow channel 1116 includes a mixing element. In some embodiments, the two side channels 1117 may have dimensions (i.e., height, width, or diameter) in a range from about 0.1 mm to about 5 mm, with a length from about 2 mm to about 100 mm. An enlarged top view 1140 of the reaction reactions chambers 1118 is shown in FIG.20C. The enlarged view 1140 includes through-holes 1142, Page 112 of 164 12865804v1Atty. Docket No.: 2013065-0954 ridge channels 1146, and through holes 1148. The reaction chambers 1118 include a U-bend design. The sample fluid flows from side channels 1117 in FIG.20B into the reaction chambers 1118 via the through-holes 1142. The ridge channels 1146 draw the fluid sample to the bottom of reaction chambers 1118, to ensure that the sample fluid reaches the through holes 1148 before filling the reaction chambers 1118. The fluid sample flows into the side channels 1119 in FIG.20B via through holes 1148. As shown in FIG.20B, the bottom surface of the device may further include a lacuna or dead space 1115 disposed therein and located between side channels 1117 and side channels 1119. The progress of a sample fluid flow through reactions chambers including a U-bend design are further illustrated in FIGs.34A-C.

[0634] Still referring to FIGs.20A and 20B, the reaction chambers 1118 are fluidly connected at their bottom to two side channels 1119. The two side channels 1119 merge into a reaction chamber fluid outlet channel 1120 which fluidly connects to the bottom of the lateral flow chamber 1124. In some embodiments, the fluid outlet channel 1120 may have dimensions (i.e., height, width, or diameter) in a range from about 0.1 mm to about 5 mm, with a length from about 2 mm to about 100 mm. In some embodiments, the two side channels 1119 may have dimensions (i.e., height, width, or diameter) in a range from about 0.1 mm to about 5 mm, with a length from about 0.5 mm to about 100 mm. An enlarged top view 1150 of the outlet channel 1120 and the lateral flow chamber 1124 is shown in FIG. 20D. The enlarged view 1150 includes diagonal hatch lines 1152 disposed within the outlet channel 1120, and fluid pins 1154 disposed within lateral flow chamber 1124. The diagonal hatch lines 1152 help with flow mixing. In some embodiments, the diagonal hatch lines 1152 include a plurality of diagonally oriented bars that protrude at roughly a 45-degree angle (for example, from about 40 degrees to about 50 degrees from a longitudinal axis of each respective channel portion, and / or from about 35 degrees to about 55 degrees). The fluid pins 1154 act as a capillary pump to help ensure rapid transfer of a sample liquid out of the outlet channel 1120 and into the lateral flow chamber 1124.

[0635] Still referring to FIGs.20A and 20B, the lateral flow chamber 1124 is configured to accommodate a lateral flow strip arranged substantially vertically, with fluid reaching the bottom of the lateral flow chamber 1124 to contact a lateral flow strip (LFS). A vent channel 1122 fluidly connects the lateral flow chamber 1124 to a PVA valve 1102 via connection point 1123. The PVA valve 1102 prevents air from escaping from the lateral flow chamber 1124. The dead airspace within the fluid outlet channel 1120 and the lateral flow chamber 1124 hold in place the sample fluid within the reaction chambers 1118, so that they Page 113 of 164 12865804v1Atty. Docket No.: 2013065-0954 do not flow out. In some embodiments, the device 1100 is designed such that the sample fluid may flow out of the reaction chamber(s) in an insignificant amount, but does not reach the lateral flow chamber 1124 during the timing delay (i.e., the sample fluid does not reach the lateral flow chamber 1124 until air is released from the lateral flow chamber 1124 via the PVA valve 1102, as described herein). The lateral flow chamber 1124 may be sized to accommodate lateral flow strips of various sizes including those with a thickness in a range from about 2 mm to about 15 mm, a width in a range from about 4 mm to about 15 mm, and a length in a range from about 15 mm to about 80 mm. Accordingly, the lateral flow chamber 1124 itself may include similar dimensions, albeit slightly larger (for example, 1-3% larger in each case) to allow for a tolerance to enable insertion and removal of a lateral flow strip into and out of the lateral flow chamber 1124.

[0636] Referring further to FIGs.20A and 20B, the reservoir 1108 is disposed on the top of the device 1100 and is used to receive a timer fluid. In some embodiments, timer fluid may be or may include water, PEG, glycerol, and surfactants such as ECOSURF™ EH-9 and Tween. In some embodiments, the reservoir 1108 may have a volume in a range from about 0.1 mL to about 5 mL and various combinations of dimensions, each in a range from about 2 mm to about 100 mm. The reservoir 1108 is fluidly connected at a bottom portion to the fluidic resistance element 1110 via through hole 1109. In some embodiments, the fluidic resistance element 1110 includes a narrow channel arranged in a series of serpentine turns connected to the serpentine timer delay channel 1112. In some embodiments, the fluidic resistance element 1110 may include dimensions (i.e., an inner diameter, or channel width) in a range from about 0.5 mm to about 1 mm. Th smaller cross-section of the fluidic resistance element 1110 may limit the amount of volume that is delivered, requiring a larger footprint. In some embodiments, the serpentine timer delay channel 1112 includes a wide channel arranged in a series of serpentine turns surrounding the reservoir 1108. In some embodiments, the serpentine timer delay channel 1112 may include an inner diameter and / or channel height / width in a range of about 0.5 mm to about 1 mm. The fluidic resistance element 1110 and the serpentine timer delay channel 1112 may have serpentine turns with rounded corners to ensure smoother timer fluid flow with minimal pinning of fluid in corners. The serpentine timer delay channel 1112 may be used as a filling chamber due to its larger cross-section. The larger cross-section of the serpentine timer delay channel 1112 may render it more vulnerable to a drop shock. The serpentine timer delay channel 1112 is fluidly connected to the PVA valve 1102 which also holds the dead airspace in the lateral flow chamber 1124. The PVA Page 114 of 164 12865804v1Atty. Docket No.: 2013065-0954 valve 1102 includes a membrane, a recess 1104 and a connection channel 1130. The recess 1104 accommodates a sponge for rupturing the membrane. The connection channel 1130 is fluidly connected to the recess 1104 via a connection point 1132. The connection channel 1130 is fluidly connected to the vent channel 1122 via the connection point 1123. Air exits the serpentine timer delay channel 1112 and fluidic resistance element 1110 via an air vent 1106. When the timer fluid reaches the PVA valve 1102 and the sponge which ruptures the membrane and air that was contained in the lateral flow chamber 1124 is able to escape (also via air vent 1106), thus allowing fluid in the fluid outlet 1120 to flow into the lateral flow chamber 1124.

[0637] FIG.21A is a CAD drawing illustrating a top view of a horizontal fluidic device 1200, according to aspects of the present embodiments. In this example, the device 1200 includes a sample inlet port and reservoir 1201, two reaction chambers 1202, a timer fluid inlet port and reservoir 1203, a fluidic serpentine timer 1204 and a lateral flow chamber 1205.

[0638] FIG.21B is a CAD drawing illustrating a top view of a horizontal fluidic device 1202, according to aspects of the present embodiments. In this example, the device 1202 includes a sample inlet port and reservoir 1201, two reaction chambers 1202, a timer fluid inlet port and reservoir 1203 and a lateral flow chamber 1205. In the embodiment of FIG.21B, the components are spatially arranged in a different configuration from that of the embodiment of FIG.21A.

[0639] FIG.21C is a CAD drawing illustrating a top view of a horizontal fluidic device 1204, according to aspects of the present embodiments. In this example, the device 1200 includes a sample inlet port and reservoir 1201, two reaction chambers 1202, a timer fluid inlet port and reservoir 1203 and a lateral flow chamber 1205. In the embodiment of FIG.21C, the components are spatially arranged in a different configuration from that of the embodiments of both FIG.21A and FIG.21B. Valves with sponges

[0640] FIG.22A is a schematic diagram illustrating a side view of a fluidic device 1220 including a fluid flow path 1221, a PVA membrane 1222 and a sponge 1224, according Page 115 of 164 12865804v1Atty. Docket No.: 2013065-0954 to aspects of the present embodiments. In this example, the sponge 1224 is not within the fluid flow path 1221, and is disposed on the opposite side of the PVA valve 1220. A fluid 1214 flows through the fluid flow path 1221 and reaches the PVA membrane 1222. Subsequently, the PVA membrane 1222 starts to dissolve and becomes porous to the liquid which passes through and begins to hydrate the sponge 1224. The sponge 1224 starts to expand from fluid intake, and further pushes the PVA membrane 1222 from outside of the fluid flow path 1221. In some embodiments, the device 1100 as described herein does not include a sponge 1224.

[0641] FIG.22B is a schematic diagram illustrating a side view of a fluidic device 1226 including a fluid flow path 1221, a PVA membrane 1222 and a sponge 1224, according to aspects of the present embodiments. In this example, the sponge 1224 is disposed within the fluid flow path 1221. A fluid 1214 flows through the fluid flow path 1221 and reaches the PVA membrane 1222 and the sponge 1224. The PVA membrane 1222 starts to dissolve while the sponge 1224 starts to expand from fluid intake, and further pushes the PVA membrane 1222 from inside the fluid flow path 1221.

[0642] FIG.22C is a schematic diagram illustrating a side view of a fluidic device 1228 including a fluid flow path 1221, a PVA membrane 1222 and a sponge 1224, according to aspects of the present embodiments. In this example, the sponge 1224 is disposed within the fluid flow path 1221, and at a distance from an air vent 1229. A fluid 1214 flows through the fluid flow path 1221 and reaches the PVA membrane 1222 and the sponge 1224. The PVA membrane 1222 starts to dissolve while the sponge 1224 starts to expand from fluid intake, and further pushes the PVA membrane 1222 from inside the fluid flow path 1221. Keeping the sponge 1224 away from the air vent 1229 may prevent the fluid 1214 from blocking the escape of air through the air vent 1229.

[0643] FIG.23A is a schematic diagram illustrating a side view of a fluidic device 1230 including a fluid flow path 1231, a PVA membrane 1232 and a sponge 1234, according to aspects of the present embodiments. The right panel shows a fluid 1214 flowing through the fluid flow path 1231. In this example, the fluid 1214 may travel along the edges of the PVA membrane 1232 and therefore not reach the PVA membrane 1232 or the sponge 1234. Stated otherwise, with the sponge 1234 located in the middle of the fluid path 1231, the possibility exists for fluid to travel around the outside edges of the fluid flow path 1231, Page 116 of 164 12865804v1Atty. Docket No.: 2013065-0954 potentially missing the PVA membrane 1232 entirely, and delaying the bursting, puncturing and / or dissolving of the PVA membrane 1232.

[0644] FIG.23B is a schematic diagram illustrating a side view of a fluidic device 1236 including a fluid flow path 1231, a PVA membrane 1232 and two sponges 1234a, 1234b, according to aspects of the present embodiments. The right panel shows a fluid 1214 flowing through the fluid flow path 1231. In this example, (and in contrast to the example of FIG.23A) the sponge 1234a ensures the fluid 1214 is wicked toward the PVA membrane 1232, while sponge 1234b expands from fluid intake, and subsequently pushes on the PVA membrane 1232, thereby encouraging the PVA membrane to burst. Horizonal serpentine timer

[0645] In some embodiments, tilting of a fluidic device may impact the flow of a fluid timer through a fluidic serpentine timer. FIGs.24A-B show a fluidic device 1270 including a fluidic serpentine timer 1273, a timer fluid reservoir 1274 and a PVA valve 1276, according to aspects of the present embodiments. The serpentine timer 1273 includes a fluidic resistance element 1271 and a serpentine timer delay channel 1272. In this example, the fluidic resistance element 1271 and the serpentine timer delay channel 1272 extend longitudinally, and distally with respect to the timer fluid reservoir 1274. FIG.24A shows the fluidic device 1270 positioned at a 0 ° tilt (i.e., no tilt). In some embodiments, a timer fluid 1214 flows through the fluidic resistance element 1271 and the serpentine timer delay channel 1272, and reaches the PVA valve 1276 in a period of fifteen minutes and 30 seconds. FIG.24B shows the fluidic device 1270 positioned at a 15 ° tilt. In some embodiments, a timer fluid 1214 flows through the fluidic resistance element 1271 while capillary forces work against gravity. The timer fluid 1214, however, stalls in the serpentine timer delay channel 1272 as gravity dominates capillary forces thereby causing the timer fluid to not reach the valve 1276.

[0646] FIGs.25A-B show a fluidic device 1280 including a fluidic serpentine timer 1283, a timer fluid reservoir 1284 and a PVA valve 1286, according to aspects of the present embodiments. The serpentine timer 1283 includes a fluidic resistance element 1281 and a serpentine timer delay channel 1282. In this example, the serpentine timer delay channel 1282 extends around and in close proximity of the reservoir 1284, while the fluidic resistance Page 117 of 164 12865804v1Atty. Docket No.: 2013065-0954 element 1281 is located laterally with respect to the reservoir 1284. FIG.25A shows the fluidic device 1280 positioned at a 0 ° tilt (i.e., no tilt). In some embodiments, a timer fluid 1214 flows through the fluidic resistance element 1281 and the serpentine timer delay channel 1282, and reaches the PVA valve 1286 in a period of eighteen minutes and 30 seconds. FIG.25B shows the fluidic device 1280 positioned at a 15 ° tilt. In some embodiments, a timer fluid 1214 flows through the fluidic resistance element 1281 and the serpentine timer delay channel 1282, and reaches the PVA valve 1286 in a period of twenty minutes and 30 seconds. The serpentine timer delay channel 1282 in close proximity of the reservoir 1284 (i.e., origin of the liquid timer 1214) minimizes the effect of gravity, and therefore enables a consistent flow through the serpentine timer delay channel 1282. Therefore, by placing the serpentine timer delay channel 1282 in close proximity to (for example, extending around) the reservoir 1284, the fluidic device 1280 can accommodate tilt angles of about 15 degrees (for example, up to about 20 degrees) with only minimal impact on the timer fluid 1214 flow period (thereby enabling the timer fluid 1214 to still reach the PVA valve 1286).

[0647] In some embodiments, one or more surfactants are added to a timer fluid to help with bursting a PVA membrane while dissolving. Presence of a surfactant in a timer fluid, however, may impact the flow rate of the timer fluid through a fluidic serpentine timing circuit. In some embodiments, at a tilted angle, a timer fluid including high surfactant may cause a premature leaky flow. In some embodiments, high surfactant includes 0.25% Ecosurf. FIGs.26A-C show two fluidic devices 1280a, 1280b positioned at a 15 ° tilt, according to aspects of the present embodiments. In each case, the device on the left includes a low surfactant timer fluid 1290 while the device on the right includes a high surfactant timer fluid. In this example, the fluidic device 1280a includes a fluidic serpentine timer 1283a, a timer fluid reservoir 1284a and a timer fluid 1290. The fluidic device 1280b includes a fluidic serpentine timer 1283b, a timer fluid reservoir 1284b and a timer fluid 1292. The timer fluids 1290, 1292 may include a surfactant. In some embodiments, the timer fluid 1290 includes 0.1% Ecosurf (i.e., low surfactant) and the timer fluid 1292 (i.e., high surfactant) includes 0.5% Ecosurf. FIG.26A shows the two fluidic devices 1280a, 1280b where the timer fluids 1290 and 1292 have flowed from the timer fluid reservoirs 1284a, 1284b, through the fluidic resistance elements 1281a, 1281b, and started flowing through the serpentine timer delay channels 1282a, 1282b. In FIG.26B the timer fluid 1290 continues to flow through the serpentine timer delay channel 1282b. The timer fluid 1292, however, continues to flow Page 118 of 164 12865804v1Atty. Docket No.: 2013065-0954 slowly through the serpentine timer delay channel 1282b, while a separate, premature leaky flow 1294 starts to flow through the serpentine timer delay channel 1282b, ahead of the timer fluid 1292. In FIG.26C the timer fluid 1290 reaches the reservoir 1284. The timer fluid 1292 continues to flow slowly through the serpentine timer delay channel 1282b, while the leaky flow 1294 reaches the reservoir 1284b. Reaction Chamber Designs

[0648] Design of reaction chambers aims to achieve three goals: filling a metered volume of fluid rapidly; robustly pining a metered volume of fluid without mixing between chambers during drops, tilting, or other shocks; and draining chambers evenly and simultaneously (for example, within about 5 seconds of each other) upon opening a valve. FIGs.27A-D are schematic diagrams of various reaction chambers designs, according to aspects of the present embodiments. FIG.27A illustrates a reaction chamber design 1300 including a splitting inlet / separate outlet arrangement. The design 1300 includes two reaction chambers 1302a, 1302b, a splitting inlet 1304, two outlets 1306a, 1306b and a lateral flow chamber 1308. In some embodiments, the design 1300 results in an unequal draining, and may require simultaneous filling to prevent premature leaking of fluid to the lateral flow chamber 1308. The design 1300 may be more vulnerable to a drop shock. FIG.27B illustrates a reaction chamber design 1310 including a fully joined arrangement. The design 1310 includes two reaction chambers 1312a, 1312b, a merged inlet 1314, a merged outlet 1316 and a lateral flow chamber 1308. In some embodiments, the design 1300 results in unequal draining, and may require simultaneous filling to prevent premature leaking of fluid to the lateral flow chamber 1308. The design 1310 may include air vents to unpin a volume of fluid. In some embodiments, unpinning air vents are vulnerable to tilting or a drop shock. FIG.27C illustrates a reaction chambers design 1320 including a fully separate arrangement. The design 1320 includes two reaction chambers 1322a, 1322b, two inlets 1324a, 1324b, two outlets 1326a, 1326b and two lateral flow chamber 1328a, 1328b. The design 1320 requires multiple lateral flow strips and multiple PVA valves. In some embodiments, the two inlets 1324a, 1324b and the two outlets 1326a, 1326b are merged via channels 1325a, 1325b, respectively. This arrangement may further require air vents for unpinning a volume of fluid. FIG.27D illustrates a reaction chambers design 1360 including a splitting inlet / merging outlet arrangement. The design 1360 includes two reaction chambers 1362a, 1362b, a splitting inlet 1364, a merging outlet 1366, and a lateral flow chamber 1368. In this example, Page 119 of 164 12865804v1Atty. Docket No.: 2013065-0954 the merging outlet 1366 includes a merging Y-junction. In some embodiments, one of the reaction chambers 1362a, 1362b, may block or cap off the other at the merging Y-junction 1366, causing air to become trapped (i.e., forms a bubble) in the slower outlet channel, preventing flow of fluid out of that reaction chamber. In some embodiments, the design 1360 results in an unequal draining of fluid from the reaction chambers 1362a, 1362b, and may require simultaneous filling to prevent premature leaking of fluid to the lateral flow chamber 1368. In some embodiment, formation of an air gap may prevent one of the reaction chambers 1362a, 1362b from draining.

[0649] FIGs.28A-D are schematic diagrams of various modified reaction chambers designs, according to aspects of the present embodiments. In connection with the above- mentioned observations, various further embodiments are disclosed herein. FIG.28A illustrates a reaction chamber design 1330 including a mixing chamber. The design 1330 includes two reaction chambers 1332a, 1332b, a merged inlet 1334, a merged outlet 1336, a mixing chamber 1337 and a lateral flow chamber 1338. In this example, the fluid is transferred to the lateral flow chamber 1338 once the full volume of both reaction chambers 1332a, 1332b has drained into the mixing chamber 1337. In some embodiments, the design 1330 may be used in connection with disclosed observations pertaining to designs 1300 and 1310, further described in connection with FIGs.27A and 27B, respectively.

[0650] FIG.28B illustrates a reaction chamber design 1340 including unpinning air vents. The design 1340 includes two reaction chambers 1342a, 1342b, a merged inlet 1344, a merged outlet 1346, two unpinning air vents 1343a, 1343b and a lateral flow chamber 1348. In this example, the air vents 1343a, 1343b are made of superhydrophobic membranes. As a result, the design 1340 may allow the filling and draining of the reaction chambers 1342a, 1342b when the air vents 1343a, 1343b are in contact with a fluid. In some embodiments, the design 1340 may be used in connection with disclosed observation pertaining to designs 1310 and 1320, further described in connection with FIGs.27B and 27C, respectively.

[0651] FIG.28C illustrates a reaction chamber design 1350 that includes features that enable the siphoning of excess sample. The design 1350 includes two reaction chambers 1352a, 1352b, a splitting inlet 1354, two outlets 1356a, 1356b, a waste metering chamber 1355, and a lateral flow chamber 1358. The waste metering chamber 1358 may be a hydrophilic metering chamber, for example, a sponge. In some embodiments, the waste metering chamber 1358 drains fluid between the reaction chambers 1352a, 1352b and Page 120 of 164 12865804v1Atty. Docket No.: 2013065-0954 therefore isolates the reaction chambers 1352a, 1352b with air plugs for drop shock robustness. In some embodiments, the design 1350 may be used in connection with disclosed observations pertaining to designs 1300, further described in connection with FIGs.27A.

[0652] FIGs.29A-D are schematic diagrams of various unpinning air vent designs, according to aspects of the present embodiments. FIG.29A illustrates an air vent design 1370 including an outlet channel 1374. In this example, a reaction chamber 1372 is firmly pinned, such that a fluid remains in place during a drop or a rotation. The draining of fluid may be constrained by an unpinning force at the outlet channel 1374, and multiple chambers may not unpin sequentially. FIG.29B illustrates an air vent design 1380 including a vent chamber 1384. In this example, the filling and draining of a reaction chamber 1382 proceeds smoothly, and multiple reaction chambers may drain synchronously. The reaction chamber 1382 may be weakly pinned, resulting in a movement of a fluid during a drop or a rotation. FIG.29C illustrates an air vent design 1390 including a membrane 1394. In some embodiments, the membrane 1394 is made of a hydrophobic (e.g., superhydrophobic) material. The superhydrophobic membrane 1394 is permeable to air, but not to water. In this example, a reaction chamber 1392 is firmly pinned, such that a fluid remains in place during a drop or a rotation. FIG.29D illustrates an air vent design 1400 including an inlet channel 1404. In this example, a reaction chamber 1402 is firmly pinned, such that a fluid remains in place during a drop or a rotation. The draining of fluid may be constrained by an unpinning due to a lateral angle of the inlet channel 1404, and multiple chambers may not unpin sequentially.

[0653] One of the challenges of designing reaction chambers is a non-simultaneous draining of separate reaction chambers. In some embodiments, a mixer chamber may be added downstream of the reaction chambers. A mixer chamber enables mixing of reaction fluids from two or more reaction chambers, and only allows flow of the mixed reaction fluid to a lateral flow chamber, after all reaction chambers are completely drained. FIGs.30-33 are schematic diagrams of various mixer chamber designs, according to aspects of the present embodiments. FIG.30 illustrates a mixer chamber 1430 featuring a serpentine design. The mixer chamber 1430 includes two separate inlets 1432a, 1432b, partial walls 1436, and an outlet 1438. Two reaction fluids 1434a and 1434b may flow into the mixer chamber 1430 through the inlets 1432a and 1432b, respectively. In some embodiments, the partial walls 1336 may guide the reactions fluids 1434a, 1434b through two separate serpentine paths, such that each of the reaction fluids 1434a, 1434b flows along the respective corresponding Page 121 of 164 12865804v1Atty. Docket No.: 2013065-0954 path without spilling into the other path. In some embodiments, a sufficient pressure may allow the reactions liquids 1434a, 1434b to penetrate the partial walls 1436 and merge together.

[0654] FIG.31 illustrates a mixer chamber 1450 featuring an X-shape design. The mixer chamber 1450 includes two separate inlets 1452a, 1452b, partial walls 1456, and an outlet 1458. Two reaction fluids 1454a and 1454b may flow into the mixer chamber 1430 through the inlets 1452a and 1452b, respectively. In some embodiments, first the reaction fluid 1454a flows through the inlet 1452a and fills half of the mixer chamber 1450 (e.g., diagonally), while the partial wall 1456 holds the reaction fluid 1454a, preventing it from reaching the outlet 1458. Subsequently, the reaction fluid 1454b flows through the inlet 1452b and fills the other half of the mixer chamber 1458, resulting in the reaction fluid 1454a to overflow and reach the outlet 1458. In some embodiments, the reaction fluids 1454a, 1454b may not be thoroughly mixed while flowing through the outlet 1458. Addition of a serpentine mixer channel may induce mixing of the reaction fluids 1454a, 1454b.

[0655] FIG.32A illustrates a mixer chamber 1470 featuring a four-turn design. The mixer chamber 1470 includes two separate inlets 1472a, 1472b, partial walls 1476, and an outlet 1478. In some embodiments, the mixer chamber 1470 is fluidly connected to a lateral flow chamber 1480 via a serpentine mixer 1479. Two reaction fluids 1474a and 1474b may flow into the mixer chamber 1430 through the inlets 1472a and 1472b, respectively. FIGs. 32B-C show the reaction fluid 1474a flowing through the inlet 1472a and along the winding path of the partial walls 1476. FIGs.32D-E show the reaction fluid 1474b flowing through the inlet 1472b and along the winding path of the partial walls 1476, while no active mixing of the reaction fluids 1454a, 1454b occurs. In some embodiments, the reaction fluids 1454a, 1454b flow through the outlet 1458, and into the serpentine mixer channel 1479 to enable mixing of the reaction fluids 1454a, 1454b before reaching the lateral flow chamber 1480.

[0656] FIG.33A-B illustrate a mixer chamber 1490 featuring a merging burst-valve design. The mixer chamber 1490 includes two separate inlets 1492a, 1492b, and an outlet 1496. Two reaction fluids 1494a and 1494b may flow into the mixer chamber 1490 through the inlets 1492a and 1492b, respectively. As shown in FIG.33A, if one of the fluids (Liquid 1 in this case) releases first, air may be trapped between the two fluids thereby pinning the second fluid (Liquid 2 in this case) in place. As shown in FIG.33B, when the fluids release Page 122 of 164 12865804v1Atty. Docket No.: 2013065-0954 at the same time (i.e., within about 5 seconds of each other (i.e., within 4-6 second)), neither fluid is pinned and fluid mixing is encouraged.

[0657] FIGs.34A-C are schematic diagrams of a U-bend reaction chambers design 1500, according to the aspects of the present embodiments. FIG.34A illustrates a front view 1501 and a side view 1502 of the U-bend reaction chambers design 1500. In some embodiments, the design 1500 includes two reaction chambers 1504, two air vent membranes 1506, a sample inlet reservoir 1508, an inlet 1510, and two outlets 1512. In this example, the inlet 1510 is a splitting inlet, which splits into two side inlets 1511. In some embodiments, the reaction chambers 1504 may each include one or more ridge channels 1505. The ridge channels 1505 may draw a fluid sample 1507 to the bottom of reaction chambers 1504, to ensure that the fluid 1507 reaches the outlets 1512 before filling the reaction chambers 1504. In FIG.34B, the fluid 1507 is flowing out of the reservoir 1508, through the inlet 1510, further through side inlets 1511 and flowing into the reaction chambers 1504, where the ridge channels 1505 draw the fluid 1507 to the bottom of reaction chambers 1504. In FIG.34C, the fluid 1507 has filled the reaction chambers 1504, and is flowing through outlets 1512.

[0658] FIG.35 are schematic diagrams of a reaction chambers design 1670 including a splitting outlet arrangement, according to the aspects of the present embodiments. FIG.35 illustrates a front view 1671 and a bottom view 1672 of the design 1670. The design 1670 includes two reaction chambers 1674, two inlets 1676, and two side outlets 1678 and a splitting outlet 1680. In this example, the splitting outlet 1680 includes a T-junction. A T- junction may prevent formation of an air gap within the two side outlets 1678. In some embodiments, the design 1670 may be used in connection with disclosed observations pertaining to the designs 1360, further described in connection with FIG. 27D.

[0659] FIGs.36A-C are schematic diagrams of various reaction chamber inlet designs, according to aspects of the present embodiments. FIG.36A illustrates a reaction chamber inlet design 1520. In some embodiments, the design 1520 includes reaction chambers 1522, a bottom face of inlet 1526, through-holes 1528, and a top face of inlet 1524. The design 1520 may enable fast simultaneous filling of the reaction chambers 1522. In some embodiments, the design 1520 may result in bubble migration between the two reaction chambers (i.e., bubble crossover). FIG.36B illustrates a reaction chamber inlet design 1530. In some embodiments, the design 1530 includes reaction chambers 1532, a bottom face of inlet 1536, through-holes 1538, and a top face of inlet 1534. The design 1530 may prevent Page 123 of 164 12865804v1Atty. Docket No.: 2013065-0954 bubble crossover. In some embodiments, the design 1530 may result in a slow simultaneous filling of the reaction chambers 1532. FIG.36C illustrates a reaction chamber inlet design 1540. In some emb...

Claims

Atty. Docket No.: 2013065-0954 CLAIMS What is claimed:

1. A device comprising a device body comprising a top surface and a bottom surface, the device comprising: a first fluid inlet port disposed in the top surface, the first fluid inlet port for receiving a fluid sample; a second fluid inlet port disposed in the top surface, the second fluid inlet port for receiving a timer fluid; at least one reaction chamber disposed in the top surface and coupled fluidly downstream of the first fluid inlet port for receiving the first fluid sample; a fluidic timing circuit disposed within the device body, the fluidic timing circuit coupled downstream of the second fluid inlet and for receiving the timer fluid from the second fluid inlet; and a release valve disposed fluidly downstream of the fluidic timing circuit for receiving the timer fluid from the fluidic timing circuit.

2. The device of claim 1, wherein the release valve is coupled fluidly downstream of the at least one reaction chamber.

3. The device of claim 2, further comprising a diagnostic area fluidly coupled downstream of the at least one reaction chamber and upstream of the release valve.

4. The device of claim 3, wherein the release valve comprises a dissolvable membrane comprising polyvinyl acid (PVA) fluidly sealing the diagnostic area from atmosphere.

5. The device of claim 4, wherein the dissolvable membrane, when contacted by the timer fluid, dissolves and fluidly couples diagnostic area to atmosphere, thereby allowing the fluid sample to flow into the diagnostic area.

6. The device of claim 1, wherein the device body is substantially planar and oriented in a horizontal plane when in use. Page 137 of 164 12865804v1Atty. Docket No.: 2013065-0954 7. The device of claim 1, wherein the fluid sample is a biological sample, and wherein the diagnostic area comprises a lateral flow chamber sized to contain at least one visible readout strip therewithin, the visible readout strip comprising at least one of a lateral flow strip (LFS) and a colorimetric reaction test strip.

8. The device of claim 1, wherein the device body comprises a continuous, single-piece body formed via 3-D printing, compression molding, or injection molding.

9. The device of claim 1, further comprising multiple features disposed within the bottom surface of the device body such that the multiple features are recessed and do not protrude beyond a plane defining the bottom surface of the device body.

10. The device of claim 9, wherein the multiple features comprise two or more of: a fluidic resistance element, a serpentine timer delay channel, a through hole, a vent channel, a lateral flow chamber, opposing side channels, a valve manifold or recess, a dead space or lacuna, and a fluid outlet channel.

11. The device of claim 7, wherein the lateral flow strip (LFS) comprises a carbon black conjugate and biotin on a first side and an oligo pulldown sequence on a second side.

12. The device of claim 1, wherein the fluidic timing circuit comprises a first plurality of serpentine channels and a second plurality of serpentine channels, wherein the first plurality of serpentine channels is fluidly coupled upstream of the second plurality of serpentine channels, wherein the first plurality of channels acts as a fluidic resistance element, and wherein the second plurality of channels acts as a serpentine timer delay channel.

13. The device of claim 12, wherein each channel of the first plurality of channels comprises a smaller internal diameter than each channel of the second plurality of channels.

14. The device of claim 12, wherein each of the first plurality of channels and the second plurality of channels comprises multiple 90-degree turns and multiple 180-degree turns. Page 138 of 164 12865804v1Atty. Docket No.: 2013065-0954 15. The device of claim 9, further comprising a bottom layer adhered to the bottom surface of the device body, wherein the bottom layer fluidly seals the multiple features disposed in the bottom surface of the device body.

16. The device of claim 15, wherein the bottom layer forms a bottom planar surface of the device devoid of any protrusions or recesses.

17. The device of claim 1, wherein each of the first inlet port, the second inlet port, and the at least one reaction chamber protrudes from the top surface of the device body.

18. The device of claim 1, further comprising a manifold housing the release valve, wherein the manifold housing the release valve protrudes from the top surface of the device body.

19. The device of claim 12, wherein the second plurality of channels protrudes from the top surface of the device body, and wherein the first plurality of channels is contained within a thickness of the device body such that it does not protrude from the top surface of the device body.

20. The device of claim 7, wherein the lateral flow chamber protrudes from the top surface of the device body.

21. The device of claim 1, comprising a lid sized and shaped to be placed over the top surface of the device body.

22. The device of claim 21, wherein the lid comprises a puncturable, at-least partially flexible container or reservoir containing timer fluid located on an underside of the lid at a location corresponding to the second sample inlet when the lid is placed over the top surface of the device body. Page 139 of 164 12865804v1Atty. Docket No.: 2013065-0954 23. The device of claim 22, further comprising at least one puncture feature disposed within, and protruding from, the second inlet port such that the puncture feature punctures the puncturable, flexible container or reservoir when the lid is placed over the top surface of the device body, thereby causing the timer fluid to flow into the second inlet port.

24. The device of claim 23, wherein the at least one puncture feature comprises at least one thread.

25. The device of claim 22, wherein the device further comprises: an O-ring disposed within the second inlet port for sealing the flexible container or reservoir to the second inlet port when the lid is placed on top of the device body, and / or a flexible sealing element integrated into the lid, wherein the flexible sealing element comprises an elastomer material over-molded onto an internal substrate or surface of the lid.

26. The device of claim 1, wherein the at least one reaction chamber comprises two reaction chambers.

27. The device of claim 26, wherein the device comprises separate inlets coupling the first inlet port to each of the two reaction chambers, and wherein the device comprises a separate fluid outlet coupled to each of the two reaction chambers.

28. The device of claim 26, wherein the device comprises a common inlet from the first inlet port that splits into two separate lines each coupled to one of the two reaction chambers, and wherein the device comprises a separate fluid outlet from each of the two reaction chambers that merge together downstream of the reaction chambers and upstream of the diagnostic area.

29. The device of claim 1, further comprising a pre-treatment chamber disposed fluidly upstream of the reaction chamber and downstream of the first inlet port, the pre-treatment chamber comprising at least one bead. Page 140 of 164 12865804v1Atty. Docket No.: 2013065-0954 30. The device of claim 1, further comprising: a valve manifold comprising at least one recess in which the release valve is disposed, the release valve comprising a PVA membrane; and at least one sponge disposed within the valve manifold positioned in the vicinity of the release valve to hydrate the PVA membrane with timer fluid.

31. The device of claim 1, comprising a common outlet line coupling the at least one reaction chamber to the diagnostic area, and wherein the common outlet line comprises at least one flow feature.

32. The device of claim 31, wherein the at least one flow feature comprises one or more diagonal hatch lines to help promote flow mixing of sample fluid from the at least one reaction chamber before reaching the diagnostic area.

33. The device of claim 1, wherein an inlet portion of the diagnostic area comprises one or more pins protruding into a flow path of the diagnostic area to promote capillary action.

34. The system of claim 1, wherein the at least one reaction chamber is coupled to the second flow channel via at least one outlet channel.

35. The system of claim 34, wherein the at least one outlet channel comprises a straight channel.

36. The system of claim 34, wherein the at least one outlet channel comprises a serpentine channel.

37. The system of claim 34, wherein the at least one outlet channel comprises a serpentine channel comprising a plurality of kite-line expansions.

38. The system of claim 34, wherein the at least one outlet channel comprises a serpentine channel comprising a plurality of diagonal hatch lines. Page 141 of 164 12865804v1Atty. Docket No.: 2013065-0954 39. The system of claim 1, wherein the second flow channel comprises a plurality of fluid pins.

40. A device comprising: a sample inlet port; at least one reaction chamber disposed fluidly downstream of the sample inlet port; a diagnostic area disposed downstream of the at least one reaction chamber, the diagnostic area comprising a vent hole at a downstream end; and a manual venting feature for opening the vent hole to atmosphere, wherein manipulating the manual venting feature such that the vent hole is open to atmosphere causes sample fluid contained in the at least one reaction chamber to flow into the diagnostic area.

41. The device of claim 40, wherein the manual venting feature comprises an adhesive pull tab.

42. The device of claim 40, wherein the manual venting feature comprises two adhesive pull tabs comprising: a first adhesive pull tab, removal of which vents the at least one reaction chamber to atmosphere and causes sample fluid to flow from the sample inlet port into the at least one reaction chamber; and a second adhesive pull tab covering the vent hole, removal of which vents the diagnostic area to atmosphere and causes sample fluid to flow from the at least one reaction chamber into the diagnostic area.

43. A method of using a device comprising: providing a device, the device comprising: a sample inlet port; at least one reaction chamber disposed fluidly downstream of the sample inlet port; the at least one reaction chamber comprising a first vent hole on a side; a diagnostic area disposed downstream of the at least one reaction chamber, the diagnostic area comprising a second vent hole at a downstream end; a first adhesive pull tab covering the first vent hole; and Page 142 of 164 12865804v1Atty. Docket No.: 2013065-0954 a second adhesive pull tab covering the vent hole; providing a fluid sample to be disposed within the sample inlet port; removing the first adhesive pull tab, venting the at least one reaction chamber to atmosphere and causing the sample fluid to flow from the sample inlet port into the at least one reaction chamber; and removing the second adhesive pull tab, venting the diagnostic area to atmosphere and causing sample fluid to flow from the at least one reaction chamber into the diagnostic area.

44. The method of claim 43, where removing the first adhesive pull tab further comprises allowing the lapse of a pre-determined amount of time.

45. A method of using a device comprising: providing a device body, the device body comprising: a first fluid inlet port disposed in a top surface; a second fluid inlet port disposed in the top surface; at least one reaction chamber disposed in the top surface and coupled fluidly downstream of the first fluid inlet port; a fluidic timing circuit disposed within the device body, the fluidic timing circuit coupled downstream of the second fluid inlet; and a release valve disposed fluidly downstream of the fluidic timing circuit; a diagnostic area fluidly coupled downstream of the at least one reaction chamber and upstream of the release valve; providing a fluid sample to be disposed within the first fluid inlet port; and providing a fluid timer to be disposed within the second fluid inlet port; wherein the release valve, when contacted by the timer fluid, dissolves and fluidly couples the at least one reaction chamber to atmosphere, thereby allowing the fluid sample to flow into the diagnostic area.

46. The method of claim 45, wherein providing the fluid comprises providing a lid comprising a puncturable reservoir containing the timer fluid located on an underside of the lid at a location corresponding to the second fluid inlet port when the lid is placed over the top surface of the device body, and Page 143 of 164 12865804v1Atty. Docket No.: 2013065-0954 wherein the device body further comprises at least one puncture feature disposed within the second inlet port such that the puncture feature punctures the puncturable reservoir when the lid is placed over the top surface of the device body, thereby causing the timer fluid to flow into the second inlet port.

47. The system of claim 157, comprising a reagent bottle for collecting the biological sample and eluting the biological sample therein thereby creating the fluid sample prior to introduction of the fluid sample to the sample inlet port.

48. The system of claim 157 or 47, further comprising at least one filter for filtering the biological sample.

49. The system of claim 48, wherein the at least one filter is disposed within the sample inlet port and / or the reagent bottle.

50. The device of claim 1, wherein the first fluid inlet port is positioned at a higher vertical height than the at least one reaction chamber when the device is in use.

51. The device of claim 22, wherein the puncturable, at-least partially flexible container or reservoir comprises a pierceable membrane or film.

52. The system of claim 116, wherein the piercing feature comprises at least one of a needle, pointed protrusion, and a knife-like protrusion.

53. A diagnostic method comprising: collecting a biological sample; providing a device as described herein; adding one or more drops of a sample fluid containing the biological sample to a sample inlet port of the device; adding timer fluid to a timer fluid inlet port of the device; waiting for a pre-specified period of time; and reading results of the diagnostic method via a diagnostic area of the device. Page 144 of 164 12865804v1Atty. Docket No.: 2013065-0954 54. The method of claim 53, wherein adding timer fluid to the timer fluid inlet port comprises placing a lid containing the timer fluid on top of the device.

55. The method of claim 53, comprising filtering the sample fluid via at least one filter prior to adding one or more drops of the sample fluid to the sample inlet port of the device, wherein the at least one filter is disposed within the sample inlet port and / or the reagent bottle.

56. The method of claim 53, wherein the pre-specified period of time comprises a time period in a range from about 5 minutes to about 30 minutes.

57. A system comprising: a device body comprising: a sample inlet port for receiving a sample; a first flow channel fluidly coupled to the sample inlet port at an upstream end of the first flow channel, the first flow channel extending to a downstream end; at least one reaction chamber fluidly coupled to the first flow channel; a passively actuated valve disposed at the downstream end of the first flow channel; and a second flow channel disposed downstream of the first flow channel, wherein the passively actuated valve, when opened, is configured to allow flow of the sample from the first flow channel to the second flow channel.

58. The system of claim 57, further comprising: at least one lyophilized reaction bead contained within the at least one reaction chamber.

59. The system of claim 57, wherein the at least one reaction chamber is coupled to the first flow channel between the upstream end and the downstream end via at least one side channel.

60. The system of claim 57, wherein the second flow channel is disposed downstream of the passively actuated valve, and Page 145 of 164 12865804v1Atty. Docket No.: 2013065-0954 wherein the passively actuated valve fluidly couples the first flow channel to the second flow channel.

61. The system of claim 57, wherein the passively actuated valve is disposed downstream of the second flow channel, and wherein the second flow channel fluidly couples the first flow channel to the passively actuated valve.

62. The system of claim 57, wherein the at least one reaction chamber is fluidly coupled to the first flow channel downstream of the sample inlet port via at least one side channel.

63. The system of claim 57, wherein the passively actuated valve comprises a soluble membrane.

64. The system of claim 63, wherein the soluble membrane comprises at least one of polyvinyl alcohol (PVA) and a hydrogel.

65. The system of claims 63-64, wherein the soluble membrane dissolves upon contact with a fluid.

66. The system of any of claims 57-65, further comprising: air contained within the second flow channel; and sample contained within the reaction chamber.

67. The system of claim 66, further comprising air contained within the first flow channel downstream of the reaction chamber.

68. The system of claim 66, wherein air in at least one of the first flow channel and the second flow channel prevents a biological solution containing the sample from flowing into the second flow channel.

69. The system of claim 68, wherein, upon dissolution of the soluble membrane, the passively actuated valve opens and at least a portion of the air in the second flow channel Page 146 of 164 12865804v1Atty. Docket No.: 2013065-0954 exits the second flow channel, thereby allowing the biological solution to flow into the second flow channel.

70. The system of claim 69, wherein the biological solution flows into the second flow channel via gravity feed.

71. The system of any of claims 57-70, wherein at least a portion of the at least one reaction chamber is disposed at a lower vertical location than that of at least a portion of the sample inlet port when the device body is in an upright position, thereby allowing the biological solution to flow from the sample inlet port into the at least one reaction chamber.

72. The system of claim 71, wherein the biological solution flows from the sample inlet port to the reaction chamber via gravity and via the first flow channel.

73. The system of any of claims 57-72, wherein the biological solution comprises at least one active nucleotide.

74. The system of any of claims 57-73, wherein the at least one reaction chamber comprises multiple reaction chambers.

75. The system of claim 74, wherein the multiple reaction chambers comprise two reaction chambers comprising a first reaction chamber and a second reaction chamber.

76. The system of claim 75, wherein the first reaction chamber is located on a first side of the first flow channel and the second reaction chamber is disposed on a second side of the first flow channel.

77. The system of claims 64-70, wherein the soluble membrane dissolves upon contact with water molecules in an aqueous solution.

78. The system of claim 77, wherein hydroxyl groups of the PVA interact with the water molecules, thereby forming hydrogen bonds. Page 147 of 164 12865804v1Atty. Docket No.: 2013065-0954 79. The system of claims 77-78, wherein the PVA comprises at least one of a degree of hydrolysis in a range from about 60% mol hydrolysis to about 99% hydrolysis and a molecular weight in a range from about 26,000 to about 200,000, thereby producing enhanced solubility of the PVA with the water molecules.

80. The system of claim 79, wherein the PVA comprises a degree of hydrolysis in a range from about 65% mol hydrolysis to about 85% mol hydrolysis.

81. The system of claim 80, wherein the PVA comprises a degree of hydrolysis in a range from about 72% mol hydrolysis to about 78% mol hydrolysis.

82. The system of claims 77-81, wherein the PVA comprises a viscosity in a range from about 4 mPas to about 12 mPas.

83. The system of claims 77-82, wherein the PVA comprises a molecular weight in a range from about 5,000 to about 200,000.

84. The system of any of claims 57-83, wherein the lyophilized reaction beads, upon contact with a biological solution containing the sample cause amplification to occur on at least one active component of the sample.

85. The system of claim 84, wherein amplification occurs for a predetermined period of time.

86. The system of claim 85, wherein the predetermined period of time concludes when the passively actuated valve opens; and wherein the predetermined period of time comprises a period from about 5minutes to about 30 minutes.

87. The system of any of claims 57-86, further comprising: a third flow channel fluidly coupled to the passively actuated valve; and at least one air diffusion component fluidly coupled to the third flow channel downstream of the passively actuated valve. Page 148 of 164 12865804v1Atty. Docket No.: 2013065-0954 88. The system of claim 87, wherein the at least one air diffusion component allows air to flow therethrough, from the third flow channel, at a controlled rate.

89. The system of claims 87-88, wherein the at least one air diffusion component is composed of a material with an average pore diameter of about 0.025 microns (i.e., for example, in a range from about 0.01 microns to about 0.05, or from about 0.015 microns to about 0.04, from about 0.015 microns to about 0.035, or from about 0.02 microns to about 0.03 microns).

90. The system of claims 87-89, wherein the at least one air diffusion component comprises a bulk density in a range from about 62% to about 82% (i.e., from about 67% to about 77%, i.e., from about 70% to about 75%).

91. The system of claims 87-90, wherein the at least one air diffusion component is composed of nitrocellulose (i.e., pyroxylin, i.e., cellulose nitrate, i.e., a mixture of nitric esters of cellulose).

92. The system of claims 87-91, wherein flow of air through the at least one air diffusion component enables air to be evacuated from the first flow channel, thereby enabling flow of the biological solution into and through at least one of the passively actuated valve and second flow channel.

93. The system of claim 89-92, wherein the average pore diameter restricts the flow of air through the at least one air diffusion component in order to slow down the flow of biological solution into and through the passively actuated valve and / or the second flow channel, thereby allowing the biological solution to remain in the at least one reaction chamber for a predetermined period of time.

94. The system of claims 87-93, wherein the at least one air diffusion component comprises a thin layer of the material adhered to the surface of the device such that air diffuses from the third flow channel through the thin layer of the material and out at external edges of the thin layer of the material. Page 149 of 164 12865804v1Atty. Docket No.: 2013065-0954 95. The system of claim 94, wherein the at least one air diffusion component comprises a porous membrane sandwiched between a doubled-sided adhesive on one side and a single- sided adhesive on the other side.

96. The system of claims 57-86, further comprising a continuous flow path fluidly coupled to the passively actuated valve.

97. The system of claim 96, wherein the continuous flow path comprises at least one of a sufficient distance and a sufficient resistance to flow to enable fluid (i.e., timing fluid) flowing within the continuous flow path to remain flowing therethrough for at least a pre- determined amount of time.

98. The system of claim 97, wherein the pre-determined amount of time comprises from about 5 to about 30 minutes.

99. The system of claim 96, wherein the continuous flow path comprises at least one serpentine flow path comprising at least one turn.

100. The system of claim 99, wherein the at least one turn comprises a 180-degree turn.

101. The system of claims 99-100, wherein the at least one serpentine flow path comprises multiple turns, at least one turn of the multiple turns comprising a 180-degree turn, thereby resulting in multiple, substantially parallel passes (or portions, or channels) of the at least one serpentine flow path.

102. The system of claims 99-101, wherein the continuous fluid path comprises: a first serpentine flow path; and a second serpentine flow path downstream of the first serpentine flow path.

103. The system of claim 102, wherein the first serpentine flow path comprises a first flow path nominal diameter, wherein the second serpentine flow path comprises a second flow path nominal diameter, and wherein the second nominal diameter is larger than the first nominal diameter. Page 150 of 164 12865804v1Atty. Docket No.: 2013065-0954 104. The system of claim 103, wherein the first flow path nominal diameter (or channel height / width) is in a range from about 2 microns to about 500 microns.

105. The system of claim 104, wherein the second flow path nominal diameter (or channel height / width) is in a range from about 1 mm to about 10 mm.

106. The system of claims 57-86 and 96-105 further comprising a timing fluid inlet port.

107. The system of claim 106, wherein the timing fluid inlet port is fluidly coupled upstream of the continuous flow path and configured to allow a fluid to gravity flow into the continuous flow path.

108. The system of any of claims 57-107, comprising at least one lateral flow strip (LFS) fluidly coupled downstream or upstream of the passively actuated valve.

109. The system of any of claims 57-108, wherein the second flow channel comprises the lateral flow strip.

110. The system of claims 57-86 and 96-109, comprising a timing fluid contained within the timing fluid inlet port.

111. The system of claim 110, wherein the timing fluid does not comprise a sample.

112. The system of claim 110 or 111, wherein the timing fluid is different than the sample.

113. The system of claims 106-112, wherein the timing fluid inlet port is fluidly uncoupled from the second flow channel when the passively actuated valve is closed, and wherein the timing fluid inlet port is fluidly coupled to the second flow channel when the passively actuated valve is opened.

114. The system of claims 110-113, wherein, when the timing fluid contacts the passively actuated valve, it causes the passively actuated valve to open. Page 151 of 164 12865804v1Atty. Docket No.: 2013065-0954 115. The system of claim 114, wherein the timing fluid causes the passively actuated valve to open by dissolving the passively actuated valve.

116. The system of claim 114, wherein the timing fluid causes the passively actuated valve to open by being absorbed into a wicking layer that expands and forces the soluble membrane against a piercing feature, thereby causing the soluble membrane to be punctured.

117. The system of any of claims 57-116, wherein the second flow channel comprises or connects to a lateral flow strip (LFS) used for testing the sample for the presence of one or more target nucleic acids.

118. The system of any of claims 57-117, further comprising a waste shunt coupled to the sample inlet port upstream of the first flow channel, the waste shunt comprising: a fourth flow channel connecting to the sample inlet port; a U-bend connecting to a downstream end of the fourth flow channel; and a vent disposed at the downstream end of the fourth flow channel.

119. The system of claim 118, wherein the waste shunt prevents overfilling of the system.

120. The system of any of claims 57-119, wherein the system further comprises: at least one bulb disposed adjacent to the at least one reaction chamber; and a vertical fill line fluidly connected to a bottom portion of the at least one reaction chamber.

121. The system of claim 120, comprising at least one side channel, wherein an upstream end of the vertical fill line is fluidly coupled to a downstream end of the at least one side channel, and wherein a downstream end of the vertical fill line is fluidly coupled to the bottom of portion of the at least one reaction chamber.

122. The system of claim 121, wherein the vertical fill line is at least partially oriented in a vertical direction.

123. The system of claim 122, wherein the vertical fill line is oriented vertically. Page 152 of 164 12865804v1Atty. Docket No.: 2013065-0954 124. The system of claim 122, wherein the vertical fill line is oriented at an angle from a vertical direction (or at an angle from a longitudinal dimension of the device body).

125. The system of claims 120-124, wherein the at least one bulb comprises a substantially spherical chamber.

126. The system of claims 120-125, wherein the at least one bulb is fluidly connected to the at least one reaction chamber via at least one of a ridge feature and a permeable membrane disposed in a top portion of the at least one reaction chamber.

127. The system of claims 120-126, wherein the bottom portion of the at least one reaction chamber comprises a bottom 50% of the at least one reaction chamber, and wherein the top portion of the at least one reaction chamber comprises the top 50% of the at least one reaction chamber.

128. The system of claims 126-127, wherein the ridge feature (in connection with surface tension) and / or the permeable membrane allows air to pass through, but prevents or restricts liquid from passing through.

129. The system of claims 120-128, wherein the vertical fill line enables the at least one reaction chamber to be filled with the biological solution (i.e., a solution containing the sample) from the bottom up.

130. The system of claims 120-129, wherein the vertical fill line enables biological solution to flow vertically in order to flow into the at least one reaction chamber.

131. The system of claims 120-130, wherein the at least one bulb enables gas (i.e., air) disposed within the at least one reaction chamber to exit the system.

132. The system of claims 126-131, wherein each of the at least one bulb and the at least one reaction chamber is substantially spherical; wherein an interface between the at least one bulb and the at least one reaction chamber comprises a substantially circular opening; and wherein the permeable membrane spans the substantially circular opening, or Page 153 of 164 12865804v1Atty. Docket No.: 2013065-0954 wherein the ridge feature extends around the substantially circular opening.

133. The system of claims 126-131, wherein each of the at least one bulb and the at least one reaction chamber is substantially cylindrical; wherein each of the cylinders defining the respective at least one bulb and the at least one reaction chamber comprises a larger diameter than height; wherein an interface between the at least one bulb and the at least one reaction chamber comprises a substantially rectangular opening; and wherein the permeable membrane spans the substantially rectangular opening, or wherein the ridge feature extends around the substantially rectangular opening.

134. The system of claim 58, wherein the soluble membrane covers (or spans, or extends across) a downstream end of the first flow channel.

135. The system of claim 134, wherein the passively actuated valve comprises: a hub defining a downstream end of the first flow channel, the soluble membrane attached (or adhered) to the hub; and a wicking layer disposed between the hub and the soluble membrane.

136. The system of claim 134 or 135, further comprising a piercing feature for puncturing the soluble membrane.

137. The system of claim 136, wherein the wicking layer comprises a hole disposed therethrough.

138. The system of claim 137, wherein the hole is disposed concentrically within the center of the wicking layer, with, in a closed position of the passively actuated valve, the soluble membrane disposed (or laid) thereover.

139. The system of claim 138, wherein the needle is positioned such that, in a closed position of the passively actuated valve, a tip of the needle is centered approximately over the center of the hole a distance from the soluble membrane on an opposite side of the soluble membrane from wicking layer. Page 154 of 164 12865804v1Atty. Docket No.: 2013065-0954 140. The system of claims 136-139, wherein the wicking layer expands as it absorbs the biological solution, thereby pushing the soluble membrane into the needle allowing the needle to puncture the soluble membrane and opening the passively actuated valve.

141. The system of any of claims 57-140, wherein the system is power free.

142. The system of any of claims 57-141, wherein the system comprises no external or internal power source.

143. The system of claim 142, wherein the device body is configured such that each of the first channel and the second channel are oriented in a substantially vertical direction, wherein gravity causes the biological solution to flow vertically downward through the first flow channel, and wherein capillary action causes the biological solution to flow vertically upward through the second flow channel.

144. The system of any of claims 57-143, wherein the system comprises no moving parts.

145. The system of any of claims 57-144, wherein the device body is 3D printed of a transparent material.

146. The system of claim 145, wherein the transparent material comprises at least one of PLA (polylactic acid), PLA+ (i.e., PLA plus at least one of an impact modifier (i.e., methacrylate butadiene styrene (MBS) terpolymer, acrylate polymethacrylate copolymer (acrylic), chlorinated polyethylene (CPE), ethylene vinyl acetate copolymer (EVA), acrylonitrile butadiene styrene terpolymer (ABS), etc., a flexibilizing agent (i.e., a curing agent), and a plasticizer (i.e., phthalate esters, ethanolamine, caproic acid, lauric acid and glycerol triacetate (triacetin)), and PETG (polyethylene terephthalate glycol).

147. The system of any of claims 57-146, wherein the device body is compression molded, injection molded, or machined from a transparent material. Page 155 of 164 12865804v1Atty. Docket No.: 2013065-0954 148. The system of claim 147, where the transparent material comprises at least one of polycarbonate, acrylic, nylon, poly(methyl methacrylate) (PMMA), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polycarbonate (PC), polypropylene (PP), acrylonitrile butadiene styrene (ABS), and polystyrene (PS).

149. The system of any of claims 57-148, wherein the biological solution comprises a surfactant or a detergent.

150. The system of claim 149, wherein the biological solution comprises surfactant on a weight percent basis in a range from about 0.01% to about 1.0%.

151. The system of claims 149 and / or 150, wherein the surfactant comprises at least one of alcohol ethoxylate, polysorbate 20, sodium lauryl sulfate, sodium dodecyl sulfate, lithium dodecyl sulfate, and sodium laureth sulfate.

152. The system of any of claims 57-151, wherein the at least one reaction chamber comprises a first reaction chamber and a second reaction chamber, wherein the second flow channel comprises or connects to a lateral flow strip (LFS) used for testing the sample for the presence of one or more conditions, wherein the at least one lyophilized reaction bead comprises a first bead contained within the first reaction chamber and a second bead contained within the second reaction chamber, and wherein the first bead is configured for a first condition of the one or more conditions and the second bead is configured for a second condition of the one or more conditions.

153. The system of claim 152, wherein the lateral flow strip (LFS) comprises multiple capture regions configured to provide indication of the presence of each of the first condition and the second condition in the biological solution.

154. The system of claims 152-153, wherein the first condition is gonorrhea and the second condition is chlamydia.

155. The system of claim 108, wherein the device body is composed of a transparent material, Page 156 of 164 12865804v1Atty. Docket No.: 2013065-0954 wherein the lateral flow strip is disposed internally between a front surface of the device body and a rear surface of the device body, and wherein the lateral flow strip is configured to provide at least one indication that is visible from a perspective external to the device body, the indication indicating the result of at least one of a biological assay and a chemical assay.

156. The system of any of claims 57-155, wherein the device body comprises a continuous, 3-dimensional body with voids disposed therein, the voids composing at least a first void defining the first flow channel, a second void or voids defining the at least one reaction chamber into which the at least one lyophilized bead can be inserted, and a third void into which a lateral flow strip (LFS) can be inserted.

157. A system comprising: a device body comprising: a sample inlet port for receiving a fluid sample; a first flow channel fluidly coupled to the sample inlet port at an upstream end of the first flow channel, the first flow channel extending to a downstream end; at least one reaction chamber fluidly coupled to the first flow channel; a passively actuated valve disposed at the downstream end of the first flow channel; and a second flow channel disposed downstream of the first flow channel, wherein the passively actuated valve, when opened, is configured to allow flow of the fluid sample from the first flow channel to the second flow channel.

158. The system of claim 138, wherein the passively actuated valve comprises at least one of a soluble membrane and a puncturable membrane.

159. The system of claims 138-139, wherein the second flow channel comprises a lateral flow strip.

160. A method of tuning a fluidic timer comprising: providing a device body of the fluidic timer, the device body comprising: a fluid inlet port; Page 157 of 164 12865804v1Atty. Docket No.: 2013065-0954 a first fluid channel fluidly connected downstream of the fluid inlet port, the first fluid channel comprising an upstream end and a downstream end; and a passively actuated valve disposed downstream of the first fluid channel and coupled (directly or indirectly) thereto; providing a fluid sample to be disposed within the fluid inlet port; and adjusting at least one parameter of the device body and / or the fluid sample such that the fluid sample flows from the fluid inlet port to the downstream end of the first fluid channel for a duration of time to match or exceed a pre-determined amount of time.

161. The method of claim 160, wherein adjusting at least one parameter of the device body and / or the fluid sample comprises at least one of: adjusting a length of the first fluid channel; adjusting an internal dimension (i.e., a diameter) of the first fluid channel; adjusting a weight percent of surfactant in the fluid sample in a range from about 0.01% to about 10%; adjusting a length of an air diffusion device disposed downstream of the downstream end of the first flow channel; and adjusting at least one of a length and an internal dimension of a continuous flow path fluidly connected to the passively actuated valve.

162. The method of claims 160 or 161, wherein the pre-determined amount of time comprises a period from about 1 minute to about 30 minutes (i.e., from about 5 minutes to about 25 minutes, i.e., from about 5 minutes to about 20 minutes, i.e., from about 7 minutes to about 20 minutes, i.e., from about 10 minutes to about 20 minutes).

163. A fluidic device, comprising: a device body arranged substantially vertically; a sample fluid input reservoir disposed at a top end of the device body; a sample flow channel fluidly connected to the sample fluid input reservoir; at least one reaction chamber fluidly connected to the sample flow channel; an outlet fluidly connected to the sample flow channel; a fluidic valve fluidly connected to and controlling flow through the outlet; and Page 158 of 164 12865804v1Atty. Docket No.: 2013065-0954 a fluidic timing element configured to control a duration of time for the sample fluid to remain in the at least one reaction chamber and in the sample flow channel before opening the fluidic valve and causing the sample fluid to flow out of the outlet, wherein operation of the fluidic device to perform a reaction in the at least one reaction chamber does not use electrical power.

164. The fluidic device of claim 163, wherein each reaction chamber of the at least one reaction chamber is fluidly connected to the sample flow channel via a side channel.

165. The fluidic device of claim 163, wherein each reaction chamber of the at least one reaction chamber is fluidly connected to an air vent.

166. The fluidic device of claim 165, wherein the air vent comprises an air pinning chamber adjacent to the reaction chamber.

167. The fluidic device of claim 163, wherein each of the at least one reaction chambers is configured to contain reagents for performing a reaction.

168. The fluidic device of claim 167, wherein the device is configured to contain reagents in a lyophilized reaction bead configured to be rehydrated by the sample fluid.

169. The fluidic device of claim 163, wherein the fluidic timing element comprises at least one of a slow air diffusion timer and a serpentine fluidic timer.

170. The fluidic device of claim 169, wherein the slow air diffusion timer comprises: an air channel fluidly connected to the sample flow channel and open to an external surface of the device body at an air channel opening; an air diffusion pad comprising: a porous membrane including a thin sheet of a porous material; an opening in a center of the thin sheet that is at least as large as the air channel opening; and a coating on an exterior planar surface of the porous membrane that is not permeable to air, Page 159 of 164 12865804v1Atty. Docket No.: 2013065-0954 wherein the air diffusion pad is adhered on an uncoated planar surface of the thin sheet to the surface of the device body exterior surrounding the air channel opening, such that air can diffuse from the air channel through an interior of the thin sheet to an edge of the thin sheet.

171. The fluidic device of claim 170, wherein an area of the thin sheet of porous material controls venting of air from the air channel and the sample flow channel.

172. The fluidic device of claim 169, wherein the serpentine fluidic timer comprises: a timer fluid input reservoir disposed at the top of the fluidic device; a timer fluid flow channel fluidly connected to the timer fluid input reservoir; a fluidic resistance element fluidly connected to the timer fluid flow channel; a serpentine fluid channel fluidly connected to the fluidic resistance element; and a timer exit channel fluidly connected between the serpentine fluid channel and the fluidic valve.

173. The fluidic device of claim 172, wherein the fluidic resistance element comprises a thin channel arranged with a plurality of turns.

174. The fluidic device of claim 172, wherein the serpentine fluid channel comprises a channel arranged with a plurality of turns.

175. The fluidic device of claim 163, wherein the fluidic valve comprises a valve membrane comprising a material dissolvable by the sample fluid, wherein the valve membrane is adhered to an exterior surface of the device body covering an opening of the sample flow channel, and wherein the valve membrane prevents flow of air through the sample flow channel.

176. The fluidic device of claim 175, wherein the fluidic valve further comprises a thin sheet of absorbent material disposed between the valve membrane and the exterior surface of the device body, and wherein the absorbent material covers the opening of the sample flow channel, and wicks sample fluid from the sample flow channel to the valve membrane. Page 160 of 164 12865804v1Atty. Docket No.: 2013065-0954 177. The fluidic device of claim 175, wherein the fluidic valve further comprises a hygroscopic sponge disposed between the valve membrane and the exterior surface of the device body, wherein the hygroscopic sponge covers the opening of the sample flow channel, and expands when absorbing sample fluid, the hygroscopic sponge pushing outward against the valve membrane, and wherein the device body comprises a counterbore recess to fit the hygroscopic sponge.

178. The fluidic device of claim 177, wherein the hygroscopic sponge further comprises a hole through an interior of the hygroscopic sponge, wherein the hole axis is perpendicular to the exterior surface of the device body, and wherein the fluidic valve further comprises a piercing feature disposed at an exterior surface of the valve membrane to puncture the valve membrane when the hygroscopic sponge pushes against the valve membrane.

179. The fluidic device of claim 178, wherein the needle is attached to a device base configured to accept the fluidic device in a vertical orientation, and wherein the needle is positioned with a pointed end adjacent to the exterior surface of the valve membrane when the fluidic device is accepted in the device base.

180. The fluidic device of claim 163, wherein the sample fluid flows substantially by gravity flow.

181. The fluidic device of claim 163, wherein the sample fluid flows out of the outlet port into at least one of a downstream reaction chamber, a readout mechanism, and a lateral flow chamber comprising a lateral flow strip.

182. The system of claim 57, wherein the sample comprises a target nucleic acid.

183. The system of claim 58, wherein the at least one lyophilized reaction bead comprises a composition configured for carrying out an amplification process. Page 161 of 164 12865804v1Atty. Docket No.: 2013065-0954 184. The system of claim 183, wherein the amplification process comprises strand displacement amplification (SDA).

185. The system of claim 183, wherein the composition comprises at least one of an oligonucleotide binder, a ligase, a reverse transcriptase, a cleavage enzyme, a restriction enzyme, a single-strand binding protein, a nickase, a strand displacing polymerase, and a dNTP.

186. The system of claim 183, wherein the composition comprises an oligonucleotide binder comprising at least one of an SDA primer binding sequence, a first and / or second nucleic acid sensor part, modified nucleotides, a reverse primer, and a forward primer.

187. The system of claim 58, wherein the at least one lyophilized reaction bead comprises a composition configured for lysing the sample.

188. The system of claim 187, wherein the composition configured for lysing the sample comprises at least one of sodium hydroxide (NaOH), at least one enzyme, a low pH level, and potassium hydroxide (KOH).

189. The system of claim 157, further comprising a base for supporting the device body, wherein the second flow channel comprises a lateral flow chamber sized and shaped to allow insertion of a lateral flow strip therein.

190. The system of claim 189, wherein the base comprises: a reservoir disposed therein for containing the fluid sample; and a plenum disposed therein for collecting the fluid sample and delivering it to the reservoir; wherein the reservoir and the plenum fluidly connect the first flow channel to the second flow channel when the passively actuated valve is opened.

191. The system of claim 189, wherein the base is integral with the device body. Page 162 of 164 12865804v1Atty. Docket No.: 2013065-0954 192. The system of claim 189, wherein the device body is configured to be inserted into (i.e., seated within) the base.

193. The method of claim 53, further comprising eluting the biological sample with at least one reagent and / or solution thereby creating the sample fluid prior to adding one or more drops of the sample fluid to the sample inlet port. Page 163 of 164 12865804v1

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