Systems and Methods for Improved Detection and Quantization of Analytes

Abstract

To provide a suitable system and method for enhanced detection and quantification of an analyte.SOLUTION: A device, a system, and a method for detecting molecules of interest within a collected sample are described in the present specification. In a certain embodiment, self-contained sample analysis system is disclosed, which includes a reusable reader component, a disposable cartridge component, and a disposable sample collection component. The reader component may communicate with a remote computing device for the digital transmission of a test protocol and test results. According to various disclosed embodiments, the system, components, and method are configured so as to identify the presence, absence, and / or quantity of particular nucleic acids, proteins, or other analytes of interest, for example, in order to test for the presence of one or more pathogens or contaminants in a sample.SELECTED DRAWING: Figure 10A

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims priority to U.S. Provisional Application No. 62 / 194,101, filed on Jul. 17, 2015, the entire contents of which are incorporated herein by reference.

[0002] This technology generally relates to the field of molecular detection. Specifically, this technology relates to microfluidic devices, systems, and methods for detecting the presence, absence, and / or amount of one or more specific analytes within a collected sample.

Background Art

[0003] Conventional techniques for identifying the presence, absence, and / or amount of nucleic acids, proteins, and / or other molecules of interest within a sample often require expensive laboratory equipment and the expertise of highly trained medical professionals. As a result, such analyses are typically performed within a laboratory or medical facility. Such molecular detection can be important, for example, for detecting the presence of pathogens, diseases, contaminants, over - ingestion, and poisoning within an individual or other animal, or within the environment. Unfortunately, currently, an individual may face long waiting times before being able to perform an appropriate test and before being able to generate and analyze the results. Due to the long waiting times and the inconvenience of traveling to a laboratory or medical facility, diseases and contaminants can often spread and cause significant harm even before the presence of the disease or contaminant is identified.

Summary of the Invention

Means for Solving the Problems

[0004] There is a significant need for improved molecular detection and quantification techniques. Described herein are devices that can detect a molecule of interest in less time and with less technical expertise than currently used conventional devices. The devices may be utilized by consumers in non-clinical settings, such as schools, workplaces, and homes. Additionally, the devices can be used by consumers visiting a pharmacy or medical facility and can generate results quickly so that the results are available by the time the consumer has spoken with a pharmacist or healthcare provider. The devices herein may also be configured to minimize biosafety risks.

[0005] One aspect of the present disclosure is directed to a system for detecting a molecule. In various embodiments, the system includes a cartridge device, a reader device removably coupled to the cartridge device, and a sample collection device.

[0006] Sample preparation reagents may be used within the system and may include a plurality of magnetic particles each having a surface-binding affinity molecule, a plurality of detection agents each of which may include a signaling substance, a plurality of amplification reagents, and / or a plurality of agents for facilitating access to the target analyte and binding between the target analyte, the surface-binding affinity molecule, and the detection agent.

[0007] According to one aspect, a sample analysis cartridge is provided for detecting at least one of the presence, absence, or amount of one or more analytes. The sample analysis cartridge may include an input tunnel, a reservoir, a reagent shuttle, and / or a sensor, each of which may be within the housing of the cartridge. The input tunnel may extend from an opening and may be configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to the sample. The reservoir may be configured to hold a fluid that may or may not be pre-filled within the reservoir. The reagent shuttle may be disposed between the reservoir and the opening in a first position. The reagent shuttle may have a first end and a second end. The reagent shuttle may be configured to store a reagent ball (e.g., a sample preparation reagent) containing a reagent between the first end and the second end. The first end may be configured to seal the reservoir from the input tunnel in the first position. The reagent shuttle may be designed to move within the input tunnel to a second position when a force exceeding a threshold force is received, such that the reagent ball and the sample are designed to move into the reservoir. The reservoir may be continuously sealed from the proximal input tunnel of the reagent shuttle during movement from the first position to the second position. The sensor may be configured to analyze a fluid mixed with the reagent ball and the sample and may be further configured to generate a signal indicative of at least one of the presence, absence, or amount of one or more analytes in the sample.

[0008] The second end of the shuttle has an opening sized to wipe excess sample from the tip of the sample collection device, such that a predetermined volume of the sample, at most, may be mixed into the fluid within the reservoir. The reservoir may be sealed via a sample collection device that is partially inserted into the second end of the shuttle during movement from the first position to the second position. The cartridge may have one or more locking members configured to irreversibly lock the sample collection device within the input tunnel in the second position.

[0009] The shuttle may include one or more sample compartments configured to store a predetermined volume of a sample, and one or more reagent ball compartments configured to store reagent balls and optionally additional reagent balls. In some embodiments, one or more sample compartments and one or more reagent ball compartments are not exposed to the fluid in the reservoir in the first position. One or more sample compartments and one or more reagent ball compartments may be exposed to the fluid in the reservoir in the second position. The shuttle may have a compartment divider configured to separate at least one sample compartment from at least one reagent ball compartment. The compartment divider may have slots configured to facilitate mixing when disposed within the sample preparation reservoir in the second position.

[0010] The reagent may include one or more of a plurality of solid particles, a plurality of affinity molecules, and / or a plurality of signaling substances. The reagent may include a plurality of magnetic particles configured to be magnetically held across the working electrode of the sensor. At least one magnetic particle of the plurality of magnetic particles may be configured to be indirectly coupled to a signaling substance.

[0011] The cartridge may include, for example, an analysis channel within the cartridge housing. At least a portion of the sensor may be disposed within the analysis channel, and the fluid mixed with the reagent balls and the sample may travel through the analysis channel to at least a portion of the sensor.

[0012] The cartridge may include a contact switch, a seal material configured to fluidly seal the fluid within the reservoir, and / or a seal piercer, and each of these components may be within the cartridge housing. Insertion of the sample collection device into the input tunnel may (i) move the shuttle from a first position to a second position, (ii) cause the seal piercer to pierce the seal material and vent the fluid within the reservoir, and / or (iii) cause activation of the contact switch.

[0013] According to another aspect, a sample analysis cartridge is provided for detecting at least one of the presence, absence, or amount of one or more analytes. The sample analysis cartridge may include an input tunnel, a reservoir, a seal material, and / or a seal piercer, each of which may be within the housing of the cartridge. The input tunnel may extend from an opening and may be configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to the sample. The reservoir may be configured to hold a fluid that may or may not be pre-filled. The seal material may be configured to fluidly seal the fluid within the reservoir. The seal piercer may be partially disposed within the input tunnel and may be configured to be contacted by a sample collection device within the input tunnel and to move in response to a force applied by the sample collection device to pierce the seal material and vent the fluid within the reservoir.

[0014] The seal piercer may be configured to move in a first direction and a second direction different from the first direction to pierce the seal material. The first direction may be substantially parallel to the movement of the sample collection device within the input tunnel, and the second direction may be substantially perpendicular to the first direction. The seal piercer may include one or more piercers. The seal piercer may include a slider configured to move in the first direction, and one or more piercers may be configured to move in a second direction different from the first direction to pierce the seal material.

[0015] The reservoir may be a sample preparation reservoir and may be configured to hold sample preparation reagents that may be in the fluid and / or may be introduced, for example, via the introduction of reagent balls. The cartridge may also include a cleaning agent reservoir and / or a substrate reservoir. The seal piercer may be configured to pierce the seal material and vent to the individual fluids within the sample preparation reservoir, the cleaning agent reservoir, and the substrate reservoir. The seal piercer may include an engaging device disposed within the input tunnel, and the engaging device may be configured to engage the engagement zone of the sample collection device when the sample collection device is within the input tunnel.

[0016] The cartridge may include a contact switch that may be disposed on a circuit board within the housing. The contact switch may be configured to be activated in response to the insertion of the sample collection device into the input tunnel. The movement of the seal piercer may cause the activation of the contact switch. The seal piercer may be configured to sequentially pierce the seal material across the sample collection reservoir, the cleaning agent reservoir, and the substrate reservoir in any order. The seal piercer may be configured to move from the one or more holes pierced into the seal material after piercing and venting to the fluid within the reservoir.

[0017] The cartridge may include a contact switch and a shuttle disposed between the reservoir and the aperture in the first position. The shuttle may have a first end and a second end, and the first end may be configured to seal the reservoir from the input tunnel in the first position. The reagent shuttle may be configured to move within the input tunnel to the second position, where the sample is moved into the reservoir. Insertion of the sample collection device into the input tunnel may (i) move the shuttle from the first position to the second position, (ii) cause the seal piercer to pierce the seal material and vent the fluid in the reservoir, and / or (iii) cause activation of the contact switch. The cartridge may include one or more locking members configured to irreversibly lock the sample collection device within the input tunnel in the second position. The one or more locking members may irreversibly lock the sample collection device within the input tunnel during partial and / or full insertion of the sample collection device into the input tunnel.

[0018] Insertion of the sample collection device into the input tunnel may cause the seal piercer to pierce the seal material and vent the fluid in the reservoir before the shuttle moves from the first position to the second position. Alternatively, or in addition, insertion of the sample collection device into the input tunnel may cause the seal piercer to pierce the seal material and vent the fluid in the reservoir during movement of the shuttle from the first position to the second position.

[0019] According to yet another aspect, a sample analysis cartridge is provided for detecting at least one of the presence, absence, or amount of one or more analytes. The sample analysis cartridge may include an input tunnel, a reservoir, an analysis channel, and / or a circuit board, each of which may be within the housing of the cartridge. The input tunnel may extend from an opening and may be configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to the sample. The reservoir may be configured to hold fluid and may be configured to receive a sample on the distal portion of the sample collection device. The analysis channel may be configured to receive from the reservoir a fluid having a sample and a reagent that includes a plurality of magnetic particles mixed therein. The circuit board may include a sensor having a working electrode, the sensor being exposed to the fluid mixed in the analysis channel and configured to generate a signal indicative of at least one of the presence, absence, or amount of one or more analytes in the sample. The working electrode may be masked with a plurality of fine grooves configured to facilitate a homogeneous distribution of the plurality of magnetic particles across the working electrode and to facilitate resistance to movement of the plurality of magnetic particles away from the working electrode.

[0020] According to yet another aspect, a kit is provided for detecting at least one of the presence, absence, or amount of one or more analytes. The kit may include a sample collection device, a sample analysis cartridge, and / or a sample analysis reader. The sample collection device may have a distal portion adapted to be exposed to the sample. The sample analysis cartridge may include an input tunnel, a reservoir, an analysis channel, and / or a circuit board, each within the cartridge housing. The input tunnel may extend from an opening and may be configured to allow insertion of the sample collection device. The reservoir may be configured to hold fluid and may be configured to receive the sample on the distal portion of the sample collection device. The analysis channel may be configured to receive from the reservoir a fluid having the sample and reagents with a plurality of magnetic particles mixed therein. The circuit board may include a sensor that is exposed to the fluid mixed in the analysis channel and is configured to generate a signal indicative of at least one of the presence, absence, or amount of one or more analytes in the sample. The sample analysis reader may be configured to receive the sample analysis cartridge. The sample analysis reader may have first and second magnetic generators configured to be disposed adjacent to a single working electrode of the sensor when the sample analysis cartridge is inserted into the sample analysis reader. The first and second magnetic generators may be configured to generate a magnetic field across the length of the single working electrode and to facilitate a homogeneous distribution of the plurality of magnetic particles across the length of the single working electrode.

[0021] The reception of a sample analysis cartridge by a sample analysis reader may cause an electrical coupling between the sample analysis cartridge and the sample analysis reader. The sample analysis cartridge may be configured to transmit to the sample analysis reader a signal indicating at least one of the presence, absence, or amount of one or more analytes in the sample for processing. The sample analysis reader may be configured to transmit to a computer a processed signal indicating at least one of the presence, absence, or amount of one or more analytes in the sample. The kit may include a computer-readable medium with instructions that, when executed by a processor of the computer, cause the computer display to display information indicating the presence, absence, and / or amount of one or more target analytes.

[0022] The housing of the sample analysis cartridge may include a bottom surface having a magnetic generator recess. The reception of the sample analysis cartridge into the sample analysis reader may cause the first and second magnetic generators to move partially within the magnetic generator recess. The magnetic generator recess may be disposed adjacent to a single working electrode.

[0023] According to another aspect, a sample analysis cartridge is provided for detecting at least one of the presence, absence, or amount of one or more analytes. The sample analysis cartridge may include an input tunnel, a reservoir, a heater, an analysis channel, and / or a sensor, each of which may be within the housing of the cartridge. The input tunnel may extend from an opening and may be configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to the sample. The reservoir may be configured to hold fluid and may be configured to receive the sample on the distal portion of the sample collection device. The reservoir may include an outlet having a phase change material therein for closing the entire cross-section thereof. The heater may be configured to heat the phase change material such that the phase change material does not close the entire cross-section of the outlet. The analysis channel may be configured to receive a fluid having a sample and a reagent, including a plurality of magnetic particles mixed therein, from the reservoir through the outlet. The sensor may be exposed to the fluid mixed within the analysis channel and may be configured to generate a signal indicative of at least one of the presence, absence, or amount of one or more analytes in the sample. The heater may be masked with a masking material configured to electrically insulate the heater from the sensor. The masking material may be a solder mask.

[0024] The cartridge may include a detergent reservoir and a detergent reservoir heater. The detergent reservoir may be configured to hold a cleaning fluid and may include a detergent reservoir outlet having a phase change material therein for closing the entire cross-section thereof. The detergent reservoir heater may be configured to heat the phase change material within the detergent reservoir outlet such that the phase change material does not close the entire cross-section of the detergent reservoir outlet, and thus the cleaning fluid may be configured to enter the analysis channel and proceed to the sensor. The detergent reservoir heater may be masked with a masking material configured to electrically insulate the detergent reservoir heater from the sensor.

[0025] The cartridge may include a substrate reservoir and a substrate reservoir heater. The substrate reservoir may be configured to hold a substrate fluid and may include a substrate reservoir outlet having a phase change material for closing the entire cross-section of the substrate reservoir outlet therein. The substrate reservoir heater is configured to heat the phase change material within the substrate reservoir outlet such that the phase change material does not close the entire cross-section of the substrate reservoir outlet, and thus the substrate fluid may be configured to enter the analysis channel and proceed to the sensor. The substrate reservoir heater may be masked with a masking material configured to electrically insulate the substrate reservoir heater from the sensor.

[0026] According to yet another aspect, a sample analysis cartridge is provided for detecting at least one of the presence, absence, or amount of one or more analytes. The sample analysis cartridge may include a sample preparation reservoir, a substrate reservoir, an analysis channel, a fluid breaker, and / or one or more heaters, each of which may be within the housing of the cartridge. The sample preparation reservoir may be configured to hold fluid and may be configured to receive a sample from a sample collection device. The sample preparation reservoir may include a sample preparation reservoir outlet having a phase change material therein for sealing the sample preparation reservoir outlet. The substrate reservoir may be configured to hold a fluid comprising a chemical substrate. The substrate reservoir may include a substrate reservoir outlet having a phase change material therein for sealing the substrate reservoir outlet. The sample preparation and substrate reservoirs may each be in fluid communication with the analysis channel at least as needed. The fluid breaker may be a phase change material. At least one of the one or more heaters is configured to heat the phase change material within the sample preparation reservoir outlet, such that the phase change material unseals the sample preparation reservoir outlet and enables fluid to mix the sample therein and flow into the analysis channel. At least one of the one or more heaters is configured to heat the phase change material of the fluid breaker after unsealing the sample preparation reservoir outlet, such that the phase change material of the fluid breaker is configured to flow into the analysis channel and fluidly isolate the sample preparation reservoir from the substrate reservoir. At least one of the one or more heaters is configured to heat the phase change material within the substrate reservoir outlet after the fluid breaker has fluidly isolated the sample preparation reservoir from the substrate reservoir, such that the phase change material unseals the substrate reservoir outlet and enables the fluid comprising the chemical substrate to flow into the analysis channel but not into the sample preparation reservoir.

[0027] One or more heaters may include a sample preparation reservoir heater, a fluid breaker heater, and / or a substrate reservoir heater. The sample preparation reservoir heater may be configured to heat a phase change material within the sample preparation reservoir outlet. The fluid breaker heater of the fluid breaker may be configured to heat a phase change material. The substrate reservoir heater may be configured to heat a phase change material within the substrate reservoir outlet. The sample preparation reservoir heater, the fluid breaker heater, and the substrate reservoir heater may each be masked with a masking material configured to electrically insulate the individual heater from a sensor within the analysis channel.

[0028] The cartridge may include a detergent reservoir configured to hold a cleaning fluid. The detergent reservoir may include a detergent reservoir outlet having a phase change material therein to seal the detergent reservoir outlet. At least one of the one or more heaters may heat a phase change material within the detergent reservoir outlet after the fluid breaker fluidically isolates the sample preparation reservoir from the substrate reservoir, but before the one or more heaters heat a phase change material within the substrate reservoir outlet, such that the phase change material unseals the detergent reservoir outlet and enables the cleaning fluid to flow into the analysis channel and wash away signal transduction materials not bound to magnetic particles from the sample preparation reservoir from a sensor within the analysis channel. A fluid having a chemical substrate may be configured to wash away signal transduction materials not bound to magnetic particles from the sample preparation reservoir from a sensor within the analysis channel.

[0029] According to another aspect, a sample analysis cartridge is provided for detecting at least one of the presence, absence, or amount of one or more analytes. The sample analysis cartridge may include an input tunnel, a reservoir, a shuttle, and / or a sensor, each of which may be within the housing of the cartridge. The input tunnel may extend from an opening and may be configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to the sample fluid. The reservoir may be configured to hold a fluid that may or may not be pre-filled. The shuttle may be disposed between the reservoir and the opening in a first position. The shuttle may have a first end and a second end and may define a sample compartment between the first end and the second end. The sample compartment may be configured to receive sample fluid compressed from the distal portion of the sample collection device. The shuttle may be configured to move within the input tunnel to a second position when subjected to a force exceeding a threshold force, such that the sample compartment having the sample fluid is configured to be moved into the reservoir. The sensor may be configured to be exposed to a fluid mixed with the sample fluid and may further be configured to generate a signal indicative of at least one of the presence, absence, or amount of one or more analytes in the sample fluid.

[0030] The shuttle may further define a reagent ball compartment between the first end and the second end. The reagent ball compartment may be configured to store one or more reagent balls comprising a reagent. The reagent ball compartment may be outside the reservoir in the first position and may be within the reservoir in the second position. The sample compartment may be configured to receive a predetermined volume of sample fluid compressed from the distal portion of the sample collection device, at most. The cartridge may include an overflow compartment configured to receive a volume of sample fluid exceeding the predetermined volume from the sample compartment.

[0031] The reagent ball is provided with the reagents necessary for amplifying the target sample. The reagent ball can be of any suitable size and shape, and non-limiting examples thereof include a diameter of about 1 mm to about 7 mm, or alternatively, about 2 mm to about 5 mm, or alternatively, about 3 mm, or alternatively, less than about 7 mm, or alternatively, less than about 5 mm, or alternatively, less than about 4 mm. The reagent ball can be of any suitable shape, such as spherical, cylindrical, conical, or elliptical.

[0032] The components of the reagent ball are pre-selected for the sample as well as for the method of amplification and subsequent detection and / or quantification thereof. In one aspect, the components of the reagent ball are provided with reagents for the detection or quantification of hormones, other small molecules, or proteins or fragments. In another aspect, the components of the reagent ball are provided with reagents for the detection and / or quantification of nucleic acids by a method including the step of amplifying the nucleic acids.

[0033] The kit may be provided to include a sample analysis cartridge and a sample collection device. The sample collection device may include a suction portion at the distal portion. The suction portion may be configured to suck up and absorb the sample fluid. The suction portion may be compressed to discharge the sample fluid into the sample compartment. The sample compartment may be configured to receive a predetermined volume of the sample fluid compressed from the distal portion of the sample collection device at most. The sample analysis cartridge may further include an overflow compartment configured to receive a volume of the sample fluid exceeding the predetermined volume from the sample compartment. The suction portion of the sample collection device may be configured to suck up and absorb a sample fluid exceeding the predetermined volume, enabling the user to measure the amount of the sample fluid compressed into the sample compartment and the overflow compartment. At least a part of the suction portion may be slidably disposed within the shroud of the sample collection device.

[0034] The sample analysis cartridge may further include one or more locking members configured to irreversibly lock the sample collection device within the input tunnel when the sample collection device is fully inserted into the input tunnel. The sample collection device may further include a sample collection indicator configured to visually alert the collector based on the volume of the collected sample fluid. The sample collection indicator may be a colored thread embedded within the suction portion that becomes increasingly visually exposed as the volume of the collected sample increases.

[0035] According to another aspect, a composition and method are provided for detecting and / or quantifying at least one of the presence, absence, or amount of a target analyte in a sample within a cartridge. The method includes mixing a sample having in a fluid within a reservoir of the cartridge a plurality of affinity molecules, a plurality of release agents, a plurality of signaling agents, a plurality of competitor molecules pre-bound to competitor binding molecules, each having a label, and a plurality of sample target analytes pre-bound to sample binding molecules; using at least one release agent of the plurality of release agents to release at least one competitor molecule from the competitor binding molecule to which it is pre-bound; using at least one release agent of the plurality of release agents to release at least one sample target analyte from the sample binding molecule to which it is pre-bound; binding the label of the released competitor molecule to a signaling agent of the plurality of signaling agents; binding the released competitor molecule to an affinity molecule of the plurality of affinity molecules; and / or generating a signal indicative of at least one of the presence, absence, or amount of a sample target analyte within the cartridge. The reagent balls can be of many sizes, non-limiting examples of which include having a diameter of from about 1 mm to about 7 mm, or alternatively from about 2 mm to about 5 mm, or alternatively about 3 mm, or alternatively less than about 7 mm, or alternatively less than about 5 mm, or alternatively less than about 4 mm. The reagent balls are illustrated as spherical, but the present disclosure is not limited thereto, and many shapes may be used, and a plurality of reagent balls, each containing the same or different reagents, may also be used.

[0036] According to another aspect, the compositions and methods are provided for amplifying, detecting, and / or quantifying at least one of the presence, absence, or amount of a target analyte, such as a target nucleic acid (deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)), in a sample within a cartridge. The method includes, or alternatively consists essentially of, preparing a plurality of amplicons with a plurality of capture elements by mixing in a fluid within a reservoir of the cartridge a plurality of enzymes that facilitate an amplification reaction, such as a polymerase, a reverse transcriptase, a plurality of magnetic beads having an affinity molecule bound thereto, and a plurality of forward and reverse primers that may or may not be labeled.

[0037] In a further aspect, provided herein are a composition and a method of using the same, wherein the reagent balls further contain a plurality of forward primers selected to amplify a target nucleic acid and having a spacer element and a capture element respectively bound thereto, a plurality of reverse primers selected to amplify a target nucleic acid and having a spacer element and a signal transducer or signal transduction capture element respectively bound thereto, and a plurality of nucleotides or analogs thereof (dNTPs) for the amplification reaction.

[0038] In a further aspect, the reagent balls further contain a reverse primer selected to bind to a target nucleic acid, the reverse primer comprising a spacer element directly or indirectly conjugated to a reporter capture element. In a further aspect, the reagent balls further contain an effective amount of reverse transcriptase effective to facilitate the amplification reaction.

[0039] In yet a further aspect, the reagent balls also contain a plurality of reporter affinity elements conjugated to a reporter element.

[0040] In a further aspect, the reagent balls also contain a single-stranded binding protein known to those skilled in the art, such as, but not limited to, SSB from E. coli or GP32 from a phage, containing, for example, from about 9 to about 18 amino acids.

[0041] On one side, the reagent ball further contains a plurality of DNA template control nucleic acids. On another side, the reagent ball alternatively or additionally contains a plurality of RNA template control nucleic acids.

[0042] In a further aspect, the reagent ball contains a plurality of reverse transcriptase-specific primers and can facilitate reverse transcription from RNA to cDNA. In yet a further aspect, the reagent ball also contains a plurality of reverse primers selected to serve as internal controls having a spacer element and a signaling substance respectively bound thereto, and a plurality of forward primers selected to serve as internal controls having a spacer element and a capture element respectively bound thereto.

[0043] In addition to the labeled primer, the reagent ball can contain an effective amount of a plurality of unlabeled primers designed to amplify at least the same target region and optionally adjacent sequences of the target sequence. The presence of the unlabeled primers can make the amplification more efficient because the labeled primer can be more sterically hindered as it binds to other elements, such as solid particles or signaling substances.

[0044] The reagent ball further contains an effective amount of one or more solubilizing agents and can release the target nucleic acid, template, and / or control from cells, microorganisms, or viruses in the sample.

[0045] In a further aspect, the reagent ball contains an effective amount of helicase to unwind dsDNA for loading primers or its analogs such as RecA or UvsX or RAD51. Additionally, the reagent ball may contain mutL, RecFOR enzymes, UvsY.

[0046] On one side, the amplification reagent is selected for any one or more other amplification reactions, such as PCR or isothermal amplification. The reporter element and / or capture element is located along the 5'-end of the nucleic acid or within the nucleic acid sequence, i.e., internal to the 5'-end. The reporter and / or capture element is covalently or non-covalently attached to the nucleic acid.

[0047] On one side, the element is provided within the sample reagent ball and mixed with the sample in the reservoir. In response to decomposition, the reagent contacts the target nucleic acid, and a series of enzyme-driven melting and reconstitution of the hybridization of the primer to the target nucleic acid and of the DNA or, in the case of target RNA, complementary DNA (cDNA) molecules results in the amplification of the target, initially generating an RNA target nucleic acid containing the target sequence and double-stranded cDNA, which then serves as a template for further amplification. The reagent ball can be of many sizes, non-limiting examples of which include having a diameter of about 1 mm to about 7 mm, or alternatively, about 2 mm to about 5 mm, or alternatively, about 3 mm, or alternatively, less than about 7 mm, or alternatively, less than about 5 mm, or alternatively, less than about 4 mm. The reagent ball is illustrated as spherical, but the present disclosure is not limited thereto, and many shapes may be used, and multiple reagent balls, each containing the same or different reagents, may also be used. The spacer element that separates the primer from the label comprises a polymer, such as hexaethylene glycol or triethylene glycol. Alternatively, it can be a linear carbon polymer containing about 1 to about 18 carbon atoms or more, such as hexane, pentane.

[0048] As will be apparent to those skilled in the art, combinations of the foregoing embodiments necessary to facilitate specific amplification of the target nucleic acid are also intended to be within the scope of the present disclosure.

[0049] The reagents and their amounts are pre-selected to facilitate specific amplification of the target nucleic acid. For illustrative purposes, non-limiting examples of reverse transcriptases are Moloney murine leukemia virus (MMLV) or its derivatives or avian myeloblastosis virus (AMV) or its derivatives. Preferred reverse transcriptases are target-dependent and may not be identical between pills for different targets, and as can be understood, the reagent balls are modified for different test applications by the primer sequence and capture element, primer concentration, particle concentration, affinity agent, solubilizer, polymerase, reverse transcriptase, and other enzymes (e.g., HIV quantification vs. influenza detection may have different reaction conditions) that best match the optimal conditions for each type of target.

[0050] The polymerase can include several different types within the strand displacement polymerase category, and optionally, for example, fragments thereof such as BsuDNA polymerase or the large fragment of BsuDNA polymerase, BstDNA polymerase or the large fragment of DNA polymerase, phi29DNA polymerase. It should be understood that for polymerases and reverse transcriptases, in many cases, it is a desired property to have a variant such as a polymerase lacking exonuclease activity or, for reverse transcriptases, a polymerase lacking RNase H activity.

[0051] The preferred reaction temperature for isothermal reactions can depend on the method and reaction conditions and can often include about 65 degrees Celsius as applicable to LAMP and 55 degrees Celsius as applicable to nicking enzyme amplification reactions. Preferably, but not limited to, the reaction temperature range is about 37 - 42 degrees Celsius for the reverse transcriptase portion of the amplification reaction when the target nucleic acid is RNA. Helicase-dependent amplification, strand displacement amplification, recombinase polymerase amplification can all occur at about 37 degrees Celsius or between 37 degrees Celsius and 42 degrees Celsius. Figure 20 shows the temperature profile of an isothermal reaction occurring in a reservoir at about 40 degrees Celsius.

[0052] To facilitate some isothermal amplification techniques, it may be desirable to include single-stranded binding proteins (SSBs), which help to stabilize the unwinding of complementary strands and strand displacement polymerization during amplification. Examples include, but are not limited to, RB49 GP32, RB69 GP32, T4 GP32, the SSB protein of Escherichia coli, and others.

[0053] In some aspects, the reagent kit and method further use a helicase to unwind dsDNA to load primers. Non-limiting examples include enzymes such as uvrD helicase from Escherichia coli, T4 Gene41 helicase, and many others. Recombinases that facilitate primer loading into dsDNA for enzymatic melting of double-stranded DNA for primer annealing include RecA from Escherichia coli, RAD51 human recombinase, DMC1 human meiotic recombinase, or T4 UvsX, RB49 UvsX, RB69 UvsX, and analogs from many other phages can be included. As is known to those skilled in the art, the combination of helicase and SSB is useful for facilitating isothermal amplification. Accessory factors such as MutL can be added to facilitate helicase-dependent amplification. The combination of recombinase and SSB can be useful in RPA, and sometimes accessory factors such as RecFOR from Escherichia coli and / or UvsY from various phages are also employed to facilitate the reaction by assisting the primary enzymes (RecA) or (uvrD) in recombinase polymerase amplification and helicase-dependent amplification, respectively. As will be understood by those skilled in the art, the reagent kit and / or reservoir can further contain any one or more of the aforementioned reagents, as needed, to facilitate the specific amplification of the target nucleic acid.

[0054] Primer concentrations for isothermal amplification reactions such as SDA, HDA, and RPA can be from 0.01 to 10 micromoles, preferably closer to 0.5 micromoles. The LAMP primer mixture can be prepared using all 4 or 6 (with loops) primers. The 10x primer mixture can contain 16 μM FIP, 16 μM BIP, 2 μM F3, 2 μM BE, 4 μM LoopF, 4 μM LoopB. dNTPs can be provided at a concentration of 1 micromole to 500 micromoles, preferably about 200 micromoles. SDA, HDA, and RPA may require a large amount of ATP because some of the enzymes that enable enzyme melting and primer loading into dsDNA require ATP functionalization, and thus, as much as 100 micromoles to 4 millimoles of ATP can be used in the reaction.

[0055] The amount of polymerase can vary depending on the target but can be in the range of 1 unit to 1000 units per reaction. Reverse transcriptase can also be provided in such a range for normal reactions. Magnesium is an essential cofactor for polymerase activity and can be provided in the reagent ball or reservoir in an amount well known in the art, such as 5 to 50 millimoles, typically about 10 millimoles.

[0056] Methods and compositions for non-covalent binding of molecules that also provide a label or signal for detection are known in the art. Non-limiting examples thereof include avidin or streptavidin-biotin conjugates. Modifications of biotin are known in the art and are intended to be within the scope of the present disclosure. Non-limiting examples thereof include biotin dT, biotin-TEG, dual biotin, PC biotin, and desthiobiotin-TEG commercially available from Integrated DNA Technologies (see idtdna.com / pages / decoded / decoded-articles / core-concepts / decoded / 2012 / 09 / 20 / which-biotin-modification-to-use-(last accessed July 16, 2016)). The present disclosure also includes the use of additional conjugation chemistries for the binding of nucleic acids and proteins, such as when a primer is directly conjugated to a signaling agent, and in one aspect, the signaling agent is an enzyme such as HRP. Non-limiting examples of covalent binding of a protein or polypeptide to another moiety include the binding of the moiety to a crosslinking reactive group, such as carbodiimide, imidoester, and maleimide. See, for example, Bioconjugate Techniques, 3rd Ed, Hermanson, G.T. (2013).

[0057] As will be apparent to those skilled in the art, the components of the reagent ball are selected to facilitate the amplification of the target nucleic acid and / or target and / or control by an appropriate method. In one aspect, the reagent is selected for rolling circle amplification (RCA) or loop-mediated isothermal amplification. In another aspect, it is selected for amplification by the loop-mediated isothermal amplification (LAMP) method. In another aspect, it is selected for strand displacement amplification (SDA). In another aspect, it is selected for recombinase polymerase amplification (RPA). In another aspect, it is selected for helicase-dependent amplification (HDA). In another aspect, it is selected for polymerase spiral reaction (PSR). In another aspect, it is selected for nicking enzyme amplification reaction (NEAR). As described above, each specific reaction type enables the efficient and selective amplification of the target and / or multiple targets and / or internal control nucleic acids and has its own preferred combination of enzymes and components known to those skilled in the art.

[0058] Among the plurality of affinity molecules, each affinity molecule may be bound to a solid particle. The solid particle may be formed from a magnetic-responsive material and / or may be formed from a non-magnetic-responsive material. The non-magnetic-responsive material may be gold nanoparticles.

[0059] The plurality of sample target analytes pre-bound to the sample binding molecule may include 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 molecules pre-bound to vitamin D binding protein molecules. The plurality of competitor molecules pre-bound to the sample binding molecule may be labeled with biotin and may include 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 molecules pre-bound to vitamin D binding protein molecules. At least one of the plurality of affinity molecules, the plurality of dissociating agents, the plurality of signal transmitters, and the plurality of competitor molecules pre-bound to the competitive binding molecule may be stored within the reagent ball.

[0060] As described above, primers (forward and reverse for the target nucleic acid and control template), if present, are designed based on the nucleotide sequence of the target nucleic acid to be amplified and detected. Methods for designing optimal primers based on the target sequence are known in the art (see, for example, simgene.com / Primer3; quill.com, molbiol-tools.caPCR, and ncbi.nlm.nih.gov / tools / primer-blast / (each accessed most recently on July 15, 2016)), and vary with the amplification method utilized, such as rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), recombinase polymerase amplification (RPA), helicase-dependent amplification (HDA), polymerase spiral reaction (PSR), and nicking enzyme amplification reaction (NEAR).

[0061] According to yet another aspect, a sample analysis cartridge is provided for detecting at least one of the presence, absence, or amount of one or more analytes. The sample analysis cartridge may include reagent balls, reservoirs, and / or sensors, each of which may be within the housing of the cartridge. The reagent balls may include a plurality of competing molecules pre-bound to competing binding molecules, and each of the plurality of competing molecules may carry a label or be bound to a signaling substance. In another aspect, the reagent balls comprise or alternatively consist essentially of the reagents necessary for amplification and detection of a target nucleic acid. The reservoir may be configured to hold a reservoir fluid that may or may not be pre-filled within the reservoir. The reservoir is configured to allow mixing of the fluid within the reservoir, a plurality of affinity molecules, a plurality of releasing agents, a plurality of signaling substances, a plurality of competing molecules pre-bound to competing binding molecules, and a sample from a sample collection device having a plurality of sample target analytes pre-bound to sample binding molecules. Some of the plurality of releasing agents may be configured to release the competing molecules from the competing binding molecules pre-bound thereto or the sample target analytes from the sample binding molecules pre-bound thereto. The label of the released competing molecules may be configured to bind to a signaling substance, and the released competing molecules may be configured to bind to some of the plurality of affinity molecules. In another aspect, the reservoir is configured to hold a reservoir fluid that may or may not be pre-filled within the reservoir.The reservoir is configured to allow mixing of a fluid within the reservoir, a plurality of enzymes for promoting an amplification reaction, such as polymerase, reverse transcriptase, a plurality of magnetic beads having an affinity molecule bound thereto, a plurality of forward primers selected to amplify a target nucleic acid and each having a spacer element bound thereto, a plurality of reverse primers selected to amplify a target nucleic acid and each having a spacer element and a signal transducer or signal transduction capture element bound thereto, a plurality of nucleotides or analogs thereof (dNTP) for the amplification reaction, a plurality of DNA template control nucleic acids, a plurality of reverse primers selected to serve as an internal control and each having a spacer element and a signal transducer bound thereto, and a plurality of forward primers selected to serve as an internal control and each having a spacer element and a capture element bound thereto. In one aspect, the amplification reagent is selected for any one or more other PCR methods or isothermal amplification methods.

[0062] In a further aspect, the reagent ball and / or reservoir contains an effective amount of a lysing agent that lyses a sample comprising cells, such as a bacterial sample, and releases intracellular DNA, RNA, and / or protein that serves as an analyte. Non-limiting examples of lysing agents include NP-40, CHAPS, deoxycholic acid, Triton X-100, NP40, and Tween 20.

[0063] In a further aspect, the reagent ball and / or reservoir contains an RNase inhibitor and / or DNase inhibitor and / or protease inhibitor in an amount that inhibits native or endogenous RNase, DNase, or protease activity in a sample added for analysis.

[0064] The spacer element may be advantageous in cases where the nucleic acid portion of the primer is further away from the label, where other steric hindrance events can occur or have already occurred, such as the primer being bound to the particle or to the signaling substance (both of which can be difficult to handle), and in some cases, because the primer can participate better in the amplification reaction. Spacer elements that separate the primer from the label include polymers such as hexaethylene glycol, triethylene glycol, C3 spacer phosphoramidite, PC spacer, hexanediol, and are commercially available from Integrated DNA Technologies (see idtdna.com / site / Catalog / Modifications / Category / 6 (last accessed July 16, 2016)).

[0065] The sensor may be configured to be exposed to the mixed reservoir fluid, and the sensor may further be configured to generate a signal indicating at least one of the presence, absence, or amount of the sample target analyte in the sample. For example, particles from the mixed reservoir fluid comprising the signaling substance may be localized within the analysis channel across the sensor, and the localized signaling substance may react with the substrate from the substrate solution to generate an electrical signal sensed by the sensor. The sensor may use the electrical signal from the reaction to transmit a signal indicating at least one of the presence, absence, or amount of the sample target analyte in the sample.

[0066] The reservoir may further be configured to allow mixing of a plurality of solid particles into the reservoir fluid. Each solid particle may be pre-bound to a certain affinity molecule among the plurality of affinity molecules. The plurality of solid particles may be formed from a magnetic responsive material and / or from a non-magnetic responsive material. The non-magnetic responsive material may be gold nanoparticles. The plurality of sample target analytes pre-bound to the sample binding molecule may include 25-hydroxyvitamin D3 and / or 25-hydroxyvitamin D2 molecules pre-bound to the binding protein molecule. The magnetic responsive material may be magnetically retained across the sensor.

[0067] According to yet another aspect, a sample analysis cartridge is provided. The sample analysis cartridge may include an input tunnel, a reservoir, a shuttle, and / or a collet, each of which may be within the housing of the cartridge. The input tunnel may extend from an opening and be configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to a sample. The reservoir may be configured to hold fluid. The shuttle may be disposed within the input tunnel between the reservoir and the opening in a first position. The collet may be disposed within the input tunnel and coupled to the shuttle in the first position. The collet may be detached from the shuttle during insertion of the sample collection device into the input tunnel. The shuttle may move within the input tunnel from the first position to a second position after the collet is detached from the shuttle, and thus, the shuttle may be disposed at least partially within the reservoir in the second position.

[0068] The sample analysis cartridge may include a sensor configured to be exposed to a fluid mixed with a sample. The sensor may generate a signal indicative of at least one of the presence, absence, or amount of one or more analytes in the sample. The shuttle may have a first end and a second end disposed proximally to the first end within the input tunnel. The second end of the shuttle may be configured to be disposed within the lumen of the collet in the first position. The first end of the shuttle may form the wall of the reservoir in the first position.

[0069] The collet may have one or more locking arms configured to couple the collet to the shuttle in the first position. The one or more locking arms may be configured to be deflected to detach the one or more locking arms from the shuttle in response to a force applied on the one or more locking arms by the sample collection device during insertion of the sample collection device into the input tunnel.

[0070] The sample analysis cartridge may include a seal material configured to fluidly seal the fluid within the reservoir and a seal piercer partially disposed within the input tunnel. The seal piercer may be contacted by a sample collection device within the input tunnel and configured to move in response to a force applied by the sample collection device to pierce the seal material and vent the fluid within the reservoir. The collet may have a slot, and a portion of the seal piercer may extend into the input tunnel through the slot to enable contact between the seal piercer and the sample collection device.

[0071] The sample analysis cartridge may include a contact switch. The collet may have a deflector portion disposed adjacent to the contact switch and configured to deflect in response to a force applied by the sample collection device onto the deflector portion during insertion of the sample collection device into the input tunnel to activate the contact switch. The deflector portion of the collet may include an arm configured to deflect downward to activate the contact switch. The contact switch may be positioned within the input tunnel such that activation of the contact switch indicates complete insertion of the sample collection device into the input tunnel.

[0072] The shuttle may be configured to store a reagent ball containing a reagent between a first end and a second end of the shuttle. Preferably, the reagent ball is not exposed to the fluid within the reservoir in the first position and is exposed to the fluid within the reservoir in the second position.

[0073] According to yet another aspect, a sample analysis cartridge is provided. The sample analysis cartridge may include an input tunnel, a reservoir, a collet, and / or a contact switch, each of which may be within the housing of the cartridge. The input tunnel may extend from an opening and may enable insertion of a sample collection device having a distal portion adapted to be exposed to a sample. The reservoir may be configured to hold a fluid. The collet may be disposed within the input tunnel between the reservoir and the opening. The collet may have a lumen sized to receive the deflector portion and the distal portion of the sample collection device therein. The contact switch may be disposed adjacent to the deflector portion of the collet. The deflector portion may be configured to deflect in response to a force applied on the deflector portion by the sample collection device during insertion of the sample collection device into the input tunnel, so as to activate the contact switch.

[0074] The sample analysis cartridge may include a shuttle disposed within the input tunnel and configured to store a reagent ball containing a reagent between a first end and a second end of the shuttle. The deflector portion of the collet may be an arm configured to deflect downwardly to activate the contact switch. The contact switch may be positioned such that activation of the contact switch indicates complete insertion of the sample collection device into the input tunnel.

[0075] The sample analysis cartridge may include a sensor configured to be exposed to a fluid mixed with a sample. The sensor may generate a signal indicating at least one of the presence, absence, or amount of one or more analytes in the sample.

[0076] According to another aspect, a sample analysis cartridge is provided. The sample analysis cartridge may include an input tunnel, a reservoir, a shuttle, and / or an ultrasonic generator, each of which may be within the housing of the cartridge. The input tunnel may extend from an opening and may enable insertion of a sample collection device having a distal portion adapted to be exposed to a sample. The reservoir may be configured to hold a fluid. The shuttle may define a first compartment and a second compartment. The first and second compartments may be configured to be disposed within the reservoir in a mixing position. The ultrasonic generator may be configured to emit acoustic waves and move the fluid within the reservoir between the first and second compartments in a wave pattern to mix the fluid within the reservoir.

[0077] The shuttle may include a compartment divider configured to divide the first compartment from the second compartment. The compartment divider may be a flange. The fluid flowing around the compartment divider may facilitate the formation of a wave pattern. The compartment divider may have slots configured to allow fluid to flow through the compartment divider via the slots during mixing. The first compartment may be a reagent ball compartment configured to store a reagent ball containing a reagent, and the second compartment may be a sample compartment configured to receive a sample from a sample collection device, for example, discharged from and / or onto the distal portion of the sample collection device. The reagent ball may contain reagents for amplification of a target nucleic acid, such as polymerase, primer, and signaling substances, as described above. The first and second compartments are preferably not disposed within the reservoir in a pre-mixing position.

[0078] The ultrasonic generator may be a piezoelectric transducer such as a piezoelectric ceramic disk. The ultrasonic generator may form part of the wall of the reservoir, for example, a part of the bottom wall of the reservoir. The reservoir may be symmetric. The walls of the reservoir may abut at an angle greater than a predetermined angle such as 60°, which can promote cavitation of the fluid passing through the outlet of the reservoir. The ultrasonic generator may be positioned offset from the center of the reservoir, which can promote mixing of the fluid within the reservoir.

[0079] The sample analysis cartridge may include a printed circuit board that is coupled to the ultrasonic generator via one or more spring contacts. The ultrasonic generator may be electrically coupled to the printed circuit board via only one or more spring contacts. The ultrasonic generator may be activated in response to a signal from a processor, for example, a processor of a reader.

[0080] The sample analysis cartridge may include a temperature sensor configured to sense a temperature indicative of the temperature of the fluid within the reservoir. The temperature sensor may be disposed on the printed circuit board positioned adjacent to the ultrasonic generator.

[0081] The sample analysis cartridge may include a contact switch configured to indicate insertion of a sample collection device into the input tunnel. The ultrasonic generator may be configured to emit acoustic waves after activation of the contact switch. For example, the reader may instruct the ultrasonic generator to emit acoustic waves after receiving an electrical signal indicating that the contact switch has been activated.

[0082] The acoustic waves emitted by the ultrasonic generator may be configured to isothermally amplify the reaction of the fluid mixed within the reservoir.

[0083] According to another aspect, a method for isothermal amplification of a target nucleic acid when present within a sample analysis cartridge is provided. The method includes, or alternatively consists of, contacting, in a reservoir, a reagent ball containing a plurality of reagents preselected for amplification and detection of the target nucleic acid as described above, with a sample for acidifying an amplicon-signal transducer complex bound to solid particles. The amplicon comprises a nucleic acid duplex comprising a reverse primer complex comprising a nucleic acid comprising a target nucleic acid bound to a spacer element and thus to a signal transducer, and a forward primer complex comprising a nucleic acid comprising a target sequence bound at one end to a capture element. In a further aspect, the reverse primer complex further comprises a signal transduction affinity element conjugated to the signal transducer and the spacer element. The amplicon-signal transducer complex can thus be conjugated to an affinity element on a solid particle and thus bound or retained on a sensor surface across a magnetic field. The amplicon-signal transducer complex can thus be conjugated to an affinity element on a solid particle and thus retained on a sensor surface across a magnetic field. When an analyte is present, the sensor detects and / or quantifies the signal transducer-labeled amplicon.

[0084] The ultrasonic generator may emit acoustic waves towards the reservoir to facilitate amplification of the target nucleic acid within the reservoir. As described above, the amplicon-signal transducer complex may be reacted with a substrate from the substrate reservoir. For example, the reaction may occur across a sensor within an analysis channel. A signal indicative of at least one of the presence, absence, or amount of the amplified nucleic acid may be generated. The signal may be transmitted from the cartridge to another device such as a reader.

[0085] The reagent ball may be held within the shuttle. The shuttle may be disposed within the input tunnel of the cartridge. The reagent ball may comprise reagents for amplification of a target nucleic acid by isothermal reaction as described above. The reagents may comprise a polymerase, primers for amplification of the target nucleic acid, and a signaling substance for detection of amplification of the target nucleic acid. One or more affinity molecules may be covalently or non-covalently bound to the solid particles for detection of the target nucleic acid.

[0086] According to another aspect, a kit is provided. The kit may include a reservoir, an ultrasonic generator, a temperature sensor, and / or a processor. The reservoir may be configured to hold fluid and receive a sample collected by a sample collection device. The ultrasonic generator may be configured to emit acoustic waves to mix the fluid and the sample within the reservoir. The temperature sensor may be configured to generate a signal indicative of the temperature of the fluid within the reservoir. The processor may be configured to activate the ultrasonic generator to emit acoustic waves and monitor the signal from the temperature sensor. The processor may further be configured to modify the emission of acoustic waves from the ultrasonic generator if the signal indicates a temperature of the fluid within the reservoir outside a threshold. The sample analysis cartridge may include a reservoir, an ultrasonic generator, and / or a temperature sensor, each within the housing of the cartridge, and the reader may include a processor. The reader may be configured to be electrically coupled to the sample analysis cartridge.

[0087] The sample analysis cartridge may include a printed circuit board, and the temperature sensor may be disposed on the printed circuit board positioned adjacent to the ultrasonic generator. The sample analysis cartridge may include a contact switch configured to generate a signal indicating insertion of the sample collection device into the input tunnel of the sample analysis cartridge. The processor of the reader may be configured to receive the signal from the contact switch and activate the ultrasonic generator after receiving the signal from the contact switch. The processor may modify the emission of acoustic waves from the ultrasonic generator by decreasing the duty cycle of the ultrasonic generator if the signal indicates that the temperature of the fluid in the reservoir exceeds a threshold. The processor may modify the emission of acoustic waves from the ultrasonic generator by increasing the duty cycle of the ultrasonic generator if the signal indicates that the temperature of the fluid in the reservoir is below the threshold. The processor may modify the emission of acoustic waves from the ultrasonic generator by deactivating the ultrasonic generator if the signal indicates that the temperature of the fluid in the reservoir exceeds a threshold.

[0088] The acoustic waves emitted by the ultrasonic generator may be configured to isothermally amplify the reaction of the fluid mixed in the reservoir. The sample analysis cartridge may include a reagent ball disposed within the sample analysis cartridge. The ultrasonic generator may be configured to emit acoustic waves to mix the fluid, the reagent ball, and the sample in the reservoir. The reagent ball may include polymerase, primer, and signaling substances. The sample analysis cartridge may include a shuttle configured to store the reagent ball.

[0089] According to another aspect, a sensor for use in a microfluidic cartridge is provided. The sensor may include a positive reference working electrode, a working electrode, and / or a negative reference working electrode. The positive reference working electrode may include an affinity molecule pre-bound to the positive reference working electrode. For example, the affinity molecule may be pre-bound to the surface of the positive reference working electrode disposed within the analysis channel of the cartridge. The positive reference working electrode may be configured to generate a first signal based on a reaction between a signaling agent and a chemical substrate that are directly or indirectly bound to the affinity molecule. The signaling agent may be from a reagent ball. The chemical substrate may be from a fluid stored in a substrate reservoir. The working electrode may be configured to generate a second signal based on a reaction between a signaling agent localized to the working electrode and a chemical substrate. The negative reference working electrode may include a self-assembled monolayer. For example, the self-assembled monolayer may be on the surface of the negative reference working electrode disposed within the analysis channel of the cartridge. The negative reference working electrode may be configured to generate a third signal based on a reaction between a signaling agent localized to the negative working electrode and a chemical substrate. As should be understood, "first", "second", and "third" are terms used to distinguish and do not necessarily mean order.

[0090] The second signal may indicate the presence, absence, or amount of one or more analytes in the sample. The first signal may indicate the reliability of the test. For example, the test may be determined to be reliable if the first signal indicates an amount of reaction within a predetermined range. The third signal indicates the reliability of the test. For example, the test may be determined to be reliable if the third signal indicates that the amount of reaction is below a threshold.

[0091] The cartridge may include the sensor. The cartridge may have an analysis channel, and the positive reference working electrode, the working electrode, and the negative reference working electrode may be disposed within the analysis channel.

[0092] A kit is also provided that includes a cartridge. The kit may include a processor configured to process a second signal and generate information indicative of the presence, absence, or amount of at least one analyte in a sample, where the amount is one or more. The processor may process a first signal and determine whether the first signal indicates an amount of reaction within a predetermined range. The processor may generate an alert if the amount is outside the predetermined range. The processor may process a third signal and determine whether the third signal indicates that the amount of reaction is below a threshold. The processor may generate an alert if the amount exceeds the threshold. The processor may be a component of a reader.

[0093] The working electrode may be masked with a plurality of fine grooves configured to facilitate a homogeneous distribution of a plurality of magnetic particles directly or indirectly bound to a signal transducer localized across the working electrode and to facilitate resistance to movement of the plurality of magnetic particles away from the working electrode.

[0094] The working electrode may include a self-assembled monolayer. For example, the self-assembled monolayer may be on the surface of the working electrode disposed within the analysis channel of the cartridge.

[0095] The working electrode may include an affinity molecule pre-bound to the working electrode. For example, the affinity molecule may be pre-bound to the surface of the working electrode disposed within the analysis channel of the cartridge.

[0096] Such methods and devices may be used, for example, among other things, to determine a disease a person is suffering from, among other things, a drug or poison a person is reacting adversely to, or among other things, a chemical substance contaminating water. Other examples, without limitation, include quantifying the concentration of various agents, including vitamins, hormones, proteins, or other analytes of interest in the body, waterborne and foodborne pathogens, microbial growth and / or contamination of medical devices, and other potential disease-causing contaminants from pets and livestock. Examples of contaminants include, without limitation, viral, bacterial, and fungal pathogens, bloodstream infections (BSIs), pneumonia (e.g., ventilator-associated pneumonia [VAP]), urinary tract infections (UTIs), and surgical site infections (SSIs), Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Candida / albicans, Pseudomonas aeruginosa, Acinetobacter / baumannii, Stenotrophomonas / maltophilia, Clostridium difficile, tuberculosis, gastroenteritis, vancomycin-resistant enterococci, Legionellosis, puerperal fever, MRSA, and Escherichia coli. Examples of foodborne pathogens include, without limitation, Shigella, Salmonella, Vibrio spp., Yersinia spp., Listeria, Escherichia coli, and Campylobacter. The uses of the present technology are not limited to pathogens or analytes important to the health of human patients and also include the health and preservation of pets and livestock, e.g., veterinary uses. The present invention provides, for example, the following. (Item 1) A sample analysis cartridge for detecting at least one of the presence, absence, or amount of one or more analytes, An input tunnel extending from an opening, the input tunnel being configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to a sample, the input tunnel; A reservoir configured to hold a fluid; A reagent shuttle disposed between the reservoir and the opening at the first position, the reagent shuttle having a first end and a second end, the reagent shuttle configured to store a reagent ball that holds a reagent between the first end and the second end, the first end configured to seal the reservoir from the input tunnel at the first position, the reagent shuttle configured to move within the input tunnel to a second position when subjected to a force exceeding a threshold force, such that the reagent ball and the sample move into the reservoir and the reservoir is continuously sealed from the proximal input tunnel of the reagent shuttle during movement from the first position to the second position, a reagent shuttle; A sensor configured to analyze a fluid mixed with the reagent ball and the sample, the sensor further configured to generate a signal indicative of the presence, absence, or amount of at least one analyte in the sample, one or more analytes; A sample analysis cartridge comprising. (Item 2) The sample analysis cartridge according to item 1, wherein the second end of the shuttle has an opening sized to wipe excess sample from the tip of the sample collection device, such that at most a predetermined volume of the sample is mixed into the fluid in the reservoir. (Item 3) The sample analysis cartridge according to item 1, wherein the reservoir is sealed via the sample collection device that is partially inserted into the second end of the shuttle during movement from the first position to the second position. (Item 4) The sample analysis cartridge according to item 1, further comprising one or more locking members configured to irreversibly lock the sample collection device within the input tunnel at the second position. (Item 5) The shuttle comprises one or more sample compartments configured to store, at most, a predetermined volume of the sample, and one or more reagent ball compartments configured to store the reagent balls and optionally additional reagent balls. The one or more sample compartments and the one or more reagent ball compartments are not exposed to the fluid in the reservoir at the first position. The one or more sample compartments and the one or more reagent ball compartments are exposed to the fluid in the reservoir at the second position. The sample analysis cartridge according to item 1. (Item 6) The sample analysis cartridge according to item 5, wherein the shuttle comprises a compartment divider configured to separate at least one sample compartment from at least one reagent ball compartment. (Item 7) The sample analysis cartridge according to item 6, wherein the compartment divider comprises slots configured to promote mixing when disposed in the sample preparation reservoir at the second position. (Item 8) The reagent of the sample analysis cartridge according to item 1 comprises one or more of a plurality of solid particles, a plurality of affinity molecules, or a plurality of signaling substances. (Item 9) The reagent of the sample analysis cartridge according to item 1 comprises a plurality of magnetic particles configured to be magnetically held across the working electrode of the sensor. (Item 10) At least one magnetic particle of the plurality of magnetic particles of the sample analysis cartridge according to item 9 is configured to be indirectly coupled to a signaling substance. (Item 11) Further comprising an analysis channel, at least a part of the sensor is disposed within the analysis channel, and the fluid mixed with the reagent ball and the sample travels through the analysis channel to at least a part of the sensor, the sample analysis cartridge according to item 1. (Item 12) Further comprising a contact switch, a sealing material configured to fluidly seal the fluid in the reservoir, and a seal piercer, Insertion of the sample collection device into the input tunnel of the sample collection device causes (i) the shuttle to move from the first position to the second position, (ii) the seal piercer to pierce the seal material and vent the fluid in the reservoir, and (iii) activation of the contact switch. The sample analysis cartridge according to item 1. (Item 13) A sample analysis cartridge for detecting at least one of the presence, absence, or amount of one or more analytes, An input tunnel extending from an opening, the input tunnel configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to a sample, the input tunnel; A reservoir configured to hold fluid; A sealing material configured to fluidly seal the fluid in the reservoir; A seal piercer partially disposed within the input tunnel, the seal piercer being contacted by a sample collection device within the input tunnel and configured to move in response to a force applied by the sample collection device to pierce the seal material and vent the fluid in the reservoir; Comprising a sample analysis cartridge. (Item 14) The seal piercer is configured to move in a first direction and a second direction different from the first direction to pierce the seal material, the sample analysis cartridge according to item 13. (Item 15) The sample analysis cartridge according to item 14, wherein the first direction is substantially parallel to the movement of the sample collection device within the input tunnel, and the second direction is substantially perpendicular to the first direction. (Item 16) The sample analysis cartridge according to item 13, wherein the seal piercer comprises one or more piercers. (Item 17) The sample analysis cartridge according to item 16, wherein the seal piercer further comprises a slider configured to move in a first direction, and the one or more piercers move in a second direction different from the first direction and are configured to pierce the seal material. (Item 18) The reservoir is a sample preparation reservoir, further comprising a cleaning agent reservoir and a substrate reservoir, The seal piercer is configured to pierce the seal material and vent to the individual fluids within the sample preparation reservoir, the cleaning agent reservoir, and the substrate reservoir. The sample analysis cartridge according to item 13. (Item 19) The sample analysis cartridge according to item 13, wherein the seal piercer comprises an engaging device disposed within the input tunnel, and the engaging device is configured to engage an engagement zone of the sample collection device when the sample collection device is within the input tunnel. (Item 20) The sample analysis cartridge according to item 13, further comprising a contact switch configured to be activated in response to insertion of the sample collection device into the input tunnel. (Item 21) The sample analysis cartridge according to item 20, wherein the movement of the seal piercer causes activation of the contact switch. (Item 22) The sample analysis cartridge according to item 18, wherein the seal piercer is configured to sequentially pierce the seal material across the sample collection reservoir, the cleaning agent reservoir, and the substrate reservoir in any order. (Item 23) The sample analysis cartridge according to item 13, wherein the seal piercer is configured to pierce and, after venting the fluid in the reservoir, move through one or more holes pierced into the seal material. (Item 24) Further comprising a contact switch and a shuttle disposed between the reservoir and the opening in a first position, the shuttle having a first end and a second end, the first end configured to seal the reservoir from the input tunnel in the first position, the reagent shuttle configured to move within the input tunnel to a second position where the sample is moved into the reservoir, Insertion of the sample collection device into the input tunnel of the sample analysis cartridge causes (i) the shuttle to move from the first position to the second position, (ii) the seal piercer to pierce the seal material and vent the fluid in the reservoir, and (iii) activation of the contact switch. The sample analysis cartridge according to item 13. (Item 25) The sample analysis cartridge according to item 24, further comprising one or more locking members configured to irreversibly lock the sample collection device within the input tunnel in the second position. (Item 26) Insertion of the sample collection device into the input tunnel of the sample analysis cartridge causes the seal piercer to pierce the seal material and vent the fluid in the reservoir before the shuttle moves from the first position to the second position, as described in item 24. (Item 27) Insertion of the sample collection device into the input tunnel causes the seal piercer to pierce the seal material and vent the fluid in the reservoir during movement of the shuttle from the first position to the second position, the sample analysis cartridge of claim 24. (Item 28) A sample analysis cartridge for detecting at least one of the presence, absence, or amount of one or more analytes, An input tunnel extending from an opening, the input tunnel being configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to a sample, the input tunnel; A reservoir configured to hold fluid and configured to receive a sample on the distal portion of the sample collection device; An analysis channel configured to receive from the reservoir a fluid having the sample and a reagent, the fluid comprising a plurality of magnetic particles mixed therein; A circuit board comprising a sensor having a working electrode, the sensor being exposed to the fluid mixed in the analysis channel and configured to generate a signal indicative of at least one of the presence, absence, or amount of one or more analytes in the sample, the circuit board; Comprising The working electrode is masked with a plurality of fine grooves configured to promote a homogeneous distribution of the plurality of magnetic particles across the working electrode and to promote resistance to movement of the plurality of magnetic particles from the working electrode; Sample analysis cartridge. (Item 29) A kit for detecting at least one of the presence, absence, or amount of one or more analytes, A sample analysis cartridge, An input tunnel extending from an opening, the input tunnel being configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to a sample, the input tunnel; A reservoir configured to hold fluid and configured to receive a sample on a distal portion of the sample collection device, An analysis channel configured to receive from the reservoir a fluid having the sample and a reagent, the fluid comprising a plurality of magnetic particles mixed therein, A circuit board comprising a sensor configured to be exposed to the fluid mixed in the analysis channel and configured to generate a signal indicative of at least one of the presence, absence, or amount of one or more analytes in the sample, A sample analysis cartridge comprising: A sample analysis reader configured to receive the sample analysis cartridge, the sample analysis reader having first and second magnetic generators configured to be disposed adjacent to a single working electrode of the sensor when the sample analysis cartridge is inserted into the sample analysis reader, the first and second magnetic generators further configured to generate a magnetic field across the length of the single working electrode and to promote a homogeneous distribution of the plurality of magnetic particles across the length of the single working electrode, A kit comprising: (Item 30) The kit of item 29, wherein receiving the sample analysis cartridge by the sample analysis reader causes an electrical coupling between the sample analysis cartridge and the sample analysis reader. (Item 31) The kit of item 30, wherein the sample analysis cartridge is configured to transmit to the sample analysis reader a signal indicative of at least one of the presence, absence, or amount of one or more analytes in the sample for processing. (Item 32) The kit of item 31, wherein the sample analysis reader is configured to transmit to a computer a processed signal indicative of at least one of the presence, absence, or amount of one or more analytes in the sample. (Item 33) The kit according to item 32, further comprising a computer-readable medium with instructions that, when executed by a processor of the computer, cause the computer's display to display information indicating the presence, absence, and / or amount of one or more target analytes. (Item 34) The housing of the sample analysis cartridge has a bottom surface and has a magnetic generator recess. Receiving the sample analysis cartridge into the sample analysis reader causes the first and second magnetic generators to move partially within the magnetic generator recess. The kit according to item 29. (Item 35) The kit according to item 34, wherein the magnetic generator recess is disposed adjacent to the single working electrode. (Item 36) A sample analysis cartridge for detecting at least one of the presence, absence, or amount of one or more analytes, comprising: An input tunnel extending from an opening, the input tunnel being configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to a sample. A reservoir configured to hold fluid and configured to receive a sample on the distal portion of the sample collection device, the reservoir having a phase change material therein for closing its entire cross-section and including an outlet. A heater configured to heat the phase change material so that the phase change material does not block the entire cross-section of the outlet. An analysis channel configured to receive the fluid having the sample and reagents, including a plurality of magnetic particles mixed therein, from the reservoir through the outlet. A sensor exposed to the fluid mixed in the analysis channel and configured to generate a signal indicating at least one of the presence, absence, or amount of one or more analytes in the sample. Comprising The heater is masked with a masking material configured to electrically insulate the heater from the sensor. Sample analysis cartridge. (Item 37) The masking material comprises a solder mask, and the sample analysis cartridge according to item 36. (Item 38) The sample analysis cartridge according to item 36 further comprises a cleaning agent reservoir and a cleaning agent reservoir heater, wherein the cleaning agent reservoir is configured to hold a cleaning fluid and has a phase change material therein for closing the entire cross-section of the cleaning agent reservoir outlet, including the cleaning agent reservoir outlet. The cleaning agent reservoir heater is configured to heat the phase change material in the cleaning agent reservoir outlet so that the phase change material does not close the entire cross-section of the cleaning agent reservoir outlet. Thus, the cleaning fluid enters the analysis channel and proceeds to the sensor. The sample analysis cartridge according to item 36. (Item 39) The masking material comprises a solder mask, and the sample analysis cartridge according to item 38, wherein the cleaning agent reservoir heater is configured to electrically insulate the cleaning agent reservoir heater from the sensor. (Item 40) The sample analysis cartridge according to item 36 further comprises a substrate reservoir and a substrate reservoir heater, wherein the substrate reservoir is configured to hold a substrate fluid and has a phase change material therein for closing the entire cross-section of the substrate reservoir outlet, including the substrate reservoir outlet. The substrate reservoir heater is configured to heat the phase change material in the substrate reservoir outlet so that the phase change material does not close the entire cross-section of the substrate reservoir outlet. Thus, the substrate fluid enters the analysis channel and proceeds to the sensor. The sample analysis cartridge according to item 36. (Item 41) The masking material comprises a solder mask, and the sample analysis cartridge according to item 40, wherein the substrate reservoir heater is configured to electrically insulate the substrate reservoir heater from the sensor. (Item 42) A sample analysis cartridge for detecting at least one of the presence, absence, or amount of one or more analytes, comprising: A sample preparation reservoir configured to hold a fluid and configured to receive a sample from a sample collection device, the sample preparation reservoir including a sample preparation reservoir outlet having a phase change material therein for sealing the sample preparation reservoir outlet; A substrate reservoir configured to hold a fluid comprising a chemical substrate, the substrate reservoir including a substrate reservoir outlet having a phase change material therein for sealing the substrate reservoir outlet; An analysis channel, wherein each of the sample and substrate reservoirs is in fluid communication with the analysis channel at least as needed; A fluid breaker comprising a phase change material; One or more heaters, at least one of the one or more heaters being configured to heat the phase change material within the sample preparation reservoir outlet, such that the phase change material unseals the sample preparation reservoir outlet and enables the fluid to mix the sample therein and flow into the analysis channel; Comprising: At least one of the one or more heaters is configured to heat the phase change material of the fluid breaker after unsealing the sample preparation reservoir outlet, such that the phase change material of the fluid breaker flows into the analysis channel and fluidly isolates the sample preparation reservoir from the substrate reservoir; At least one of the one or more heaters is configured to heat a phase change material within the substrate reservoir outlet after the fluid breaker fluidly isolates the sample preparation reservoir from the substrate reservoir; thus, the phase change material unseals the substrate reservoir outlet and enables fluid comprising the chemical substrate to flow into the analysis channel but not into the sample preparation reservoir. Sample analysis cartridge. (Item 43) The one or more heaters comprise a sample preparation reservoir heater, a fluid breaker heater, and a substrate reservoir heater. The sample preparation reservoir heater is configured to heat a phase change material within the sample preparation reservoir outlet. The fluid breaker heater is configured to heat a phase change material of the fluid breaker. The substrate reservoir heater is configured to heat a phase change material within the substrate reservoir outlet. The sample analysis cartridge according to item 42. (Item 44) The sample analysis cartridge according to item 43, wherein the sample preparation reservoir heater, the fluid breaker heater, and the substrate reservoir heater are each masked with a masking material configured to electrically insulate the individual heaters from sensors within the analysis channel. (Item 45) The sample analysis cartridge according to item 42, further comprising a cleaning agent reservoir configured to hold a cleaning fluid, the cleaning agent reservoir including a cleaning agent reservoir outlet having a phase change material therein for sealing the cleaning agent reservoir outlet. (Item 46) At least one of the one or more heaters is configured to heat the phase change material in the detergent reservoir outlet after the fluid breaker fluidically isolates the sample preparation reservoir from the substrate reservoir, but before the one or more heaters heat the phase change material within the substrate reservoir outlet, such that the phase change material unseals the detergent reservoir outlet and enables the wash fluid to flow into the analysis channel to wash away signal transduction material not bound to magnetic particles from the sample preparation reservoir from the sensor within the analysis channel, the sample analysis cartridge of item 45. (Item 47) The fluid comprising the chemical substrate is configured to wash away signal transduction material not bound to magnetic particles from the sample preparation reservoir from the sensor within the analysis channel, the sample analysis cartridge of item 42. (Item 48) A sample analysis cartridge for detecting at least one of the presence, absence, or amount of one or more analytes, An input tunnel extending from an opening and configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to sample fluid, the input tunnel; A reservoir configured to hold fluid; A shuttle disposed between the reservoir and the opening in a first position, the shuttle having a first end and a second end and defining a sample compartment therebetween, the sample compartment being configured to receive sample fluid compressed from the distal portion of the sample collection device, the shuttle being configured to move within the input tunnel to a second position when subjected to a force exceeding a threshold force, such that the sample compartment having the sample fluid is moved into the reservoir, the shuttle; A sensor configured to be exposed to a fluid mixed with the sample fluid, the sensor further configured to generate a signal indicative of at least one of the presence, absence, or amount of one or more analytes in the sample fluid. A sample analysis cartridge comprising the same. (Item 49) The shuttle further defines a reagent ball compartment between the first end and the second end, the reagent ball compartment being configured to store one or more reagent balls comprising reagents. The reagent ball compartment is outside the reservoir at the first position and inside the reservoir at the second position. The sample analysis cartridge according to item 48. (Item 50) The sample analysis cartridge according to item 48, wherein the sample compartment is configured to receive a predetermined volume of the sample fluid compressed from the distal portion of the sample collection device at most. (Item 51) The sample analysis cartridge according to item 50, further comprising an overflow compartment configured to receive a volume of the sample fluid exceeding the predetermined volume from the sample compartment. (Item 52) A kit comprising: The sample analysis cartridge according to item 48; and The sample collection device, the sample collection device comprising a suction portion at the distal portion, the suction portion being configured to suck and absorb the sample fluid. A kit comprising the same. (Item 53) The kit according to item 52, wherein the suction portion is compressed to discharge the sample fluid into the sample compartment. (Item 54) The sample compartment is configured to receive, at most, a predetermined volume of the sample fluid compressed from the distal portion of the sample collection device. The sample analysis cartridge further includes an overflow compartment configured to receive a volume of the sample fluid that exceeds the predetermined volume from the sample compartment. The suction portion of the sample collection device is configured to suck up and absorb sample fluid that exceeds the predetermined volume, enabling a user to measure the amount of sample fluid compressed into the sample compartment and the overflow compartment. The kit according to item 52. (Item 55) The kit according to item 52, wherein at least a portion of the suction portion is slidably disposed within a shroud of the sample collection device. (Item 56) The kit according to item 52, wherein the sample analysis cartridge further includes one or more locking members configured to irreversibly lock the sample collection device within the input tunnel when the sample collection device is fully inserted into the input tunnel. (Item 57) The kit according to item 52, wherein the sample collection device further includes a sample collection indicator configured to visually alert a collector based on the volume of the collected sample fluid. (Item 58) The kit according to item 57, wherein the sample collection indicator is a colored thread embedded within the suction portion that becomes increasingly visually exposed as the volume of the collected sample increases. (Item 59) A method for detecting at least one of the presence, absence, or amount of a target analyte in a sample within a cartridge, A step of mixing a sample having a plurality of affinity molecules, a plurality of binding release agents, a plurality of signal transmitters, a plurality of competing molecules pre-bound to competing binding molecules, each having a label, and a plurality of sample target analytes pre-bound to sample binding molecules in a fluid in a reservoir of the cartridge; A step of releasing at least one competing molecule from the pre-bound competing binding molecule using at least one binding release agent among the plurality of binding release agents; A step of releasing at least one sample target analyte from the pre-bound sample binding molecule using at least one binding release agent among the plurality of binding release agents; A step of binding the label of the released competing molecule to a certain signal transmitter among the plurality of signal transmitters; A step of binding the released competing molecule to a certain affinity molecule among the plurality of affinity molecules; A step of generating a signal indicating at least one of the presence, absence, or amount of a sample target analyte in the cartridge; A method comprising the above steps. (Item 60) The method according to item 59, wherein the affinity molecules among the plurality of affinity molecules are each bound to solid particles. (Item 61) The method according to item 60, wherein the solid particles comprise a magnetic responsive material. (Item 62) The method according to item 60, wherein the solid particles comprise a non-magnetic responsive material. (Item 63) The method according to item 62, wherein the non-magnetic responsive material comprises gold nanoparticles. (Item 64) The method according to item 59, wherein the plurality of sample target analytes pre-bound to the sample binding molecule comprise 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 molecules pre-bound to vitamin D binding protein molecules. (Item 65) The method according to item 59, wherein the plurality of competing molecules pre-bound to the sample binding molecule are labeled with biotin and comprise 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 molecules pre-bound to vitamin D binding protein molecules. (Item 66) The method according to item 59, wherein at least one of the plurality of affinity molecules, the plurality of dissociation agents, the plurality of signaling substances, and the plurality of competing molecules pre-bound to the competitive binding molecule is stored in a reagent ball. (Item 67) A sample analysis cartridge for detecting at least one of the presence, absence, or amount of a target analyte, A reagent ball comprising a plurality of competing molecules pre-bound to a competitive binding molecule, each of the plurality of competing molecules carrying a label or being bound to a signaling substance, A reservoir configured to hold a reservoir fluid, the reservoir being further configured to enable mixing of a sample comprising a plurality of affinity molecules, a plurality of dissociation agents, a plurality of signaling substances, a plurality of competing molecules pre-bound to a competitive binding molecule, and a sample from a sample collection device, the sample having a plurality of sample target analytes pre-bound to sample binding molecules, One of the plurality of dissociation agents is configured to dissociate a competing molecule from the pre-bound competitive binding molecule or a sample target analyte from the pre-bound sample binding molecule, The label of the dissociated competing molecule is configured to bind to a signaling substance, and the dissociated competing molecule is configured to bind to one of the plurality of affinity molecules, A reservoir, A sensor configured to be exposed to the mixed reservoir fluid, the sensor being further configured to generate a signal indicative of at least one of the presence, absence, or amount of a sample target analyte in the sample, A sample analysis cartridge comprising. (Item 68) The reservoir is further configured to allow mixing of a plurality of solid particles into the reservoir fluid, the sample analysis cartridge according to item 67. (Item 69) Each solid particle is pre-bound to a certain affinity molecule among the plurality of affinity molecules, the sample analysis cartridge according to item 68. (Item 70) The plurality of solid particles comprise a magnetic responsive material, the sample analysis cartridge according to item 68. (Item 71) The plurality of solid particles comprise a non-magnetic responsive material, the sample analysis cartridge according to item 68. (Item 72) The non-magnetic responsive material comprises gold nanoparticles, the sample analysis cartridge according to item 71. (Item 73) The plurality of sample target analytes pre-bound to the sample binding molecules comprise 25-hydroxyvitamin D3 and / or 25-hydroxyvitamin D2 molecules pre-bound to binding protein molecules, the sample analysis cartridge according to item 67. (Item 74) The magnetic responsive material is magnetically retained across the sensor, the sample analysis cartridge according to item 70. (Item 75) A sample analysis cartridge, An input tunnel extending from an opening, the input tunnel being configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to a sample, the input tunnel, and A reservoir configured to hold a fluid, and A shuttle disposed within the input tunnel between the reservoir and the opening at a first position, At the first position, a collet disposed within the input tunnel and coupled to the shuttle, the collet being configured to disconnect from the shuttle during insertion of the sample collection device into the input tunnel. Comprising The shuttle is configured to move within the input tunnel from the first position to a second position after the collet has been disconnected from the shuttle, and thus the shuttle is at least partially disposed within the reservoir at the second position. Sample analysis cartridge. (Item 76) The sample analysis cartridge according to item 75, further comprising a sensor configured to be exposed to a fluid mixed with the sample, the sensor being further configured to generate a signal indicating at least one of the presence, absence, or amount of one or more analytes in the sample. (Item 77) The shuttle has a first end and a second end disposed proximal to the first end within the input tunnel, the second end of the shuttle being configured to be disposed within the lumen of the collet at the first position. The sample analysis cartridge according to item 75. (Item 78) The first end of the shuttle forms the wall of the reservoir at the first position. The sample analysis cartridge according to item 77. (Item 79) The collet according to item 77, comprising one or more locking arms configured to couple the collet to the shuttle at the first position. (Item 80) The one or more locking arms are configured to deflect the one or more locking arms away from the shuttle in response to a force applied on the one or more locking arms by the sample collection device during insertion of the sample collection device into the input tunnel, for the sample analysis cartridge according to item 79. (Item 81) The sample analysis cartridge according to item 75 further comprises a seal material configured to fluidly seal the fluid in the reservoir and a seal piercer partially disposed in the input tunnel, the seal piercer being contacted by a sample collection device in the input tunnel and moving in response to a force applied by the sample collection device to pierce the seal material and vent to the fluid in the reservoir. (Item 82) The collet comprises a slot, and a part of the seal piercer extends into the input tunnel through the slot, for the sample analysis cartridge according to item 81, enabling contact between the seal piercer and the sample collection device. (Item 83) Further comprising a contact switch, The collet comprises a deflector portion disposed adjacent to the contact switch, the deflector portion being configured to deflect to activate the contact switch in response to a force applied on the deflector portion by the sample collection device during insertion of the sample collection device into the input tunnel. For the sample analysis cartridge according to item 75. (Item 84) The deflector portion of the collet is configured to deflect downward and comprises an arm to activate the contact switch, for the sample analysis cartridge according to item 83. (Item 85) The contact switch is positioned such that activation of the contact switch indicates complete insertion of the sample collection device into the input tunnel, for the sample analysis cartridge according to item 83. (Item 86) The shuttle is configured to store a reagent ball containing a reagent between a first end and a second end of the shuttle. The reagent ball is not exposed to the fluid in the reservoir at the first position and is exposed to the fluid in the reservoir at the second position. The sample analysis cartridge according to item 75. (Item 87) A sample analysis cartridge, An input tunnel extending from an opening, the input tunnel being configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to a sample. A reservoir configured to hold a fluid, A collet disposed in the input tunnel between the reservoir and the opening, the collet having a lumen sized to receive a deflector portion and the distal portion of the sample collection device therein. A contact switch disposed adjacent to the deflector portion of the collet, comprising The deflector portion is configured to deflect so as to activate the contact switch in response to a force applied to the deflector portion by the sample collection device during insertion of the sample collection device into the input tunnel. Sample analysis cartridge. (Item 88) Further comprising a shuttle disposed in the input tunnel, the shuttle being configured to store a reagent ball containing a reagent between a first end and a second end of the shuttle. The sample analysis cartridge according to item 87. (Item 89) The deflector portion of the collet is provided with an arm configured to deflect downward to activate the contact switch. The sample analysis cartridge according to item 87. (Item 90) The sample analysis cartridge according to item 87, wherein the contact switch is positioned such that activation of the contact switch indicates complete insertion of the sample collection device into the input tunnel. (Item 91) The sample analysis cartridge according to item 87, further comprising a sensor configured to be exposed to a fluid mixed with the sample, the sensor being further configured to generate a signal indicating at least one of the presence, absence, or amount of one or more analytes in the sample. (Item 92) A sample analysis cartridge, An input tunnel extending from an opening, the input tunnel being configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to a sample. A reservoir configured to hold a fluid. A shuttle defining a first compartment and a second compartment, the first and second compartments being configured to be disposed within the reservoir at a mixing position. An ultrasonic generator configured to emit acoustic waves to move the fluid within the reservoir in a wave pattern between the first and second compartments to mix the fluid within the reservoir. A sample analysis cartridge comprising. (Item 93) The sample analysis cartridge according to item 92, wherein the shuttle comprises a compartment divider configured to divide the first compartment from the second compartment. (Item 94) The sample analysis cartridge according to item 93, wherein the fluid flowing around the compartment divider facilitates formation of the wave pattern. (Item 95) The compartment divider of the sample analysis cartridge according to item 93 is provided with slots configured to allow the fluid to flow through the compartment divider via the slots during mixing. (Item 96) The compartment divider of the sample analysis cartridge according to item 93 is a flange. (Item 97) The sample analysis cartridge according to item 92 includes a reagent ball compartment configured to store a reagent ball containing a reagent, and a sample compartment configured to receive a sample from the sample collection device in the second compartment. (Item 98) The reagent ball of the sample analysis cartridge according to item 97 includes a polymerase, a primer, and a signaling substance. (Item 99) The first and second compartments of the sample analysis cartridge according to item 92 are not disposed in the reservoir at the pre-mixing position. (Item 100) The ultrasonic generator of the sample analysis cartridge according to item 92 includes a piezoelectric ceramic disk. (Item 101) The ultrasonic generator forms the wall of the reservoir in the sample analysis cartridge according to item 92. (Item 102) The reservoir of the sample analysis cartridge according to item 92 is symmetric. (Item 103) The walls of the reservoir each abut at an angle greater than 60°, promoting cavitation of the fluid through the outlet of the reservoir, in the sample analysis cartridge according to item 92. (Item 104) The ultrasonic generator is positioned offset from the center of the reservoir, promoting mixing of the fluid within the reservoir, in the sample analysis cartridge according to item 92. (Item 105) The sample analysis cartridge according to item 92, further comprising a printed circuit board coupled to the ultrasonic generator via one or more spring contacts. (Item 106) The sample analysis cartridge according to item 105, wherein the ultrasonic generator is electrically coupled to the printed circuit board only via the one or more spring contacts. (Item 107) The sample analysis cartridge according to item 92, wherein the ultrasonic generator is activated in response to a signal from a processor. (Item 108) The sample analysis cartridge according to item 92, further comprising a temperature sensor configured to sense a temperature indicative of a temperature of a fluid within the reservoir. (Item 109) The sample analysis cartridge according to item 108, wherein the temperature sensor is disposed on a printed circuit board positioned adjacent to the ultrasonic generator. (Item 110) The sample analysis cartridge according to item 92, further comprising a contact switch configured to indicate insertion of the sample collection device into the input tunnel, wherein the ultrasonic generator is configured to emit the acoustic wave after actuation of the contact switch. The sample analysis cartridge according to item 92. (Item 111) The sample analysis cartridge according to item 92, wherein the acoustic wave emitted by the ultrasonic generator is configured to isothermally amplify a reaction of a fluid mixed within the reservoir. (Item 112) A kit, A sample analysis cartridge, comprising a reservoir configured to hold a fluid and receive a sample collected by a sample collection device, an ultrasonic generator configured to emit an acoustic wave and mix the fluid and the sample within the reservoir, A temperature sensor configured to generate a signal indicative of the temperature of the fluid within the reservoir; A sample analysis cartridge comprising; A reader configured to be electrically coupled to the sample analysis cartridge, A processor configured to activate the ultrasonic generator, emit the acoustic wave, and monitor the signal from the temperature sensor, the processor further configured to modify the emission of the acoustic wave from the ultrasonic generator if the signal indicates a temperature of the fluid within the reservoir outside of a threshold; A reader comprising; A kit comprising. (Item 113) The kit according to item 112, wherein the sample analysis cartridge further comprises a printed circuit board, and the temperature sensor is disposed on the printed circuit board positioned adjacent to the ultrasonic generator. (Item 114) The sample analysis cartridge further comprises a contact switch configured to generate a signal indicative of insertion of the sample collection device into the input tunnel of the sample analysis cartridge, The processor of the reader is configured to receive the signal from the contact switch and activate the ultrasonic generator after receiving the signal from the contact switch. The kit according to item 112. (Item 115) The kit according to item 112, wherein the processor modifies the emission of the acoustic wave from the ultrasonic generator by decreasing the duty cycle of the ultrasonic generator if the signal indicates that the temperature of the fluid within the reservoir exceeds the threshold. (Item 116) The kit according to item 112, wherein the processor modifies the emission of the acoustic wave from the ultrasonic generator by increasing the duty cycle of the ultrasonic generator if the signal indicates that the temperature of the fluid within the reservoir is below the threshold. (Item 117) The kit according to item 112, wherein when the signal indicates that the temperature of the fluid in the reservoir exceeds the threshold value, the processor modifies the emission of the acoustic wave from the ultrasonic generator by deactivating the ultrasonic generator. (Item 118) The kit according to item 112, wherein the acoustic wave emitted by the ultrasonic generator is configured to isothermally amplify the reaction of the fluid mixed in the reservoir. (Item 119) The kit according to item 112, further comprising a reagent ball disposed in the sample analysis cartridge, wherein the ultrasonic generator is configured to emit the acoustic wave and mix the fluid, the reagent ball, and the sample in the reservoir. (Item 120) The kit according to item 119, wherein the reagent ball comprises a polymerase, a primer, and a signaling substance. (Item 121) The kit according to item 119, wherein the sample analysis cartridge further comprises a shuttle configured to store the reagent ball. (Item 122) A sensor for use in a microfluidic cartridge, A positive control working electrode comprising an affinity molecule pre-bonded to a positive control working electrode, wherein the positive control working electrode is configured to generate a first signal based on a reaction between a signaling substance and a chemical substrate directly or indirectly bonded to the affinity molecule. A positive control working electrode, A working electrode configured to generate a second signal based on a reaction between a signaling substance localized on the working electrode and the chemical substrate. A negative control working electrode comprising a self-assembled monolayer, wherein the negative control working electrode is configured to generate a third signal based on a reaction between a signaling substance localized on the negative working electrode and the chemical substrate. A negative control working electrode, A sensor comprising. (Item 123) The sensor according to item 122, wherein the second signal indicates at least one of the presence, absence, or number of the one or more analytes in the sample. (Item 124) The sensor according to item 122, wherein the first signal indicates the reliability of the test. (Item 125) The sensor according to item 124, wherein the test is determined to be reliable when the first signal indicates an amount of reaction within a predetermined range. (Item 126) The sensor according to item 122, wherein the third signal indicates the reliability of the test. (Item 127) The sensor according to item 126, wherein the test is determined to be reliable when the third signal indicates that the amount of reaction is below a threshold value. (Item 128) A cartridge comprising the sensor according to item 122, wherein the cartridge further comprises an analysis channel, The positive control working electrode, the working electrode, and the negative control working electrode are disposed within the analysis channel. Cartridge. (Item 129) A kit comprising the cartridge according to item 128, wherein the kit further comprises a processor configured to process the second signal and generate information indicating at least one of the presence, absence, or number of the one or more analytes in the sample. (Item 130) The kit according to item 129, wherein the processor is further configured to process the first signal and determine whether the first signal indicates an amount of reaction within a predetermined range. (Item 131) The kit according to item 130, wherein the processor is configured to generate an alert when the amount is outside the predetermined range. (Item 132) The kit according to item 129, wherein the processor is further configured to process the third signal and determine whether the third signal indicates that the amount of the reaction is below a threshold value. (Item 133) The kit according to item 132, wherein the processor is configured to generate an alert when the amount exceeds the threshold value. (Item 134) The kit according to item 129, wherein the processor is a component of the reader. (Item 135) The sensor according to item 122, wherein the working electrode is masked with a plurality of fine grooves configured to promote a homogeneous distribution of a plurality of magnetic particles directly or indirectly bound to the signaling substance localized across the working electrode and to promote resistance to movement of the plurality of magnetic particles from the working electrode. (Item 136) The sensor according to item 122, wherein the working electrode comprises a self-assembled monolayer. (Item 137) The sensor according to item 122, wherein the working electrode comprises an affinity molecule pre-bound to the working electrode. (Item 138) A method for isothermal amplification of a target nucleic acid in a sample analysis cartridge according to item 92, comprising: contacting an amplicon and a signaling substance directly or indirectly within the reservoir to form an amplicon-signaling substance complex, wherein the ultrasonic generator is configured to emit the acoustic wave towards the reservoir and to facilitate amplification of the target nucleic acid within the reservoir; reacting the amplicon-signaling substance complex with a substrate from a substrate reservoir; generating a signal indicating at least one of the presence, absence, or amount of the amplified nucleic acid; and a method comprising the steps of: (Item 139) The sample analysis cartridge according to item 92, further comprising a reagent ball held within the shuttle, wherein the reagent ball comprises a reagent for amplification of a target nucleic acid by isothermal reaction. (Item 140) The sample analysis cartridge according to item 139, wherein the reagent comprises a polymerase, a primer for amplification of the target nucleic acid, and a signaling substance for detection of amplification of the target nucleic acid. (Item 141) The sample analysis cartridge according to item 140, further comprising one or more affinity molecules covalently or non-covalently bound to solid particles for detection of the target nucleic acid.

Brief Description of the Drawings

[0097] Exemplary embodiments are described below with reference to the accompanying drawings, wherein like numerals indicate like elements.

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Figure 32-4

Figure 32-5

[0166]

Figure 33

[0167] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. The embodiments described in the drawings and the specification are illustrative and not intended to be limiting. As used herein, the term "exemplary" means "serving as an example or illustration" and should not necessarily be construed as preferred or advantageous over other embodiments. Other embodiments may be utilized and modifications may be made without departing from the spirit or scope of the subject matter presented herein. Aspects of the disclosure described and illustrated herein can be arranged, combined, and designed in a variety of different configurations, all of which are explicitly contemplated and form part of the disclosure.

[0168] The various devices, systems, kits, and methods disclosed herein are intended to isolate, tag, and detect a target analyte in a sample taken from a specimen. In certain embodiments, chemical reactions are employed to enable such detection.

[0169] Various embodiments of the systems described herein are designed to generate a self - contained environment in which any of the chemical reactions occur in an automated fashion, with little or no human intervention, as described, for example, in U.S. Patent Publication No. 2014 / 0336083 to Khattak, U.S. Patent No. 9,034,168 to Khattak, U.S. Patent No. 9,052,275 to Khattak, U.S. Patent No. 9,086,417 to Khattak, U.S. Patent No. 9,207,244 to Khattak, U.S. Patent No. 9,207,245 to Khattak, and U.S. Patent No. 9,360,491 to Sever, the entire contents of each of which are incorporated herein by reference. In some of the designs described herein, one or more chemical reactions proceed without any need for an operator to add or remove reagents to or from the system. In one embodiment, the system is enclosed so as to minimize biohazard risks such as the risk of overflowing samples collected from a specimen. In various embodiments, such a system includes at least a sample collection device, a cartridge device, and a reader device. Some exemplary embodiments of such devices are described in detail below.

[0170] Figures 1A and 1B illustrate an exemplary specimen detection system constructed in accordance with the principles of the present disclosure. The detection system 100 may include a sample collection device 200, a cartridge device 300, a reader device 400, a charger 500, and / or a software - based detection interface system 600. The detection system 100 may be used to detect the presence, absence, and / or amount of one or more target specimens.

[0171] The sample collection device 200 is configured to be exposed to a sample for analysis. For example, the sample collection device 200 may be exposed to a biological sample such as, but not limited to, blood, plasma, urine, saliva, mucosa, cell material, and / or other biological materials to determine the presence, absence, and / or amount of one or more target analytes in the sample. Additionally, or alternatively, the sample collection device may be exposed to a solid or other surface suspected of carrying the target analyte, for example, a foodborne pathogen and the surface is a cooking or food preparation surface.

[0172] The cartridge device 300 is configured to analyze a sample collected using the sample collection device 200. The cartridge device 300 may include an input tunnel 301 that extends into the cartridge housing from an opening 302. The input tunnel 301 is configured to allow insertion of the sample collection device 200, as shown in FIG. 1B, such that the collected sample can be analyzed within the cartridge device 300. Based on the analysis, the cartridge device 300 is configured to generate an electrical signal indicative of the presence, absence, and / or amount of one or more target analytes in the sample.

[0173] The reader 400 is configured for electrical coupling with the cartridge device 300 to enable transmission of an electrical signal indicative of the presence, absence, and / or amount of one or more target analytes in a sample generated by the cartridge device 300. The cartridge device 300 may be electrically coupled to the reader 400 by inserting the cartridge device 300 into the reader opening 401 of the reader 400 as shown in FIG. 1B such that the individual electrical connectors of the cartridge device 300 and the reader 400 come into contact with each other. The reader 400 may comprise a computer-readable medium that, when executed by a processor of the reader 400, causes the electrical components of the cartridge 300 to perform steps for analyzing a sample on the sample collection device 200, accompanied by instructions. Preferably, the instructions are not executed until, for example, as shown in FIG. 1B or 4A, the cartridge device 300 is electrically coupled to the reader 400 and the sample collection device 200 is suitably disposed within the cartridge device 300.

[0174] In one embodiment, the sample collection device 200 and the cartridge device 300 are each disposable and designed for single use, while the reader 400 is designed for multiple use and for receiving a number of different cartridge devices throughout the life of the reader 400 such that a number of samples are analyzed by the reader 400 to determine the presence, absence, and / or amount of one or more target analytes in an individual sample. Such a configuration is expected to facilitate hygienic use of the system since the components exposed to the sample are disposable, while reducing costs since components involving more expensive electronics, e.g., the reader 400, can be reused repeatedly.

[0175] The charger 500 is configured to charge one or more batteries within the reader 400 via, for example, an individual induction coil disposed within the housings of the charger 500 and the reader 400. The charger 500 may be plugged into a conventional outlet via, for example, a cord or a cord with an AC / DC power converter to charge components within the charger 500 and enable charging of the reader 400.

[0176] As will be readily apparent to those skilled in the art, the detection system does not require a charger. For example, referring to FIG. 1C, the detection system 100' is constructed similarly to the detection systems 100 of FIGS. 1A and 1B, and like components are identified by like primed reference numerals. Thus, for example, the cartridge device 300' in FIG. 1C corresponds to the cartridge devices 300 of FIGS. 1A and 1B. As will be observed by comparing FIGS. 1C and 1B, the detection system 100' does not include the charger 500. In such an embodiment, the reader 400' may be plugged into a conventional outlet via, for example, a cord or a cord with an AC / DC power converter to power the reader 400', and / or the components of the reader 400' may include a suitable battery such as a replaceable battery or a rechargeable battery, and the reader 400' may include a circuit and a removable power cord for charging the rechargeable battery.

[0177] In FIGS. 1A and 1B, a software-based detection interface system 600 is installed and launched on a computing device 601, enabling a user to review analyte detection test results, for example, on a display 602 of the computing device 601. The computing device 601 may be, for example, a smartphone, smartwatch, tablet, wearable device, laptop, or other computer. As shown in FIGS. 1A and 1B, a reader 400 may communicate wirelessly with the computing device 601 and transmit data indicating the presence, absence, and / or amount of one or more target analytes based on electrical signals generated within a cartridge device 300. Additionally, or alternatively, a removable wired connection, such as a cable connection, may be provided between the reader 400 and the computing device 601. The software-based detection interface system 600 may comprise a computer-readable medium that, when executed by a processor of the computing device 601, includes instructions to cause the display 602 to display information indicating the presence, absence, and / or amount of one or more target analytes. Sample Collection Devices and Cartridges

[0178] Sample collection devices of various embodiments are configured to collect samples from specimens. The sample collection devices may be configured to collect cells and other biological materials from any desired area or location, such as the inner cheek, throat, nasal cavity, ear, urine, blood, plasma, saliva, or another body part. One exemplary sample collection device includes a unit that sucks small droplets of blood or urine into a small capillary channel. In other embodiments, the sample collection devices may be configured to collect biological materials, particulate matter, or other chemical substances from the environment, such as from air or water, or from a physical surface or other structure.

[0179] Sample collection devices of various embodiments are sized and shaped to collect a sufficiently large sample from an appropriate location of a sample so as to be able to detect the presence, absence, and / or amount of a target analyte in and / or on the sample using other devices described below. For example, for some target analytes such as those associated with colds or influenza-like symptoms caused by viruses, the sample collection device may be a nasal swab, and the swab is sized and shaped to collect a sufficient amount of sample from an individual's nasal cavity so as to enable detection of a target analyte associated with a cold or influenza-like symptom caused by a virus if present in the individual. For example, for other target analytes such as those associated with streptococcal pharyngitis, the sample collection device may be a throat swab shaped to scrape a sufficient number of cells from an individual's throat or mouth. As another example, a sample collection device appropriate for collecting a target analyte associated with HIV may include a blood lancet. In another example, a sample collection device configured to collect urine may be appropriate for collecting target analytes for various tests, including tests for tracking testosterone levels, drug levels, vitamin levels, and / or fertility. A sample collection device for collecting a fluid such as urine, blood, plasma, or saliva may include features for expelling the sample absorbed on the suction portion for analysis by compressing the suction portion of the device. In yet a further aspect, the sample collection device is shaped to collect a sample from a solid surface, for example, from a medical device, a medical instrument, or the surface of a food preparation surface such as a cutting board or a flat surface.

[0180] Referring to FIGS. 2A and 2B, a sample collection device 200 is illustrated. The sample collection device 200 is configured to collect a small amount of sample to be analyzed and, after sample collection, is configured for full or partial insertion into a cartridge device 300. The sample collection device 200 may include a distal portion 201, a proximal portion 202, and a shaft 203 extending therebetween. The distal portion 201 may include a tip 204 having a tube 205 therein. The sample collection device 200 may also include a handle 206, a proximal seal zone 207, a distal seal zone 208, and / or an engagement zone 209.

[0181] The distal portion 201, including the tip 204, is configured to be exposed to the sample such that, at most, a predetermined volume of the sample is disposed within the tube 205 for analysis. Collection of the predetermined volume of the sample is expected to facilitate the accuracy of specimen analysis since a substantially known amount of the sample will be analyzed. The tip 204 may be transparent and may enable the collector to verify that the sample is disposed within the tube 205. The tip 204 may have a rounded end as illustrated, although various shapes, including any blunt or substantially blunt tip shape, may be used. The tip 204 may be configured to collect a sample from any desired area or location, for example, from the inner cheek, throat, mouth, nasal cavity, ear, urine, blood, plasma, saliva, or another body part.

[0182] The proximal portion 202 may include a handle 206 sized and shaped to be held by the collector's hand. The handle 206 may include gripping protrusions, as shown. The handle 206 may further lock the sample collection device 200 within the input tunnel of the cartridge device 300. The sample collection device 200 may also include a proximal seal zone 207 configured to seal the input tunnel of the cartridge device 300 when the sample collection device 200 is inserted into the input tunnel. The proximal seal zone 207 may extend around the shaft 203 and may include a protrusion sized to extend beyond the input tunnel opening to seal the input tunnel. Thus, the protrusion may further lock the sample collection device within the input tunnel of the cartridge device 300. The handle 206 may be detachable from the remainder of the sample collection device 200 in a breakable or otherwise removable manner following insertion of the sample collection device 200 into the remainder of the cartridge device 300.

[0183] The shaft 203 is elongated to facilitate easy and hygienic collection with the collector's hand removed from the collection site. For example, the shaft 203 may be elongated such that the tip 204 is exposed to the sample, such as from the inner cheek, throat, mouth, nasal cavity, ear, urine, blood, plasma, saliva, etc., and can collect fluids, cells, and other biological materials, while the handle 206 is not exposed to the sample. The shaft 203, tip 204, and handle 206 may be formed from the same material or different materials. The shaft 203, tip 204, and handle 206 may be formed from plastic. The sample collection device 200 may be pre-packaged within a sterile wrapper and is preferably configured for single use.

[0184] The sample collection device 200 may have a distal seal zone 208 to facilitate the formation of a liquid-tight seal between the sample collection device 200 and the cartridge device 300 after insertion of the sample collection device 200 into the cartridge device 300 of the sample collection device 200. For example, the distal seal zone 208 may be sized and shaped to seal the collected sample on the tip 204 and the fluid in the sample preparation reservoir of the cartridge device 300 within the cartridge device 300. The distal seal zone 208 may have a radial size that exceeds the tip 204. For example, the distal seal zone 208 may include a shoulder that extends further from the longitudinal axis of the sample collection device 200 than the tip 204 such that the shoulder abuts against a part of the cartridge device 300, such as a shuttle, to form a liquid-tight seal. In this way, the sample may be sealed within the cartridge device 300, reducing leakage and exposure of the sample outside the cartridge. Additionally, the shoulder may be used to move a seal piercer and vent one or more reservoirs within the cartridge device 300 before, during, or after the formation of the liquid-tight seal.

[0185] The sample collection device 200 may include an engagement zone 209 configured for engagement with one or more components of the cartridge device 300. For example, the engagement zone 209 may be permanently or temporarily coupled to a seal piercer of the cartridge device and configured to move the seal piercer within the cartridge device in response to movement of the sample collection device 200. The engagement zone 209 may also facilitate a fixed engagement between the sample collection device 200 and the cartridge device such that when the sample collection device 200 is inserted into the input tunnel of the cartridge to a predetermined distance, the sample collection device 200 meshes with the cartridge irreversibly and immovably. The engagement zone 209 may be a groove around the shaft 203, as illustrated, or a plurality of grooves extending a short distance from the longitudinal axis than the normal shaft surface, and / or one or more protrusions extending a greater distance from the longitudinal axis of the sample collection device 200.

[0186] In various embodiments, the cartridge is formed of a housing that defines an enclosed space and enables the cartridge to receive a sample with a target analyte from the sample collection device, store the sample with a sample preparation reagent, provide a space for mixing and binding the target analyte with the sample preparation reagent, provide an analysis zone where the bound target analyte localizes over a sensor for detection, provide a fluid medium for transporting the bound target analyte to the analysis zone, receive a detectable reaction when introduced to the bound target analyte, store and provide a substrate, provide a liquid medium for transporting the substrate to the bound target analyte within the analysis zone, and provide a waste collection zone where waste is stored, having various features enabling one or more of the foregoing.

[0187] In various embodiments, the cartridge is a substantially closed system in which the reactions necessary to detect the presence, absence, and / or amount of one or more target analytes within the cartridge occur. A cartridge of such an embodiment is said to be "substantially closed" because the only inputs required into the cartridge system are one or more of a sample from a specimen, energy to facilitate mixing and binding, and magnetic forces to facilitate localization of bound target analytes within the analysis zone, and the only output from the cartridge is an electrical signal. In various embodiments, the cartridge is analyte specific, with included sample preparation reagents selected to detect one or more specific target analytes. Different cartridge types contain different reagents, intended to identify different target analytes. For example, different cartridge types may include inflammation, influenza, testosterone, fertility, HIV, and vitamin D, each containing use-specific reagents intended to identify different target analytes.

[0188] Referring now to FIG. 3, an exemplary cartridge is illustrated. Cartridge device 300 may include an input tunnel 301 that extends into cartridge housing 304 from an opening 302 on front surface 303. Cartridge housing 304 may have a generally rectangular prism shape as illustrated, although the present disclosure is not limited thereto. Cartridge housing 304 has a front surface 303, an upper surface 305, a right side surface 306, a left side surface 307 (shown in FIG. 4A), a bottom surface 308 (shown in FIG. 4B), and a back surface 309 (shown in FIG. 4B). Cartridge housing 304 may be formed from a single component or multiple components. For example, cartridge housing 304 may include a first cover component 310 configured to be laterally coupled to a second cover component 311 such that the internal components of cartridge device 300 are housed therein.

[0189] Figures 4A and 4B illustrate the sample collection device 200 inserted into the input tunnel 301 of the cartridge device 300 at the mixing position. At the mixing position, the proximal seal zone 207 of the sample collection device 200 may seal the input tunnel 301 at the opening 302, reducing or eliminating leakage from the input tunnel 301. The input tunnel 301 may be integrally formed with the housing 304 of the cartridge device 300 or removably coupled to the cartridge housing 304. As shown in FIG. 4B, the cartridge device 300 may include an electrical connector 312 configured, for example, for electrical coupling with a reader via the electrical connector of the reader. Thus, a signal indicating the presence, absence, and / or amount of one or more target analytes may be transmitted from the cartridge device 300 to the reader via the electrical connector 312. The electrical connector 312 is positioned on the bottom surface 308 and the back surface 309 and may facilitate coupling with the electrical connector within the opening of the reader.

[0190] The bottom surface 308 of the cartridge device 300 may include a first lamp portion 313, a second lamp portion 314, and a magnetic generator recess 315. The first lamp portion 313 is configured to gradually depress one or more magnetic generators of the reader during insertion of the cartridge device 300 into the opening of the reader. The first lamp portion 313 may start from a recess of the cartridge housing 304 where the electrical connector 312 is positioned and descend to the bottom surface 308. As the cartridge device 300 is inserted beyond the first lamp portion 313, the magnetic generator rides into the magnetic generator recess 315 and remains in the depressed position until it contacts the second lamp portion 314. The second lamp portion 314 is configured to gradually guide one or more magnetic generators of the reader into the magnetic generator recess 315. The magnetic generator recess 315 is arranged directly below one or more working electrodes of the cartridge device 300 such that when the cartridge device 300 is fully inserted into the reader, one or more magnetic generators of the reader move upward into the magnetic generator recess 315 and are arranged adjacent to one or more working electrodes. The second lamp portion 314 also facilitates removal of the cartridge device 300 from the reader by gradually depressing one or more magnetic generators of the reader during cartridge removal.

[0191] Referring now to FIG. 5, an exploded view of the cartridge device 300 is shown. The cartridge device 300 may include an internal component 316 that may include a sample preparation reservoir 317, a cleaning agent reservoir 318, and a substrate reservoir 319, a sealing material 320, a seal piercing device 321 that may include a slider 322 and a piercer 323, a shuttle 324, a desiccant 325, an input tunnel component 326, an ultrasonic generator element 327, an absorption pad 328, a layer 329, an analysis channel 330, and a circuit board 331 electrically coupled to a memory 332. The internal component may be disposed within a housing 304, for example, between first and second cover components 310 and 311. Alternatively, one or more internal components may be disposed within one housing while other internal components may be disposed within a separate housing. In the case of multiple housings, such separate housings may be configured to couple to each other.

[0192] The internal component 316 is configured to define one or more reservoirs, by way of example, a sample preparation reservoir 317, a cleaning agent reservoir 318, and a substrate reservoir 319. The internal component 316 may further define a portion of the analysis channel 330, such as by creating an upper boundary of the analysis channel 330 when the cartridge device 300 is assembled. The internal component 316 may store other internal components, such as the absorption pad 328. Further, the internal component 316 may be formed from a suitable material such as plastic and may have a base dimensioned in a generally rectangular shape to seat over the circuit board 331.

[0193] The sample preparation reservoir 317 is configured to hold a fluid, preferably a liquid having a sample preparation reagent. For example, the fluid may be water, an aqueous saline solution, magnetic particles, affinity molecules, linker molecules, signaling substances, competitive binding molecules, competitor molecules, labels, and / or a water / aqueous saline solution mixed with one or more of the signaling substances, as will be described in more detail below. The sample preparation reservoir 317 is positioned adjacent to the distal end of the input tunnel 301 such that the input tunnel 301 is connected to the sample preparation reservoir 317. As will be further described below, the sample preparation reservoir 317 may be formed, in part, for example as part or all of the bottom surface, with an ultrasonic generator element 327, which facilitates mixing of the fluid and additional particles in the fluid. Additionally, the sample preparation reservoir 317 is formed, in part, with the end of the shuttle 324 during the pre-mixing state and, in part, with another portion of the shuttle 324 during the mixing state when one or more reagent balls and a sample are placed within the sample preparation reservoir 317. Thus, the sample preparation reservoir 317 is fluidically sealed by the shuttle 324 both in the pre-mixing state and in the mixing state and remains continuously fluidically sealed throughout the transition from the pre-mixing state to the mixing state so that fluid does not leak proximally past the shuttle 324. The sample preparation reservoir 317 is positioned such that, in response to insertion of the sample collection device 200 into the input tunnel 301 of the sample preparation reservoir 317, for example, the distal portion 201 having the sample enters the sample preparation reservoir 317 at the tip 204 and / or the tube 205. When the sample collection device 200 enters the sample preparation reservoir 317, the sample preparation reservoir 317 becomes further filled with sample particles, including one or more target analytes in the sample if present. The fluid may be gently mixed, for example via the ultrasonic generator element 327, with one or more reagent balls and the sample to suspend and hybridize the particles within the sample preparation reservoir 317.The target analyte in the sample can at least hybridize to and / or bind to magnetic particles and / or affinity molecules present between the sample preparation reagents, forming a magnetic particle-bound complex and / or an affinity molecule-target complex. The sample preparation reservoir 317 is configured to release, for example, via an outlet, a fluid having the sample and the sample preparation reagents mixed therein into the analysis channel 330 to analyze the presence, absence, and / or amount of one or more target analytes in the sample. The outlet of the sample preparation reservoir 317 may be sealed with a thermally actuated valve. When the valve is opened, the fluid from the sample preparation reservoir 317 acts as a transport medium to flow the magnetic particle-bound complex and / or the affinity molecule-target complex and other particles from the sample preparation reservoir 317 into the analysis channel 330. Advantageously, the fluid serving as the mixing medium and storage medium within the sample preparation reservoir 317 also acts as a flow medium for transporting the contents of the sample preparation reservoir 317 to the analysis zone within the analysis channel 330 without the need for a pump.

[0194] The cleaning agent reservoir 318 is configured to hold a fluid, preferably a liquid configured as a cleaning solution. The cleaning agent reservoir 318 is further configured to release the cleaning solution, for example, through an outlet, into the analysis channel 330 and to move particles or pre-bound surface affinity molecules in the previously released mixed fluid from the sample preparation reservoir 317 that are not bound to the magnetic particles away from the working electrode and / or the positive control working electrode within the analysis channel. The outlet of the cleaning agent reservoir 318 may be sealed with a thermally actuated valve. When the valve is opened, the cleaning solution flows from the cleaning agent reservoir 318 into the analysis channel 330, thereby removing all or substantially all of the unbound detector and / or unbound competitive binding agents from the analysis channel 330. On one aspect, most or all of the floating unbound molecules from the sample preparation reservoir 317 are washed from the analysis channel 330, reducing the likelihood that any significant non-specific binding and / or floating signaling agents, for example, significant non-specific signals generated by HRP, occur within the analysis zone of the analysis channel 330.

[0195] The substrate reservoir 319 is configured to hold a fluid, preferably a substrate solution comprising a substrate such as a chemical substrate. The fluid in the substrate reservoir 319 may contain a substrate that undergoes a reaction in the presence of a signaling substance from the sample preparation reservoir 317. For example, the substrate in the substrate reservoir 319 may undergo an oxidation reaction in the presence of an oxidase from the sample preparation reservoir 317. The fluid may be a substrate solution containing a substrate and a receptor molecule such as hydrogen peroxide, and an enzyme substrate such as tetramethylbenzidine (TMB) and / or o-phenylenediamine dihydrochloride (OPD) molecules. As an example, the substrate may be a commercially available enzyme-linked immunosorbent assay (ELISA) substrate. Preferably, the substrate is oxidizable and / or reducible. The receptor molecule may be configured to accept electrons taken from the substrate by the signaling substance during the reaction between the substrate and the signaling substance (thereby oxidizing the substrate). For example, when the receptor molecule is hydrogen peroxide, the oxidase reaction between the substrate, e.g., TMB, OPD, and the signaling substance, e.g., HRP, SBP, causes the receptor molecule to convert to another molecule (e.g., water) by taking electrons from the substrate and donating them to the receptor molecule (e.g., hydrogen peroxide) during the oxidase reaction. In some embodiments, hexacyanoferrate is used as the substrate (and reacted with a signaling substance that can be an oxidation dye such as methylene blue from the sample preparation reservoir 317). The substrate reservoir 319 is further configured to allow the fluid with the substrate to be released into the analysis channel 330, for example, via an outlet. The outlet of the substrate reservoir 319 may be sealed with a thermally actuated valve. When the valve opens, the fluid from the substrate reservoir 319 acts as a transport medium and flows the chemical substrate from the substrate reservoir 319 into the analysis channel 330.

[0196] One skilled in the art will understand that although three reservoirs are depicted, in various embodiments, the plurality of reservoirs may include two reservoirs or four or more reservoirs, and alternative spatial configurations may be employed. For example, the detergent reservoir 318 and the substrate reservoir 319 may be combined within a reservoir configured to hold a fluid that acts as a cleaning solution and has a chemical substrate. Additionally, the reservoirs are preferably pre-filled with the aforementioned individual fluids, but the present disclosure is not limited thereto, and one or more reservoirs may be empty in a non-use state and filled with the individual fluids during mixing.

[0197] A seal material 320 is configured to fluidically seal the fluid within one or more reservoirs. For example, the seal material 320 may fluidically seal the individual fluids within the sample preparation reservoir 317, the detergent reservoir 318, and the substrate reservoir 319. The seal material 320 may be a single piece of material configured to cover all the reservoirs as shown, or may be separate pieces, each configured to cover one or more reservoirs within the cartridge device 300. The seal material 320 may be any material capable of fluidically sealing a fluid, such as a foil. Preferably, the seal material 320 is a liquid-impermeable membrane.

[0198] The seal piercer 321 is configured to pierce the seal material 320 and vent to the fluid in the sample preparation reservoir 317, the cleaning agent reservoir 318, and / or the substrate reservoir 319. The seal piercer 321 may be contacted by the distal portion 201, for example, at the shoulder or engagement zone 209 of the sample collection device 200 within the input tunnel 301, and move within the housing 304 in response to the force applied by the sample collection device 200 to pierce the seal material 320 and vent to the fluid in the sample preparation reservoir 317, the cleaning agent reservoir 318, and / or the substrate reservoir 319. The seal piercer 321 may be disposed within the housing 304 and partially within the input tunnel 301. In one embodiment, the seal piercer 321 is configured to move in a first direction, for example, laterally, and a second direction, for example, perpendicularly, in response to insertion of the sample collection device 200 into the input tunnel 301 and pierce into the seal material 320.

[0199] The seal piercer 321 may be a single component or may include multiple components. By way of illustration, the seal piercer 321 includes a slider 322 and a piercer 323. The slider 322 is disposed within the housing 304 and may be partially disposed within the input tunnel 301. For example, the slider 322 may have an engaging element adapted to be disposed within the input tunnel 301. The engaging element is configured to temporarily or permanently couple to the sample collection device 200, for example, at the shoulder or engagement zone 209, and to enable movement of the slider 322 in response to insertion of the sample collection device 200 into the input tunnel 301. The engaging element may be sized to fit within the groove of the U-shaped engagement zone 209, for example, as illustrated, or to receive the protrusion of the engagement zone 209. The slider 322 is configured to move within the housing 304 in response to a force applied by the sample collection device 200 such that a collector distally pushes the sample collection device into the input tunnel 301, causing the seal material 320 to be pierced by the piercer 323 and resulting in venting of the fluid within the sample preparation reservoir 317, the cleaning agent reservoir 318, and / or the substrate reservoir 319. The piercer 323 may be one or more piercing elements with an end sharp enough to cut and open the seal material 320. As will be described in detail below, the piercer 323 may include three different piercers, each disposed within the housing 304 above one of the sample preparation reservoir 317, the cleaning agent reservoir 318, or the substrate reservoir 319. As the slider 322 moves within the input tunnel 301 as a result of insertion of the sample collection device 200, the slider 322 contacts the piercer 323 and moves the piercer 323 in a direction to pierce the seal material 320. In one embodiment, the slider 322 is configured to move in a first direction, for example, laterally / approximately parallel to the movement of the sample collection device 200, in response to insertion of the sample collection device 200 into the input tunnel 301, and to move the piercer 323 in a second direction, for example, vertically, to pierce into the seal material 320.

[0200] The shuttle 324 is configured to be disposed within the housing 304, preferably between the sample preparation reservoir 317 and the opening 302. For example, the shuttle 324 may be disposed within the input tunnel 301 such that when the cartridge is in an unused state, the distal end of the shuttle 324 forms the wall of the sample preparation reservoir 317 and seals fluid therein. The shuttle 324 may define one or more compartments configured to receive the collected sample from the sample collection device 200 when inserted into the input tunnel 301. The shuttle 324 may also define one or more additional compartments configured to store one or more reagent balls. The shuttle 324 may be configured to move within the housing 304 to a second position when subjected to a threshold force, which may occur, for example, by contacting the sample collection device 200 with the shuttle 324, such that one or more sample compartments having a sample and / or one or more reagent ball compartments having one or more reagent balls therein are disposed within the fluid in the sample preparation reservoir 317. The proximal end of the shuttle 324 may, together with the sample collection device 200, reform the wall of the sample preparation reservoir 317 and seal fluid therein when the sample and / or one or more reagent balls are within the sample preparation reservoir 317. Thus, the sample preparation reservoir 317 remains fluidly sealed by the shuttle 324 both in the unused state and in the mixed state. Additionally, unlike a frangible membrane that stores a reagent, the shuttle 324 may remain intact as the sample is moved into the sample preparation reservoir 317 for analysis.

[0201] The desiccant 325 may be disposed within the housing 304 and in fluid communication with one or more reagent balls stored within the shuttle 324. The desiccant 325 is configured to absorb moisture entering into the housing 304 and reduce moisture exposure to one or more reagent balls in the non-use state. The desiccant 325 may be a pad and may be disposed at least partially within the input tunnel 301. The desiccant 325 may have a lumen sized to allow a sample collection device to be inserted therethrough.

[0202] The input tunnel component 326 forms a part of the input tunnel 301 and is sized and shaped to secure the shuttle 324 within the input tunnel 301. For example, the input tunnel component 326 may have a U shape and may house a generally cylindrical shuttle.

[0203] The ultrasonic generator element 327 is disposed within the housing 304, preferably adjacent to or integrally formed with the sample preparation reservoir 317, to enable mixing of fluids therein. The ultrasonic generator element 327 is configured to transmit a controlled amount of energy into the sample preparation reservoir and may include a piezoelectric component. The ultrasonic generator element 327 may be disposed on or form the bottom wall of the sample preparation reservoir 317. The ultrasonic generator element 327 may be electrically insulated from other electrical components within the housing 304, such as components on a circuit board 331 that include sensors and heaters, for example, via a relay. The ultrasonic energy may be controlled to achieve mixing and binding of components within the sample preparation reservoir 317 while limiting damage to fragile DNA probes or other molecules such as antibodies and enzymes. The ultrasonic generator element 327 may include a piezoresistive piezoelectric disk. The ultrasonic generator element 327 may also include a high moisture content blister disposed between the sample preparation reservoir 317 and the piezoelectric disk. Such a high moisture content blister may be attached under the sample preparation reservoir 317 in the cartridge production process. The high moisture content blister may facilitate delivery of ultrasonic energy from the ultrasonic generator element 327 to the sample preparation reservoir 317 with minimal attenuation. The blister may be replaced with another suitably conductive ultrasonic medium, and the component serving as the ultrasonic medium may be dry on the outside and free of liquid residue.

[0204] The absorption pad 328 is disposed within the housing 304 at the most downstream end of the analysis channel 330. The absorption pad 328 sucks up the fluid from the analysis channel 330, thereby promoting the fluid to flow downstream of the absorption pad 328. The absorption pad 328 may act as a waste receptacle and collect all waste fluid and waste particles after flowing through the analysis channel 330. The size and degree of absorbency of the absorption pad 328 may be selected to meter the flow rate of the fluid and particles within the analysis channel 330. For example, the volume of fluid that the absorption pad 328 can suck up must be large enough to drain all the fluid from the sample preparation reservoir 317 and the cleaning agent reservoir 318 and draw in the fluid carrying the chemical substrate from the substrate reservoir 319. Such conditions may serve as a lower limit for absorbency.

[0205] The layer 329 is disposed between the internal component 316 and the circuit board 331 and forms part of the analysis channel 330. The layer 329 may be an adhesive layer configured to couple the internal component 316 to the circuit board 331. For example, the layer 329 may be a double-sided adhesive tape that may be hydrophilic to support capillary flow of the fluid.

[0206] The analysis channel 330 may be defined by the walls of the internal component 316, the walls of the layer 329, and / or the walls of the circuit board component 331. For example, the upper wall of the analysis channel 330 may be defined by the internal component 315, the side walls of the analysis channel 330 may be defined by the layer 329, and the bottom wall of the analysis channel 330 may be defined by the circuit board 331. Additionally, each reservoir 317, 318, 319 includes an outlet that connects the reservoir to the analysis channel 330. Thus, the fluid within each of the reservoirs can flow into the analysis channel 330 through its respective outlet. The analysis channel 330 may extend from the reservoir to the absorption pad 328. Preferably, one or more sensors on the circuit board 331 are positioned at least partially within the analysis channel 330.

[0207] The circuit board 331 is disposed within the housing 304 and may be coupled to the internal component 316, for example, via the layer 329. The circuit board 331 includes electrical components, such as one or more than one of resistors, electrical conductors, vias, and sensors required for detection of the target analyte. Although described separately, it should be understood that the electrical components of the circuit board 331 need not be distinct structural elements. One or more than one electrical component and / or circuit may perform some or all of the roles of the various components described herein.

[0208] The memory 332 is disposed within the housing 304 and electrically coupled to the circuit board 331. The memory 332 may be any type of memory suitable for storing data related to the cartridge device 300, such as an EPROM, EEPROM, flash memory, or the like. The memory 332 may store data such as cartridge type (e.g., inflammation, influenza, testosterone, fertility, vitamin D), cartridge identification information (e.g., manufacturing number), and / or information regarding calibration information. When the cartridge device 300 is electrically coupled to the reader device 400, the reader device 400 may receive data transmitted from the memory 332 and use such data to facilitate determination of the presence, absence, and / or amount of one or more target analytes. In one embodiment, one or more cartridge devices of a selected group of cartridges (e.g., a common lot of production cartridges) may be tested using a known amount of a target analyte and the electrical characteristics associated with one or more target analytes sensed by the sensors of the device being tested may be determined. Calibration information based on the test results may be stored in the memory 332 within the selected group of cartridges and the electrical signals generated by the sensors of the cartridges and the calibration information may be used to accurately and consistently determine the presence, absence, and / or amount of one or more target analytes. The memory 332 may also store test result reliability information of a predetermined range of parameters, such as voltage, current, etc., that can be compared with the electrical signals generated by the positive control working electrode and determine whether the parameters are within a predetermined range.

[0209] Referring now to FIGS. 6A, 6B, and 6C, an exemplary circuit board and layers are illustrated, FIG. 6A depicts the circuit board 331, FIG. 6B depicts the layer 329, and FIG. 6C depicts the layer 329 disposed on and coupled to the circuit board 331.

[0210] As shown in FIG. 6A, the circuit board 331 may include a sensor 338 that may include heating elements 333, 334, 335, 336, and / or 337, and reference electrodes 339, working electrode 340, counter electrode 341, background working electrode 342, and / or reference electrode 343. The working electrode 340 may be masked by one or more than one narrow groove 344, and the background working electrode 342 may be masked by one or more than one narrow groove 345. The circuit board 331 may further include contacts 346 and 347 for electrically coupling the circuit board 331 to the ultrasonic generator element 327 via wires. However, the ultrasonic generator element 327 may also be electrically coupled to the circuit board 331 using spring contacts, as described below.

[0211] The heating elements 333, 334, 335, 336, and 337 are configured to generate heat in the housing 304 based on an electrical signal transmitted from the reader 400 at a time specified by a protocol stored in the memory of the reader 400, for example. Each of the heating elements 333, 334, 335, 336, and 337 may form part of the circuit board 331. For example, the heating elements 333, 334, 335, 336, and 337 may be resistive heating elements that appear as serpentine traces located on the bottom side of the circuit board 331 surrounding vias. In other embodiments, the heating elements are located, for example, on the reader, outside the cartridge. In various embodiments where resistive heating elements are used to generate heat, for example, current is allowed to flow through the resistive heating elements through the operation of transistors. The current passing through the resistive heating elements generates heat through Joule heating. The heat is conducted to the vias by physical contact between the resistive heating elements and the vias. The heating elements 333, 334, 335, 336, and 337 may be masked, for example, with a solder mask to promote electrical insulation from the sensor 338 and maintain heat transfer while minimizing interference with the electrical signals sensed by the sensor 338.

[0212] The heating element 333 may be positioned adjacent to the outlet of the sample preparation reservoir 317. The outlet may have a phase change material therein for closing the entire cross-section of the outlet, thereby fluidly sealing the outlet. The heating element 333 may be configured to heat the phase change material within the outlet of the sample preparation reservoir 317 such that the phase change material unseals the outlet of the sample preparation reservoir 317 and enables the fluid to mix the sample held within the sample preparation reservoir 317 therein and flow into the analysis channel 330. The heating element 333 may be caused to heat the phase change material at a time specified by a protocol stored in the memory of the reader 400, for example, after the reader 400 detects the cartridge device 300 to which it is electrically coupled thereto and after the reader 400 detects proper insertion of the sample collection device 200 into the cartridge device 300.

[0213] The heating element 334 may be positioned adjacent to the outlet of the cleaning agent reservoir 318. The outlet may have a phase change material therein for closing the entire cross-section of the outlet, thereby fluidly sealing the outlet. The heating element 334 may be configured to heat the phase change material within the outlet of the cleaning agent reservoir 318 such that the phase change material unseals the outlet of the cleaning agent reservoir 318 and enables the cleaning solution held within the cleaning agent reservoir 318 to flow into the analysis channel 330. The heating element 334 may be caused to heat the phase change material at a time specified by a protocol stored in the memory of the reader 400, for example, after a predetermined time after the reader 400 causes the heating element 333 to heat and / or after a predetermined time after the reader 400 causes the heating element 336 to heat.

[0214] The heating element 335 may be positioned adjacent to the outlet of the substrate reservoir 319. The outlet may have a phase change material therein to block the entire cross-section of the outlet, thereby fluidly sealing the outlet. The heating element 335 may be configured to heat the phase change material within the outlet of the substrate reservoir 319 such that the phase change material unseals the outlet of the substrate reservoir 319 and enables the fluid retained within the substrate in the substrate reservoir 319 to flow into the analysis channel 330. The heating element 335 may heat the phase change material at a time defined by a protocol stored in the memory of the reader 400, for example, after a predetermined time from when the reader 400 heats the heating element 334.

[0215] The heating element 336 may be positioned adjacent to a fluid breaker, which may comprise a phase change material. The heating element 336 may be configured to heat the phase change material of the fluid breaker after the outlet of the sample preparation reservoir 317 is unsealed such that the phase change material of the fluid breaker flows into the analysis channel 330 and fluidly isolates the sample preparation reservoir 317 from the substrate reservoir 319. The heating element 336 may heat the phase change material at a time defined by a protocol stored in the memory of the reader 400, for example, after a predetermined time from when the reader 400 heats the heating element 333.

[0216] The heating element 337 may be positioned adjacent to a pocket of gas, such as air, within the analysis channel 330. The heating element 337 may be configured to heat the pocket of air, expand the air, apply pressure to the phase change material, thereby facilitating the movement of the phase change material within the analysis channel 330. The heating element 336 may heat the phase change material at a time defined by a protocol stored in the memory of the reader 400, for example, after a predetermined time from when the reader 400 heats the heating element 333. The placement of the heating element 337 downstream of the heating elements 333, 334, and 335 is expected to reduce bubble formation within the analysis channel 330.

[0217] The electrical conductors (shown in FIG. 8) of the circuit board 331 may be provided to establish electrical connection and continuity with the reader device. The electrical conductors may be electrically coupled to the sensor 338, which includes heating elements 333, 334, 335, 336, 337, reference electrode 339, working electrode 340, counter electrode 341, background working electrode 342, and reference electrode 343, contacts 346, 347, as well as to the memory 332. Thus, such components may receive current when activated by the reader device. Advantageously, the electrical conductors are exposed at the electrical connector portion on the bottom surface of the circuit board 331 (shown in FIG. 4B), but the electrical conductors that electrically couple the connectors to the components may be traceless on the upper surface of the circuit board 331, as shown in FIG. 6A. The traceless configuration of the circuit board 331 between the electrical connector portion and these components creates a smooth upper surface of the circuit board 331, reduces bonding interference, thereby facilitating adherent bonding with the layer 329. Bonding interference can cause leakage when fluid enters the analysis channel 330 due to deformation of the layer 329 caused by such interference.

[0218] The heating elements 333, 334, 335, 336, 337 may be formed from a conductor and may each include a via. Vias are standard products on a printed circuit board and are typically used to enable electrical continuity between signal traces on one layer of the circuit board and another layer. Vias provide electrical continuity through multiple layers. Such vias are excellent conductors of heat, and because the surrounding materials that make up most of the circuit board are excellent insulators, they enable heat transfer to very precise locations without affecting the surrounding area. Thus, in various embodiments, a plurality of vias are provided as heating elements within the circuit board 331, and each via is disposed under, above, or adjacent to a phase change heat actuated valve disposed within the reservoir outlet to generate a valve actuating element. The accuracy of heat transfer associated with the vias allows for minimal crosstalk between valves positioned in close proximity to each other, and thus, the timing of valve actuation can be carefully controlled for each valve. The valve may be formed from a solder, such as a phase change material like a hydrophilic solder, and the via acts as a conductor of heat to melt the solder at a precise point as controlled by the reader device. In response to a phase transition, such as the melting of a solder valve disposed within the outlet of the reservoir, the outlet is no longer blocked and the reservoir has an opening through which its fluid contents can be discharged into the analysis channel. The holes within the via may be filled with a filling material, such as solder, and the via may be masked, for example, with a solder mask, to promote electrical insulation from the sensor 338 and maintain heat transfer while minimizing interference with the electrical signal sensed by the sensor 338.

[0219] To ensure complete melting of the solder at precise timings, in various embodiments, the solder valve is carefully constructed within the outlet of the reservoir. For example, in some embodiments, it is preferred that the solder valve has a minimum height necessary to block the outlet of the reservoir, and the minimum height minimizes the distance that heat must travel to melt the solder. One exemplary method for achieving a solder barrier with such characteristics involves applying the melted solder to a preheated via. Advantageously, when the via is preheated, it takes longer for the solder valve to solidify against the room-temperature via, and thus the solder has more time to flatten and expand outwardly before hardening. The "pancaking" of the solder is desirable to minimize the height, which will maximize the chance of proper melting operation of the valve. Additionally, heating the via promotes a greater level of contact area between the solder and the via such that most of the solder receives heat and maximizes the chance of proper valve operation. The method of heating the via prior to deposition of the solder is further enhanced by the following method. When the solder is applied to the preheated via such that the molten solder adheres to the plurality of inner walls of the reservoir and the via itself simultaneously as the solder hardens, the opening at the bottom of the reservoir is aligned to cover the via such that the opening of the reservoir is spatially close to the via. This is advantageous for increasing the manufacturing yield of a pristine valve that completely blocks the opening to the analysis channel so that inadvertent fluid flow from the reservoir does not occur.

[0220] Sensor 338 may be configured to be exposed to the fluid within the analysis channel 330 and generate a signal indicative of the presence, absence, and / or amount of one or more analytes in the sample. Sensor 338 may detect an electrical signal resulting from a chemical reaction across sensor 338. For example, the mixed fluid from sample preparation reservoir 317 may be introduced into analysis channel 330, and signaling agents in the mixed fluid may localize across sensor 338 (e.g., in response to a magnetic field holding magnetic particles directly or indirectly bound to the signaling agent, if present). The chemical reaction may occur when the fluid from substrate reservoir 319 reacts with particles of the mixed fluid from sample preparation reservoir 317 localized across sensor 338. For example, a substrate solution having a substrate may be introduced from substrate reservoir 319, and sensor 338 may detect an electrical signal resulting from a reaction between a substrate (e.g., TMB, OPD) localized across sensor 338 and a signaling agent (e.g., HRP, SBP). The reaction may cause electrons to be stripped from the substrate by the signaling agent (the electrons may be donated to a receptor molecule from the substrate solution), thereby generating an electrical signal detectable by sensor 338. Such detected electrical signals may be used to generate a signal indicative of the presence, absence, and / or amount of one or more analytes in the sample. The signal may be transmitted to reader device 400, for example, via individual electrical connectors of cartridge device 300 and reader device 400.

[0221] Sensor 338 may include a reference electrode 339, a working electrode 340, a counter electrode 341, a background working electrode 342, and / or a reference electrode 343. Sensor 338 is disposed within analysis channel 330, and the area of analysis channel 330 above sensor 338 may be referred to as the "analysis zone". Sensor 338 is included within cartridge 300 in which circuit board 331 is assembled, and is strategically positioned such that when the surface of circuit board 331 forms one wall of analysis channel 330, sensor 338 is disposed at least partially within analysis channel 330. Although one sensor is illustrated, a plurality of sensors may be provided, spaced apart from each other, and preferably all aligned within analysis channel 330. Additionally, working electrode 340 and background working electrode 342 may be arranged upstream and downstream of each other or vice versa, and sensor 338 may include additional working electrodes other than working electrode 340 and background working electrode 342.

[0222] Sensor 338 may be an electrochemical sensor that forms an electrochemical cell within analysis channel 330. Reference electrode 339 may be configured to generate a voltage difference between itself and working electrode 340. Counter electrode 341 may provide electrons (e.g., electrons taken from a substrate by a signaling agent) that collect on working electrode 340 when the electrical environment generated by reference electrode 339 and working electrode 340 brings positive charge across working electrode 340. As described above, an oxidation reaction can occur at sensor 338 when an oxidase (e.g., HRP, SBP, or other signaling agents described herein that may be introduced from sample preparation reservoir 317 into analysis channel 330) that is indirectly bound to particles (e.g., magnetic particles that may be introduced from sample preparation reservoir 317 into analysis channel 330) is present at the sensor, and an appropriate chemical substrate (e.g., TMB, OPD) is introduced into analysis channel 330 (e.g., from substrate reservoir 319). In such an embodiment, working electrode 340 emits electrons and replenishes the electrons taken from the substrate by the oxidase in an amount proportional to the amount of oxidase present. The emission of electrons from working electrode 340 (e.g., from a substrate that reacts with a signaling agent across working electrode 340) is a current that may be detectable as a signal within a circuit connected to sensor 338. Sensor 338 can thereby indirectly detect the presence, absence, and / or amount of oxidase localized within the analysis zone. For example, a processor within a reader device described below can then correlate the presence, absence, and / or amount of one or more target analytes with the presence, absence, and / or amount of the oxidase. The functionality of such a processor is described in further detail below. One or more magnetic fields may be used to facilitate the localization of an enzyme or other signaling agent within the analysis zone. Advantageously, in such embodiments, no affinity molecules need to be pre-bound to sensor 338 to achieve localization, which would otherwise significantly slow the analyte quantification process due to the limits of diffusion-based hybridization reaction rates. Details of the magnetic fields are also provided below.

[0223] Sensor 338 may include a gold surface fabricated through an ENIG process. In other embodiments, a gold or gold-plated sensor not fabricated through the ENIG process is used. One of ordinary skill in the art can understand that there are many plating processes for the catalytic and autocatalytic deposition of gold utilized to generate electrically active pads in the printed circuit board industry. Working electrode 340 may have a surface chemistry formed from a self-assembled monolayer such as dithiols, such as thiolated ethylene glycol and / or hexaethylene glycol dithiol, for additional stability. The hydrophilic nature of the head groups of such surface chemistries promotes fluidity and protein resistance. Additionally, or alternatively, the surface of one or more of the electrodes may be backfilled with mercaptoundecanoic acid, mercaptohexanol, or any other backfilling agent. The surface of one or more of the electrodes within sensor 338 may be formed at a non-high temperature through the continuous addition and incubation of ethylene glycol dithiol and a backfilling agent.

[0224] Background working electrode 342 may be a surrounding electrochemical noise sensor spaced within analysis channel 330 away from the site of magnetic particle localization. Background working electrode 342 may be used to quantify background noise downstream or upstream of working electrode 340 depending on the selected order of working electrode 340 and background working electrode 342 within analysis channel 330. Such noise may result, for example, from the presence of nonspecifically bound enzymes. During processing of the detection results, the processor in reader 400 may apply an algorithm to remove the background working electrode signal (from background working electrode 342) from the detection sensor signal (from working electrode 340), address, and / or eliminate system noise, thereby enabling proper quantification or detection of one or more target analytes. The signal from background working electrode 342 may be used for error detection and diagnosis of a malfunctioning cartridge, such as being evident by a signal from background working electrode 342 having an electrical value outside a predetermined range.

[0225] The reference electrode 343 may be configured to generate a voltage difference between itself and the background working electrode 342. The counter electrode 341 may also provide electrons that collect on the background working electrode 342 when the electrical environment generated by the reference electrode 343 and the background working electrode 342 results in a positive charge across the background working electrode 342.

[0226] In some embodiments, detection is performed using a standard electrochemical circuit that utilizes the bias potential generated at the background working electrode 342 for the oxidation / reduction reaction to proceed. The potential is maintained at the reduction potential of the chemical substrate (low enough such that there is some non-specific reduction of reducible species in solution) so that the flow of current to the oxidizing molecules can be quantified using an operational amplifier-based current / voltage (op-amp) circuit topology within a reader device 400 electrically connected to the working electrode 340.

[0227] A common substrate molecule, namely, tetramethylbenzidine, may be used for HRP. When present, HRP oxidizes the TMB molecules, which are then reduced by the working electrode 340. This event occurs in proportion to the amount of HRP present and, thus, in proportion to the amount of the target analyte present, resulting in a change in the current / voltage op-amp measurement. An analog / digital converter can be used to deliver the actual signal to a processor for processing. As will be described in more detail below, in various embodiments, the processor and signal processing components are provided within the reader device.

[0228] The working electrode 340 is masked with a plurality of fine grooves 344, for example, solder masked, configured to facilitate the homogeneous distribution and retention of a plurality of magnetic particles released from the sample preparation reservoir 317 across the working electrode 340. The accuracy of the analyte detection may be adversely affected by the premature washing of the magnetic particles during the analysis, for example, due to the force exerted on the fluid with the particles and / or chemical substrate from the substrate reservoir 319 in the analysis channel flow direction caused by the release of the washing solution from the detergent reservoir 318 exceeding the magnetic force towards the magnetic generator of the reader 400 disposed directly below the working electrode 340 during the analysis. Such fine grooves 344 facilitate the resistance to the movement of the plurality of magnetic particles from the working electrode 340. Additionally, the background working electrode 342 is masked with a plurality of fine grooves 345, for example, solder masked, although such fine grooves are not necessary on the background working electrode 342 and are merely exemplary of an alternative embodiment.

[0229] In an alternative embodiment, the sensor 338 may be configured to analyze the fluid within the analysis channel 330 and generate a signal indicative of the presence, absence, and / or amount of one or more analytes in the sample, and the signal is visible. For example, the housing of the cartridge device includes a window that enables the user to visually inspect, for example, using a camera, fluorescence, quantifying the fluorescence, and determining the presence, absence, and / or amount of one or more analytes in the sample.

[0230] Referring now to FIG. 6C, layer 329 is disposed on the upper surface of circuit board 331 such that sensor 338 is at least partially disposed within analysis channel 330. Heating elements 333, 334, 335, 336, and 337 may be partially or fully covered by masks 348, 349, 350, 351, and 352, respectively. Masks 348, 349, 350, 351, and 352 may be solder masks. Mask 348 promotes electrical insulation between heating element 333 and sensor 338 and maintains heat transfer from heating element 333 to the phase change material within the outlet of sample preparation reservoir 317 at an energy level sufficient to cause a phase change of the material while minimizing interference with the electrical signal sensed by sensor 338 due to heating element 333. Mask 349 promotes electrical insulation between heating element 334 and sensor 338 and maintains heat transfer from heating element 334 to the phase change material within the outlet of detergent reservoir 318 at an energy level sufficient to cause a phase change of the material while minimizing interference with the electrical signal sensed by sensor 338 due to heating element 334. Mask 350 promotes electrical insulation between heating element 335 and sensor 338 and maintains heat transfer from heating element 335 to the phase change material within the outlet of substrate reservoir 319 at an energy level sufficient to cause a phase change of the material while minimizing interference with the electrical signal sensed by sensor 338 due to heating element 335. Mask 351 promotes electrical insulation between heating element 336 and sensor 338 and maintains heat transfer from heating element 336 to the phase change material of the fluid breaker at an energy level sufficient to cause a phase change of the material while minimizing interference with the electrical signal sensed by sensor 338 due to heating element 336. Mask 352 promotes electrical insulation between heating element 337 and sensor 338 and maintains heat transfer from heating element 337 to the pocket of gas within analysis channel 330 above heating element 337 at an energy level sufficient to cause downstream movement of the phase change material within analysis channel 330 while minimizing interference with the electrical signal sensed by sensor 338 due to heating element 337.

[0231] Referring now to FIGS. 7A, 7B, and 7C, circuit board 331' and layer 329' are constructed in a similar manner to circuit board 331 and layer 329 of FIGS. 6A, 6B, and 6C, except that heating elements 333', 334', 335', 336', and 337' are positioned in a different configuration on circuit board 331', and analysis channel 330' is reshaped as appropriate. Additionally, FIG. 7C depicts absorption pad 328, which is coupled to layer 329' at the downstream end of analysis channel 330'.

[0232] Referring now to FIGS. 7D and 7E, an alternative exemplary sensor that can be used within the cartridge described herein is provided. Sensor 338'' may include reference electrode 339'', working electrode 340'', counter electrode 341'', negative control working electrode 342'' (also referred to herein as a background working electrode), and / or positive control working electrode 376. Sensor 338'' may detect an electrical signal generated by a chemical reaction in sensor 338'' in the same manner as described above with respect to sensor 338. Sensor 338'' is disposed within the analysis channel in the same manner as the aforementioned sensor 338. Although one sensor is illustrated, a plurality of sensors may be provided, spaced apart from one another, and preferably, all are aligned within the analysis channel. Preferably, fluid flows from a reservoir within the analysis channel and progresses across the electrodes in the order of positive control working electrode 376, reference electrode 339'', counter electrode 341'', working electrode 340'', and negative control working electrode 342''.

[0233] Sensor 338'' may be an electrochemical sensor that forms an electrochemical cell within the analysis channel. The positive control working electrode 376 may have an affinity molecule pre-bound to the surface of the positive control working electrode 376 and achieve localization of an oxidase or other signaling substance across the positive control working electrode 376. The affinity molecule may be a surface-bound antibody. The positive control working electrode 376 may be configured to detect a current generated by a reaction between an oxidase or other signaling substance indirectly bound to the affinity molecule and an appropriate chemical substrate introduced into the analysis channel, for example, from a substrate reservoir. In such an embodiment, the positive control working electrode 376 emits electrons and replenishes the electrons taken from the substrate by the oxidase in an amount proportional to the amount of oxidase present. The emission of electrons from the positive control working electrode 376 may be a current that can be detected as a signal within a circuit connected to the sensor 338''. For example, a processor within a reader device described below may process the signal to determine whether the signal indicates that the amount of oxidase or other signaling substance is within a predetermined range that can be stored in the memory of the cartridge and / or the reader device. If the detected amount is within the range, the processor may verify the cartridge, continue signal processing, and determine the presence, absence, and / or amount of one or more target analytes in the sample. The signal from the positive control working electrode 376 may be used for error detection and diagnosis of a malfunctioning cartridge, for example, to be evident from a signal from the positive control working electrode 376 having an electrical value outside a predetermined range. For example, the processor of the reader may generate an error alert if the signal from the positive control working electrode 376 is outside a predetermined range and / or may consider the read value from the working electrode 340'' acceptable if it is within a predetermined range.

[0234] The reference electrode 339'' may be configured to generate a voltage difference between itself and the working electrode 340''. The counter electrode 341'' may provide electrons that collect on the working electrode 340'' when the electrical environment generated by the reference electrode 339'' and the working electrode 340'' brings positive charge across the working electrode 340''. The reference electrode 339 and / or the counter electrode 341'' may have surface chemistry formed from self-assembled monolayers of dithiols such as thiolated ethylene glycol and / or hexamethylene glycol dithiol for additional stability. The hydrophilic nature of the head groups of such surface chemistries promotes fluidity and protein resistance. Additionally, or alternatively, the surface of the reference electrode 339 and / or the counter electrode 341'' may be backfilled with mercaptoundecanoic acid, mercaptohexanol, or any other backfilling agent.

[0235] Sensor 338'' may be used when magnetic particles are not present in the cartridge. For example, working electrode 340'' may have an affinity molecule pre - bound to the surface of working electrode 340'', and may achieve localization of an oxidase or other signaling substance across working electrode 340''. The affinity molecule may be a surface - bound antibody. Working electrode 340'' may be configured to detect a current generated by a reaction between an oxidase or other signaling substance indirectly bound to the affinity molecule and a suitable chemical substrate introduced, for example, from a substrate reservoir into the analysis channel. In such an embodiment, working electrode 340'' emits electrons and replenishes the electrons taken from the substrate by the oxidase in an amount proportional to the amount of oxidase present. The emission of electrons from working electrode 340'' may be a current that can be detected as a signal within a circuit connected to sensor 338''. Sensor 338 can thereby indirectly detect the presence, absence, and / or amount of oxidase localized within the analysis zone. For example, a processor within a reader device, described below, can then correlate the presence, absence, and / or amount of one or more target analytes with the presence, absence, and / or amount of the oxidase. The function of such a processor is described in more detail below.

[0236] As an illustration, working electrode 340'' does not include a plurality of fine grooves because sensor 338'' can be used to detect the presence, absence, and / or amount of one or more target analytes without using magnetic particles.

[0237] The negative control working electrode 342'' may be a surrounding electrochemical noise sensor spaced apart within an analysis channel away from the site of localization. The negative control working electrode 342'' may be used to quantify background noise downstream of the working electrode 340''. Such noise may result, for example, from the presence of non-specifically bound enzymes. During processing of the detection results, a processor in the reader 400 applies an algorithm to remove the negative control working electrode signal (from the negative control working electrode 342'') from the detection sensor signal (from the working electrode 340''), to address and / or eliminate system noise, thereby enabling proper quantification or detection of one or more target analytes. The signal from the negative control working electrode 342'' may be used for error detection and diagnosis of a malfunctioning cartridge, such as being evident by a signal from the negative control working electrode 342'' having an electrical value outside a predetermined range. For example, the processor of the reader may generate an error alert if the signal from the negative working control electrode 342'' exceeds a threshold and / or may consider the read value from the working electrode 340'' acceptable if it is below the threshold.

[0238] The negative control working electrode 342'' may have surface chemistry formed from a self-assembled monolayer of dithiols such as thiolated ethylene glycol and / or hexamethylene glycol dithiol for additional stability. The hydrophilic nature of the head groups of such surface chemistry promotes fluidity and protein resistance. Additionally or alternatively, the surface of the negative control working electrode 342'' may be backfilled with mercaptoundecanoic acid, mercaptohexanol, or any other backfilling agent.

[0239] Referring now to FIG. 7E, sensor 338''' may be used when magnetic particles are present within the cartridge. Similar to sensor 338'', sensor 338''', which is constructed in a similar manner to the similarly primed components of FIG. 7D described above, includes a positive reference working electrode 376', a reference electrode 339''', a counter electrode 341''', and a negative reference working electrode 342'''. Working electrode 340''' may be structurally similar to working electrode 340 described above with respect to FIG. 6A. Thus, sensor 338''' is particularly well-suited for target analyte detection that promotes the localization of enzymes or other signaling species within the analysis zone, using one or more magnetic fields in particular.

[0240] Referring now to FIG. 7F, an alternative exemplary sensor that may be used within the cartridge described herein is provided. Sensor 338'''' may be constructed in the same manner as sensor 338''', but the negative reference working electrode 342'''' may be positioned between the positive reference working electrode 376'' and the reference electrode 339'''. Preferably, fluid flows from a reservoir within the analysis channel and proceeds across the electrodes in the order of the positive reference working electrode 376'', the negative reference working electrode 342'''', the reference electrode 339'''', the counter electrode 341'''', and the working electrode 340'''.

[0241] Referring to FIG. 8A, the internal component 316 is coupled to the circuit board 331, for example, via a layer 329 positioned therebetween. The internal component 316 includes a sample preparation reservoir 317 having an outlet 353 with a valve 354 positioned adjacent to a heating element 333, a cleaning agent reservoir 318 having an outlet 355 with a valve 356 positioned adjacent to a heating element 334, and a substrate reservoir 319 having an outlet 357 with a valve 358 positioned adjacent to a heating element 335. The reservoirs 317, 318, and 319 are each in fluid communication with the analysis channel 330 such that when an individual valve is opened, at least optionally, fluid exits the reservoir, for example, through its respective outlet, and flows into the analysis channel 330. Additionally, a fluid breaker 359 may be positioned adjacent to the heating element 336 and in fluid communication with the analysis channel 330.

[0242] The valves 354, 356, and 358 may each be positioned within the outlets 353, 355, and 357, respectively, at the bottoms of the reservoirs 317, 318, and 319 of the cartridge 300. The outlets 353, 355, and 357 may each be formed from holes in the bottom wall of the internal component 316 above the analysis channel 330. The valves 354, 356, and 358 may each be formed from a heat-sensitive phase change material such as a hydrophilic wax, for example. Prior to actuation, the wax or other heat-sensitive material of the valve is in a solid or semi-solid state and is sized and shaped to fill the entire cross-section of the outlet such that fluid cannot escape from the individual reservoir into the analysis channel 330. The valves 354, 356, and 358 may be aligned directly above one or more heating elements (with a solder mask therebetween) or other localized heat-conductive elements. Such alignment allows for the localized application of heat and induces a phase change of the valve without causing a phase change of any neighboring valves. In various embodiments, the phase change does not result in a complete blockage of the outlet and instead melts or otherwise transforms the heat-sensitive material such that fluid within the individual reservoir can flow into the analysis channel 330.

[0243] In addition, the fluid breaker 359 may also be formed from a heat-sensitive phase change material such as, for example, hydrophilic wax. Prior to operation, the wax or other heat-sensitive material is in a solid or semi-solid state and is disposed outside the flow path between the outlet of the individual reservoir and the sensor 338 within the analysis channel 330. The fluid breaker 359 may be sized and shaped to block the flow path on the analysis channel 330 between the outlet of one reservoir, for example, the sample preparation reservoir 317, and the outlet of another reservoir, for example, the substrate reservoir 319, when the fluid breaker is activated, for example, by heating the heating element 336. The fluid breaker 359 may be aligned directly above the heating element 336 (with a solder mask therebetween) or other localized thermally conductive element. Such alignment allows for a localized application of heat and induces a phase change within the fluid breaker 359 without causing a phase change in any adjacent valves. In various embodiments, the phase change melts or otherwise converts the heat-sensitive material to flow into the analysis channel, blocking the outlet 353 of the sample preparation reservoir 317 from the analysis channel 330. In this way, fluid from the substrate reservoir 319, when released into the analysis channel 330, cannot enter the sample preparation reservoir 317 and cannot interact with the remaining signaling material from the sample preparation reservoir 317.

[0244] The solder material that is disposed on the via or solder mask across the via and closes the openings of the individual reservoirs or isolates the analysis channels may be a hydrophilic material such as hexadecanol or octadecanol. This advantageously does not impede the flow of fluid after actuation, but rather promotes flow over any solder pieces that solidify within any area of the analysis channel. These materials also preferably have a melting temperature of 50 to 100 degrees Celsius, which allows for operation with reasonable power consumption for battery-operated devices, but remains non-operational in general handling and storage environments and / or during ultrasonic protocols. The amount of solder per valve may be less than 1 microliter in its liquid state, the amount may be less than or equal to 0.5 microliter, and the amount may be greater than 2 nanoliters. Using a minimal amount of solder within the valve is one way to reduce any blockage of the analysis channel and maximize full valve operation when heat is applied. The valve may also have a feedback and control system that allows for a consistent thermal profile to be achieved at the via for consistent valve operation. Further, this feedback and control system may incorporate sensing elements to allow the system to confirm that each valve is operating properly.

[0245] As shown in FIG. 8A, the circuit board 331 may have exposed conductors 360 to the electrical connectors 312. In one embodiment, the conductors 360 are exposed only on the bottom surface of the circuit board 331, but the conductors 360 may also be exposed on the upper surface of the circuit board 331 and are preferably trace-less as shown. As described above, the electrical connector 312 enables the cartridge device 300 to transmit a signal indicating the presence, absence, and / or amount of one or more target analytes in the collected sample sensed by the sensors of the cartridge device 300 to the corresponding electrical connector of the reader device 400.

[0246] The internal component 316 may include an absorbent pad housing 361 that is sized and shaped to hold the absorbent pad 328. The absorbent pad housing 361 may include a plurality of ventilation holes 362 and may allow exposure of the absorbent pad 328 within the absorbent pad housing 361 to the environment within the housing 304 of the cartridge device 300.

[0247] The input tunnel 301 of the cartridge device 300 may include a slot 363 configured to allow the seal piercer 321 to be at least partially disposed within the input tunnel 301. The slot 363 may be at the proximal end of the input tunnel component 326 as shown. The slot 363 may be sized and shaped to allow the engagement member 324 of the slider 321 to be disposed within the input tunnel 301. Additionally, the slot 363 may have a length sufficient to allow the slider 322 to slide distally from the pre-ventilation position to the ventilation position when the engagement member 324 contacts the sample collection device within the input tunnel 301.

[0248] Referring now to FIGS. 8B and 8C, enlarged views of the circuit board 331 and certain components of the valve are shown. FIG. 8B depicts a resistor 365, e.g., an aluminum resistor, coupled to the heating element 333, and a conductor 364. FIG. 8C further shows a mask 348 disposed between the heating element 333 and the valve 354. As will be apparent to those skilled in the art, details of the heating element 333, mask 348, and valve 354 are illustrated, but such a configuration may be utilized with respect to heating elements 334, 335, 336, and 337 with their respective masks and valves, fluid breakers, or air pockets. Current from the reader device 400 may pass through the resistor 365 from the conductor 364 and generate heat through Joule heating. The heat is conducted to the heating element 333 due to physical contact between the resistor 365 and the heating element 333. The heating element 333 promotes electrical insulation from the sensor 338, generates heat through the mask 348 while minimizing interference with the electrical signal sensed by the sensor 338 by the heating element 333, and causes a phase change of the phase change material of the valve 354.

[0249] Referring now to FIG. 8D, an enlarged view of the circuit board 331' and alternative components of the valve is shown. FIG. 8D depicts a resistor 365', e.g., an aluminum resistor, and a conductor 364', e.g., a solder pad, coupled thereto. Different from the configurations shown in FIGS. 8B and 8C, the conductor 364' is not coupled to the heating element 333' (including vias within the circuit board 331') such that a mask disposed between the heating element 333' and the valve 354' is not required. As will be apparent to those skilled in the art, details of the heating element 333' and the valve 354' are shown, but such a configuration may be utilized with respect to the heating elements 334, 335, 336, and 337 with their respective valves, fluid breakers, or air pockets. Current from the reader device 400 may pass from the conductor 364' through the resistor 365' to generate heat through Joule heating. The vias of the heating element 333' are disposed between the conductors 364' coupled to the resistor 365' and are electrically insulated therefrom. Heat is conducted to the heating element 333' through indirect contact between the resistor 365' and the heating element 333'. The heating element 333' promotes electrical insulation from the sensor and generates heat while minimizing interference with the electrical signals sensed by the sensor by the heating element 333', causing a phase change of the phase change material of the valve 354'.

[0250] FIGS. 9A and 9B illustrate various shuttles that may be disposed within the input tunnel of the cartridge device. Referring to FIG. 9A, the shuttle 324 may include a first end 366, a reagent ball compartment 367, a compartment divider 368 having a slot 369, a sample compartment 370, a second end 371 having an opening 372 therethrough, and / or beams 373, 374.

[0251] The shuttle 324 is configured to be disposed within the input tunnel in a pre - mixing state, preferably within the cartridge housing and preferably within the input tunnel between the sample preparation reservoir and the opening that defines the opening of the input tunnel. In such a pre - mixing state, the first end 366 forms the wall of the sample preparation reservoir and may seal the fluid within the reservoir. The first end 366 may include one or more seal members, such as an O - ring, which may be made at least in part of chlorobutyl, to improve the liquid - tight seal. The area between the first end 366 and the compartment divider 368 may be referred to as the reagent ball compartment 367. The reagent ball compartment 367 is configured to store one or more reagent balls, such as reagent ball 375. In the pre - mixing state, the reagent ball compartment 367 is preferably sealed from the fluid within the sample preparation reservoir. The compartment divider 368 may be used to divide the compartments within the shuttle 324, for example, into a reagent ball compartment 367 and a sample compartment 370. The compartment divider 368 may include a slot 369 that allows fluid to flow through the compartment divider 368 when the compartment divider 368 is disposed in the fluid of the sample preparation reservoir in the mixing state. Allowing fluid to flow through the slot 369 can improve the mixing of the sample, reagent balls, and fluid within the sample preparation reservoir. Additionally, the slot 369 allows for improved fluid communication between the reagent ball 375 and the desiccant 325 disposed within the cartridge housing in the pre - mixing state. Although not explicitly stated, it should be understood that the compartments may contain the same or different reagent balls from each other and can be pre - selected for the detection and / or quantification of a target analyte.

[0252] In the pre-mixing state, the second end 371 may be disposed between the first end 366 and the opening defining the opening of the input tunnel. The second end 371 may include an opening 372 configured to allow the collected sample to be inserted therethrough into the sample compartment 370. For example, the opening 372 may be dimensioned slightly larger than the tip of the sample collection device such that the tip can advance through the opening 372 while the opening 372 wipes excess sample from the tip. Thus, at most, a predetermined volume of the sample is inserted into the sample compartment 370. The area between the second end 371 and the compartment divider 368 may be referred to as the sample compartment 370. The shuttle 324 may be configured to couple the first end 366 to the second end 371 and may include one or more beams 373, 374 coupled to the compartment divider 368. The beams 373, 374 are preferably positioned closer to the upper surface of the cartridge housing to avoid interference with fluid mixing in the mixed state when within the input tunnel.

[0253] The shuttle 324 may move from a first position in the pre-mixing state to a second position in the mixing state in response to the application of a force that exceeds a threshold force applied onto the shuttle 324 by, for example, a sample collection device. In the mixing state, the first end 366, the reagent ball compartment 367, and the sample compartment 370 may be disposed within the sample preparation reservoir. Thus, the sample and the reagent balls may be mixed in the fluid within the sample preparation reservoir. The second end 371 of the shuttle 324 may reform the wall of the sample preparation reservoir and seal the fluid therein when the sample and / or one or more reagent balls are within the sample preparation reservoir. Preferably, the distal portion of the sample collection device fluidly seals the opening 372 such that in the mixing state, fluid cannot escape from the sample preparation reservoir into the input tunnel. The second end 371 may include one or more sealing members, such as an O-ring, to improve the liquid-tight seal. Thus, the sample preparation reservoir remains fluidly sealed by the first end 366 in the pre-mixing state and by the second end 371 and the sample collection device in the mixing state during the transition from the pre-mixing state to the mixing state. Additionally, unlike the frangible membrane housing reagent, the shuttle 324 may remain intact as the sample is moved into the sample preparation reservoir 317 for analysis.

[0254] The reagent ball 375 may contain one or more than one of magnetic particles, affinity molecules, linker molecules, signaling substances, competitive binding molecules, competitor molecules, labels, signaling substances, primers, nucleic acid probes, and / or polymerases, as well as other enzymes or components as described in more detail herein, and the components, encapsulating materials, and dimensions may be the same as or different from each other. In one aspect, the reagent ball 375 is formed by freezing to a temperature that induces a phase change in the liquid (i.e., reducing the temperature of a certain volume of liquid (such as 5 microliters to 30 microliters, etc.)). The temperature can vary depending on the components of the liquid. In a further aspect, the liquid may additionally undergo a process of freezing and drying, and as it becomes the reagent ball 375, it may be sensitive to temperature in the liquid volume, for example, cryoprotectants for the functional preservation of nucleic acid and / or protein components, etc., and excipients known to those skilled in the art of lyophilization are provided. Stabilizers such as disaccharides like sucrose and trehalose, or other cryoprotectants such as polyethylene glycols of various molecular weights, as well as bulking agents or solidifying agents such as mannitol, glycine, povidone, and others known in the art, in addition to the reagents described herein, can constitute some of the final components of the reagent ball 375. The (w / v) percentage of the excipient in the volume of the liquid to be lyophilized to form the reagent ball 375 can vary widely from about 0.1% to about 30%, and in many cases, combinations of disaccharides such as cryoprotectants and solidifying agents or bulking agents for adding structure can be combined in various ways. The reagent ball 375 can be of many sizes, and non-limiting examples thereof include having a diameter of about 1 mm to about 7 mm, or alternatively, about 2 mm to about 5 mm, or alternatively, about 3 mm, or alternatively, less than about 7 mm, or alternatively, less than about 5 mm, or alternatively, less than about 4 mm. Although the reagent ball 375 is illustrated as spherical, the present disclosure is not limited thereto, and many shapes may be used, and a plurality of reagent balls, each containing the same or different reagents, may also be used.As is normal in the art, such liquids can be frozen through instant freezing in liquid nitrogen or through shelf freezing in a freeze dryer when formed to contain components and excipients. After freezing, the frozen volume is potentially subjected to annealing treatment, primary drying, and secondary drying for crystallization of the solidifying agent, and water is removed until a sufficiently small percentage (e.g., <8%, preferably <5%, preferably about 1%) remains in the final freeze-dried product, which is the reagent ball 375.

[0255] Referring now to FIG. 9B, the shuttle 324' is constructed similarly to the shuttle 324 of FIG. 9A, but the compartment divider 368' is solid without slots, and the beams 377, 378, and 379 are positioned on the shuttle 324' in different orientations.

[0256] As will be readily apparent to those skilled in the art, FIGS. 9A and 9B illustrate a shuttle having one sample compartment and one reagent ball compartment, but the present disclosure is not limited thereto, and the shuttle may define one or more compartments configured to receive the sample collected from the sample collection device when inserted into the input tunnel 301, and one or more additional compartments configured to store one or more reagent balls.

[0257] Referring now to FIGS. 10A - 10P, the insertion of a sample into a cartridge device by a sample collection device is described. Prior to insertion into the input tunnel 301 of the cartridge device 300, the sample collection device 200 is exposed to samples, for example, from the inner cheek, throat, mouth, nasal cavity, ear canal, urine, blood, plasma, saliva, etc. The tip 204 of the sample collection device 200 retains a portion of the sample and is designed to enable the analysis of the presence, absence, and / or amount of one or more target analytes in the sample using the cartridge device 300 and the reader device 400. The cartridge device 300 may be electrically coupled to the reader device 400 either before or after the sample collection device 200 is inserted into the cartridge device 300.

[0258] Referring to FIG. 10A, the distal end of the sample collection device 200 is first inserted into the opening that defines the opening of the input tunnel 301. The shuttle 324 is disposed within the input tunnel 301 in the pre - mix position, and the first end 366 of the shuttle 324 forms the wall of the sample preparation reservoir 317 and fluidly seals the fluid within the sample preparation reservoir 317. In this pre - mix position, the reagent ball 375 stored by the shuttle 324 is within the input tunnel 301 and is not exposed to the fluid within the sample preparation reservoir 317. As the distal end of the sample collection device 200 moves distally into the input tunnel 301, the sample collection device 200 may contact the engagement member 380 of the slider 322. For example, the distal seal zone 208 may contact the engagement member 380 as shown. The distal seal zone 208 may be angled to facilitate movement of the engagement member 380 beyond the distal seal zone 208 as the sample collection device 200 is moved further distally, or the distal seal zone 208 may be a shoulder sized to cause movement of the slider 322.

[0259] As shown in FIG. 10B, the tip 204 (which has the sample thereon and / or within the tube 205) enters the sample compartment 370 of the shuttle 324 through the opening 372 of the second end 371. Preferably, as the tip 204 enters the sample compartment 370, the shuttle 324 remains substantially in place within the input tunnel 301, and the first end 366 of the shuttle 324 forms the wall of the sample preparation reservoir 317 and continues to seal fluid within the sample preparation reservoir 317. Additionally, the first end 366 of the shuttle 324 may form the wall of the sample preparation reservoir 317 and continue to seal fluid within the sample preparation reservoir 317 until the distal seal portion 208 contacts the second end 371 and fluidly seals the opening 372 (and the shuttle 324 may remain substantially in the pre-mixing position). Application of a force to the shuttle 324 by the sample collection device 200 (e.g., at the second end 371) that exceeds a threshold force (at the distal seal portion 208) may move the shuttle 324 from the pre-mixing position to the mixing position, where the sample and reagent balls 375 on the sample collection device 200 are mixed in the fluid of the sample preparation reservoir 317.

[0260] Additionally, as the sample collection device 200 (e.g., the tip 204) is inserted into the shuttle 324 (e.g., through the opening 372), the shuttle 324 may wipe excess sample from the sample collection device 200, thereby preventing the wiped sample from entering the shuttle 324. For example, the wall of the second end 371 that defines the opening 372 may wipe excess sample from the tip 204 as the tip 204 is inserted through the opening 372. All or substantially all of the sample on the outer surface of the tip 204 may be wiped, and only the sample may be disposed within the tube 205. The tube 205 may hold a maximum of a predetermined volume of sample, e.g., about 2 μl. Wiping of excess sample from the sample collection device 200 may improve the accuracy, precision, and / or consistency of the analysis because a maximum of a predetermined volume of sample is inserted into the sample preparation reservoir 317 at the mixing position.

[0261] Referring now to FIGS. 10C - 10K, an exemplary process for piercing a seal material disposed across one or more reservoirs within the cartridge device through the interaction between the sample collection device and the seal piercer within the cartridge device is described. As described above, the seal piercer 321 may include a slider 322 and a piercer 323.

[0262] FIG. 10C is a top view of a portion of the cartridge device 300, illustrating a possible orientation of the piercing elements across the reservoirs. The piercer 323 may have a first piercing element 381 having a piercing end disposed across the sample preparation reservoir 317, a second piercing element 382 having a piercing end disposed across the detergent reservoir 318, and / or a third piercing element 383 having a piercing end disposed across the substrate reservoir 319. In the pre - mixing state, the piercing elements 381, 382, and 383 are disposed across their respective reservoirs, do not pierce the seal material, and seal the fluid within the individual reservoirs. The piercing elements 381, 382, and 383 may be coupled to the housing of the cartridge device (e.g., at the opposite end of the piercing end).

[0263] FIG. 10D is a perspective view of the sample collection device 200 within the input tunnel of the cartridge device 300, with half of the cartridge housing removed to show the slider 322 for clarity. The slider 322 may include a first track 384 configured to engage the first piercer 381 and move it to a piercing position, a second track 385 configured to engage the second piercer 382 and move it to a piercing position, and / or a third track 386 configured to engage the third piercer 383 and move it to a piercing position. As the sample collection device 200 is moved distally through the input tunnel 301, preferably, the slider 322 remains within the cartridge housing and in a pre-ventilation position until the sample collection device 200 fixedly engages with the slider 322. The slider 322 may fixedly engage with the sample collection device 200 by temporarily or permanently coupling the engaging member 380 of the slider 322 to the engaging zone 209 of the sample collection device 200. The engaging member 380 is disposed within the input tunnel 301 (e.g., at least at any time, hanging below the slot 363 of FIG. 8A). The engaging member 380 may be sized to fit within a groove of the engaging zone 209, such as a protrusion and / or a U-shaped engaging zone 209, or to receive a protrusion of the engaging zone 209.

[0264] Figure 10E is a cross-sectional side view depicting the seal trocar in the pre-puncture position and the shuttle in the pre-mixing position. As shown, the sample collection device 200 has been distally moved within the input tunnel 301 such that the slider engagement member 380 engages the engagement zone 209 of the sample collection device 200 and the distal seal zone 208 of the sample collection device 200 fluidly seals the opening of the second end 371 of the shuttle 324. Preferably, the first end 366 continues to fluidly seal fluid within the sample preparation reservoir 317 at least until the sample collection device 200 fluidly seals the second end 371 of the shuttle 324. Thus, one or more reagent balls stored by the shuttle 324 and the collected sample within the shuttle 324 remain out of fluid contact with the fluid within the sample preparation reservoir 317. The slider 322 and the shuttle 324 may be positioned within the cartridge housing such that the engagement member 380 of the slider 322 engages the engagement zone 209 of the sample collection device 200 at the same time or substantially at the same time as the distal seal zone 208 of the sample collection device contacts and fluidly seals the second end 371 of the shuttle 324. In such an embodiment, the slider 322 and / or the shuttle 324 may not yet be movable within the cartridge device 300 at this point.

[0265] Figures 10F and 10G further illustrate the positioning of the slider and the puncturer at the pre-ventilation position shown in Figure 10E. As shown in Figure 10F, when the engage-ment member 380 of the slider 322 engages the engagement zone 209 of the sample collection device 200 at the pre-ventilation position, the puncturing elements 381, 382, and 383 have not yet been deflected downward by the slider 322 towards their respective reservoirs. At such a pre-ventilation position, the tracks 384, 385, and 386 cannot yet contact the contact puncturing elements 381, 382, and 383, respectively. As shown in Figure 10G, at the pre-ventilation position, the puncturing element 381 is disposed upward but has not yet punctured the sealing material 320 that seals fluid within the sample preparation reservoir 317. In Figure 10G, the track 384 of the slider 322 has not yet contacted the puncturing element 381 of the puncturer 323.

[0266] Referring now to FIG. 10H, the sample collection device 200 is further distally moved within the input tunnel 301 from the pre-mixing and pre-ventilation position towards the mixing and ventilation position. As the collector distally pushes the sample collection device 200, the shuttle 324 is partially moved within the sample preparation reservoir 317. The shuttle 324 may be moved within the cartridge, for example, by the application of a force that exceeds a threshold force by the sample collection device 200 (e.g., at the distal seal zone 208) onto the shuttle 324 (e.g., at the second end 371). As the shuttle 324 moves distally, the first end 366 may unseal so that one or more reagent balls within the shuttle 324 and the collected sample within the shuttle 324 are exposed to the fluid within the sample preparation reservoir 317. Advantageously, before the first end 366 of the shuttle 324 is unsealed, the second end 371 of the shuttle 324 is fluidly sealed by the sample collection device 200 so that fluid from the sample preparation reservoir 317 does not leak into the input tunnel 301 beyond the second end 371. Additionally, as the distal movement of the sample collection reservoir 200 causes a transition from the pre-mixing position to the mixing position, the second end 371 of the shuttle continuously fluidly seals the fluid within the sample preparation reservoir 317 from the input tunnel 301.

[0267] As the collector distally pushes the sample collection device 200, the seal piercer 321 may also be moved from the pre-ventilation position toward the ventilation position. The seal piercer 321 may be moved within the cartridge, for example, by the application of a force exceeding a threshold force by the sample collection device 200 (e.g., at the engagement zone 209) onto the seal piercer 321 (e.g., at the coupler 380 of the slider 322). As the sample collection device 200 is moved distally, the seal piercer 321 pierces the seal material across the sample preparation reservoir 317, the cleaning agent reservoir 318, and / or the substrate reservoir 319, venting the fluid within the reservoir. In response to insertion into the input tunnel 301 of the sample collection device 200, the seal piercer 321 is moved within the input tunnel 301 in a generally lateral direction in a first direction, e.g., generally parallel to the movement of the sample collection device 200, and downward in a second (different) direction, e.g., generally perpendicular to the individual reservoir, to pierce into the seal material 320. For example, the slider 322 may move in the first direction and the piercer 323 may move in the second direction.

[0268] As shown in FIG. 10I, as the sample collection device 200 distally moves the slider 322 in a direction generally parallel to the movement of the sample collection device 200, the slider 322 contacts the contact piercer 323 and may move the piercer 323 in a different direction, e.g., generally perpendicular to the movement of the slider 322. As the slider 322 moves distally, the tracks 384, 385, and 386 of the slider 322 may each contact the piercing elements 381, 382, and 383 of the piercer 323. The distal movement of the slider 322 may cause the piercer 323 to pierce the seal material and vent the reservoir.

[0269] FIG. 10J illustrates the piercing element 381 piercing the seal material 320 across the sample preparation reservoir 317 and venting the sample preparation reservoir 317. As shown, the track 384 contacts the piercing element 381 and moves it downward to the piercing position.

[0270] Figure 10K shows the shuttle 324 in the mixing position and the seal piercer in the venting position. In the mixing position, the first end 366 of the shuttle 324, one or more reagent ball compartments of the shuttle that store one or more reagent balls, and / or one or more sample compartments that store the sample to be analyzed may be disposed within the sample preparation reservoir 317. As described above, the shuttle 324 and the sample collection device 200 (e.g., via the tip 204 inserted into the opening 372 and the distal seal zone 208) may also fluidly seal the sample preparation reservoir 317 at the mixing position such that the collected sample, reagent balls, and fluid within the sample preparation reservoir 317 are sealed within the reservoir. Thus, the collected sample, reagent balls, and fluid may be mixed within the sample preparation reservoir 317.

[0271] Advantageously, the configuration in which insertion of the sample collection device 200 vents the sample preparation reservoir 317, the cleaning agent reservoir 318, and / or the substrate reservoir 319 ensures that the reservoirs remain fluidly sealed prior to insertion of the sample collection device 200 and facilitates the discharge of the reservoirs into the analysis channel when the outlet of an individual reservoir allows fluid to flow therethrough.

[0272] At the mixing position, the seal piercer 321 may move from the pierced hole, open the pierced hole, and promote ventilation. As shown in FIG. 10K, the engagement zone 209 of the sample collection device 200 may be distally moved beyond the engagement device 380 such that the engagement device 380 disengages from the engagement zone 209 of the sample collection device 200 at the ventilation position. Additionally, at the mixing position, the proximal seal zone 207 of the sample collection device 200 is configured to seal the input tunnel 301 at the opening 302. Thus, the proximal seal zone 207 provides additional structure for minimizing or eliminating, for example, liquid leakage from the cartridge device 300 at the opening 302.

[0273] The cartridge device 300 may further include a locking member 387 configured to irreversibly lock the sample collection device 200 within the cartridge device 300. The locking member 387 may be biased inwardly within the input tunnel 301 such that the locking end of the locking member 387 engages the sample collection device 200 at the mixing position. The locking end may engage the engagement zone 209. The locking end may be a protrusion sized to fit within a groove of the engagement zone 209 as shown. The locking member 387 may also define a portion of the input channel 301 and may be coupled to the cartridge housing at the opposite end of the locking end. Advantageously, locking the sample collection device 200 within the input tunnel 301 (e.g., longitudinally and / or axially) promotes the seal of the sample preparation reservoir 317 over time such that once the test is initiated, the user cannot inadvertently pull the sample collection device 200 out of the cartridge device 300, and once locked, the sample collection device 200 cannot be retracted, promoting safe disposability and test consistency.

[0274] FIGS. 10L and 10M further depict, for clarity, the sample collection device 200 (FIG. 10L) and internal components 316 (FIG. 10M) at the mixing position within the cartridge device 300.

[0275] Referring now to FIGS. 10N, 10O, and 10P, a process for improved mixing of the contents within the sample preparation reservoir 317 is described. At the mixing location, the fluid within the sample preparation reservoir 317 is mixed with the collected sample and one or more reagent balls (if provided). The mixing may be enhanced via an ultrasonic generator element 327, which can be a piezoelectric transducer such as a piezoelectric ceramic disk. The ultrasonic generator element 327 vibrates in response to an electrical signal (e.g., transmitted from a reader device) and is configured to further mix the contents within the sample preparation reservoir 317. For example, the ultrasonic generator element 327 can facilitate mixing of the fluid held within the sample preparation reservoir 317, which may be pre-filled with the reagent ball 375 and the collected sample and / or filled during the mixing process. The ultrasonic generator element 327 may flow the fluid in a wave pattern as shown in FIGS. 10O and 10P. Such a wave pattern may be between compartments defined by the shuttle, e.g., between the reagent ball compartment 367 and the sample compartment 370. For example, the ultrasonic generator element 327 may flow the fluid in one or more directions (e.g., generally up and down as shown in FIG. 10O) within the reagent ball compartment 367 during a portion of the wave cycle. Such flow is expected to accelerate and improve mixing within the sample preparation reservoir 317. The wave motion of the fluid within the sample preparation reservoir 317 may also facilitate removal of the sample from the distal portion of the sample collection device 200 and improve mixing and homogenization.

[0276] The ultrasonic generator element 327 may be configured to emit acoustic waves that move the fluid within the sample preparation reservoir 317 in a wave pattern between the reagent ball compartment 367 and the sample compartment 370 to mix the fluid within the sample preparation reservoir 317. Such mixing can produce a fluid mixture of the sample, the fluid from the reservoir, and the fluid of the dissolved reagent ball. The acoustic emission from the ultrasonic generator element 327 can heat the fluid within the sample preparation reservoir 317 and mix the contents of the sample preparation reservoir 317 at the macro and micro levels for amplification such as isothermal amplification. The reagent ball may include, for example, polymerase, primers, and signaling substances for isothermal amplification. The shuttle 324 may have a compartment divider 368 that can be a flange configured to divide the reagent ball compartment 367 and the sample compartment 370. The fluid flowing around the compartment divider 368 can facilitate the formation of the wave pattern. The compartment divider 368 may have slots configured to allow fluid to flow through the compartment divider 368 through the slots during mixing. The ultrasonic generator element 327 may form part of a wall, such as the bottom wall of the sample preparation reservoir 317, and may be positioned offset from the center of the sample preparation reservoir 317 to facilitate mixing of the fluid within the sample preparation reservoir 317. For example, the ultrasonic generator element 327 may be positioned offset from the center with respect to the central axis of the sample preparation reservoir 317 that extends perpendicular to the longitudinal axis extending through the center of the input tunnel of the cartridge. Such an offset positioning of the ultrasonic generator element 327 from the center also facilitates improved mixing. The ultrasonic generator element 327 may be electrically coupled to a printed circuit board via one or more spring contacts as will be described in detail below.

[0277] Referring now to FIGS. 11A - 11E, an alternative seal piercer is shown. FIGS. 11A and 11B show the seal piercer 321' in the pre - ventilation and pre - contact positions. The seal piercer 321' includes a slider 322' that is slidably coupled to the piercer 323' throughout the piercing process. The seal piercer 321' may further include a post 388 that is configured to press against the contact switch 389 of the circuit board 331'. The recess of the contact switch 389 may complete a circuit such that an electrical signal may be transmitted to, for example, a reader device and / or a computing device to activate a software - based user interface system. Thus, proper insertion of the sample collection device 200' into the input tunnel generates an electrical signal that is transmitted to the reader device and / or the computing device to notify the reader device and / or the computing device of such proper insertion. The post 388 may be coupled to the piercer 323' or may be integral with the piercer 323'. As the sample collection device 200' moves the slider 322' distally in a direction substantially parallel to the movement of the sample collection device 200' (e.g., by the force applied to the coupler 380' by the engagement zone 209' and / or the engagement zone 210), the slider 322' may move the piercer 323 / post 388 in a different direction, e.g., substantially perpendicular to the movement of the slider 322'. The slider 322' may have an angled surface 390 that is configured to contact the angled surface 391 of the piercer 323' as the distal movement of the sample collection device 200' causes a distal movement of the slider 322'. As the distal movement of the sample collection device 200' continues, thereby causing a distal movement of the slider 322', the piercer 323' and the post 388 are moved downward such that the piercer 323' pierces the seal material on an individual reservoir and the post 388 activates the contact switch 389, as shown in FIGS. 11D and 11E at the contact position.

[0278] After contacting the contact switch 389 and piercing the seal material, the seal piercer 321' may move such that the support 388 no longer depresses the contact switch 389 and the piercer 323' moves from the hole pierced in the seal material for venting to an individual reservoir. Insertion into the input tunnel of the sample collection device may cause the seal piercer to pierce the seal material before the shuttle starts to move from the pre-mixing position to the mixing position or during the movement of the shuttle from the pre-mixing position to the mixing position.

[0279] Figures 11A-11E illustrate an ultrasonic generator element 327' coupled to a spring contact 392. The spring contact 392 is coupled to a circuit board 331' and is a conductor such that the ultrasonic generator element 327' is electrically coupled to the circuit board 331' via the spring contact 392. Advantageously, the spring contact 392 absorbs the movement of the ultrasonic generator element 327' such that when the ultrasonic generator element 327' is activated in response to a signal transmitted, for example, by a reader device, the circuit board 331' vibrates minimally in a suitable manner, and the spring contact 392 allows for ease and reproducibility of assembly as compared to solder, which can adversely affect the ultrasonic generator element 327'.

[0280] As will be readily understood by those skilled in the art, Figures 11A and 11D do not depict the shuttle and / or reagent balls within the input tunnel, although such features may be included in these embodiments.

[0281] Referring now to FIGS. 12A - 12E, an alternative configuration for collecting and analyzing fluid samples is described. The cartridge device 300'' may be constructed similarly to the aforementioned cartridge device 300, but the cartridge device 300'' may be modified for improved collection of a relatively large volume of fluid. Additionally, the sample collection device 200'' may be constructed similarly to the aforementioned sample collection device 200, but the sample collection device 200'' may have a modified collection area for improved collection of a relatively large volume of fluid. For example, the sample collection device 200'' and the cartridge device 300'' may be particularly useful for collecting and analyzing saliva, blood, plasma, urine, or the like.

[0282] The sample collection device 200'' may include a distal seal zone 208'', a suction portion 211, an intermediate seal zone 212, and a shroud 213, and may include a distal portion 201'' modified for improved collection of a relatively large volume of fluid (e.g., about 10 to 100 microliters). The distal portion 201'' of the sample collection device 200'' is exposed to the sample, preferably a liquid sample, absorbs at least a portion of the sample, and is adapted to be compressed to discharge the collected sample from the distal portion 201'' into the cartridge device 300'' for analysis of the discharged sample. The suction portion 211 may be configured to suck up and absorb the sample and may be formed from a suction material. The suction portion 211 may be coupled to the intermediate seal zone 212. The intermediate seal zone 212 may be slidably disposed within the shroud 213 and may include one or more seal members, such as an O-ring, configured to create a liquid-tight seal that reduces or prevents fluid absorbed on the suction portion 211 from moving proximally within the shroud 213 past the intermediate seal zone 212. The suction portion 211 may be at least partially disposed within the shroud 213 and may slide within the lumen of the shroud 213. The suction portion 211 may be configured to become transparent when exposed to fluid such that an increasing amount of the suction portion 211 becomes transparent as an increasing amount of sample fluid is collected. The sample collection device 200'' may include a sample collection indicator 214 configured to visually alert the collector based on the volume of the collected sample fluid. In one embodiment, the sample collection indicator 214 optionally visually alerts the collector that at least a predetermined volume of sample fluid has been collected as the volume of the collected sample increases. For example, the sample collection indicator 214 may change color when a predetermined volume or more of the sample has been collected. As another example, an increasing amount of the sample collection indicator 214 may become visible as the deposition of the collected sample increases.For example, the sample collection indicator 214 may be a colored thread embedded in the suction portion 211 such that as the volume of the collected fluid sample gradually becomes more visually exposed, replacing the surrounding suction material with a more transparent one as the collector monitors the progress of fluid collection and determines when a sufficient volume of the sample has been collected. In one embodiment, the sample collection indicator 214 includes a transparent area on the shroud 213. Additionally or alternatively, the sample collection indicator 214 may change color as the volume of the collected sample increases.

[0283] The shuttle 324'' may include a first end 366'', a reagent ball compartment 367'', a compartment divider 368'', and a sample compartment 370'', which are constructed in the same manner as those individual components described above. Preferably, the compartment divider 368'' does not have slots, similar to the compartment divider 368', such that the compartment divider 368'' fluidly seals the reagent ball compartment 367'' from the sample compartment 370'' in the pre-mixing position. The second end 371'' of the shuttle 324'' may be modified to include a distal flange 393 and a proximal flange 394 and have a cavity 395. Additionally, unlike the opening 372 of the shuttle 324, the opening 372'' is configured to allow the flow of the discharged sample to be compressed from the sample collection device 200'' into the sample compartment 370'', rather than being part of the sample collection device 200''. The second end 371'' (e.g., at the distal flange 393) may be configured to fluidly seal the sample compartment 370'' both in the pre-mixing position and the mixing position, and continuously during the transition therebetween. The distal flange 393 may include one or more seal members, such as O-rings, configured to create a liquid-tight seal that reduces or prevents fluid from flowing proximally from the sample compartment 370''. Additionally, the compartment divider 368'' may be configured to fluidly seal the sample compartment 370'' in the pre-mixing position. The compartment divider 368'' may include one or more seal members, such as O-rings, configured to create a liquid-tight seal that reduces or prevents fluid from flowing distally from the sample compartment 370''.

[0284] Prior to insertion into the input tunnel 301'' of the cartridge device 300'', the sample collection device 200'' is exposed to samples such as, for example, from the inner cheek, throat, mouth, nasal cavity, ear canal, urine, blood, plasma, saliva, etc. The suction portion 211 of the sample collection device 200'' retains a portion of the sample and is designed to enable analysis of the presence, absence, and / or amount of one or more target analytes in the sample using the cartridge device 300'' and the reader device. The cartridge device 300'' may be electrically coupled to the reader device either before or after the sample collection device 200'' is inserted into the cartridge device 300''.

[0285] Referring to FIG. 12A, the distal end of the sample collection device 200'' is first inserted into the opening 302'' that defines the opening of the input tunnel 301''. The shuttle 324'' is disposed within the input tunnel 301'' in the pre-mixing position, and the first end 366'' of the shuttle 324'' forms the wall of the sample preparation reservoir 317'' and fluidly seals the fluid within the sample preparation reservoir 317''. In this pre-mixing position, the reagent ball 375'' stored by the shuttle 324'' is within the input tunnel 301'' and is not exposed to the fluid within the sample preparation reservoir 317''.

[0286] Referring to FIG. 12B, as the sample collection device 200'' moves distally within the input tunnel 301'', the sample collection device 200'' may contact the shuttle 324''. For example, the distal end and / or the distal seal zone 208'' may contact the second end 371'' as shown (e.g., at the cavity 395 and / or the proximal flange 394). The cavity 395 may be dimensioned slightly larger than the outer surface of the suction portion 211 such that the distal end of the suction portion 211 fits snugly within the cavity 395. The distal seal zone 208'' may be configured to fluidly seal the sample collection device 200'' to the shuttle 324'' such that fluid discharged from the sample collection device 200'' travels into the sample compartment 370''. In this pre-mixing position, the sample compartment 370'' is within the input tunnel 301'' and is not exposed to the fluid within the sample preparation reservoir 317''.

[0287] As shown in FIG. 12C, as the sample collection device 200'' moves further distally within the input tunnel 301'', the sample collection device 200'' may discharge the collected fluid sample into the sample compartment 370'' of the shuttle 324'', for example, through the opening 372'' of the second end 371''. As the sample collection device 200'' is moved distally, the suction portion 211 may be compressed to discharge the collected sample, and the distal end of the suction portion 211 may remain substantially in place during the compression. The intermediate seal zone 212 and / or the shroud 213 may move distally in proportion to the movement of the handle of the sample collection device 200'' during such compression. Preferably, as the suction portion 211 is compressed and the sample is discharged therefrom, the discharged sample travels through the opening 372'' into the sample compartment 370''. During the compression, the shuttle 324'' may remain substantially in place within the input tunnel 301'', and the first end 366'' of the shuttle 324'' may continue to form the wall of the sample preparation reservoir 317'' and seal the fluid within the sample preparation reservoir 317''. The cartridge device 300'' may include a proximal step 396 configured to hold the shuttle 324'' in the pre-mixing position during the compression of the suction portion 211. The proximal step 396 may engage the proximal flange 394 and hold the shuttle 324'' in place.

[0288] A sample of a predetermined volume may be configured to be held, at most, within the sample compartment 370''. The cartridge device 300'' may include an overflow compartment 397 and an overflow lumen 398. The overflow compartment 397 and the overflow lumen 398 may be part of the cartridge housing or internal components within the cartridge. If the amount of sample introduced into the sample compartment 370'' exceeds a predetermined volume, e.g., exceeds about 20 μl, the excess sample may travel to an overflow compartment 397 that is fluidly connected to the sample compartment 370'' via, e.g., the overflow lumen 398. Limiting the volume of the sample within the sample compartment 370'' can improve the accuracy, precision, and / or consistency of the analysis because, at the mixing position, at most a sample of a predetermined volume is inserted into the sample preparation reservoir 317. The overflow compartment 397 may otherwise be sealed to prevent or reduce leakage of the excess sample within the overflow compartment 397.

[0289] Figure 12D shows the sample collection device 200'' and the cartridge device 300'' in the mixing position, and Figure 12E shows an enlarged view of a part of Figure 12D for clarity. As the collector distally pushes the sample collection device 200'' from the pre-mixing position to the mixing position, the shuttle 324'' is partially moved within the sample preparation reservoir 317''. Application of a force to the shuttle 324'' by the sample collection device 200'' (e.g., preferably at the second end 371'' within the cavity 395) that exceeds a threshold force (at the distal seal portion 208'' and / or the distal end of the suction material 211) may move the shuttle 324'' from the pre-mixing position to the mixing position, where the discharged sample and the reagent ball 375'' within the sample compartment 370'' are mixed in the fluid of the sample preparation reservoir 317''. For example, the threshold force may be the force required to distally push the shuttle 324'' beyond the proximal step 396. As the shuttle 324'' moves distally, the first end 366'' may unseal so that one or more reagent balls within the shuttle 324'' and the sample collected within the shuttle 324'' are exposed to the fluid in the sample preparation reservoir 317''. Advantageously, before the first end 366'' of the shuttle 324'' is unsealed, the second end 371'' of the shuttle 324'' is fluidly sealed by the sample collection device 200'' so that fluid from the sample preparation reservoir 317'' does not leak into the input tunnel 301'' past the second end 371'' and / or the intermediate seal zone 212. Additionally, as the distal movement of the sample collection device 200'' causes a transition from the pre-mixing position to the mixing position, the distal flange 393 of the shuttle 324'' continuously fluidly seals the fluid in the sample preparation reservoir 317'' from the input tunnel 301''. The cartridge device 300'' may include a distal step 399 configured to hold the shuttle 324'' in the mixing position and prevent further distal movement. The distal step 399 may engage the proximal flange 394 and hold the shuttle 324'' in place.

[0290] At the mixing position, the first end 366'' of the shuttle 324'', one or more reagent ball compartments of the shuttle that store one or more reagent balls, and / or one or more sample compartments that store the sample to be analyzed may be disposed within the sample preparation reservoir 317''. As described above, the shuttle 324'' and the sample collection device 200'' (e.g., via the distal seal zone 208'' inserted into the cavity 395) (e.g., at the second end 371'') may also fluidly seal the sample preparation reservoir 317'' at the mixing position such that the collected sample, the reagent balls, and the fluid within the sample preparation reservoir 317'' are sealed within the reservoir. Thus, the collected sample, the reagent balls, and the fluid may be mixed within the sample preparation reservoir 317''.

[0291] The cartridge device 300'' may further include a locking member 387'' configured to irreversibly lock the sample collection device 200'' within the cartridge device 300''. The locking member 387'' may be biased inwardly within the input tunnel 301'' such that, in the mixing position, the locking end of the locking member 387'' engages the sample collection device 200''. The locking end may lock to an engagement zone of the sample collection device 200''. The locking end may be a protrusion sized to fit within a groove on the shaft of the sample collection device 200'' or within a groove on the shroud 213, as illustrated. The locking member 387'' may also, as illustrated, define a portion of the input channel 301'' and may be coupled to the cartridge housing at an end opposite its locking end. Advantageously, locking the sample collection device 200'' within the input tunnel 301'' (e.g., longitudinally and / or axially) prevents the user from inadvertently pulling the sample collection device 200'' out of the cartridge device 300'' once the test has started, promoting the seal of the sample preparation reservoir 317'' over time and facilitating safe disposal and test consistency such that the sample collection device 200'' cannot be retracted once locked.

[0292] As will be readily understood by those skilled in the art, FIGS. 12A-12E do not depict the seal piercer within the cartridge device, although the seal piercer may be included in these embodiments. For example, the sample collection device 200'' (e.g., at the distal seal zone 208'') may contact the seal piercer and be moved from the pre-ventilation position to the ventilation position in the manner described above with respect to FIGS. 10A-10J and / or FIGS. 11A-11E. The sample collection device 200'' may contact the shuttle 324'' and contact the seal piercer before, during, and / or after moving the shuttle 324'' from the pre-mixing position to the mixing position, moving it from the pre-ventilation position to the ventilation position. Additionally, insertion of the sample collection device 200'' may cause activation of the contact switch, as described above with respect to FIGS. 11A-11E. For example, the sample collection device 200'' may cause movement of the seal piercer, which in turn causes activation of the contact switch.

[0293] Referring now to FIG. 13A, an alternative cartridge for analyzing a sample is described. Cartridge device 300''' may be constructed similarly to the aforementioned cartridge device 300 and / or cartridge device 300'', and like components are identified by like primed reference numerals. Cartridge device 300''' is a general-purpose configuration that includes components that can be used for different types of samples. For example, many of the components within cartridge device 300''' may be used for different types of samples without modification, and in some embodiments, only the shuttle, collet, and reagent ball may vary for the analysis of different indications. Thus, cartridge device 300''' may be general-purpose, and the shuttle, collet, and / or reagent ball may be selected for use in cartridge device 300''' based on the target analyte to be analyzed. For example, cartridge device 300''' may be fitted with a shuttle, such as shuttle 324 or 324' described above with respect to FIGS. 9A and 9B, designed for relatively small sample collection, e.g., for nasal, ear, blood, and a collet designed for relatively small sample collection, or cartridge device 300''' may be fitted with a shuttle, such as shuttle 324'' described above with respect to FIGS. 12A-12E, designed for relatively large fluid sample collection, e.g., for saliva, blood, plasma, urine, and a collet designed for relatively large fluid sample collection. Cartridge device 300''' may further be fitted with a reagent ball intended to identify different target analytes that may indicate, for example, inflammation, influenza, testosterone, fertility, HIV, or vitamin D. Thus, cartridge device 300''' is highly compatible for different types of samples and indications, which reduces cost and manufacturing burden.

[0294] In FIG. 13A, an exploded view of the cartridge device 300''' is shown. The cartridge device 300''' may include an internal component 316''', a seal material 320''', a seal piercer 321''', a shuttle 324''' having a first end 366''' and a seal member 614, a desiccant 325''', an ultrasonic generator element 327''', an absorption pad 328''', a layer 329''', an analysis channel 330''', a circuit board 331''', a memory 332''', a sensor 338''', a heating element, a contact switch 389''', a spring contact 392''', a reagent ball 375''', a temperature sensor 616, and / or a collet 618. The internal component may be disposed within the housing 304''', for example, between the first and second cover components 310''' and 311'''. Alternatively, one or more internal components may be disposed within one housing while other internal components may be disposed within another housing. In the case of multiple housings, such separate housings may be configured to couple to each other. The sample preparation reservoir 317''', the cleaning agent reservoir 318''', the substrate reservoir 319''', the input tunnel component 326''', the overflow compartment 397''', and / or the struts 610 and 612 may also be included.

[0295] In FIG. 13A, the shuttle 324''' is illustrated as being substantially similar to the shuttle 324'' of FIGS. 12A-12E, although a shuttle such as that of the shuttle 324 in FIG. 9A or the shuttle 324' in FIG. 9B may be used depending on the type of sample to be collected. The collet 618 may also be substituted with the collet 618' described below based on the type of sample to be collected. Similarly, various reagent balls 375''' may also be substituted within the cartridge device 300'''.

[0296] The struts 610 and 612 are configured to be coupled to the seal piercer 321''' and to enable the seal piercer 321''' to be moved from the pre-ventilation position to the ventilation position. The struts 610 and 612 may be integrally formed with the internal component 316''' or may be separate parts.

[0297] The temperature sensor 616 may be configured to sense a temperature indicative of the temperature of a fluid within a reservoir, such as the sample preparation reservoir 317'''. For example, the temperature sensor 616 may sense a temperature change adjacent to the reservoir indicative of the temperature within the reservoir. The temperature sensor 616 may be a thermistor and may be disposed on the circuit board 331''' and may enable an electrical coupling with a reader device via one or more conductors within the circuit board 331'''. Preferably, the temperature sensor 616 is positioned adjacent to the ultrasonic generator element 327''' on the circuit board 331''' such that the temperature sensor 616 senses the temperature within the sample preparation reservoir 317''' via the ultrasonic generator element 327''' during mixing. Advantageously, the temperature indicative of the temperature within the sample preparation reservoir 317''' is monitored during mixing of the fluid within the reservoir with the reagent from the reagent ball and the sample to ensure that the temperature within the sample preparation reservoir 317''' is within a predetermined range. If outside the predetermined range, the emission of acoustic waves into the sample preparation reservoir 317''' via the ultrasonic generator element 327''' may be modified, for example, in response to an electrical signal transmitted to the ultrasonic generator element 327''' by a reader. The temperature sensor 616 may generate a signal indicative of the temperature of the fluid within the reservoir that may be transmitted to the reader via conductors within the circuit board 331''' for processing. The temperature sensor 616 may be positioned directly below the ultrasonic generator element 327''' on the circuit board 331''' and adjacent to one or more spring contacts, for example, between the first and second spring contacts 392'''.

[0298] The collet 618 is preferably disposed within the input tunnel 301''' between the aperture 302''' and the sample preparation reservoir 317'''. The collet 618 may also be disposed at least partially proximal to the shuttle 324''' within the input tunnel 301'''. For example, in the pre-mixing position, an end, e.g., the second end of the shuttle, may be disposed within the collet 618. The collet 618 may also be configured to hold the shuttle 324''' in the pre-mixing position and disconnect from the shuttle 324''' during insertion of the sample collection device into the input tunnel 301'''. Thus, the collet 618 may hold the shuttle 324''' in the pre-mixed state until a force applied from the sample collection device disconnects the collet 618 from the shuttle 324''' while the shuttle 324''' moves from the pre-mixing position to the mixing position and the collet 618 remains in a fixed position within the input tunnel 301'''. The collet 618 has a lumen sized to allow insertion of the distal portion of the sample collection device therethrough. The collet 618 may have a generally tubular shape as illustrated in FIG. 13A. The collet 618 may also be configured to activate the contact switch 389'''. For example, the collet 618 may activate the contact switch 389''' in response to a force applied on the collet 618 by the sample collection device during insertion of the sample collection device into the input tunnel 301'''.

[0299] Referring now to FIGS. 13B-13SS, various exemplary sample collection devices that may be used within the detection system 100 are illustrated.

[0300] Referring to FIG. 13B, the sample collection device 200''' may be constructed in the same manner as the aforementioned sample collection device 200'', but the sample collection device 200''' may have a modified shaft for locking the sample collection device 200''' within the cartridge during partial and full insertion of the sample collection device 200'''. For example, the sample collection device 200''' includes, by way of illustration, an engagement zone 209''' having a plurality of grooves and protrusions that are distal to and spaced from the proximal seal zone 207'''. The plurality of grooves and protrusions are configured for engagement with one or more components of the cartridge device for non-retractability of the sample collection device 200''' during partial and full insertion of the sample collection device 200''' into the cartridge. For example, the cartridge may successively engage the grooves of the plurality of grooves within the engagement zone 209''' in a distal-to-proximal direction as the sample collection device 200''' is moved distally within the input tunnel. The engagement zone 209''' may be permanently or temporarily coupled to a seal piercer of the cartridge device and may be configured to move the seal piercer within the cartridge device in response to movement of the sample collection device 200'''. Additionally, or alternatively, the engagement zone 209''' may be configured to activate a contact switch during sample collection device insertion. For example, a shoulder 220 at the distal end of the engagement zone 209''' may be coupled to the seal piercer and / or may be configured to activate the contact switch.

[0301] Similar to the sample collection device 200'', the sample collection device 200''' may include a distal seal zone 208''', a suction portion 211''', an intermediate seal zone, and / or a shroud 213''', and may include a distal portion 201''' modified for improved collection of a relatively large volume of fluid (e.g., about 10 to 100 microliters, preferably about 20 microliters). The sample collection device 200''' may also include a proximal portion 202''', a shaft 203''' extending between the distal portion 201''' and the proximal portion 202''', a handle 206''', a proximal seal zone 207''', and / or an engagement zone 209''' having a shoulder 220, similar to the same primed reference numbers as described above. The sample collection device 200''' is configured for complete or partial insertion into the cartridge device 300''' after sample collection. The sample collection device 200''' and the cartridge device 300''' may be particularly useful for collecting and analyzing saliva, blood, plasma, urine, or the like. The shoulder 220 of the engagement zone 209''' preferably extends further from the longitudinal axis of the sample collection device 200''' than the shoulder of the distal seal zone 208''' such that the shoulder 220 contacts the seal piercer, moves it, vents it to one or more reservoirs within the cartridge device 300''', and / or contacts the collet and activates the contact switch, while the shoulder of the distal seal zone 208''' may be sized to move distally through the input tunnel without moving the seal piercer and / or without activating the contact switch.

[0302] Figures 13C, 13D, 13E, 13F, 13G, and 13H are, respectively, rear, side, front, rear, side, and front views of the sample collection device 200'''.

[0303] Referring to FIG. 13I, the sample collection device 200'''' may be constructed in the same manner as the aforementioned sample collection device 200, but the sample collection device 200'''' may have an engagement zone 209'''' similar to the engagement zone 209''' of FIG. 13B, and the tip 204'''' does not include a tube. The tip 204'''' may have a rounded end as shown, and may be configured to collect a sample from any desired area or location, but the tip 204'''' may be particularly useful when collecting a sample from the nasal area. FIGS. 13J, 13K, 13L, 13M, 13N, and 13O are, respectively, the rear, side, front, rear, side, and front views of the sample collection device 200''''.

[0304] Referring to FIG. 13P, the sample collection device 200''''' may be constructed in the same manner as the sample collection device 200'''' shown in FIG. 13I, but the tip 204''''' of the sample collection device 200''''' includes a tube 205''''' (like the tubes shown in FIGS. 2A and 2B). The distal portion 201''''' including the tip 204''''' is configured to be exposed to the sample such that a predetermined volume of the sample (e.g., 10 microliters, preferably less than about 2 microliters) is placed within the tube 205''''' for analysis at most. Collection of a predetermined volume of the sample is expected to facilitate the accuracy of specimen analysis since a substantially known amount of the sample will be analyzed. The tip 204''''' may have a rounded end as shown, and may be configured to collect a sample from any desired area or location, but the tip 204''''' may be particularly useful when collecting a sample of blood. FIGS. 13Q, 13R, 13S, 13T, 13U, 13V, and 13W are, respectively, the side, rear, side, front, rear, side, and front views of the sample collection device 200'''''

[0305] Referring to FIG. 13X, the sample collection device 200'''''' may be constructed similarly to the sample collection device 200''' shown in FIG. 13P, but the tip 204'''''' of the sample collection device 200'''''' includes a slot 222 rather than a tube. The distal portion 201'''''', including the tip 204'''''', is configured to be exposed to the sample such that a predetermined volume of the sample (e.g., 10 microliters, preferably less than about 5 microliters) is disposed within the slot 222 for analysis at most. Collection of a predetermined volume of the sample is expected to facilitate the accuracy of specimen analysis because a substantially known amount of the sample will be analyzed. The tip 204'''''' may have a rounded end as shown and may be configured to collect the sample from any desired area or location, but the tip 204'''''' may be particularly useful when collecting a blood sample. FIGS. 13Y, 13Z, 13AA, 13BB, 13CC, 13DD, and 13EE are side, rear, side, front, rear, side, and front views, respectively, of the sample collection device 200''''''.

[0306] Referring to FIG. 13FF, the sample collection device 200''''''' may be constructed similarly to the sample collection device 200''''' shown in FIG. 13P, but the tip 204''''''' of the sample collection device 200''''''' includes a ring 224 instead of a tube. The distal portion 201''''''', including the tip 204''''''', is configured to be exposed to the sample such that a predetermined volume of the sample (e.g., 10 microliters, preferably less than about 2 microliters) is disposed within a groove formed by the ring 224 for analysis at most. Collection of a predetermined volume of the sample is expected to facilitate the accuracy of specimen analysis since a substantially known amount of the sample will be analyzed. The tip 204''''''' may have a rounded end as shown and may be configured to collect the sample from any desired region or location, but the tip 204''''''' may be particularly useful when collecting a sample of blood. FIGS. 13GG, 13HH, 13II, 13JJ, 13KK, and 13LL are rear, side, front, rear, side, and front views of the sample collection device 200''''''', respectively.

[0307] Referring to FIG. 13MM, the sample collection device 200'''''''' may be constructed similarly to the sample collection device 200''' shown in FIG. 13P, but the tip 204'''''''' of the sample collection device 200'''''''' includes a first ring 226 and a second ring 228 rather than a tube. The distal portion 201''''''', including the tip 204''''''', is configured to be exposed to the sample such that, at most, a predetermined volume of the sample (e.g., 10 microliters, preferably less than about 5 microliters) is disposed within the grooves formed by the first ring 226 and the grooves formed by the second ring 228 for analysis. Collection of a predetermined volume of the sample is expected to facilitate the accuracy of specimen analysis because a substantially known amount of the sample will be analyzed. The tip 204'''''''' may have a rounded end as shown and may be configured to collect the sample from any desired area or location, but the tip 204'''''''' may be particularly useful when collecting a sample of blood. FIGS. 13NN, 13OO, 13PP, 13QQ, 13RR, and 13SS are, respectively, rear, side, front, rear, side, and front views of the sample collection device 200''''''''.

[0308] Referring now to FIGS. 14A and 14B, an exemplary collet for use in a cartridge is described. The collet 618 may be specifically designed for relatively large volume fluid sample collection, such as saliva, blood, plasma, urine, when the sample is compressed from the distal portion of the sample collection device, as described above and below with respect to FIGS. 12A-12E, 13B. The collet 618 may include a proximal end 620, a distal end 622, and a lumen 624 extending between the ends 620 and 622. The lumen 624 may be sized to allow insertion of the distal portion of the sample collection device into the lumen 624. The collet 618 is positioned within the input tunnel such that the distal portion of the sample collection device first enters the lumen 624 at the proximal end 620. The collet 618 may also include a slot 626, for example, on the upper surface of the collet 618. The slot 626 is sized to receive a portion of the seal piercer therethrough. For example, the engagement member of the seal piercer may extend through the slot into the input tunnel, allowing contact between the seal piercer and the sample collection device.

[0309] The lumen 624 of the collet 618 may also be sized to receive a portion of the shuttle therein. For example, the proximal end of the shuttle may be disposed within the lumen 624 through the distal end 622 of the collet 618. The collet 618 may also be configured to hold the shuttle within the input tunnel in a pre-mixing position. For example, the collet 618 may include one or more locking arms configured to couple the collet 618 to the shuttle in a pre-mixing position. By way of illustration, the collet 618 includes a first locking arm 628 and a second locking arm 630 on opposing lateral sides of the collet 618. Advantageously, when using a sample collection device having a compressible distal portion for collecting a fluid sample, the collet 618 may hold the shuttle in a fixed position within the input tunnel during compression of the distal portion and discharge sample fluid into the shuttle. Each locking arm may include a ramp and a protrusion, shown in FIG. 14B as a ramp 632 and a protrusion 634 for the locking arm 630. The protrusion 634 may be coupled to the shuttle and hold the shuttle in a fixed position in a pre-mixing position. For example, the protrusion may hold the proximal flange of the shuttle during compression of the distal portion of the sample collection device. The first and second locking arms 628 and 630 may also be disengaged from the shuttle during insertion of the sample collection device into the input tunnel 301. The first and second locking arms 628 and 630 may be deflected in response to a force applied on the first and second locking arms 628 and 630 by the sample collection device during insertion of the sample collection device into the input tunnel, disengaging the first and second locking arms 628 and 630 from the shuttle. For example, the shoulder of the sample collection device may contact the ramp of the locking arm and deflect the protrusion outward as the sample collection device moves distally within the input tunnel along the ramp. The ramp is shaped such that the protrusion disengages from the proximal flange of the shuttle, unlocking the shuttle and allowing the shuttle to move from the pre-mixing position to the mixing position, where the shuttle is shaped to be partially disposed within the sample preparation reservoir.

[0310] The collet 618 may include a deflector portion 636 configured to deflect within the cartridge to activate a contact switch. Preferably, the deflector portion 636 is disposed on the bottom surface of the collet 618 and positioned above the contact switch within the input tunnel. The deflector portion 636 may deflect and activate the contact switch in response to a force applied to the deflector portion 636 by the sample collection device during insertion of the sample collection device into the input tunnel. For example, the shoulder of the sample collection device may contact the deflector portion 636 as the sample collection device moves distally within the input tunnel, pushing the deflector portion 636 downward and activating the contact switch. By way of illustration, the deflector portion 636 is an arm configured to deflect downward.

[0311] Referring now to FIGS. 14C and 14D, an alternative collet for use in a cartridge is described. The collet 618' may be designed specifically for relatively small sample collection, such as nasal, ear, blood, when it is not necessary for the sample to be compressed from the distal portion of the sample collection device. As will be appreciated by comparing FIGS. 14C and 14D with FIGS. 14A and 14B, the collet 618' is similar to the collet 618, but the collet 618' does not have a locking arm. The collet 618' has one or more protrusions 637 configured to be disposed at the distal end 622' and contact the proximal end of the shuttle in the pre-mixing position. For example, the one or more protrusions 637 may contact a seal member, such as an O-ring, at the second end 371, 371' of the shuttles 324, 324' and hold the seal member in place. The one or more protrusions may have an angled lead-in configured to direct the distal portion of the sample collection device into the opening at the second end of the shuttle.

[0312] Referring now to FIGS. 15A - 15D, a cartridge 300''', with the upper surface of the housing removed for clarity, is shown in various positions. FIGS. 15A and 15B are cross - sectional views through the center of the input tunnel for further clarity. In FIG. 15A, the cartridge 300''' is shown in the pre - mixing, pre - venting, storage position, and the sample collection device has not yet been inserted into the input tunnel 301'''. As shown, the seal piercer 321''' is in the pre - venting position and has not yet pierced the seal material 320''' across the reservoir. The proximal end of the shuttle 324''' is disposed within the distal end of the collet 618, while the distal end of the shuttle 324''' forms the wall of the sample preparation reservoir 317'''. The deflector portion 636 of the collet 618 is in the pre - deflected position, where the deflector portion 636 has not activated the contact switch 389'''. In FIG. 15B, the cartridge 300''' is shown in the mixing, venting, analysis position, where the sample collection device is fully inserted into the input tunnel 301'''. As shown, the seal piercer 321''' is in the venting position and has pierced the seal material 320''' across each of the reservoirs. The shuttle 324''' has been moved distally from the collet 618 such that the sample and reagent balls 375''' are mixed with the fluid in the sample preparation reservoir 317'''. The deflector portion 636 of the collet 618 is in the deflected position, where the deflector portion 636 has activated the contact switch 389''', and the locking member 387''' locks the sample collection device within the input tunnel 301'''. In FIG. 15C, the cartridge 300''' is still in the pre - mixing, pre - venting position because the sample collection device is only partially inserted into the input tunnel 301'''. FIG. 15D shows the cartridge 300''' in the venting position.

[0313] Referring back to FIG. 15A, an exemplary process for piercing seal material disposed across one or more reservoirs within the cartridge device is described through the interaction between the sample collection device and the seal piercer within the cartridge device.

[0314] The seal piercer 321''' is configured to pierce the seal material 320''' and vent the fluid in the sample preparation reservoir 317''', the cleaning agent reservoir 318''', and / or the substrate reservoir 319'''. Preferably, the seal piercer 321''' is configured to continuously pierce the seal material 320''' across the reservoir during piercing to reduce the resistance onto the sample collection device. The seal piercer 321''' is contacted by a distal portion of the sample collection device within the input tunnel 301''', for example, at the shoulder, in response to the force applied by the sample collection device, moves within the housing 304''', pierces the seal material 320''', and may be configured to vent the fluid in the sample preparation reservoir 317''', the cleaning agent reservoir 318''', and / or the substrate reservoir 319'''. By way of illustration, the seal piercer 321''' is a single piece. The seal piercer 321''' may be disposed within the housing 304''' and partially within the input tunnel 301'''. For example, the engagement member 380''' of the seal piercer 321''' may be disposed within the input tunnel 301''' through the slot 626 of the collet 618, for example. The seal piercer 321''' has one or more piercing el...

Claims

1. 1. A sample analysis cartridge comprising: A housing and an input tunnel extending from an opening in the housing, the input tunnel configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to a sample; a reservoir disposed within the housing adjacent to the input tunnel, the reservoir configured to hold a fluid, the reservoir further configured to receive the sample collected by the sample collection device; a sealing material configured to fluidly seal the fluid within the reservoir; and a seal piercer comprising an engager, a slot, and a piercer, the seal piercer disposed within the housing such that the piercer is adjacent to the reservoir and the engager is within the input tunnel, the engager being configured to be contacted by the sample collection device in the input tunnel and to, in response to a force applied by the sample collection device, slide the seal piercer through the slot during insertion of the sample collection device within the input tunnel such that the piercer moves in a first direction and a second direction different from the first direction to pierce the seal material and vent the fluid in the reservoir; A sample analysis cartridge comprising:

2. a second reservoir disposed within the housing adjacent to the reservoir, the second reservoir configured to hold a second fluid; 2. The sample analysis cartridge of claim 1, wherein the seal piercer further comprises a second piercer disposed adjacent to the second reservoir, the second piercer configured to pierce the seal material across the second reservoir during insertion of the sample collection device within the input tunnel to vent the second fluid in the second reservoir.

3. The sample analysis cartridge of claim 1 , wherein the first direction is generally parallel to a longitudinal axis of the input tunnel and the second direction is generally perpendicular to the first direction.

4. The sample analysis cartridge of claim 1 , wherein the seal piercer further comprises one or more additional piercers.

5. 10. The sample analysis cartridge of claim 1, further comprising one or more ramps configured to deflect the piercer in the second direction toward the sealing material to pierce the sealing material.

6. 2. The sample analysis cartridge of claim 1, wherein the seal piercer further comprises a slider configured such that movement of the slider in the first direction causes the piercer to move in the second direction to pierce the seal material.

7. the reservoir is a sample preparation reservoir; The device further comprises a cleaning agent reservoir and a substrate reservoir, the cleaning agent reservoir being disposed within the housing and configured to hold a cleaning fluid, and the substrate reservoir being disposed within the housing and configured to hold a substrate fluid; the sealing material is further configured to fluidly seal the cleaning fluid in the cleaning agent reservoir and the substrate fluid in the substrate reservoir; The sample analysis cartridge of claim 1 , wherein the seal piercer is configured to pierce the seal material to vent the fluid in the sample preparation reservoir, the cleaning fluid in the cleaning agent reservoir, and the substrate fluid in the substrate reservoir.

8. The sample analysis cartridge of claim 7 , wherein the seal piercer is configured to sequentially pierce the seal material across the sample preparation reservoir, the detergent reservoir, and the substrate reservoir, in any order.

9. a first lamp positioned adjacent to the sample preparation reservoir, a second lamp positioned adjacent to the detergent reservoir, and a third lamp positioned adjacent to the substrate reservoir; the seal piercer further comprises a second piercer and a third piercer; the first ramp is configured to deflect the piercer toward the sealing material to pierce the sealing material across the sample preparation reservoir; the second ramp is configured to deflect the second piercer towards the sealing material to pierce the sealing material across the irrigant reservoir; The sample analysis cartridge of claim 7 , wherein the third ramp is configured to deflect the third piercer towards the sealing material to pierce the sealing material across the substrate reservoir.

10. 10. The sample analysis cartridge of claim 9, wherein the distances between each ramp and each piercer are different in a pre-vent position such that the seal piercer is configured to sequentially pierce the seal material across the sample preparation reservoir, the detergent reservoir, and the substrate reservoir, in any order.

11. The sample analysis cartridge of claim 1 , wherein the engager is configured to engage an engagement zone of the sample collection device when the sample collection device is in the input tunnel.

12. The sample analysis cartridge of claim 1 , wherein the engager is U-shaped.

13. The sample analysis cartridge of claim 1 , further comprising a contact switch positioned within the input tunnel to be activated during insertion of the sample collection device within the input tunnel.

14. The sample analysis cartridge of claim 1 , wherein the seal piercer is configured to move out of one or more holes pierced in the seal material after piercing and venting the fluid in the reservoir.

15. 2. The sample analysis cartridge of claim 1, further comprising a shuttle disposed within the input tunnel between the reservoir and the opening at a first position, the shuttle comprising a body having a first end and a second end, the shuttle configured to seal the reservoir from the input tunnel at the first end at the first position, the shuttle configured to receive the sample via the sample collection device at the second end, and the shuttle configured to move within the input tunnel to a second position where the sample is moved into the reservoir.

16. The sample analysis cartridge of claim 15, further comprising one or more locking members configured to couple to the sample collection device to irreversibly lock the sample collection device within the input tunnel in the second position.

17. The sample analysis cartridge of claim 15 , wherein the seal piercer is configured to pierce the seal material to vent the fluid in the reservoir before the shuttle moves to the second position.

18. 16. The sample analysis cartridge of claim 15, wherein the shuttle is configured to store a reagent ball formed from a component containing a reagent in a compartment between the first end and the second end such that in the first position the reagent ball is not exposed to the fluid in the reservoir and in the second position the reagent ball is exposed to the fluid in the reservoir.

19. a collet coupled to the shuttle at the first position and disposed within the input tunnel, the collet configured to be decoupled from the shuttle during insertion of the sample collection device within the input tunnel; The sample analysis cartridge of claim 15 , wherein the shuttle is configured to move within the input tunnel from the first position to the second position while the collet remains in a fixed position within the input tunnel.

20. 20. The sample analysis cartridge of claim 19, wherein the collet comprises a collet slot dimensioned to receive a portion of the seal piercer therethrough to enable contact between the sample collection device and the seal piercer within the input tunnel.

21. the reservoir is configured such that the fluid therein is mixed with the sample and reagents within the reservoir; The sample analysis cartridge of claim 18, further comprising a sensor positioned within the housing so as to be exposed to the sample from the reservoir and the fluid mixed with the reagent, the sensor configured to generate a signal indicative of at least one of the presence, absence, or amount of one or more analytes in the sample.

22. 22. The sample analysis cartridge of claim 21, wherein the reservoir is configured such that the fluid therein is mixed with the reagent comprising a plurality of solid particles, a plurality of affinity molecules, and a plurality of signal transduction substances, and the sample.

23. 22. The sample analysis cartridge of claim 21, wherein the reservoir is configured such that the fluid therein is mixed with the sample and the reagent comprises a plurality of magnetic particles configured to be magnetically held across a working electrode of the sensor.

24. 22. The sample analysis cartridge of claim 21, further comprising an analytical channel disposed within the housing, the analytical channel configured to receive the sample from the reservoir and the fluid mixed with the reagent, at least a portion of the sensor is disposed within the analytical channel, and the fluid mixed with the sample and the reagent advances to at least the portion of the sensor via the analytical channel.

25. 20. The sample analysis cartridge of claim 18, wherein the reservoir is configured to allow amplification of one or more nucleic acids within the sample.

26. the reservoir is configured such that the fluid therein is mixed with the sample and reagents within the reservoir; The sample analysis cartridge of claim 18 , further comprising a piezoelectric transducer configured to emit energy into the reservoir to mix the fluid, the reagent, and the sample.

27. The sample analysis cartridge of claim 1 , wherein the sample analysis cartridge is sized to be electrically coupled to and at least partially disposed within a reader, the reader configured to receive and process electrical signals from the sample analysis cartridge.

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