Consumable microfluidic molecular diagnostic devices, and associated systems and methods of manufacture, including roll-to-roll manufacturing processes

Abstract

The present disclosure is directed to examples of low-cost, point-of-care molecular diagnostic system architectures, herein termed "SIMPLE chip" systems. The disclosure includes readers, fluidic consumable devices, open R&D platforms, and applications for the SIMPLE chip systems. The disclosure describes roll-to-roll and other manufacturing methods of the fluidic consumables. The SIMPLE chip systems are configured to combine the advantages of conventional antigen / antibody-based lateral flow assay (low-cost, quick results) with conventional PCR (high-sensitivity). The SIMPLE chip systems of the present disclosure are capable of providing PCR-like sensitivity and specificity on-site, with multiple pathogens tested concurrently within minutes, and at a low-cost compared to conventional PCR testing. A SIMPLE chip molecular diagnostic device can include a first layer and a second layer sealingly coupled to the first layer, the first layer having raised portions spaced apart from the second layer to form a fluidic circuit between the first and second layers.

Description

CONSUMABLE MICROFLUIDIC MOLECULAR DIAGNOSTIC DEVICES, AND ASSOCIATED SYSTEMS AND METHODS OF MANUFACTURE, INCLUDING ROLL-TO-ROLL MANUFACTURING PROCESSESTECHNICAL FIELD

[0001] The present disclosure generally relates to diagnostic sensing systems of biological targets.BACKGROUND

[0002] Traditional methods of pathogen identification include numerous drawbacks. These traditional testing methods can result in high expense to the healthcare provider performing the test, which is passed through to the patient or a health insurance provider; can extend the waiting period for test results, delaying diagnosis and increasing the risk of pathogen spread; can require highly trained technicians to accurately process a specimen sample; and / or can require expensive, large equipment to analyze the sample. As a result of these and other drawbacks, patients and / or healthcare providers may elect to forego testing for pathogens, which can lead to poorer patient health and under-informed healthcare providers.

[0003] Lateral flow assays have been the dominant tool for first line infectious disease screening in point-of-care settings due to their low cost and simplicity. However, the lateral flow assays can have lower accuracy, inferior detection limits, and need a longer postinfection window for detection compared to molecular diagnostics (e.g., PCR / isothermal amplification). These conventional lateral flow assay tools are also limited by low multiplexing ability (testing multiple targets simultaneously). In contrast, conventional PCR systems rely on a centralized, batched lab testing model, which has high cost and low turnaround time, and therefore cannot be used in a point-of-care environment. Recent point- of-care molecular diagnostic systems are too high cost to replace lateral flow assays as standard technology. Therefore, a need exists for a point-of-care molecular diagnostic tool that has cost parity' to lateral flow assays, is as easy to use as lateral flow assays, has accuracy at least equivalent to PCR, and can conduct multi-target testing similar to PCR panels.DESCRIPTION OF THE DRAWINGS

[0004] The foregoing aspects and many of the attendant advantages of the claimed subject matter will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

[0005] FIGURES 1A-1D include front, rear, and detail perspective views of one embodiment of a roll-to-roll (hereinafter “R2R”) manufactured Gen2 SIMPLE chip and one embodiment of a peripheral sample collection system, in accordance with embodiments of the present disclosure;

[0006] FIGURE IE shows several embodiments of R2R Gen2 SIMPLE chip molds having different main chamber shapes;

[0007] FIGURE IF is an embodiment of an R2R SIMPLE chip with filled micro chambers containing acoustic mix-enhancing air pockets;

[0008] FIGURE 1G is an embodiment of an R2R SIMPLE chip with arches and ribs for structural support and enhanced acoustic mixing;

[0009] FIGURES 2A-2E show embodiments of the swab insertion procedure for the Gen2 SIMPLE chip, showing the peripheral sample collection system of FIGURE ID;

[0010] FIGURES 3A-3C are flow charts depicting steps in the roll-to-roll mass production manufacturing of the R2R Gen2 SIMPLE chip of FIGURES 1 A-1D;

[0011] FIGURE 3D shows one embodiment of an R2R mold used to thermoform SIMPLE chips, with the left side having negative / engraved features, and the right side having positive / protruding features;

[0012] FIGURE 3E shows one embodiment of a pressure sealing plate and gasket used to seal the sample prep module of a Genl SIMPLE chip;

[0013] FIGURE 4A is a view of a machine for piercing and dispensing liquid reagents in microliter to milliliter volumes into a Genl SIMPLE chip, which can be optionally adapted to be used in-line with the R2R process;

[0014] FIGURE 4B is a view of one embodiment of a wax / high viscosity' liquid dispensing system configured for use in-line on an R2R consumable chip manufacturing process;

[0015] FIGURE 4C shows one embodiment of a roll-to-roll manufacturing machine configured to produce R2R consumable chips;

[0016] FIGURE 4D shows one embodiment of a non-quick change mold.

[0017] FIGURES 4E-4F show embodiments of a quick-change mold adaptor component used to switch to different embodiments of SIMPLE chip molds, such as for the thermoforming station of the roll-to-roll machine of FIGURE 4C;

[0018] FIGURES 4G-4J are views of the Gen2 SIMPLE chip of FIGURES 1A-1D at different stages in the R2R manufacturing process;

[0019] FIGURE 5 A is a view of one embodiment of a reader system used to process the R2R Gen2 SIMPLE chip of FIGURES 1A-1D;

[0020] FIGURE 5B shows one embodiment of the internal components of an R2R Gen2 SIMPLE chip reader system of FIGURE 5A that utilizes an acoustic transducer to mix solutions at various steps during processing;

[0021] FIGURE 5C shows the backplate assembly for the Gen2 SIMPLE chip reader system of FIGURE 5 A;

[0022] FIGURE 5D is a view of one embodiment of a sliding mechanism and base of the reader system of FIGURE 5 A;

[0023] FIGURES 5E and 5F show detail views of the backplate assembly for the Gen2 SIMPLE chip reader system of FIGURE 5 A;

[0024] FIGURE 5G is an embodiment of a backplate assembly using piezo surface speakers (PiezoListen) to mix solutions for the Gen2 SIMPLE chip reader system of FIGURE 5 A;

[0025] FIGURE 5H shows the piezo listen-based acoustic mixing and heater assembly cross-section for the Gen2 SIMPLE chip reader system of FIGURE 5 A;

[0026] FIGURE 51 shows an alternative embodiment of the gen2 SIMPLE chip reader of FIGURE 5A with a haptic bar for acoustic mixing;

[0027] FIGURES 5J and 5K show the reader system of FIGURE 5A and one embodiment of a fluorescent optical imaging system embodiment inside the reader;

[0028] FIGURES 5L and 5M show an alternative embodiment of a fluorescent optical imaging system inside the reader system of FIGURE 5 A that reduces cost and space;

[0029] FIGURES 6A-6C show an embodiment of a hard plastic (non-R2R) SIMPLE chip reader that utilizes a haptic piezo component for mixing;

[0030] FIGURE 7 shows a cassette embodiment of a hard plastic (non-R2R) SIMPLE chip reader;

[0031] FIGURES 8A-8D shows another embodiment of a hard plastic (non-R2R) SIMPLE chip reader with various embodiments of backplate support;

[0032] FIGURE 8E is a cross-sectional view of a concept allowing simple and easy insertion and removal of hard plastic (non-R2R) SIMPLE chips into a reader;

[0033] FIGURES 9A-9E show various embodiments of hard plastic (non-R2R) SIMPLE chips;

[0034] FIGURE 9F shows a non-wax valve embodiment of a rigid 2D SIMPLE chip, using a mechanical puncture needle to open the valve;

[0035] FIGURE 10 shows an alternative embodiment view of a concept allowing horizontal insertion and removal of rigid (non R2R) SIMPLE chips into a reader;

[0036] FIGURES 11A-11E show concepts of various embodiments of rigid (nonI R) SIMPLE chips that can be produced via an injection molding manufacturing process:

[0037] FIGURES 12A and 12B show cross-sectional and detail views of concepts of various embodiments of rigid (non-R2R) SIMPLE chips that can be produced via an injection molding manufacturing process;

[0038] FIGURE 13A shows an internal view of the open reader platform of the Genl reader;

[0039] FIGURES 13B-1 and 13B-2 show the revolving four tool gantry of the open reader platform of FIGURE 13 A;

[0040] FIGURES 13C and 13D shows the backplate assembly of the open reader platform of FIGURE 13 A;

[0041] FIGURE 14 is a view of an embodiment of a Genl R2R SIMPLE chip, showing burst valve designs and an integration pin;

[0042] FIGURES 15A-15C are views of a '‘free-floating” backplate design that enhances acoustic mixing;

[0043] FIGURE 16 shows a downstream silicone microfluidic chip with vacuum battery technology;

[0044] FIGURES 17A and 17B are graphs that shows the effect of without mixing (FIGURE 17A) and with mixing (FIGURE 17B) for isothermal amplification;

[0045] FIGURE 17C shows that the Genl SIMPLE chip system can achieve a 200 DNA copy / swab limit of detection with a complete workflow, showing the average isothermal amplification only required 4.3 minutes; and

[0046] FIGURES 18A and 18B show clinical testing results for 102 patients.DETAILED DESCRIPTION

[0047] The detailed description set forth above in connection with the appended drawings, where like numerals reference like elements, are intended as a description of various embodiments of the present disclosure and are not intended to represent the only embodiments. Each embodiment described in this disclosure is provided merely as an example or illustration and should not be constmed as preferred or advantageous over other embodiments. The illustrative examples provided herein are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed.

[0048] As will be described in more detail below, the present disclosure provides examples of systems, devices, and methods, each with capabilities to collect, process, and produce results to inform patients and healthcare providers about the presence or absence of a pathogen. Embodiments of the present disclosure can include one or more of severalcomponents: a single-use cartridge (“SIMPLE chip”) shown in FIGURES 1A-1C, a reader that processes the chip to produce a result, an optional smart device (such as a smartphone, tablet, or computer), an optional app, and peripherals necessary to run the test (swab, sample tube, packaging, instruction manual, etc.).

[0049] The following is a listing of the sections in the detailed description for purposes of organization:1, Gen2 SIMPLE chip design compatible with roll-to-roll manufacturing a. Overview b. Alternate embodiments of the wax valve c. Alternative detection embodiments of the SIMPLE chip d. Acoustic mixing details and alternative embodiments e. Microwell and fluidic design details of the gen2 SIMPLE chip f. Additional large wells for adding spatially and time-independent processes g. Example embodiments2, Roll-to-roll manufacturing methods for ultra-low-cost SIMPLE chips a. Overview b. Mold quick change adaptor assembly c. Example embodiments3, Gen2 SIMPLE chip reader a. Overview b. Sliding mechanism and base c. Alternative embodiments of reader mixing mechanism d. Standard optical imaging system e. Alternative low-cost optical imaging system f. Example embodiments4, Automated inlet sealing module for the SIMPLE chip a. Overviewb. Example embodiments , Two-dimensional rigid SIMPLE chips a. Overview b. Example embodiments , Reader for rigid 2D SIMPLE chips a. Overview b. Example embodiments , Injection-molded 3D SIMPLE chips a. Overview b. Example embodiments , Genl roll-to-roll manufactured SIMPLE chips integrated with vacuum batten- technology' a. Overview b. Design and manufacturing of the genl SIMPLE chip c. Example embodiments , Automated filling machine a. Overview b. Example embodiments 0, Roll-to-roll manufactured burst valves a. Overview b. Example embodiments 1, Fluidic Integration pins a. Overview b. Example embodiments 2, Open reader platform for rapid cartridge R&D a. Overview b. Hardware design c. Software designd. Example embodiments13, loT and Al applications a. Overview b. Internet of Things (loT) applications c. Telemedicine applications d. Analytics applications e. Research applications and ad-hoc clinical trials f. Public health and Al applications g. Integration with other third-party software and services h. Example embodiments14, Other applications a. Overview b. Example embodiments

[0050] 1. Gen2 SIMPLE chip design compatible with roll-to-roll manufacturing a. Overview

[0051] Some expected advantages of the embodiments of the Gen2 SIMPLE chip include, but are not limited to:1) Ultra-low-cost roll-to-roll manufacturing is more efficient than conventional multipart construction injection molding, which most competing technologies use. It is possible to reach cost parity with rapid antigen tests using this architecture;2) As few as two major assembly parts are needed (layer with fluidic channels and backing cover layer);3) Vacuum-based loading is faster than capillary-based loading and has fewer geometrical and surface material restrictions — capillary' loading needs hydrophilic surface materials;4) Vacuum-based loading is much faster than elastomer (e g., PDMS) based degas loading, which speeds the assay time up;) Stored vacuum pumping removes the need for external pumping or pressure sources to drive liquid, further reducing system cost; ) Vacuum-based loading allows tunable trapping of air bubbles in the microwells, which enhances acoustic mixing effects; ) Vacuum loading format allows less reagent usage because of less dead volume; ) Does not require elastomers, which is a difficult material to work with for R2R processes; ) Simple and low-cost spatial multiplexing possible; 0) No external pumps, vacuum, or pressure sources are needed; 1) No robotic motion automation is needed; 2) Active acoustic microfluidic mixing enhances biochemistry reaction kinetics significantly; 3) Simple multiplexing. Spatial (microwell) multiplexing instead of colorbased multiplexing reduces the need for extra sets of optical filters, which decreases cost. No complex primer design is needed (vs. one pot multiplexing where multiple primers are in the same chamber); 4) Easier to multiplex and faster speed than paper strip format; 5) One-step sample prep chemistry enables faster speed and lowers engineering complexity (cost); 6) Extreme limit of detection possible (better than 20 genomic copies equivalent (gee) per swab); 7) No (plastic or elastomer) injection molded parts required; and 8) Relatively fast assay speed compared to conventional technologies; coupling one-step sample prep and next-gen isothermal amplification technologies, the total assay time from sample to answer can be less than-lOmins.

[0052] Embodiments of the present disclosure will now' be described in detail. In use, for a single test in one embodiment, the user of the system will receive a package of singleuse components, including one consumable chip in sealed vacuum packaging, one swab in a sealed package, and a sealed dropper tube.

[0053] The first step of using the system is to load the chip onto the reader FIGURES 5A-5C. Specifically, the chip is placed on a chip load plate 506 of the reader and secured by the chip catch 523. The chip load plate’s alignment perimeter 522 matches the geometry of the chip’s alignment ridge 104 (see FIGURE 1A) to ensure that positioning is consistent when the chip is loaded. The chip load plate with the loaded chip is then rotated back against the processing backplate 505 using a hinge 514. Clasping magnets 513 firmly clamp the chip against the processing equipment of the backplate. The mating magnets allow the chip to maintain thermal contact with the heaters and enable improved acoustic vibrational energy transfer. The acoustic transducer 511 induces vibration for fluid mixing. The processing backplate and chip load plate can be designed with less mass to minimize vibrational damping. They should be made of rigid material that transfers vibrational energy well with minimal damping. The backplate assembly 510 (all parts in FIGURE 5B except the sliding mechanism) rests on a sliding mechanism 504.

[0054] The next step is to collect a biological sample. One embodiment of accomplishing this is shown in FIGURE ID as a swab 1 11. In one embodiment, the swab 111 can be a nasal swab, a nasal pharyngeal swab, a mid-turbinet swab, or a throat swab. Alternatively, it could be any type of other swab. In one embodiment shown in FIGURES 2A and 2B. the swab tip 111 is placed into a dropper tube 113 containing an initial lysis (or elution / hydration) solution. The dropper tube may have a foil lidding material 114 (see FIGURE ID) to store liquids long-term. The foil is removed, and the swab tip could be broken off and kept in the dropper tube. The lysis solution, for example, includes some or all but is not limited to, chaotropic buffers, salts, chelating agents, Rnase inhibitors, pH buffers, ethanol or IP A, enzymes, protease k, lysozyme, surfactants, and or detergents. The lysis buffer is designed to lyse bacteria, fungi, cells, and / or viruses to release their DNA / RNA, control ionconcentrations, reduce inhibitor effects, and not significantly inhibit downstream amplification reactions. After the lid 115 of the dropper tube is closed and the tester shakes to mix the dropper tube, the tip 116 (see FIGURES 2 A and 2B) of the dropper tube 113 is inserted into the insertion port 110 (FIGURE 2C) of the single-use cartridge “chip” that is loaded on the reader and the solution is squirted into the chip 100. As shown in FIGURE 2C. the door can have a tab 202 that can be pushed out to serve as a pull tab to pull open the door. The tab 202 can be spring loaded to fall back into the natural flush closed state, or use gravity (in this case hinge is on top side) so it naturally falls back flush. The tab 202 has a hinge that it pivots around. The top side can be pushed in to get the bottom side out, then the user grabs the protruding section and pulls the door open.

[0055] In an alternative embodiment shown in FIGURES 2D and 2E of the sample collection method, the swab 111 is put directly into the insertion port 110 of the single-use cartridge chip 100 after collecting the sample. In this embodiment, the swab tip will be snapped off and into the chip 100 at a weakened breakpoint 112. The user will dispense a dropper tube containing the lysis solution (or hydration / elution buffer) in the chip’s insertion port while the chip is loaded on the reader. This alternative embodiment is expected to provide controlled and enhanced mixing with acoustic mixing to elute the sample off the swab.

[0056] The next step is for the user to push the reader door 503 (FIGURE 5A) into the main reader body 501. As the user pushes the door closed, the roll of tape 109 (FIGURE IB) on the back of the chip 100 contacts the reader's tape roller 508, which causes the sealing tape roll 109 to unroll across the open insertion port of the chip 110 as shown in FIGURE 1C, sealing its contents and preventing external biological contamination. The tape roller material may optionally be a fluoropolymer or alternative material that does not stick to the tape. The tape roller may include brushes or flexible sleeves to press down on the PSA to make a more uniform seal. This mechanical design allows for automatic, consistent sealing of the chip every time and removes manual -induced variability from the user, ultimately reducing the risk of seal leaks. The sealing tape roll 109 is flexible (and optionally elastic material), and the relative air volume above the micro wells is much larger than within the micro wells (providinggas compressibility as a pressure buffer), so even without a vent, it does not impact the vacuum loading of the microwells negatively. The sealing tape roll also minimizes evaporation. Excessive evaporation is a risk during heating processes. Evaporation can be detrimental to biochemical processes such as isothermal amplification, as relative concentrations of enzymes need to be constant at specific ranges. Other embodiments may seal the chip instead of a sealing tape roll. For example, a flap of tape can be used, or a mechanical locking cap or a cap with adhesive sealing may be used to seal the chip. In an alternative embodiment, the reader door may be closed using electrical actuators such as motors, solenoids, or linear actuators instead of manual closing.

[0057] Once the reader's door is closed, the automatic processing of the chip by the reader is ready to begin. In one embodiment, the chip processing is initiated by a touchscreen user interface 502 of the reader 500. An alternative embodiment may include interfacing with the reader by connecting to the reader with a centralized device that can control several readers simultaneously, e.g., several of the reader 500 linked together. Another alternative embodiment may include controlling single or multiple readers from a computer that has a communication connection to the reader via Bluetooth or another communication protocol. In this embodiment, the readers may not have an embedded screen interface.

[0058] The first step of automatic processing is to (optionally) heat and mix the contents of the main loading chamber 101. Components on the backplate (FIGURES 5E and 5F) enable heating and acoustic mixing. FIGURES 5E and 5F show one embodiment of the processing backplate and its components. The main chamber heating unit 543 can be turned on to heat the main chamber contents. In one embodiment, this heater is a Peltier-type heater 547 with a surface-mounted temperature sensor 548 and an aluminum cover plate 549. The aluminum cover plate 549 protects the sensor and creates a flat surface and transfer heat to the chip. The system can precisely control the surface temperature from the onboard electronics. Other types of heaters can be used, for example, and not limited to, resistive heaters, polyimide heaters, optical heaters, quartz heaters, laser heaters, infrared heaters,silicone rubber heaters, polyester flexible heaters, PET metallic electric films, Mica heaters, mylar heaters, Kapton heaters, etc.

[0059] To mix the contents of the main loading chamber, the acoustic transducer 511 is turned on, which causes the acoustic transducer’s oscillation plate 541 to vibrate back and forth at a specified frequency. The oscillation plate is directly connected to the processing backplate 505, and the processing backplate is unrestricted to move in the horizontal direction. Therefore, the chip contacting the processing backplate is part of the system oscillating at the acoustic transducer’s set frequency.

[0060] This oscillation energy will be transferred into the fluid, and the contents in the main chamber 101 of the chip 100 will be mixed. To further enhance mixing in the main chamber, air bubble capture mixing structures are on the surface 102 of the chip 100. These shapes are designed to capture a specific volume (such as lul~1000ul) of air bubbles that will have a resonance that matches the oscillation frequency of the acoustic transducer (20hz~200Mhz).

[0061] After the contents of the main chamber are thoroughly heated and mixed (note that this step is optional or can be done manually inside the sample dropper tube with lysis buffer), the wax valve heater 544 is turned on to its temperature set point. In the depicted embodiment, the construction of the wax valve heater can be the same as the main chamber heater: a Peltier-type heater with a surface-mounted temperature sensor and an aluminum cover plate. The aluminum cover plate serves to distribute the heat more evenly. As the wax valve heater’s temperature increases, the solid wax in the chip’s wax valve channel 105 melts. As this happens, the liquid contents in the main chamber are pulled through the wax valve channel 105, then through a wax cooling chamber 106, and then travel through a micro channel distribution network 107 to their final destination of micro wells 108. The wax cooling chamber 106 is designed in a shape that allows the wax to cool and solidify while allowing liquid to pass through. There is an expansion of space, which allows for slower flow rates to cool the wax, and then there is a lateral offset so the wax does not plug the outlet of the cooling chamber upon cooling. The mechanism that causes the fluid to be pulled into thesystem is a pre-stored vacuum in all the chip channels and microwell spaces below the wax valve. This vacuum is created during the chip manufacturing process and remains stored in the chip until the wax valve is melted. The chip’s manufacturing process, including the wax valve creation and the vacuum, will be discussed in greater detail below.

[0062] Once the liquid transfer process into the microwells is complete, the third and final heater, the amplification hearing unit 545 (FIGURE 5F), is turned on to its temperature set point. Additionally, the contents in the microwells are mixed by turning on the acoustic transducer. In the depicted embodiment described herein, the construction of the amplification heating unit is the same as the main chamber heater and the wax valve heater. It is a Peltier-type heater with a surface-mounted temperature sensor and an aluminum cover plate. The mechanism of mixing the contents in the microwells 108 is also the same as the mechanism described above regarding mixing the contents in the main chamber, i.e., by the acoustic transducer oscillating the backplate. Again, small air bubbles 151 are trapped in the microwells 108 (FIGURE IF) to enhance acoustic mixing of the liquid 150. The size of these air bubbles 151 can be modulated by how strong a vacuum the chip is stored in during manufacturing. If a weaker vacuum is used during manufacturing and bag sealing, the air bubbles will increase in size. The acoustic mixer must be tuned so that the resonant frequency that induces maximum mixing matches the air bubble size. Since there can be slight variations in the air bubble sizes, a looped sweep in acoustic frequency can be used to accommodate the variations and increase mixing in general. It is also possible for mixing to occur without air bubbles, though a different resonance frequency will likely be needed.

[0063] Acoustic mixing in microwells is essential to increase the kinetics of the isothermal amplification. Without mixing, isothermal amplification can be slow and have jagged amplification curves due to the high viscosity of the master mix and poor diffusion kinetics. Also, due to a lack of mixing, isothermal amplification at a lower starting template concentration shows a lower maximum fluorescence plateau at the end of the reaction. We have shown that mixing significantly increases the amplification speed and recovers the fluorescence signal plateau at lower template concentrations. FIGURES 17A and 17B showthis effect. A double titration of lowering the starting template DNA concentration was tested in duplicates, with and without mixing. Labels denote the DNA copy number per reaction. At the lowest tested level at 3.9 DNA copies / reaction (solid line), double reaction speed was achieved when mixing was applied, and the exponential inflection point moved from ~7.5mins to- 3.7mins. The 3.9 DNA copies / reaction condition also showed -fivefold increased fluorescence signal strength achieved after mixing.

[0064] As the contents in the microwells are heated and mixed, a molecular amplification reaction occurs that will multiply a target biological marker if the target is present in the sample. Isothermal amplification runs in the microwell. Isothermal amplification chemistries include and are not limited to EXPAR, HAD, SDA, RPA, Rapid Amp, LAMP, NASBA, WGA, NEAR, Sherlock, Detectr, TMA, RCA, MDA, SMAP2, SPIA, SMART, ICAN, PHAMP, HCR, CRISDA, BAD AMP, EIC, and any new isothermal formulations, etc. Alternatively, PCR can also be used. Non-enzymatic amplification may also be used. A fluorescent probe (such as molecular probes, FRET probes, FISH probes, fluorophore-quencher pairs, aptamer probes, RPA exo, RPA nfo probes, CRISPR probes, CAS9 probes, CAS12 probes), or dye (e.g., SYBR green) can be used to generate a fluorescence optical signal that will allow the amplification process to be captured in an image by the reader’s optical system (FIGURE 5J and 5K). Quantitative or qualitative analysis of fluorescence strength is possible. If the target is present, the amplification reaction occurs. The fluorescent signal in the microwells will increase, which will be captured by the optical system by taking images at consistent intervals. A positive signal can be differentiated from a negative signal by comparing it with internal controls, or the ramp rate of fluorescence intensity.

[0065] At this point, the testing is complete, and the system will notify the user of the result on the reader’s touchscreen 502 or, optionally, the app. The user then can open the reader by pulling the reader’s door out 503, rotating the chip load plate down 506, and removing the consumable chip 100. The chip is then placed in a sealable bag that is included with the testing contents, and the bag is discarded. The whole consumable chip is self-contained, and the sealable bag provides a secondary containment to low er the amplicon or biohazard contamination risk. b. Alternate embodiments of the wax valve

[0066] The wax valve temporarily holds the vacuum in downstream fluidic circuits after removing the chip from its vacuum bag. The valve should keep the vacuum in the fluidic circuits at operational levels for at least 10 minutes so the user has adequate time to run the test. Instead of wax valves, other valves may be used to release the stored vacuum on demand. For example, normally closed mechanical valves can be opened on demand by mechanical forces, magnetic forces, external pressure forces, heating sources, and puncture forces. For example, a film or thin area of the SIMPLE chip could be used as a valve, and the film / thin area could be punctured by a sharp mechanical tip, allowing ambient air pressure regions to flow liquid toward vacuumed areas. The reader’s automation process could drive the puncturing actions. Alternatively, it could be driven by the user. Alternatively, the film structure could be athermal meltable film that can be opened by laser irradiation. For example, parafilm could be used to separate the ambient pressure and vacuum regions and be opened by a heat source (laser, heating pads, optical lamps, etc.).

[0067] In other embodiments, the valve material can be made of (and not limited to) wax, paraffin, hydrocarbon polymers, fluorocarbon polymers, grease, etc., that can be thermally melted. The valve can be made of soluble materials that dissolve and open upon contact with liquids. The valve can be engineered to have a specific time delay factor to open during the dissolving process. c. Alternative detection embodiments of the SIMPLE chip

[0068] Instead of using fluorescence optics to detect the test results, the reader may use other optical methods to detect assay results. Other methods include colorimetric methods to sense color change or optical absorption methods detecting spectrum absorption changes. Note that the reader can have a minimal footprint if colorimetric methods are used because the fluorescence detection unit is unnecessary. The reader can also utilize near-field or far- field optics. The reader can also use total internal reflection fluorescence (TIRF), surface-enhanced ramen scatering (SERS), chemiluminescence, interferometer-based techniques, surface plasmon resonance (SPR), darkfield optics / microscopy, or other optical methods to sense scatering. For example, the reader can use scattering-based optical sensing methods for agglutination assays.

[0069] Alternatively, electrical chemistry may be used instead of optics to sense test results. For example, electrodes may be printed onto the cover layer to provide electrochemistry-based methods to detect test results. Electrical sensors placed outside of the SIMPLE chip may also be used to measure capacitive, magnetic, acoustic resonance, or impedance changes.

[0070] Alternatively, electrodes inside the chip cover layer could move liquids with electric-weting-on-dielectric (EWOD) technology7. The electrodes may also be located outside the chip but proximal to the cover layer. The cover layer could be made of hydrophobic material to facilitate EWOD.

[0071] Alternatively, to molecular amplification assays, other chemistries may be used in the microwells, including, but not limited to, agglutination assays, immunoassays, enzymatic assays, chemistry7panels, aptamer assays, and various chemistry7assays. Other assays described in various SIMPLE chip embodiments in this patent may also be used. d. Acoustic mixing details and alternative embodiments

[0072] We tested many different embodiments of the roll-to-roll consumable chip. These versions have experimented with geometry features that have produced different results. FIGURE 1A shows one embodiment of an R2R gen2 SIMPLE chip with ribs 102 to catch air bubbles to enhance acoustic mixing. In other embodiments, micro-texture pores (see, e.g.. FIGURES 9A-9C) can be located on the surface of the chamber 101 to similarly catch air bubbles to enhance acoustic mixing. FIGURE IE shows the forming molds of several embodiments of the R2R SIMPLE chip with shape variation of the main chamber. A “spike’' design mold 130 shows spikes 132 in the chamber 131 to capture air bubbles to enhance acoustic mixing. The “wide" design mold 140 shows the liquid-air interface surface area can be increased using a wide main chamber 141, increasing the mixing kinetics.

[0073] As noted above, FIGURE 1A shows a ribbed design with ribs 102 to capture air bubbles to enhance acoustic mixing. Spikes can be seen on the side as well (FIGURE IE). FIGURES 9A-9C shows a hybrid of spikes and micropores 908, 902, and 909 to capture air bubbles to enhance acoustic mixing in the main chamber. Note that these air bubble-capturing structures can be manufactured in injection molded and thin film R2R designs. In general, there may be many shapes to capture air bubbles, for example: a rib, a divot, a dimple, a surface etch, a trough, or a protrusion. The key is to design abruptly changing geometries that trap air bubbles while the liquid is loading. In general, the larger the air bubble, the better mixing is achieved. Other shapes or other asymmetric shapes may also help increase acoustic mixing in the microwells by creating an imbalance in local fluid pressure, creating a net continuous flow inside the microwells. Within the microwells, the air bubble is created from residue air in the microwell during the manufacturing vacuum process. The size of the air bubbles in the microwells can be modulated by the initial strength of the vacuum that the consumable was exposed to during manufacturing (and storage). A weaker vacuum stored initially results in larger air bubbles and vice versa.

[0074] In an alternative embodiment, materials with higher density than the liquid can be prepacked and utilized for inertia or magnetic-based mixing. For example, steel bead(s) can be placed in microwells. An external force can force the bead(s) to move at contrasting speeds relevant to the fluidic and induce mixing. The external force can be shaking, vibrating, acoustic, or magnetic. e. Microwell and fluidic design details of the gen2 SIMPLE chip

[0075] There could be one or more microwells for spatial multiplexing. The microwell (or macrow ell) diameter could range from 1pm to 2cm. Different primers and / or molecular probes may be put into each microwell for multiplexing. Other chemistries may also be put into each mi crow-ell for multiplexing. Positive and / or negative controls may be put into the microwells. Optionally, the microw-ell(s) may be used for color-based multiplexing. In other embodiments, the microwell(s) are suitable for use with both colorbased and spatial multiplexing, concurrently.

[0076] For the fluidic channel design, it would be better to have equal fluidic resistance to level volumetric loading into the microwells. We have found that smaller channels work better to help level volumetric loading consistency. When the consumables are orientated vertically, the microwells sometimes load unevenly due to gravity effects. We have found that using smaller channels helps alleviate this problem. The fluidic channels and microwells may be microfluidic or macrofluidic in size, with cross-sectional widths and heights running anywhere from 1 pm~2cm. The microwells can be placed at the terminal end of the fluidic channel, or can be placed at an intermediate location along the fluidic channel, such as in embodiments where a different type of chamber is positioned at the terminal end of the fluidic channel (e.g.. a vacuum chamber, etc.).

[0077] Small arched or ribbed designs can prevent 3D structures such as fluidic channels and blisters from being crushed or deformed under atmospheric pressure storage. FIGURE 1G shows a positive mold with these features, such as perimeter and chamber ribs 161 and an arch structure 1 2. The structures experience an external load due to the vacuum inside and higher atmospheric pressure outside the bag. Note that the ribbed structures 161 in the sample loading area can also serve as air bubble capture areas to enhance acoustic mixing. Arch structures 162 can further increase structural rigidity.

[0078] The thin film material forming the fluidic circuit layer needs to be of sufficient thickness so that it has enough rigidity that the fluidic features are not crushed during the vacuum packaging, storage, and assay process. Films thickness can be lpm~4mm. The film may be even thicker, although the fluidic layer material might not be stored as ’‘rolls” due to its thickness. Nevertheless, a thicker material (optionally cut into specific, manageable length units instead of a continuous big roll) may still be fed in-line continuously through the R2R process described in the manufacturing section. See below for a description of the 2-D rigidSIMPLE chips.

[0079] Notably, one improvement of the gen2 SIMPLE chip 100 over the genlSIMPLE chip (see, e.g., FIGURE 14) is using a direct vacuum to draw in liquids instead of the vacuum battery system, which relies primarily on slower vacuum diffusion (explained inanother section). The improvement allows much faster liquid loading since there is no need to wait for degas-based fluidic loading. Another improvement is that gen2 allows the SIMPLE chip 100 to leave a controllable and constant air bubble size that does not vanish within the microwells after loading, which is critical for acoustic mixing. Gen2 SIMLE chip can be entirely made by the R2R process and does not require a separate coupling with the vacuum battery chip described in other sections.

[0080] Vacuum loading also allows very easy multiplexing, fast loading, and less reagent usage. If more multiplexing is needed, more vacuum microwells can be added. Vacuum loading has no dead volume, so less reagent is needed. Vacuum loading can quickly and easily load tiny amounts of liquids, so less reagent is needed. For example, gravity-loaded systems need a critical mass to allow the reagents to flow into fluidic chambers. Fluidic systems that require external pressure sources will have sizeable dead volumes and cannot handle small volumes easily. Capillary fluidic systems have material limitations (surface angle needs to be low) and geometric limitations (small capillary channels needed). It is hard to mix in capillary systems, whereas our vacuum loading concept with trapped air bubbles allows for excellent mixing.

[0081] While all figures show a vertical orientation of the SIMPLE chip, the SIMPLE chip can also be operated horizontally or any other orientation. For example, a syringe-like sample injector can inject fluid samples vertically (optionally, the sample injector may or may not stay attached to the SIMPLE chip) while the SIMPLE chip lies horizontally to receive the liquids. The SIMPLE chip may also be horizontal, slightly inclined to encourage fluid to pool naturally near the wax valve channel. A horizontal chip may use a reader that receives the SIMPLE chip horizontally (FIGURE 10). f. Additional large wells for adding spatially and time-independent processes

[0082] FIGURES 9D and 9E show that one or more additional large sample wells 91 1 ,912, 921 can be placed upstream of the microwells. The large sample wells 911, 912, 921 can be separated by normally closed wax valves that can be opened on demand. Additionallarge wells can be made for injection molded or R2R-manufactured consumable chips. Additional large wells allow the chip to run more time-dependent, spatially separated steps sequentially. For example, additional large wells can include functions such as lysis buffer neutralization agents, pre-amplification, nested amplification, pH neutralization agents, or ion chelators.

[0083] One embodiment, for example, is that the lysis buffer can be neutralized in the upper large well 911. Hence, the lysis buffer no longer inhibits downstream isothermal amplification processes. This neutralizing can be done by storing dried or lyophilized pellets of neutralizing chemistry (for example, but not limited to pH stabilizing buffers, chelators, detergent removers, etc.) in the large well that neutralizes the lysis buffer. Once the lysis buffer is neutralized, then the normally closed wax valve 914 opens (via heating), cools at the cooling region 915, and the neutralized lysis buffer gets sucked via stored vacuum into the lower large well for an optional first-stage isothermal amplification. In the lower large well 912, one or more sets of primers can be lyophilized along with the isothermal master mix and pre-loaded onto the chip in dry form.

[0084] A first stage of isothermal amplification allows the device to achieve a better limit of detection levels by boosting target pathogen amplicon levels in a first stage first before dividing the samples into smaller microwells. If there were no first stage amplification, the samples would be divided when they enter multiple microwells, effectively making the limit of detection worse. After the first stage amplification is completed after a set time, the bottom large well’s wax valve opens and distributes the amplicons into multiple microwells. In the microwells, it can optionally have independent molecular probes to test for amplificons directly, or it could have a complete second stage isothermal reaction with individual primers and probes, which can further amplify the amplicons and enhance the fluorescence signal output. With 2-stage amplification, it is possible to achieve ~20 genomic copy equivalent / swab limit of detection performance, making it one of the most sensitive systems to date. The chip can be fully sealed by melting the normally open wax valve to a closed state in channel 913. The wax valve during storage can have a shape that does not block the ventingchannel. After melting and solidifying, it blocks the venting channel via gravity or capillary flow. While the wax valve is open, the channel structure 913 serves as an air vent to neutralize air pressure. g. Example embodiments

[0085] A collection of example embodiments providing additional description of a variety7of embodiment ty pes in accordance with the concepts described herein are provided below. These examples are not meant to be mutually exclusive, exhaustive, or restrictive; and the claimed subject matter is not limited to these example embodiments but rather encompasses all possible modifications and variations within the scope of the issued claims and their equivalents.

[0086] Independent Claim:A point-of-care diagnostic system design comprising: a consumable that has all one-time-use components physically located on the consumable, wherein the consumable is made by' a roll-to-roll manufacturing process, the consumable further comprising: a pressure-induced mechanism to trap gas bubbles within the consumable.

[0087] Independent Claim:A roll-to-roll manufactured device, comprising: pre-stored vacuum in its fluidic circuit(s); one or more valves that can be opened so the vacuum can be released on demand; vacuum drives fluidic movement upon valve opening; and can be manufactured with as few as two major assemblies (formed channel layer plus backing layer).

[0088] Independent Claim:A fluidic cartridge, comprising: manufactured by a roll-to-roll manufacturing process; a cover layer;a fluidic layer; a normally closed valve that can be opened on demand; vacuum prestored in the fluidic circuit downstream of the valve; microwells; and air bubbles are trapped in the microwells during fluid loading.

[0089] Independent Claim:A fluidic cartridge, comprising: roll-to-roll manufactured cartridge; requires only two major assemblies (3D formed layer and cover layer); stores vacuum; a wax valve that can be opened on demand and allow vacuum pumping; microwells; and the microwells trap tiny air bubbles for enhanced acoustic mixing.

[0090] Dependent Claims (based on Independent Claims):Can have multiple microwells for spatial multiplexing.Different primers may be put into each microwell for multiplexing.Different molecular probes may be put into each microwell for multiplexing.Different chemistries may be put into each microwell for multiplexing.Positive and / or negative controls may be put into the microwells.Microwells have equal fluidic resistance to level volumetric loading.Smaller channels leading up to microwells help to make consistent volumetric loading among the micro wells.Passive vacuum fluidic loading, no external pressure sources such as pumps required.It has no vents.It has a sealing tape roll.Long-term vacuum storage can be done in an aluminum-lined vacuum pouch.The wax valve maintains vacuum transiently and can be melted by direct contact with heating elements or optical heating.Valve(s) could be made of thermally meltable materials such as wax, paraffin, hydrocarbon polymers, fluorocarbon polymers, grease, etc.Valve(s) can be made of soluble materials that dissolve and open upon contact.Includes a wax cooling area to prevent clogging downstream channels.The wax cooling area has an expansion of fluidic cross section compared to incoming fluidic channels.The wax-cooling area has an offset to allow further wax cooling.The wax valve can be substituted with a mechanical valve that can be opened by external actuation.The valve can be a film material that is punctured by a mechanical force.The device is used for diagnostics.The device is used for molecular diagnostics.The device is used for point-of-care molecular diagnostics. no (plastic or elastomer) injection molded parts required.It has less than three plastic components (or layers).It has no on-chip liquid storage.It does not require robotic motion automation.It has an acoustic mixing compatible design.The mi crowells automatically trap air bubbles.The air bubbles' size can be modulated by vacuum storage strength in the microwells.It can trap additional bubbles by geometrical design (dimples, ribs, holes, etc.), which is helpful for acoustic mixing.The device can run isothermal amplification chemistries, including and are not limited to, EXPAR. HD A, SDA, RPA. Rapid Amp, LAMP, NASBA, WGA, NEAR,Sherlock, Detectr, TMA, RCA, MDA, SMAP2, SPIA, SMART, ICAN, PHAMP, HCR, CRISDA, BAD AMP, and EICThe device can run PCR reactions.A fluorescent probe (such as molecular probes, FRET probes, FISH probes, fluorophore-quencher pairs, aptamer probes, RPA exo, RPA nfo probes. CRISPR probes, CAS9 probes, CAS 12 probes), or dye (e.g., SYBR green) can be used to generate a fluorescence optical signal that will allow the amplification process to be captured in an image by the reader's optical system.A multi-step process is possible (2-stage amplification or nested amplification).It can achieve better than 100 gce / reaction limit of detection performance.It has a tape-sealed inlet to prevent evaporation and or contamination.Compatible with optical detection methods such as fluorescence methods, colorimetric methods, optical absorption methods, spectroscopy, near-field optics, far- field optics, total internal reflection fluorescence (TIRF), surface-enhanced ramen scattering (SERS), surface plasmon resonance (SPR), darkfield optics / microscopy, chemiluminescence, Interferometers-based techniques, and other optical methods to sense scattering.Compatible with electrical sensing methods such as electrochemistry, capacitive sensing, magnetic sensing, and impedance sensing.Alternatively, to molecular amplification assays, other chemistries may be used in the microwells, including, but not limited to, agglutination assays, immunoassays, enzymatic assays, and various chemistry assays.Use of small arch designs to prevent 3D structures such as fluidic channels and blisters from being crushed or deformed under atmospheric pressure storage.Can be made in microfluidic or macrofluidic format.No vents are required in this design, which further lowers evaporation problems and reduces cross-contamination risks.The relative air volume above the microwells is much larger than within the microwells, so even without a vent, it does not negatively impact the vacuum loading of the microwells.The thin film material forming the fluidic circuit layer is of sufficient thickness so that it has enough rigidity that the fluidic features are not crushed during the vacuum packaging and storage process.The web can be made from a wide variety of thermoplastics, including PolyPropylene (PP), Polyvinyl chloride (PVC), Cyclo Olefin Polymer (COP), Cyclic olefin copolymers (COC), polyethylene terephthalate (PET), polyvinylidene chloride (PVDC), Poly chlorotrifluoroethylene (PCTFE), PETG, HIPS, fluoropolymers, Aclar, and many other varieties that can include proprietary blends and multi-ply structures. It could also be made of paper, cold-form foils (such as aluminum), or a blend of any of the above as a combined multilayer laminate.Materials with higher density than the liquid can be prepacked and utilized for inertia or magnetic-based mixing.Electrodes inside (or proximal to) the chip cover layer could be used to move liquids with electric-wetting-on-dielectric (EWOD) technology.

[0091] 2, Roll-to-roll manufacturing methods for ultra-low-cost SIMPLE chips a. Overview

[0092] Some expected advantages of the embodiments of the R2R manufacturing methods include, but are not limited to:1) Lower cap-ex and op-ex cost to setup manufacturing line compared to conventional injection molding and multi-part assembly;2) More efficient and less materials used than injection molding, resulting in lower consumable costs and lower waste volume;3) Lower tooling costs due to simplicity compared to injection molding;4) Quick change tooling sets for forming allow rapid R&D iteration at a lower cost;5) Molding and assembly are achieved in-line and do not need separate processes like injection molding; and6) No photoresist is required during manufacturing.

[0093] Embodiments of the present disclosure will now be described in detail. FIGURES 3A-3C show an overview diagram of two R2R consumable SIMPLE manufacturing process embodiments. The following description will be based on the process diagrammed in FIGURE 3A. Alternative embodiment examples are shown in FIGURE 3B and FIGURE 3C. Dashed lines depict the main differences. The following describes the gen2 SIMPLE chip manufacturing process, such as the chip 100, though some or all of the process steps and / or materials may be cross-compatible with all other SIMPLE chip embodiments.

[0094] One of the significant advantages is that the consumable chip can be manufactured with as few as two major assemblies (layer 100a with 3D fluidic structures plus backing cover layer 100b), reducing cost.

[0095] The base machine for the manufacturing is a customized roll-to-roll machine 420 which one embodiment of is shown in FIGURE 4C. In alternative embodiments, the base machine can be a custom modified film converter, blister packaging machine, food packaging machine, thermoforming machine, tray sealing machine, form-fill-seal machine, blister sealing machine, skin packaging machine, or shrink wrapping machine, etc. Functional components can be modified and / or added / removed per need. The user may control the machine using a human-machine interface 423. The process starts with a roll of thermoforming material called a web 427. The web can be made from a wide variety of including and not limited to thermoplastics, Poly Propylene (PP), Polyvinyl chloride (PVC). Cyclo Olefin Polymer (COP), Cyclic olefin copolymers (COC), polyethylene terephthalate (PET), APET (Amorphous Polyethylene Terephthalate), polyvinylidene chloride (PVDC). PS (Polystyrene), Poly chlorotrifluoroethylene (PCTFE), PETG, HIPS, fluoropolymers, Aclar, polyolefins, silicones, elastomers, thermoplastic elastomers, and many other varieties that can include proprietary blends and multi-ply structures. It could also be made of paper, biodegradable materials, cold-forming materials (such as aluminum), thermal-formingmaterials, or a blend of any of the above as a part of a multilayer laminate. Note that any material used should be selected for compatibility with the (bio)chemistry application. In general, it is better to use low-surface energy materials because they nonspecifically bind to DNA / RNA / proteins less.

[0096] To form 3-dimensional geometry (fluidic layer) from the raw flat web material, a section of the web is brought through a heating station 421 that brings the material to a softened state, then the web is then indexed forward so the softened section is brought to the forming station 422 which contains a mold of the 3D geometry that is being formed. Automated clamps pull the web material for a defined distance in a defined time interval and stop for a defined duration for all the stations to perform their tasks simultaneously.

[0097] Some examples of pressure-forming molds can be seen in FIGURE IE. Both negative and positive molds can be used. FIGURE 3D shows one embodiment of a SIMPLE chip mold 390 used to examine the results of using positive extruding geometries 393 versus negative inset features 392. Channel cross sections have better fidelity with the positive mold features, but the negative mold 392 released the webbing material after forming better. The (webbing) plastic material shrinks slightly, typical for all molding processes. When the materials shrink, they release themselves better from negative molds and sometimes “grab” onto positive molds. The insert mold has a securing shelf 394 to counter the grab force. This securing shelf helps the mold insert to lock into adapter plate 468a (see FIGURE 4F). Draft angles 395 help release the web from the mold. The softened material is forced against the mold shape by vacuum or compressed air, and as the material cools, it sets to take on the form of the mold. Vent holes 396 allow air to vent. A chip after it has been formed at this stage can be seen in FIGURE 4G.

[0098] Returning to FIGURE 4C, in the next step, the web with formed geometry is indexed forward to a pick-and-place robot station 429 or dispensing station that places dried or wet reagents onto the chip. An index is the unit cycle for the R2R process, where the entire web is moved forward and stops for processing (forming, dispensing, sealing, cutting, packaging, etc.). The index typically lasts for only a few seconds per cycle. With basicoptimization, we have routinely run 4-second cycles, which is much faster than injection molding, and the cycle time can be reduced further. Each cycle applies all the manufacturing processes simultaneously but on SIMPLE chips at each various process stage. The number of SIMPLE chips that can be made each cycle depends on how many SIMPLE chips can be made on a mold.

[0099] FIGURES 4G-4J show various stages of the devices lOOa-d, viewing from the transparent cover layer, towards the fluidic layer, i.e., viewing the inside surface of the fluidic layer. During the reagent / pellet dispensing step, lyophilized amplification reaction pellets can be placed into the microwells and valve material, such as wax pellets on the wax loading stage 472 shown in FIGURES 4G and 4H. The wax loading stage 472 (see also wax loading stage 103 of FIGURE 1A) can be aflat area or shape-conforming area on the SIMPLE chip fluidic layer to temporarily secure the wax pellet before the wax is melted to close the wax valve. The wax loading design feature is optional. Optionally, the wax pellet (or other valve material) can be made by punching pellets out of thin layers of material, or the valve material could be dispensed directly as a liquid state using a heated dispenser and optionally allowed to cool and solidify at the desired location.

[0100] Next, a cover layer is sealed over the fluidic layer to create a closed system. The cover layer (lidding material) can be dispensed from a rolled format 425 and overlaid onto the fluidic layer in a continuous R2R process.

[0101] This material may be one or a combination of an optically transparent material, a reflective material (enhance fluorescence signal), an opaque material, a dark material (decrease fluorescence background), PCR tape, a pressure-sensitive adhesive (PSA), a silicone adhesive PSA, a polyolefin backing based PSA, a PP backing based PSA, a peelable lidding material, an acrylic adhesive PSA, a biodegradable material a barrier layer such as aluminum foil based lidding material, or a layer that requires heat to seal. The cover and fluid layers should have biocompatibility and / or chemistry compatibility with the reactions. For sealing films to the fluidic layer, a heat or pressure station is used 424. Heat sealing, ultrasonic sealing, or pressure (adhesive) sealing may be used to seal the cover layer.

[0102] Next, optionally, a puncture hole 474 is made through the backing material in the area of the wax cooling chamber to allow for gas transfer during wax valve heating shown in FIGURE 4H and later vacuuming steps. The puncture hole can be anywhere downstream of the wax valve. Additionally, at this step, the sample insertion port 110 at the top of the chip can be cut into the backing layer 100b. Optionally the sample insertion port at the top of the chip and or puncture hole at the wax cooling chamber can be precut on the cover layer, so when the cover layer is sealed to the thermoformed (or cold-formed) material, there is already a sample inlet hole (see FIGURE 3B). In this case, the cover layer must be aligned precisely with the fluidic layer during each indexing cycle. The puncture hole and cover layer’s sample insertion port act as vents and prevent the wax from being pushed out of the wax valve channel undesirably during the manufacturing melting step described below, allowing both sides of the melted wax plug to maintain the same air pressure. If the vents are absent, the wax can be pushed out of the wax valve area very easily, which is undesirable. An imbalance of pressure causes this wax dislocation.

[0103] Next, the web is indexed forward into a station that heats the solid wax puck, which causes it to liquefy and enter the wax valve channel 472 shown in FIGURE 41 by reflow. Optionally, gravity, capillary forces, or slight air pressure may be used to ensure the wax flows into the wax valve channel. For example, gravity or capillary action can allow the wax to flow downward from the wax loading stage toward the wax cooling chamber. The sudden expansion from the wax valve channel to the wax loading stage creates a natural stop valve (capillary burst valve) so that wax does not flow beyond the wax valve channel during manufacturing. The chip then progresses forward into an unheated zone where the wax cools and solidifies in the wax channel to create a seal. Optionally, the valve material, such as wax, is dispensed as melted or malleable (possibly heated) state into the wax valve channel directly instead of the wax loading stage, and the cover layer is placed and sealed directly. The wax automatically seals off the stop valve area in one single action, so no additional wax heating and reflow is needed. This method could reduce manufacturing steps.

[0104] At this point, optionally, the inlet sealing tape roll 109 and a tape flap are placed in position shown in FIGURE 4J. The inlet sealing tape roll 109 is located at the inlet 110. The tape flap 476 is located at the puncture hole 474. The tape flap 476 may have a slight kink, so it naturally avoids sealing the puncture hole 474. The tape flap is placed in a position so that when it is pressed down in a later step (during the vacuum sealing process), it will cover the vent hole 474 that was created earlier in FIGURE 4H over the wax cooling chamber. The tape flap can be made of pressure-sensitive adhesive (PSA).

[0105] In an alternative embodiment outlined in FIGURE 3C, the tape flap and cover layer are a single piece as the cover layer. In this embodiment, there is no need to puncture the puncture hole and use an additional PSA to seal the puncture hole on the cover layer. During the wax pellet melting process, the cover layer can partially seal the thermoformed (or cold-formed) fluidic layer. Hence, no trapped air can cause undesirable expansion and movement of the wax valve manufacturing process. The partial seal can avoid complete sealing of the cover layer to the fluidic layer downstream of the wax valve. The partial seal serves as a vent instead of the puncture hole, keeping both sides of the wax under equal air pressure by not fully sealing the fluidic circuit. The partially sealed cover layer can be fully sealed during the vacuum packing process (without the additional tape flap), by applying external pressure. In this embodiment, the device only has one layer of 3D fluidic structure, one cover layer, and a sample inlet hole cover. So, only two main structural assembly layers are needed with an additional inlet hole cover. The minimal use of parts is advantageous to lower the cost of manufacturing.

[0106] The sealing tape roll 109 can be placed near the inlet together or in any step after the cover layer is applied. The sealing tape roll can be made of rolled-up PSA material. It should be secured near the inlet and can be unrolled while closing the reader door, which causes the inlet hole cover to seal the consumable chip. The PSA material can be flexible and / or elastic. It can be a single-sided PSA material. The flexible or elastic backing material can help reduce the microwell loading variation due to less impact on the downstream vacuum suction from the microwell area once the wax valve melts. The sealing tape roll can deformslightly and help maintain pressure in the main loading chamber 101 near ambient pressure once sealed. The sealing tape roll may optionally have a solid core for structural support.

[0107] Next, the chips are cut from the web into individual consumable chips using the cutting station 428. The cutting station may have a die that custom cuts each chip to the desired shape, similar to cookie cutters. The rewind scrap station collects all the excess web material 426. The chips are placed in a vacuum-sealable bag. Note that liquid (such as lysis buffer) is not stored in the vacuum bag but in external containers, such as the sample collection tube, as liquid vapor pressure can degrade vacuum pressure during storage and reduce lyophilized reagents' shelf life. Chips then pass through the open entry door of the in-line vacuum chamber, where the entry and exit doors of the chamber then close, and the inside of the chamber is brought to a vacuum pressure. The puncture hole 474 allow air inside the chip to escape, forming a “pre-stored vacuum.” While inside the chamber, the vacuum bag is heat sealed so the vacuum pressure is maintained inside the bag. The entry and exit doors of the chamber then open and expose the chip in its vacuum-sealed bag to ambient pressure. The vacuum-sealed bag can be made with (aluminum) foil backed with heat-sealable polymer coatings or high barrier (MVTR, OTR) bags (that are optionally transparent). The bags should retain the vacuum throughout the shelf life of the consumable chip. Ambient air pressure causes the vacuum-sealable bag to tightly wrap around the chip, which forces the tape flap (or cover layer) that was placed earlier to be pushed down flat and seal the vent (hole) over the wax cooling chamber, forming an entirely closed-off vacuum environment inside the microwells, and wax cooling area, and connected channels to these parts. This vacuum is the basis that drives fluid flow and controls the air bubble sizes in subsequent operations. The advantage of this design is that the consumable is a passive system for fluid actuation and does not require complex and costly external equipment such as pumps, valves, and actuators to drive the fluid flow, which helps to reduce the overall system cost.

[0108] In a different embodiment, the vacuum sealing process can be a batch rather than an inline process. For example, many consumable chips may be pooled on a tray and simultaneously sealed inside a large vacuum chamber.

[0109] In another embodiment, the large vacuum may optionally have actuators that press down the cover layer (or puncture hole tape) while under the vacuum to help form a fully sealed chip that retains the vacuum. The large vacuum chamber can also have heat sealers that seal the external vacuum seal bag material.

[0110] At this point, the construction of the consumable chip is complete, and the secondary' packaging phase can begin, where the consumable chip is put into a package with the other required testing components such as a swab, a sample dropper tube containing (lysis / elution) buffer, a waste bag, instructions, and other items. The chip is stored at an adequate temperature and pressure until it is ready for use by the tester.[OHl] In a different embodiment, the consumable chip can be formed via cold forming instead of thermoforming. Cold forming materials include, for example, but are not limited to, aluminum films, aluminum foil with heat sealable backing, aluminum foil with various polymer backing, aluminum foil with polypropylene backing, aluminum foil with Aclar backing, aluminum foil with peelable seal backing, etc.

[0112] In another embodiment, thermoforming could be done by positive air pressure, vacuum pressure, mechanical pressure, or a combination.

[0113] In another embodiment, the consumable chip can be made with hot embossing.

[0114] In a different embodiment, reagents can be dried in-line onto the consumable during manufacturing. For example, instead of dropping lyophilized reagents into the microwells, liquid reagents may be placed in the microwells and dried during the R2R process. Instead of lyophilization, a drying process can be used to dry and stabilize the reagents.

[0115] In a different embodiment, the cover layer may be made of a rigid material, or the 3D structured fluidic layer may be made of a rigid material. Both parts may be made of ngid materials, which will be discussed below.

[0116] In a different embodiment, the sealing station 424 brings two plates together to increase pressure and seal the cover layer to the fluidic layer. The sealing station may be room temperature or heated. One of the plates can be flat and smooth (or optionally with a knurled texture to increase sealing strength). The other plate has seal molds with raised metalor rubber gaskets around the fluidic channel features, FIGURE 3E shows one embodiment of a pressure sealing plate 397 and gasket 398 used to seal the sample prep module of a Genl SIMPLE chip. The sealing plate 397 can have cutouts 399 for various features of the SIMPLE chip. The gasket 398 can be made from an elastic material, such as a rubber gasket, or can be a unibody metallic raised section. This helps increase pressure on the areas that need sealing to ensure a better leak-resistant seal. Rubber gaskets increase the evenness of the pressure distributed. Metal gaskets are easier to prototype and more durable. An extra layer of soft material, such as rubber, placed underneath the seal molds can further increase pressure evenness. Alternatively, the extra raised metal or rubber gaskets can be on the top plate, and a simple negative mold, having opposite negative features of those of the mold shown in FIGURE IE, can be on the bottom plate.

[0117] In another embodiment, large radius turns in FIGURE IE can be made for the wax valve channel 105 (see FIGURE 1A) to allow better endmill access during mold manufacturing. To make the SIMPLE chip more rigid, more prominent profile perimeter ridges 104 (see FIGURE 1 A) can be made on the mold for forming.

[0118] In a different embodiment, the valve material can be made of soluble materials that dissolve and open upon contact with liquid. Dissolvable valves may have a time delay and open after a specified time range after they come into contact with liquid.

[0119] In a different embodiment, the mixing element, such as piezo material, can be embedded directly into the SIMPLE chip. b. Mold quick change adaptor assembly

[0120] The embodiments of the present disclosure include a quick change mold assembly allowing rapid, low-cost, and iterative R&D of R2R-made devices. This method can also be used for manufacturing purposes. Previously, swapping traditional R2R molds required substantial effort to disassemble the whole molding assembly. The whole molding assembly is typically attached to the R2R line itself, and removing the whole assembly can further involve complex disassembly of the R2R line components (such as pneumatic linear guides). The new quick-change method allows for very efficient changing of new molds.With this process, a new mold can be designed, made, and implemented in-house within just a few hours of work, which makes R&D processes extremely fast. Since the insert mold 456 (FIGURE 4E) is much smaller than the non-quick change mold 448 (FIGURE 4D), it is faster and has a lower manufacturing cost.

[0121] FIGURES 4D-4F show standard thermoforming mold assemblies, with FIGURE 4D showing a “non-quick change” mold adapter assembly 440 and FIGURES 4E and 4F showing an embodiment of a “quick change” mold adaptor assembly 450. In the non- quick change mold adapter assembly 440, pressurized air enters a top plate 442 through a pneumatic fitting 441 and is ejected through an orifice 444 on the bottom of the top plate 442. In the quick change mold adapter assembly 450, pressurized air enters a top plate 452 through a pneumatic fitting 451 and is ejected through an orifice 454 on the bottom of the top plate 452. During machine operation, this pressurized air is forced against a softened plastic web material, causing the material to take on the shape of a mold 448 (FIGURE 4D) and 456 (FIGURE 4E).

[0122] In the non-quick change version shown in FIGURE 4D, the mold 448 is made from a larger block of material and, during fabrication, requires more material removal on each face of the mold. The large mold is costly to fabricate. Additionally, the entire assembly must be removed to switch mold plates. In the quick change mold adapter assembly 450 shown in FIGURE 4E, the insert mold 456 is a single thin plate insert with features cut from only the top surface, causing it to be more cost-effective to make. The insert mold has small diameter (~0.5mm) vent holes in or around the fluidic features to allow air pressure venting, allowing better-forming fidelity. In the quick change version, the insert mold 456 fits into the opening on the surface of the mold adaptor plate 458. The mold adaptor plate has grooves 457b to allow air to vent out and ridges 457a to support the mold 456. The grooves 457b have multiple small venting holes so air can vent out, or a vacuum can be applied. Additionally, this insert mold 456 can be inserted and removed without removing the rest of the assembly.

[0123] At the bottom of the assembly, in both the standard and quick change embodiments, is a plate that circulates chilled fluid 449, 459 to keep the mold assembly fromgetting too hot, which can negatively impact forming results. Alternatively, warm fluid may be supplied to elevate the mold temperature. In some cases, an elevated mold temperature can increase molding fidelity. The ridges 457a assist heat transfer from plate 459 to the insert mold. The insert mold can be made by CNC machining or 3D printing.

[0124] FIGURE 4F shows detail of an embodiment of a quick change mold adaptor assembly 460. In this embodiment, the insert mold 466 has a shelf 467 cut into its surface on one side that is used to help secure the mold 466 in the adapter plate 468. The adapter plate 468 has a matching undercut shelf 468a cut into it and securing screws 465 on the opposite side to secure the mold 466 and prevent it from being ejected from the assembly 460 during operation. The pictured mold adaptor plate embodiment can also have a heating or cooling pad 463 and temperature sensor 464, allowing mold temperature control. The embodiment may also have a plate 469 that circulates chilled or hot fluid to regulate the insert mold’s temperature. In another embodiment, cold forming instead of thermal forming may be used. c. Example embodiments

[0125] A collection of example embodiments providing additional description of a variety of embodiment types in accordance with the concepts described herein are provided below. These examples are not meant to be mutually exclusive, exhaustive, or restrictive; and the claimed subject matter is not limited to these example embodiments but rather encompasses all possible modifications and variations within the scope of the issued claims and their equivalents.

[0126] Independent Claim:A roll-to-roll manufacturing process that comprises the steps below: forming a 3D structured first layer that includes a fluidic circuit; placing valve material for forming a valve at an outlet of a loading chamber and reagents into an assay well of the first layer; operably coupling a second layer to the first layer to seal the fluidic circuit therebetween, wherein during the step of operably coupling air is removed from the fluidic circuit to create a vacuum; andclosing the valve such that the valve occludes the fluidic circuit between the outlet of the loading chamber and the assay well to entrap the vacuum in at least the assay well.

[0127] Independent Claim:A quick change mold adaptor assembly for roll-to-roll manufacturing, comprising: a mold adaptor plate having vents and a supporting structure for an insert mold; an insert mold is secured to the adaptor plate; wherein the interfaces with the supporting structure of the adaptor plate during the roll-to-roll manufacturing.

[0128] Dependent Claims (based on Independent Claims):Forming step can be cold forming or thermoforming.Vacuum sealing and closing off the cover layer so the consumable device retains vacuum internally.This manufacturing process requires as little as two major film layer assemblies (the fluidic layer and cover layer) to produce a vacuum fluidic consumable device.Valve materials can be wax. paraffin, hydrocarbon polymers, fluorocarbon polymers, grease, etc.Valve material can be melted upon heating.The valve can be made of soluble materials that dissolve and open upon contact with liquid.Dissolvable valves may have a time delay and open after a specified time range after it gets into contact with liquid.It can have pick-and-place machines.The second layer can have pressure-release vents.Adding an inlet sealing apparatus (sequence can change for this step).Can have liquid or solid dispensing stations.Has quick change thermoform or cold form stations.Has seal molds with raised rubber gaskets around the fluidic structures.It has seal molds with raised metal gaskets around the fluidic structures.The first layer can be made from one or more of the following materials: thermoplastics, Poly Propylene (PP), Polyvinyl chloride (PVC), Cyclo Olefin Polymer (COP), Cyclic olefin copolymers (COC). polyethylene terephthalate (PET), APET (Amorphous Polyethylene Terephthalate), polyvinylidene chloride (PVDC), PS (Polystyrene), Polychlorotrifluoroethylene (PCTFE), PETG, HIPS, fluoropolymers, Aclar, polyolefins, silicones, elastomers, thermoplastic elastomers, proprietary blends, multi-ply structures, paper, biodegradable materials, cold-forming material, thermoforming material, or a blend of any of the above as a part of a multilayer laminate.The second layer material may be one or a combination of the following- an optically transparent material, a reflective material (enhance fluorescence signal), an opaque material, a dark material (decrease fluorescence background), PCR tape, pressure-sensitive adhesive (PSA), silicone adhesive PSA, polyolefin backing based PSA, PP backing based PSA, a peelable lidding material, acrylic adhesive PSA, a biodegradable material, a barrier layer such as aluminum foil based lidding material, or a layer that requires heat to seal.Dried or wet reagents can be placed onto the chip.Reagents can be dried in-line onto the consumable during manufacturing. For example, instead of dropping lyophilized reagents into the assay well, liquid reagents may be placed in the assay well and dried during the R2R process.The steps include storing the chip in a vacuum bag for long-term storage.The second layer may be made of a flexible or rigid material.The 3D structured first layer may be made of flexible or a rigid material.No photoresist is required during manufacturing.Reagents can be dried in-line onto the consumable during manufacturing.The mixing element, such as piezo material, can be embedded directly into theSIMPLE chip.

[0129] 3, Gen2 SIMPLE chip reader a. Overview

[0130] Some expected advantages of the embodiments of the Gen2 SIMPLE chip reader include, but are not limited to:1) All reusable components (optics / electronics / heaters, etc.) placed on the reader side to amortize cost and minimize SIMPLE chip cost;2) Reader design has no moving robotic parts to minimize cost;3) Stand-alone mode and multi-reader mode for low and high throughput settings;4) Acoustic mixing allows relatively faster kinetics for bio / chemical reactions such as isothermal amplification; and5) Use of audible range acoustic transducers and amplifiers significantly reduces cost compared to using ultrasonic transducers and high- frequency amplifiers (or piezo drivers).

[0131] Embodiments of the present disclosure will now be described in detail. The optical reader 500 shown in FIGURE 5A is designed to allow ultra-low-cost diagnostics. All reusable components, such as optics, electronics, heaters, acoustic transducers, etc., are placed on the reader side, and all the single-used components are on the SIMPLE chip consumable. This design allows the consumable to be manufactured at an extremely low cost. Components have been carefully selected to minimize cost, including audible range audio transducers that standard speaker amplifiers can power. Acoustic mixing is included, which dramatically increases kinetic speeds. For isothermal amplification-based chemistry, good mixing directly translates to a better limit of detection, faster assay time, and higher fluorescence signals. There are no robotic or moving motor parts, which further reduces cost. It can be used as a standalone system, where the smart device (smartphone / tablet / computer) app is optional, orit can be used in a parallel format, where an optional app communicates with each reader and unifies all user interaction to a single interface on an app on the smart device.

[0132] Uniquely, the reader allows the SIMPLE chip to be loaded and unloaded without using any adhesives to secure the SIMPLE chip permanently. We found that audio range transducers coupled with audio amplifiers provide sufficient vibrational energy to effectively mix SIMPLE chip liquid contents without permanently securing the SIMPLE chip to the vibrational element. We found that audio amplifiers with auto impedance matching features best drive different acoustic transducers (including audible range transducers, haptic elements, and piezo-based transducers).

[0133] Design-wise, it is possible to use a complete sheet metal construction for the shell casing, significantly reducing its cost. The reader can include a touchscreen as a human interface with operators. Alternatively, the shell casing can be made by injection molding, metal forming, hydroforming, thermal forming, and vacuum forming. It is designed to be smooth on the surface to disinfect and clean easily. The screen has a slight angle to increase ergonomics. It has no pressure or vacuum sources to drive fluidics within the SIMPLE chip. The touch screen 502 and the inclined plane holding the screen can be situated above the door opening side instead of the current side shown in FIGURE 5A. This would allow multiple readers stacked like a row' of books on a shelf, w here all the doors and screens are on the same side for easier access. The readers shape profile can also be redesigned to be stacked both vertically and horizontally.

[0134] In an alternative embodiment, an electrical sensing-based version reader is possible without optical components. The reader may be compatible with electrochemistry, capacitive, magnetic, and impedance sensing methods.

[0135] In an alternative embodiment, other types of optical sensing instead of fluorescence may be used, for example, colorimetric methods, optical absorption methods, spectroscopy, near-field optics, far-field optics, total internal reflection fluorescence (TIRF), surface-enhanced ramen scattering (SERS), surface plasmon resonance (SPR), darkfieldoptics / microscopy, chemiluminescence. Interferometers-based techniques, and or other optical methods to sense scattering.

[0136] In another embodiment, the reader may have air pressure sensors to monitor ambient air pressure. Variances in ambient air pressure may influence the air bubble size in the microwell, affecting reaction kinetics and mixing optimal resonance frequency. If the air pressure is out of range for the SIMPLE chip to run successfully, the pressure sensor can notify the user, prevent, or halt the test routine. Alternatively, the acoustic resonance frequency for mixing may vary according to ambient air pressure conditions. Optionally, the reader may have air pressure regulation to maintain a constant internal pressure range while the SIMPLE chip is being tested.

[0137] Some or all reader design features are cross-compatible with other reader embodiments and the open reader. b. Sliding mechanism and base

[0138] FIGURES 5A and 5D shows the sliding mechanism 504 and base design. The purpose of the design is to allow the user to slide the processing backplate assembly 510 (FIGURE 5C) in and out of the reader 500 consistently. It allows the automated inlet sealing module (described in another section) to work by providing structural support. The sliding mechanism is designed to hold positions in either a closed or open state. It can slide outside of the reader door to enable user access. It is made of low-cost components. The base 531 attaches to the bottom of the reader and can be made of sheet metal. The sliding mechanism 504 can include a fixed sleeve bearing carriage 532, a position-hold magnetic pair 533, a sliding sleeve bearing carriage 534, and sleeve bearing guide rails 535. The backplate assembly 510 attaches the sliding sleeve bearing carriage via the backplate assembly’s base 525. The position-hold magnet pairs allow the carriages to be latched temporarily in the fully extended or retracted state. Rubber feet 536 may be included at the bottom of the base. The rubber feet 536 provide extra acoustic and vibrational isolation to the external surface that the reader is placed on. c. Alternative embodiments of reader mixing mechanism

[0139] As shown in FIGURE 5B. the gen2 prototype reader’s backplate assembly 510 utilizes an audible acoustic transducer 511 to mix contents inside the consumable chip. These acoustic transducers are similar to loudspeakers, although the diaphragm part is absent. FIGURES 5E and 5F show that an electromagnetic coil converts electrical signals into vibrational movement. That movement translates to an (optional) acoustic transducer oscillation plate 541, which in turn shakes the processing backplate 505, which in turn shakes the SIMPLE chip 100. When tiny air bubbles are inside the SIMPLE chip’s fluid features, a vigorous enhanced mixing is possible when the vibration approaches resonance with the air bubbles. The air bubble’s meniscus air-liquid interface vibrates vigorously, translating to fluidic mixing.

[0140] An alternative embodiment of the reader processing system uses thin-layer piezo haptic generators or piezo surface speakers (e.g., Piezolisten) on the processing backplate 505. Piezo surface speakers allow flat-screen TVs to create sound using the screen without a separate speaker diaphragm. They are attached behind the screen and are shallow profile. FIGURE 5G shows piezo surface speakers 533 on the surfaces of the Peltier heaters 545 to mix the contents inside the consumable chip instead of the acoustic generator. An aluminum (or other metal) cover plate 552 that houses the thermistor 554 is placed between the peltier and piezo surface speaker. It helps to spread the heat uniformly on the heater surface. Wiring 542 can optionally come out from the side to keep the profile low. The chip load plate 506 can be kept clamped closed to the processing backplate 505 during testing using the clasping magnets 513, and the chip load plate can be opened after testing. These piezo haptic generators function through the entire audible frequency range (some can operate in ultrasonic range) and can be fine-tuned to resonate at a frequency that maximizes mixing in the chambers and microwells. The advantages are that a smaller form factor can be achieved, the target area is directly in contact with the mixing device, less weight, and a regular audio amplifier can also power it. Disadvantages are possibly higher cost and possibly more noise produced.

[0141] Another embodiment is bar-shaped piezo haptic elements 610 (e.g., Haptic Bars) to drive vibration (see FIGURE 6C). Other types of transducers may be used to induce acoustic mixing, for example, and not limited to. piezo disks, stacked piezo transducers, bone conductors, ultrasonic transducers, atomizers, ultrasonic horns, speakers, other piezo transducers, and audio transducers. Resonance frequencies depend on the size of the air bubbles and transducer / amplifier ty pe. We have found that 20hz~200Mhz works. For the acoustic transducer, 20hz~20khz works well, and lower frequencies of 50hz~4khz work best. The acoustic mixing mechanism works well on R2R and rigid (e.g., injection molded) consumables.

[0142] FIGURE 51 shows an alternative embodiment with a backplate assembly that uses a haptic bar 610, such as the one shown in FIGURE 6C. The haptic bar 610 does not need to be located at a central location in relation to the SIMPLE chip 100, nor does it need to be strictly under the fluidic features that need mixing. A proximal location is sufficient because the chip itself can transfer vibrational energy. There can be sufficient mixing if there is good physical contact with the chip. The haptic bar has one or more contact points with the chip. In this case, a single round dome-like contact point 611 (see FIGURE 6B). FIGURE 51 shows a gen2 SIMPLE chip 100 overlaid on the backplate assembly. The sample is mixed and heated with heater 543 for lysis (with optional pre-amplification) in the main loading chamber 101. The wax valve 105 is heated with heater 544 to open, and the vacuum pulls the sample into the microwells. The heaters 545 heat the microwells, and isothermal amplification commences. There can be several (3) independent heaters 543, 544, 545. The heaters may be slightly protruded, so the SIMPLE chip has minimal contact with the rest of the backplate, to avoid dampening vibrations. Electrical wiring can route from the side to enable a slimmer backplate assembly profile. The backplate assembly sits on a sliding mechanism and base 535. d. Standard optical imaging system

[0143] FIGURES 5 J and 5K show the standard optical embodiment in the gen2 reader used for the R2R SIMPLE chip 100. It includes an optical cube 560 with a light source 561that emits light at a wavelength that matches the fluorescent dye's excitation wavelength used in the molecular amplification reaction. The optical reader images the microwells of the SIMPLE chip. The light source can be an LED(s) or laser. Optionally, diffusers, lenses, and reflectors can be placed in front of the light source to shape and disperse the light so that light is illuminated evenly onto the SIMPLE chip. There is an excitation filter 562, an emission filter 563, a dichroic mirror 567, and a camera 565 to capture the fluorescent image. Optionally, a lens system could be in front of the camera to allow for proper magnification and / or field of view.

[0144] The optical cube 560 could have an extended chamber 566 placed on the opposite side of the light source 561. The extended chamber helps reduce light scattering. Light scattering impacts the signal-to-noise ratio. Using an optical cube optimized for green fluorescence detection as an example, when the (dominantly blue) light source hits the dichroic mirror, some of the light source’s (fringe green) light can pass straight through the dichroic mirror if the excitation filter does not filter 100%(green) light. The (green) light can scatter around the optical cube and undesirably pass through the (green) emission filter, causing a higher background. The extended chamber is a space that is further recessed away from the camera, so most of the undesirable scattered light passes into the extended chamber cavity7and does not reflect toward the camera’s direction. The entire inside surface of the optical cube should be matte black and highly absorptive to scattering light.

[0145] In another embodiment, the extended chamber’s wall 564 facing the light source 561 is slanted at an angle (such as 45 degrees) designed to reflect stray light from the light source out of the optical cube 560. Optionally, in this case, one of the sides of the extended chamber does not have a wall to allow the stray light to exit the extended chamber.

[0146] In another embodiment, the optical cube wall facing the light source is not present (or has a hole), so the light source’s stray light exits the optical cube without reflecting back to the camera.

[0147] The optical reader cube 560 is specifically designed for a vast field of view (so all microwells can be imaged in a single shot), extremely compact, high signal-to-noise ratio,but low-cost fluorescent imaging. Embodiments of the optical reader cube 560 can have improved optical performance compared to conventional technologies. For example, compared to the Zeiss fluorescence stereoscope (Axiozoom) system, the optical reader cube 560 achieved a field of view of 27mm vs. 15mm for the Axiozoom, the optical reader cube 560 has a cost reduced more than 200 times, the optical reader cube 560 has a volume decreased more than 100 times, and the optical reader cube 560 has a signal to noise ratio 1.33 vs. 1.09 for the Axiozoom. The field of view extents of the cube 560 is significantly improved compared to the Axiozoom, with substantially equivalent image quality.

[0148] The reader’s software allows time series capture of fluorescence images. It can briefly turn on the light source during each imaging event to minimize photobleaching. The reader has optical analysis software, so the microwell’s fluorescence can be automatically analyzed quantitatively. A mathematical, statistical model automatically determines positives and negatives by comparing the test and control microwell signals. It is possible to determine target pathogen concentration quantitatively, semi-quantitatively, or qualitatively. A baseline reading may be used as a reference to calibrate the optical signal. Markers or stickers with standard fluorescence intensity on the SIMPLE chip may also be used as a fluorescence intensity standard reference. e. Alternative low-cost optical imaging system

[0149] An alternative embodiment of the optics system is shown in FIGURES 5L and 5M. It includes a ring or array of (optionally narrow band) LEDs 571, an emission filter 572, and a camera 573. Instead of a ring shape, the LEDs can be arranged in other shapes as long as the illuminated area is uniform. Again, optional optical diffusers may be used to create uniform illumination, and an optional lens system can be used in front of the camera. The purpose of this optical system is the same as the previous explanations of the optics system: to take pictures of the microwells at a consistent interval during the amplification process to observe whether there is an increase in fluorescent signal intensity, which would indicate the presence of a target. This alternative embodiment omits the excitation filter with narrow-band blue LEDs and the dichroic mirror with angled lighting. The LEDs are placed at an angle(such as 45 degrees or any angle between 5 and 85 degrees) to the sample so that reflected stray light is minimized and to not go back to the camera. If the lights are shining at the sample from the camera’s direction, there would be lots of reflected stray light. If rigid SIMPLE chips (described in other sections) are used, side illumination (0 degrees) can be used because the transparent bulk material of the SIMPLE chip itself can be a light guide. The emission filter size is smaller than the standard optical imaging system. Overall, product casing can be smaller due to more compact optics. Overall, the cost could be lower with this embodiment. The optical filters may be absorptive, interference, or dichroic filters on both the standard and alternative embodiment. In particular, absorptive filters can drive the cost down significantly, although at a slightly diminished optical performance. Again, the optical system can achieve a vast field of view like the standard one. It may also have an extended chamber to reduce stray light scattering issues. Some or all design features may be crosscompatible between the low-cost and standard optical systems. f. Example embodiments

[0150] A collection of example embodiments providing additional description of a variety of embodiment types in accordance w ith the concepts described herein are provided below. These examples are not meant to be mutually exclusive, exhaustive, or restrictive; and the claimed subject matter is not limited to these example embodiments but rather encompasses all possible modifications and variations within the scope of the issued claims and their equivalents.

[0151] Independent Claim:A reader for a molecular diagnostic device manufactured with a roll-to-roll manufacturing process, comprising: an acoustic transducer for microfluidic mixing, the acoustic transducer configured to receive and release the consumable without adhering to it permanently; a heating element configured to melt a wax valve contained in the molecular diagnostic device; anda heating element to provide isothermal amplification in an assay well of the molecular diagnostic device; and a control interfacing having a standalone mode without a linked control application, and a multiplexed mode with a linked control application.

[0152] Independent Claim:A reader that has all physical components that can be reused located on the reader, the reader comprising: an active mixing system.

[0153] Independent Claim:An optical fluorescence imager, to capture fluorescence images, comprising: a camera; an emission filter in front of the camera; no dichroic filter; no excitation filter; an optical window of at least 5mm in diameter: andLED illumination comes in from the side of the imaged sample at 0 to 85 degrees angle.

[0154] Independent Claim:A sliding base mechanism, comprising: a base portion having two latched positions controlled by magnets; a first latched position is a fully retracted position; a second latched position is a fully extended position; enables the backplate assembly to slide in and out of the reader door; and enables the automated inlet sealing module, defined in section 4. to work by providing structural support.

[0155] Independent Claim:An optical cube with the following components: an emission filter;an excitation filter; a dichroic mirror; and an extended chamber to control stray light scattering.

[0156] Dependent Claims (based on Independent Claims):The reader contains an automated inlet sealing module that seals the diagnostic device’s inlet.The reader works with the gen2 SIMPLE chip, defined in section 1.The reader works with other embodiments of the SIMPLE chip, defined in sections other than section 1.It can also work with injection-molded consumables.Can use a standard optical system.The reader uses an optical imaging system or non-optical electrical method for readout.Includes fluorescence filters and an imaging system.An electrical sensing-based version reader — the reader may be compatible with electrochemistry, capacitive, magnetic, or impedance sensing methods.Other types of optical sensing instead of fluorescence may be used, for example, colorimetric methods, optical absorption methods, spectroscopy, near-field optics, far-field optics, total internal reflection fluorescence (TIRF), surface-enhanced ramen scattering (SERS), surface plasmon resonance (SPR), darkfield optics / microscopy, chemiluminescence, Interferometers-based techniques, and or other optical methods to sense scattering.Complete sheet metal enclosure.Optional touch screen for human interface.Different technologies can be used for the acoustic transducer, such as piezo disks, stacked piezo transducers, bone conductors, ultrasonic transducers, atomizers, ultrasonic horns, speakers, piezo speakers, PiezoListen elements, haptic generators, power hap bars, piezo transducers, or audio transducers.The acoustic transducer is designed to mix fluids in the microw ells by causing vibrations on the air bubble’s liquid-air interface.Acoustic range for mixing (0~20kHz).Ultrasonic range for mixing (20kHz+).The reader works with a consumable that has pre-stored vacuum to drive fluids, the reader does not need pressure sources, vacuum sources, robotics, or motorized moving mechanical parts.Optical filters may be made of absorptive filters, interference, or dichroic filters.It has an audio range amplifier.It has an audio range amplifier with auto impedance matching functions.The reader has some or all cross-compatible design features with the reader for all other SIMPLE chip reader embodiments, including Genl SIMPLE chip.The reader has air pressure sensors.The reader has air pressure regulation to maintain a constant range of internal pressure.

[0157] 4, Automated inlet sealing module for the SIMPLE chip a. Overview7

[0158] Some expected advantages of the embodiments of the Automated Inlet Sealing Module for the SIMPLE chip include, but are not limited to:1) Prevents cross-contamination and reagent evaporation;2) Automatically seals the SIMPLE chip to make it fully self-contained, minimizes user error and variability7in the sealing process; and3) Low-cost design with minimal parts required for both consumable and reader.

[0159] Embodiments of the present disclosure will now7be described in detail. The sealing tape roll 109 is designed to reduce user-induced error, reduce manual steps needed, and seal the inlet 110 of the SIMPLE chip 100 with high consistency at a very low cost. It iscompatible with all SIMPLE chip embodiments. This design may be used for other consumables for different applications as well. For nucleic acid amplification applications, it is essential to make sure all consumables are fully sealed and self-contained because there will be a very high concentration of amplicons post-reaction, which could cause false positives in future assays if there is cross-contamination of the amplicons between SIMPLE chip devices. Amplicons can linger and stay in the environment for a long time and may render the testing site unusable for a long time. Conventional device inlet sealing is done by users manually placing seals or caps onto the diagnostic device, which risks failed seals due to manual error, requires extra steps, and is typically higher cost due to the complexity and material used.

[0160] The sealing tape roll can be a piece of single-sided PSA that is pre-rolled and secured nearby the SIMPLE chip inlet and is unrolled by a mechanical device (such as a roller) 508 when the SIMPLE chip is loaded, and the reader door is closed. The mechanical device may exert pressure on the PSA to induce a better seal with the SIMPLE chip. The unrolling and pressure sealing action may also be done by two separate mechanical devices, such as two rollers. The rollers may have a hard or soft surface. The roller(s) may be spring-loaded to maintain contact pressure. The roller that unrolls the PSA should be made with material that does not stick to the PSA itself, for example, using fluoropolymer material. A soft surface for the pressure-inducing roller can help distribute the roller’s pressure more evenly for a better seal.

[0161] As an alternative to the second pressure-inducing roller, adjacent to the first roller may also include a brush or flexible squeegee-like flap that exerts extra pressure onto the PSA once the PSA is unrolled and fully seals the PSA to the SIMPLE chip inlet.

[0162] In this system, the mechanical action variation is limited, reducing manually caused variation such as alignment or pressure placed on the sealing process. The only significant variable is how fast the door is closed. This variation could be further mitigated by having a motor-based reader door opening / closing system. Alternatively, motors, solenoids, and linear actuators may also be used to close the reader door. The speed of thedoor closing might not affect sealing performance, and if so, no electrical-based actuator will be needed.

[0163] The sealing tape roll PSA may be made of flexible or elastic material. An elastic material further reduces the microwell liquid loading variation during the vacuum loading process.

[0164] An alternative embodiment is that instead of a rolled-shaped PSA, it could be a flap or cap (optionally with PSA) that seals the SIMPLE chip automatically upon closing the reader’s door. Optionally, the flap or cap could be placed manually to cover the inlet, and the roller / brush / squeegee on the reader door exerts pressure to seal the flap or cap automatically.

[0165] Optionally, the rolled PSA may have a rigid inner core material that provides additional stability to prevent rolled PSA from being crushed during the vacuum bag sealing process.

[0166] Optionally, the exposed PSA has a removable liner that prevents the PSA from sticking to the vacuum bag.

[0167] Optionally the sealing tape roll (or cap) is secured to the SIMPLE chip and stored together inside the vacuum bag during manufacturing, or it could be a separate piece stored outside the SIMPLE chip, with the user placing the sealing tape roll (or cap) on the SIMPLE chip during operation.

[0168] Optionally the sealing tape roll, cap, or flap can be sealed directly manually without involving the reader.

[0169] In other embodiments, the inlet sealing action can be done before, during, or after the assay. For example, an immiscible, inert fluid, such as mineral oil can be used to cover the sample fluid in the main chamber, and sealing of the chip’s inlet is performed after amplification. b. Example embodiments

[0170] A collection of example embodiments providing additional description of a variety of embodiment types in accordance with the concepts described herein are providedbelow. These examples are not meant to be mutually exclusive, exhaustive, or restrictive; and the claimed subject matter is not limited to these example embodiments but rather encompasses all possible modifications and variations within the scope of the issued claims and their equivalents.

[0171] Independent Claim:An inlet sealing apparatus, comprising: a mechanical structure on a reader instrument that is configured to seal the consumable with a sealing member when the consumable is loaded onto a loading dock and a door is closed; and closing the door causes the sealing member to press and automatically seal the inlet of the consumable to prevent air ingress into a fluidic circuit of the consumable.

[0172] Dependent Claims (based on Independent Claim):The mechanical roller portion is configured to interface a sealing member that is secured on a consumable during manufacturing.The sealing member is a pressure sensitive adhesive (PSA) roll.The PSA roll is single-sided PSA.The roller portion having a material that may be a fluoropolymer or alternative material that does not stick to the tape.There may be two roller portions on the reader, one to unroll the tape and another to apply pressure on the tape.The roller (s) is spring loaded.There is a roller on the reader to unroll the tape, coupled with a brush or squeegee to apply sealing pressure on the PSA.It works with the roll-to-roll manufactured SIMPLE chip consumable defined in claim set in section 1.It works with some or all embodiments of the SIMPLE chip consumable defined in claim sets other than claim set in section 1.It works with injection-molded consumables.The reader door can be closed manually.The reader door can be closed automatically with electrical actuators, such as motors, solenoids, or linear actuators.The sealing tape roll is flexible (and optionally elastic material).The sealing member can be a flap of tape, a mechanical locking cap, or a cap with adhesives may be used to seal the chip.The sealing tape roll (or cap) may be secured onto the SIMPLE chip during manufacturing and packed within the vacuum bag.The sealing tape roll (or cap) may be outside the vacuum bag and placed onto the SIMPLE chip manually during operation.The sealing tape roll, cap, or flap can be sealed manually without the reader.

[0173] Two-dimensional rigid SIMPLE chips a. Overview

[0174] Some expected advantages of the embodiments of the 2-dimensional rigid SIMPLE chip include, but are not limited to:1) All the same advantages as the R2R manufactured chip, except with a potentially higher cost than the R2R processes, but still straightforward in construction, making it lower cost than other conventional systems;2) As few as two main assembly parts are needed (molded rigid fluidic layer and cover layer);3) Higher fidelity and resolution features possible than R2R manufactured parts; and4) Compatible with injection molding, compression molding, and hot embossing.

[0175] Embodiments of the present disclosure will now be described in detail. An alternative form for the single-use consumable chip is a rigid plastic chip (or elastomeric polymer chip) made via injection molding or other processes (such as hot embossing, stamping, or even R2R manufacturing). Instead of a “film” based roll-to-roll manufacturedchip, a “block / slab” of bulk material is used. Instead of rolled films, it is also possible to input blank extruded plate-shaped material into an R2R manufacturing format and use techniques such as hot embossing on a heated roll drum to form fluidic features on the plates in a continuous manner (and also continuously laminate cover layers like label applicators). While we call these SIMPLE chips two-dimensional, in reality, they could have 3D structures as well, as long as it is compatible with the same 2D manufacturing processes. For example, with injection molding, the same type of mold that parts two ways, as in the 2D design, can be used to create 3D structures by modifying the mold features to be 3D, such as shown in FIGURE 9F.

[0176] Higher resolution features may be made with these methods, although possibly costing more than film-based R2R-manufactured chips. An example of this type of flat rigid plastic chip can be seen in FIGURES 9A-9F. These chips function in the same way as the processing of the chips described in the earlier section regarding film-based R2R SIMPLE chip processing, in that they contain (wax) valves that separate chambers that can store vacuum pressure, reagents or other contents. They can be manufactured with as little as two major assemblies: a rigid fluidic layer and a cover layer. The cover layer may be rigid or flexible. It may also be PSA.

[0177] Some or all of the designs, materials, and manufacturing methods of the 2D rigid version and other SIMPLE chip embodiments are cross-compatible and can be crossimplemented. For example, features such as fluidic channels, microwells, sealing tape roll, etc., can be made in either manufacturing format. However, the detailed design specifications must fit within manufacturing constraints (such as aspect ratio, resolution, channel crosssection, draft angle, etc.).

[0178] The fluidic layer and or cover layer can be made with a variety of materials, such as and not limited to silicon, glass, quartz, ceramics, elastomers, PDMS, silicones, thermoset polyester, thermal plastic elastomer, thermal plastic polymers, thermal set materials, polypropylene, polystyrene, polycarbonate, poly-methyl methacrylate, poly-ethylene glycol diacrylate, perfluorinated compounds, PVC, polyurethane, Cyclic-olefin copolymer (COC),cyclic olefin polymers (COP), etc. It can be made of a blend of these materials as well. For optical detection, at least one between the cover layer or fluidic layer should be optically transparent (and low autofluorescence if fluorescent readout). The fluidic layer can be transparent, nontransparent, or reflective.

[0179] FIGURES 9A-9C show chips 900a-c. For example, chip 900b has an inlet 901 on the top of the chip, which can be tape-sealed or cap-sealed. Optionally, the inlet can also follow an in-plane format with the gen2 SIMPLE chip cover layer 100b, which is compatible with the sealing tape roll design. FIGURE 9B shows the microholes 902 and side pockets that trap air bubbles in the sample collection chamber, in which the trapped air bubbles enhance acoustic mixing. The microholes can be through-holes or blind holes manufactured into the chip’s material. The microholes can have a cylindrical shape or other shapes configured to aid trapping air bubbles. If through-holes are made, the outside of the chip can be sealed with a cover layer, such as PSA, to prevent liquid leakage. The microholes’ volume can be modulated to change the acoustic resonance frequency. We demonstrated acoustic mixing using magnetic beads for visualization. A liquid mixture containing magnetic beads is initially injected into the sample collection chamber. A magnet is used to concentrate beads. Once the acoustic transducer is turned on, acoustic vibrations cause the beads to mix immediately within 1-2 seconds.

[0180] FIGURE 9B shows the junction 903 where the wax valve is located and the wax cooling area 904. It is designed with the same principles as the R2R SIMPLE chip 100; specifically, it has a sudden expansion so the wax does not flow beyond the narrow channel above due to capillary pinning. It has a larger channel cross-section and optionally shifts to the side horizontally to allow the melted wax during processing to cool down and solidify before entering the microwells 905 that have vacuum pre-stored in them. Multiple shapes of the microwells 905 can be used to test the best effect for acoustic mixing. Air bubbles are trapped in the microwells and enhance acoustic mixing. Some of the mi crow-ells have features (such as small side channels) to enhance air bubble capture. Optionally the small side channels can be orientated in a pattern that promotes mixing circulation in one direction (suchas pointing the channels at a tangent or angle to the microwell). Returning to FIGURE IF, a trapped air bubble 151 can be seen in the microwell 108. The size of the air bubbles can be modulated on the level of vacuum pre-stored in the consumable during manufacturing. Optionally, the microwell shapes, such as the microwells 905, can have an asymmetrical design to enable better acoustic mixing (generate a net directional force, such as a circular mixing pattern inside the microwell). FIGURE 9A shows a rigid SIMPLE chip 900a with three fluidic sections: sample loading chamber 907, second chamber 908, and microwells. In this design, two-step amplification or nested amplification can be done. The sample loading chamber 909 of chip 900c in FIGURE 9C can include a narrowing slope towards the bottom wax valve area to minimize unwanted large air pockets being accidentally trapped near the wax valve inlet during sample loading. FIGURE 9C also shows a standard round-shaped microwell 908 format. Microwells can be used for isothermal amplification or PCR.

[0181] FIGURES 9D and 9E show alternative 2D rigid SIMPLE chip designs that allow for multi-step reactions (such as two-step amplification or nested amplification) with extra large fluidic wells (sample collection well 911 and another extra fluidic well 912). The upper sample collection well can be used to neutralize the lysis buffer (optionally neutralization reagents can be pre-stored here). The lower fluidic well can be used for first stage isothermal amplification. Multiple sets of primers may be used for multiplexing. It has two wax valves normally closed until melted open 914 and two small wax cooling areas 915. FIGURE 9E shows an alternative wax cooling area shape with a larger chamber 921 and lateral displacement. FIGURE 9D includes a heat-to-close valve 913 that is normally open. These valves contain a wax puck that does not obstruct the vent initially, and after melting from a heat source on the reader, melted wax flows and wicks into a channel blocking the vent (chip is orientated vertically). The vent is designed to allow air to vent out while the sample is loading if the sample inlet 916 is loaded with a device that does not allow air to vent out, such as a needle, syringe, or an injection device that fits perfectly with the inlet. The same principle of the heat-to-close valve can be used to completely seal a chip after sample loading to prevent cross-contamination and is an alternative design to the sealing tape rolldesign. FIGURE 9E shows an alternative heat-to-close valve 922. In this case, the vent is a puncture through the cover layer at or upstream of the wax valve instead of a physical channel to the side of the chip. After first stage amplification, the post reaction fluid is sucked into the microwell area 918 for second stage amplification or probe-based detection. The microwell shapes can be varied to evaluate their effect on acoustic mixing.

[0182] In another embodiment shown in FIGURE 9F, other types of valves, instead of a wax valve, may also be used as specified in the R2R SIMPLE chip. The general concept uses a puncture action to open a film or thin-walled section that serves as a valve (similarly to the integration pin concept described in other sections). For example, instead of wax valves, a puncture valve 931 may create holes through the chip's films or thin wall sections 936.

[0183] In another embodiment, the 2D SIMPLE chip may also be coupled with other fluidic circuits, such as an additional sample tube 932 that contains the sample, and the user places it onto the SIMPLE chip’s inlet. A cap 933 can be screwed on the sample tube, forming a seal, and that action compresses a sharp needle-like structure 934, which moves own and punctures through a cork section 935 and also a thin-walled section (or film) 936 at the inlet of the vacuum fluidic circuit. At this point, the fluid can freely flow from the sample tube into the fluidic circuit and microwells 937, which have a pre-stored vacuum. Again, air bubbles can be trapped with the same principles described in other sections. The cover layer (e.g., PSA) can be on the bottom 938. Note that the vacuum chip is operating in a horizontal position here, though other positions may be used, too.

[0184] The cork section serves as a fluid stop so that when the sample buffer is loaded, it does not leak out of the sample tube prematurely. The cork can be made in one piece with the same material as the sample tube (just being a thinner walled section), or it can be a separate piece such as an elastomeric plug-type material or lidding material (e.g., foil). The needle can be optionally secured on the cork (but not puncturing the cork) to provide stability and alignment. Other designs may be done, for example, having the cover layer on top instead of on the bottom of the SIMPLE chip. The cap may optionally have a hydrophobic membrane-covered vent 939 to allow air pressure to balance inside the sample tube duringthe vacuum loading process. The cap can still work without the hydrophobic vent, as the internal pressure inside the sample tube will still be higher than the vacuumed areas in the fluidic circuit, so the fluid flow will still commence. The screw cap may be a push cap. The cap and the sample tube may have mechanical structures (such as reverse barbs) that only allow assembly once and cannot be reversely opened again. The sample tube can optionally be screwed into the SIMPLE chip or pushed into the SIMPLE chip.

[0185] The embodiment shown in FIGURE 9F can be easily made with compression molding or injection molding with very simple molds. The needle may be a single unified part within the sample tube or the SIMPLE chip. The needle can be an integration pin (described in other sections). The fluidic coupling mechanism is designed so that there is minimal dead volume. Minimal dead volume is possible because fluid pools in the sample tube pool down towards the puncture valve, and the channel leading to the puncture valve has a slight taper. All or most of the liquid can be transferred to the SIMPLE chip.

[0186] Again, all or parts of the design features, materials, and manufacturing methods may be cross-compatible with other embodiments in this patent and may be combined in part with each other to form new designs. The sample tube and SIMPLE chip can be stored separately in different bags if the sample tube contains liquid reagents, as liquid reagents do not store well in vacuum bags. Optionally, the sample tube and SIMPLE chip can be a unibody design, so storing them in separate bags is unnecessary. In this case, the liquid will be dispensed into the sample tube using a separate dropper tube or syringe.

[0187] The SIMPLE chip is stored in a vacuum bag for long-term storage and taken out right before operation, and the sample tube is inserted into the SIMPLE chip.

[0188] Loading many microwells using a vacuum loading format is generally possible, making multiplexing very easy. Vacuum loading is also much quicker than lateral flow- systems. b. Example embodiments

[0189] A collection of example embodiments providing additional description of a variety of embodiment types in accordance with the concepts described herein are providedbelow. These examples are not meant to be mutually exclusive, exhaustive, or restrictive; and the claimed subject matter is not limited to these example embodiments but rather encompasses all possible modifications and variations within the scope of the issued claims and their equivalents.

[0190] Independent Claim:A consumable device that has the following features: can be made with as little as two major layers (a cover layer and a rigid molded fluidic layer); has a portion of the device retaining vacuum; has a valve that can be opened on demand and allows fluids to flow toward the sections with retained vacuum; and has microwells that can trap air bubbles.

[0191] Independent Claim:A SIMPLE chip that has the following features: has pre-stored vacuum on chip; and a heat-to-close vent.

[0192] Independent Claim:A fluidic cartridge that has the following features: has a puncture valve to activate the vacuum loading process; has microwells that can trap air bubbles; and has pre-stored vacuum on chip.

[0193] Dependent Claims (based on Independent Claims)The fluidic layer may be manufactured by injection molding, hot embossing, or compression molding.The cover layer may be made of a rigid or flexible material.Some or all of the claims listed in section 1 -g.

[0194] 6, Reader for rigid 2D SIMPLE chips a. Overview

[0195] Some expected advantages of the embodiments of the 2-dimensional rigidSIMPLE chip include, but are not limited to:1) Low-cost optical system;2) Cross-compatible design features with other SIMPLE chip reader embodiments;3) Includes acoustic mixing;4) Vibrational damping reducing balancing systems included; and5) Fewer steps to load the SIMPLE chip with the clamp mechanism.

[0196] Embodiments of the present disclosure will now be described in detail. Examples of different embodiments of these reader systems that can process the rigid SIMPLE chips can be seen in FIGURES 6A-6C, 7, 8 A-8E, and 10. These readers contain some or all of the same components as the gen2 SIMPLE chip reader. For example, components include heaters for heating contents and melting wax valves, an acoustic or piezo vibration mixing system, and an optics system to image microwells. Some or all reader design concepts are cross-compatible with the gen2 SIMPLE chip reader (and open reader platform described in other sections), as it can have the same components that sen e the same functions. The design concepts can also be translated for a reader for 3D SIMPLE chips described in other sections. The detailed design specifications need to be tailored for each scenario.

[0197] One embodiment of the hard plastic chip reader is shown in a cut-out view in FIGURES 6A-6C. The reader can utilize a haptic piezo bar (e.g., PowerHap Bar) 610 to vibrate and transfer energy into the rigid plastic chip that mixes its contents. A haptic bar’s advantage is that it can make the reader’s form factor very small (versus audio transducers) while operating with low-cost audible range amplifiers. A haptic bar enables much larger vibrational displacement than regular piezo disks because the bar’s mechanical structure amplifies the haptic bar’s piezo movements. The original use for a haptic bar is for human tactile sensory feedback on electronics, so it is designed specifically to transmit significant displacement vibrations. The reader may also utilize Peltier heaters 602. 603, 604 for heating. The Peltier heaters are located on the processing backplate 601. The Peltier heaters may havea temperature sensor to create a feedback loop for precise temperature control. The heaters can be designed to be slightly protruded from the backplate so only the heaters stay in contact with the chip, ensuring thermal contact and maximizing all the vibrational energy transfer to a few local points on the chip to mix desired areas in the chip maximally. A slightly sloped protrusion 605 can be added in front of the raised Peltier heaters so that when the rigid SIMPLE chip 606 slides in from the top side of the reader, the sloped protrusion guides and prevents the chip from getting stuck on the protruded Peltier heater(s).

[0198] FIGURES 7 and 8A show one embodiment of the loading mechanism in detail for the SIMPLE chip. The external casing is not shown here. In the cassette embodiment 700 shown in FIGURE 7, the chip is placed on the processing backplate 706, and then the user closes a chip clasp door 703, which sandwiches the SIMPLE chip 704 and secures the chip in place. Magnets may allow a releasable locking mechanism to lock the chip clasp door and processing backplate. The optical windows 707, 708 allow the optical system to view the chip 704. The optical windows are cutouts or transparent windows for the camera to view the chip. Then, the assembly of the processing backplate, chip, and chip clasp door can be flipped up to the optical fluorescence imaging system 701, which can be again secured by magnets that can be pried open after processing. The optical cube 709 can be removed from the optical fluorescence imaging system, so different optical filter sets can be swapped out quickly or serviced. During prototyping stage, the optical cube 709 can be a modular part, allowing custom mechanical outer shells 701 for the optical fluorescence imaging system, to accommodate it into each new prototype iteration mechanically. For example, the same internal optical cube 709 was used for all three generation readers described herein (genl. gen2, rigid chip reader). The vibrational element, in this case, an acoustic transducer 702 mounted on top of the reader, provides vibrational energy to mix the contents inside the chip. The acoustic transducer can be placed anywhere in the reader if vibrational energy can be adequately transferred to the chip. An electronic enclosure is inside the reader 705, which can be placed in other locations as well.

[0199] In another embodiment, to further enhance acoustic mixing, an extension spring embodiment 800 is shown in FIGURE 8A and can be used to avoid excessively damping the vibrational energy. The backplate assembly with the chip 802 can be held with extension springs 809 that attach to the optical reader 806 (or anywhere else rigid in the reader, not on the backplate assembly). The backplate assembly can be secured on a pivot pin 808. The extension springs prevent the backplate from having too much contact with the rest of the reader so that vibrational energy is minimally damped. The pivot allows the chip to move freely and retain vibrational movement. Optionally, the acoustic mixing transducer 807 can be placed directly on the back of the processing back plate.

[0200] In other embodiments shown in FIGURE 8B, thin sheet metal brace supports 810 may enhance acoustic mixing further and reduce damping caused by friction or contact. The backplate assembly is preferably not in contact with the fluorescence imaging system 801. Two braces are shown in the figure, although just one brace on the bottom or the top is effective, too. The thin sheet metal braces secure the backplate assembly but allow some flex for it to vibrate and minimize damping during acoustic mixing. The SIMPLE chip 802 is inserted from the top. The acoustic mixing transducer is optionally on the backplate assembly.

[0201] Alternatively, as shown in FIGURE 8C. the pivot pin 831 can be at the top. and a magnetic base balancing system 830 is at the bottom. The magnetic base balancing system acts similarly to a spring system but just without physical contact. Magnets on both sides of the backplate assembly can be placed so that the magnets on the backplate themselves repel each other so that the resting position of the backplate is neutral. However, once vibration starts, the backplate can swing between the repulsing magnets with minimal friction and vibrational damping. In another embodiment shown in FIGURE 8D, a spring-based balancing system 840 can replace the magnetic base balancing system. The pivot and the balancing systems (spring, magnetic, sheet metal) can be upright or upside down. For example, the balancing system can be located on the top or bottom of the backplate assembly.

[0202] Turning to FIGURE 8E, the processing backplate 803 and the chip clasp door802 can be preassembled into a single unit with a clamp mechanism 820, 822. The advantageis that users can simply slide the SIMPLE chip in and out of the backplate instead of flipping and closing multiple parts in the cassette embodiment (FIGURE 8A). The clamp mechanism uses springs 820 that push the processing backplate and chip clasp door together. The springs 820 are retained by fasteners 822. Screws can secure the backplate and chip clasp door so it does not over-extend and fall apart. An expanding spring can create an expanding force on the screw and backplate. The screw is secured to the chip clasp door. The result is that the spring pushes the backplate and chip clasp door together so it fits over the SIMPLE chip snugly. The snug fit helps the chip maintain good thermal contact with the heaters and reduces vibrational damping losses. Alternatively, magnets may replace the spring’s function as well. A ramp structure 605 (FIGURE 6B) can allow the SIMPLE chip to slide into the backplate without getting caught on the slightly protruding heating elements.

[0203] The external casing shell can surround the embodiments shown in FIGURES 8A-8E and can have a cap that flips open to receive the SIMPLE chip and closes afterward to prevent external light from coming in, which could interfere with the optical readout. A fully closed enclosure ensures the optical environment is stable across different tests to minimize or eliminate the need for optical recalibration. The casing can be made by injection molding, sheet metal forming, vacuum forming, thermal forming, etc.

[0204] In another embodiment, the SIMPLE chip can be loaded horizontally in the device 1000 as shown in FIGURE 10. The acoustic mixer 1001 and / or the heaters 1002 can be suspended by springs and “free-floating.’' This design minimizes the vibrational damping effect. The SIMPLE chip can be slotted in via the slot guides 1003, which correctly aligns the chip with the optics module 1004. Once the chip slots fully in. it causes the mechanical chip ejector button 1005 to protrude and temporarily lock the chip in place. After the test, the chip ejector button can be pushed to eject the SIMPLE chip. The chip ejector button is coupled with the loading clamps 1006. The loading clamps are spring-compressed, so the natural state is to remain closed. There are inclined slots at the chip entry position so that the clamps open as the chip is pushed into the optical position. Once the chip reaches the fullyinserted position, the clamps fall back down and clamp the chip in place until the user pushes the ejector button to release the SIMPLE chip. b. Example embodiments

[0205] A collection of example embodiments providing additional description of a variety of embodiment types in accordance with the concepts described herein are provided below. These examples are not meant to be mutually exclusive, exhaustive, or restrictive; and the claimed subject matter is not limited to these example embodiments but rather encompasses all possible modifications and variations within the scope of the issued claims and their equivalents.

[0206] Independent Claim:A reader for rigid SIMPLE chips that have the following features: acoustic mixing transducers; heating elements; and an optical system.

[0207] Independent Claim:A reader for fluidic cartridges that have the following features: acoustic mixing transducers; heating elements; an optical system; and a backplate assembly design that minimally damps vibrational energy transfer.

[0208] Dependent Claims (based on Independent Claims):The reader has cross-compatible design features with the reader for 3D SIMPLE chips or R2R-manufactured SIMPLE chips.The reader includes uses a haptic piezo bar for SIMPLE chip mixing.The reader includes a cassette embodiment for chip loading.The reader includes an extension spring embodiment for chip loading.The reader includes a sheet metal brace balancing design.The reader includes a magnetic base balancing design.The reader includes a spring base balancing design.The clamp mechanism allows the SIMPLE chip to be slipped in and out of the backplate with minimal user steps.Fully enclosed shell to keep the optical environment consistent across tests.Some or all of the independent and dependent claims listed in Section 3-f:Gen2 SIMPLE chip reader.

[0209] 7, Injection-molded 3D SIMPLE chips a. Overview

[0210] Some expected advantages of the embodiments of the Injection-molded 3D SIMPLE chip include, but are not limited to:1) All the same advantages as the R2R manufactured chip, except that it is higher cost than R2R processes, still straightforward in construction, making it much lower cost than other peer systems;2) As few as two main injection molded parts are needed (main body and cork);3) Higher fidelity and resolution features possible than R2R manufactured parts;4) Cork design makes it easier to dispense liquid reagents and seal off SIMPLE chip;5) Less manual steps since the cork sealing and reagent dispensing action is combined;6) Less user error risk;7) More design flexibility compared to 2D SIMPLE chips; and8) Compatible with injection molding manufacturing.

[0211] Embodiments of the present disclosure will now be described in detail. A third concept family of a SIMPLE chip 1 100 embodiment is show n in FIGURES 11 A-l 1 E. These are 3-dimensional and would be formed by an injection molding process. The process of using this type of chip is similar to the process described in the above sections regarding theroll-to-roll chips and the 2D rigid chips, but some nuances of this variety will be discussed here. Some or all design concepts, materials, and applications can be transplanted across the R2R version. 2D rigid version, and 3D rigid version of the SIMPLE chips. Design and manufacturing elements may be combined to form new designs. Detailed design specifications will need to be tailored for each format, however.

[0212] One embodiment of the sample collection method for this type of chip is a single body that contains a ‘'cork’’ 1101 with an optionally attached swab 1102. The user procures the biological sample with the swab and then plugs the cork / swab unit into the main body cylinder 11 10. The top of the main body 111 1 has a funnel shape that mates with the cork and creates a seal once the cork is pushed down far enough for the cork's rubber o-ring 1107 to seat into an optional internal recess of the main body funnel 1112. The o-ring is optionally a soft gasket rubber o-ring-like material 1107, or it could just be the same material and is unibody with the cork itself. Optionally, the cork can be screwed into the body cylinder instead of being pushed in. The cork can optionally be designed to be level or shorter than the main body after insertion to prevent users from removing the cork once it is inserted. Alternatively, the cork and main body have other locking mechanisms (such as reverse hook structures), allowing the cork to be assembled but not dissembled. Once the cork is inserted into the main body, the system becomes fully self-contained, lowering the amplicon contamination risk.

[0213] As the user is pressing the cork into the main body, a protruding structure such as a cylindrical plunger 1106 on the main body pushes a cylinder 1113 up inside the cork. The other side of the cylinder can be pre-filled with a liquid reagent (lysis buffer or elution buffer). As the plunger moves the cylinder up the cavity7, the reagent is ejected from a port at the bottom of the cork 1105 through an internal fluidic channel 1 104. The lysing or elution reagents coat the swab as it rests at the bottom of the main body tube. The cylinder 1113 may optionally have an o-ring or a rubber plunger design (similar to syringe internal plungers) to prevent fluid leakage during storage and dispensing. The cylinder’s natural state during storage is at the bottom of the cork, and liquid can be stored in the cavity above. As thecylinder is pushed during cork-main body assembly, the stored liquid is forced out via the internal fluidic channel. At the bottom port 1 105 of the internal fluidic channel 1104, there may be a foil, wax valve, grease valve, burst valve, membrane, or other feature that functions to hold the liquid and prevent evaporation during storage but allows the liquid to move through the feature once the cylinder is moved up. Optionally, the feature that holds liquid may be mechanically punctured open during assembly of the cork 1101.

[0214] The main advantage of this design is that there are fewer parts (no separate dropper tube needed, such as the components shown in FIGURE ID). Another advantage is fewer steps for the user because the reagent injection step is unified with the cork capping step. Fewer steps also reduce manual user error risk. Injection-molded parts also allow finer features with higher consistency and fidelity. The disadvantage is that this system requires injection molding, which is a higher cost when compared to R2R manufacturing.

[0215] An alternative, more compact embodiment of the cork, swab sample collection unit, and SIMPLE chip is shown in FIGURE HE. A brief process flow is to put the tube cartridge chip in the reader, perform the nasal swab, break the swab stick, and drop the swab into the cartridge tube, close the cork top onto the tube cartridge, then the automatic reader heats and mixes the reaction tube sample, melts the wax valve to suck sample into the reaction chambers, and heats / mixes / images the microwells for isothermal reaction. This component uses a weakened breakaway swab and an optional notch feature 11 16 on the cork to remove most of the swab’s stick, and the sample end of the swab is dropped into the main chamber tube. The swab may optionally be attached to the cork during manufacturing or be a standalone swab. The notch is optional and can be placed inside the main body cylinder or the cork.

[0216] Once the swab and lysis solution are in the main chamber tube, the contents are mixed using an acoustic or piezo method, as described in earlier sections. FIGURE HA shows one embodiment utilizing a perforated sleeve on the main body cylinder 1 1 10 to capture air bubbles to enhance mixing. The size of the perforations is proportional to the size of captured air bubbles. However, if the perforations are too large, they cannot capture airbubbles. The acoustic mixing frequency is finely tuned to air bubbles’ resonance frequency to induce more turbulent mixing. The perforated sleeve can be a separate part 1103 inserted into the main body cylinder as shown in FIGURE 1 IB. Alternatively, the perforations can be directly in the main body cylinder (manufactured together during the injection molding as a unibody assembly).

[0217] An alternative embodiment is to have the perforated sleeve in 2D format 1108 instead of a cylindrical format 1110 for easier injection molding so the perforations and main body can be manufactured in one step. The perforated sleeve can be sealed off with pressuresensitive adhesives (or heat-sealed lidding material) to prevent fluid from leaking out of the device while retaining small air bubbles that enhance microfluidic acoustic mixing. The sample in the tube is then optionally heated (acoustically) and mixed, and subsequently, a wax valve 1114 is melted and solidifies in the cooling chamber 1115, and the contents are sucked into the microwells 1109 by a stored vacuum inside the chip. The wax valve inlet can be connected to the sample receiving chamber via a fluidic through-hole port. As noted in the 2D SIMPLE chip section, a puncture-ri pe valve can be implemented alternatively. Again, air bubble size can be controlled inside the microwells by modulating initial vacuum storage conditions during manufacturing. Isothermal amplification reactions can commence inside the microwells.

[0218] The entire main body may be manufactured as one injection molded piece (or multiple pieces), with PSA films (or heat-sealed films) sealing off all the perforations and fluidic channels. The cork may also be manufactured as one injection molded piece (or multiple pieces). Materials that can be used are the same ones discussed in the R2R version and 2D version SIMPLE chip designs. The main body is stored inside a vacuum bag during long-term storage, similar to R2R SIMPLE chips. The main body is removed from the vacuum bag right before the operation. The cork, if containing liquid, should be stored in a separate non-vacuum bag.

[0219] In another embodiment, FIGURES 12A and 12B show a hybrid concept 1200 between 2D and 3D rigid body SIMPLE chips. An example procedure is also shown. Thecork section 1201 can be a 3D design that is inserted (or screwed) onto a 2D SIMPLE chip main body 1202. Note that the perforated section 1203 can be a planer rectangular shape instead of a cylindrical one for easier inj ection molding. Note that the cork structure is vertical, while the 2D main SIMPLE chip main body is horizontal. Optionally, screw threads 1204 on the 2D SIMPLE chip can interface with the cork assembly. A cover layer 1206 can seal the base of the main body 1202. The cover layer 1206 can be a thick material or a thin film material (such as PSA).

[0220] In other embodiments, a SIMPLE chip (all variations), can contain gas permeable material. These chips can optionally be stored in a vacuum before testing. A pressure differential can be induced across the gas-permeable material to drive the fluidic movement and control the air bubble size retained in the microwells. The pressure differential may be positive or negative (or both), and it can be administered internally or externally on the chip. For example, positive pressure can be applied to the inlet to push liquids into microwells, or negative pressure can be applied externally to the chip to suck liquid into the microwells. The gas-permeable material allows for excess gas to vent so fluids can be moved.

[0221] In all embodiments, the SIMPLE chip can be made by injection molding, compression molding, blow molding, transfer molding, thermoforming, injection blow molding, and / or hot embossing techniques, among other suitable manufacturing processes. b. Example embodiments

[0222] A collection of example embodiments providing additional description of a variety of embodiment types in accordance with the concepts described herein are provided below. These examples are not meant to be mutually exclusive, exhaustive, or restrictive; and the claimed subject matter is not limited to these example embodiments but rather encompasses all possible modifications and variations within the scope of the issued claims and their equivalents.

[0223] Independent Claim:A consumable device that has the following features:made with as litle as two major assemblies (a rigid molded fluidic part and a rigid molded cork part) and a cover layer; a portion of the device retaining vacuum; a valve that can be opened on demand and allows fluids to flow toward the sections with retained vacuum; and microwells that trap air bubbles.

[0224] Independent Claim:A consumable device that has the following features: a cork design; cork can store liquid buffers; the cork seals the consumable device and dispenses the stored liquid during the joining process of the cork and the assembly that contains fluidic circuits; a valve to maintain the vacuum; and fluidic circuit has stored vacuum and sucks fluid in after a valve is opened.

[0225] Dependent Claims (based on Independent Claims):The parts are manufactured by injection molding.The parts are manufactured by hot embossing.The parts are manufactured by compression molding.The consumable contains gas permeable material.Some or all of the dependent claims in section 1-g: “Gen2 SIMPLE chip design compatible with roll-to-roll manufacturing”

[0226] 8, Genl roll-to-roll manufactured SIMPLE chips integrated with vacuum batery technology a. Overview

[0227] Some expected advantages of the embodiments of the Genl R2R manufactured SIMPLE chip include, but are not limited to:1) Compatible with versatile sample prep chemistry, can use the Boom method to extract both DNA and RNA from bacteria and viruses;2) Not as low cost as gen2, but still lower cost than other competing systems on the market;3) Extreme limit of detection possible (200 genomic copies per swab); and4) R2R fluidic module can be integrated, upstream or downstream to other fluidic circuits, forming a versatile platform that can be used for many life science applications.

[0228] Embodiments of the present disclosure will now' be described in detail. The precursor to the second generation of the R2R-manufactured SIMPLE chip was a Genl R2R SIMPLE chip 1400 shown in FIGURE 14. It involves a design that performs multi-step sample preparation inside an R2R consumable chip (sample prep module) with a downstream microfluidic module that uses vacuum battery technology. It is coupled with an open testing platform (FIGURE 13 A) that can test and perform different protocols of many different designs of the R2R chip because the robotic gantry (FIGURES 13B-1 and 13B-2) can move and be programmed for various geometries and protocols. The example shown here shows that it is feasible for an R2R manufactured part (sample prep module) to be combined with other types of microfluidic systems (vacuum battery microfluidics). It also shows that complex, multi-step, sequenced fluidic processing may be done with R2R consumables, making it a very' versatile technology' platform.

[0229] Instead of using a single-step sample prep chemistry' as in gen2, the genl SIMPLE chip uses a multiple-step method, such as, but not limited to, the Boom method. The Boom method involves silica-coated magnetic beads and chaotropic lysis buffers with multiple buffer washes to wash and elute the DNA and RNA. FIGURE 14 shows several of the chambers on the chip containing washing buffers that are brought across a biological sample for cleaning. DNA / RNA is kept in place by a magnetic silicon bead capture method. The mechanism of moving fluid around the system is by applying force to the surface of a burst pouch 1412 and forcing fluid flowing into the rest of the channel system, coupling with vacuum pressure to drain liquid (positive pressure may alternatively be used).

[0230] The sample prep module can achieve -50% DNA recovery yield, similar to standard manual off-shelf sample prep products. We spiked target bacterial DNA into simulated swab samples and ran sample prep on the consumable chip, then took out the elution for isothermal amplification testing (on a qPCR machine). We found much higher consistency using the automated system versus manual sample prep, probably due to fewer inherent variations while running the protocol. Without full optimization, sample prep recovery7is already 50%. Higher efficiency is expected beyond 50% when full optimization is done.

[0231] An example step-by-step process is described herein with reference to FIGURE 14. The user first takes a biological swab (e.g., nasal swab) and elutes the swab in a sample lysis buffer, which could contain some or all of the following, and not limited to chaotropic lysis buffer(s), salts, AVL buffer, guanidinium chloride, guanidine hydrochloride, ethanol or IPA, detergents, and silica-coated magnetic beads. Enzymes such as protease k or lysozy me may be present as well. RNase inhibitors and / or carrier RNA may be included as well. Bacteria and viruses are lysed while their DNA / RNA binds to the silica-coated magnetic beads. A sliding magnet is raised by an actuator (such as a linear actuator) to the mixing pouch position to interact with beads 1431, and the vacuum port 1426 from the reader is turned on, then the sample / lysis mixture is injected into the inlet port 1421a. The vacuum draws the lysis buffer into the waste chamber 1428, and the sliding magnet retains all the magnetic beads 1431 in the mixing pouch 1430 while the vacuum is on. Vacuum port 1426 is turned off, and then the fluidic path under the inlet pouch is heat-sealed. Heat sealing spots 1402 are depicted, with rectangular shapes denoting heat-sealed areas (at separate steps).

[0232] Then, the washl buffer in the pouch 1422 is pressed by the reader gantry push pin so that the washl buffer is expelled when the burst valve 1412 opens. The washl buffer can be a guanidine based buffer in the base of the pouch 1422. An optional heat sealing step near the burst valve 1412 prevents fluid backflow. Optionally, the area of the burst valve can be heated to weaken the valve as well. Washl buffer contains a buffer that washes the lysis buffer away (such as Qiagen AW1 buffer). Some lysis buffers contain chaotropic reagents such as guanidine chloride, which is highly detrimental to downstream isothermalamplification reactions and must be thoroughly washed away. The washl buffer pools and covers magnetic beads 1431. Washl buffer can move forward into the mixing pouch even though the vacuum 1 port 1426 is not turned on because there is a vent2 1403. Vents can be made by a pinhole (such as a needle piercing the SIMPLE chip). Fluid resistance can be modulated with the pinhole’s diameter.

[0233] The second vent 1403 is at the top of a liquid trap 1410. The liquid trap has a broader cross-section than the inflow fluidic channel from below. It helps retain any overflow liquid during the sample loading or pouch bursting process and helps liquid pool strictly in the mixing pouch. The broad cross-section allows a large volume to buffer and fill up before liquid flows into the multi-channel junction (three-way channels) immediately to the left of the trap. The multi-channel junction is designed to be slightly higher than the trap so the liquid does not overflow undesirably but flows into the trap first. Wash buffers accidentally entering the mix 2 pouch 1435 can be detrimental to subsequent isothermal amplification reactions. Any liquid that flows into the trap automatically drains downwards back toward the mixing pouch 1430. The low er part of the trap has a narrow channel that connects to the mix pouch 1430. The narrow channel minimizes unintentional splashing of mix pouch 1430 contents (especially during mixing), which reduces reagent cross-contamination risk between washes. Vent2 and vacuuml act in conjunction to remove all liquid in the mix pouch and liquid trap.

[0234] Washl buffer pools in the mixing pouch 1430, the sliding magnet is lowered away from the mixing pouch, and ultrasonic mixing is commenced on the mixing pouch so the magnetic beads mix well with the washl buffer. Then, the sliding magnet is raised to capture the beads again, and the vacuuml port is turned on again to drain all of washl.

[0235] The above process with the ultrasonic magnetic beads 1431 is repeated for wash2 1424 and wash3 1423. Different buffers can be used depending on the exact chemistry. For example, Wash2 and Wash3 can be Tris-based ethanol buffers (such as Qiagen AW2). The same buffers can be used more than once in repeats to wash all previous residue from the magnetic beads thoroughly. Once the beads are thoroughly washed, vacuum 1 can be turnedon for an extended period to dry any liquid residue completely. Optional heating (such as optical heating) may be included to accelerate the drying process. At this point, contaminants or inhibitors to downstream assays are removed, and only nucleic acid remains attached to the beads.

[0236] Next, an elution step is done. The elution pouch 1425 contains a buffer that releases the nucleic acids from the magnetic bead surface. Example buffers include AVE, AE, TE buffer, and / or water. Carrier RNA and / or RNase inhibitors may also be in the buffer. The elution buffer is pushed into the mixing pouch 1430, and ultrasonic mixing is repeated. At this point, heating may be conducted to increase elution efficiency further. An optical heater with halogen lamp(s) or LED(s) may be used. The infrared wavelength is desirable for fluidic heating. Elalogen lamps have intense infrared wavelength optical emission, and water solutions have strong optical absorption at the same spectrum, so the solutions are rapidly heated. The black magnetic beads also absorb optical heating particularly well and contribute to the high efficiency of nucleic acid elution. DNA / RNA is released from the beads' surface and eluted into the elution buffer. Ultrasonic and / or acoustic mixing may also be done to further increase elution kinetics.

[0237] Next, the fluidic path to the waste chamber (at the multi-channel junction 1407) and the right entry7channel to the mixing pouch 1430 is heat sealed, and vacuum 1427 is turned on to transfer the elution into the mix2 pouch 1435. Vacuum? is located on a side branch higher than the mix2 pouch and is offset to the side from the channel that brings elution into the mix2 pouch. This design enables the elution fluid to naturally follow the contour of the pouch and pool at the bottom, and no elution is accidentally sucked away when vacuum2 is turned on. Mix2 pouch can contain dried or lyophilized reagents. For example, dried isothermal master mix buffer, isothermal master mix, magnesium acetate, and or other reagents may be optionally stored here. Reagents that should not be in the microwells' lyophilized pellets or not interact with upstream workflows can be stored in the mix2 pouch. For example, if salts are added to the mix pouch, these reagents may interfere with the elutionstep (or cause DNA / RNA to rebind to the magnetic bead surface, as high salt concentrations can form binding conditions).

[0238] The mix2 pouch is connected to vent3 1401. Vent3 prevents unwanted liquid (lysis buffer, Washl, Wash2, Wash3) from being accidentally sucked into the mix2 pouch during the vacuum 1 suction process because it creates a unidirectional airflow towards the waste chamber. Vent3 has a smaller diameter than vent 2’s pinhole, effectively creating a flow resistance differential, which enables prioritizing fluid and air movement through the channels connected directly to the mixing pouch. If vent3 were too large, the air would bypass any fluid movement, and air would be sucked into the waste pouch (from mix2 pouch vent3) instead of liquid being sucked into the waste chamber 1428 (from mixing pouch 1430), which is undesirable.

[0239] All fluidic channels to the mix2 pouch are heat sealed (heat seals shown in rectangular outlines, such as heat seal 1402), then ultrasonic mixing is commenced again in the mix2 pouch to reconstitute the pre-stored dried reagents with the elution liquid. The next step is integration, which transfers the elution fluid from the sample prep module fluidic circuit to the vacuum batten chip. Mix2 pouch contains an integration pin 1434, that has a sharp point at an end. The sharp point is located at the bottom of the mix2 pouch near air bubble traps 1406, so pooled fluid (elution) covers the integration pin 1434. Once an external force (e.g., push pin) crushes the mix2 pouch, the sharp point punctures the PSA film at the bottom of the sample prep module and also punctures the vacuum battery chip’s packaging material. This action creates a pathway that connects the sample prep module’s fluidic circuit to the vacuum battery chip’s 1411 fluidic circuit. The vacuum battery chip 1411 is located behind the sample prep module. The mix2 pouch, integration pin sharp point, and the vacuum battery chip's inlet 1436 are all aligned.

[0240] As shown in FIGURE 16, the vacuum battery chip’s 1600 primary function is to perform multiplexed isothermal amplification. It draws the liquid in using vacuum battery technology, described in another section. After integration, the elution fluid is sucked into the microwells 1603 that contain lyophilized isothermal amplification pellets (or PCR pellets).All isothermal chemistries, including PCR, mentioned in other SIMPLE chip sections can be used. General design ideas and device functions are cross-compatible with the other types of SIMPLE chips described herein. Different primer sets may be placed in the lyophilized isothermal amplification pellet so each microwell becomes an individual separate reaction for multiplexing. Various molecular probes and dyes described in this patent may also be used. The microwells may also serve as positive or negative controls.

[0241] The elution fluid and isothermal amplification pellets are mixed using acoustic mixing. A constant temperature (or thermal cycling) is applied, and the isothermal amplification (or PCR) commences. When a target amplicon is present, a fluorescence signal appears. A time-lapsed fluorescence image series is taken, and test microwell signals are compared with negative and positive control microwell baseline signals to determine whether there is a true positive signal. All vents are heat sealed so the genl SIMPLE chip becomes fully self-contained. After processing, the genl SIMPLE chip is placed in an airtight bag (such as a Ziplock) to reduce amplicon contamination risk and discarded according to local regulations.

[0242] Using the genl SIMPLE chip and the open format reader described below, we have shown a limit of detection at 200 genomic copies of DNA per swab (in single-step amplification format), which is similar to full workflow conventional PCR processes that include sample preparation (FIGURE 17C). The samples named "‘QC” were human nasal pharyngeal swabs spiked with 200 genomic copies of DNA per swab. 1CFU equals 1 genomic copy. The " P " samples were different patient samples with a bacterial DNA target present, confirmed by a Biofire assay. The entire process for sample prep takes less than 12 minutes in the sample prep module. Using one of the isothermal chemistries mentioned herein, the downstream amplification part can be completed in an average of 4.3 mins (FIGURE 17C). So the total sample-to-assay time can be less than 17 minutes.

[0243] FIGURES 18A and 18B show clinical testing results with the fully automated workflow. Using 102 patient samples, we have shown that this SIMPLE chip system can process bacterial DNA and viruses (SARS-Cov-2, RNA) using the same system. It showed98-100% accuracy, 96-100% clinical sensitivity, and 100% clinical specificity' compared with FDA-approved PCR assays. The Biofire system was used as the bacterial DNA test comparator, and the Thermo qPCR FDA EUA assay was used as the RNA test comparator. Note that we received leftover samples, which were nasopharyngeal swabs diluted in transport media, so the sample was already 7.5~15x diluted and possibly somewhat degraded. That is potentially why there was a false negative for the bacterial assay. Our system is designed to receive dry swabs instead of diluted transport media. The dry swabs would not have any dilution effect as all of the target template material would go through our system. Nevertheless, this data proved that our system could also receive transport media samples. This flexibility is due to the utility of the Boom sample prep method, and it can receive a wide range of samples in different matrixes.

[0244] In other embodiments, different sequences of protocols can be used to process the SIMPLE chip. The R2R-made sample prep module (repurposed with buffers and / or beads within and redesigned with alternative fluidic circuits) can have applications other than point- of-care diagnostics. For example, it could be used as a standalone sample prep module for nucleic acid preparation. Alternatively, it can be adapted to, for example, and not limited to, sequencing library prep work, immunoassays, DNA arrays, protein extraction protocols, cell staining, electroporation, protein engineering, forensic application sample prep, etc. It could be used for next-generation sequencing applications such as nucleic acid isolation, purification, size selection and clean-up, and library normalization.

[0245] In other embodiments, the R2R-made sample prep module (repurposed with different buffers, protocol sequence, and fluidic circuits) can be coupled with the vacuum battery module and or other (micro)fluidic systems downstream for applications such as and not limited to electrophoresis modules, other R2R fluidic circuits, sequencing modules, flow cytometry', cell staining, electroporation, directed evolution, droplet microfluidics, lateral flow assays, paper microfluidics, ELISA, immunoassays, capillary^ microfluidics, DNA arrays, pH sensors, gas sensors, exosome purification, exosome detection, PCR, qPCR, digital PCR, cancer detection, next generation sequencing, etc. The combined system can also be used forany of the applications in the previous paragraph. It may also be cross-compatible with some or all of the other life science and non-life science applications mentioned in other sections.

[0246] In another embodiment, instead of silica-coated magnetic beads and boom chemistry', charge switch surface-coated magnetic beads or magnetic beads with capture probes may be used for nucleic acid extraction (buffer sets must also be tailored to each surface chemistry). Magnetic beads may be conjugated with antibodies, enzy mes, aptamers, and other custom ligands. The repurposed sample prep module can be used for exome sequencing applications. Using streptavidin-coated magnetic beads to capture exon fragments or specific genes hybridized to biotinylated complementary' sequences is possible. The sample prep module can run immunoprecipitation (IP) and co-IP assays to study protein interactions using Protein A / G-conjugated magnetic beads. The magnetic beads can be carboxylate-coated and used for DNA fragment size selection for NGS. Alternatively, all magnetic bead functions could be replaced by stationary filter-type materials or columns with various corresponding surface chemistries mentioned above (e.g., similar to silica spin columns or HPLC columns).

[0247] In another embodiment, doing an electrical, magnetic, or electrochemical readout is possible instead of an optical readout.

[0248] In another embodiment, nested or two-step amplification can be done to improve the limit of detection. For example, the (isothermal) amplification reagents can be stored in mix pouch 2 and optionally be a one-pot multiplexed reaction with multiple primer sets. After amplification commences in mix pouch 2, a secondary amplification can be done in the vacuum chip microwells, which could be pre-loaded with alternative sets of isothermal amplification reagents, probes, and / or primers. Alternatively, the vacuum chip can have just different molecular probes. Using a two-step amplification format, we have shown an extreme limit of detection possible down to 20 DNA copies / reaction. Two identical negative controls and six identical test reactions were lyophilized into the vacuum battery' chip. Note that two- step amplification and / or nested amplification are compatible with all other SIMPLE chip embodiments (especially gen2 SIMPLE chips).

[0249] In summary', the R2R upstream module can be repurposed with different buffers, reagents, and or fluidic circuit designs to be used alone or in combination with one or more upstream or downstream modules (such as vacuum battery chip and others mentioned above), coupled with different reader protocols, to run any of the mentioned applications in the above sections. It is a highly’ versatile technology platform for biotechnology R&D and diagnostic workflows. b. Design and manufacturing of the genl SIMPLE chip

[0250] The genl SIMPLE chip is made of two modules. There is an upstream sample prep module 1400 and a downstream vacuum battery chip 1411. The upstream sample prep module performs sample preparation. The downstream vacuum battery chip runs multiplexed isothermal amplification. The two modules are stuck together (FIGURE 14), using PSA to form an airtight seal. An integration pin 1434 connects the two fluidic circuits on demand through the inlet 1436.

[0251] The upstream sample prep module is manufactured by an R2R process. It comprises two layers, a formed layer with fluidic circuits and a sealing cover layer. Some or all materials and manufacturing methods are compatible with the other embodiments of SIMPLE chip manufacturing processes described previously. The sealing cover layer can be PSA, adhesively sealed film, thermally sealed film, or ultrasonically sealed film. It should be inert and not interfere with biochemical reactions inside the module.

[0252] The sample prep module fluidic circuit includes some or all of the follow ing and is not limited to: fluidic channels, blister pouches for reagent storage 1422, 1423, 1424, burst valves 1412, vents 1403, 1401, liquid traps 1410, waste chamber(s) 1428, alignment markers 1405, heat seal sections 1402, mixing pouches 1430, crushable structures, integration areas with pin 1434, air bubble traps 1431. vacuum / pressure ports 1426, 1427, multi-channel junctions 1407, liquid ladder traps 1408, fluidic pooling areas, and built-in labeling 1409, etc. Note that a wide variety7of other shapes and fluidic designs that are standard in (micro)fluidics can be added as long as they can be formed in the fluidic layer.

[0253] Some of the features are already described in the previous section. The Alignment markers serve as a visual and mechanical structure that mates with the backplate, so the genl SIMPLE chip is always loaded consistently. Heat seal sections are normally open channels but can be closed on demand by applying heat and pressure externally. These serve as valves. Crushable structures, for example, include the reagent containing blister pouches and mix2 pouch where the integration pin lies. The structure can be crushed to provide subsequent functions such as fluid release or integration pin action. Air bubble traps are deadend channel structures that trap air when liquid is present, and this helps to increase acoustic mixing efficiency significantly. Air bubble traps can be orientated at a slight offset so a net circular flow can be achieved for mixing. We design a special multi-channel Y-j unction 1407. which allows the wash buffers to flow7towards the waste chamber via the larger channel without any accidental flow into the smaller channel towards the mix2 chamber. This feature is achieved by creating a differential fluid resistance via modulating cross-channel size, vent2, and vent3 size. Elution can flow towards the mix2 pouch after the larger channel is sealed and vacuum2 is turned on.

[0254] A liquid ladder 1408 is designed to minimize liquid residue in channels. It allows the liquid to “climb and rest and climb over the summit.” When vacuum 1 is applied and stopped, sometimes there is liquid residue in the vertical section, and that residue tends to pool back at the closest areas due to gravity (in this case, the bottom of the ladder and bottom of the mix pouch), vacuuml can be stopped, to allow the pooling, and restarted again to draw all the remaining residue out. Fluidic pooling areas are specifically designed to allow the liquid to pool using gravity, including, for example, the integration pin pouch. The liquid needs to pool over the pin for it to function. Built-in labeling may be manufactured in one step using the same mold along with all the fluidic features if the labeling structure is engraved into the mold (e.g., thermoforming mold).

[0255] The downstream vacuum battery chip module 141 1, 1600 has the following design features: it has interconnected large voids 1601 , and the voids are also connected to the auxiliary vacuum channels 1602. These areas contain pre-stored vacuum and serve as areservoir to continuously draw air out from the microfluidic circuit via air diffusion through the thin microwell wells, which results in fluid movement. The vacuum battery chip is wholly or partially made of gas permeable material to allow gas diffusion. The microfluidic circuit includes (micro)well(s) 1603, an inlet 1604, and connecting channels 1605. The whole vacuum battery chip is stored under a vacuum inside a clear vacuum bag. The bag material may have very low MVTR and OTR to hold the vacuum for extended periods. It may have a high barrier against gas diffusion. The bag may also exhibit low autofluorescence and minimally affect optical readout.

[0256] After integration, fluid from the sample prep module flows into the vacuum battery chip, initially by the direct vacuum within the microwells and channels, and then gradually, all residue air pockets are degassed via the bulk silicone material. Residue air can also diffuse through the thin walls of the microwells into the auxiliary channels, which contain more vacuum. In this design, no air pockets are left inside the chip after a few seconds to minutes, and the microwells are completely loaded fully before the isothermal amplification is finished. Compared to the gen2 prototype, the advantage of this is that there is always guaranteed very high fluid volume loading consistency. All the microwells will be completely loaded without any air bubbles left. However, to enable good acoustic mixing, the acoustic mixer needs to continuously form a sweep of frequencies that continuously resonates with the shrinking air bubble within the microwells. The mixing effect diminishes after the air bubble disappears, which is a disadvantage compared to Gen2 SIMPLE chips.

[0257] The vacuum battery chip also includes optical alignment markers 1606, which are helpful for the assembly process to align the vacuum battery module to the sample prep module. It is also helpful during the optical detection process, where it helps serve as geometric reference points to establish coordinates for the microwells so the software correctly defines the region of interest to quantify the fluorescence in the microwells. The optical markers may be designed to work in bright field, dark field, or fluorescent optical conditions. For example, it can create distinct shadows, scattering patterns, or be loaded with fluorescent material. Sharp points and abrupt changes in geometry are beneficial forscattering mode detection (see triangle shape and disk shape). Round cavities may be filled with matching diameter fluorescence beads to serve as a geometrical reference (and a baseline fluorescence intensity calibration marker), Other fluorescence calibration markers may be slotted into the optical alignment markers, For example, fluorescent plastic pieces can be dropped into the optical alignment cavities. In another embodiment, an autofluorescent tape material can be placed on the vacuum battery’ chip as a fluorescence intensity' reference. To maximize the fluorescence signal-to-noise ratio, a reflective material (such as glossy aluminum foil) can be placed on the back of the micro wells 1603. This effectively creates a reflection of the excitation light source, causing the fluorophores to get twice a chance to emit. The emission light is also reflected toward the camera, further increasing signal intensity. Good filter sets will filter out increased excitation light effectively. The aluminum foil also serves to distribute isothermal heating more evenly. The vacuum battery chip 1600 may include a slope 1607 at the side of the chip to allow fewer wrinkles on the soft vacuum bag and easier insertion into a rigid shell enclosure.

[0258] The vacuum battery chip can be made with a material compatible with injection molding, compression molding, casting, or R2R forming. Gas-permeable properties of the material are needed to allow the vacuum battery to function correctly. The material does not have to be transparent but needs low autofluorescence if fluorescence is used for detection. Material examples include silicones, PDMS, thermoplastic elastomers, fluoropolymers, etc. For assembly, a formed piece of the fluidic portion is filled with lyophilized pellets of isothermal master mix in the microwells. Then, an optically transparent PSA cover layer is placed to cover and seal the fluidic features. The PSA layer should have low autofluorescence if optical detection is used. The reflective material (e.g., glossy aluminum foil) is placed on the backside. A small piece of double-sided PSA is placed at the inlet; this allows the inlet area to be fully sealed with the vacuum bag and prevents liquid from accidentally transferring between the pierced vacuum bag / enclosure and vacuum batten chip. The vacuum battery’ chip is then placed into an enclosure, such as a vacuum bag. and placed in a vacuum environment to degas all the remaining air in the chip. The gas diffuses outgradually from the gas-permeable material (such as silicone). Once this is complete, a heat sealing process is used to seal the enclosure under a vacuum, permanently keeping the enclosure's interior environment under a vacuum.

[0259] In another embodiment, the vacuum battery chip is sealed in a transparent vacuum high barrier rigidly formed 3D shell instead of a soft bag material. One side of the 3D enclosure could be a rigidly formed plastic material with transparent vacuum barrier properties that form a snug fit over the vacuum battery-formed chip’s profile. The 3D enclosure can be sealed with foil-type heat-sealable lidding material (or transparent high vacuum barrier material). This approach removes all wrinkles that form with the soft bag type approach.

[0260] Alternatively, the 3D shell material can be rigid or soft but conforms and fits snuggly with the chip, and another peelable vacuum barrier material, such as aluminum foil, covers the 3D shell material (the 3D enclosure’s open side is still sealed with a flat lidding material). The peelable vacuum barrier material keeps high vacuum barrier properties, and the shell material can be optimized for optical transparency instead of needing to have both excellent vacuum barrier and optical properties at once. The peelable foil laminate can be peeled off right before operation. Once peeled, it would be sufficient if the 3D shell material could hold the vacuum for a reasonable amount of time (at least 20 minutes).

[0261] The manufacturing steps are described below. First, the fluidic layer is formed using the R2R process described in other sections. Some or all manufacturing methods, materials, and design features are cross-compatible with the Gen2 SIMPLE chip. Then, the vents are punctured. Next, the integration pin. (optionally burst valve grease), (optionally liquid reagents), and dried reagents (such as isothermal amplification buffers) are placed onto the chip. Pick-and-place machines or dispensers can do this placement action. The next step is to seal the cover layer via the R2R process onto the fluidic layer (PSA can be used here). Then, the PSA covering the vacuum ports is punctured to create openings. If double-sided PSA is used, a part of the backing liner is removed, and a vacuum battery chip is stuck onto the exposed PSA. The inlet of the vacuum chip and integration pin on the sample prep moduleshould be aligned. The chip can have an optical capture side and a sample processing side where the reader’s gantry tools process the chip. The manufacturing process is straightforward and can be done entirely on an R2R line. Much less material is used compared to injection molding methods. The consumable’s construction is simple and costs less than other competing systems.

[0262] In another embodiment, the fluid reagents are injected via needle-like injectors after the cover layer and fluidic layer are sealed. Then, fluid reagents are injected by piercing the cover layer into the blister pouch. Optionally, a secondary' vent to vent out the air during the fluidic injection process in the blister pouches can be made at the blister pouch area. The secondary’ vent can be optionally created simultaneously when the fluidic needle injector is inserted into the blister by placing a second needle alongside the fluidic needle injector. During fluid injection, the vent prevents sudden over-pressuring and damaging of the burst valves. After injecting the fluid, another second cover layer (or small local covers such as PSA stickers) can cover the vent and fluidic injection holes. The second cover layer is then sealed. Optionally, the second cover layer is a double-sided PSA that can be used to stick later additional fluidic circuits, such as the vacuum battery' chip, to the R2R manufactured portion. All of these processes can be made in line with the R2R process.

[0263] In other embodiments, some, or all of the components on the upstream R2R module can be rearranged and tailored for other applications. For example, fluidic channels, blister pouches for reagent storage, burst valves, vents, liquid traps, waste chambers, heat seal sections, mixing pouches, crushable structures, integration pin areas, air bubble traps, vacuum / pressure ports, multi-channel junctions, liquid ladder traps, fluidic pooling areas, built-in labeling, and other (micro)fluidic features may be rearranged to new fluidic circuits for different applications, in an analogy that basic electrical elements such as capacitors, CPUs, resistors, wiring connections, can be rearranged for many different applications. Various applications have been discussed in other sections already. Other types of common fluidic features not discussed may also be included in the R2R module as long as they are compatible with the manufacturing process.

[0264] In other embodiments, the upstream R2R module may integrate with nonvacuum battery modules downstream, such as and not limited to the 2D rigid SIMPLE chips, R2R gen2 SIMPLE chips, R2R manufactured downstream modules, injection molded / hot- embossed downstream modules. Various applications have been discussed in other sections already.

[0265] In other embodiments, the R2R module and / or vacuum battery chip may be used as a standalone module, or it can be used as an upstream, downstream, or in-between module integrated with other fluidic modules.

[0266] In summary , we have demonstrated that R2R modules can be integrated with other fluidic circuits (e.g.. vacuum battery chip) and perform complex multi-step fluidic functions. The general concept also allows many other life science applications discussed herein to be possible. It also allows non-life scrence applications to be possible. c. Example embodiments

[0267] A collection of example embodiments providing additional description of a variety of embodiment E pes in accordance with the concepts described herein are provided below. These examples are not meant to be mutually exclusive, exhaustive, or restrictive; and the claimed subject matter is not limited to these example embodiments but rather encompasses all possible modifications and variations within the scope of the issued claims and their equivalents.

[0268] Independent Claim:A roll-to-roll manufactured fluidic module, comprising: a device that can be used as a stand-alone format independently; optionally be coupled upstream, downstream, or in between other fluidic circuits; and multistep, sequenced protocols can be run on the module.

[0269] Independent Claim:A consumable for point-of-care diagnostics, comprising:a device that has an upstream roll-to-roll manufactured part with fluidic channels; has a downstream part that uses vacuum battery technology; and the two parts are integrated by a valve that is opened on demand.

[0270] Independent Claim:A manufacturing process that entails: roll-to-roll manufacturing of a first fluidic circuit; and assembling a second fluidic circuit onto the first circuit in the same R2R line.

[0271] Independent Claim:A vacuum battery chip with these features: it uses vacuum batten technology7to drive fluid flow passively. The chip using this technology includes a gas permeable material, a void section to store vacuum, an auxiliary7channel, and a thin walled area to allow for gas diffusion; it is designed to be coupled with other fluidic circuits; andIntegrates with other fluidic circuits.

[0272] Independent Claim:A shell enclosure to store the vacuum battery chip under the vacuum with these features: a transparent plastic shell shape conforming to the vacuum battery chip (first layer); and sealed with a peelable lidding layer (second layer) with high vacuum barrier properties.

[0273] Independent Claim:A method for processing a roll-to-roll manufactured consumable with an automated workflow, the method comprising: at least two steps of the following: vacuum draining, acoustic mixing, heating, blister pushing, venting, liquid trapping, heat sealing, mixing, air bubble trapping, liquid ladder steps, and fluidic pooling areas.

[0274] Dependent Claims (based on Independent Claims):It has at least one of the following design features: blister pouch(s) and vacuum draining vents, and can be made with as few as two layers.The upstream roll-to-roll manufactured part is designed to perform sample prep for nucleic acid extraction.For the above claim, the extraction method is the boom method.For the above claim, the boom method uses nano or micro-sized magnetic beads with silica coating to capture nucleic acid.The fluidic module can run nucleic acid extraction with one or more components in the following for its reagents: a lysis buffer, chaotropic lysis buffer(s). salts, ethanol or IP A, detergents, silica-coated magnetic beads, enzymes, protease k, lysozyme, RNase inhibitors, carrier RNA, AW1 buffer, AW2 buffer, guanidine chloride, AVE buffer, TE buffer, water, DI water, charge switch beads, and AVL buffer.Alternatively, the magnetic beads above have a charge switch chemistry.The upstream roll-to-roll manufactured part has at least one of these components: fluidic channels, blister pouches for reagent storage, burst valves, vents, liquid traps, waste chamber(s), alignment markers, heat seal sections, mixing pouches, crushable structures, integration areas, air bubble traps, vacuum / pressure ports, multichannel junctions, liquid ladder traps, fluidic pooling areas, and built-in labeling.The consumable device has microfluidic parts and / or macro-fluidic parts.The consumable can be used with the "open development reader platform / ’ Pressure-sensitive adhesives join together the upstream and downstream parts. The valve area has overlapping fluidic channels that are separated by film material.The integration area has an integration pin that punctures the film that separates the upstream and downstream fluidic channels.The vacuum force in the downstream module allows fluid loading into microwells.Isothermal amplification is run in the consumable and can include one of these chemistries: EXPAR, HD A, SDA, RPA, Rapid Amp, LAMP, NASBA, WGA, NEAR, Sherlock, Detectr, TMA, RCA, MDA, SMAP2, SPIA, SMART, ICAN, PHAMP. HCR, CRISDA, BAD AMP, and EIC.PCR can be run on the consumable.At least some of the design features, materials, and / or manufacturing methods are cross-compatible with gen2 SIMPLE chip designs, defined in section 1.It is integrated with a downstream vacuum battery fluidic circuit.The R2R module and or vacuum battery chip can optionally be coupled upstream, downstream, or in between other fluidic circuits.It is used for life science applications.It is used for non-life science applications.It is used for one of these applications: point-of-care diagnostics, nucleic acid preparation, sequencing library prep work, immunoassays, DNA arrays, protein extraction protocols, cell staining, electroporation, protein engineering, forensic application sample prep, next-generation sequencing applications, nucleic acid isolation, purification, size selection, clean-up, library normalization, electrophoresis, integration with other R2R fluidic circuits, flow cytometry', cell staining, electroporation, directed evolution, droplet microfluidics, lateral flow assays, paper microfluidics. ELISA, immunoassays, capillary microfluidics, DNA arrays, pH sensors, gas sensors, exosome purification, exosome detection, PCR, qPCR, digital PCR. and cancer detection.Fluidic circuits, buffers, and protocols can be designed and tailored for application-specific workflows.Magnetic beads are conjugated with other surface chemistries such as antibodies, enzymes, aptamers, other custom ligands, protein A / G, and carboxylate coatings.It is used with a reader or test platform that has at least one of these functions: heat sealing, acoustic mixing, ultrasonic mixing, heating, optical readout, electrical readout, electrochemical readout, push pin crushing, repositioning gantries, reprogrammable workflows.The vacuum battery chip has alignment markers located on its surface.Nested or two-step amplification can be done to improve the limit of detection.Some or all of the manufacturing processes, materials, and design features are cross-compatible with other variations of the SIMPLE chip.

[0275] Automated filling machine a. Overview

[0276] Some expected advantages of the embodiments of the automated filling machine include, but are not limited to:1) Can be integrated and automated into the SIMPLE chip manufacturing R2R line;2) Can dispense accurate amounts of liquid and solids into the SIMPLE chip;3) Can create vents, cut hole ports, or remove unwanted material;4) All steps above can be done in a single step or separate steps; and5) Minimizes spilling with an optional tilted loading tray.

[0277] Embodiments of the present disclosure will now be described in detail. A filling machine shown in FIGURE 4A was developed for the Genl SIMPLE chip to dispense wash buffers (or other reagents such as wax pellets or lyophilized pellets) and other required reagents into the R2R consumable chip cartridge at microliter to milliliter volumes. The system uses a loading tray 405 that orients an empty R2R chip 406 in a consistent position, flat side facing up, and keeps the chip's alignment to the injection needles. The figure showsthe loading tray at an optional ~30-degree angle relative to the ground, so reagents will be less likely to spill during and after injection. In this case, the cover layer is already sealed onto the fluidic layer, and the inclination angle allows fluid to pool towards the bottom side of the blister pouches, leaving the air vent and liquid injection site in the blister pouches above the liquid level, in effect reducing spill risk. Depending on the application, the loading tray can be horizontal or at other angles (vertical or almost vertical). The loading tray angle should be adjusted to minimize spilling risk.

[0278] The loading tray door hinge is optionally closed, clasped via magnets, and inserted into the main structure of the machine. An optional safety lock pin 408 is placed to secure the loading tray in the machine. The user then activates the machine via an external push button, which causes a linear pneumatic cylinder 401 to extend and push a series of needles 403 on the sliding plate 404 down. The sliding plate can be on linear bearings 409. Optionally, the pneumatic cylinder could be replaced by electric motors or solenoids. As the needles move down, they pass through alignment holes on the surface of the loading tray 407 and then pierce the backing material of the genl SIMPLE chip's R2R sample prep module’s blister pouches and come to a stop so the needle orifice is inside the chamber of the chip. Optionally, an extra needle can simultaneously create a side vent in the same blister pouch to prevent overpressuring and damaging the burst valves during the liquid injection.

[0279] An external (multichannel) peristaltic pump (other types of pumps may be used too, such as screw pumps, syringe pumps, microfluidic pumps, air pressure pumps, piezo pumps, etc.) that is connected to the needles at a connection point 402 is then activated and dispenses specific reagents for each channel at a specified volume. In the genl SIMPLE chip case, that includes washl / 2 / 3 buffers. After the liquid dispensing process, the linear pneumatic cylinder is de-pressurized, and the needle plate is pulled out of the chip. The chip can then be removed from the system the same way it was inserted, and a lidding layer can then be applied to seal the needle puncture holes.

[0280] In a fully integrated format in an R2R line, the filling machine would be integrated in-line with the R2R line (FIGURE 4C). The tray would receive the SIMPLE chipscontinuously in-line automatically, so the tray itself would not need to be removed each time manually (or the tray would move with the film according to each index forward. An index is the unit step cycle forward in an R2R line). The SIMPLE chips would continuously feed into a tray that allows stability7and alignment of the chip during needle piercing. Again, the chip filling station may be at any angle that minimizes fluid spilling risk. The chips would exit the filling machine station opposite the way it entered. The activation of the needle piercing motion and liquid dispensing can be electronically programmed and triggered by syncing with the R2R index cycle.

[0281] In addition to liquid reagents, viscous grease, or wax material for burst valves can be dispensed using a modified machine version. In particular, screw pumps can pump high-viscosity material precisely and effectively.

[0282] In another embodiment, a solid pellet dispenser may be integrated into the R2R line (parallel or serial to the liquid dispensers). For example, all lyophilized pellets in each of the microwells can be dropped simultaneously using a solid dispensing mechanism instead of a needle format. The mechanism should only allow one (or a differently defined amount) unit of solid material to be dispensed per channel (such as one pellet into one microwell) per R2R index cycle. Using the same concept, another solid pellet dispenser placing integration pins can also be added to the R2R line, and so on. Alternatively, extra vent holes or inlet holes may be made with the chip-filling machine simultaneously with a puncture-type tool. A sharp cutting die or blade with custom shapes can cut through the fluidic layer or cover layer to make vents, inlet ports, and outlet ports or remove any undesired material.

[0283] In another embodiment, the injection dispenser 410 can be angled and have multiple movement axes (FIGURE 4B). This could allow reagents to be “laid” down in a pattern similar to squeezing writing onto cakes. It could allow the fluid injection needle further travel distance into the blister pouch or a fluidic channel. For example, it could allow the cover layer to be sealed on the fluidic layer first, then inject wax or grease material into a narrow, normally open channel to make that into a wax valve. There will be less risk of the wax valve material causing cover layer sealing problems with the fluidic layer since thesealing process is done first. Alternatively, it could lay down a strip of the desired pattern shape of the material. Optionally, a screw pump 411 may be used for high-viscosity materials, such as fluoropolymer grease. The pump may be actuated by stepper motors 419. It may optionally have a heated hopper to reduce the viscosity' 412. It may have needles 413 and multiple units. It can be combined with other dispenser units using a different technology 414. It may be mounted on linear rails parallel to the production line 415. It can be fully adjustable in the x, y, z, and rotational positions 416. One or more linear actuators 417 may be used to allow movement of the dispensing head in relation to the (fluidic) cartridge 418. All adjustable joints can be motorized to provide custom movement in the X, Y, Z, and rotational direction.

[0284] The chip-filling machine can dispense unit amounts of liquid, solids, lyophilized pellets, and / or integration pins. It can create holes and vents or remove unwanted material. All of these actions may be combined with one another or be separated into different filling machines. The actions can optionally be done together in a single index cycle step. The chip-filling machine can be used in all generations and embodiments of the SIMPLE chips. The chip-filling machine can work in an in-line R2R manufacturing format or as a standalone semi-automated station. The chip-filling machine is not limited to filling R2R- made SIMPLE chips. It can be used for general (micro)fluidic chip filling of reagents or parts. It can fill other non-R2R-made chips (such as injection molded chips). b. Example embodiments

[0285] A collection of example embodiments providing additional description of a variety of embodiment ty pes in accordance with the concepts described herein are provided below. These examples are not meant to be mutually exclusive, exhaustive, or restrictive; and the claimed subject matter is not limited to these example embodiments but rather encompasses all possible modifications and variations within the scope of the issued claims and their equivalents.

[0286] Independent Claim:A filling machine, comprising:a loading tray, a dispenser, and an alignment feature; an exit that chips would move out the filling station from the opposite end they entered the filling station, allowing continuous in-line filling; configured to dispense liquids or solids into SIMPLE cartridges in manufacturing lines; can punch holes, vents, and or remove material; can combine some or all above actions into a single station and actuate in a single index cycle step; and the loading tray is orientated at an angle to minimize liquid spilling.

[0287] Dependent Claims (based on Independent Claim):It can be used for R2R-manufactured SIMPLE chips.It can be used for none R2R manufactured SIMPLE chips.It can be used for fluidic cartridges.It can be used for microfluidic cartridges.Dispenses microliter to milliliter volumes.The loading tray is orientated at an angle range of 0 to 90 deg to minimize liquid spilling.It uses at least one of these pumps in the following peristaltic pumps: screw pumps, syringe pumps, microfluidic pumps, air pressure pumps, or piezo pumps.Can be multi-channel.Can dispense high-viscosity material.Can dispense wax and or grease material.Loading tray can move with the R2R line’s index.The filling machine has liquid dispensers with needles.The filling machine has solid material dispensers.The filling machine can dispense grease and or wax.The filling machine can be semi-automated or fully automated.Can fill material into all embodiments of SIMPLE chips.The machine has multiple movement axis.The machine can dispense specified patterns.The machine is heated.

[0288] 10, Roll-to-roll manufactured burst valves a. Overview

[0289] Some expected advantages of the embodiments of the R2R manufactured burst valves include, but are not limited to:1) Releases fluid and / or gas inside a blister on demand, gently without an intense burst (a strong burst can be disruptive for downstream fluidics);2) Simple to integrate into roll-to-roll manufacturing, conventional microfluidic blisters require more complex integration;3) Simpler construction than conventional microfluidic blisters, hence the lower cost;4) Conventional microfluidic blisters use a separate aluminum-formed pouch that is adhered to a fluid receiving port — the fluidic layer often needs molded sharp (plastic) pins to rupture the blister pouch, which is not needed in these embodiments; and5) Can entirely dispense without fluid residue, which is difficult to accomplish with traditional microfluidic blister systems.

[0290] Embodiments of the present disclosure will now be described in detail. Roll- to-roll manufactured burst valves, such as the burst valves 1412 shown in FIGURE 14, can store liquid reagents and release liquid on demand when the blister pouches 1422, 1423, 1424. etc. are crushed. The R2R-burst valves have a simple chip construction design and are easy to manufacture. In contrast to conventional microfluidic blister systems, which typically involve a separate blister foil pouch attached to a rigid body (plastic) fluidic circuit that has puncture pins, the R2R burst valves are unibody in design and do not require extra parts or a rigid body construction to work. This design does not add any separate complexmanufacturing steps not already included with the R2R manufacturing process described herein (or any new steps that can be directly and easily integrated into the R2R line).

[0291] The burst valve can have a narrower cross-section than the fluidic channel (or optionally equal or larger to the fluidic channel if the burst valve channel cross-section is sufficiently small). The burst valve is located between the blister pouch and the fluidic channel. It has fluoropolymer grease to act as a normally closed valve that bursts open upon compressing the blister pouch. The location of the grease valve may have a smaller fluidic channel cross section or have no channel structure at all to increase flow resistance. The burst valve is fluidically sealed on the sides of the valve, the seals continuously following the sealed portions of the sides of the fluidic channels towards the blister pouch to prevent undesirable leakage to the side. Grease material also increases fluidic resistance. The valve material (such as grease) holds the liquid in the blister within an internal pressure range. Once the pressure exceeds a certain threshold, the grease material deforms and is pushed out of the burst valve, allowing the liquid to flow through.

[0292] The burst valve’s fluid flow through area does not have the cover layer sealed to the formed layer unless it is a nongreasy valve format (such as a two-step heat seal format discussed below). The valve material can be fluoropolymer grease, which is high viscosity, inert, and does not interact with the stored liquids. Fluoropolymer grease also does not affect cover layer sealing or heat sealing.

[0293] The burst valve can facilitate the gradual release of the liquid contents without causing a sudden burst (which is often the case with other types of burst valves). Avoiding a sudden “burst” is desirable because, for example, sudden bursting can cause subsequent fluidic actions, such as in the genl SIMPLE chip’s magnetic beads overflowing into the waste chamber accidentally. After bursting, a significant portion of the grease that was originally inside the blister pouch moves into the other side of the valve and stays in the fluidic channel side. It conforms to the channel, and after the fluid contents flow through, the valve material can plug the smaller cross-section of the burst valve, effectively preventing backflow. Backflow prevention is possible because the grease reflows and plugs the smaller cross-section with any reverse flow pressure. Preventing backflow is desirable because often, there is back pressure created by other fluidic steps, which can cause unwanted backflow, resulting in residue or cross-contamination problems. Optionally, to further reduce any risk of backflow, a heat sealing process can permanently close off the fluidic channel leading to the blister pouch after the burst valve is opened. In this case, the grease still functions as a temporary backflow stopper before the heat-sealing action can be done. Without the grease stopping backflow, often, when the external pressure force such as the push pin is released from the blister pouch, the blister pouch may have a shape memory' effect and try to wholly or partially reform its shape, which can cause expelled fluid to flow in reverse back into the blister pouch.

[0294] The burst valve holding strength, defined as the internal pressure threshold that needs to reach until the valve is open, can be modulated and made higher by decreasing the burst valve cross-sectional area, increasing grease viscosity (or structural integrity for solid wax-type valves), lowering the temperature during the bursting process, increasing the fluidic flowpath's fluid resistance, and vice versa. For example, the burst valve can be made with an extra high viscosity' grease and a small cross-section, so there is a high holding power burst threshold, but it can be heated to weaken the grease (which lowers its viscosity) so the burst threshold is decreased. When the burst threshold is decreased, there is less risk of a sudden ■‘burst” action, where fluids rush out suddenly, which can be undesirable for downstream processes. For example, a sudden burst may cause downstream magnetic beads to be accidentally expelled into the waste chamber in the genl SIMPLE chip.

[0295] Alternatively, the burst valve may be made of a two-step heat sealing process without grease (weaker seals can be made with lower sealing temperatures than surrounding channel stronger seal areas). In this embodiment, the burst valve area has the cover layer and fluidic layers sealed together. This seal area has a weaker seal than surrounding fluidic channel perimeter seals. The burst valve could be made with a two-step heat sealing process that could be integrated directly into the R2R process that is implemented sequentially. Alternatively, a weaker seal section can be made by coating the specific weak area withmaterial that weakens its sealing strength (e.g., oils, grease, hydrophobic coatings, surface energy reducing coatings, oleophobic coatings, hydrocarbon materials, fluoropolymer material, other polymers, solvents, fine particles, and others). FIGURE 4C shows an embodiment of this setup in 424. There are two heating stations. One is designed to seal the fluidic channels, and the other is designed to seal the burst valve. In this setup, heating pressure, time, sealing plate knurl texture, and temperature can all be modulated. Stronger (following the sides of the fluidic path) and weaker seal (directly at the burst valve where it stops the flow) sections can be created by modulating the heat sealing process’ heating time, pressure, temperature, and sealing plate knurl surface texture. Higher heating time, pressure, temperature, and more sealing plate knurl texture generally create more robust seals and vice versa. The cover lidding material must be matched to work with the heating process modulation. For example, the cover material may create more robust seals when more heating and pressure are applied.

[0296] Alternatively, instead of heat, a two-step sealing process can create weak and strong seals by modulating the pressure applied when PSA cover layers are used (or adding some combination of the factors mentioned above). The PSA should have a seal strength dependent on the sealing pressure and duration of sealing applied.

[0297] For the grease-ty pe embodiment, fluoropolymer greases can be used. It is inert and does not interfere with the buffer reactions. Fluoropolymer generally does not interfere significantly with the cover layer bonding process to the fluidic layer (including heat sealing and PSA sealing) as long as adequate pressure is applied. Alternatively, the grease can be made of hydrocarbon-based greases such as VASELINE® or other petroleum-based grease. We have found that Vaseline does not interfere with our sample prep reagents. Alternatively, hydrocarbon or fluoropolymer solid wax-type material may be used instead of grease. For example, paraffin or certain fluoropolymer ski waxes may be used. Optionally, the grease, wax, or weak heat seal at the burst valve can be melted to lower the burst pressure threshold during bursting.

[0298] The blister pouch may be any shape if it can contain liquids and be crushed easily while directing the fluid toward the burst valve. For example, a teardrop-like shape with a small liquid channel at the bottom near the burst valve works well. The blister pouches should be easily crushable by external forces, so their footprint must be sufficient. When the blister pouch’s footprint (diameter) is too small, it can form a rigid dome-like structure that is difficult to crush (thinner forming material or larger diameters can be used to mitigate this problem).

[0299] The blister pouch volume is optionally larger than the stored fluidic volume so that when the fluid is injected, it does not overflow during manufacturing. A smaller liquid volume means an air volume will be trapped inside the pouch. This air volume’s compressibility allows enhanced functionality w ith the blister valve design. If less air volume is left during manufacturing in the blister pouch, then the pouch releases liquid as fast or slow as the push pin is pushing due to the incompressibility of the fluid inside the pouch. This effect helps modulate and control the flow rate during valve opening. For example, using this format, “bursting” is avoided, and a slow and gentle flow can be achieved during valve opening (wash2 1424, wash3 1423 pouches).

[0300] When complete liquid dispensing with minimal residue or dead volume (remaining in the blister pouch) is desired, more air can be inside the pouch during manufacturing. A faster push pin action is used, and this effectively compresses the air inside the pouch slightly, so it has a “spring” effect where when the liquid starts to expel through the valve, the remaining compressed air uncompresses and pushes all remaining fluids entirely out of the blister pouch. Due to a combination of air compression and faster external pressure application (by push pin), the bursting speed is increased. Hence, the bursting speed of the fluid can be modulated by the volumetnc ratio of air versus liquid stored in the blister before bursting. Optionally, along narrow-shaped blister pouch 1425 can be used so that liquid does not retain residue on the side w alls of the pouch during fluid bursting since the receding fluid meniscus draws out all surface liquid residue. An even smaller cross-section of the burst valve channel size can increase fluidic resistance further and allow a more pronounced aircompression effect to minimize fluid residue. A smaller cross-section of the burst valve also reduces the flow rate during fluid release.

[0301] In another embodiment, two immiscible liquids may be placed in the blister pouch during manufacturing to reduce further post-dispensing fluid residue remaining in the blister pouch. For example, after the reagent buffers are added, fluorinated oil or hydrocarbon oils / solvents that are immiscible with water may be placed in the same blister pouch with a water-miscible type buffer. If a gentle fluid release (slow push pin action) without burst but minimal buffer residue is desired, then one can use this format. Note that fluorinated oils typically have a higher density than water, and hydrocarbon oils / solvents have a lower density than water. In a vertical chip format, the burst valve needs to be orientated on the bottom for hydrocarbon systems. The burst valves need to be orientated on top for fluorinated oil systems so that the water-based buffer is always the first one being expelled out of the valve (when the SIMPLE chip is used in a vertical format as in the genl reader). While there could be residue or dead volume left from the fluorinated / hydrocarbon fluids, those fluids are selected not to interfere or participate in downstream reactions, so the dead volume of these liquids does not affect the overall reaction, while the desired water miscible fluid is entirely dispensed at its total volume.

[0302] The manufacturing process can be integrated entirely in-line with the R2R process described in other sections. For example, the blister pouches and burst valves can be formed at the same thermo-forming process with the fluidic layer in a unified single step by including all these features in the same mold. The grease can be applied before, concurrently, after, or in sequence to lyophilized pellet and buffer loading, all on the same R2R line (at the same or different index steps). The cover layer sealing process can be done in a unified mold to seal all the burst valves and fluidic channels in a single step (or optionally in two steps for weak and robust seal sections). All the materials for the cover layer and fluidic layers mentioned in other sections are cross-compatible. The burst valve can be manufactured with as little as two significant layers, including a fluidic layer and a cover layer.

[0303] Fluidic storage and on-demand release are fundamental in many microfluidic and macrofluidic systems, especially in life science applications such as diagnostics. These burst valves can be implemented in many types of fluidic consumables, including but not limited to all the life science applications described in other sections. It may also be used for non-life science applications. b. Example embodiments

[0304] A collection of example embodiments providing additional description of a variety of embodiment types in accordance with the concepts described herein are provided below. These examples are not meant to be mutually exclusive, exhaustive, or restrictive; and the claimed subject matter is not limited to these example embodiments but rather encompasses all possible modifications and variations within the scope of the issued claims and their equivalents.

[0305] Independent Claim:A valve that permits liquid and or gases to be released on-demand from one part of a fluidic circuit to another fluidic circuit, comprising: a blister pouch structure to store liquid and or gas; a normally closed valve; the valve having a frangible section that can be opened when the internal pressure in the blister structure is increased; fluid and or gases transfer from the blister to the fluidic circuit on the other side of the valve once the valve is opened; and the entire burst valve assembly can be manufactured with roll-to-roll processes, along with other microfluidic or macrofluidic fluidic circuits and features.

[0306] Independent Claim:A burst valve with the following features: a highly viscous valve material; the valve material and channels act as a normally closed valve that can be opened on demand;the valve material can prevent backflow; it releases fluid contents without a sudden burst; it is coupled with a blister pouch; and the entire burst valve assembly can be manufactured with roll-to-roll processes, along with other microfluidic or macrofluidic fluidic circuits and features.

[0307] Independent Claim:A burst valve blister design with the following features: it can fully dispense all liquid within the blister pouch, leveraging the compressibility effect of gas within the blister pouch; it can modulate the bursting speed by modulating the air / fluid ratio within the blister; and it can be manufactured with a roll-to-roll process.

[0308] Independent Claim:A burst valve-blister design with the following features: it holds two immiscible fluids within the blister; it allows full dispensing with no dead volume left for one of the fluids; and it can be manufactured with a roll-to-roll process.

[0309] Dependent Claims (based on Independent Claims):The valve can be manufactured with as few as two major layers, a fluidic layer, and a cover layer.The valve opens without causing the blister's liquid (or gas) to “burst” suddenly, and the liquid (or gas) release is gentle and gradual.Said valve has a decreased cross-sectional area compared to the blister.The valve's narrow section does not have any preformed channel structures. just a frangible section that can be delaminated so fluid (and or gas) can pass-through.The valve has preformed channel structures that are continuously open but has a frangible material blocking the channel structures.The valve uses fluorocarbon-based grease, hydrocarbon-based grease, solid hydrocarbon materials, or solid fluorocarbon materials for the frangible section.The valve has paraffin wax or fluorocarbon wax in the frangible section.The valve can be weakened (and or opened) by heating.The valve area has a weaker seal compared to the surrounding seals around all the fluidic components, so the weaker seal is ruptured when the blister pouch’s internal pressure is increased on demand.The valve is made with a two-step heat and or pressure sealing process, creating a weaker seal at the valve and a firm seal around fluidic channels to contain the fluidic flow path.The valve can be used for both microfluidic and macrofluidic circuits.The valve can be combined with heat sealing to prevent backflow.The burst valve holding strength can be modulated by the burst valve cross- sectional area, grease viscosity, heat, and or channel fluidic resistance.The valve is integrated inside the SIMPLE chip.The fluid bursting speed of the fluid can be modulated by the volumetric ratio of gas versus fluidic stored in the blister before bursting.Fluid can be fully expelled from the blister pouch if there is a large enough air volume present and sufficient speed in applying external pressure to the blister pouch.The valve can utilize gas compression effects to expel liquid from the blister pouch fully.The fluid bursting speed can be modulated by controlling the external pressure force on the valve.The valve cross-sectional area modulates the fluid bursting flow rate.The burst valve does not need any mechanical sharp puncturing devices to open the burst valve.Air volume remaining in the blister pouch can modulate fluidic burst speed.A gentle burst speed can be achieved with more liquid and less air in the burst pouch.Two different immiscible liquids may be placed in the blister pouch.The manufacturing process is cross-compatible with SIMPLE chip manufacturing.Materials are cross-compatible with SIMPLE chip manufacturing.It can be used for life science applications.It can be used for non-life science applications.It can be incorporated into microfluidic or macrofluidic designs.

[0310] 11, Fluidic Integration pins a. Overview

[0311] Some expected advantages of the embodiments of the fluidic integration pins include, but are not limited to:1) Simple, low-cost device that allows two fluidic circuits to be joined together;2) Compatible with roll-to-roll devices or injection-molded devices;3) Impacts fluidic functions (i.e., mixing) and chemistries minimally (i.e.. nucleic acid extraction); and4) Retains minimal fluidic residue.

[0312] Embodiments of the present disclosure will now be described in detail. The fluidic integration pin 1434 (FIGURE 14) can serve as a mechanism that connects two distinct, previously unconnected fluidic circuits together on demand, such as the sample prep module and vacuum battery chip in the Genl SIMPLE chip. It is located within the fluidic circuit, such as inside the mix pouch 1435. It retains minimal fluidic residue across different washes due to its thin wire profile shape. Fluid residue tends not to stick to the structure. Due to its low cross-section profile, it also impacts fluidic mixing minimally. Fluid can flow and mix around the pin freely. At one end, the pin has a sharp point that can puncture a membrane valve between two fluidic circuits 1436. The membrane valve can be a film(s) or a thin-walled section(s) that is normally closed. It can have a flat shape at its stability base at the other end to partially wedge itself between the cover and fluidic layers so the pin does not move out of place or rotate out of the integration site undesirably. The stability base secures the pin into a safe resting position so that during manufacturing or transport, the pin does not accidentally puncture the membrane valve prematurely. The pin 1434 may have one or more bends 1434a between the sharp point and flat wedge to conform to the fluidic chamber shape and point the sharp point towards the membrane to be punctured. The sharpness of the puncture tip can be modulated so that it is not so sharp that simple vibrations from transport will cause the membrane to be punctured but sharp enough when an external force is applied that it can puncture the membrane valve on demand. An external force can be applied manually or robotically on the pouch holding the pin. The pin material and design should provide enough structural integrity to puncture through the membrane valve. The integration pin sits on one side of the first fluidic circuit (such as the sample prep module) and has its puncture point aligned to the outlet of the first fluidic circuit and the inlet of the second fluidic circuit (such as the vacuum battery chip 1411). Initially, the two fluidic circuits are kept independent by a membrane valve. The membrane valve can be one or a combination of the following: thin films, PSA, vacuum bag material, and thin-walled section(s). Once the integration pin is pushed down, it punctures the membrane valve and creates a continuous flow path connecting the two fluidic systems.

[0313] There are several methods for the stability base to secure the pin. Examples include wedging the pin’s stability base between the cover layer and fluidic layer, wedging the pin’s stability base into fluidic features in the fluidic layer itself (such as small fluidic channels), sticking the pin directly onto the material of the consumable (such as PSA layer), sticking or molding the pin into the bulk material itself (such as bulk silicone in vacuum battery chip, or the rigid body SIMPLE chip), or placing the stability base in shape conforming sections (such as creating inverse cavity shapes on the rigid body SIMPLE chip to hold the pin). Optionally, instead of a flat wedge at one end, the pin could have a stability base made of a continuous wire bent multiple times like a paper clip to provide alignment and stabilityto secure the pin. The paper clip-like 2D stability base can conform to the pouch’s shape or stick to the PSA cover layer to provide even more stability. Optionally, the wire bends can be further wedged into small fluidic structures (instead of between the cover layer and fluidic layer) to provide further stability. Alternatively, part of an unbend pin can be wedged into small fluidic channel structures.

[0314] In another embodiment, the shape of the pin does not have to be a wire. If injection molding or compression molding is used to create the pin, other form factors may be used. The stability base can be a shape that wedges to predetermined fluidic shapes or cavities on the chip side so the sharp tip end does not move out of alignment. Alternatively, if sheet metal manufacturing (and other molding techniques) are used to create a flat profile pin, the pin can have one or more protrusions at the stability base that is fitted or wedged into small fluidic structures that secures the alignment position of the pin. With these alternative manufacturing techniques making non-wire shapes, the pin could have little side protrusion arms that wedge in small side fluidic cavities to provide stability. The pins can be shaped and bent in a one-step process, such as using cold forming, hydroforming, pressure forming, hydraulic pressing techniques, etc. The pin may be shaped in less than three steps. Other methods not discussed may be used, too. The general concept is that the stability base should allow the pin to stay at a resting position that does not cause accidental puncture during manufacturing, storage, or transportation, aligns the pin to the integration site, and can allow a displacement such that the integration pin can puncture and open the membrane valve consistently.

[0315] The pin can have a short ~90-degree bend at the sharp point to point the sharp point toward the membrane valve 1436 and to prop the sharp point slightly up away from the membrane valve so that it will have sufficient travel distance to puncture the films.

[0316] Instead of a wire-based manufacturing process, the integration pin can also be made from sheet metal processing, laser cutting, waterjet cutting, injection molding, stamping, die cutting, compression molding, thermoforming, cold forming, hot embossing, embossing, and bending processes. A combination of these processes can be used. Using the wire shape,it could adopt fast continuous processes used in paper clip manufacturing to bend the pin into desired shapes very efficiently.

[0317] In other embodiments, the sharp puncture point does not need to be needle- or wire-shaped. It can be blade-shaped, screw-shaped, drill-shaped, hollow-shaped, hole puncher-shaped, star-shaped, starfruit-shaped, football-shaped, round-shaped, Phillipsshaped, hex-shaped, cross-shaped, coconut opener shaped, all common other screwdriver tip shapes, and other shapes. In principle, the sharp point shape should allow the integration pin to puncture the membrane valve without causing a blockage of the fluid flow. For example, this can be done by designing the pin to follow a starfruit shape so that there is a sharp tip to puncture, and the starfruit protrusions expand the puncture hole larger but leave small channel gaps for fluid to flow through.

[0318] In another embodiment, the integration pin may have a “spring back” mechanism built in to prevent blockage of the flow. “Spring back” means the pin can return to its original resting position (or move out of the puncture site) after a puncture, minimizing the risk of the pin blocking fluid flow. For example, the stability base and bends allow a pin made with material that has shape memory to spring back to the resting state after external pressure is applied, effectively removing the pin from the puncture point so it does not block flow'. In another embodiment, a none w ire-ty pe pin (such as a sheet metal or injection molded integration pin) can be a straight or “v” shape, with hinges in the middle serving as the stability base so that the integration pin works similarly as a seesaw, where it can rotate slightly at the hinge. The stability' bases are side protrusion arms that conform to fluidic cavities, acting like hinges with some rotational flexibility. The external pressure can press one end of the seesaw containing the sharp point to puncture the membrane valve, then press the other end to remove the pin from the puncture site, avoiding fluidic blockage. In another embodiment, the integration pin has magnetic or ferromagnetic material and can be removed from its puncture side using an external magnetic force. The external magnetic force may also actuate it.

[0319] In another embodiment, acoustic vibrations can facilitate the membrane valve's puncture action. For example, ultrasonic transducers or horns may be applied to vibrate the pin and cause a puncture.

[0320] In another embodiment, the integration pin can be in non-R2R manufactured fluidic circuits, for example, the needle 934 (FIGURE 9F), which is part of a sample tube in 932. It can also integrate downstream fluidic circuits that are non-vacuum battery technologybased. The upstream and downstream fluidic circuit does not need to be limited to the sample prep module, R2R manufactured, injection molded. SIMPLE chip, or vacuum battery chip. The integration pin is versatile and can be used in any type of other fluidic circuit.

[0321] Various materials can be used to make the integration pin. Here are some examples, but not limited to stainless steel, galvanized steel, plastic, polymers, epoxy, titanium, aluminum, steel, alloys, other metals, magnetic materials, non-magnetic materials, ferrous materials, non-ferrous materials, low or high magnetic coercivity- materials, low- or high surface energy materials, hydrophilic / hydrophobic / oleophobic materials, and fluoropolymer material, etc. The integration pin can be made with single or multiple materials combined. The pin material must be carefully selected for biological or chemical reactions to avoid interfering with the reactions. We have found that titanium material works well in biochemical reactions. For example, titanium pins can be used for nucleic acid sample prep applications and do not reduce the DNA / RNA recovery yield. Due to titanium’s non-ferrous properties and low magnetic coercivity, it does not interfere with the reaction by undesirably retaining magnetic beads. Polypropylene-made pins also work well for biochemical reactions due to their inertness and low surface energy.

[0322] The two fluidic circuits may be on two distinct assemblies on top of one another, as shown in FIGURE 14. Alternatively, the two fluidic circuits may be on the same assembly, such as all on the R2R sample prep module side, but separated by a membrane valve. In this case, additional layers can be added, such as adding another fluidic layer beneath the cover layer so that it has “u” bridge connectors, which connect the two top fluidic circuitsoutlets and inlets, but are separated by membrane valves that can be punctured with integration pins.

[0323] In another embodiment, at least one of the fluidic layers is made by the R2R process, silicone molding process, vacuum battery' technology', injection molding process, compression molding process, hot embossing, and SIMPLE chip technology.

[0324] Other applications include using the integration pin to connect more than two fluidic circuits with one or more pins. The integration pin assembly can serve as a normally stopped valve that can be opened on demand, which can be very' useful in a wide range of microfluidic or macrofluidic applications. b. Example embodiments

[0325] A collection of example embodiments providing additional description of a variety of embodiment types in accordance with the concepts described herein are provided below. These examples are not meant to be mutually exclusive, exhaustive, or restrictive; and the claimed subject matter is not limited to these example embodiments but rather encompasses all possible modifications and variations within the scope of the issued claims and their equivalents.

[0326] Independent Claim:A device that connects two independent fluidic circuits, comprising: a membrane valve that separates the two fluidic circuits; the membrane valve is a film or a thin-walled section; a pin with a sharp point to perform a puncture action that connects the two fluidic circuits; the pin has a stability base; andCan be actuated with an external force.

[0327] Independent Claim:A device that opens membrane valve structures to connect one fluidic system with higher pressure to another fluidic system that is under vacuum when an external force is applied to the pouch, comprising:a pin made of formed material; located inside a fluidic structure; one end has a stability base, which secures the pin to restrict movement except for the puncture action; the pin has a sharp point to puncture membrane valve(s): and the membrane valve is a thin film or thin wall section that separates the two fluidic circuits.

[0328] Dependent Claims (based on Independent Claims):At least one of the fluidic circuits is made by the R2R process, silicone molding process, vacuum battery technology, or SIMPLE chip technology.The pin is made of sheet metal.The pin is a wire that is folded.The pin is shaped in a one-step process.The pin is shaped in less than three steps.The pin is made of titanium or stainless steel.The membrane valve can be film-based or a thin-walled section.The integration pin has a stability base to secure the pin from misalignment from the desired puncture location.A s tabi 1 i ty base securing structure can be a flat portion that wedges in between two layers of supporting structures.The stability base may be wire folded on a 2D plane, like a paper clip.The stability base may be a wire flattened at one end.Connects a roll-to-roll manufactured fluidic circuit to another fluidic circuit.Connects a roll-to-roll manufactured fluidic circuit to a vacuum batten -driven fluidic circuit.Pin material does not interfere with biochemical reactions.The pin material is made of titanium.Pin material is made of one of these materials: stainless steel, galvanized steel, plastic, polymers, epoxy, titanium, aluminum, steel, other metals, magnetic materials, non-magnetic materials, ferrous materials, non-ferrous materials, and fluoropolymer material.The pin is made of a wire material.The pin is made into a non-wire shape.The pin is manufactured by metal processing, injection molding, stamping, die cutting, compression molding, thermoforming, cold forming, hot embossing, embossing, and bending processes.The sharpness of the puncture tip can be modulated so that it is not so sharp that simple vibrations from transport will cause the membrane to be punctured but sharp enough when an external force is applied that it can puncture the membrane valve on demand.It retains minimal fluidic residue across different washes due to its thin wire profde shape.The integration pin sits on one side of the first fluidic circuit (such as the sample prep module) and has its puncture point aligned to the outlet of the first fluidic circuit and the inlet of the second fluidic circuit (such as the vacuum battery' chip).The pin tip can be blade-shaped, screw-shaped, drill-shaped, hollow-shaped, hole puncher-shaped, star-shaped, starfruit-shaped, football-shaped, round-shaped, Phillips-shaped, hex-shaped, coconut opener shaped, all common other screwdriver shapes, and other shapes.The pin has a spring back mechanism, where material shape memory' allows the pin to spring back to the resting state shape after external pressure is applied and released.The pin’s piercing action can be actuated with acoustic vibrations.At least one of the fluidic circuits is made of the R2R process, silicone molding process, vacuum battery technology, injection molding process, compression molding process, hot embossing, and or SIMPLE chip technology.More than two fluidic circuits with one or more pins can be connected.

[0329] 12, Open reader platform for rapid cartridge R&D a. Overview

[0330] Some expected advantages of the embodiments of the open reader platform for rapid cartridge R&D include, but are not limited to:1) Allows extremely fast R&D iterations due to the open format of the reader. Different test routines and tool movements can be programmed and tested using the same reader while updating the consumable designs continuously;2) Using the same consumable design, multiple routines can be tested and optimized, further increasing overall analytical performance;3) Can utilize various tools heads, including but not limited to contact heaters, optical heaters, magnetic tool heads, push pins, heat sealers, ultrasonic horns, vacuum ports, and pressure ports;4) Can use this reader platform to obtain analytical and clinical testing data on the consumable chip;5) Reader is future compatible with new designs of the cartridge; and6) Useful for a wide range of life science and non-life science applications. b. Hardware design

[0331] Embodiments of the present disclosure will now be described in detail. While developing the Genl SIMPLE chip, we developed an open reader platform to accelerate SIMPLE chip cartridge development. Specifically, the open reader can intake unlimited variations of SIMPLE chip designs for fast iterative testing. Once the SIMPLE chip design is set. the open reader can optimize test routines further to improve chip performance and / or reduce speed. Finally, the reader could be used to acquire analytical and clinical performancespecs for the SIMPLE chip. Analytical testing included characterization of the limit of detection (LOD), analytical sensitivity, analytical specificity, sample prep DNA / RNA recovery yield, and assay time. Clinical testing included clinical sensitivity and specificity.

[0332] R&D is majorly accelerated by the ability’ to quickly test many R2R consumable designs using the same open reader. It is crucial to find the optimal design for the consumable first and then build the reader around it to save R&D time and cost. Once the exact tools and routines are set, a new “fixed’’ lower-cost reader can be custom-made to perform the open reader's tasks and for mass production. Lower costs can be achieved by removing unnecessary components (such as tools, positioning, and automation hardware).

[0333] The open reader is shown in FIGURE 13 A. The reader contains a gantry with four onboard tools shown in FIGURES 13B-1 and 13B-2. The gantry 1301 can move horizontally, vertically, and rotate 360 degrees in either direction. The individual onboard tools are connected to linear motor systems 1309 that allow- them to move closer or away from the SIMPLE chip / cartridge individually. The four tools on the gantry include the following: a push pin with a magnet (or electromagnet) 1311 that is used to both compress chambers to induce fluid motion and to manipulate the DNA capture magnetic beads magnetically, a heated sealer 1312 that can be used to both preheat burst valves for more effortless opening and to compress and seal channels to prevent reflow', an ultrasonic hom 1313 that is used to fluidic mixing, and an optical heater 1314 that is used to heat a solution inside a chamber of the chip in a non-contact manner.

[0334] The push pin can compress the blister pouches or integration pin. It may also move and drag or hold magnetic beads during washes. The heated sealer may have a temperature control system to maintain a constant temperature. The heated sealer’s contact head should be a shape that effectively seals fluidic channels. For example, “line” shapes, “x” shapes, “o” shapes, or other shapes could be used. The sealer’s contact head should be made of highly heat-conductive material such as aluminum or copper. The ultrasonic hom is connected to a piezo ultrasonic transducer on the back. The hom amplifies and focuses thevibrational energy from the piezo transducer to a locally defined point, which can help with liquid mixing.

[0335] The ultrasonic horn 1313 contacts the SIMPLE chip and can help disperse (magnetic) beads in solution for enhanced washing, elution, and mixing. It can also help increase the mixing of elution and the dried reagents in the SIMPLE chip’s mix2 pouch 1435. It can also help the integration pin 1434 puncture membrane valves by vibrational energy. It can also enhance mixing kinetics in the vacuum battery chip. If more substantial power or vibration duration is applied, it can also cut open ports (such as vents) on the SIMPLE chip. Some tools, such as the heat sealer and the ultrasonic horn, have spring cushioning mechanisms 1306 that help maintain contact and prevent the tools from over-pressuring the SIMPLE chip, which can cause accidental ruptures or leaks. There may be subtle variations in the distance of the tool to the SIMPLE chip each time (due to natural tolerance variations). The spring system helps buffer those variations for sensitive operations. For example, if there was just a slight variation in the travel distance of the ultrasonic horn to the SIMPLE chip, there could be a big difference in the pressure applied to the SIMPLE chip if there was no spring cushioning present, which could lead to accidental rupturing of the SIMPLE chip by overpressure. The spring system helps the tools remain in flush contact with the SIMPLE chip, applies consistent pressure that can be modulated gradually based on Hooke’s law' and prevents overpressure. This spring system allows high consistency sealing with the heat sealer.

[0336] The optical heater can be a halogen lamp(s) or LED(s) that emits strongly in the infrared spectrum, which makes it very efficient at heating water solutions quickly without contact, as water absorbs optical energy efficiently in the infrared wavelength. The optical heater can heat the magnetic beads and elution to enhance DNA / RNA recovery' yield for the Genl SIMPLE chip. Optical heating can achieve faster heating than contact-based heating for bulk liquid pouches due to a better heat transfer than contact-type heaters. In alternative embodiments, other spectrums and / or types of light sources can be used according to the application's needs. One or multiple limit switches 1307 can limit or calibrate (home) motor position within the reader's system.

[0337] The gantry head and its internal brackets can all be made by sheet metal manufacturing processes, which can decrease the cost. The gantry head can have a revolving system 1318, which helps to turn 360 degrees both clockwise and counterclockwise. It can be mounted on lazy susan type or similar turntable-like bearing systems and driven by stepper motors with a belt. The gantry head 1301 sits on linear guides 1319 to move up and down vertically by the vertical translation drive system 1302, which is driven by two stepper motors.

[0338] The sliding backplate 1303 holds the Genl SIMPLE chip cartridges vertically, so gravity can pool liquid correctly for the integration site, mixing pouch, and burst valves. If liquid pooling is unnecessary, the backplate can be at any angle. The backplate also has linear guides that can slide horizontally by the horizontal translation drive system 1304. A stepper motor drives the horizontal translation drive system. Notably, the sliding backplate is on telescope linear guides and can extend out of the reader door for the user's chip loading and unloading access. This design allows the automatic presentation of the backplate to the user and automatic retraction without adding much more cost or design complexity to the reader. The sliding mechanism may be mechanically coupled with the reader's door, so the door automatically opens and closes in synchronization with the backplate’s extension and retraction movement. The reader has a fluorescence imaging system 1305. It is designed with the same principles as the imaging systems described in other sections. The reader also contains a custom control system PCB 1306 to house its electronics. The electronics placement may be any here where there is space available. We placed it on the upper section of the reader to enable easy access. To sense the movement axes' position, the reader has multiple limit switches 1307. such as hall effect switches.

[0339] As shown in FIGURES 13C and 13D, an R2R-manufactured cartridge (e.g., Genl SIMPLE chip) 1321 is first placed onto a bottom frame 1332. The bottom frame has alignment features 1333 that help align the cartridge so that the cartridge / chip is positioned precisely at the exact location each time. The alignment features are located at opposing far edges of the SIMPLE chip, which helps increase positional alignment consistency. Optionally, there is a “cross shape” and a “C” shape (or any other shape) that mates snugly with the chip’sfeatures. Within the extent of the open area of the bottom backplate, the chip can take on more variations of designs. The bottom frame may also be reconfigured to allow larger or smaller extents for even more flexibility of chip design. The entire back plate assembly (top frame 1334 + bottom frame 1332 + back frame 1327) can be redesigned to allow even more flexibility in cartridge design. An unlimited variation of cartridge designs can be compatible with the open reader.

[0340] The top frame 1334 is then closed by the user, and magnetic latches 1335 may hold the top and bottom frame together snuggly. The top frame may have a soft backing 1336, which helps improve heat sealing consistency. The soft backing could be an elastomer, rubber, or other soft material. The soft backing material ensures an even pressure distribution of heated sealer while sealing fluidic channels. The soft backing material may also create some buffer, lowering the risk of the tool overpressunng the chip (which can lead to chip ruptures). The top frame, chip, and bottom frame assembly are then closed to the back frame 1327. The back frame has linear rail carriages to connect to the horizontal translation system. The back frame may have vacuum or pressure ports 1338 connected to pressure or vacuum sources. The ports have o-rings to prevent gas or fluid leakage. Vacuum ports may be connected to a fluidic trap to prevent fluidic overflow into the pump. For the ports, the pressure / vacuum sources connected to these ports may have one-way valves to ensure uni-directional gas / liquid movement. Again, these vacuum ports may be relocated in different backplate designs to allow for more cartridge design flexibility. The back frame may have cut-out windows to allow for optical sensing or adding sensors that ensure the user has correctly loaded and closed the frames. In an alternative embodiment, there are only two frames. The top frame and back frame can be combined into one single frame.

[0341] The completely chip-loaded position is shown in FIGURE 13C. The backplate may have retractable magnets 1322 mounted on linear actuators. In the Genl SIMPLE chip, the retractable magnets, when extended, capture and secure magnetic beads on demand. Alternatively, it can retract to allow the beads to be released so they can be mixed freely when necessary . The backplate may have contact heaters (optionally with feedback control) 1324.In the Genl SIMPLE chip, the contact heaters facilitate isothermal amplification inside the vacuum battery chip. Heat transfers through the thin film of the R2R sample prep module side into the vacuum battery chip.

[0342] The backplate may also have acoustic transducers 1325, which can vibrate the SIMPLE chip and facilitate fluidic mixing. The transducer can be mounted on a floating bracket 1323, and the bracket can be directly secured to the contact heater. The acoustic transducer does not touch the backplate but is only in contact with the floating bracket. This design allows all of the acoustic vibrational energy to transfer to the contact heater first, in which the contact heater is primarily in contact with the vacuum battery chip. Maximum acoustic vibrational energy is transferred to the microwells in the vacuum battery chip, facilitating much better mixing, faster reaction kinetics, and high saturation of end-point fluorescence signals during isothermal amplification.

[0343] The components shown in FIGURES 13A-13D can be confined within a case. All primary frame structural supports and shell components of this case can be made with sheet metal processes, effectively lowering the cost. Sheet metal can be made of any metals, preferably stainless steel, and aluminum. Stainless steel is easy to clean and durable, while aluminum is lighter and cheaper. The outer surface is designed to be smooth to enable easy cleaning and disinfection. Protrusions and crevices are minimized to enable easy cleaning. The exterior shell casing is designed to be easily removed so technical repairs or maintenance can be done with easy manual access. The reader may have interface ports, for example, an on / off switch, power cord, screen connections, HDMI, and USB ports. It may have a touchscreen for human interface use. A vertical orientation is shown, but the screen may be optionally in a slanted degree like Gen2 reader for easier viewing and access. For both the open reader and gen2 reader embodiments, the screen and door can be on the same side. This design allows multiple readers to be stacked side to side like books, allowing more accessible screen and door access. The casing may be designed in a way that vertical stacking is possible. A door can be manually or automatically opened and closed. It can rotate open or optionally slide open like elevator doors. The door may also have magnetic latches to secure it. Thecasing may have handlebars at the top to allow for easy transport. None of the prototypes disclosed herein has undergone a dedicated industrial design process. Further humancentered design implementation can improve ergonomics, aesthetics, and user experience. Further industrial design can optimize it for manufacturing.

[0344] The electronics and computing system includes some or all of these subsystems: Stepper motor control, stepper motor, stepper motor driver, servo motor, servo motor control, temperature control, system cooling, airflow management, air filtering, power supply, screen connection, USB connection, Wi-Fi connection, Bluetooth connection, cellular connection, GPS, switching, relays, computing, LED driver, LEDs, optical lamp, camera, PCB board, wiring, circuit breaker, display, ultrasonic transducer driver / amplifier, acoustic transducer amplifier, MOSFET, microcontroller, human machine interface, UI / UX interface, system control software, onboard data storage, cloud data storage, imaging analysis software, limit switches, position homing systems, reprogrammable script for workflows, Raspberry' Pi computing, Arduino computing, g-Code software, Marlin software, C software, Python software, Open CV software. IOT software, API, security software, electronic medical records software, telemedicine related software, App software, or software to connect to apps. Other electronics and computing systems may be added as needed. Some or all of these components are cross-compatible with other embodiments of readers discussed in other sections. c. Software design

[0345] The reader can perform very precise, repeatable workflows where all tools and actions (e.g., temperature set points, ultrasonic mixing duration, acoustic mixer frequency, etc.) can be done in very' defined settings and sequences of steps. All movement is precise and repeatable. These workflows can be reprogrammable easily to allow for fast iterative R&D. The workflow can be a human-machine interface (graphic user interface) for R&D mode. This interface is for internal R&D, not customer-facing. The user can select to run automated full routines, which process the SIMPLE chip entirely from start to finish, or also subroutines such as, for example, just the Washl step. This feature is handy for optimizingsubcomponents on the SIMPLE chip. For example, many chips can be loaded with varying amounts of gas / liquid ratio in the washl blister pouch. Then, the washl subroutine can be used repeatedly to optimize that ratio. The user can also select different run modes. The “move-to” mode moves the gantry' towards each subroutine’s starting position. This mode helps check alignment. The “practice” mode runs the selected subroutine (or entire routine) through, without any physical contact of the tools to the chip or actuating heaters and camera. All actions are accelerated. It allows a quick practice run-through of the chip without processing it. This feature is helpful to verify that all positions are at the correct locations after a new- chip or new routine is implemented without sacrificing a chip. The “process” mode performs the full / subroutine task and processes the chip. This mode is useful to optimize the chip and routine iteratively.

[0346] All 7 axes' movements can be manually controlled precisely by pressing the jog control tabs with up, down, left, right, counterclockwise, clockwise, etc. Movements include rotation, X-axis (horizontal back frame movement), Y-axis (vertical gantry movement), pushpin (PP), soldering iron (heat sealer), ultrasonic hom, and heat lamp. Preset fine to coarse jog increments can move smaller or larger distances. Pressing the “extend” and “retract” buttons can fully move the corresponding tools to their resting position or preset fully extended position. All 7 axes can be moved to manually inputted coordinates and be homed. Homing means returning the tool or axis to a limit switch, which recalibrates the starting coordinate position. Homing is useful in stepper motor systems so the reader “knows” the moveable components' position. All position coordinates are fed back in real-time for view. There can be independently controllable devices (for example, eleven devices), including the soldering iron (heat sealer), pump (vacuum), vacuum valve that directs the vacuum to waste chamber or mix2 chamber, heater, sliding magnet, ultrasonic mixer, heat lamp, internal light, acoustic mixer, optics LED, and camera image taking. A real-time w indow can show the current actions and status the reader is undertaking. All of the above features are very useful to help adjust existing subroutines or create new subroutines, then stitch the subroutine together to a full routine that can process cartridges end-to-end. The usercan manually create subroutines by manually moving to the desired positions and performing desired actions, then note the coordinates and sequence and write that into a new programming script to automate the subroutine in the future. Different subroutines can be combined to form new full routines. Inside the programming script, more detail customization is possible, including, for example, but not limited to, shutter time control for the camera, time-lapse period, pump strength, heater setpoint, ultrasonic transducer vibration duration, frequency of acoustic mixer, etc. Essentially, with this setup, an unlimited number of variations of workflow routines can be created and tested.

[0347] In another embodiment, the open reader may also have an “assay mode” human-machine interface with minimal buttons for the user to run full processing workflows. It may provide simple user instructions. Assay mode would be the customer-facing mode that allows straightforward use of the open reader.

[0348] In another embodiment, stepper motors and limit switches may be replaced with servo motors with internal positioning feedback control.

[0349] In another embodiment, more or fewer motion axes can be implemented. More or fewer tools can be implemented. Different tool sets can be implemented. New tool sets can be added for expanded functions (for example, pipettes or cooling tools can be added).

[0350] In another embodiment, instead of using screw -type linear actuators, stepper motors (or servo motors) coupled with “rack and pinion” gears or “worm gears” may be used to create systems with high force (beneficial for the push pin and soldering iron), to replace the mechanism that moves tools inside the gantry head. These alternative systems may reduce cost and / or make the gantry more compact.

[0351] In another embodiment, the open reader concept can be used for non-SIMPLE chip cartridges or non-R2R manufactured cartridges. It can also be used for different variations of SIMPLE chip cartridges.

[0352] In another embodiment, the ultrasonic horn can be used for heat sealing.

[0353] In another embodiment, the open reader can be tailored for different (micro) fluidic cartridges or consumables. For example, it can be used as a test bed to develop newtypes of cartridges for many applications, including but not limited to various life science applications or non life science applications. It can be used for all various applications discussed in the genl SIMPLE chip section.

[0354] In another embodiment, the open reader can work on workflows that automated robot pipettors can perform, but not be limited to well plate (e.g.. 96well) type formats. It is unique that the consumable and reader backplate can be designed to fit together so an unlimited number of cartridge designs can be used.

[0355] In another embodiment, all backplate components can be redesigned and repositioned for different applications. For example, moving the vacuum port and contact heater, adding additional contact heaters, etc.

[0356] In another embodiment, the loading tray can be oriented not vertically but at any other angle, such as horizontally, to receive cartridges, which gives more design flexibility.

[0357] In another embodiment, as shown in FIGURES 15A-15C, the backplate assembly can be a '‘free-floating” design 1500, where the first backframe 1501 holding the chip 1502 is suspended by springs 1503. This design allows minimal vibrational damping, which can help with the acoustic mixing process. The acoustic transducers 1054 can be mounted directly on the backframe. The backframe’ s springs can fully compress and allow the backframe to rest entirely on a second backframe 1505. This feature is helpful to allow zero play when high compression forces are applied to the SIMPLE chip, such as push pin actions on the blister pouches. There may be one or more force sensors 1506 between the two backframes, allowing a force / pressure readout. This feature would be helpful with SIMPLE chip R&D processes. For example, different blister pouch shapes can be tested, and the pressure needed to crush the blister pouch can be optimized. Another example is that burst valve threshold pressures can be optimized as well.

[0358] In another embodiment, the motion axis can be placed in other spots as specified in FIGURE 13 A. For example, both the vertical and horizontal translation drive systems can be placed on the gantry head itself (move gantry but do not move backplate).Altematively. the drive systems can be both on the backplate (move the backplate but do not move the gantry head).

[0359] In another embodiment, non-optical sensors can be used for detection or readout, such as using electrochemical, capacitive, or magnetic readout methods.

[0360] In another embodiment, the backplate may have only one frame or two frames (like the gen2 reader). With the one-frame embodiment, the single frame may have a chip catch as in the gen2 reader.

[0361] In another embodiment, the open reader can be used as a teaching tool for students to test and run different cartridge designs. A microfluidics lab course could be built on this.

[0362] In another embodiment, other t pes of optical detection methods can be used. These methods have been discussed in other sections herein in this patent for SIMPLE chip readout.

[0363] In another embodiment, the same open reader may be compatible with future generations of cartridges for similar applications. For example, the same reader can be used for newer variations of SIMPLE chips. The reader can allow new workflows to be run according to each generation of SIMPLE chips. This flexibility is essential because the customer only needs to invest in the reader hardware once, and it is future-proof for new models of products for the cartridge. The reader may have a mechanism to scan barcodes and update software to run new routines according to each use case.

[0364] In another embodiment, the open reader can be used for life science and nonlife science applications.

[0365] In another embodiment, the open reader can develop or run workflows for various applications on various macrofluidic and microfluidic cartridges. For example, and not limited to the following applications: infectious disease testing, cell analysis, -omics research, chromatography, protein purification, nucleic acid purification, exosome applications, circulating tumor cell applications, digital PCR, real time PCR, digital isothermal amplification, nucleic acid quantification, electrophoresis, blotting, cell patterning,acoustic liquid dispensing, liquid metering, lyophilization, flow cytometry. NGS, CRISPR applications, single cell analysis, multiplexed assays, library prep, immunoassays, gene expression, directed evolution, strain engineering, transfection, cell incubation, hematology, blood chemistry diagnosis, infectious disease diagnosis, neuroscience, pharma applications, drug development, drug screening, in-vitro tests, oncology, clinical chemistry, digital health, hemostasis, coagulation, lab automation, urinalysis, histopathology, forensics, blood screening, sample collection, sample concentration, library quantification, immunohistochemistry, HPLC, DNA arrays, aptamer development, companion diagnostics, blood gas and electrolyte testing, diabetes testing, donor screening, lab automation, nucleic acid testing, serology’, rapid antigen tests, ELISA, westem / norther / southem blots, EWOD. gene therapy, clinical genomics, spectroscopy, cloning, tissue culture, stem cell research, synthetic biology, epigenetics research, ncRNA research, gene expression analysis, genoty ping, genome editing, microarray, nucleic acid development, nucleic acid synthesis, protein biology, RNAi, structural biology7, chromatography, microbiology, biopharma, allergy diagnostics, autoimmunity diagnostics, mass spectroscopy, diagnostic tool development, diagnostic testing, HLA biomarkers, precision medicine, companion diagnostics, public health applications. cfDNA isolation and analysis, biomarker discovery, mass cytometry, cell segmentation, pharmacogenomics, single cell analysis, cannabis and hemp testing, droplet microfluidics, mutagenesis, library generation, liquid biopsies, hematoxylin & Eosin, staining, hybridization, flow cytometry7, FISH probes, gas chromatography, protein expression, sepsis, veterinarian diagnostics, agro-food testing, bioconjugation, and drug monitoring. The open reader may also be used for elemental analysis, isotope analysis, bioprocessing, electron microscopy applications, the food industry’, manufacturing, materials science, radiation detection, air quality monitoring, water quality monitoring, nanoparticle synthesis, polymer synthesis, semiconductor analysis, security7threat detection, chemistry development, sanger sequencing, forensics, automated liquid handling, and quality control.

[0366] Some or all applications mentioned above can be cross-compatible with otherSIMPLE chip embodiments described in other sections. The open reader may also be adoptedfor cartridges that are not SIMPLE chip embodiments. Some or all of the open reader’s design, manufacturing method, applications, or components may be cross-compatible with other SIMPLE chip readers described in other sections (and vice versa). d. Example embodiments

[0367] A collection of example embodiments providing additional description of a variety7of embodiment ty pes in accordance with the concepts described herein are provided below. These examples are not meant to be mutually exclusive, exhaustive, or restrictive; and the claimed subject matter is not limited to these example embodiments but rather encompasses all possible modifications and variations within the scope of the issued claims and their equivalents.

[0368] Independent Claim:A device that serves as an “open” platform that can be used for rapid fluidic cartridge testing and development, comprising: at least one gantry7head; the gantry head has at least one tool; the gantry head tool includes at least of one these tools: heater, ultrasonic horn, heat sealer, and push pin; the device has a configurable backplate that can intake various designs of fluidic cartridges; the gantry head, and tools, can move in relation to the cartridge and perform automated routines; and the device can be reconfigured to run different steps and movements to accommodate various applications and cartridge designs.

[0369] Independent Claim:An R&D software to control the open reader, which has the following features: allows full routine run-through; allows selected subroutine run-through; displays the coordinates of each tool in real-time;can control the coordinates (and rotation) of each tool manually; it can turn on and off each tool manually; and it can be reprogrammed to run custom routines.

[0370] Dependent Claims (based on Independent Claims):The device is used for rapid iterative testing of (micro)fluidic cartridges.The fluidic cartridge is roll-to-roll manufactured.The fluidic cartridge is not roll-to-roll manufactured.The cartridge is a SIMPLE chip.The cartridge is a SIMPLE chip with vacuum batten technology.The gantry head tools can precisely move X, Y. Z, rotational, closer, or further relative to the consumable.The gantry head can be adapted to have other tools.The heater tool can be an optical heater.The heater tool can be a contact-based heater.The optical heater is a halogen lamp or LED that emits in the infrared wavelength.The device has contact heaters.Some or all of the open reader’s manufacturing, components, applications, and design elements are cross-compatible with other SIMPLE chip readers (and vice versa).The push pin tool has magnetic or electromagnetic properties.The ultrasonic horn can be used for mixing, heat sealing, and or actuating the integration pin.At least one tool has a spring cushioning mechanism to prevent overpressure, where the mechanism has a spring that helps the tool to maintain contact and provide pressure buffering against slight variations of the travel distance of the tool to the fluidic cartridge.The heat sealer’s contact head is a “line,” “x,” or “o” shape.The device has at least one pressure port to generate positive pressure to push liquids in the consumable.The device has at least one vacuum port to generate negative pressure to draw liquids into the consumable.The vacuum port includes a fluid trap, which is a wider cross sectional area in the fluidic circuit that retains excess fluid by gravity, and allows air to pass through.The pressure / vacuum port connection has a one-way valve(s).The sliding backplate is vertical.The sliding backplate is not vertical.The sliding backplate can automatically extend outside of the casing door (and retract) to allow user access.The backplate has a bottom frame, top frame, and backframe.The backplate aligns the cartridge precisely upon loading.The backplate has a soft backing layer.The device has at least one of these components- sliding magnets, acoustic mixers, or contact heaters.The acoustic transducer is mounted on a floating bracket, where the acoustic transducer does not touch the backplate but is only in contact with the floating bracket, and where the floating bracket is mounted on the backplate.Most structural components and casing are made of sheet metal.The door and screen are on the same side.The electronics and computing system includes one or more of these subsystems: Stepper motor control, stepper motor, stepper motor driver, servo motor, servo motor control, temperature control, system cooling, airflow management, power supply, screen connection, USB connection, Wi-Fi connection, Bluetooth connection, cellular connection, GPS, switching, relays, computing, LED driver, LEDs, optical lamp, camera, PCB board, wiring, circuit breaker, display, ultrasonic transducer driver / amplifier, acoustic transducer amplifier, MOSFET, microcontroller, humanmachine interface, UI / UX interface, system control software, onboard data storage, cloud data storage, imaging analysis software, limit switches, position homing systems, reprogrammable script for workflows, Raspberry’ Pi computing. Arduino computing, g-Code software, Marlin software, C software, Python software, Open CV software, IOT software, API, security software, electronic medical records software, telemedicine related software, App software, or software to connect to apps.The device has an R&D mode human-machine interface (HMI).The R&D mode allows manual control of subcomponents and tools.The R&D mode displays tool position coordinates in real time.The R&D mode can be used to create subroutines.The device has an assay mode human-machine interface.More or fewer motion axes can be implemented.Servo motors can be used instead of stepper motors.Screw-type linear actuators, ‘Tack and pinion" actuators, or “worm gears” is used to move the gantry’ tools.The device can be used for life science applications and or non-life science applications.The device can work on similar workflows that automated robotic pipettors can perform.The backplate can be reconfigured to adopt various cartridge designs.The device can use electrochemical, capacitive, or magnetic readout methods instead of optical readout.The device is compatible with future models of cartridges.The device has one. two, or three frames on the backplate.Coupling with application specific cartridges, the device can be used for one or more of the following applications: cell analysis, -omics research, chromatography, protein purification, nucleic acid purification, exosome applications, circulating tumor cell applications, digital PCR, real time PCR, digital isothermal amplification, nucleicacid quantification, electrophoresis, blotting, cell patterning, acoustic liquid dispensing, liquid metering, lyophilization, flow cytometry, NGS, CRISPR applications, single cell analysis, multiplexed assays, library prep, immunoassays, gene expression, directed evolution, strain engineering, transfection, cell incubation, haematology, blood chemistry diagnosis, infectious disease diagnosis, neuroscience, pharma applications, drug development, drug screening, in-vitro tests, oncology, clinical chemistry, digital health, hemostasis, coagulation, lab automation, urinalysis, histopathology, forensics, blood screening, sample collection, sample concentration, library quantification, immunohistochemistry, HPLC, DNA arrays, aptamer development, companion diagnostics, blood gas and electrolyte testing, diabetes testing, donor screening, lab automation, nucleic acid testing, serology7, rapid antigen tests, ELISA, westem / norther / southem blots, EWOD, gene therapy, clinical genomics, spectroscopy, cloning, tissue culture, stem cell research, synthetic biology', epigenetics research, ncRNA research, gene expression analysis, genoty ping, genome editing, microarray, nucleic acid development, nucleic acid synthesis, protein biology, RNAi, structural biology', chromatography, microbiology, biopharma, allergy' diagnostics, autoimmunity diagnostics, mass spectroscopy, diagnostic tool development, diagnostic testing, HLA biomarkers, precision medicine, companion diagnostics, public health applications, cfDNA isolation and analysis, biomarker discovery, mass cytometry, cell segmentation, pharmacogenomics. single cell analysis, cannabis and hemp testing, droplet microfluidics, mutagenesis, library generation, liquid biopsies, hematoxylin & Eosin, staining, hybridization, flow cytometry, FISH probes, gas chromatography, protein expression, sepsis, veterinarian diagnostics, agro-food testing, bioconjugation, and drug monitoring, elemental analysis, isotope analysis, bioprocessing, electron microscopy applications, food industry, manufacturing, materials science, radiation detection, air quality monitoring, water quality monitoring, nanoparticle synthesis, polymer synthesis, semiconductor analysis, security’ threatdetection, chemistry development, sanger sequencing, forensics, automated liquid handling, and or quality control.The device has an optical module that includes a camera and fluorescence filters.The device can work with fluidic cartridges made with SIMPLE chip manufacturing processes, and non SIMPLE chip manufacturing processes.Some or all of the open reader’s design, manufacturing method, applications, or components may be cross-compatible with other SIMPLE chip readers described in other sections (and vice versa).Programmable steps include one or more of the following: heating, moving the gantry head, rotating the gantry head, mixing, inducing pressure changes inside the consumable, pressing the consumable mechanically, and or heat sealing.Mixing can be acoustic-based methods.Acoustic transducers can be used for mixing.Ultrasonic transducers can be used for mixing.Ultrasonic horns can be used to enhance mixing.The device can run sample prep routines for nucleic acid extraction on the consumable.The device can run isothermal amplification routines on the consumable.The device can take fluorescence images of the biochemical reaction.The device can run a routine for Boom-based magnetic nucleic acid extraction and sample prep on the consumable.

[0371] 13, loT and Al applications a. Overview

[0372] Some expected advantages of the embodiments of the loT and Al applications include, but are not limited to:1) SIMPLE chip system can be connected to the internet for various applications;2) Al can be used to enhance captured data utility;3) Telemedicine, public health, analytics, and research applications possible;4) Ad hoc clinical trials can be set up quickly with reduced costs and time; and5) Can integrate with third-party services for enhanced functionality'. b. Internet of Things (loT) applications

[0373] Embodiments of the present disclosure will now be described in detail. The SIMPLE chip system can be connected to the internet, essentially becoming an loT device. The SIMPLE chip system includes all embodiments and variations of the SIMPLE chip / readers disclosed above, including the open reader platform and peripherals (everything to make the system work, such as swabs, instructions, packaging, etc.). The SIMPLE chip system can optionally include one or more of the following: a smart device with a dedicated app (a smart device may already be present in the user setting), a cloud database, UI / UX software, analytics software, and artificial intelligence(AI) software. Mass distribution of the SIMPLE chip system makes it a distributed sensor network that can gather real-time data. The reader may be able to connect to the intemet / cloud database. Internet connection could be made by communication protocols such as Wi-Fi and / or cellular networks. Alternatively, the reader could communicate via a smart device via a dedicated app (Bluetooth, LAN, WiFi, or other wired / wireless protocols). Smart device examples include smartphones, computers, and tablets. It is possible to leverage the smart device’s native capabilities to connect to the intemet / cloud (such as Wi-Fi, Bluetooth. LTE, satellite communication, cellular network connectivity, and other communication protocols).

[0374] Furthermore, the smart device’s other native capabilities, such as GPS geotagging, computational power, touchscreen for UI / UX interface, microphone, speaker, local data storage, camera, and apps, could be leveraged. Leveraging the capabilities of the smart device is an excellent way to keep reader costs low, as the reader’s system requirements can be kept very basic. For example, the smart device’s camera could scan barcodes to verity'the user and the cartridge, verify the SIMPLE chip expiration date, or verify that the SIMPLE chip is an authentic product. The SIMPLE chip reader and or smart device’s internet connectivity could be used for local or cloud data storage, push message alerts, recall notifications, over-the-air software updates, telemedicine integration, user manual updates, training video updates, patient data logging, reader maintenance, calibration, HIPAA compliance, and or electronic medical record (EMR) integration. The smart device may have a dedicated app that manages all the functions above.

[0375] The real-time reporting, app, and internet connection allow for real-time scheduling of reader availability when there are multiple readers at the same site. When there is a high volume of patients, readers that have completed testing and are ready to be used again can be notified. Dynamic access can be achieved, which means tests can be done individually in any order immediately if there is an unused reader. In contrast to PCR, No batch processing is required, reducing test-to-result turnaround time. Anticipated and forecasted wait times for testing can also be reported. Real-time scheduling of reader availability may also be done across multiple sites. For example, patients can log into a website to view where testing sites are available and which locations have low or no wait times for testing. The reader's availability and testing site’s appointment availability (such as Minute clinics in pharmacies or primary care clinics) can be aggregated and presented to patients via an integrated online scheduling system, so patients can book and get immediate testing by going to available locations. These scheduling reporting features can help manage the inflow of patients and maximize processing efficiency. c. Telemedicine applications

[0376] There are many telemedicine applications possible by coupling the SIMPLE chip system with an app and smart device with the internet. For example, automated messages on test results may be sent to the patient or medical provider immediately after testing. In another example, the patient can connect via an online video call with a health provider for step-by-step guidance on performing a test. Patients can also consult with medical professionals online on test results and get prescriptions. Alternatively, a survey algorithmmay ask the patients for various background information related to the illness and determine a prescription based on the combined test results plus background information. The survey algorithm may be a simple decision flow chart or based on artificial intelligence (Al). The Al survey algorithm may also be coupled with natural language generative Al (e.g., ChatGPT, PaLM-E, ERNIE BOT®, LLaMA, etc.) to provide a more natural human-like interactive experience. Natural language Al may also provide medical advice, forecast likely prognosis, and provide more background information about the pathogen, making the experience similar to a visit with a human doctor. Video recordings could be made for documentation and liability purposes. Automated audio-to-text scripting can also be made. The app allows AI- targeted online ads to reach patients and suggest relevant products. For example, depending on test results, medications, and supportive peripheral products can be marketed to the patient. The app can serve as an online marketplace for relevant third-party businesses. For example, a pharmaceutical company could advertise prescription medicine and supportive supplements to the patient while paying a fee (or credit) to the patient (and / or seller of the SIMPLE chip system). Similar business methods may be used for companion diagnostic applications.

[0377] The real-time sensing network allows other applications such as real-time medication, testing services, and / or SIMPLE chip-related product delivery. For example, after a test result, through the app, patients can order immediate delivery7of medication (or other products) via logistics providers such as for example and not limited to- mobile clinics, (online) taxi services, UBER®, LYFT®, delivery7services, DOORDASH®, FEDEX®, USPS®, UPS®, drone delivery services, and other similar services, etc. d. Analytics applications

[0378] Leveraging the IOT connectedness and Al, it is possible to perform various analytics with the SIMPLE chip system. Data can be uploaded to a cloud database for (Al) analysis via the reader or the coupled smart device app. Example applications included and are not limited to customer buying trend analytics, testing logs (yvho performed the test, which patients got the test, test results, time, location, etc.), testing frequency analytics, testing cartridge type analysis, seasonal use analytics, location use analytics, demographic useanalytics, and location setting use analytics (e.g., home vs. clinic vs. hospital). In addition, consumable stock monitoring can be done in real time, and automated reordering can be prompted. An Al-based cost-benefit analysis can be provided to the user, justifying the purchase of more readers if high-volume testing is done in short periods. Maintenance schedules can be notified to the user. Reader / cartridge failures, failure type, failure rate, failure part analysis, and average reader life can all be automatically notified to the manufacturer, which helps with servicing and the iterative product improvement process. Finally, the analytics data can also be integrated with personal health apps (e.g., Apple Health app), third party apps, medical care providers, primary care providers, sequencing data (e.g., 23&me), and become part of the patient's health history data. e. Research applications and ad-hoc clinical trials

[0379] Due to the IOT nature, the SIMPLE chip system can support various research efforts efficiently. A dedicated app or website interface can be provided to researchers to analyze the data aggregated into the cloud database. For example, medical intervention outcomes can be tracked and correlated to the test results (optionally with Al automation). A medication’s (or intervention’s) efficacy can be tracked based on the test results by collaborating with the medical staff at the testing site. Statistical analytics can be done to track if patients take a specific drug at a particular time after pathogen exposure (or onset of symptoms), what is the recovery speed and rate, how many patients proceed into in-hospital care, what is the mortality' rate, what is the side effect rate, coinfection rate.

[0380] Test results can be correlated with geography, season, underlying health conditions, health risk factors, co-infection incidence, and demographics of the patients. Surveys to collect additional patient and / or test site information can be pushed over the air through the app.

[0381] Clinical trials can be done more efficiently and at a lower cost. For example, researchers can create over-the-air electronic patient consent forms at multiple testing sites using the smart device app, a website, messaging, or email (or a combination of these). Researchers can set up multiple clinical research sites over the air instantly. Alternatively,clinical research can be set up and executed by partnering with a third party contract research organization, so patient consent and / or clinical research sites can be set up directly over the air. Optionally, third-party contract research organizations may utilize the app to set up contracts and push paperwork to customers for clinical trials.

[0382] The consumable may have, for example, a panel of 6 pathogen tests that the FDA approves and only reports 6 test results back to the medical provider (and / or patient). However, the consumable has extra microwells loaded with additional primer sets for new pathogen clinical trial testing. The primers for the clinical trial can test for new targets, such as alternative pathogens, or the efficacy of new variants of primers. An “ad hoc" clinical trial can be performed on the existing network of customers efficiently, in the sense that the customers are getting their regular test results. However, the researchers are getting additional real-world clinical trial data (with patient consent) without the significant effort of restarting a completely whole new clinical trial at new sites, without purchasing additional clinical samples, and without completely remanufacturing dedicated consumables. They can ensure the samples are fresh and tested immediately (instead of transporting clinical samples (possibly cold chain) to centralized testing sites, which risks sample degradation issues). Since the customers are already (or willing to) operating the SIMPLE chip system, the readers are already distributed, and the customers are used to operating the system. There is little friction to starting a new clinical site. Any ad hoc clinical trial will minimally affect the customer’s workflow. Customers may be incentivized to sign on the ad hoc clinical trials via credit rebates, gift cards, future product discounts, and / or cash payments. The manufacturer benefits from continuous profit from sales, saves clinical trial costs, and saves time.

[0383] Monitoring customers running tests enables acquiring real-world user behavioral analytics data to improve the product further iteratively. For example, human factors research can be done on user error risks analysis, failure mode analysis, user environment analysis, UI / UX improvement, workflow, etc. Furthermore, the smart device may take videos (under patient consent) to record the entire user workflow during testing for human factors research and ensure samples are correctly tested. The videos can be uploadedto a cloud database for further analytical purposes. The videos can also support clinical trial work.

[0384] In another example, sample-ty pe performance clinical research could be done. For example, studying test performance differences among nasal, midturbinet, nasal pharyngeal, and / or throat swabs is possible. During purchasing the diagnostic product, the user may elect to participate in ad hoc clinical trials via an e-commerce website, email link, or app. The standard SIMPLE chips can be shipped with extra swabs for each sample type to be collected. The user can take multiple types of sample swabs and test them concurrently. Test results are uploaded and analyzed by clinical trial researchers. f. Public health and Al applications

[0385] The Internet-connected SIMPLE chip system has many public health applications. The ability for geolocation and time stamping of test results allows for real-time incidence mapping. All data can be uploaded to a centralized cloud data system. With this data, an (optionally Al-based) forecasting and tracking system for epidemic / pandemic outbreaks can be modeled. Within this model, several analytics and correlations can be tracked and forecasted, such as rate of infectious disease spread, geolocation of spread, hot spots of outbreaks, correlation with weather patterns, historical / future epidemiological patterns, strain variants, coinfection trends, basic reproduction number, transmission routes, incubation period, correlation with patient demographics, policy intervention outcome, medical intervention outcome, prevalence, hospitalization rate, mortality' rate, contact tracing, warning alerts for at-risk populations, vaccine efficacy, cost-benefit analysis, companion diagnostic medication efficacy, pathogen mutation rates, variant detection, herd immunity rate, economic impact correlation with the number of infections, and outbreak origin tracing.

[0386] In some cases, extra primer sets can be added in microwells on the SIMPLE chip for the above applications. The SIMPLE chip can be made with extra microwells to accommodate future assays. For example, a standard FDA-approved respiratory' panel for the SIMPLE chip is already in market circulation, and the SIMPLE chip has extra empty unused microwells. In the case of a future respiratory-type pandemic, the SIMPLE chip can bequickly adopted for emergency use (FDA EUA) by adding specific primer sets for the new type of pathogen. The original panel plus the new assay becomes a valuable tool for differential diagnosis. The SIMPLE chip system can be used for pooled screening in other use cases.

[0387] In summary, the SIMPLE chip system can help with better medical response and medical resource triage. If public health agencies can forecast where new hotspots will be, the resources can be quickly appropriated to relevant locations. Best interventions can be quickly determined based on statistical analytics. g. Integration with other third-party software and services

[0388] The SIMPLE chip system can integrate with other third-party software and services. For example, and not limited to - real-time delivery logistical services (e.g., DOORDASH®, UBER®, etc. for consumable or medication delivery), mobile clinics (e.g., door-to-door on-demand testing), pharmacy websites (can show the availability of readers), retail clinics (e.g. MINUTE CLINIC®), pharma companies (e.g., ads for medications), telemedicine services, health network systems (e.g., integrate with KAISER PERMANENTE® KP online system and alike), public health databases (e.g., CDC, Johns Hopkins Coronavirus Resource Center and alike), contract research organizations, clinical trial partners, and EMR systems, etc. h. Example embodiments

[0389] A collection of example embodiments providing additional description of a variety of embodiment types in accordance with the concepts described herein are provided below. These examples are not meant to be mutually exclusive, exhaustive, or restrictive; and the claimed subject matter is not limited to these example embodiments but rather encompasses all possible modifications and variations within the scope of the issued claims and their equivalents.

[0390] Independent Claim:A point-of-care diagnostic system, comprising:a chip consumable, a corresponding reader, and a coupled smart device with a dedicated app; it can connect to the internet and a cloud database; it can create ad hoc clinical trials using existing customer sites; and it can be used for at least one of the applications in the following: Internet of Things (loT), artificial intelligence, telemedicine, analytics, research, and public health applications.

[0391] Independent Claim:A molecular diagnostic system, comprising: a consumable, defined in claim set of section 1. a corresponding reader, and or optionally coupled smart device with an app; it has the capability to connect to the internet and a cloud database; and it can be used for at least one of the applications in the following: Internet of Things (loT), Al, telemedicine, analytics, research, clinical trials, and public health applications.

[0392] Dependent Claims (based on Independent Claims):The diagnostic system can be integrated with third party software and services.The diagnostic system is a SIMPLE chip system.The reader itself can connect to the internet and cloud without the smart device and app.The smart drive and app can act as a bridge to connect the reader to the internet and cloud.The smart device’s other native capabilities could be leveraged as well, such as GPS geotagging, computational power, touchscreen for UI / UX interface, microphone, speaker, local data storage, camera, and apps. loT applications include at least one of the following- local or cloud data storage, push message alerts, recall notifications, over-the-air software updates, telemedicine integration, user manual updates, training video updates, patient datalogging, reader maintenance, calibration, HIPPA compliance, real-time scheduling, and or electronic medical record (EMR) integration.The smart device has a dedicated app that communicates with the reader and manages various loT-related functions.Automated prescriptions can be done.Al-assisted automated prescriptions and ads can be done.Video meetings with healthcare providers can be done.Real-time medication delivery' can be done.Analytics applications include at least one of the following: customer buying trend analytics, testing logs, testing frequency analytics, testing cartridge type analysis, seasonal use analytics, location use analytics, demographic use analytics, and setting use analytics (e.g., home vs. clinic vs. hospital), consumable stock monitoring, automated reordering, cost-benefit analysis, maintenance schedules notification, Reader / cartridge failure stats, failure type, failure rate, failure part analysis, average reader life analysis, and integration with personal health apps / third party apps / medical care providers / primary care providers.Research applications include at least one of the following: medical intervention tracking, medication efficacy tracking, recovery' speed / rate, in-hospital care transition rate, mortality rate, and side effect rate.Research applications include at least one of the following: correlations between coinfection rate, geography, season, underlying health conditions, health risk factors, and demographics.Clinical trials can be performed ad hoc.Patient consent can be pushed over the air.Clinical trials can be set up over the air.Third party contract organizations can set up clinical trials and patent consent directly over the air via the app.SIMPLE chips may have extra microwells that are dedicated to ad hoc clinical trials.The extra microwells can be loaded with new primers.The SIMPLE chip system reports FDA-approved results to patients and reports clinical trial results to the research organization.Ad hoc clinical trials minimally affect customer workflow.Customers participating in clinical trials may receive incentives such as one or more of the following: credit rebates, gift cards, future discounts on products, and or cash payments.The diagnostic system can monitor user behavior while running tests and provide analytics such as error risks analysis, failure mode analysis, user environment analysis, UI / UX improvement, and workflow.The diagnostic system can take videos of the user workflow (under consent) for human factors research.Videos can be uploaded to the cloud for analytical purposes.Users may participate in ad hoc clinical trials while purchasing the diagnostics via an e-commerce website, email link, or app.Public health applications include tracking / forecasting / modeling one or more of the following: geolocation stamping, time stamping, real-time incidence mapping, modeling epidemic / pandemics outbreaks, rate of infectious disease spread, geolocation of spread, hot spots of outbreaks, coinfection trends, correlation with weather patterns, historical / future epidemiological patterns, strain variants, basic reproduction number, transmission routes, incubation period, correlation with patient demographics, policy intervention outcome, medical intervention outcome, prevalence, hospitalization rate, mortality' rate, contact tracing, warning alerts for at- risk populations, vaccine efficacy, cost-benefit analysis, companion diagnostic medication efficacy, pathogen mutation rates, variant detection, herd immunity’ rate,economic impact correlation with the number of infections, and outbreak origin tracing.Extra primer sets can be added in microwells on the diagnostic. The diagnostic can be made with extra microwells to accommodate future assays.The diagnostic can be quickly adopted for emergency use (FDA EUA) by adding specific primer sets for the new type of pathogen.The diagnostic can integrate with third party software and other services.The diagnostic can integrate with real-time delivery logistical services (e.g., DOORDASH®, UBER®, etc., for consumable or medication delivery), mobile clinics (e.g., door-to-door on-demand testing), retail clinics (e.g. CVS®) pharmacy websites (can show the availability of readers), pharma companies (e.g., ads for medications), telemedicine services, health network systems (e.g., integrate with KAISER PERMANENTE® KP online system and alike), public health databases (e.g., CDC, Johns Hopkins Coronavirus Resource Center and alike), contract research organizations, clinical trial partners, and EMR systems, etc.

[0393] 14, Other applications a. Overview

[0394] Some expected advantages of the embodiments of the other applications include, but are not limited to:1) Various pathogen panels types possible;2) Can be used in various decentralized settings; and3) Replaces rapid antigen / antibody tests and conventional PCR.

[0395] Embodiments of the present disclosure will now be described in detail. By driving the cost down to near parity with rapid antigen tests, the SIMPLE chip system can replace rapid antigen / antibody tests. The SIMPLE chip system solves the problems associated with rapid antigen / antibody tests. These problems include low accuracy, sensitivity, selectivity, multiplexing, and delayed post-exposure detection window. The SIMPLE chip system retains the rapid antigen / antibody test’s advantage of being very low cost, immediateonsite testing capability, and PCR’s high accuracy. The SIMPLE chip system can also replace conventional batched PCR testing, traditionally done in centralized settings. Conventional PCR is high cost, and the requirement for batching causes time-to-result delays.

[0396] The SIMPLE chip system can be used for the following applications, for example, and is not limited to respiratory panels, sexually transmitted disease panels, cancer panels, companion diagnostics, tropical fever panels, urinary tract infection panels, veterinary- panels, gastrointestinal panels, companion animal diagnostics (e.g., cats / dogs), and outbreak panels. There are various other applications besides infectious testing.

[0397] The SIMPLE chip system can be used in the following settings, for example, and not limited to homes, primary care settings, physician offices, pharmacies, minute clinics, retail clinics, drive-through testing sites, emergency response camps, refugee camps, mobile clinics, nursing homes, group settings, companies, schools, cruise ships, sports clubs, fleets, military bases, clinics, public health labs, testing labs (e.g., Quest / LabCorp). ports of entry, airports, hospital labs, prisons, emergency departments, etc.

[0398] The SIMPLE chip system is designed to be the future platform standard for first line infectious disease testing, replacing conventional standards of rapid antigen / antibody tests and PCR. b. Example embodiments

[0399] A collection of example embodiments providing additional description of a variety of embodiment ty pes in accordance with the concepts described herein are provided below. These examples are not meant to be mutually exclusive, exhaustive, or restrictive; and the claimed subject matter is not limited to these example embodiments but rather encompasses all possible modifications and variations within the scope of the issued claims and their equivalents.

[0400] Independent Claim:A diagnostic system that is designed to replace rapid antibody / antigen tests and PCR systems for first line testing, which comprises: A SIMPLE chip system.

[0401] Dependent claims (based on Independent Claim):The SIMPLE chip system can be used for one or more of the following testing applications- respiratory panels, sexually transmitted disease panels, cancer panels, companion diagnostics, tropical fever panels, urinary tract infection panels, veterinary panels, gastrointestinal panels, companion animal diagnostics (e.g., cats / dogs), and outbreak panels.The SIMPLE chip system can be used in one or more of the following settings- homes, primary care settings, physician offices, pharmacies, minute clinics, retail clinics, drive-through testing sites, emergency response camps, refugee camps, mobile clinics, nursing homes, group settings, companies, schools, cruise ships, sports clubs, fleets, military bases, clinics, public health labs, testing labs (e.g., Quest / LabCorp). ports of entry, airports, hospital labs, prisons, and emergency departments.

[0402] In the foregoing description, specific details are set forth to provide a thorough understanding of exemplary' embodiments of the present disclosure. It will be apparent to one skilled in the art, however, that the embodiments disclosed herein may be practiced without embodying all of the specific details. In some instances, well-known process steps have not been described in detail in order not to unnecessarily obscure various aspects of the present disclosure. Further, it will be appreciated that embodiments of the present disclosure may employ any combination of features described herein.

[0403] The present application may reference quantities and numbers. Unless specifically stated, such quantities and numbers are not to be considered restrictive, but exemplary of the possible quantities or numbers associated with the present application. Also in this regard, the present application may use the term "plurality" to reference a quantity or number. In this regard, the term “plurality” is meant to be any number that is more than one, for example, two, three, four, five, etc. The terms “about,” “approximately,” “near.” etc., mean plus or minus 10% of the stated value. For the purposes of the present disclosure, the phrase “at least one of A and B” is equivalent to “A and / or B” or vice versa, namely “A” alone, “B” alone or “A and B.” Similarly, the phrase “at least one of A. B, and C,” for example,means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C), including all further possible permutations when greater than three elements are listed.

[0404] It should be noted that for purposes of this disclosure, terminology such as “upper,” “lower,” “vertical,” “horizontal,” “fore,” “aft,” “inner,” “outer,” “front,” “rear,” etc., should be construed as descriptive and not limiting the scope of the claimed subject matter. Further, the use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless limited otherwise, the terms “connected,” “coupled,” and “mounted” and variations thereof herein are used broadly and encompass direct and indirect connections, couplings, and mountings.

[0405] Throughout this specification, terms of art may be used. These terms are to take on their ordinary meaning in the art from which they come, unless specifically defined herein or the context of their use would clearly suggest otherwise.

[0406] The principles, representative embodiments, and modes of operation of the present disclosure have been described in the foregoing description. However, aspects of the present disclosure, which are intended to be protected, are not to be construed as limited to the particular embodiments disclosed. Further, the embodiments described herein are to be regarded as illustrative rather than restrictive. It will be appreciated that variations and changes may be made by others, and equivalents employed, without departing from the spirit of the present disclosure. Accordingly, it is expressly intended that all such variations, changes, and equivalents fall within the spirit and scope of the present disclosure as claimed.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A molecular diagnostic device, comprising: a first layer and a second layer sealingly coupled to the first layer, the first layer having raised portions spaced apart from the second layer to form a fluidic circuit between the first and second layers, the fluidic circuit comprising: a loading chamber configured to receive a solution having a biological sample through an insertion aperture; an assay well positioned in the fluidic circuit; and a valve positioned at an outlet of the loading chamber, the valve having an initial closed state fluidly disconnecting the loading chamber from the assay well, and a secondary open state fluidly coupling the loading chamber and the assay well to permit flow of the solution through the fluidic circuit, wherein, when the valve is in the initial closed state, the assay well contains a vacuum, and wherein when the valve transitions to the secondary open state, the vacuum draws solution from the loading chamber into the assay well.

2. The molecular diagnostic device of Claim 1, wherein the valve is a wax valve configured to transition from the initial closed state to the secondary open state with thermal energy, and wherein the fluidic circuit further comprises: a wax valve channel fluidly coupled to the outlet of the loading chamber; and a wax cooling chamber positioned at a terminal end of the wax valve channel, the wax cooling chamber positioned upstream of the assay well, wherein the wax cooling chamber has a larger fluidic cross-section than the wax valve channel to capture wax material of the wax valve such that the solution is permitted to flow to the assay well.

3. The molecular diagnostic device of Claim 2, wherein the fluidic circuit further comprises a distribution channel fluidly coupling the wax cooling chamber and the assay well.

4. The molecular diagnostic device of Claim 3, wherein the assay well comprises a first assay well and a second assay well separated from the first assay well, and wherein the distribution channel directs a portion of the solution to the first assay well and a portion of the solution to the second assay well.

5. The molecular diagnostic device of Claim 3. wherein the terminal end of the wax valve channel is offset from an inlet end of the distribution channel such that the wax material is captured within the wax cooling chamber.

6. The molecular diagnostic device of Claim 1 , wherein the assay well comprises a molecular amplification assay, an agglutination assay, an immunoassay, an enzymatic assay, or a chemistry assay.

7. The molecular diagnostic device of Claim 1, wherein the assay well is a molecular amplification assay, and wherein the molecular amplification chemistry' is isothermal amplification. PCR amplification, or non-enzymatic amplification.

8. The molecular diagnostic device of Claim 1, wherein the valve is a mechanical valve, a fluidly soluble valve, a thermally meltable valve, a magnetic valve, a pressure valve, a pierceable film valve, or a thermal film valve.

9. The molecular diagnostic device of Claim 1 , wherein the loading chamber and the assay well have an arched shape for structural rigidity' under atmospheric pressure storage.

10. The molecular diagnostic device of Claim 1, wherein the assay well is configured to entrap an air bubble therein as the vacuum draws solution from the loading chamber into the assay well.

11. The molecular diagnostic device of Claim 10, wherein the size of the air bubble is altered by the strength of the vacuum in the assay well.

12. The molecular diagnostic device of Claim 1, wherein the device is manufactured using a roll-to-roll manufacturing process.

13. The molecular diagnostic device of Claim 1, further comprising a sealing tape positioned to selectively occlude the insertion aperture.

14. The molecular diagnostic device of Claim 1. wherein the loading chamber further comprises a structure configured to entrap air bubbles in the loading chamber, wherein the structure is a rib, a divot, a dimple, a surface etch, a trough, or a protrusion.

15. The molecular diagnostic device of Claim 1, wherein the fluidic circuit is sealed to the atmosphere except through the insertion aperture.

16. A molecular diagnostic device for identification of a pathogen, the molecular diagnostic device consisting of: a first layer: a second layer sealingly coupled to the first layer; and a fluidic circuit extending between the first and second layers, the fluidic circuit comprising: a loading chamber configured to receive a solution having a biological sample of the pathogen through an insertion aperture; a plurality of isothermal amplification wells positioned in the fluidic circuit; a thermal valve positioned at an outlet of the loading chamber, the thermal valve having an initial closed state fluidly disconnecting the loading chamber from the plurality of isothermal amplification wells, and a secondary open state fluidly coupling the loading chamber and the plurality' of isothermal amplification wells to permit flow of the solution through the fluidic circuit; anddistribution channels directing a portion of the solution to each of the plurality of isothermal amplification wells, wherein, when the thermal valve is in the initial closed state, the plurality of isothermal amplification wells contain a vacuum, and wherein when the thermal valve transitions to the secondary open state, the vacuum draws solution from the loading chamber, through the distribution channels, and into the plurality of isothermal amplification wells.

17. The molecular diagnostic device of Claim 16, wherein the thermal valve is a wax valve configured to transition from the initial closed state to the secondary open state with thermal energy, and wherein the fluidic circuit further comprises: a wax valve channel fluidly coupled to the outlet of the loading chamber; and a wax cooling chamber positioned at a terminal end of the wax valve channel, the wax cooling chamber positioned upstream of distribution channels, wherein the wax cooling chamber has a larger fluidic cross-section than the wax valve channel to capture wax material of the wax valve such that the solution is permitted to flow to the plurality7of isothermal amplification wells.

18. A method of identifying a pathogen with a molecular diagnostic device, the molecular diagnostic device having: a first layer and a second layer sealingly coupled to the first layer, the first layer having raised portions spaced apart from the second layer to form a fluidic circuit between the first and second layers, the fluidic circuit comprising: a loading chamber; an assay well containing a vacuum and being positioned at a terminal end of the fluidic circuit; and a valve positioned at and occluding an outlet of the loading chamber to fluidly disconnect the loading chamber from the assay well, the method comprising:-reflowing a solution having a biological sample through an insertion aperture into the loading chamber; and transitioning the valve to a secondary’ open state fluidly coupling the loading chamber and the assay well such that the vacuum in the assay well draws solution from the loading chamber into the assay well.

19. The method of Claim 18, wherein the valve is a wax valve configured to transition from the initial closed state to the secondary’ open state with thermal energy', and wherein the fluidic circuit further comprises: a wax valve channel fluidly coupled to the outlet of the loading chamber; and a wax cooling chamber positioned at a terminal end of the wax valve channel, the wax cooling chamber positioned upstream of the assay well, wherein the wax cooling chamber has a larger fluidic cross-section than the wax valve channel, wherein transitioning the valve to the secondary open state comprises heating the wax valve, and wherein the method further comprises capturing the wax material of the wax valve such that the solution is permitted to flow to the plurality' of isothermal amplification wells.

20. The method of Claim 18, wherein the assay well is configured to entrap an air bubble therein as the vacuum draws solution from the loading chamber into the assay well, and wherein the method further comprises applying an acoustic transducer to the molecular diagnostic device to mix the solution in the assay well by' movement of the solution and the air bubble.

21. The method of Claim 20, wherein during the step of applying an acoustic transducer to the molecular diagnostic device, the method further comprises heating the solution in the assay well.

22. The method of Claim 18, wherein the loading chamber is configured to entrap air bubbles therein, and wherein the method further comprises applying an acoustic transducerto the molecular diagnostic device to mix the solution in the loading chamber by movement of the solution and the air bubbles.

23. The method of Claim 22, wherein during the step of applying an acoustic transducer to the molecular diagnostic device, the method further comprises heating the solution in the loading chamber.

24. The method of Claim 18, further comprising sealing the insertion aperture to seal the fluidic circuit from the atmosphere.

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