Disposable diagnostics device system

Abstract

A disposable diagnostic device system is disclosed for rapid detection of biomolecules in biological or environmental samples. The device can include an inlet port, microfluidic channels, and one or more sealed detection chambers containing reagents for nucleic acid or protein analysis. Sample introduction and cap closure can drive fluid into the detection chambers, where amplification reactions such as LAMP, SDA, NASBA, TMA, HDA, RPA, or related methods can occur. Detection may be performed by fluorescence, absorbance, or electrochemical measurement using integrated optical components and sensors. The chambers can be vented through gas-permeable membranes to ensure proper filling and mixing with dried or liquid reagents. The device can incorporate onboard electronics, including heaters, temperature sensors, photodetectors, and circuitry powered by a single battery, with optional NFC communication for wireless data transfer. Results may be displayed on the device or transmitted to a smartphone or electronic health system. The system can provide low-cost, portable, point-of-care testing with turnaround times of less than one hour, eliminating reliance on centralized laboratories. Applications can include veterinary diagnostics, livestock management, environmental monitoring, human diagnostics, and companion animal testing, enabling rapid and accessible screening for pathogens at the point of need.

Description

TITLEDISPOSABLE DIAGNOSTICS DEVICE SYSTEMCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63 / 700,225, filed 27 September 2024, which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Current diagnostic testing for pathogens is largely performed in centralized laboratories. In this process, samples are collected from a human or animal patient, either in a clinical setting or in the field, and then transported to a laboratory for analysis. Sample types may include biological fluids (e.g., blood, urine, saliva), swabs (e.g., nasal, rectal, vaginal), fecal matter, or tissue. Once collected, the samples are shipped to the laboratory, where trained personnel conduct testing and subsequently return results to the healthcare provider or end user. This process can take several days, is labor-intensive, and can be costly, due in part to shipping expenses. More importantly, delayed results can postpone treatment decisions and, in cases requiring immediate intervention, may lead to poor outcomes, including disease progression, increased mortality, and further transmission of the pathogen.

[0003] There is a critical need for cost-effective, point-of-care diagnostic devices that eliminate reliance on centralized laboratories. Current solutions, such as in clinic benchtop instruments, are not portable and practical for field use, require intensive training and maintenance for standard use, and are costly. Additionally, there is increasing demand for integration of diagnostic data with electronic health monitoring systems. The widespread use of smartphones allows for digital tracking, storage, and transmission of results, which are valuable both to consumers and healthcare providers.

[0004] Therefore, there is a clear unmet need for inexpensive, disposable, self-contained diagnostic devices that can provide accurate diagnostic information. These devices need to be easy to use and provide rapid results (i.e. less than 1 hour). They should not rely on external equipment, which will eliminate the need for additional personnel as well as costs associated with purchase and maintenance. The devices need to be able to integrate with consumer electronics, like smartphones, to transfer, monitor, and store health data. Such a device would overcome the limitations of existing diagnostic approaches while enabling rapid, accurate, and accessible testing at the point of care.Levine Bagade Han LLP 1 GTSCZOIOOOWOSUMMARY

[0005] A disposable device for detecting biomolecules in a sample is disclosed. The biomolecules could be proteins or nucleic acids. The proteins could be antibodies. The biomolecules could be released from organisms for analysis or could be detected while still a functional part of the organism. The biomolecules could be on the outer portion of an antigen and used to detect the antigen. The device can have an inlet port where a sample can be introduced, the device may have one or more channels that connect the inlet port to one or more detection chambers. The detection chambers may hold reagents needed for performing a nucleic acid reaction. The nucleic acid may be detected by performing isothermal nucleic acid amplification or it may be a thermocycled amplification method. The isothermal amplification methods may include but are not limited to Loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), nucleic acid sequencebased amplification (NASBA), transcription mediated amplification (TMA), whole genome amplification (WGA), helicase dependant amplification (HD A), strand invasion based amplification (SIB A) or recombinase polymerase amplification (RPA). The detection from a nucleic acid reaction may be a fluorescent detection or it may be electrochemical detection methods or it may be absorbance measurement. The device may have a cap that seals to the inlet port. The cap may be attached with a string or a mechanical member that can accommodate the cap being closed. The cap may seal in such a manner that it creates a pressure in the inlet port such that the closing of the cap may directly or indirectly drive fluid from the inlet port into one or more of the detection chambers. The detection chambers may have a gas-permeable membrane in one or more areas such that air can escape from the chambers but such that liquid will stay in the chambers. The gas permeable membrane may be in the top portion of the chambers such that air will escape from the top. The detection chambers may have a gas-permeable plug or an outlet channel that can evacuate air. Each detection chamber may have a separate gas-permeable membrane or multiple detection chambers may share a gas-permeable membrane, the gas-permeable membrane may be connected through a channel. A liquid sample can be introduced in the inlet port and the closing of the cap can push the liquid through channels into one or more detection chambers where it fills the detection chambers and mixes with reagents and pushes out the air through one or more gas-permeable membranes. The device may have a lid on the detection chambers that can be applied after other operations have been performed on the detection chamber, for example the detection chambers may have one or more lids that may be snapped onto the detection chambers after reagents have been loaded into the detection chambers, this wouldLevine Bagade Han LLP 2 GTSCZOIOOOWOfacilitate loading reagents into the device after many or most other assembly steps have been performed on the device thereby accommodating shipping reagent free devices to a customer or to another site to be filled with reagents and then shipped further to end users. The lid for the detection chambers may have a gas-permeable membrane for venting. The reagents described in the device may be dried reagents like lyophilized reagents or gel or they may be liquid reagents or a combination of liquid and dried or gel reagents. There may be reagents in the inlet port. Each detection chamber may hold one or more dried reagent beads or chunks or coatings or cake or powders. Lyophilized, gel or dried reagents may be applied in the detection chambers as spherical or semispherical or non- spherical beads. There may be one or more lyophilized reagent beads in the detection chambers. The reagents may be separated into different lyophilized, dried or gel beads, chunks portions, process or areas, for example, one bead may contain primarily enzymes and another bead may contain primarily oligos to avoid nonspecific product amplification in nucleic acid amplification reactions. Reagents may be placed as lyophilized beads in the reagent chambers or reagents may be loaded into the reaction chambers before the lyophilization process thereby lyophilizing reagents with the detection chambers or the reagents may be a combination of lyophilized beads and inchamber lyophilized reagents. In order for reagents to not flow out of the detection chambers when loaded as liquid reagents before lyophilization, the detection chambers may be held below the freezing point of the reagents thereby freezing the liquid reagents in place before they can flow out of the detection chamber. A separate liquid like water or a buffer may be frozen into the bottom of the reagent chambers before adding other reagents for lyophilization.

[0006] A case may be fitted around the fluidic structure, the case may be constructed such that it can be snapped around the internal components and fluidic structure. The case may be light tight such that ambient light does not interfere with the detection system used for detecting pathogens. The case may be constructed such that the it has several parts that can be snapped together or clicked together by hand or by a press such that the device can be loaded with reagents at a separate location and the case can be snapped in place after reagents and / or other components have been assembled into the device.

[0007] The sample can be a biological fluid, a tissue sample, a hair sample, a fecal sample, an environmental sample, a swab containing a biological fluid, a swab containing an environmental sample, a swab containing a tissue sample, a swab containing a fecal sample, a vaginal swab, a rectal swab, an oral swab, a nasal swab, a nasopharyngeal swab. The sample could be a water sample. The biological fluid could be blood, urine, semen, vaginalLevine Bagade Han LLP 3 GTSCZ01000WOdischarge, cerebrospinal fluid, synovial fluid, or saliva. The sample could come from an animal, human, plant or environmental source. The biomolecule being detected could be nucleic acid from a pathogenic organism, an antibody formed by an organism as a result of a pathogenic infection, genomic DNA from an organism not associated with any infection, messenger RNA from an organism. Pathogenic organisms could include viruses, viroids, prions, fungi, spores, parasites or bacteria. Examples of pathogenic organisms include but are not limited to Equine herpes virus 1,2,3 and 4, Streptococcus equi, Equine influenza, African swine fever, Classical swine fever, Porcine epidemic diarrhea virus, Classical swine fever, Human immunodeficiency virus, SARS C0V2 virus, Human influenza virus, Avian influenza virus, Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Streptococcus pyogenes, Staphylococcus aureus, hop latent viroid, and methicillin-resistant Staphylococcus aureus.Applications:

[0008] This device can be a useful tool for large animal veterinarians making farm calls, as they are able to screen for infection and get results at the animal’s side. This can allow for treatment and handling of a diseased animal.

[0009] Some applications of this device include testing / screening of horses upon arrival to showgrounds, racetracks, breeding facilities, boarding and training facilities, etc. The information obtained from the device can be used to decide if a horse needs to be quarantined away from other horses, or if the horse is able to be released back into the herd. The device can be used in outbreak situations to determine if exposed horses have developed disease, as well as to monitor disease progression of infected horses so they can be cleared from quarantine.

[0010] Additional applications of this device include the testing of fecal matter from scouring pigs to detect pathogens such as Porcine Epidemic Diarrhea Virus. This can allow for the veterinarian or farm manager to know what pathogens are present within their flock and make decisions, such as culling, appropriately. The device can also be used to test environmental surfaces to determine what pathogens are present on the farm or on a trailer, allowing for evaluation of cleaning protocols and overall safety of the environment. The device can be useful when transporting livestock between barns, feedlots, and slaughterhouses, as diseased animals can be identified before they leave or arrive at the facilities.

[0011] This device can be used by companion animal owners on their pets in veterinary telemedicine situations. This can include testing for things like respiratory panels in cats orLevine Bagade Han LLP 4 GTSCZ01000WOdiarrhea panels in dogs. It can also be a useful tool at boarding facilities and pet daycare centers to screen animals before they come into contact with other animals.

[0012] The device may have a circuit board that holds a microcontroller as well as a NFC communication circuit, the microcontroller and the NFC circuit may be one IC. An antenna for NFC communication may be embedded on the circuit board using conductive traces. The circuit board may be a rigid circuit board or it may be a flexible board or it may be a rigid- flex circuit board. The circuit board may be constructed from FR4 material or from polyimide material or other materials that can have conductive traces. Optical components such as LEDs and Photo Diodes or Photo Transistors or Photo Detectors may be on the circuit board. Heater traces may be part of the circuit board such that the reaction wells or other wells can be heated. The heating traces may be copper traces or traces made from other conductive materials. The heating can also be generated from one or more semiconductors or a combination of conductive and semiconductor materials. Thermal sensors such as thermistors or RTD or semiconductor sensors may be on the circuit board to measure temperature. Thermal sensors may provide feedback for controlling the temperature of the reaction chambers or other chambers. The circuit board may have an interface to a power source such as a battery. The device may be powered from a single battery. The circuit board may have circuitry to increase the voltage potential from the power source such that enough voltage potential is provided to adequately power the circuitry in the device. The circuit board may have a boost converter or a step-up converter or a voltage doubler or other circuitry to increase the voltage potential from the power source. The device may be powered from a power source that provides adequate voltage potential for adequately powering the device. The device may be powered from one or more battery cells. The one or more batteries may be one time use or rechargeable. The battery may be Alkaline, NiMH, Lithium, Carbon Zinc or other battery technologies. It may be an advantage to power the device from a single battery cell in the disposable device to reduce the amount of waste, it may be an advantage for the power source to be an Alkaline or a Carbon-Zinc battery due to cost and the ease of disposing and / or recycling of the used battery. The device may have a holder or cover that can easily be removed for disposing or recycling the battery or batteries separate from the rest of the device since some part of the device other than the battery may be contaminated with biological fluid. The device may be designed such that the battery is not exposed to biological fluid or reaction fluid in the device. The device may be designed such that the battery is not exposed to biological fluid or reaction fluid when disposing of the battery. The battery may be situated in the device along with dried reagents during storage and shipment before use. The deviceLevine Bagade Han LLP 5 GTSCZOIOOOWOwith battery may be enclosed in a vapor barrier such as a foil pouch such that ambient humidity will not significantly affect the lyophilized reagents during storage and shipment. The Battery holder may be designed in a way or have a barrier between the battery and the rest of the device in order to minimize the evaporation of liquid or emission of gases from the battery affecting the lyophilized or dried reagents. Batteries such as Alkaline and Lithium and Carbon-Zinc and NiMH can be stored with lyophilized reagents along with desiccant to absorb moisture. The desiccant can be of type silica gel, clay (like bentonite), molecular sieves, activated alumina, calcium chloride, and activated carbon. Lyophilized reagents can be stored with batteries for 1 month or for 3 months or for 6 months or for 1 year or for 2 years or for any amount of time those reagents have shown to be stable in such environment. Some batteries have the outer case electrically connected to the positive terminal such as for example Duracell AA coppertop batteries, the outer case extends to the negative terminal side such that both a positive and negative terminal is at one end of the battery. A battery may be acquired such that the one end of the battery exposes both the positive and negative terminal, if such a battery is used in the device then the connection to the battery can be simplified by only require simple and / or short contacts from the circuit board to the battery for powering of the device, such a system reduces the material needed in the disposable device and makes the device more manufacturable and thereby lower cost.

[0013] The device may track temperature and / or humidity periodically during storage to check that the device is stored under correct environmental conditions. If the device detects that temperature and / or humidity is outside pre-set ranges then the device indicates to the user that the device is unusable or it may indicate that the results are unreliable.

[0014] The electrical circuitry may perform liquid sensing in order to detect when sample has been applied and thereby when to initiate starting of the test, instead of or in addition, the starting of the test can be initiated by a user pressing a button or by a user removing an isolation between the battery and a battery connection or by the user inserting a battery or by a button or switch being actuated when closing the cap or by an optical sensor or mechanical sensor or other sensor detecting the closing of the cap or by sensing pressure inside the fluidic chamber. The liquid sensing can be capacitive sensing or conductive sensing or optical sensing. The device may be enclosed in a light tight pouch such as a foil pouch so that when the bag is opened to retrieve the device then a light sensor may sense the opening of the bag and thereby start the detection process or it may set up the device to detect when sample has been applied or when the lid has been closed. The closing of the lid can be detected by a light sensor inside the device positioned such that it detects light when the lid is open and whereLevine Bagade Han LLP 6 GTSCZOIOOOWOthe light sensor does not detect light when the lid is closed. The light sensor may be placed in the bottom of the inlet chamber or other places inside the device. In a version of the device a light sensor can be placed in the bottom of the transparent inlet chamber thereby detecting when the device is removed from a foil pouch and the inlet cap is open and thereby starting to detect when sample has been applied, once sample has been applied and the cap closed then the light sensor detects the closing of the cap and the device starts the detection process. The starting of the detection process can be initiated by the device observing a fluorescent signal change from the detection chambers when one or more of the detection chambers are filled with liquid and thereby dissolving dried or lyophilized material. The device may be triggered to start the test by taking optical readings of the fluidic chip. The device may continuously capture optical readings once the device has been removed from the foil pouch by the user. The optical readings may change once a sample has been added and the cap or lid has been closed and secured. This sequence may alter the optical readings and trigger for the device to start the test sequence. The device may take optical readings every second or up to every 10 seconds to monitor when to start the test sequence. Figure28 shows an example of the optical readings difference between when the device is empty and when the sample and cap have been added to change the baseline readings of the device.

[0015] The cap for the inlet chamber may be made of polypropylene or other material, it may be attached to the case or it may be attached to the fluidic structure with a flexible or articulating member. The cap may include an O-ring for sealing the cap. The cap may be molded such that it seals to the inside of the inlet chamber by a tight fit.

[0016] The detection of the signal from amplifying nucleic acid may be done with a fluorescent detector consisting of a LED light source and a photo detector. An excitation filter may be placed between the LED light source and the detection chamber. An emission filter may be placed between the detection chamber and the photo detector. There may not be a lens between the photo detector and the detection chamber. There may be a lens between the photo detector and the detection chamber. There may not be a lens between the LED and the detection chamber. There may be a lens between the LED and the detection chamber. The LED may have a built in lens for directing the light toward one primary direction. The detection chamber may have molded- in light guides such that light will be guided from the LED light source to the detection chamber reaction. The detection chamber may have molded in light guides such that light will be guided from the detection chamber reaction to the photo detector. Light may be distributed from a LED to one or more detection chambers. Light may be distributed from one or more detection chambers to a photo detector. Light guides mayLevine Bagade Han LLP 7 GTSCZ01000WDfunction such that optical detectors and emitters may be mounted on a circuit board in one plane where the light guides direct the light into and out from a detection chamber that is offset from the mounting plane of the optical detectors and emitters, by using light guides the placement and assembly of the optical components is more manufacturable and cost effective. The light guides and / or the fluidic structure may be molded out of clear polypropylene such as YUNGSOX PP 5090T or Flint Hills P9M7R-056 or any other clear or clarified polypropylene. The light guides and / or the fluidic structure may be molded out of other clear plastics such as polycarbonate, PMMA, polystyrene, COG or COP. Parts or all of the light guides and / or fluidic structure may be coated or flame polished to enhance the clarity and optical functionality of the light guides and / or fluidic structure. The device may correct for ambient light leakage by subtracting a photo detector reading when a LED is turned off from a photo detector reading when a LED is turned on. The one or more LEDs may be turned on constantly or they may be blinking on and off sequentially or in sync. The blinking frequency may be from less than 1 micro second to more than 100 seconds. The detection circuit may consist of a photodiode and an amplifier circuit. The amplifier circuit may have a resistive feedback loop with a high value resistor for high gain, the feedback loop may also have a capacitor in the feedback for stability. The amplifier circuit may have an integrating capacitor where the light on the photo diode creates a voltage increase across the integrating capacitor, where the integrating capacitor may be reset using a voltage from a low leakage diode, where the signal is read as a slope of the integrated signal on the integration capacitor or as a final level on the integrating capacitor or as an end capacitor level minus the beginning capacitor level or as an average of capacitor level at the end minus an average of capacitor level at the beginning of the reset pulse period. Using an integrating capacitor as an amplifier for the photo diode may be lower cost than a high resistance feedback resistor. The amplification circuit may be coated with a coating to protect the circuitry from moisture or dirt, the coating may be Corona Dope, Silicone coating, or polyurethane coatings. Select areas can be chosen to be coated to optimize the coating cost (seen in Figure 21), and other areas to be avoided to prevent coating from interfering with the optical system (seen in Figure 22). The coating can cure prior to assembly, especially since it could affect the lyophilized reagents present in the fluidics ship. The photo detector may be an integrated photo detector where optical levels can be read via serial communication. The photo detector may be an integrated photo detector where detected optical levels generate changes in frequency of an output signal.Levine Bagade Han LLP 8 GTSCZ01000WD

[0017] The device may be shaped such that it fits in a standard laboratory test tube rack. The device may have a lower portion be less than 18mm diameter such that it fits in a standard laboratory test tube rack.

[0018] The readout of the result from a test may be by wireless communication via NFC or it may be read directly on the device by a LED or by a thermal indicator, where the thermal indicator is a thermal sensitive paper or material that changes color upon heating, where the heating can be accomplished by a resistor or a conductive trace or a semiconductor component adjacent to the thermal sensitive material. A thermal sensitive label can enable the result to be a permanent visual indication. The result may only be available via wireless communication and not be communicated to the user but be transmitted to a 3rdparty via a smartphone or a computer or other communication device.

[0019] The inlet port may lead to an inlet chamber wherein a sample may be initially deposited. The sample may be a swab or a liquid sample. The inlet chamber may contain liquid or liquid reagents. The inlet chamber may have features on the inside walls, a cap may close the inlet port by screwing the cap on the device, the cap may have features like baffles that can mix the sample and reagents in the inlet chamber, features on the inside of the inlet chamber may interact with features on the cap such that enhanced mixing is achieved when screwing the cap on the device by creating vibration in the baffles when they pass by features on the inside of the inlet chamber. The cap may be a snap type cap. The cap may have features that engage with the rest of the device such that it prevents the cap from being opened once the cap is closed.

[0020] The inlet chamber may hold liquid or liquid reagents during shipment where the inlet chamber may be covered by a foil seal to prevent the liquid from escaping during transport before use, where the foil seal may be removed or punctured by the user. The inlet chamber may have a foil seal at the bottom of the inlet camber to prevent liquid from going into the detection chambers until a sample has been added and until, the foil seal may be punctured by features in the cap such as baffles when the cap is screwed or pushed onto the inlet port; a bottom foil seal also prevents liquid from affecting dried reagents in the detection chambers during transport. A foil seal in the bottom of the inlet chamber also prevents dried reagents in the inlet chamber from moving into detection chambers until a liquid sample has been added.

[0021] The device may have an assembly with an optics configuration that can have an optical filter stacked on top of an LED or an optical filter stacked on top of a fluorescent detector sensor. The optical filters, LEDs, and fluorescent detector sensors may all be secured by a metal heating element. The metal heating element may be bonded to the circuit boardLevine Bagade Han LLP 9 GTSCZ01000WDusing double-sided adhesive, UV glue, or other bonding material. The device may capture optical readings every second for 20 minutes to make a determination on the result of the test. The device may have an algorithm that measures and analyzes the second derivative of the optical data to capture an inflection point where fluorescence increases and triggers a positive result. The device may determine a positive result when, at any point, the second derivative is above the Limit of Blank threshold, set by the manufacturer of the device. Otherwise the device may call the result negative. The device may display the result of the test using LEDs, or a heat activated paper indicator, or the result may be transmitted via wireless communication to a smartphone.

[0022] The battery clip can connect a standard battery to a rigid or flexible circuit board without the need for additional wiring or for a flexible circuit board to reach the bottom of the battery to connect. The battery clip can be a compression element to ensure consistent contact by pushing the battery toward the flex board, while also extending the opposite terminal without requiring extra components. The clip can have angled wings at the base, which can secure the clip within the casing, preventing movement, rotation, or detachment. Additionally, the battery clip can help keep the battery stable, minimizing movement and preventing premature ejection. The battery clip can be made from formed sheet metal and can be plated with nickel for added durability and enhanced electrical conductivity.

[0023] The device may communicate with a mobile device app that can process the data and generate a results readout report, listing the pathogens or other targets that the device tested for. The app may have the option to notify the required state animal health official depending on the geographical location where the test was conducted. The app may prevent viewing of results until the results have been transmitted to state animal health officials or to another authority or database for storage. The app may have the option to add additional information to the result report, such as geographical location, animal identification like a code or a number or a name, animal owner, test conductor (such as a veterinarian or technician), as well as health history like symptoms exhibited, vaccination history, travel history, etc of the animal. The app may have the ability to acquire and store a picture of the animal. The app may have the ability to read an identifying tag on the animal such as an RFID tag or a barcode or other type of identifying code or number or name. The app may have the ability to link to external management systems such as veterinary records management software and systems, laboratory management software and systems, and practice management software and systems. The app may also be able to link to external reporting systems, such as disease outbreak dashboards, or other mobile device apps, such as mail or messaging apps.Levine Bagade Han LLP 10 GTSCZ01000WD

[0024] The device may be available in a test kit with extra swabs, vials, and / or tubes for collection of additional samples to be sent for simultaneous testing in a diagnostic laboratory.

[0025] The kit may contain a swab-acceptance tube that accepts the swab after sampling of the bodily fluid. The swab acceptance tube may contain a liquid. This liquid may be an aqueous buffer that elutes the target from the swab and can be used directly in a nucleic acid amplification reaction. This aqueous buffer may contain some or all of the reagents necessary to run a nucleic acid amplification reaction. This aqueous buffer may also contain a portion of the reagents necessary to run a nucleic acid amplification reaction and be mixed with either wet or dried remaining reagents later in an assay protocol to run the nucleic acid amplification reaction.

[0026] The swab-acceptance tube may have a lid that is placed on the tube. This lid may have an outlet port so that when the tub is inverted, the liquid may exit the outlet port. The lid may contain a filter with a pore size between 0.1 microns to 50 microns that the liquid must pass through to exit the outlet port. The filter material used may be polyethylene, polyester, polycarbonate, or polypropylene, (filter examples).

[0027] The swab-acceptance tube may also contain a solid phase that can selectively adsorb substances from the liquid phase before exiting the outlet port of the lid. These removed substances may be inhibitors of nucleic acid amplification reactions for example humic acid, dust, glycosylated proteins, hemoglobin, lactoferrin, lysozymes, nucleases, proteinases, polysaccharides, smoke. These substances may also be analytical target biomolecules to be isolated and used later in the assay workflow. The solid phase may be a powder or beads. The solid phase may be separated from the liquid by passing the liquid through the filter and outlet port of the swab acceptance tube. The solid phase may be polyvinylpolypyrrolidine (PVPP). The solid phase may be carbon. The solid phase may be silica. The solid phase may be positively or negatively ionically charged. The solid phase may have analyte specific binding molecules. The binding molecules may be nucleic acids, proteins, peptides, or antibodies. The binding molecules may be carboxylic acids or amino functional groups.

[0028] To use this diagnostic system, the user can start by opening the sample collection swab or other collection device. Next, they open the sealed buffer tube containing the liquid reagents and add the sample. After securely closing the tube, which has a dropper, they dispense the correct amount into the device’s inlet. Once this is done, they close the device cap and wait for the results to be displayed.

[0029] A disposable diagnostic device for detecting a biomolecule in a sample is disclosed that can have an inlet port configured to receive the sample, one or more microfluidicLevine Bagade Han LLP 11 GTSCZ01000WOchannels fluidly coupled to the inlet port, one or more detection chambers in fluid communication with the channels, the detection chambers containing one or more reagents configured to interact with the biomolecule, a cap configured to seal the inlet port and apply pressure to drive the sample into the detection chambers, at least one gas-permeable membrane associated with the detection chambers to vent air while retaining liquid, and an onboard detection system configured to monitor a signal generated by the interaction of the biomolecule and the reagents. The biomolecule can have or be a nucleic acid, a protein, or an antibody. The reagents can be dried, lyophilized, or gel-form reagents. The amplification reaction can be selected from the group consisting of loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), helicase-dependent amplification (HD A), or recombinase polymerase amplification (RPA). The detection system can have one or more LEDs, photodiodes, phototransistors, or optical detectors configured to detect a fluorescent signal. The detection system can have a heating element and temperature sensor configured to control reaction temperature within the detection chambers. The disposable diagnostic device can have an electronic circuit with near-field communication (NFC) capability for wireless data transfer to an external device. The onboard power supply can have a single disposable or rechargeable battery. The sample can be blood, saliva, urine, fecal matter, tissue, an environmental swab, water, or combinations thereof. The biomolecule can be associated with a pathogenic organism selected from the group consisting of viruses, bacteria, fungi, parasites, or prions. The test results can be displayed on a display area of the device via a light indicator or thermal indicator, or transmitted wirelessly to a smartphone or electronic health system via the electronic circuit.

[0030] A method of detecting a biomolecule in an animal or human upper respiratory sample is disclosed. The method can include introducing the sample into the inlet port of fluidic structure, and sealing the inlet port with the cap on the fluidic structure such that a pressure is created in the inlet port of the fluidic structure that pushes the sample into one or more detection chambers through one or more fluidic channels. The air in the detection chambers can be emitted from the detection chambers through a gas permeable membrane or a gas permeable plug in the one or more detection chambers. The one or more detection chambers can be filled with the sample mixed with lyophilized or dried reagents in the detection chambers.

[0031] The method can include initiating an amplification and detection reaction in the detection chambers. The amplification and detection reaction can be selected from loop-Levine Bagade Han LLP 12 GTSCZ01000WDmediated isothermal amplification (LAMP), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), or polymerase chain reaction (PCR).

[0032] The method can include detecting a signal indicative of the presence or absence of the biomolecule. The method can further include contacting the sample with a solid-phase material selected from polyvinylpolypyrrolidone (PVPP), carbon, silica, or an ionically charged resin to remove one or more inhibitors of the amplification reaction prior to introduction into the detection chambers.

[0033] A diagnostic device for detecting a biomolecule in a sample can have a swab acceptance tube or inlet chamber configured to receive the sample, a liquid or buffer solution disposed within the chamber, and a solid phase material selected from polyvinylpolypyrrolidone (PVPP), carbon, silica, or an ionically charged resin. The solid phase material can selectively adsorb one or more inhibitors of a nucleic acid amplification reaction from the sample prior to the sample entering a detection chamber. The solid phase material can be PVPP in bead, powder, or particulate form. The inhibitors can have or be humic acid, proteins, polysaccharides, dust, glycosylated proteins, hemoglobin, lactoferrin, lysozymes, nucleases, proteinases, polysaccharides, smoke or other reaction-interfering molecules. The solid phase material can have a surface functionalized with nucleic acids, peptides, or antibodies configured to selectively bind inhibitory substances. The solid phase material can be upstream of a filter having a pore size between 0.1 and 50 microns, such that the sample can be filtered after inhibitor removal. The solid phase material can be in a swabacceptance tube, buffer reservoir, or channel in fluid communication with the detection chambers. The solid phase material can be incorporated into a removable cartridge or insert for replacement or customization of inhibitor-removal properties.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figures 1A and IB illustrate an example configuration of a diagnostics device with a push in cap or snap cap.

[0035] Figures 2A and 2B illustrate an example configuration of a diagnostics device with a screw on cap.

[0036] Figure 3 illustrates a fluidic structure.

[0037] Figure 4 illustrates a subassembly of the diagnostics device with fluidic structure and optical components.Levine Bagade Han LLP 13 GTSCZ01000WD

[0038] Figure 5A illustrates how a single cell battery can connect to a circuit board in the diagnostics device.

[0039] Figure 5B illustrates a diagram of the circuitry in the diagnostics device.

[0040] Figure 6 illustrates a low noise integrating amplification circuit for detecting light.

[0041] Figure 7 illustrates the curves from an integrating amplification circuit.

[0042] Figure 8 illustrates a classic photo diode detection circuit.

[0043] Figure 9 illustrates a device using a flexible circuit.

[0044] Figure 10 illustrates a diagnostics device fitting into a standard laboratory test tube rack.

[0045] Figure 11 illustrates an amplification plot of a no template control of a lamp assay run.

[0046] Figure 12 illustrates an amplification plot of a positive lamp assay run.

[0047] Figure 13 illustrates an amplification plot of a positive lamp assay run with ambient light subtracted from results.

[0048] Figure 14 Illustrates a sample kit.

[0049] Figures 15A - 15D illustrate a battery clip.

[0050] Figure 16 illustrates the placement of the battery clip inside the diagnostics device.

[0051] Figure 17 shows an exploded view of a variation of the diagnostics device.

[0052] Figures 18A - 18B illustrate a variation of the diagnostics device.

[0053] Figure 19 illustrates a flexible circuit board where the circuit board is unfolded flat.

[0054] Figure 20 illustrates the resulting fluorescent signal from an SDA reaction when reagents are stored with an active battery.

[0055] Figure 21 illustrates the amount of moisture released from batteries over time.

[0056] Figure 22 illustrates a portion of the diagnostics device where the fluidic structure fits into an optics block.

[0057] Figures 23A 23B illustrate how the light guides in the fluidic structure guides light from LEDs and to optical detectors.

[0058] Figure 24 illustrates the resulting fluorescent signal from a Positive and Negative Lamp reaction.

[0059] Figure 25 illustrates the change in fluorescent signal in a detection chamber when it is filled with liquid.

[0060] Figure 26 is a flow diagram of the assembly process of the diagnostics device.

[0061] Figure 27 illustrates a swab acceptance tube that may be part of the sample kit.Levine Bagade Han LLP 14 GTSCZ01000WO

[0062] Figure 28 shows amplification plots from a Lamp assay of untreated vs PVPP treated sample.

[0063] Figures 29 A 29B illustrate variations of the cap.

[0064] Figure 30 illustrates a flow diagram describing user steps.

[0065] Figure 31 illustrates a flow diagram describing device startup and run sequence.

[0066] Figures 32A - 32C illustrate systems where one or more detection devices can be attached to a power source device.

[0067] Figures 33A - 33C illustrate fluidic modules with caps that can be inserted into a separate reader device.

[0068] Figure 34 illustrates a reader device that accepts one or more fluidic modules.DETAILED DESCRIPTION

[0069] Figure 1A illustrates device 100 that can have a lower casing 107 and an upper casing 101 that has an inlet port 103 that can receive a sample to be analyzed, a cap 102 to close the inlet port 103, result indicators 105 and a battery cover 104 that can be detached for disposing of a battery 106 (not shown).

[0070] Figure IB shows an exploded view of the device 100 in figure 1A, where the cap 102 has a cap seal 114 that seals to the inside of the inlet chamber. The device 100 has a fluidic structure 108 that has a bottom seal that also functions as thermal contact to the circuit board 113 for heat transfer to the detection chambers. The fluidic structure 108 is capped with a detection chamber lid 109 that has a gas-permeable membrane 110 attached to the top such that the detection chambers can be vented. The fluidic structure 108 is in thermal proximity to the circuit board 113 where heating traces heats the detection chambers to the right reaction temperature using a thermal sensor feedback (not shown). The circuit board 113 also has NFC circuitry and antenna traces to create an NFC antenna. The circuit board 113 also has optical detectors and emitters for detecting a fluorescent signal in the detection chambers. There are also optical excitation filters and emission filters to filter the light from the LEDs and to the optical detectors, the optical filters are held in place using an optics block, also known as an Optics holder 111, the Optics holder 111 also functions to block light from one detection chamber to another and from ambient light by having it made of optically dense material, the optics holder may also function to distribute the heat from the circuit board 113 to the detection chambers in fluidics structure 108, the optics holder may be made of aluminum or other thermally conductive material, the optics holder may be anodized black to reduce light scatter. The detection chamber lid 109 may also function to block light from oneLevine Bagade Han LLP 15 GTSCZ01000WDdetection chamber to another and from ambient light by having it made of optically dense material. The circuit board 113 may have contacts for connecting to a battery 106. The circuit board 113 also has Result LEDs 105 for indicating the result of a test. The device has an upper case 101, a lower case 107 for protecting internal components as well as for blocking of ambient light and for labeling and indicator indication. The device also has a battery cover 104 for holding the battery 106 in place, where the battery cover 104 can be easily removed by a simple fractional turn for release of the battery 106.

[0071] Figure 2A illustrates device 100 that can have a lower casing 107 and an upper casing 101 that has an inlet port 103 that can receive a sample to be analyzed, a screw cap 201 to close the inlet port 103, result indicators 105 and a battery cover 104 that can be detached for disposing of a battery 106 (not shown).

[0072] Figure 2B shows an exploded view of the device 100 in figure 2A, where the screw cap 201 has a cap seal piece 202 that goes into the cap, the cap seal also has mixing baffles 203 for mixing the sample when the cap 201 gets screwed onto the inlet port 103. The device 100 has a fluidic structure 108 that has a bottom seal that also functions as thermal contact to the circuit board 113 for heat transfer to the detection chambers. The fluidic structure 108 is capped with a detection chamber lid 109 that has a gas-permeable membrane 110 attached to the top such that the detection chambers can be vented. The fluidic structure 108 is in thermal proximity to the circuit board 113 where heating traces heats the detection chambers to the right reaction temperature using a thermal sensor feedback (not shown). The circuit board 113 also has NFC circuitry and antenna traces to create an NFC antenna. The circuit board 113 also has optical detectors and emitters for detecting a fluorescent signal in the detection chambers. There are also optical excitation filters and emission filters to filter the light from the EEDs and to the optical detectors, the optical filters are held in place using an optics block, also known as an Optics holder 111, the Optics holder 111 also functions to block light from one detection chamber to another and from ambient light by having it made of optically dense material, the optics holder may also function to distribute the heat from the circuit board 113 to the detection chambers in fluidics structure 108, the optics holder may be made of aluminum or other thermally conductive material, the optics holder may be anodized black to reduce light scatter. The detection chamber lid 109 may also function to block light from one detection chamber to another and from ambient light by having it made of optically dense material. The circuit board 113 may have contacts for connecting to a battery 106. The circuit board 113 also has Result EEDs 105 for indicating the result of a test. The device has an upper case 101, a lower case 107 for protecting internal components as well as for blockingLevine Bagade Han LLP 16 GTSCZ01000WDof ambient light and for labeling and indicator indication. The device also has a battery cover 104 for holding the battery 106 in place, where the battery cover 104 can be easily removed by a simple fractional turn for release of the battery 106.

[0073] Figure 3 illustrates a fluidic structure 108 which is part of the device 100 where the inlet port 103 leads to an inlet chamber 301 that is connected to multiple detection chambers 302 via channels 303. Liquid can flow from the inlet port 103 through the inlet chamber 301 into the detection chambers 302. The structure also has light guides 304 such that optical emission and detection components can be mounted flat on a surface and light will be directed into the detection chambers 302. The fluidic structure 108 also has features 305 on the inside of the inlet chamber 301 for enhanced mixing when a screw cap 201 is screwed into the inlet port 103 with mixing baffles 203 for closing the inlet port 103; such that the mixing baffles 203 interferes with the features 305 to create impulses in the mixing baffles 203 which enhances mixing.

[0074] Figure 4 illustrates a sub-assembly 400 of the device 100 where the fluidic structure 108 is on a circuit board 113 with optical detectors 402 and LEDs 401 for measuring the optical signal from the detection chambers 302. The figure also shows excitation filters 403 and emission filters 404 for filtering the light as well as light guides 304 for directing the light to and from the detection chambers 302 to the optical components on the circuit board 113. The figure shows a detection chamber lid 109 with a gas permeable membrane 110. The detection chamber lid 109 may be snapped onto the detection chambers 302 or it may be adhered or heat staked or ultrasonically welded or laser welded or pressed on. The gas permeable membrane 110 may be heat staked onto the detection chamber lid 109 or it may be press fit onto or it may be ultrasonically welded onto or it may be laser welded onto the detection chamber lid 109 or directly onto the detection chambers 302. Result LEDs 105 on the circuit board 113 indicate the result of a test. The circuit board 113 can have heating areas 405, the heating areas 405 can be formed by copper traces on the circuit board 113. The circuit board has an NFC antenna 406 as a copper trace. The inlet port 103 accepts a sample into the inlet chamber 301.

[0075] Figure 5A illustrates a portion 500 of device 100 which shows how a single cell battery 106 can easily connect to the circuit board 208. The battery 106 has a positive metal case 501 and the case 501 is not covered with electrical isolation at the negative end 502 of the battery, therefore both a positive 505 and negative 506 connection can be made to the battery 106 at one end of the battery 106 using contacts on the circuit board 113, where theLevine Bagade Han LLP 17 GTSCZ01000WDcircuit board 113 can have a positive contact 503 and a negative contact 504 to connect to the battery end 502.

[0076] Figure 5B illustrates a diagram of a variation of the circuitry in the diagnostics device as follows. A Microcontroller 507A controls the different functions in the diagnostics device 100. Detection LEDs 401 illuminate the detection chambers needed to detect fluorescent signals with the Optical detectors 402. Heaters 405 are used to heat detection chambers for creating a reaction, for example the detection chambers are heated to 65 °C for a lamp reaction or to 60°C for a SDA reaction. Temperature sensors 507B are used as feedback for the Heaters 405 to get accurate control of temperature in the detection chambers.Temperature sensors 507B can also be used along with Humidity sensor 507C for monitoring environmental conditions during shipping and storage to make sure that the reagents are maintained at acceptable environmental conditions. Indicator LEDs 105 are used to indicate status and results of reactions. NFC circuitry and antenna 507D is used to transfer results and status and raw data to a reader external to the diagnostics device like to a smartphone or a tablet or a computer. Ambient light sensor 507E is used to initiate starting a device run. The battery 106 powers the device. The heaters may be powered directly from the battery, but other circuitry may need voltage boost circuitry 507F to generate enough voltage if a single cell battery is used.

[0077] Figure 6 illustrates a schematic of a detection circuit 600 used to detect light from a detection chamber 302. Light is captured by the photodiode 601 and the amplifier 602 will integrate the current onto the capacitor 603 where it can be read by an Analog-to-Digital- Converter on the circuit board 113 or in a microcontroller on the circuit board 113.Periodically the capacitor 603 can be reset by applying a positive pulse on the Reset pin 606 on diode 605. The positive Reset pulse may be current-limited by a resistor in series with the diode 605. The Output 604 may be sampled multiple times during the integration time and the samples may be compared to each other to determine the rate of voltage increase on the Output 604. The rate of voltage increase on the Output 604 is proportional to the intensity of light on the photodiode 601. The integrating circuit is low cost and compact and avoids the Johnson noise from a feedback resistor when compared to a classic photodiode amplification circuit 800 shown in Figure 8. The detection circuit 600 in Figure 6 can be used to detect or amplify any kind of electrical current signal, such as sensor signals or sound signals or electrochemical signals, by removing the photodiode and applying the particular signal to be amplified to the negative input on the amplifier 604.Levine Bagade Han LLP 18 GTSCZ01000WD

[0078] Figure 7 illustrates a curve of the Output signal 604 compared to the Reset pulse 606 for circuit 600; as can be seen the output signal 604 increases linearly with a constant light intensity until a Reset pulse 606 is high then the output signal 604 goes low and then after the Reset pulse 606 goes low then the signal rise on Output signal 604 starts over. The Slope 701 is proportional to the light intensity.

[0079] Figure 8 illustrates a classic photodiode amplification circuit 800 for detecting optical light captured by photodiode 601. The current generated by photodiode 601 is amplified by amplifier 602 with resistor 801 and capacitor 802 as feedback to generate a voltage on the Output 604 which can be read by an Analog-to-Digital-Converter on the circuit board 113.

[0080] Figure 9 illustrates a version 900 of the device 100 where a flexible circuit 901 can be used as a circuit board. The device can have a cover removed such that some of the internal components can be seen like the fluidic structure 108, also seen is the battery cover 104, the inlet port 103 is on the top with a snap cap. The flexible circuit 901 can be wrapped around the fluidic structure 108 so as to accommodate the LEDs and Optical detectors being placed close to the side walls of the detection chambers 302.

[0081] Figure 10 illustrates a setup 1000 that shows how a device 100 can be placed in a standard laboratory test tube rack 1001. By having the diagnostics device 100 fit into a standard laboratory test tube rack 1001 it is possible to easily have one or multiple devices 100 perform test in parallel with low risk of the devices falling over during the test.

[0082] Figure 11 illustrates an amplification plot of fluorescence vs time (minutes) of a no template control of a lamp assay run in the diagnostics device 100 confirming no amplification since the curves plateau and do not show a sigmoidal or exponential increase. One curve is from a Equine Herpes Virus 1 (EHV-1) assay 1101, the other curve is from a control assay 1102.

[0083] Figure 12 illustrates an amplification plot of fluorescence vs time (minutes) of a positive sample of a lamp assay run in the diagnostics device 100, the amplification curve shows an exponential increase 1201 then a plateau or the beginning of a plateauing in both the assay 1101 and endogenous control curve 1102, confirming the positive signal for both the assay and endogenous control. One curve is from a Equine Herpes Virus 1 (EHV-1) assay 1101, the other curve is from a control assay 1102.

[0084] Figure 13 illustrates an amplification plot of fluorescence vs time (minutes) of a positive sample similar to Figure 12 but with the ambient light subtracted from the signal when the LEDs are turned on giving a smoother signal that allows for a more accurate determination of the amplification curve. One curve is from a Equine Herpes Virus 1 (EHV-Levine Bagade Han LLP 19 GTSCZ01000WD1) assay 1101, the other curve is from a control assay 1102. By subtracting ambient light readings from the detected signal from the detection chambers then the resulting signal is less affected by ambient light. The ambient light readings can be acquired with the same detector that is reading the signal from the detection chambers 302 but the ambient reading is taken when the detection LEDs 401 is off.

[0085] Figure 14 illustrates a sample kit 1400 containing the diagnostic device 100, swab acceptance tube or buffer tube 1401, sample collection medium 1402, and rack 1403 to hold the device and the buffer tube. The rack 1403 can prevent any tilting of the device 100 or any spillage or contamination both from the device 100 and buffer tube 1401. The sample collection medium 1402 may be a swab.

[0086] Figure 15A - 15D illustrates a battery clip 1500 with dimensions in millimeters and angles in degree. The clip may be used to make a connection from battery 106 to circuit board 113. The bottom part of the clip has a compression element 1502 to ensure the battery is consistently connected to the circuit board 113, along with wings 1501 that hold it in place, preventing movement or dislodging from the battery casing 104 after assembly. The top part of the battery clip extends to the circuit board 113 to connect the opposite side of the battery to the same board. This top section is also designed with a feature 1503 that holds it in place during assembly, and it has its own spring contact element 1504 to maintain consistent contact with the board. The battery clip 1500 may be used for connecting AA, AAA AAAA, C, D or other size batteries, it may be used to connect one or multiple batteries in series to the circuit board 113. Figure 15A shows a perspective illustration of the battery clip 1500, Figure 15B illustrates a side view of the battery clip 1500, Figure 15C illustrates a back view of the battery clip 1500, Figure 15D illustrates a top view of the battery clip 1500.

[0087] Figure 16 illustrates the placement of the battery clip 1500 inside the diagnostics device 100, positioned at the bottom of the battery casing 104. The wings 1501 hold it in place. The compression element 1502 pushes the battery 106 towards the circuit board 113 to ensure contact. The battery clip's extension exceeds the height of the battery casing 104, allowing it to connect the opposite side of the battery 106 to the circuit board 113.

[0088] Figure 17 shows an exploded view 1700 of the diagnostics device 100. The top part of the device, from left to right, includes the back casing 1701 with clips for attachment 1702, an optics block 111 that holds the flex circuit board 113 in place when folded with a fluidics chip 108 with a tight fit for optimal and consistent thermal transfer, preventing detachment during initial assembly. The board also has a switch 1704 positioned to trigger the device when the cap 102 is closed. Attached to the flex circuit board 113 is a thermal paper 1705Levine Bagade Han LLP 20 GTSCZ01000WOthat changes color when heated, there are 3 heating regions for the thermal paper 1705 corresponding to the positive, negative, and indeterminate result indicators on the front top casing 1703. The bottom part of the device can have three components: a bottom casing 104 that screws into the top casing 1701 and 1703, a battery 106, and a battery clip 1500 that compresses the battery 106 and connects the opposite side of the battery 106 to the circuit board 113.

[0089] Figure 18A: illustrates a variation of the diagnostics device 100 with an open cap 102, allowing the user to add sample and / or reagents to the inlet 103. The diagnostics device 100 can have a display area 1806. The display area 1806 can be positioned at an end of the device and angled such the surface of the display area 1806 is easily viewable by a user. The display area 1806 can comprise a display which indicates positive 1801, negative 1802, and undetermined 1803 results through intuitive signs located below the round holes, which correspond to the areas of the thermal paper 1705 that change color as results are displayed. A feature 1804 on the cap engages with the device 100 such that the cap 102 is locked and / or prevented from opening once closed and cannot easily be opened. A feature 1804 on cap 102 activates the switch 1704 (not shown) when the cap 102 is closed thereby starting the test. Figure 18B illustrates the size of the diagnostics device 100, where the battery cover 104 inner diameter is 14.5mm minimum to fit one AA battery. The battery cover 104 can have other dimensions appropriate to fit other battery sizes and quantity.

[0090] Figure 19 illustrates a flexible circuit board 113 where the circuit board 113 is unfolded flat. The circuit board 113 has an area 1901 for connecting to a battery 106 using a battery clip 1500. The circuit board 113 has heating areas 405 for heating detection chambers 302 (not shown). The circuit board 113 has an antenna area 406 for wireless communication of results and / or other data and / or commands and / or for calibrating the device 100. The circuit board 113 has connector or connector pads 1902 for communication of results and / or other data and / or commands and / or for programming the device 100 and / or for calibrating the device 100. The circuit board 113 has areas for detection LEDs 401 and Photo Detectors 402 for fluorescence detection. The circuit board 113 has areas 1903 for Heating resistor and / or heating traces for heating thermal paper 1705 indicators for indicating results of a test. The circuit board 113 has an area for a switch 1704 for starting the test. The circuit board 113 has an area for an ambient light sensor 1904 for starting the test. To maintain low noise and interference the circuit board 113 may be coated with corona dope or other silicone or acrylic or epoxy coating or other insulating coating around the optical detector areas and / or the amplifier areas and / or other areas. When coating the circuit board 113 there may be areas thatLevine Bagade Han LLP 21 GTSCZ01000WDshould not be coated since they may interfere with the function of the circuit board 113, for example the optical detectors 402 themselves as well as the LEDs 401 themself as well as the switch 1704 itself may not be coated.

[0091] Figure 20 illustrates the resulting fluorescent signal from a SDA reaction where the vertical axis is fluorescent signal intensity and the horizontal axis is time in minutes for a 20 minute run when reagents are stored with an active battery. The various lines are from reagents that have been stored with AA batteries in sealed pouches for 6 months to observe the effect of active AA batteries on lyophilized SDA reagents. This experiment includes a bench control with fresh reagents 2001, an experimental control without batteries 2002, and tests with active alkaline batteries 2003 and lithium batteries 2004. The graph shows minimal effect of the batteries on the SDA reagents which indicates that the batteries can be enclosed with lyophilized reagents. Desiccant is stored along with the lyophilized reagents to absorb any moisture released from the batteries and / or moisture that may migrate into the storage pouch or container from ambient air.

[0092] Figure 21 illustrates a plot of delta weight (gram) over time in days of multiple brands of AA batteries. This experiment was performed to quantify the amount of moisture being released from the batteries over time since most of the weight loss is due to moisture like water release or water evaporation. The batteries were stored in a 70°C oven. Results are shown for Alkaline battery brand A 2101, Alkaline battery brand B 2102, Eithium battery 2103, nickel metal hydride battery 2104, carbon zinc battery 2105. The weight of the batteries were measured over time and subtracted the initial weight at the beginning of the experiment. The results of this experiment is used to determine how much desiccant is needed to absorb the moisture from the batteries when stored with lyophilized or gel or dried reagents such that the moisture from the battery does not increase the humidity around the reagents and thereby degrade the reagents to an unacceptable performance. The results can also be used to select batteries that have acceptable moisture release.

[0093] Figure 22 illustrates a portion 2200 of device 100 which shows how the fluidic structure 108 can fit into the optics and heating block 111 in contact with the circuit board 113. The circuit board 113 has indicator EEDs 113 to indicate the results from a test. This variation of the fluidic structure can have 8 detection wells (not shown) that are in contact with the optics block. The fluidic structure 108 has an inlet port 103 for sample introduction. The detection wells (not shown) can be capped with a lid 109 with a gas permeable membrane for venting.Levine Bagade Han LLP 22 GTSCZ01000WD

[0094] Figure 23A illustrates a section 2300 of device 100 which shows how the fluidic structure 108 fits into the optics and heating block 111. The illustration can be sectioned such that the detection chambers 302 are shown with light guides 304 that directs lights from LEDs 401 into the chambers 302 after passing through an excitation filter 403. The section 2300 also shows the positive 503 and negative 504 battery terminal on the circuit board 113 for connecting to a battery 106 (not shown). Figure 23B illustrates a section 2301 of device 100 which shows how the fluidic structure 108 fits into the optics and heating block 111. The illustration is sectioned such that the detection chambers 302 are shown with light guides 304 that directs lights to the photo detector(s) 402 from the chambers 302 after passing through an emission filter 404. The section 2301 also shows the positive 503 and negative 504 battery terminal on the circuit board 113 for connecting to a battery 106 (not shown).

[0095] Figure 24 illustrates the resulting fluorescent signal from a Lamp reaction where the vertical axis is fluorescent signal intensity and the horizontal axis is time in minutes for a 20 minute run. The graph shows the signal from a No Template Control (NTC) i.e. a Negative reaction 2401, it also shows the signal from a positive signal 2402.

[0096] Figure 25 illustrates the fluorescent signal from a detection well when it changes from an empty well with dry lyophilized reagents to being filled with liquid. As can be seen, the signal has a significant increase 2501 then liquid fills the detection chamber. This can be used to start the test i.e. start the heaters and the fluorescence detection to determine which detection chambers have a positive or negative reaction. The sequence for starting the test may be as follows: detect the device has been removed from the storage pouch by an ambient light sensor then determine that the sample has been loaded into the device by detecting the change in fluorescence in one or more detection wells.

[0097] Figure 26 is a flow diagram 2600 of the assembly process of the diagnostics device 100. The fluidic structure assembly 2601 may be assembled by applying a bottom seal of the fluidic channels and then loading with lyophilized or dried reagents in the detection chambers 302 and possibly the inlet chamber. The circuit assembly 2602 may be assembled by soldering electronic components onto the circuit board 113 and then attaching the optics block 111 to the circuit board 113 using mechanical latches or pins or fasteners or adhesive or adhesive film, then the circuit board 113 with optics block 111 is placed in the lower case 107 using mechanical latches or pins or fasteners or adhesive or adhesive film. The circuit assembly 2602 may be combined with the battery 106 and the battery cover 104 into lower assembly 2604. The upper case assembly 2605 may be assembled by attaching a gas permeable 110 membrane to the detection chamber lid 109using ultrasonic welding, heatLevine Bagade Han LLP 23 GTSCZ01000WOstaking, heat sealing or adhesive or mechanical interference, the assembly of the detection chamber lid 109 and the gas permeable membrane 110 may be inserted into the upper case 101. The cap assembly 2606 may be assembled by inserting the cap seal 202 into the cap 201, an O-ring (not shown) may also need to be located onto the cap 201 or cap seal 202. The Upper assembly 2607 may be created by attaching the cap assembly 2606 to the upper case assembly 2606. The final step of the assembly of device 100 may be to insert the fluidic structure assembly 2601 into the lower assembly 2604 and then press the upper assembly 2607 onto the rest of the assembly. Labels may need to be applied. Software may need to be loaded. Calibrations of temperature and / or optical systems may need to be performed. QC steps may need to be performed. The assembly steps can use any of mechanical fastening, screw or thread engagement, press fit, ultrasonic welding, laser welding, adhesion, heat sealing, heat staking, mechanical latches.

[0098] Figure 27 illustrates a swab acceptance tube or buffer tube 1401 that may be part of the sample kit 1400. The swab acceptance tube 1401 may have an upper portion that may have a filter 2704 such that a liquid sample would pass through the filter 2704 before exiting the outlet port 2702. The swab acceptance tube may have a liquid 2701 for diluting and / or dissolving material from a swab or from a solid or a liquid sample. The liquid 2701 may be a buffer. The liquid 2701 may contain reagents for lysing of the sample. The liquid 2701 may contain reagents that are needed for a nucleic acid reaction. The swab acceptance tube 1401 may contain a solid phase 2703 that can selectively adsorb substances from the liquid phase before exiting the outlet port of the lid 2702; These removed substances may be inhibitors of nucleic acid amplification reactions for example humic acid, dust, glycosylated proteins, hemoglobin, lactoferrin, lysozymes, nucleases, proteinases, polysaccharides, smoke; These substances may also be analytical target biomolecules to be isolated and used later in the assay workflow. The solid phase 2703 may be a powder or beads. The solid phase 2703 may be separated from the liquid 2701 by passing the liquid through the filter 2704 and outlet port 2702 of the swab acceptance tube 1401. The solid phase 2703 may be polyvinylpolypyrrolidine (PVPP). The solid phase 2703 may be carbon. The solid phase 2703 may be silica. The solid phase 2703 may be positively or negatively ionically charged. The solid phase 2703 may have analyte specific binding molecules; The binding molecules may be nucleic acids, proteins, peptides, or antibodies; The binding molecules may be carboxylic acids or amino functional groups. The swab acceptance tube may ship with a foil seal on top of the tube that will be removed by the user. Once the user has applied a sample into the tube then the top piece may be pressed onto the top of the tube and sample is squeezed out throughLevine Bagade Han LLP 24 GTSCZ01000WOthe outlet port 2702 in the top piece. The sample may be dispensed into the diagnostics device 100. The dispensing of sample into the diagnostics device 100 may be by the user applying a number of drops of sample into the inlet port 103 of the diagnostics device 100. The outlet port 2702 may be designed to promote certain drop sizes to form such that a certain number of drops correspond to a certain sample volume.

[0099] Figure 28. shows amplification plot 2800 of fluorescence vs time (minutes) of a positive sample using a Lamp assay, the amplification curves shows an exponential increase then a plateau or the beginning of a plateauing in an untreated sample 2801 and PVPP treated sample 2802. The result confirms that PVPP helps absorb inhibitory or unwanted material of a sample (like dirt) since the signal from the curve that has been treated with PVPP has an earlier inflection point and a stronger end signal than the curve that is untreated.

[0100] Figure 29 A illustrates a variation of the cap 102 where the sealing surface 114 is part of the cap 102 by having an interference fit with the inlet chamber of the diagnostics device 100. A feature 1804 on the cap engages with the device 100 such that the cap 102 is locked and / or prevented from opening once closed and cannot easily be opened. A feature 1804 on cap 102 activates the switch 1704 (not shown) when the cap 102 is closed thereby starting the test. Figure 29B illustrates a variation of the cap 102 where the sealing surface is created with one or multiple O-rings 2901 applied on the cap 102. The O-rings 2901 seals to the inside of the inlet chamber of the diagnostics device 100, the design allows for placement of multiple O-ring positions to create the right amount of pressure for the liquid sample to fill the detection chambers 302. Cap is designed to be able to allow for a defined amount of pressure such that when the liquid sample and / or reagents are placed in the inlet chamber 301 it would flow into the detection chambers 302 when the cap 102 is closed by the user. An ideal amount of pressure is calculated and tested to properly fill the 8 detection chambers 302 at the right level. A tolerance is calculated to achieve the balance between too little pressure, which would cause the chambers to not fill properly, and too much pressure, which could lead to leaks at the bottom seal or membrane. Additionally, the design allows for the placement of two additional O-rings 2901 at different levels to serve as a fallback in case the design does not perform as expected with the molds due to material properties or imperfections. Also, this may be applied later on in the design to decrease the amount of filling if necessary relating to optimizing the reagents volumes in the chambers or in case the number of chambers needed to be decreased which may help save up on reagents. Cap 102 is able to be trimmed to only allow either or both o-rings 2901 to be issued instead of the sealing surface 114 of the cap 102.Levine Bagade Han LLP 25 GTSCZ01000WO

[0101] Figure 30 illustrates a flow diagram describing user steps as follows. 3001A the user removes the diagnostics device 100 from the pouch it is stored in. 3001B The user removes the foil seal from the swab acceptance tube 1401. 3001C the user applies a swab sample into the swab acceptance tube 1401. 3001D the user removes the swab while squeezing the tube 1401 to retain liquid on the swab. 3001E the user closes the lid on the swab acceptance tube 1401 and shakes the tube. 3001F the user applies 5 drops from the swab acceptance tube 1401 into the diagnostics device 100. 3001G the user closes the lid 102 on the diagnostics device 100. 3001H when the result LED(s) light up then the user notes if the run was successful. 3001J the user opens an app on a smartphone and scans the diagnostics device 100 with the smartphone to acquire results. 300 IK the user enters supporting information in the app and then uploads results to state veterinary authority and to Practice Management System (PMS). 3001L the user removes the battery 106 from the device and disposes the battery 106, the rest of device 100 along with the swab and swab acceptance tube 1401 in accordance with regulatory requirements.

[0102] Figure 31 illustrates a flow diagram describing device 100 startup and run sequence as follows. A starting point 3101 A can be as the device is shipped from the factory. A timer can be reset and started 3101B. The device can measure 3101C the ambient Temperature and / or Humidity. 310 ID the device checks that the Temperature and / or Humidity reading is within range; if the Temperature and / or Humidity is not within range then it proceeds to 3101E where the device is rendered unusable and displays an error condition on the device followed by 3101N where the error condition is transferred to a NFC reader or smartphone. If the Temperature and / or Humidity is within range in step 3101D then it proceeds to 3101F where the device checks the ambient light sensor to determine if the device has been removed from its package or pouch. If Ambient light is not detected then the device proceeds to step 3101G where the Timer is checked to see if enough time has passed for doing another Temperature and / or humidity check. The amount of time between Temperature and / or humidity checks can be 5 minutes or it can be from a microsecond to 24 hours. If the timer in 3101G has not exceeded the time limit then the device goes back to step 310 IF to check the light sensor. If the Timer has exceeded the time limit in step 3101G then the device goes back to step 3101B and repeats the follow-on steps from there. If Ambient light is detected in step 3101F then the device proceeds to step 3101H where the device measures fluorescence in one or more detection chambers. Then the device proceeds to step 3101 J where the device determines if the fluorescent signal measured in step 3101H has risen enough which would indicate that liquid has entered the one or more detection chambers; if the fluorescent signal has not risenLevine Bagade Han LLP 26 GTSCZ01000WOenough to indicate liquid in the one or more detection chambers then the device goes back to step 3101H and proceeds from there. If in step 3101 J the device determines that the fluorescent signal has risen enough to indicate liquid in the one or more detection chambers then the device continues to step 310 IK where the device will perform the amplification and detection of pathogens in the detection chambers by applying heat and measuring fluorescent signals. After the amplification and detection process in step 3101K then the device continues to step 310 IL where the device calculates and / or determines the reaction results based on the fluorescent signals from step 310 IK. Thereafter the device proceeds to step 310 IM where the results are displayed on the device or the device may just display that results are available. Thereafter the device can proceeds to step 310 IN where results can be transferred to a NFC reader or smartphone.

[0103] Figure 32A illustrates a system 3200 where a detection device 3203 comprising an inlet port 103 a cap 102 for the inlet port 103 such that when the cap 102 is closed into the inlet port 103 a liquid sample and / or reagents are pushed into one or more detection chambers with the pressure generated from pushing the cap 102 into the inlet port 103. The detection device 3203 can have a display area 3210 extending circumferentially about the detection device 3203. The display area 3210 can comprise indicators 105 for displaying results of one or more reactions. The indicators 105 can be equiangularly spaced around the detection device 3203 within the display area 3210, thereby forming a uniform distribution of indicators 105 about the display area 3210. The cap 102 has a feature 1804 that engages with a feature 3208 on the detection device 3203 such that the cap is locked in place once closed. The detection device 3203 comprises detection chambers with heating capability and optical detection capability as well as a microcontroller and circuitry for controlling the detection device 3203. The detection device 3203 may comprise reagents in dried or wet or lyophilized or gel form to perform one or more reactions. Reagents may be located in detection chambers in the detection device 3203. The reactions may be nucleic amplification reactions of target pathogens. The detection device 3203 may comprise wireless communication circuitry for communicating results. The detection device 3203 comprises power connection for powering the detection device 3203 to perform a test on the sample for pathogens. The detection device 3203 can engage with a power device 3204 via a port 3205 such that the power device 3204 can power the detection device 3203. The power device 3204 has connections for delivering power to detection device 3203. The power device 3204 may also have communication connections for communicating with detection device 3203. The power device 3204 may contain one or more batteries; the batteries may be rechargeable. The power device 3204 mayLevine Bagade Han LLP 27 GTSCZ01000WOcomprise a connector 3206 for powering or for charging the power device 3204. The connector 3206 may be a USB port. The connector 3206 may also function as a communication port for communicating with the power device 3204 and possibly with the detection device 3203. The detection device 3203 may be one time use and may be disposed of after use.

[0104] Figure 32B illustrates a system 3201 where the detection device 3203 is engaged with the power device 3204 via a port 3205 such that the power device 3204 is powering the detection device 3203.

[0105] Figure 32C illustrates a system 3202 where a power device 3207 can engage with and power multiple devices 3203 by having multiple ports 3205 for accepting detection devices 3203. The power device 3207 may contain one or more batteries; the batteries may be rechargeable. The power device 3207 may comprise a connector 3206 for powering or for charging the power device 3207. The connector 3206 may be a USB port. The connector3206 may also function as a communication port for communicating with the power device3207 and possibly with the detection devices 3203. For example the connector 3206 may connect to a PC or a tablet or a smartphone for result data upload. The detection devices 3203 may perform tests in parallel or they may perform tests in sequence. Some ports 3205 may be free while other ports 3205 are powering detection devices 3203.

[0106] Figure 33A illustrates a fluidic device 3300 comprising a fluidic structure 108 with a cap 102. The fluidic device 3300 comprises one or more detection chambers 302 connected to an inlet chamber with fluidic channels 303. To create the fluidic channel structure 303 a sealing material is applied over the channels 303 to close them off. The sealing material may be a film made of plastic. The cap 102 may have a locking feature 1804 such that when the cap is closed then the locking feature 1804 engages with a corresponding feature 3302 on the fluidic structure such that the cap 102 is prevented from opening once closed.

[0107] Figure 33B illustrates a fluidic device 3301 similar to 3300 but where the detection chambers 302 have been closed with a lid 109 with a gas permeable member 110. The gas permeable member 110 may be in the form of a membrane or a plug or a sheet or a filter or a plate. The detection chambers 302 may contain reagents in dried or wet or lyophilized or gel form to perform one or more reactions. The reactions may be nucleic amplification reactions of target pathogens. The fluidic device 3301 has an inlet port 103 for accepting a sample and / or reagents. The sample may be in liquid form such that it can flow into the detection chambers 302 when the pressure is created from closing the cap 102 into the inlet port 103. The sample may be in solid form and may be fully or partially dissolved in liquid containedLevine Bagade Han LLP 28 GTSCZ01000WOin the fluidic device 3301 or the in a separate tube where the content of the separate tube is applied into the inlet port 103. The fluidic device 3301 may be one time use and may be disposed of after use.

[0108] Figure 33C illustrates the fluidic device 3301 where the cap 102 has been closed thereby distributing a sample from the inlet port 103 to the detection chambers 302.

[0109] Figure 34 illustrates a reader device 3400 that accepts one or more fluidic devices 3301 for performing tests of the samples in the one or more fluidic devices 3301. The reader device 3400 comprises one or more compartments 3402 for accepting fluidic devices 3301. The one or more compartments 3402 may have a moveable cover 3403. The reader device 3400 may comprise a screen 3401 or indicators for displaying results of one or more reactions. The reader device 3400 may comprise one or more heaters or thermoelectric devices for heating or cooling or cycling the temperature of the fluidic devices 3301. The reader device 3400 may comprise optical detection capability for detecting reaction results from the one or more fluidic devices 3301. The reader device 3400 may comprise a microcontroller and circuitry for controlling the test procedure for the fluidic devices 3301. The reader device 3400 may comprise wireless communication circuitry for communicating results. The reader device 3400 may comprise a connector 3206 for powering or for charging the reader device 3400. The connector 3206 may be a USB port. The connector 3206 may also function as a communication port for communicating with the reader device 3400. For example the connector 3206 may connect to a PC or a tablet or a smartphone for result data upload. The reader device 3400 may perform tests on the fluidic devices 3301 in parallel or it may perform tests in sequence. Some compartments 3402 may be free while other compartments 3402 are actively testing fluidic devices 3301. The reader device 3400 may contain one or more batteries; the batteries may be rechargeable.Levine Bagade Han LLP 29 GTSCZ01000WO

Claims

CLAIMSWE CLAIM:

1. A disposable diagnostic device for detecting a biomolecule in a sample, comprising: an inlet port configured to receive the sample; a microfluidic channel fluidly coupled to the inlet port; a detection chamber in fluid communication with the channel, the detection chamber comprising a reagent for interacting with the biomolecule; a cap configured to seal the inlet port and apply pressure to drive the sample into the detection chamber; a gas-permeable membrane associated with the detection chamber, wherein the membrane is for venting air while retaining liquid; and an onboard detection system for monitoring a signal generated by the interaction of the biomolecule and the reagent.

2. The device of claim 1, wherein the biomolecule comprises a nucleic acid, a protein, and / or an antibody.

3. The device of any of the previous claims, wherein the reagent comprises dried, lyophilized, and / or gel-form reagents.

4. The device of any of the previous claims, wherein the interaction is an amplification reaction selected from the group consisting of loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), helicase-dependent amplification (HD A), and recombinase polymerase amplification (RPA).

5. The device of any of the previous claims, wherein the onboard detection system comprises one or more LEDs, photodiodes, phototransistors, and / or optical detectors configured to detect a fluorescent signal.

6. The device of any of the previous claims, wherein the onboard detection system comprises a heating element and temperature sensor configured to control reaction temperature within the detection chamber.

7. The device of any of the previous claims, further comprising an electronic circuit with near-field communication (NFC) capability for wireless data transfer to an external device.

8. The device of claim 7, wherein the electronic circuit is for transmitting a result wirelessly to a smartphone or electronic health system.Levine Bagade Han LLP 30 GTSCZOIOOOWO9. The device of any of the previous claims, further comprising an onboard power supply, wherein the onboard power supply comprises a battery.

10. The device of any of the previous claims, wherein the sample comprises blood, saliva, urine, fecal matter, tissue, an environmental swab, and / or water.

11. The device of any of the previous claims, wherein the biomolecule is associated with a pathogenic organism, wherein the pathogenic organism comprises viruses, bacteria, fungi, parasites, and / or prions.

12. The device of any of the previous claims, further comprising a display area wherein results are displayed on the display area of the device via a light indicator or thermal indicator.

13. A method of detecting a biomolecule in an animal or human upper respiratory sample, comprising: introducing the sample into an inlet port of a fluidic structure; sealing the inlet port with a cap on the fluidic structure such that a pressure is created in the inlet port of the fluidic structure that pushes the sample into one or more detection chambers through one or more fluidic channels; emitting air from the one or more detection chambers through a gas permeable membrane or a gas permeable plug in the one or more detection chambers; mixing the sample with lyophilized or dried reagents in the one or more detection chambers, thereby filling the one or more detection chambers; initiating an amplification and detection reaction in the one or more detection chambers, wherein the amplification and detection reaction is loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), helicase-dependent amplification (HD A), recombinase polymerase amplification (RPA), or polymerase chain reaction (PCR); and detecting a signal indicative of a presence or absence of the biomolecule.

14. The method of claim 13, further comprising contacting the sample with a solid-phase material selected from polyvinylpolypyrrolidone (PVPP), carbon, silica, or an ionically charged resin to remove one or more inhibitors of the amplification and detection reaction prior to introduction into the one or more detection chambers.

15. A diagnostic device for detecting a biomolecule in a sample, comprising: a swab acceptance tube or inlet chamber configured to receive the sample; a liquid or buffer solution disposed within the chamber;Levine Bagade Han LLP 31 GTSCZOIOOOWOone or more detection chambers in fluid communication with the inlet chamber; and a solid phase material selected from polyvinylpolypyrrolidone (PVPP), carbon, silica, or an ionically charged resin, the solid phase material being configured to selectively adsorb one or more inhibitors of a nucleic acid amplification reaction from the sample prior to the sample entering the one or more detection chambers.

16. The device of claim 15, wherein the solid phase material is PVPP in bead, powder, or particulate form.

17. The device of either of claims 15 or 16, wherein the one or more inhibitors comprise humic acid, proteins, polysaccharides, dust, glycosylated proteins, hemoglobin, lactoferrin, lysozymes, nucleases, proteinases, polysaccharides, smoke, or other reaction-interfering molecules.

18. The device of any of claims 15-17, wherein the solid phase material comprises a surface functionalized with nucleic acids, peptides, or antibodies configured to selectively bind inhibitory substances.

19. The device of any of claims 15-18, wherein the solid phase material is disposed upstream of a filter having a pore size between 0.1 and 50 microns, such that the sample is filtered after inhibitor removal.

20. The device of any of claims 15-19, wherein the solid phase material is located in a swabacceptance tube, buffer reservoir, or channel in fluid communication with the one or more detection chambers.

21. The device of any of claims 15-20, wherein the solid phase material is incorporated into a removable cartridge or insert for replacement or customization of inhibitor-removal properties.Levine Bagade Han LLP 32 GTSCZOIOOOWO

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