System and method for enhanced detection and quantification of analyte

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 July 17, 2015, the entire contents of which are incorporated herein by reference.

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

[0003] Conventional techniques for identifying the presence, absence, and / or quantity of nucleic acids, proteins, and / or other molecules of interest in a sample often require expensive laboratory equipment and the expertise of highly trained medical professionals.As a result, such analyses are typically performed in laboratories or medical facilities.Such molecular detection can be important, for example, for detecting the presence of pathogens, diseases, contamination, overdose, and poisoning in individuals or other animals, or in the environment.Unfortunately, individuals currently face long waiting times before being able to perform the appropriate test and before results can be generated and analyzed.Due to long waiting times and the inconvenience of traveling to laboratories or medical facilities, diseases and contaminations often spread and can cause significant harm even before the presence of the disease or contamination is identified. Summary of the Invention [Means for solving the problem]

[0004] There is a significant need for improved molecular detection and quantification technologies. Described herein is a device that can detect molecules of interest in less time and with less technical expertise than conventional devices currently in use. The device may be utilized by consumers in non-clinical settings, such as at school, work, and home. In addition, the device can be used by consumers visiting pharmacies or medical facilities and can generate results quickly so that the results are available by the time the consumer speaks with a pharmacist or medical professional. The device herein may also be configured to minimize biohazard risks.

[0005] One aspect of the present disclosure is directed to a system for detecting molecules. 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-bound affinity molecule, a plurality of detection agents, each of which may include a signal transduction agent, 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-bound 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 cartridge housing. 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, which may or may not be pre-filled within the reservoir. The reagent shuttle may be disposed between the reservoir and the opening in the first position, and the reagent shuttle may have a first end and a second end. The reagent shuttle may be configured to store a reagent ball comprising a reagent (e.g., a sample preparation 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 configured to move within the input tunnel to the second position upon receiving a force above a threshold force, thereby moving the reagent ball and sample 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 the 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, so that up to a predetermined volume of sample may be mixed into the fluid in the reservoir. The reservoir may be sealed via the sample collection device being 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 in the input tunnel in the second position.

[0009] The shuttle may include up to one or more sample compartments configured to store a predetermined volume of sample and one or more reagent ball compartments configured to store a reagent ball and optionally additional reagent balls. In some embodiments, the one or more sample compartments and the one or more reagent ball compartments are not exposed to fluid in the reservoir in the first position. The one or more sample compartments and the one or more reagent ball compartments may be exposed to fluid in the reservoir in the second position. The shuttle may have a compartment divider configured to divide at least one sample compartment from at least one reagent ball compartment. The compartment divider may have a slot configured to promote mixing when placed in 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 agents. The reagent may include a plurality of magnetic particles configured to be magnetically held across a working electrode of the sensor. At least one magnetic particle of the plurality of magnetic particles may be configured to be indirectly bound to the signaling agent.

[0011] The cartridge may, for example, include 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 ball and sample may travel via the analysis channel to at least a portion of the sensor.

[0012] The cartridge may include a contact switch, a sealing material configured to fluidly seal the fluid in the reservoir, and / or a seal piercer, each of which 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 sealing material and vent the fluid in 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 cartridge housing. The input tunnel may extend from the opening, and the input tunnel 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, which may or may not be pre-filled. The seal material may be configured to fluidically seal the fluid within the reservoir. The seal piercer may be partially disposed within the input tunnel, and the seal piercer may be configured to be contacted by a sample collection device within the input tunnel and move in response to a force applied by the sample collection device to puncture 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 generally parallel to the movement of the sample collection device within the input tunnel, and the second direction may be generally 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 the one or more piercers may be configured to move in the 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 a sample preparation reagent, which may be in fluid and / or may be introduced, for example, via the introduction of a reagent ball. The cartridge may also include a detergent reservoir and / or a substrate reservoir. The seal piercer may be configured to pierce the seal material and vent the individual fluids in the sample preparation reservoir, detergent reservoir, and substrate reservoir. The seal piercer may include an engager disposed in the input tunnel, which may be configured to engage an engagement zone of the sample collection device when the sample collection device is in the input tunnel.

[0016] The cartridge may include a contact switch, which may be disposed on a circuit board within the housing. The contact switch may be configured to be activated upon insertion of the sample collection device into the input tunnel. Movement of the seal piercer may cause activation of the contact switch. The seal piercer may be configured to sequentially pierce the seal material across the sample collection reservoir, the irrigant reservoir, and the substrate reservoir in any order. The seal piercer may be configured to pierce and vent the fluid in the reservoirs before moving from one or more holes pierced in the seal material.

[0017] The cartridge may include a contact switch and a shuttle disposed between the reservoir and the opening 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 a 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 puncture the seal material and vent the fluid in the reservoir, and / or (iii) activate 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 cartridge housing. The input tunnel may extend from an opening, and the input tunnel 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 and to receive the sample on the distal portion of the sample collection device. The analysis channel may be configured to receive a fluid having a sample and a reagent, the fluid comprising a plurality of magnetic particles mixed therein, from the reservoir. The circuit board may include a sensor having a working electrode, and the sensor may be configured to be exposed to the mixed fluid in the analysis channel and 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 grooves configured to promote homogeneous distribution of the plurality of magnetic particles across the working electrode and promote resistance to movement of the plurality of magnetic particles 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 a sample. 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 cartridge housing. The input tunnel may extend from an opening and be configured to allow insertion of the sample collection device. The reservoir may be configured to hold a fluid and to receive a sample on the distal portion of the sample collection device. The analysis channel may be configured to receive a fluid having a sample and a reagent, the fluid comprising a plurality of magnetic particles mixed therein, from the reservoir. The circuit board may include a sensor exposed to the mixed fluid 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 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 positioned adjacent to the single working electrode of the sensor when the sample analysis cartridge is inserted into the sample analysis reader, and the first and second magnetic generators may be configured to generate a magnetic field across the length of the single working electrode to promote a homogeneous distribution of the plurality of magnetic particles across the length of the single working electrode.

[0021] Reception of the sample analysis cartridge by the sample analysis reader may result in an electrical coupling between the sample analysis cartridge and the sample analysis reader. The sample analysis cartridge may be configured to transmit signals indicative of at least one of the presence, absence, or amount of one or more analytes in the sample to the sample analysis reader for processing. The sample analysis reader may be configured to transmit processed signals indicative of at least one of the presence, absence, or amount of one or more analytes in the sample to a computer. The kit may include a computer-readable medium with instructions that, when executed by a processor of a computer, cause a display of the computer to display information indicative of 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. 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 the 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 cartridge housing. The input tunnel may extend from an opening, and the input tunnel 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 and 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 occluding its entire cross section. The heater may be configured to heat the phase change material so that the phase change material does not occlude the entire cross section of the outlet. The analysis channel may be configured to receive a fluid having a sample and a reagent, the fluid comprising a plurality of magnetic particles mixed therein, from the reservoir through the outlet. A sensor may be exposed to the mixed fluid 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 heater may be masked with a masking material configured to electrically isolate the heater from the sensor. The masking material may be a solder mask.

[0024] The cartridge may include a cleaning agent reservoir and a cleaning agent reservoir heater. The cleaning agent reservoir may be configured to hold a cleaning fluid and may include a cleaning agent reservoir outlet having a phase change material therein for blocking an entire cross-section of the cleaning agent reservoir outlet. The cleaning agent reservoir heater may be configured to heat the phase change material in the cleaning agent reservoir outlet such that the phase change material does not block an entire cross-section of the cleaning agent reservoir outlet, thereby allowing the cleaning fluid to enter the analysis channel and proceed to the sensor. The cleaning agent reservoir heater may be masked with a masking material configured to electrically insulate the cleaning agent 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 therein for blocking an entire cross-section of the substrate reservoir outlet. The substrate reservoir heater may be configured to heat the phase change material in the substrate reservoir outlet such that the phase change material does not block an entire cross-section of the substrate reservoir outlet, thereby allowing the substrate fluid 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 disconnector, and / or one or more heaters, each of which may be within the cartridge housing. The sample preparation reservoir may be configured to hold a 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 from time to time. The fluid disconnector may be a phase change material. At least one of the one or more heaters may be configured to heat a phase change material in the sample preparation reservoir outlet, so that the phase change material unseals the sample preparation reservoir outlet and allows fluid to mix with the sample therein and flow into the analysis channel. At least one of the one or more heaters may be configured to heat a phase change material in the fluid disconnector after unsealing the sample preparation reservoir outlet, so that the phase change material in the fluid disconnector flows into the analysis channel and fluidically isolates the sample preparation reservoir from the substrate reservoir. At least one of the one or more heaters may be configured to heat a phase change material in the substrate reservoir outlet after the fluid disconnector fluidically isolates the sample preparation reservoir from the substrate reservoir, so that the phase change material unseals the substrate reservoir outlet and allows fluid comprising the chemical substrate to flow into the analysis channel but not into the sample preparation reservoir.

[0027] The one or more heaters may include a sample preparation reservoir heater, a fluid disconnector heater, and / or a substrate reservoir heater. The sample preparation reservoir heater may be configured to heat a phase change material in the sample preparation reservoir outlet. The fluid disconnector heater of the fluid disconnector may be configured to heat the phase change material. The substrate reservoir heater may be configured to heat a phase change material in the substrate reservoir outlet. The sample preparation reservoir heater, the fluid disconnector heater, and the substrate reservoir heater may each be masked with a masking material configured to electrically isolate the respective heater from a sensor in the analysis channel.

[0028] The cartridge may include a cleaning agent reservoir configured to hold a cleaning fluid. The cleaning agent reservoir may include a cleaning agent reservoir outlet having a phase change material therein for sealing the cleaning agent reservoir outlet. At least one of the one or more heaters may be configured to heat the phase change material in the cleaning agent reservoir outlet after the fluid disconnector fluidically isolates the sample preparation reservoir from the substrate reservoir but before the one or more heaters heat the phase change material in the substrate reservoir outlet, so that the phase change material unseals the cleaning agent reservoir outlet and allows the cleaning fluid to flow into the analysis channel, flushing signaling substances from the sample preparation reservoir that are not bound to the magnetic particles from the sensor in the analysis channel. The fluid with the chemical substrate may be configured to flush signaling substances from the sample preparation reservoir that are not bound to the magnetic particles from the sensor in 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 cartridge housing. The input tunnel may extend from an opening, and the input tunnel may be configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to sample fluid. The reservoir may be configured to hold a fluid, which 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 upon receiving a force above a threshold force, thereby moving the sample compartment with the sample fluid into the reservoir. The sensor may be configured to be exposed to a fluid mixed with the sample fluid, and the sensor 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 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 within the reservoir in the second position. The sample compartment may be configured to receive up to a predetermined volume of sample fluid compressed from a distal portion of the sample collection device. The cartridge may include an overflow compartment configured to receive a volume of sample fluid in excess of the predetermined volume from the sample compartment.

[0031] The reagent balls comprise the reagents necessary to perform amplification of the target analyte. The reagent balls can be of any suitable shape, non-limiting examples of which include diameters 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 balls can be of any suitable shape, for example, spherical, cylindrical, conical, or elliptical.

[0032] The components of the reagent bowl are preselected for the analyte and its method for amplification and subsequent detection and / or quantification. In one aspect, the components of the reagent bowl comprise reagents for the detection or quantification of hormones, other small molecules, or proteins or fragments. In another aspect, the components of the reagent bowl comprise reagents for the detection and / or quantification of nucleic acids by a method that includes amplifying the nucleic acid.

[0033] A kit may be provided that includes a sample analysis cartridge and a sample collection device. The sample collection device may include a wicking portion at a distal portion. The wicking portion may be configured to wick up and absorb sample fluid. The wicking portion may be compressed to expel the sample fluid into the sample compartment. The sample compartment may be configured to receive up to a predetermined volume of sample fluid compressed from the distal portion of the sample collection device. The sample analysis cartridge may further include an overflow compartment configured to receive a volume of sample fluid in excess of the predetermined volume from the sample compartment. The wicking portion of the sample collection device may be configured to wick up and absorb sample fluid in excess of the predetermined volume, allowing a user to meter the amount of sample fluid compressed into the sample compartment and the overflow compartment. At least a portion of the wicking portion may be slidably disposed within a 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 in 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 a collector based on the volume of sample fluid collected. The sample collection indicator may be a colored thread embedded within the wicking portion that becomes increasingly visually exposed as the volume of sample collected increases.

[0035] According to another aspect, compositions and methods are provided for detecting and / or quantifying at least one of the presence, absence, or amount of a target analyte in a sample in a cartridge. The method may include mixing a sample having, in a fluid reservoir of the cartridge, a plurality of affinity molecules, a plurality of debinding agents, a plurality of signal transduction agents, a plurality of competitor molecules pre-bound to competitive binding molecules, each competitor molecule carrying a label, and a plurality of sample target analytes pre-bound to sample binding molecules, using at least one debinding agent of the plurality of debinding agents to debind at least one competitor molecule from the pre-bound competitive binding molecule, using at least one debinding agent of the plurality of debinding agents to debind at least one sample target analyte from the pre-bound sample binding molecule, binding the label of the debound competitor molecule to a signal transduction agent of the plurality of signal transduction agents, binding the debound 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 the sample target analyte in the cartridge. The reagent balls can be 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. Although the reagent balls are illustrated as spherical, 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.

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

[0037] In a further aspect, provided herein are compositions and methods of use thereof, wherein the reagent ball further contains a plurality of forward primers selected to amplify a target nucleic acid, each having a spacer element and a capture element attached thereto, a plurality of reverse primers selected to amplify the target nucleic acid, each having a spacer element and a signal transduction agent or signal transduction capture element attached thereto, and a plurality of nucleotides or analogs thereof (dNTPs) for the amplification reaction.

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

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

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

[0041] In one aspect, the reagent bowl further contains a plurality of DNA template control nucleic acids. In another aspect, the reagent bowl alternatively or also contains a plurality of RNA template control nucleic acids.

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

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

[0044] The reagent bowl may further contain an effective amount of one or more lysing agents to liberate 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 a helicase to unwind the dsDNA for loading a primer or an analog thereof, such as RecA or UvsX or RAD51. Additionally, the reagent ball may contain mutL, RecFOR enzyme, UvsY.

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

[0047] In one aspect, the elements are provided in a sample reagent bowl and mixed with the sample in the reservoir. Upon degradation, the reagent contacts the target nucleic acid, and amplification of the target occurs through hybridization of the primer with the target nucleic acid and a series of enzyme-driven melting and reassembly of DNA or, in the case of target RNA, complementary DNA (cDNA) molecules, which are generated first from the RNA target nucleic acid containing the target sequence and double-stranded cDNA, which then serves as a template for further amplification. The reagent bowl 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. While the reagent bowl is illustrated as spherical, the present disclosure is not limited thereto; many shapes may be used, and multiple reagent bowls, each containing the same or different reagents, may also be used. The spacer element separating the primer from the label comprises a polymer such as hexaethylene glycol or triethylene glycol. Alternatively, it can be a linear carbon polymer containing from about 1 to about 18 carbon atoms or more, such as hexane or pentane.

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

[0049] The reagents and their amounts are preselected to promote 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. The preferred reverse transcriptase depends on the target and may not be the same between pills for different targets. As can be understood, the reagent ball can be used for a variety of different test applications by modifying the primer sequence and capture element, primer concentration, particle concentration, affinity agent, lysis agent, polymerase, reverse transcriptase, and other enzymes that best match the optimal conditions for each type of target (e.g., HIV quantification versus influenza detection may have different reaction conditions).

[0050] The polymerase can include several different types within the strand displacement polymerase category, optionally including, for example, Bsu DNA polymerase or a fragment thereof, such as Bsu DNA polymerase large fragment, Bst DNA polymerase or a fragment thereof, such as DNA polymerase large fragment, phi29 DNA polymerase, etc. It should be understood that with respect to polymerases and reverse transcriptases, it is often a desirable property to have a polymerase that lacks exonuclease activity, or with respect to reverse transcriptases, a polymerase that lacks RNase H activity, etc.

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

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

[0053] In some aspects, the reagent ball and method further utilize a helicase to unwind the dsDNA for primer loading. Non-limiting examples include enzymes such as uvrD helicase from E. coli, T4 Gene41 helicase, and many others. Recombinases that facilitate primer loading into the dsDNA for enzymatic melting of double-stranded DNA for primer annealing include RecA from E. coli, RAD51 human recombinase, DMC1 human meiotic recombinase, or T4 UvsX, RB49 Recombinases and SSBs can include analogs from phages such as UvsX, RB69 UvsX, and many others. As known to those skilled in the art, combinations of helicases and SSBs are useful for promoting isothermal amplification. Cofactors such as MutL can be added to promote helicase-dependent amplification. Combinations of recombinases and SSBs can be useful in RPA, and sometimes cofactors such as RecFOR from E. coli and / or UvsY from various phages are also employed to promote the reaction by assisting the primary enzymes (RecA) or (uvrD) in recombinase polymerase amplification and helicase-dependent amplification, respectively. As will be appreciated by those skilled in the art, the reagent bowl and / or reservoir can further contain any one or more of the aforementioned reagents, as needed, to promote specific amplification of target nucleic acids.

[0054] Primer concentrations for isothermal amplification reactions such as SDA, HDA, and RPA can be 0.01 to 10 micromolar, preferably closer to 0.5 micromolar. A LAMP primer mix can be prepared using a total of four or six (looped) primers. A 10x primer mix can contain 16 μM FIP, 16 μM BIP, 2 μM F3, 2 μM BE, 4 μM Loop F, and 4 μM Loop B. dNTPs can be provided at concentrations of 1 μM to 500 μmolar, preferably about 200 μmolar. SDA, HDA, and RPA can require large amounts of ATP because some of the enzymes that enable enzymatic melting and primer loading into dsDNA require ATP for functionalization; therefore, as much as 100 μmol to 4 millimolar ATP can be used in the reaction.

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

[0056] Methods and compositions for non-covalent attachment of molecules that can also provide a label or signal for detection are known in the art. Non-limiting examples include avidin or streptavidin-biotin conjugates. Modifications of biotin are known in the art and are intended to be within the scope of this disclosure. Non-limiting examples include biotin-dT, biotin-TEG, dual biotin, PC-biotin, and desthiobiotin-TEG, 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 binding nucleic acids and proteins, such as when a primer is directly conjugated to a signaling agent; in one aspect, the signaling agent is an enzyme such as HRP. Non-limiting examples of covalent attachment of a protein or polypeptide to another moiety include attachment of the moiety to a cross-linking reactive group, such as carbodiimide, imidoester, and maleimide. See, e.g., Bioconjugate Techniques, 3rd Ed., Hermanson, GT (2013).

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

[0058] Each of the affinity molecules of the plurality of affinity molecules may be bound to a solid particle. The solid particle may be formed of a magnetically responsive material and / or a non-magnetically responsive material. The non-magnetically responsive material may be a gold nanoparticle.

[0059] The plurality of sample target analytes pre-bound to the sample binding molecules 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 molecules may include 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 molecules labeled with biotin and pre-bound to vitamin D binding protein molecules. At least one of the plurality of affinity molecules, the plurality of unbinding agents, the plurality of signal transduction substances, and the plurality of competitor molecules pre-bound to the competitive binding molecules may be stored in a reagent bowl.

[0060] As mentioned above, primers (forward and reverse for target nucleic acid and control template) are designed based on the nucleotide sequence of the target nucleic acid to be amplified and detected, if any.The method of designing optimal primers based on target sequence is known in the art (see, for example, simgene.com / Primer3; quill.com, molbiol-tools.caPCR, and ncbi.nlm.nih.gov / tools / primer-blast / (each last accessed July 15, 2016)), and varies according to the amplification method used, 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 a reagent bowl, a reservoir, and / or a sensor, each of which may be located within the cartridge housing. The reagent bowl may include a plurality of competitor molecules pre-bound to a competitive binding molecule, and each of the plurality of competitor molecules may carry a label or be bound to a signaling agent. In another aspect, the reagent bowl comprises, or alternatively consists essentially of, reagents necessary for amplification and detection of a target nucleic acid. The reservoir may be configured to hold a reservoir fluid, which may or may not be pre-filled within the reservoir. The reservoir may be configured to allow mixing of the fluid in the reservoir, the plurality of affinity molecules, the plurality of debinding agents, the plurality of signaling agents, the plurality of competitor molecules pre-bound to the competitive binding molecules, and a sample from a sample collection device, the sample having a plurality of sample target analytes pre-bound to the sample binding molecules. A debinding agent of the plurality of debinding agents may be configured to debind a competitor molecule from a pre-bound competitor binding molecule or a sample target analyte from a pre-bound sample binding molecule. The label of the debound competitor molecule may be configured to bind to a signaling agent, and the debound competitor molecule may be configured to bind to an affinity molecule of the plurality of affinity molecules. In another aspect, the reservoir is configured to hold a reservoir fluid, which may or may not be pre-filled into the reservoir.The reservoir is configured to allow mixing of a fluid within the reservoir, a plurality of enzymes for facilitating the amplification reaction, e.g., a polymerase, a reverse transcriptase, a plurality of magnetic beads having affinity molecules bound thereto, a plurality of forward primers selected to amplify a target nucleic acid, each having a spacer element bound thereto, a plurality of reverse primers selected to amplify a target nucleic acid, each having a spacer element and a signal transduction or signal capture element bound thereto, a plurality of nucleotides or analogs thereof (dNTPs) for the amplification reaction, a plurality of DNA template control nucleic acids, a plurality of reverse primers selected to serve as internal controls, each having a spacer element and a signal transduction agent bound thereto, and a plurality of forward primers selected to serve as internal controls, each having a spacer element and a capture element bound thereto. In one aspect, the amplification reagents are selected for any one or more other PCR or isothermal amplification methods.

[0062] In a further aspect, the reagent bowl and / or reservoir contains an effective amount of a lysing agent to lyse a sample comprising cells, e.g., a bacterial sample, and release intracellular DNA, RNA, and / or proteins, which serve as analytes. Non-limiting examples of lysing agents include NP-40, CHAPS, deoxycholate, Triton X-100, NP40, and Tween 20.

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

[0064] A spacer element can be advantageous because it places the nucleic acid portion of the primer further away from the label, allowing the primer to better participate in the amplification reaction if other steric hindrance events, such as being bound to a particle or a signaling agent (both of which can be cumbersome), occur or have already occurred. 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 be further configured to generate a signal indicative of 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 a signaling agent may localize in the analysis channel across the sensor, and the localized signaling agent may react with a substrate from the substrate solution to generate an electrical signal that is sensed by the sensor. The sensor may use the electrical signal from the reaction to transmit a signal indicative of at least one of the presence, absence, or amount of the sample target analyte in the sample.

[0066] The reservoir may be further configured to allow mixing of a plurality of solid particles into the reservoir fluid. Each solid particle may be pre-bound to an affinity molecule from the plurality of affinity molecules. The plurality of solid particles may be formed from a magnetically responsive material and / or a non-magnetically responsive material. The non-magnetically responsive material may be gold nanoparticles. The plurality of sample target analytes pre-bound to the sample binding molecules may include 25-hydroxyvitamin D3 and / or 25-hydroxyvitamin D2 molecules pre-bound to binding protein molecules. The magnetically responsive material may be magnetically held 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 cartridge housing. The input tunnel may be configured to allow insertion of a sample collection device extending from an opening and having a distal portion adapted to be exposed to a sample. The reservoir may be configured to hold a 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 decouple from the shuttle during insertion of the sample collection device into the input tunnel. The shuttle moves within the input tunnel from the first position to a second position after the collet decouples from the shuttle, such that the shuttle may be at least partially disposed 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 the 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 proximal 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 a 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, and the one or more locking arms may be configured to be deflected to decouple 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 in the reservoir and a seal piercer partially disposed within the input tunnel. The seal piercer may be configured to be contacted by a sample collection device in 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 in the reservoir. The collet may have a slot, and a portion of the seal piercer may extend through the slot into the input tunnel to allow 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 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. The deflector portion of the collet may include an arm configured to deflect downward and activate the contact switch. The contact switch may be positioned within the input tunnel such that activation of the contact switch indicates full insertion of the sample collection device into the input tunnel.

[0072] The shuttle may be configured to store a reagent ball comprising a reagent between a first end and a second end of the shuttle, preferably 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.

[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 cartridge housing. The input tunnel may extend from an opening and may 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 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 a deflector portion and a 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 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.

[0074] The sample analysis cartridge may include a shuttle disposed within the input tunnel and configured to store a reagent ball comprising 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 downward and activate a contact switch. The contact switch may be positioned such that activation of the contact switch indicates full 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 the sample, and 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.

[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 cartridge housing. The input tunnel may extend from an opening and may 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. 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 to move fluid in the reservoir between the first and second compartments in a wave pattern and mix the fluid in the reservoir.

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

[0078] The ultrasonic generator may be a piezoelectric transducer, such as a piezoelectric ceramic disc. The ultrasonic generator may form part of a wall of the reservoir, for example, the bottom wall of the reservoir. The reservoir may be symmetrical. The reservoir walls may meet at an angle greater than a predetermined angle, such as 60°, to facilitate emptying of the fluid through the reservoir outlet. The ultrasonic generator may be positioned off-center of the reservoir to facilitate mixing of the fluid within the reservoir.

[0079] The sample analysis cartridge may include a printed circuit board 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 in the reservoir, and the temperature sensor may be disposed on a printed circuit board located adjacent to the ultrasonic generator.

[0081] The sample analysis cartridge may include a contact switch configured to indicate insertion of the sample collection device into the input tunnel. The ultrasound generator may be configured to emit acoustic waves after actuation of the contact switch. For example, the reader may instruct the ultrasound generator to emit acoustic waves after the reader receives 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 a reaction of the mixed fluids in the reservoir.

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

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

[0085] The reagent bowl may be held within the shuttle. The shuttle may be disposed within the input tunnel of the cartridge. The reagent bowl may comprise reagents for isothermal amplification of the target nucleic acid, as described above. The reagents may comprise a polymerase, primers for amplification of the target nucleic acid, and a signaling substance for detection of the 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 a fluid and receive a sample collected by a sample collection device. The ultrasonic generator may be configured to emit acoustic waves and mix the fluid and the sample in the reservoir. The temperature sensor may be configured to generate a signal indicative of the temperature of the fluid in the reservoir. The processor may be configured to activate the ultrasonic generator, emit acoustic waves, and monitor a signal from the temperature sensor. The processor may be further configured to modify the emission of acoustic waves from the ultrasonic generator if the signal indicates a temperature of the fluid in the reservoir outside a threshold. The sample analysis cartridge may include the reservoir, the ultrasonic generator, and / or the temperature sensor, each of which may be within the cartridge housing, 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 located 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 when the signal indicates that the temperature of the fluid in the reservoir is above a threshold. The processor may modify the emission of acoustic waves from the ultrasonic generator by increasing the duty cycle of the ultrasonic generator when the signal indicates that the temperature of the fluid in the reservoir is below a threshold. The processor may modify the emission of acoustic waves from the ultrasonic generator by deactivating the ultrasonic generator when the signal indicates that the temperature of the fluid in the reservoir is above a threshold.

[0088] The acoustic waves emitted by the ultrasonic generator may be configured to isothermally amplify a reaction of the fluid mixed in the reservoir. The sample analysis cartridge may include a reagent ball disposed in 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 a polymerase, a primer, and a signaling substance. 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 control working electrode, a working electrode, and / or a negative control working electrode. The positive control working electrode may include an affinity molecule pre-bound to the positive control working electrode. For example, the affinity molecule may be pre-bound to the surface of the positive control working electrode disposed in an analysis channel of the cartridge. The positive control working electrode may be configured to generate a first signal based on a reaction between a signaling substance bound directly or indirectly to the affinity molecule and a chemical substrate. The signaling substance may be from a reagent bowl. 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 the signaling substance and the chemical substrate localized on the working electrode. The negative control working electrode may include a self-assembled monolayer. For example, the self-assembled monolayer may be on the surface of the negative control working electrode disposed in the analysis channel of the cartridge. The negative control working electrode may be configured to generate a third signal based on a reaction between a signaling agent localized at the negative working electrode and a chemical substrate. As should be understood, the terms "first," "second," and "third" are used to distinguish between terms and do not necessarily imply an order.

[0090] The second signal may indicate at least one of the presence, absence, or number 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 an amount of reaction below a threshold.

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

[0092] A kit including the cartridge is also provided. The kit may include a processor configured to process the second signal and generate information indicating at least one of the presence, absence, or number of one or more analytes in the sample. The processor may process the 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 the third signal and determine whether the third signal indicates an amount of reaction below a threshold. The processor may generate an alert if the amount is above the threshold. The processor may be a component of the reader.

[0093] The working electrode may be masked with a plurality of grooves configured to promote a homogeneous distribution of a plurality of magnetic particles directly or indirectly coupled to a signaling agent localized across the working electrode and to promote resistance to movement of the plurality of magnetic particles 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 a working electrode that is disposed within the analysis channel of the cartridge.

[0095] The working electrode may include affinity molecules pre-bound to the working electrode, for example, affinity molecules may be pre-bound to the surface of a working electrode that is disposed within the analysis channel of the cartridge.

[0096] Such methods and devices may be used, for example, to determine, among other things, an illness a person is suffering from, among other things, a drug or poison to which a person is adversely reacting, among other things, or a chemical that is contaminating water, among other things. Other examples include quantifying the concentrations of various agents, including, but not limited to, vitamins, hormones, proteins, or other analytes of interest in the body, waterborne and foodborne pathogens, microbial growth and / or contamination of medical equipment, and other potentially disease-causing contaminants from pets and livestock. Examples of contaminants include, but are not limited to, 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, but are not limited to, Shigella, Salmonella, Vibrio, Yersinia, Listeria, E. coli, and Campylobacter. Applications of this technology are not limited to pathogens or specimens important to the health of human patients, but also include the health and conservation of pets and livestock, e.g., veterinary applications. The present invention provides, for example, the following. (Item 1) 1. 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 the opening, 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 configured to hold a fluid; and a reagent shuttle disposed between the reservoir and the opening at a first position, the reagent shuttle having a first end and a second end, the reagent shuttle configured to store a reagent ball having 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 upon receiving a force above a threshold force, such that the reagent ball and the sample move into the reservoir and the reservoir is continuously sealed from the input tunnel proximal to the reagent shuttle during movement from the first position to the second position; a sensor configured to analyze the reagent ball and a fluid mixed with the sample, 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; A sample analysis cartridge comprising: (Item 2) Item 1. The sample analysis cartridge of 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, so that up to a predetermined volume of the sample is mixed into the fluid in the reservoir. (Item 3) 2. The sample analysis cartridge of claim 1, wherein the reservoir is sealed via the sample collection device being partially inserted into the second end of the shuttle during movement from the first position to the second position. (Item 4) Item 10. The sample analysis cartridge of item 1, further comprising one or more locking members configured to irreversibly lock the sample collection device within the input tunnel in the second position. (Item 5) the shuttle comprises at most one or more sample compartments configured to store 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 fluid in the reservoir in the first position; the one or more sample compartments and the one or more reagent ball compartments are exposed to fluid in the reservoir in the second position; Item 1. The sample analysis cartridge according to item 1. (Item 6) 6. The sample analysis cartridge of claim 5, wherein the shuttle comprises a compartment divider configured to divide at least one sample compartment from at least one reagent bowl compartment. (Item 7) 7. The sample analysis cartridge of claim 6, wherein the compartment divider comprises a slot configured to promote mixing when disposed within the sample preparation reservoir in the second position. (Item 8) 2. The sample analysis cartridge of claim 1, wherein the reagent comprises one or more of a plurality of solid particles, a plurality of affinity molecules, or a plurality of signal transduction substances. (Item 9) Item 10. The sample analysis cartridge of item 1, wherein the reagent comprises a plurality of magnetic particles configured to be magnetically held across a working electrode of the sensor. (Item 10) 10. The sample analysis cartridge of claim 9, wherein at least one magnetic particle of the plurality of magnetic particles is configured to be indirectly bound to a signal transduction agent. (Item 11) 2. The sample analysis cartridge of claim 1, further comprising an analysis channel, wherein at least a portion of the sensor is disposed within the analysis channel, and wherein the fluid mixed with the reagent ball and the sample travels through the analysis channel to at least a portion of the sensor. (Item 12) a contact switch, a sealing material configured to fluidly seal fluid within the reservoir, and a seal piercer; Insertion of the sample collection device into the input tunnel (i) moves the shuttle from the first position to the second position, (ii) causes the seal piercer to pierce the seal material and vent fluid in the reservoir, and (iii) causes activation of the contact switch. Item 1. The sample analysis cartridge according to item 1. (Item 13) 1. 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 the opening, 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 configured to hold a fluid; and a sealing material configured to fluidly seal fluid within the reservoir; and a seal piercer partially disposed within the input tunnel, the seal piercer 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 fluid within the reservoir; and A sample analysis cartridge comprising: (Item 14) Item 14. The sample analysis cartridge of item 13, wherein 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. (Item 15) Item 15. The sample analysis cartridge of item 14, wherein the first direction is generally parallel to the movement of the sample collection device within the input tunnel, and the second direction is generally perpendicular to the first direction. (Item 16) Item 14. The sample analysis cartridge of item 13, wherein the seal piercer comprises one or more piercers. (Item 17) 17. The sample analysis cartridge of claim 16, wherein the seal piercer further comprises a slider configured to move in a first direction, and wherein the one or more piercers are configured to move in a second direction different from the first direction to pierce the seal material. (Item 18) the reservoir is a sample preparation reservoir; further comprising a detergent reservoir and a substrate reservoir; the seal piercer is configured to pierce the seal material and vent the individual fluids in the sample preparation reservoir, the detergent reservoir, and the substrate reservoir; Item 14. The sample analysis cartridge according to item 13. (Item 19) 14. The sample analysis cartridge of claim 13, wherein the seal piercer comprises an engager disposed within the input tunnel, the engager configured to engage an engagement zone of the sample collection device when the sample collection device is in the input tunnel. (Item 20) Item 14. The sample analysis cartridge of item 13, further comprising a contact switch, the contact switch configured to be activated in response to insertion of the sample collection device into the input tunnel. (Item 21) 21. The sample analysis cartridge of claim 20, wherein movement of the seal piercer causes activation of the contact switch. (Item 22) 20. The sample analysis cartridge of claim 18, wherein the seal piercer is configured to sequentially pierce the seal material across the sample collection reservoir, the irrigant reservoir, and the substrate reservoir in any order. (Item 23) Item 14. The sample analysis cartridge of item 13, wherein the seal piercer is configured to move out of one or more holes pierced in the seal material after piercing and aerating fluid in the reservoir. (Item 24) 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, and 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 (i) moves the shuttle from the first position to the second position, (ii) causes the seal piercer to pierce the seal material and vent fluid in the reservoir, and (iii) causes activation of the contact switch. Item 14. The sample analysis cartridge according to item 13. (Item 25) 25. The sample analysis cartridge of claim 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) 25. The sample analysis cartridge of claim 24, wherein insertion of the sample collection device into the input tunnel causes the seal piercer to pierce the seal material and vent fluid in the reservoir before the shuttle moves from the first position to the second position. (Item 27) 25. The sample analysis cartridge of claim 24, wherein insertion of the sample collection device into the input tunnel causes the seal piercer to pierce the seal material and vent fluid in the reservoir during movement of the shuttle from the first position to the second position. (Item 28) 1. 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 the opening, 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 configured to hold a fluid and configured to receive a sample on a distal portion of the sample collection device; an analytical channel configured to receive a fluid having the sample and a reagent, the fluid having a plurality of magnetic particles mixed therein, from the reservoir; a circuit board comprising a sensor having a working electrode, the sensor configured to be exposed to the mixed fluid in the analysis channel and to generate a signal indicative of at least one of the presence, absence, or amount of one or more analytes in the sample; Equipped with the working electrode is masked with a plurality of 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) 1. A kit for detecting at least one of the presence, absence, or amount of one or more analytes, comprising: 1. A sample analysis cartridge comprising: an input tunnel extending from the opening, 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 configured to hold a fluid and configured to receive a sample on a distal portion of the sample collection device; an analytical channel configured to receive a fluid having the sample and a reagent, the fluid having a plurality of magnetic particles mixed therein, from the reservoir; a circuit board comprising a sensor exposed to the mixed fluid 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 positioned 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 a length of the single working electrode to promote a homogeneous distribution of the plurality of magnetic particles across the length of the single working electrode; A kit comprising: (Item 30) 30. The kit of claim 29, wherein receipt of the sample analysis cartridge by the sample analysis reader results in an electrical coupling between the sample analysis cartridge and the sample analysis reader. (Item 31) 31. The kit of claim 30, wherein the sample analysis cartridge is configured to transmit a signal indicative of at least one of the presence, absence, or amount of one or more analytes in the sample to the sample analysis reader for processing. (Item 32) 32. The kit of claim 31, wherein the sample analysis reader is configured to transmit a processed signal to a computer indicative of at least one of the presence, absence, or amount of one or more analytes in the sample. (Item 33) 33. The kit of claim 32, further comprising a computer-readable medium with instructions that, when executed by a processor of the computer, cause a display of the computer to display information indicative of the presence, absence, and / or amount of one or more target analytes. (Item 34) the sample analysis cartridge housing has a bottom surface with a magnetic generator recess; receiving the sample analysis cartridge into a sample analysis reader causes the first and second magnetic generators to move partially within the magnetic generator recess; Item 30. The kit according to item 29. (Item 35) 35. The kit of claim 34, wherein the magnetic generator recess is positioned adjacent to the single working electrode. (Item 36) 1. 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 the opening, 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 configured to hold a fluid and configured to receive a sample on a distal portion of the sample collection device, the reservoir including an outlet having a phase change material therein for occluding an entire cross section thereof; a heater configured to heat the phase change material such that the phase change material does not block an entire cross section of the outlet; and an analysis channel configured to receive a fluid having the sample and a reagent, the fluid having a plurality of magnetic particles mixed therein, from the reservoir through the outlet; a sensor exposed to the mixed fluid 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; Equipped with the heater is masked with a masking material configured to electrically isolate the heater from the sensor; Sample analysis cartridge. (Item 37) 37. The sample analysis cartridge of claim 36, wherein the masking material comprises a solder mask. (Item 38) further comprising a cleaning agent reservoir and a cleaning agent reservoir heater, the cleaning agent reservoir including a cleaning agent reservoir outlet configured to hold a cleaning fluid and having a phase change material therein for blocking an entire cross section of the cleaning agent reservoir outlet; the cleaning agent reservoir heater is configured to heat the phase change material in the cleaning agent reservoir outlet such that the phase change material does not block an entire cross section of the cleaning agent reservoir outlet, thereby allowing the cleaning fluid to enter the analysis channel and proceed to the sensor; 37. The sample analysis cartridge according to item 36. (Item 39) Item 39. The sample analysis cartridge of item 38, wherein the detergent reservoir heater is masked with a masking material configured to electrically isolate the detergent reservoir heater from the sensor. (Item 40) further comprising a substrate reservoir and a substrate reservoir heater, the substrate reservoir including a substrate reservoir outlet configured to hold a substrate fluid and having a phase change material therein for occluding an entire cross section of the substrate reservoir outlet; the substrate reservoir heater is configured to heat the phase change material in the substrate reservoir outlet such that the phase change material does not block an entire cross section of the substrate reservoir outlet, thereby allowing the substrate fluid to enter the analysis channel and proceed to the sensor; 37. The sample analysis cartridge according to item 36. (Item 41) Item 41. The sample analysis cartridge of item 40, wherein the substrate reservoir heater is masked with a masking material configured to electrically isolate the substrate reservoir heater from the sensor. (Item 42) 1. 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 the sample and substrate reservoirs are each in fluid communication with the analysis channel at least at some time; a fluid disconnector comprising a phase change material; one or more heaters, at least one of which is configured to heat a phase change material in the sample preparation reservoir outlet, such that the phase change material unseals the sample preparation reservoir outlet and allows the fluid to mix with the sample therein and flow into the analysis channel; Equipped with at least one of the one or more heaters is configured to heat a phase change material of the fluid disconnector after unsealing the sample preparation reservoir outlet, such that the phase change material of the fluid disconnector flows into the analysis channel and fluidically 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 in the substrate reservoir outlet after the fluid disconnector fluidically isolates the sample preparation reservoir from the substrate reservoir, such that the phase change material unseals the substrate reservoir outlet and allows 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 disconnector heater, and a substrate reservoir heater; the sample preparation reservoir heater is configured to heat a phase change material in the sample preparation reservoir outlet; the fluid disconnector heater is configured to heat a phase change material of the fluid disconnector; the substrate reservoir heater is configured to heat a phase change material in the substrate reservoir outlet; 43. The sample analysis cartridge according to item 42. (Item 44) Item 44. The sample analysis cartridge of item 43, wherein the sample preparation reservoir heater, the fluid disconnector heater, and the substrate reservoir heater are each masked with a masking material configured to electrically isolate the individual heaters from sensors in the analysis channels. (Item 45) Item 43. The sample analysis cartridge of 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) 46. ​​The sample analysis cartridge of claim 45, wherein 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 disconnector fluidically isolates the sample preparation reservoir from the substrate reservoir but before the one or more heaters heat the phase change material in the substrate reservoir outlet, such that the phase change material unseals the detergent reservoir outlet, allowing the washing fluid to flow into the analysis channel and flushing signaling substances from the sample preparation reservoir that were not bound to magnetic particles from the sensor in the analysis channel. (Item 47) 43. The sample analysis cartridge of claim 42, wherein the fluid comprising the chemical substrate is configured to wash signaling substances not bound to magnetic particles from the sample preparation reservoir away from sensors in the analysis channel. (Item 48) 1. 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 the opening, the input tunnel configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to a sample fluid; a reservoir configured to hold a fluid; and a shuttle disposed between the reservoir and the opening at a first position, the shuttle having a first end and a second end, the shuttle defining a sample compartment between the first end and the second end, the sample compartment configured to receive sample fluid compressed from a distal portion of the sample collection device, the shuttle configured to move within the input tunnel to a second position upon receiving a force above a threshold force, such that the sample compartment with the sample fluid is moved into the reservoir; 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: (Item 49) the shuttle further defines a reagent ball compartment between the first end and the second end, the reagent ball compartment configured to store one or more reagent balls comprising a reagent; the reagent bowl compartment is outside the reservoir in the first position and within the reservoir in the second position; 49. The sample analysis cartridge according to item 48. (Item 50) Item 49. The sample analysis cartridge of item 48, wherein the sample compartment is configured to receive up to a predetermined volume of the sample fluid compressed from a distal portion of the sample collection device. (Item 51) Item 51. The sample analysis cartridge of item 50, further comprising a spillover compartment configured to receive a volume of the sample fluid in excess of the predetermined volume from the sample compartment. (Item 52) A kit comprising: Item 48. A sample analysis cartridge according to item 48; the sample collection device, the sample collection device comprising a wicking portion at the distal portion, the wicking portion configured to wick and absorb the sample fluid; A kit comprising: (Item 53) Item 53. The kit of item 52, wherein the wicking portion is compressed to expel the sample fluid into the sample compartment. (Item 54) the sample compartment is configured to receive up to a predetermined volume of the sample fluid compressed from a distal portion of the sample collection device; the sample analysis cartridge further comprising a spillover compartment configured to receive a volume of the sample fluid in excess of the predetermined volume from the sample compartment; a wicking portion of the sample collection device configured to wick up and absorb sample fluid above the predetermined volume and allow a user to meter the amount of sample fluid compressed into the sample compartment and the overflow compartment; Item 53. The kit according to item 52. (Item 55) Item 53. The kit of item 52, wherein at least a portion of the wicking portion is slidably disposed within a shroud of the sample collection device. (Item 56) Item 53. The kit of item 52, wherein the sample analysis cartridge further comprises one or more locking members configured to irreversibly lock the sample collection device in the input tunnel when the sample collection device is fully inserted into the input tunnel. (Item 57) 53. The kit of claim 52, wherein the sample collection device further comprises a sample collection indicator configured to visually alert a collector based on the volume of sample fluid collected. (Item 58) 58. The kit of claim 57, wherein the sample collection indicator is a colored thread embedded within the wicking portion that becomes increasingly visually exposed as the volume of collected sample increases. (Item 59) 1. A method for detecting at least one of the presence, absence, or amount of a target analyte in a sample in a cartridge, comprising: mixing a sample having in fluid within a reservoir of the cartridge a plurality of affinity molecules, a plurality of debinding agents, a plurality of signal transduction substances, a plurality of competitor molecules pre-bound to competitive binding molecules, each of the competitor molecules carrying a label, and a plurality of sample target analytes pre-bound to sample binding molecules; debinding at least one competitor molecule from the pre-bound competitive binding molecule using at least one debinding agent of the plurality of debinding agents; debinding at least one sample target analyte from the pre-bound sample binding molecules using at least one debinding agent of the plurality of debinding agents; binding the label of the unbound competitor molecule to a signal transduction substance among the plurality of signal transduction substances; binding the unbound competitor molecule to an affinity molecule of the plurality of affinity molecules; generating a signal indicative of at least one of the presence, absence, or amount of a sample target analyte within the cartridge; A method comprising: (Item 60) 60. The method of claim 59, wherein each affinity molecule of the plurality of affinity molecules is bound to a solid particle. (Item 61) Item 61. The method of item 60, wherein the solid particles comprise a magnetically responsive material. (Item 62) Item 61. The method of item 60, wherein the solid particles comprise a non-magnetically responsive material. (Item 63) Item 63. The method of item 62, wherein the non-magnetically responsive material comprises gold nanoparticles. (Item 64) 60. The method of claim 59, wherein the plurality of sample target analytes pre-bound to sample binding molecules comprises 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 molecules pre-bound to vitamin D binding protein molecules. (Item 65) 60. The method of claim 59, wherein the plurality of competitor molecules pre-bound to the sample binding molecules comprises 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 molecules labeled with biotin and pre-bound to vitamin D binding protein molecules. (Item 66) Item 59. The method of item 59, wherein at least one of the plurality of affinity molecules, the plurality of unbinding agents, the plurality of signal transduction substances, and the plurality of competitor molecules pre-bound to the competitive binding molecules is stored in a reagent bowl. (Item 67) 1. A sample analysis cartridge for detecting at least one of the presence, absence, or amount of a target analyte, comprising: a reagent ball comprising a plurality of competitor molecules pre-bound to a competitive binding molecule, each of the plurality of competitor molecules carrying a label or bound to a signaling substance; a reservoir configured to hold a reservoir fluid, the reservoir further configured to allow mixing within the reservoir fluid of a plurality of affinity molecules, a plurality of unbinding agents, a plurality of signal transduction agents, a plurality of competitor molecules pre-bound to the competitive binding molecules, and a sample from a sample collection device, the sample having a plurality of sample target analytes pre-bound to the sample binding molecules; a debinding agent of the plurality of debinding agents is configured to debind a competitor molecule from the pre-bound competitive binding molecule or a sample target analyte from the pre-bound sample binding molecule; the label of the unbound competitor molecule is configured to bind to a signaling agent, and the unbound competitor molecule is configured to bind to an affinity molecule of the plurality of affinity molecules; a reservoir; a sensor configured to be exposed to the mixed reservoir fluid, the sensor 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) Item 68. The sample analysis cartridge of item 67, wherein the reservoir is further configured to allow mixing of a plurality of solid particles into the reservoir fluid. (Item 69) Item 69. The sample analysis cartridge of item 68, wherein each solid particle is pre-bound to an affinity molecule of the plurality of affinity molecules. (Item 70) Item 69. The sample analysis cartridge of item 68, wherein the plurality of solid particles comprises a magnetically responsive material. (Item 71) Item 69. The sample analysis cartridge of item 68, wherein the plurality of solid particles comprises a non-magnetically responsive material. (Item 72) 72. The sample analysis cartridge of claim 71, wherein the non-magnetically responsive material comprises gold nanoparticles. (Item 73) 68. The sample analysis cartridge of claim 67, wherein the plurality of sample target analytes pre-bound to sample binding molecules comprises 25-hydroxyvitamin D3 and / or 25-hydroxyvitamin D2 molecules pre-bound to binding protein molecules. (Item 74) 71. The sample analysis cartridge of claim 70, wherein the magnetically responsive material is magnetically held across the sensor. (Item 75) 1. A sample analysis cartridge comprising: an input tunnel extending from the opening, 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 configured to hold a fluid; and a shuttle disposed in the input tunnel between the reservoir and the opening in a first position; a collet disposed within the input tunnel and coupled to the shuttle at the first position, the collet configured to decouple from the shuttle during insertion of the sample collection device into the input tunnel; Equipped with the shuttle is configured to move within the input tunnel from the first position to the second position after the collet is decoupled from the shuttle, such that the shuttle is at least partially disposed within the reservoir at the second position; Sample analysis cartridge. (Item 76) 76. The sample analysis cartridge of claim 75, further comprising a sensor configured to be exposed to a fluid mixed with the sample, 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. (Item 77) Item 76. The sample analysis cartridge of item 75, wherein the shuttle has a first end and a second end positioned proximal to the first end within the input tunnel, the second end of the shuttle being configured to be positioned within the lumen of the collet in the first position. (Item 78) Item 78. The sample analysis cartridge of item 77, wherein the first end of the shuttle forms a wall of the reservoir in the first position. (Item 79) Item 78. The sample analysis cartridge of item 77, wherein the collet comprises one or more locking arms configured to couple the collet to the shuttle in the first position. (Item 80) 80. The sample analysis cartridge of claim 79, wherein the one or more locking arms are configured to be biased to decouple 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. (Item 81) 76. The sample analysis cartridge of claim 75, further comprising a sealing material configured to fluidly seal fluid in the reservoir; and a seal piercer partially disposed within the input tunnel, the seal piercer configured to be contacted by a sample collection device in the input tunnel and to move in response to a force applied by the sample collection device to puncture the sealing material and vent the fluid in the reservoir. (Item 82) Item 82. The sample analysis cartridge of item 81, wherein the collet includes a slot and a portion of the seal piercer extends through the slot into the input tunnel to allow contact between the seal piercer and the sample collection device. (Item 83) Further comprising a contact switch; the collet includes a deflector portion disposed adjacent to the contact switch, the deflector portion 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. 76. The sample analysis cartridge according to item 75. (Item 84) Item 84. The sample analysis cartridge of item 83, wherein the deflector portion of the collet comprises an arm configured to deflect downward and activate the contact switch. (Item 85) Item 84. The sample analysis cartridge of item 83, wherein the contact switch is positioned such that activation of the contact switch indicates full insertion of the sample collection device into the input tunnel. (Item 86) the shuttle is configured to store a reagent bowl comprising 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 in the first position and is exposed to the fluid in the reservoir in the second position; 76. The sample analysis cartridge according to item 75. (Item 87) 1. A sample analysis cartridge comprising: an input tunnel extending from the opening, 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 configured to hold a fluid; and a collet disposed within the input tunnel between the reservoir and the opening, the collet comprising a lumen sized to receive a deflector portion and a distal portion of the sample collection device therein; a contact switch disposed adjacent the deflector portion of the collet; Equipped with the deflector portion is 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. Sample analysis cartridge. (Item 88) Item 88. The sample analysis cartridge of item 87, further comprising a shuttle disposed within the input tunnel, the shuttle configured to store a reagent ball comprising a reagent between a first end and a second end of the shuttle. (Item 89) Item 88. The sample analysis cartridge of item 87, wherein the deflector portion of the collet comprises an arm configured to deflect downward and activate the contact switch. (Item 90) Item 88. The sample analysis cartridge of item 87, wherein the contact switch is positioned such that activation of the contact switch indicates full insertion of the sample collection device into the input tunnel. (Item 91) Item 88. The sample analysis cartridge of item 87, further comprising a sensor configured to be exposed to a fluid mixed with the sample, 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. (Item 92) 1. A sample analysis cartridge comprising: an input tunnel extending from the opening, 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 configured to hold a fluid; and a shuttle defining a first compartment and a second compartment, the first and second compartments configured to be disposed within the reservoir in a mixing position; an ultrasonic generator configured to emit acoustic waves to move fluid in the reservoir between the first and second compartments in a wave pattern and mix the fluid in the reservoir; A sample analysis cartridge comprising: (Item 93) Item 93. The sample analysis cartridge of item 92, wherein the shuttle comprises a compartment divider configured to divide the first compartment from the second compartment. (Item 94) Item 94. The sample analysis cartridge of item 93, wherein fluid flowing around the compartment divider promotes the formation of the wave pattern. (Item 95) Item 94. The sample analysis cartridge of item 93, wherein the compartment divider comprises a slot configured to allow the fluid to flow through the compartment divider via the slot during mixing. (Item 96) Item 94. The sample analysis cartridge of item 93, wherein the compartment divider is a flange. (Item 97) Item 93. The sample analysis cartridge of item 92, wherein the first compartment comprises a reagent ball compartment configured to store a reagent ball comprising a reagent, and the second compartment comprises a sample compartment configured to receive a sample from the sample collection device. (Item 98) Item 98. The sample analysis cartridge of item 97, wherein the reagent bowl comprises a polymerase, a primer, and a signal transduction substance. (Item 99) Item 93. The sample analysis cartridge of item 92, wherein the first and second compartments are not disposed within the reservoir in a pre-mixing position. (Item 100) Item 93. The sample analysis cartridge of item 92, wherein the ultrasonic generator comprises a piezoelectric ceramic disc. (Item 101) Item 93. The sample analysis cartridge of item 92, wherein the ultrasonic generator forms a wall of the reservoir. (Item 102) Item 93. The sample analysis cartridge of item 92, wherein the reservoir is symmetrical. (Item 103) Item 93. The sample analysis cartridge of item 92, wherein the reservoir walls each meet at an angle greater than 60° to facilitate emptying of fluid through the reservoir outlet. (Item 104) Item 93. The sample analysis cartridge of item 92, wherein the ultrasonic generator is positioned off-center of the reservoir to promote mixing of fluid within the reservoir. (Item 105) 93. The sample analysis cartridge of claim 92, further comprising a printed circuit board coupled to the ultrasonic generator via one or more spring contacts. (Item 106) Item 106. The sample analysis cartridge of item 105, wherein the ultrasonic generator is electrically coupled to the printed circuit board only via the one or more spring contacts. (Item 107) Item 93. The sample analysis cartridge of item 92, wherein the ultrasonic generator is activated in response to a signal from a processor. (Item 108) Item 93. The sample analysis cartridge of item 92, further comprising a temperature sensor configured to sense a temperature indicative of the temperature of fluid in the reservoir. (Item 109) Item 109. The sample analysis cartridge of item 108, wherein the temperature sensor is disposed on a printed circuit board located adjacent to the ultrasonic generator. (Item 110) a contact switch configured to indicate insertion of the sample collection device into the input tunnel; the ultrasonic generator is configured to emit the acoustic waves after activation of the contact switch; 93. The sample analysis cartridge of item 92. (Item 111) Item 93. The sample analysis cartridge of item 92, wherein the acoustic waves emitted by the ultrasonic generator are configured to isothermally amplify a reaction of the fluids mixed in the reservoir. (Item 112) A kit comprising: 1. A sample analysis cartridge comprising: a reservoir configured to hold a fluid and to receive a sample collected by the sample collection device; an ultrasonic generator configured to emit acoustic waves to mix the fluid and the sample within the reservoir; a temperature sensor configured to generate a signal indicative of a temperature of fluid in the reservoir; and 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 waves, and monitor a signal from the temperature sensor, the processor further configured to modify the emission of the acoustic waves from the ultrasonic generator if the signal indicates a temperature of fluid in the reservoir outside a threshold; a reader comprising: A kit comprising: (Item 113) Item 113. The kit of item 112, wherein the sample analysis cartridge further comprises a printed circuit board, and the temperature sensor is disposed on the printed circuit board located 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 an input tunnel of the sample analysis cartridge; a processor of the reader configured to receive the signal from the contact switch and activate the ultrasonic generator after receiving the signal from the contact switch; Item 113. The kit according to item 112. (Item 115) Item 113. The kit of item 112, wherein the processor modifies the emission of the acoustic waves from the ultrasonic generator by reducing a duty cycle of the ultrasonic generator when the signal indicates that the temperature of the fluid in the reservoir is above the threshold. (Item 116) Item 113. The kit of item 112, wherein the processor modifies the emission of the acoustic waves from the ultrasonic generator by increasing a duty cycle of the ultrasonic generator when the signal indicates that the temperature of the fluid in the reservoir is below the threshold. (Item 117) Item 113. The kit of item 112, wherein the processor modifies the emission of the acoustic waves from the ultrasonic generator by deactivating the ultrasonic generator when the signal indicates that the temperature of the fluid in the reservoir is above the threshold. (Item 118) Item 113. The kit of item 112, wherein the acoustic waves emitted by the ultrasonic generator are configured to isothermally amplify a reaction of the fluids mixed in the reservoir. (Item 119) Item 113. The kit of item 112, further comprising a reagent ball disposed within the sample analysis cartridge, wherein the ultrasonic generator is configured to emit the acoustic waves to mix the fluid, the reagent ball, and the sample within the reservoir. (Item 120) Item 120. The kit of item 119, wherein the reagent bowl comprises a polymerase, a primer, and a signal transduction agent. (Item 121) Item 120. The kit of item 119, wherein the sample analysis cartridge further comprises a shuttle configured to store the reagent ball. (Item 122) 1. A sensor for use in a microfluidic cartridge, comprising: a positive control working electrode comprising an affinity molecule pre-bound to the positive control working electrode, the positive control working electrode configured to generate a first signal based on a reaction between a signaling agent and a chemical substrate bound directly or indirectly to the affinity molecule; a working electrode configured to generate a second signal based on a reaction between a signaling agent localized at the working electrode and the chemical substrate; a negative control working electrode comprising a self-assembled monolayer, the negative control working electrode configured to generate a third signal based on a reaction between a signaling agent localized at the negative working electrode and the chemical substrate; A sensor comprising: (Item 123) Item 123. The sensor of 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) Item 123. The sensor of item 122, wherein the first signal indicates the reliability of the test. (Item 125) Item 125. The sensor of item 124, wherein the test is determined to be reliable if the first signal indicates an amount of response that is within a predetermined range. (Item 126) Item 123. The sensor of item 122, wherein the third signal indicates the reliability of the test. (Item 127) Item 127. The sensor of item 126, wherein the test is determined to be reliable if the third signal indicates that the amount of the response is below a threshold value. (Item 128) 123. 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) Item 129. A kit comprising the cartridge of item 128, the kit further comprising a processor configured to process the second signal and generate information indicative of at least one of the presence, absence, or number of the one or more analytes in the sample. (Item 130) Item 130. The kit of item 129, wherein the processor is further configured to process the first signal and determine whether the first signal indicates an amount of the reaction within a predetermined range. (Item 131) Item 131. The kit of item 130, wherein the processor is configured to generate an alert if the amount is outside the predetermined range. (Item 132) Item 130. The kit of 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 response is below a threshold. (Item 133) Item 133. The kit of item 132, wherein the processor is configured to generate an alert if the amount exceeds the threshold. (Item 134) Item 130. The kit of item 129, wherein the processor is a component of a reader. (Item 135) Item 123. The sensor of item 122, wherein the working electrode is masked with a plurality of grooves configured to promote a homogeneous distribution of a plurality of magnetic particles directly or indirectly bound to the signaling agent localized across the working electrode and promote resistance to movement of the plurality of magnetic particles from the working electrode. (Item 136) Item 123. The sensor of item 122, wherein the working electrode comprises a self-assembled monolayer. (Item 137) Item 123. The sensor of item 122, wherein the working electrode comprises an affinity molecule pre-bound to the working electrode. (Item 138) 93. A method for isothermal amplification of a target nucleic acid in a sample analysis cartridge according to item 92, comprising: contacting, directly or indirectly, an amplicon with a signaling agent in the reservoir to form an amplicon-signaling agent complex, wherein the ultrasonic generator is configured to emit the acoustic waves toward the reservoir to facilitate amplification of the target nucleic acid in the reservoir; reacting the amplicon-signal signaling substance complex with a substrate from a substrate reservoir; generating a signal indicative of at least one of the presence, absence, or amount of amplified nucleic acid; A method comprising: (Item 139) Item 93. The sample analysis cartridge of item 92, further comprising a reagent bowl held within the shuttle, the reagent bowl comprising reagents for amplification of a target nucleic acid by an isothermal reaction. (Item 140) 140. The sample analysis cartridge of claim 139, wherein the reagents comprise 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) 141. The sample analysis cartridge of claim 140, further comprising one or more affinity molecules covalently or non-covalently bound to solid particles for detection of the target nucleic acid. [Brief explanation of the drawings]

[0097] Exemplary embodiments are described below with reference to the accompanying drawings, in which like numerals refer to like elements.

[0098] [Figure 1A] 1A-1B provide a schematic depiction of an exemplary analyte detection system for analyzing the presence, absence, and / or amount of one or more target analytes in a collected sample and viewing the analysis results, with FIG. 1A showing the unbound components and FIG. 1B showing the components bound for analysis and charging. [Figure 1B] 1A-1B provide a schematic depiction of an exemplary analyte detection system for analyzing the presence, absence, and / or amount of one or more target analytes in a collected sample and viewing the analysis results, with FIG. 1A showing the unbound components and FIG. 1B showing the components bound for analysis and charging.

[0099] [Figure 1C] FIG. 1C provides a schematic depiction of another exemplary analyte detection system for analyzing the presence, absence, and / or amount of one or more target analytes in a collected sample and viewing the analysis results; a charger is not provided.

[0100] [Figure 2] 2A and 2B illustrate perspective views of an exemplary sample collection device for use in a detection system.

[0101] [Figure 3] FIG. 3 illustrates a perspective view of an exemplary cartridge device for use in a detection system.

[0102] [Figure 4] Figures 4A and 4B illustrate perspective views of a cartridge device with a sample collection device locked therein for analysis of collected samples, with Figure 4A showing the top surface of the cartridge device and Figure 4B showing the bottom surface of the cartridge device.

[0103] [Figure 5] FIG. 5 illustrates an exploded view of an exemplary cartridge device showing the internal components that may be within the cartridge housing.

[0104] [Figure 6] 6A, 6B, and 6C illustrate an exemplary circuit board and exemplary layers that may be used within the housing of a cartridge device, with FIG. 6A depicting the circuit board, FIG. 6B depicting the layers, and FIG. 6C depicting the layers disposed on and coupled to the circuit board.

[0105] [Figure 7-1] 7A, 7B, and 7C illustrate another exemplary circuit board and another exemplary layer that may be used within the housing of a cartridge device, with FIG. 7A depicting the circuit board, FIG. 7B depicting the layer, and FIG. 7C depicting the layer and absorbent pad disposed on and bonded to the circuit board.

[0106] [Figure 7-2] 7D-7F illustrate alternative exemplary sensors that may be used within the housing of the cartridge device.

[0107] [Figure 8A] FIG. 8A illustrates exemplary internal components coupled to an exemplary circuit board via layers positioned therebetween, all of which may be disposed within the housing of the cartridge device.

[0108] [Figure 8B] 8B and 8C show close-up views of certain components of a circuit board and valve for use within the housing of a cartridge device. [Figure 8C] 8B and 8C show close-up views of certain components of a circuit board and valve for use within the housing of a cartridge device.

[0109] [Figure 8D] FIG. 8D shows an expanded view of alternative components and valves of a circuit board for use within the housing of a cartridge device.

[0110] [Figure 9] 9A and 9B illustrate exemplary shuttles that may be disposed within the housing of a cartridge device, each shown housing a reagent ball.

[0111] [Figure 10A] FIG. 10A is a cross-sectional perspective view showing a sample collection device partially inserted into an input tunnel of a cartridge device.

[0112] [Figure 10B] FIG. 10B is a cross-sectional perspective view showing the tip of the sample collection device entering the shuttle positioned within the input tunnel of the cartridge device.

[0113] [Figure 10C] FIG. 10C is a top view showing an exemplary orientation of a piercing element across a reservoir within the housing of a cartridge device.

[0114] [Figure 10D]FIG. 10D is a cross-sectional perspective view showing the engagement between the sample collection device and the slider of the seal piercer within the input tunnel of the cartridge device, with a portion of the cartridge device removed.

[0115] [Figure 10E] FIG. 10E is a cross-sectional side view showing the engagement between the sample collection device and the seal piercer and the seal between the sample collection device and the shuttle, with the sample preparation reservoir remaining sealed by the shuttle.

[0116] [Figure 10F] FIG. 10F is a top view showing the engagement between the sample collection device and the seal piercer in the pre-vent position.

[0117] [Figure 10G] FIG. 10G is a cross-sectional perspective view showing the piercing element and slider of the seal piercer in a pre-vent position, with the seal material over the sample preparation reservoir in the cartridge device not yet pierced.

[0118] [Figure 10H] FIG. 10H is a cross-sectional side view illustrating the transition from a pre-mixing and pre-venting position within the input tunnel of the cartridge device toward a mixing and venting position.

[0119] [Figure 10I] FIG. 10I is a top view showing movement of the sample collection device causing movement of the seal piercer to the vent position.

[0120] [Figure 10J] FIG. 10J is a cross-sectional perspective view showing a piercing element piercing a sealing material over a sample preparation reservoir in a cartridge device.

[0121] [Figure 10K]FIG. 10K is a cross-sectional side view showing the sample collection device in the venting and mixing position, in which the sealing material over the sample preparation reservoir is vented and the collected sample and reagent ball are mixed in the fluid within the sample preparation reservoir, which has been resealed by the shuttle.

[0122] [Figure 10L] FIG. 10L is a cross-sectional perspective view showing the sample collection device in a venting and mixing position within a cartridge device.

[0123] [Figure 10M] FIG. 10M is a cross-sectional perspective view showing the sample collection device in a venting and mixing position within the sample preparation reservoir of the internal component of the cartridge device.

[0124] [Figure 10N] 10N, 10O, and 10P are cross-sectional side views showing enhanced mixing of the collected sample and reagent balls with fluid in the sample preparation reservoir via the ultrasonic generator element. [Figure 10O] 10N, 10O, and 10P are cross-sectional side views showing enhanced mixing of the collected sample and reagent balls with fluid in the sample preparation reservoir via the ultrasonic generator element. [Figure 10P] 10N, 10O, and 10P are cross-sectional side views showing enhanced mixing of the collected sample and reagent balls with fluid in the sample preparation reservoir via the ultrasonic generator element.

[0125] [Figure 11] 11A-11E illustrate an alternative seal piercer, where insertion of a sample collection device into the input tunnel of a cartridge device also activates a switch to indicate proper sample collection device insertion.

[0126] [Figure 12]12A-12E illustrate cross-sectional side views of an alternative sample collection device and an alternative cartridge device for collecting and analyzing a fluid sample.

[0127] [Figure 13A] FIG. 13A illustrates an exploded view of another exemplary cartridge device showing the internal components that may be within the cartridge housing.

[0128] [Figure 13-1] 13B-13SS illustrate various views of an exemplary sample collection device that may be used within the detection system. [Figure 13-2] 13B-13SS illustrate various views of an exemplary sample collection device that may be used within the detection system. [Figure 13-3] 13B-13SS illustrate various views of an exemplary sample collection device that may be used within the detection system. [Figure 13-4] 13B-13SS illustrate various views of an exemplary sample collection device that may be used within the detection system. [Figure 13-5] 13B-13SS illustrate various views of an exemplary sample collection device that may be used within the detection system. [Figure 13-6] 13B-13SS illustrate various views of an exemplary sample collection device that may be used within the detection system.

[0129] [Figure 14] 14A-14D illustrate an exemplary collet that may be disposed within the housing of a cartridge device, with FIGS. 14A and 14C showing perspective views and FIGS. 14B and 14D showing cross-sectional views of the collet in FIGS. 14A and 14C, respectively.

[0130] [Figure 15A] FIG. 15A illustrates a cross-sectional view through the center of the input tunnel of an exemplary cartridge in a pre-mixing, pre-aeration, storage position.

[0131] [Figure 15B] FIG. 15B illustrates a cross-sectional view of an exemplary cartridge and exemplary sample collection device fully inserted into the input tunnel in the mixing, venting, and analysis position.

[0132] [Figure 15C] 15C and 15D illustrate perspective views of an exemplary cartridge with an exemplary sample collection device inserted therein in a pre-vent position (FIG. 15C) and a vent position (FIG. 15D). [Figure 15D] 15C and 15D illustrate perspective views of an exemplary cartridge with an exemplary sample collection device inserted therein in a pre-vent position (FIG. 15C) and a vent position (FIG. 15D).

[0133] [Figure 16-1] 16A-16E illustrate cross-sectional side views showing the insertion of a sample collection device into a cartridge.

[0134] [Figure 16-2] 16F and 16G illustrate cross-sectional top views showing further distal insertion of the sample collection device into the cartridge.

[0135] [Figure 16-3] 16H-16J illustrate cross-sectional side views showing further distal insertion of the sample collection device into the cartridge, with the sample collection device fully inserted into the input tunnel in the mixing, venting, and analysis position in FIG. 16J.

[0136] [Figure 17] 17A-17D illustrate various views of an exemplary ultrasonic generator electrically coupled to a circuit board via spring contacts for use within an exemplary cartridge housing.

[0137] [Figure 18]18A and 18B are cross-sectional side and top views, respectively, of another exemplary cartridge device.

[0138] [Figure 19] FIG. 19 illustrates an exemplary process for monitoring temperature during enhanced mixing via an ultrasonic generator.

[0139] [Figure 20] FIG. 20 is a graph showing the temperature measured over time during enhanced mixing via an ultrasonic generator.

[0140] [Figure 21A] FIG. 21A illustrates a perspective view of an exemplary reader device for use in a detection system.

[0141] [Figure 21B] FIG. 21B illustrates an exploded view of the exemplary reader device of FIG. 21A, showing the internal components that may be within the reader housing.

[0142] [Figure 22A] FIG. 22A shows a cross-sectional perspective view of an exemplary reader device.

[0143] [Figure 22B] FIG. 22B is a cross-sectional side view showing a cartridge device (with a sample collection device partially inserted therein) partially inserted into an exemplary reader device.

[0144] [Figure 22C] 22C and 22D are cross-sectional perspective and side views, respectively, showing a cartridge device inserted into an exemplary reader device in an analysis position. [Figure 22D] 22C and 22D are cross-sectional perspective and side views, respectively, showing a cartridge device inserted into an exemplary reader device in an analysis position.

[0145] [Figure 23] 23A and 23B are graphs showing the magnetic field strength across the length of a single working electrode for a single magnet (FIG. 23A) versus a dual magnet (FIG. 23B) design.

[0146] [Figure 24] FIG. 24 provides a flow chart of one embodiment of a method for detecting the presence, absence, and / or amount of one or more target analytes in a sample.

[0147] [Figure 25A] FIG. 25A illustrates a perspective view of an exemplary charger that may be used in the detection system.

[0148] [Figure 25B] FIG. 25B illustrates an exploded view of the example charger of FIG. 25A, showing the internal components that may be within the charger housing.

[0149] [Figure 26-1] 26A and 26B provide a schematic depiction of the molecules and reactions found in one embodiment of the analyte detection system of the present disclosure.

[0150] [Figure 26-2] 26C and 26D provide a schematic depiction of the molecules and reactions found in another embodiment of the analyte detection system of the present disclosure.

[0151] [Figure 27] 27A and 27B provide a schematic depiction of the molecules and reactions found in yet another embodiment of the analyte detection system of the present disclosure.

[0152] [Figure 28A] FIG. 28A is a schematic depiction of molecules in a sample on a sample collection device.

[0153] [Figure 28B]FIG. 28B is a schematic depiction of molecules in two reagent bowls for reacting with molecules in the collected sample.

[0154] [Figure 28C] FIG. 28C is a schematic depiction of molecules in a single reagent bowl for reacting with molecules in a collected sample.

[0155] [Figure 28D] FIG. 28D is a molecular schematic depiction showing the collected sample being introduced into the sample preparation reservoir.

[0156] [Figure 28E] FIG. 28E is a schematic representation of molecules showing the mixing of molecules of the collected sample with molecules of the sample preparation reagent in the fluid of the sample preparation reservoir.

[0157] [Figure 28F] 28F, 28G, and 28H are schematic molecular depictions showing reactions between molecules of the collected sample and sample preparation reagent molecules in the fluid of the sample preparation reservoir. [Figure 28G] 28F, 28G, and 28H are schematic molecular depictions showing reactions between molecules of the collected sample and sample preparation reagent molecules in the fluid of the sample preparation reservoir. [Figure 28H] 28F, 28G, and 28H are schematic molecular depictions showing reactions between molecules of the collected sample and sample preparation reagent molecules in the fluid of the sample preparation reservoir.

[0158] [Figure 29] FIG. 29A shows a graph illustrating electrochemical sensor readings versus concentration of target analyte using a pre-bound competitive binding molecule, and FIG. 29B shows a graph comparing electrochemical sensor readings versus concentration when no competitive binding molecule is pre-bound.

[0159] [Figure 30A]FIG. 30A is another schematic depiction of molecules in a sample on a sample collection device.

[0160] [Figure 30B] FIG. 30B is another schematic depiction of molecules in two reagent bowls for reacting with molecules in a collected sample.

[0161] [Figure 30C] FIG. 30C is another schematic depiction of molecules in a single reagent bowl for reacting with molecules in a collected sample.

[0162] [Figure 30D] FIG. 30D is another schematic depiction of a molecule showing the collected sample being introduced into a sample preparation reservoir.

[0163] [Figure 30E] FIG. 30E is another schematic depiction of molecules showing the mixing of molecules of the collected sample with molecules of the sample preparation reagent in the fluid of the sample preparation reservoir.

[0164] [Figure 30F] 30F, 30G, and 30H are schematic molecular depictions showing reactions between molecules of the collected sample and sample preparation reagent molecules in the fluid of the sample preparation reservoir. [Figure 30G] 30F, 30G, and 30H are schematic molecular depictions showing reactions between molecules of the collected sample and sample preparation reagent molecules in the fluid of the sample preparation reservoir. [Figure 30H] 30F, 30G, and 30H are schematic molecular depictions showing reactions between molecules of the collected sample and sample preparation reagent molecules in the fluid of the sample preparation reservoir.

[0165] [Figure 31-1]Figures 31A-32H show an exemplary process for detecting the presence, absence, and / or amount of a target analyte in a sample in a cartridge. Figures 32A-32K show an exemplary process for detecting the presence, absence, and / or amount of a target analyte in a sample in a cartridge using isothermal amplification. [Figure 31-2] Figures 31A-32H show an exemplary process for detecting the presence, absence, and / or amount of a target analyte in a sample in a cartridge. Figures 32A-32K show an exemplary process for detecting the presence, absence, and / or amount of a target analyte in a sample in a cartridge using isothermal amplification. [Figure 32-1] Figures 31A-32H show an exemplary process for detecting the presence, absence, and / or amount of a target analyte in a sample in a cartridge. Figures 32A-32K show an exemplary process for detecting the presence, absence, and / or amount of a target analyte in a sample in a cartridge using isothermal amplification. [Figure 32-2] Figures 31A-32H show an exemplary process for detecting the presence, absence, and / or amount of a target analyte in a sample in a cartridge. Figures 32A-32K show an exemplary process for detecting the presence, absence, and / or amount of a target analyte in a sample in a cartridge using isothermal amplification. [Figure 32-3] Figures 31A-32H show an exemplary process for detecting the presence, absence, and / or amount of a target analyte in a sample in a cartridge. Figures 32A-32K show an exemplary process for detecting the presence, absence, and / or amount of a target analyte in a sample in a cartridge using isothermal amplification. [Figure 32-4] Figures 31A-32H show an exemplary process for detecting the presence, absence, and / or amount of a target analyte in a sample in a cartridge. Figures 32A-32K show an exemplary process for detecting the presence, absence, and / or amount of a target analyte in a sample in a cartridge using isothermal amplification. [Figure 32-5]Figures 31A-32H show an exemplary process for detecting the presence, absence, and / or amount of a target analyte in a sample in a cartridge. Figures 32A-32K show an exemplary process for detecting the presence, absence, and / or amount of a target analyte in a sample in a cartridge using isothermal amplification.

[0166] [Figure 33] FIG. 33 provides a schematic depiction of the exemplary analyte detection system of FIGS. 1A-1B, communicatively coupled to one or more servers via a network. DETAILED DESCRIPTION OF THE INVENTION

[0167] In the following detailed description, reference is made to the accompanying drawings, which form a part of this disclosure. The embodiments set forth in the drawings and description 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. The aspects of the present disclosure as described and illustrated herein can be arranged, combined, and designed in a variety of different configurations, all expressly contemplated and forming a part of this disclosure.

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

[0169] Various embodiments of the systems described herein are designed to create a self-contained environment in which any of the chemical reactions occur in an automated manner, completely or substantially without human intervention, as described, for example, in commonly assigned 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 designs described herein, one or more chemical reactions proceed without any need for an operator to add or remove reagents from the system. In certain embodiments, the system is closed to minimize biohazard risks, such as the risk of spilling sample collected from the 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] 1A and 1B illustrate an exemplary analyte detection system constructed in accordance with the principles of the present disclosure. 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. Detection system 100 may be used to detect the presence, absence, and / or amount of one or more target analytes.

[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, cellular material, and / or other biological material, to determine the presence, absence, and / or amount of one or more target analytes in the sample. Additionally or alternatively, the sample collection device is exposed to a solid or other surface suspected of carrying the target analytes, e.g., a foodborne pathogen, and the surface is a cooking or food preparation surface.

[0172] Cartridge device 300 is configured to analyze a sample collected using sample collection device 200. Cartridge device 300 may include an input tunnel 301 extending into the cartridge housing from opening 302. Input tunnel 301 is configured to allow insertion of sample collection device 200, as shown in FIG. 1B, so that the collected sample may be analyzed within cartridge device 300. Based on the analysis, 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 a reader opening 401 of the reader 400, as shown in FIG. 1B, such that the respective electrical connectors of the cartridge device 300 and the reader 400 contact each other. The reader 400 may comprise a computer-readable medium with instructions that, when executed by a processor of the reader 400, cause the electrical components of the cartridge 300 to perform steps for analyzing a sample on the sample collection device 200. Preferably, the instructions are not executed until the cartridge device 300 is electrically coupled to the reader 400 and the sample collection device 200 is suitably positioned within the cartridge device 300, for example, as shown in FIG. 1B or 4A.

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

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

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

[0177] 1A and 1B, a software-based detection interface system 600 is installed and launched on a computing device 601, allowing 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, a smartwatch, a tablet, a wearable device, a laptop, or other computer. As shown in FIGS. 1A and 1B, the reader 400 may wirelessly communicate with the computing device 601 and transmit data indicative of the presence, absence, and / or amount of one or more target analytes based on electrical signals generated within the 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 with instructions that, when executed by a processor of the computing device 601, cause the display 602 to display information indicative of the presence, absence, and / or amount of one or more target analytes. Sample Collection Devices and Cartridges

[0178] The sample collection device of various embodiments is configured to collect a sample from a specimen. The sample collection device may be configured to collect cells and other biological material 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 draws small droplets of blood or urine into a small capillary channel. In other embodiments, the sample collection device may be configured to collect biological material, particulate matter, or other chemicals from the environment, such as from air or water, or from physical surfaces or other structures.

[0179] The sample collection device of various embodiments is sized and shaped to collect a sufficiently large sample from an appropriate location of a sample so that the presence, absence, and / or amount of a target analyte in and / or on the sample can be detected using other devices described below. For example, for some target analytes, such as those associated with cold or flu-like symptoms caused by viruses, the sample collection device may be a nasal swab, the swab sized and shaped to collect a sufficient amount of sample from an individual's nasal cavity to enable detection of the target analyte associated with the cold or flu-like symptoms, if present in the individual. For other target analytes, such as those associated with strep throat, the sample collection device may be a throat swab shaped to scrape sufficient cells from the individual's throat or mouth. As another example, a sample collection device suitable 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 suitable for collecting target analytes for various tests, including, for example, tests for tracking testosterone levels, drug levels, vitamin levels, and / or fertility. A sample collection device for collecting fluids such as urine, blood, plasma, or saliva may include a feature for compressing the wicking portion of the device and expelling a sample absorbed onto the wicking portion for analysis of the expelled sample. In yet a further aspect, the sample collection device is shaped to collect a sample from a solid surface, for example, on a medical device, on a medical instrument, or on a food preparation surface such as a cutting board or flat surface.

[0180] 2A and 2B, a sample collection device 200 is illustrated. The sample collection device 200 is configured to collect a small sample to be analyzed and is configured for full or partial insertion into a cartridge device 300 after sample collection. 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] Distal portion 201, including tip 204, is configured to be exposed to the sample so that up to a predetermined volume of the sample is disposed within tube 205 for analysis. Collection of a predetermined volume of sample is expected to facilitate accuracy of specimen analysis, as a substantially known amount of sample will be analyzed. Tip 204 may be transparent, allowing the collector to verify that a sample has been disposed within tube 205. Tip 204 may have a rounded end as shown, although various shapes may be used, including any blunt or substantially blunt tip shape. Tip 204 may be configured to collect a sample from any desired area or location, such as 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 that is sized and shaped to be held by a collector's hand. The handle 206 may include a gripping protrusion, 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 that is configured to seal the input tunnel of the cartridge device 300 when the sample collection device 200 is inserted therein. The proximal seal zone 207 may include a protrusion that extends around the shaft 203 and is sized above the input tunnel opening to seal the input tunnel. The protrusion may thus further lock the sample collection device within the input tunnel of the cartridge device 300. The handle 206 may be frangible or otherwise removable from the remainder of the sample collection device 200 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 hands removed from the collection site. For example, the shaft 203 may be elongated so that the tip 204 is exposed to the sample and can collect fluids, cells, and other biological materials from the inner cheek, throat, mouth, nasal cavity, ear, urine, blood, plasma, saliva, etc., 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 in sterile packaging 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 fluid-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. For example, the distal seal zone 208 may be sized and shaped to seal the collected sample on the tip 204 and fluid within the sample preparation reservoir of the cartridge device 300 within the cartridge device 300. The distal seal zone 208 may have a radial size greater than 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 portion of the cartridge device 300, e.g., a shuttle, to form a fluid-tight seal. In this manner, 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 to vent one or more reservoirs within the cartridge device 300 before, during, or after the formation of a fluid-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 secure engagement between the sample collection device 200 and the cartridge device such that the sample collection device 200 irreversibly and non-movably mates with the cartridge when the sample collection device 200 is inserted a predetermined distance within the input tunnel of the cartridge. The engagement zone 209 may be a groove around the shaft 203, as shown, or multiple grooves that extend a shorter distance from the longitudinal axis than the typical shaft surface, and / or one or more protrusions that extend 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 has various features that enable the cartridge to do one or more of: receive a sample with target analytes from a sample collection device; store the sample with sample preparation reagents; provide a space for mixing and binding of the target analytes with the sample preparation reagents; provide an analysis zone where the bound target analytes localize across a sensor for detection; provide a fluid medium for transporting the bound target analytes to the analysis zone; store and provide a substrate that can undergo a detectable reaction when introduced to the bound target analytes; provide a liquid medium for transporting the substrate to the bound target analytes in the analysis zone; and provide a waste collection zone where waste is stored.

[0187] In various embodiments, the cartridge is a substantially closed system in which reactions required to detect the presence, absence, and / or quantity of one or more target analytes occur within the cartridge. The cartridges of such embodiments are 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 force 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 cartridges are target 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 application-specific reagents intended to identify a different target analyte.

[0188] Referring now to FIG. 3 , an exemplary cartridge is illustrated. The cartridge device 300 may include an input tunnel 301 extending from an opening 302 on a front surface 303 into a cartridge housing 304. The cartridge housing 304 may have a generally rectangular prism shape as shown, although the present disclosure is not limited thereto. The cartridge housing 304 has a front surface 303, a top 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 rear surface 309 (shown in FIG. 4B ). The cartridge housing 304 may be formed from a single component or multiple components. For example, the 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 the cartridge device 300 are stored therein.

[0189] 4A and 4B illustrate the sample collection device 200 inserted into the input tunnel 301 of the cartridge device 300 in the mixing position. In 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 may be removably coupled to the cartridge housing 304. As shown in FIG. 4B, the cartridge device 300 may include an electrical connector 312 configured for electrical coupling with a reader, for example, via the reader's electrical connector. Thus, a signal indicative of 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 may be positioned on the bottom surface 308 and the back surface 309 to facilitate coupling with an electrical connector in the opening of the reader.

[0190] The bottom surface 308 of the cartridge device 300 may include a first ramp portion 313, a second ramp portion 314, and a magnetic generator recess 315. The first ramp 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 ramp portion 313 may start from a recess in the cartridge housing 304 where the electrical connector 312 is located and descend to the bottom surface 308. As the cartridge device 300 is inserted over the first ramp portion 313, the magnetic generators ride up into the magnetic generator recess 315 and remain in the depressed position until they contact the second ramp portion 314. The second ramp 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 positioned directly below the one or more working electrodes of the cartridge device 300 such that when the cartridge device 300 is fully inserted into the reader, the one or more magnetic generators of the reader move upward into the magnetic generator recess 315 and are positioned adjacent to the one or more working electrodes. The second ramp portion 314 also facilitates removal of the cartridge device 300 from the reader by gradually depressing the one or more magnetic generators of the reader during cartridge removal.

[0191] 5 , an exploded view of cartridge device 300 is shown. Cartridge device 300 may include internal components 316, which may include a sample preparation reservoir 317, a cleaning agent reservoir 318, and a substrate reservoir 319; a seal material 320; a seal piercer 321, which may include a slider 322 and a piercer 323; a shuttle 324; a desiccant 325; an input tunnel component 326; an ultrasonic generator component 327; an absorbent pad 328; a layer 329; an analysis channel 330; and a circuit board 331 electrically coupled to memory 332. The internal components may be disposed within 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 are disposed within another housing. In the case of multiple housings, such separate housings may be configured to couple to each other.

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

[0193] 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, a saline solution, a water / saline solution mixed with one or more of: magnetic particles, affinity molecules, connecting molecules, signaling agents, competitive binding molecules, competitor molecules, labels, and / or signaling agents, as described in further detail below. Sample preparation reservoir 317 is positioned adjacent to the distal end of input tunnel 301 such that input tunnel 301 connects to sample preparation reservoir 317. As described further below, sample preparation reservoir 317 may be formed in part, e.g., as part or all of its bottom surface, with ultrasonic generator element 327, which promotes mixing of the fluid with additional particles in the fluid. Additionally, sample preparation reservoir 317 may be formed partially with an end of shuttle 324 during the pre-mixing state and partially with another portion of shuttle 324 during the mixing state when one or more reagent balls and sample are placed in sample preparation reservoir 317. In this manner, sample preparation reservoir 317 is fluidly sealed by shuttle 324 in the pre-mixing state and by shuttle 324 in the mixing state, and remains fluidly sealed continuously throughout the transition from the pre-mixing state to the mixing state such that fluid does not leak proximally beyond shuttle 324. Sample preparation reservoir 317 is positioned such that upon insertion of sample collection device 200 into input tunnel 301, for example, distal portion 201 having sample at tip 204 and / or tube 205 enters sample preparation reservoir 317. As 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 with the one or more reagent balls and sample, for example, via the ultrasonic generator element 327, to suspend and hybridize the particles within the sample preparation reservoir 317.Target analytes in the sample may hybridize and / or bind to at least the magnetic particles and / or affinity molecules present among the sample preparation reagents, forming magnetic particle-bound complexes and / or affinity molecule-target complexes. The sample preparation reservoir 317 is configured to allow a fluid having the sample and sample preparation reagents mixed therein to be released, for example, via an outlet into the analysis channel 330 for analyzing 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 opens, the fluid from the sample preparation reservoir 317 acts as a transport medium, causing the magnetic particle-bound complexes and / or affinity molecule-target complexes and other particles to flow from the sample preparation reservoir 317 into the analysis channel 330. Advantageously, the fluid serving as the mixing and storage medium in 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 in the analysis channel 330 without the need for a pump.

[0194] The wash reservoir 318 is configured to hold a fluid, preferably a liquid configured as a wash solution. The wash reservoir 318 is further configured to release the wash solution into the analysis channel 330, for example, via an outlet, allowing particles in the mixed fluid previously released from the sample preparation reservoir 317 that are not bound to the magnetic particles or pre-bound surface affinity molecules to be displaced from the working electrode and / or positive control working electrode in the analysis channel. The outlet of the wash reservoir 318 may be sealed with a thermally actuated valve. When the valve opens, the wash solution flows from the wash reservoir 318 into the analysis channel 330, thereby removing all or substantially all unbound detection agent and / or unbound competitive binding agent from the analysis channel 330. In one aspect, most or all of the free-floating, unbound molecules from the sample preparation reservoir 317 are washed from the analysis channel 330, reducing the likelihood of any significant non-specific binding and / or significant non-specific signal generated by free-floating signaling substances, e.g., HRP, occurring 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 include a substrate that undergoes a reaction in the presence of a signaling agent 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 including an acceptor molecule, such as hydrogen peroxide, and a substrate, which may be an enzyme substrate, such as a tetramethylbenzidine (TMB) and / or o-phenylenediamine dihydrochloride (OPD) molecule. 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 acceptor molecule may be configured to accept electrons stolen from the substrate by the signaling agent during the reaction between the substrate and the signaling agent (thereby oxidizing the substrate). For example, when the acceptor molecule is hydrogen peroxide, an oxidase reaction between a substrate, e.g., TMB, OPD, and a signaling agent, e.g., HRP, SBP, causes electrons to be removed from the substrate and donated to the acceptor molecule (e.g., hydrogen peroxide) during the oxidase reaction, such that the acceptor molecule is converted to another molecule (e.g., water). In some embodiments, ferricyanide is used as the substrate (and reacted with the signaling agent, which may be an oxidation dye such as methylene blue from the sample preparation reservoir 317). The substrate reservoir 319 is further configured to allow the release of a fluid with the substrate 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, allowing the chemical substrate to flow from the substrate reservoir 319 into the analysis channel 330.

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

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

[0198] The seal piercer 321 is configured to pierce the seal material 320 and vent fluids in the sample preparation reservoir 317, the detergent reservoir 318, and / or the substrate reservoir 319. The seal piercer 321 may be contacted by the distal portion 201, for example, at a shoulder or engagement zone 209 of the sample collection device 200 in the input tunnel 301, and may be configured to move within the housing 304 in response to a force applied by the sample collection device 200, piercing the seal material 320 and venting fluids in the sample preparation reservoir 317, the detergent 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, e.g., laterally, and in a second direction, e.g., vertically, to pierce into the seal material 320 in response to insertion of the sample collection device 200 into the input tunnel 301.

[0199] The seal piercer 321 may be a single piece or may include multiple pieces. Illustratively, the seal piercer 321 includes a slider 322 and a piercer 323. The slider 322 may be disposed within the housing 304 and partially within the input tunnel 301. For example, the slider 322 may have an engager adapted to be disposed within the input tunnel 301. The engager may be temporarily or permanently coupled to the sample collection device 200, for example, at a shoulder or engagement zone 209, and configured to allow movement of the slider 322 in response to insertion of the sample collection device 200 into the input tunnel 301. The engager may be sized to fit within a groove in the U-shaped engagement zone 209 or to receive a protrusion in the engagement zone 209, for example, as shown. The slider 322 may be configured to move within the housing 304 in response to a force applied by the sample collection device 200, causing a collector to push the sample collection device distally into the input tunnel 301, causing the sealing material 320 to be pierced by the piercer 323, resulting in aeration of fluid within the sample preparation reservoir 317, the detergent reservoir 318, and / or the substrate reservoir 319. The piercer 323 may be one or more piercing elements with an edge that is sufficiently sharp to cut open the sealing material 320. As described in detail below, the piercer 323 may include three different piercers, each positioned within the housing 304 above one of the sample preparation reservoir 317, the detergent reservoir 318, or the substrate reservoir 319. As the slider 322 moves within the input tunnel 301, such as occurs with the 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 sealing material 320. In one embodiment, the slider 322 is configured to move in a first direction, e.g., laterally / substantially 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, e.g., vertically, to pierce into the sealing material 320.

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

[0201] Desiccant 325 may be disposed within housing 304 and in fluid communication with one or more reagent balls housed within shuttle 324. Desiccant 325 is configured to absorb moisture that enters housing 304 and reduce moisture exposure to the one or more reagent balls when not in use. Desiccant 325 may be a pad and may be at least partially disposed within input tunnel 301. Desiccant 325 may have a lumen sized to allow a sample collection device to be inserted therethrough.

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

[0203] An ultrasonic generator element 327 is disposed within the housing 304, preferably adjacent to or integral 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 isolated from other electrical components within the housing 304, such as components on the circuit board 331, including sensors and heaters, for example, via a relay. The ultrasonic energy may be controlled to achieve mixing and bonding 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 pressure-sensitive piezoelectric disc. The ultrasonic generator element 327 may also include a high-moisture blister disposed between the sample preparation reservoir 317 and the piezoelectric disc. Such a high moisture content blister may be affixed below the sample preparation reservoir 317 during 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] An absorbent pad 328 is disposed within the housing 304 at the downstream-most end of the analytical channel 330. The absorbent pad 328 wicks up fluid from the analytical channel 330, thereby encouraging the fluid to flow downstream of the absorbent pad 328. The absorbent pad 328 may act as a waste receptacle, collecting all waste fluid and waste particles after flowing through the analytical channel 330. The size and degree of absorbency of the absorbent pad 328 may be selected to meter the flow rate of fluid and particles within the analytical channel 330. For example, the volume of fluid that the absorbent pad 328 can wick up must be large enough to expel all fluid from the sample preparation reservoir 317 and the detergent reservoir 318 and to draw fluid carrying the chemical substrate from the substrate reservoir 319. Such a condition may serve as a lower limit for absorbency.

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

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

[0207] Circuit board 331 may be disposed within housing 304 and coupled to internal components 316, for example, via layer 329. Circuit board 331 includes electrical components, such as one or more of resistors, electrical leads, vias, and sensors required for detection of target analytes. Although described separately, it should be understood that the electrical components of circuit board 331 need not be separate structural elements. One or more electrical components and / or circuits may fulfill some or all of the functions of the various components described herein.

[0208] The memory 332 is disposed within the housing 304 and is 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 information regarding cartridge type (e.g., inflammation, influenza, testosterone, fertility, vitamin D), cartridge identification information (e.g., serial number), and / or 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 determining the presence, absence, and / or amount of one or more target analytes. In one embodiment, one or more cartridge devices of a select group of cartridges (e.g., a common lot of production cartridges) may be tested using a known amount of the target analyte to determine an electrical characteristic associated with the one or more target analytes sensed by a sensor in the tested device. Calibration information based on the test results may be stored in memory 332 within select cartridges, and the electrical signals generated by the cartridge sensors and the calibration information may be used to accurately and consistently determine the presence, absence, and / or amount of one or more target analytes. Memory 332 may also store test result reliability information for predetermined ranges of parameters, e.g., voltage, current, etc., that may be compared to the electrical signal generated by a positive control working electrode to determine whether the parameter is within a predetermined range, as described below.

[0209] 6A, 6B, and 6C, exemplary circuit boards and layers are illustrated, with FIG. 6A depicting circuit board 331, FIG. 6B depicting layer 329, and FIG. 6C depicting layer 329 disposed on and bonded to circuit board 331.

[0210] 6A, circuit board 331 may include heating elements 333, 334, 335, 336, and / or 337, and sensor 338, which may include reference electrode 339, working electrode 340, counter electrode 341, background working electrode 342, and / or reference electrode 343. Working electrode 340 may be masked with one or more grooves 344, and background working electrode 342 may be masked with one or more grooves 345. Circuit board 331 may further include contacts 346 and 347 for electrically coupling circuit board 331 to ultrasonic generator element 327 via wires, although ultrasonic generator element 327 may also be electrically coupled to circuit board 331 using spring contacts, as described below.

[0211] Heating elements 333, 334, 335, 336, and 337 are configured to generate heat within housing 304 based on electrical signals transmitted from reader 400, for example, at times defined by a protocol stored in the reader's memory. Heating elements 333, 334, 335, 336, and 337 may each form part of circuit board 331. For example, heating elements 333, 334, 335, 336, and 337 may be resistive heating elements that appear as serpentine traces located on the bottom side of circuit board 331 surrounding vias. In other embodiments, the heating elements are located external to the cartridge, for example, on the reader. In various embodiments where resistive heating elements are used to generate heat, current is allowed to flow through the resistive heating elements, for example, through the operation of a transistor. Current passing through the resistive heating elements generates heat through Joule heating. Heat is conducted to the vias by physical contact between the resistive heating elements and the vias. Heating elements 333, 334, 335, 336, and 337 may be masked, for example, with solder mask, to promote electrical isolation from sensor 338 and maintain heat transfer while minimizing interference with the electrical signal sensed by 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 to occlude the entire cross section of the outlet, thereby fluidically sealing the outlet. The heating element 333 may be configured to heat the phase change material in the outlet of the sample preparation reservoir 317 such that the phase change material unseals the outlet of the sample preparation reservoir 317 and allows fluid to mix therein with the sample held in the sample preparation reservoir 317 and flow into the analysis channel 330. The heating element 333 may be caused to heat the phase change material at a time defined by a protocol stored in the memory of the reader 400, for example, after the reader 400 detects a cartridge device 300 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 irrigant reservoir 318. The outlet may have a phase change material therein to occlude 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 in the outlet of the irrigant reservoir 318 such that the phase change material unseals the outlet of the irrigant reservoir 318 and allows the wash solution held in the irrigant reservoir 318 to flow into the analysis channel 330. The heating element 334 may heat the phase change material at a time defined by a protocol stored in the memory of the reader 400, e.g., a predetermined time after the reader 400 heats the heating element 333 and / or a predetermined time after the reader 400 heats the heating element 336.

[0214] The heating element 335 may be positioned adjacent to the outlet of the substrate reservoir 319. The outlet may have phase change material therein to occlude the entire cross section of the outlet, thereby fluidically sealing the outlet. The heating element 335 may be configured to heat the phase change material in the outlet of the substrate reservoir 319 such that the phase change material unseals the outlet of the substrate reservoir 319 and allows fluid held 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, a predetermined time after the reader 400 causes the heating element 334 to heat.

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

[0216] Heating element 337 may be positioned adjacent to a pocket of gas, e.g., air, within analysis channel 330. Heating element 337 may be configured to heat the pocket of air, causing the air to expand and exert pressure on the phase-change material, thereby promoting movement of the phase-change material within analysis channel 330. Heating element 336 may heat the phase-change material at a time defined by a protocol stored in the memory of reader 400, e.g., a predetermined time after reader 400 heats heating element 333. Placement of heating element 337 downstream of heating elements 333, 334, and 335 is expected to reduce bubble formation within analysis channel 330.

[0217] Electrical leads (shown in FIG. 8 ) on circuit board 331 may be provided for establishing electrical connection and continuity with a reader device. The electrical leads may be electrically coupled to heating elements 333, 334, 335, 336, and 337, respectively, to sensor 338, including reference electrode 339, working electrode 340, counter electrode 341, background working electrode 342, and reference electrode 343, contacts 346 and 347, and to memory 332. In this manner, such components may receive electrical current when activated by the reader device. Advantageously, electrical leads are exposed at the electrical connector portion on the bottom surface of circuit board 331 (shown in FIG. 4B ), but electrical leads electrically coupling the connector to the components may be traceless on the top surface of 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, reducing mating interference and thereby promoting adhesive adhesion with the layer 329. Mating 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, and 337 may be formed from conductors and may each include a via. Vias are a standard feature on printed circuit boards and are typically used to allow signal traces on one layer of the circuit board to be electrically continuous with another layer. Vias provide electrical continuity through multiple layers. Such vias are excellent conductors of heat, and because the surrounding material that makes up most circuit boards is an excellent insulator, they allow heat to be transferred to very precise locations without affecting the surrounding area. Thus, in various embodiments, multiple vias are provided as heating elements within the circuit board 331, each located below, above, or adjacent to a phase-change thermally actuated valve located within the reservoir outlet to create a valve actuation element. The precision of heat transfer associated with the vias allows for minimal crosstalk between valves located in close proximity to one another, so the timing of valve actuation can be carefully controlled for each valve. The valve may be formed from a phase-change material such as wax, e.g., a hydrophilic wax, and the via acts as a heat conductor, causing the wax to melt at a precise time as controlled by a reader device. Upon a phase transition, e.g., melting of a wax valve disposed within the reservoir outlet, the outlet is no longer blocked and the reservoir has an opening through which its fluid contents can drain into the analysis channel. The hole within the via may be filled with a filler material, e.g., solder, and the via may be masked, e.g., with a solder mask, to promote electrical isolation from the sensor 338 and maintain heat transfer while minimizing interference with the electrical signal sensed by the sensor 338.

[0219] To ensure precise timing and complete melting of the solder, in various embodiments, the solder valve is carefully constructed within the reservoir outlet. For example, in some embodiments, it is preferable for the solder valve to have a minimum height necessary to block the reservoir outlet, which minimizes the distance heat must travel to melt the solder. One exemplary method for achieving a solder barrier with such characteristics involves applying molten solder to a preheated via. Advantageously, when the via is preheated, the solder valve takes longer to solidify relative to a room-temperature via; therefore, the solder has more time to flatten and expand outward before hardening. "Pancaking" of the solder is desirable to minimize the height, which will maximize the chances of proper valve melt actuation. Additionally, heating the via promotes a greater level of contact area between the solder and the via so that the majority of the solder receives heat, also maximizing the chances of proper valve actuation. The method of heating the via prior to solder deposition is further enhanced in the following ways. When the molten wax is applied to the preheated via, the reservoir opening is aligned over the via so that the opening at the bottom of the reservoir is spatially close to the via, such that when the wax hardens, it simultaneously adheres to the interior walls of the reservoir and the via itself. This is advantageous for enhancing the yield of fabrication of an intact 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 exposed to fluid in analysis channel 330 and configured to 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, mixed fluid from sample preparation reservoir 317 may be introduced into analysis channel 330, and a signaling agent in the mixed fluid may localize across sensor 338 (e.g., in response to a magnetic field, if present, that retains magnetic particles that are directly or indirectly bound to the signaling agent). A chemical reaction may occur when 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 the substrate (e.g., TMB, OPD) and the signaling agent (e.g., HRP, SBP) localized across sensor 338. The reaction may cause electrons to be withdrawn from the substrate by the signaling agent (electrons may be donated to an acceptor molecule from the substrate solution), thereby generating an electrical signal detectable by the sensor 338. Such detected electrical signals may be used to generate signals indicative of the presence, absence, and / or amount of one or more analytes in the sample. The signals may be transmitted to the reader device 400, for example, via respective electrical connectors on the cartridge device 300 and the reader device 400.

[0221] Sensor 338 may include reference electrode 339, working electrode 340, counter electrode 341, background working electrode 342, and / or 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 strategically positioned such that when circuit board 331 is contained within assembled cartridge 300 and a surface of circuit board 331 forms one wall of analysis channel 330, sensor 338 is at least partially disposed within analysis channel 330. Although one sensor is illustrated, multiple sensors may be provided, each spaced apart relative to the others and preferably all aligned within analysis channel 330. In addition, working electrode 340 and background working electrode 342 may be disposed 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., stolen from a substrate by a signaling agent) that collect on working electrode 340 when the electrical environment created by reference electrode 339 and working electrode 340 results in a positive charge across working electrode 340. As described above, an oxidation reaction can occur in sensor 338 when an oxidase (e.g., a signaling agent described herein, such as HRP, SBP, etc., that may be introduced into analysis channel 330 from sample preparation reservoir 317) indirectly bound to a particle (e.g., a magnetic particle that may be introduced into analysis channel 330 from sample preparation reservoir 317) is present in sensor 338 and an appropriate chemical substrate (e.g., TMB, OPD) is introduced into analysis channel 330 (e.g., from substrate reservoir 319). In such embodiments, the working electrode 340 releases electrons to replenish those taken from the substrate by the oxidase in an amount proportional to the amount of oxidase present. The release of electrons from the working electrode 340 (e.g., from the substrate reacting with the signaling agent across the working electrode 340) is a current that can be detectable as a signal in a circuit connected to the sensor 338. The sensor 338 can thereby indirectly detect the presence, absence, and / or amount of the oxidase localized in the analysis zone. For example, a processor in 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 localization of the enzyme or other signaling agent within the analysis zone. Advantageously, in such embodiments, no affinity molecules need to be pre-bound to the sensor 338 to achieve localization, which would otherwise significantly slow down the analyte quantification process due to limitations in diffusion-based hybridization kinetics. Details of the magnetic field are also provided below.

[0223] The sensor 338 may include a gold surface created through the ENIG process. In other embodiments, gold or gold-plated sensors not created through the ENIG process are used. Those skilled in the art will appreciate that there are many plating processes for catalytic and autocatalytic deposition of gold utilized to create electrically active pads in the printed circuit board industry. The working electrode 340 may have a surface chemistry formed from a self-assembled monolayer, such as a dithiol, such as thiolated ethylene glycol and / or hexaethylene glycol dithiol, for added stability. The hydrophilic nature of the head groups of such surface chemistries promotes flow and protein resistance. Additionally or alternatively, one or more surfaces of the electrodes may be backed with mercaptoundecanoic acid, mercaptohexanol, or any other backing agent. The surface of one or more electrodes in the sensor 338 may be formed through sequential addition and incubation of ethylene glycol dithiol and backing agent at a non-high temperature.

[0224] The background working electrode 342 may be an ambient electrochemical noise sensor spaced within the analysis channel 330 away from the site of magnetic particle localization. The background working electrode 342 may be used to quantify background noise downstream or upstream of the working electrode 340, depending on the selected order of the working electrode 340 and background working electrode 342 within the analysis channel 330. Such noise may be due, for example, to the presence of nonspecifically bound enzymes. During processing of the detection results, the processor in the reader 400 may apply an algorithm to subtract the background working electrode signal (from the background working electrode 342) from the detected 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 background working electrode 342 may be used for error detection and diagnosis of improperly functioning cartridges, for example, as evidenced by a signal from the background working electrode 342 having an electrical value outside a predetermined range.

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

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

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

[0228] The working electrode 340 may be masked, e.g., solder masked, with a plurality of grooves 344 configured to promote uniform distribution and retention of the plurality of magnetic particles released from the sample preparation reservoir 317 across the working electrode 340. The accuracy of analyte detection may be adversely affected by premature washing of the magnetic particles during an analysis, for example, due to the force exerted on the particles in the analysis channel flow direction caused by the release of wash solution from the wash reservoir 318 and / or fluid bearing chemical substrates from the substrate reservoir 319 exceeding the magnetic force toward the magnetic generator of the reader 400 positioned directly below the working electrode 340 during the analysis. Such grooves 344 promote resistance to migration of the plurality of magnetic particles from the working electrode 340. Additionally, the background working electrode 342 may be masked, e.g., solder masked, with a plurality of grooves 345, although such grooves are not required on the background working electrode 342 and are merely illustrative of an alternative embodiment.

[0229] In alternative embodiments, the sensor 338 may be configured to analyze the fluid in the analysis channel 330 and generate a signal indicative of the presence, absence, and / or amount of one or more analytes in the sample, where the signal is visible. For example, the housing of the cartridge device may include a window that allows a user to view, e.g., using a camera, fluorescence may be quantified to determine the presence, absence, and / or amount of one or more analytes in the sample.

[0230] 6C , layer 329 is disposed on the top 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 completely covered with 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 is configured to minimize interference by heating element 333 with the electrical signal sensed by sensor 338 while maintaining heat transfer from heating element 333 to the phase change material in the outlet of sample preparation reservoir 317 at an energy level sufficient to cause a phase change in the material. The mask 349 is configured to promote electrical isolation between the heating element 334 and the sensor 338 and maintain heat transfer from the heating element 334 to the phase change material in the outlet of the irrigant reservoir 318 at an energy level sufficient to cause a phase change of the material while minimizing interference from the heating element 334 to the electrical signal sensed by the sensor 338. The mask 350 is configured to promote electrical isolation between the heating element 335 and the sensor 338 and maintain heat transfer from the heating element 335 to the phase change material in the outlet of the substrate reservoir 319 at an energy level sufficient to cause a phase change of the material while minimizing interference from the heating element 335 to the electrical signal sensed by the sensor 338. Mask 351 promotes electrical isolation between heating element 336 and sensor 338 and is configured to maintain heat transfer from heating element 336 to the phase change material of the fluid disconnector at an energy level sufficient to cause a phase change of the material while minimizing interference by heating element 336 with the electrical signal sensed by sensor 338. Mask 352 promotes electrical isolation between heating element 337 and sensor 338 and is configured to maintain heat transfer from heating element 337 to the pocket of gas in analysis channel 330 above heating element 337 at an energy level sufficient to cause downstream movement of the phase change material in analysis channel 330 while minimizing interference by heating element 337 with the electrical signal sensed by sensor 338.

[0231] 7A, 7B, and 7C, circuit board 331' and layer 329' are constructed similarly to circuit board 331 and layer 329 of Figures 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 accordingly. In addition, Figure 7C depicts absorbent pad 328 bonded to layer 329' at the downstream end of analysis channel 330'.

[0232] 7D and 7E, an alternative exemplary sensor that may be used in the cartridges described herein is provided. Sensor 338″ may include a reference electrode 339″, a working electrode 340″, a counter electrode 341″, a negative control working electrode 342″ (also referred to herein as a background working electrode), and / or a positive control working electrode 376. Sensor 338″ may detect electrical signals generated by chemical reactions in sensor 338″, as described above with respect to sensor 338. Sensor 338″ is positioned within the analysis channel in the same manner as sensor 338 described above. While one sensor is illustrated, multiple sensors may be provided, each spaced apart relative to the others and preferably all aligned within the analysis channel. Preferably, the fluid flows from a reservoir in the analysis channel and progresses across the electrodes in the following order: 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. Positive control working electrode 376 may have an affinity molecule pre-bound to its surface to achieve localization of the oxidase or other signaling substance across the positive control working electrode 376. The affinity molecule may be a surface-bound antibody. 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, positive control working electrode 376 releases electrons to replenish those taken from the substrate by the oxidase in an amount proportional to the amount of oxidase present. The release of electrons from positive control working electrode 376 is a current that may be detectable as a signal in a circuit connected to sensor 338''. For example, a processor within the 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, which may be stored in the cartridge and / or memory of the reader device. If the detected amount is within the range, the processor may validate the cartridge and continue signal processing to 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 diagnostics of an improperly functioning cartridge, for example, as evidenced by a signal from the positive control working electrode 376 having an electrical value outside of a predetermined range. For example, the reader processor may generate an error alert if the signal from the positive working control electrode 376 is outside of a predetermined range, and / or may consider the reading from the working electrode 340'' acceptable if it is within the predetermined range.

[0234] Reference electrode 339'' may be configured to generate a voltage difference between itself and working electrode 340''. Counter electrode 341'' may provide electrons that collect on working electrode 340'' when the electrical environment generated by reference electrode 339'' and working electrode 340'' results in a positive charge across working electrode 340''. Reference electrode 339'' and / or counter electrode 341'' may have a surface chemistry formed from a self-assembled monolayer, such as a dithiol, 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 flow and protein resistance. Additionally or alternatively, the surfaces of reference electrode 339'' and / or counter electrode 341'' may be backed with mercaptoundecanoic acid, mercaptohexanol, or any other backing agent.

[0235] The sensor 338'' may be used when magnetic particles are not present in the cartridge. For example, the working electrode 340'' may have an affinity molecule pre-bound to its surface to achieve localization of the oxidase or other signaling substance across the working electrode 340''. The affinity molecule may be a surface-bound antibody. The working electrode 340'' may be configured to detect a current generated by a reaction between the oxidase or other signaling substance indirectly bound to the affinity molecule and an appropriate chemical substrate introduced into the analysis channel, e.g., from a substrate reservoir. In such an embodiment, the working electrode 340'' releases electrons to replenish those taken from the substrate by the oxidase in an amount proportional to the amount of oxidase present. The release of electrons from the working electrode 340'' is a current that may be detectable as a signal in a circuit connected to the sensor 338''. The sensor 338 can thereby indirectly detect the presence, absence, and / or amount of the 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 oxidase. The functionality of such a processor is described in more detail below.

[0236] The working electrode 340'' does not include multiple grooves, illustratively because the sensor 338'' may be used to detect the presence, absence, and / or amount of one or more target analytes without the use of magnetic particles.

[0237] The negative control working electrode 342'' may be an ambient electrochemical noise sensor spaced within the 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 be due, for example, to the presence of non-specifically bound enzyme. During processing of the detection results, the processor in the reader 400 may apply an algorithm to remove the negative control working electrode signal (from the negative control working electrode 342'') from the detected 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 improperly functioning cartridges, for example, as evidenced by a signal from the negative control working electrode 342'' having an electrical value outside a predetermined range. For example, the reader processor may generate an error alert if the signal from the negative working control electrode 342'' is above a threshold, and / or may consider the reading from the working electrode 340'' acceptable if it is below the threshold.

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

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

[0240] 7F, an alternative exemplary sensor that can be used in the cartridges described herein is provided. Sensor 338"" may be constructed in the same manner as sensor 338'", except that negative control working electrode 342"" may be positioned between positive control working electrode 376" and reference electrode 339"". Preferably, fluid flows from a reservoir in the analysis channel and progresses across the electrodes in the following order: positive control working electrode 376", negative control working electrode 342"", reference electrode 339"", counter electrode 341"", and working electrode 340"".

[0241] 8A , internal component 316 is coupled to circuit board 331, e.g., via layer 329 positioned therebetween. Internal component 316 may include sample preparation reservoir 317 having outlet 353 with valve 354 positioned adjacent heating element 336, irrigant reservoir 318 having outlet 355 with valve 356 positioned adjacent heating element 336, and substrate reservoir 319 having outlet 357 with valve 358 positioned adjacent heating element 336. Reservoirs 317, 318, and 319 are each in fluid communication with analytical channel 330, such that, at least from time to time, when the respective valve is opened, fluid exiting the reservoir, e.g., via its respective outlet, flows into analytical channel 330. Additionally, fluid disconnector 359 may be positioned adjacent heating element 336 and in fluid communication with analytical channel 330.

[0242] Valves 354, 356, and 358 may be located in outlets 353, 355, and 357, respectively, at the bottom of reservoirs 317, 318, and 319 of cartridge 300. Outlets 353, 355, and 357 may each be formed from holes in the bottom wall of internal component 316 above analysis channel 330. Valves 354, 356, and 358 may each be formed from a heat-sensitive phase-change material, such as a hydrophilic wax. Prior to actuation, the valve wax or other heat-sensitive material is in a solid or semi-solid state and is sized and shaped to fill the entire cross-section of the outlet so that fluid cannot escape from the respective reservoirs into analysis channel 330. Valves 354, 356, and 358 may be aligned directly over one or more heating elements (with a solder mask therebetween) or other localized thermally conductive elements. Such alignment allows for the localized application of heat to induce a phase change in the valve without causing a phase change in any neighboring valves. In various embodiments, the phase change melts or otherwise converts the heat-sensitive material such that it no longer causes complete occlusion of the outlet, but instead allows fluid in the individual reservoirs to flow into the analysis channel 330.

[0243] Additionally, the fluid isolator 359 may also be formed from a heat-sensitive phase-change material, such as, for example, a hydrophilic wax. Prior to activation, the wax or other heat-sensitive material is in a solid or semi-solid state and disposed outside the flow path between the outlet of the respective reservoir and the sensor 338 in the analytical channel 330. The fluid isolator 359 may be sized and shaped so that, when the fluid isolator is activated, for example, by heating the heating element 336, it blocks the flow path on the analytical channel 330 between the outlet of one reservoir, e.g., the sample preparation reservoir 317, and the outlet of another reservoir, e.g., the substrate reservoir 319. The fluid isolator 359 may be aligned directly over the heating element 336 (with a solder mask therebetween) or other localized thermally conductive element. Such alignment allows for the localized application of heat to induce a phase change in the fluid isolator 359 without causing a phase change in any nearby valves. In various embodiments, the phase change melts or otherwise converts the heat-sensitive material so that it flows 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, once released into the analysis channel 330, cannot enter the sample preparation reservoir 317 and interact with any remaining signaling agent from the sample preparation reservoir 317.

[0244] The brazing material placed on the vias or solder mask over the vias to block individual reservoir openings or isolate analysis channels may be a hydrophilic material such as hexadecanol or octadecanol. This advantageously encourages fluid flow after actuation, rather than impeding it, by flowing past any brazing pieces that solidify within any area of ​​the analysis channel. These materials also preferably have a melting temperature of 50-100 degrees Celsius, which allows for actuation with reasonable power consumption for battery-operated devices, but remains unactuated in general handling and storage environments and / or during ultrasonic protocols. The amount of brazing material per valve may be less than 1 microliter in its liquid state, less than or equal to 0.5 microliters, or greater than 2 nanoliters. Using a minimal amount of brazing material in the valve is one way to reduce any blockage of the analysis channel and maximize full valve actuation when heat is applied. The valve may also have a feedback and control system that allows a consistent thermal profile to be achieved in the vias for consistent valve actuation. Additionally, the feedback and control system may incorporate sensing elements to enable the system to verify that each valve is operating properly.

[0245] 8A, the circuit board 331 may have exposed electrical leads 360 at the electrical connector 312. In one embodiment, the electrical leads 360 are exposed only on the bottom surface of the circuit board 331, however, the electrical leads 360 may also be exposed on the top surface of the circuit board 331, and are preferably traceless as shown. As previously mentioned, the electrical connector 312 enables electrical connection with a corresponding electrical connector of the reader device 400 such that the cartridge device 300 may transmit a signal indicative of the presence, absence, and / or amount of one or more target analytes in a collected sample as sensed by a sensor of the cartridge device 300.

[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 vent holes 362 to 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] Input tunnel 301 of cartridge device 300 may include slot 363 configured to allow seal piercer 321 to be at least partially disposed within input tunnel 301. Slot 363 may be at the proximal end of input tunnel component 326, as shown. Slot 363 may be sized and shaped to allow engager 324 of slider 321 to be disposed within input tunnel 301. Additionally, slot 363 may have a sufficient length to allow slider 322 to slide distally from a pre-vent position to a vent position when engager 324 contacts a sample collection device within input tunnel 301.

[0248] 8B and 8C, expanded views of certain components of the circuit board 331 and valve are shown. FIG. 8B depicts a conductor 364 coupled to a resistor 365, e.g., an aluminum resistor, coupled to the heating element 333, and 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, although details of the heating element 333, mask 348, and valve 354 are illustrated, such configurations may be utilized for heating elements 334, 335, 336, and 337, along with their respective masks and valves, fluid disconnectors, or air pockets. Current from the reader device 400 may pass from the conductor 364 through the resistor 365 and generate heat through Joule heating. 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 generates heat through the mask 348 to cause a phase change in the phase change material of the valve 354 while promoting electrical isolation from the sensor 338 and minimizing interference by the heating element 333 with the electrical signal sensed by the sensor 338.

[0249] Referring now to FIG. 8D, an expanded view of alternative components of the circuit board 331′ and valve is shown. FIG. 8D depicts conductor 364′, e.g., a solder pad, coupled to resistor 365′, e.g., an aluminum resistor. Unlike the configuration shown in FIGS. 8B and 8C, conductor 364′ is not coupled to heating element 333′ (including vias in circuit board 331′) so that a mask disposed between heating element 333′ and valve 354′ is not required. As will be clear to one skilled in the art, although details of heating element 333′ and valve 354′ are illustrated, such a configuration may be utilized for heating elements 334, 335, 336, and 337, along with their respective valves, fluid disconnectors, or air pockets. Current from reader device 400 may pass from conductor 364′ through resistor 365′, generating heat through Joule heating. The vias in the heating element 333' are disposed between and electrically isolated from the conductors 364' that are coupled to the resistors 365'. Heat is conducted to the heating element 333' through indirect contact between the resistors 365' and the heating element 333'. The heating element 333' generates heat and causes a phase change in the phase change material of the valve 354' while promoting electrical isolation from the sensor, minimizing interference by the heating element 333' with the electrical signal sensed by the sensor.

[0250] 9A and 9B illustrate various shuttles that may be positioned within the input tunnel of a cartridge device. Referring to FIG. 9A, shuttle 324 may include a first end 366, a reagent bowl 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] In a pre-mixing state, shuttle 324 is configured to be disposed within the cartridge housing, preferably within the input tunnel between the sample preparation reservoir and the opening defining the input tunnel. In such a pre-mixing state, first end 366 may form a wall of the sample preparation reservoir and seal fluid within the reservoir. First end 366 may include one or more sealing members, e.g., O-rings, which may be composed, in part, of chlorobutyl, to enhance a fluid-tight seal. The area between first end 366 and compartment divider 368 may be referred to as reagent ball compartment 367. Reagent ball compartment 367 is configured to store one or more reagent balls, e.g., reagent ball 375. In the pre-mixing state, reagent ball compartment 367 is preferably sealed from fluid within the sample preparation reservoir. Compartment divider 368 may be used to divide a compartment within shuttle 324, e.g., into reagent ball compartment 367 and sample compartment 370. Compartment divider 368 may include slot 369 that allows fluid to flow through compartment divider 368 when compartment divider 368 is placed in the fluid of the sample preparation reservoir in a mixed state. Allowing fluid to flow through slot 369 may improve mixing of the sample, reagent balls, and fluid in the sample preparation reservoir. Additionally, slot 369 allows for improved fluid communication between reagent ball 375 and desiccant 325 disposed within the cartridge housing in a pre-mixed state. Although not explicitly stated, it should be understood that the compartments may contain reagent balls that are identical or different from one another and can be pre-selected for detection and / or quantification of target analytes.

[0252] In the pre-mixed state, the second end 371 may be disposed between the first end 366 and the aperture 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 sized slightly larger than the tip of the sample collection device so that the tip can advance through the opening 372 while the opening 372 wipes excess sample from the tip. In this manner, up to a predetermined volume of sample can be 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 include one or more beams 373, 374 configured to couple the first end 366 to the second end 371 and coupled to the compartment divider 368. Beams 373, 374 are preferably positioned closer to the top surface of the cartridge housing when in the input tunnel to avoid interfering with fluid mixing in the mixed state.

[0253] The shuttle 324 may move from a first position in the pre-mixing state to a second position in the mixing state, for example, in response to the application of a force exceeding a threshold force exerted on the shuttle 324 by the 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 mix 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 fluidically seals the opening 372 in the mixing state so that 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 O-rings, to enhance the fluid-tight seal. In this manner, the sample preparation reservoir remains fluidly sealed during transition from the pre-mixed state to the mixed state by first end 366 in the pre-mixed state and by second end 371 and the sample collection device in the mixed state. Additionally, unlike breakable membrane-enclosed reagents, shuttle 324 may remain intact as sample is moved into sample preparation reservoir 317 for analysis.

[0254] Reagent ball 375 may include one or more of magnetic particles, affinity molecules, connecting molecules, signaling agents, competitive binding molecules, competitor molecules, labels, signaling agents, primers, nucleic acid probes, and / or polymerases, as well as other enzymes or components as described in further detail herein, and the components, encapsulating materials, and dimensions may be the same or different from one another. In one aspect, reagent ball 375 is formed by freezing (i.e., lowering the temperature of a volume of liquid (e.g., 5 microliters to 30 microliters)) to a temperature that induces a phase change in the liquid. The temperature may vary depending on the components of the liquid. In a further aspect, the liquid additionally comprises excipients known to those skilled in the art of lyophilization, such as lyoprotectants for functional preservation of nucleic acid and / or protein components, that may be temperature-sensitive in the liquid volume, as it undergoes the freezing and drying process to become reagent ball 375. Stabilizers, such as disaccharides like sucrose and trehalose, or other lyoprotectants, such as polyethylene glycols of various molecular weights, and bulking or solidifying agents, such as mannitol, glycine, povidone, and others known in the art, can comprise some of the final components of reagent ball 375, in addition to the reagents described herein. The percentage (w / v) of excipients in the volume of liquid to be freeze-dried to form reagent ball 375 can vary widely from about 0.1% to about 30%, and can be combined in various ways, often using a combination of disaccharides, such as lyoprotectants, and solidifying or bulking agents to add structure. Reagent ball 375 can be in 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. Although the reagent ball 375 is illustrated as spherical, 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.As is conventional in the art, such liquids, when formed to contain ingredients and excipients, can be frozen through flash freezing in liquid nitrogen or through shelf freezing in a freeze dryer. After freezing, the frozen volume potentially undergoes an annealing process for crystallization of the solidifying agent, primary drying, and secondary drying to remove water until a sufficiently small percentage (e.g., <8%, preferably <5%, preferably about 1%) remains in the final freeze-dried product, which is reagent bowl 375.

[0255] Referring now to FIG. 9B, shuttle 324′ is constructed similarly to shuttle 324 of FIG. 9A, except that compartment divider 368′ is solid without slots, and beams 377, 378, and 379 are positioned in a different orientation on shuttle 324′.

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

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

[0258] 10A , the distal end of the sample collection device 200 is first inserted into the opening defining the opening of the input tunnel 301. In the pre-mixing position, the shuttle 324 is disposed within the input tunnel 301, and the first end 366 of the shuttle 324 forms a wall of the sample preparation reservoir 317, fluidly sealing the fluid within the sample preparation reservoir 317. In this pre-mixing position, the reagent ball 375 housed 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 engager 380 of the slider 322. For example, the distal seal zone 208 may contact the engager 380 as shown. The distal seal zone 208 may be angled to facilitate movement of the engager 380 beyond the distal seal zone 208 as the sample collection device 200 is moved more distally, or the distal seal zone 208 may be a shoulder that is sized to cause movement of the slider 322.

[0259] 10B , tip 204 (with sample thereon and / or in tube 205) enters sample compartment 370 of shuttle 324 through opening 372 in second end 371. Preferably, as tip 204 enters sample compartment 370, shuttle 324 remains substantially in a fixed position within input tunnel 301, with first end 366 of shuttle 324 continuing to form the walls of sample preparation reservoir 317 and seal fluid within sample preparation reservoir 317. Additionally, first end 366 of shuttle 324 may continue to form the walls of sample preparation reservoir 317 and seal fluid within sample preparation reservoir 317 (and shuttle 324 may remain substantially in the pre-mixing position) until distal seal portion 208 contacts second end 371 and fluidly seals opening 372. Application of a force greater than a threshold force by the sample collection device 200 (at the distal seal portion 208) to the shuttle 324 (e.g., at the second end 371) can move the shuttle 324 from a pre-mixing position to a mixing position, where the sample and reagent ball 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., tip 204) is inserted into the shuttle 324 (e.g., through 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 walls of the second end 371, which define 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 up to a predetermined volume of sample, e.g., about 2 μl. Wiping excess sample from the sample collection device 200 may improve the precision, accuracy, and / or consistency of the analysis because, in the mixing position, up to a predetermined volume of sample is inserted into the sample preparation reservoir 317.

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

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

[0263] 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 for clarity to show the slider 322. The slider 322 may include a first track 384 configured to engage the first piercer 381 and move it to the puncturing position, a second track 385 configured to engage the second piercer 382 and move it to the puncturing position, and / or a third track 386 configured to engage the third piercer 383 and move it to the puncturing position. As the sample collection device 200 is moved distally through the input tunnel 301, preferably, the slider 322 does not move within the cartridge housing and remains in the pre-vent position until the sample collection device 200 securely engages the slider 322. The slider 322 may be temporarily or permanently fixedly engaged with the sample collection device 200 by coupling an engager 380 of the slider 322 to the engagement zone 209 of the sample collection device 200. The engager 380 is disposed within the input tunnel 301 (e.g., at least at times suspended below the slot 363 in FIG. 8A ). The engager 380 may be sized to fit within a protrusion and / or a groove of the U-shaped engagement zone 209 or to receive a protrusion of the engagement zone 209, for example.

[0264] 10E is a cross-sectional side view depicting the seal piercer in a pre-puncture position and the shuttle in a pre-mixing position. As shown, the sample collection device 200 has been moved distally within the input tunnel 301 such that the slider's engager 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 the 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, the 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 shuttle 324 may be positioned within the cartridge housing such that the engager 380 of the slider 322 engages the engagement zone 209 of the sample collection device 200 at or near the same time that the distal seal zone 208 of the sample collection device contacts and fluidly seals the second end 371 of the shuttle 324. In such embodiments, the slider 322 and / or shuttle 324 cannot yet be moved within the cartridge device 300 at this point.

[0265] 10F and 10G further illustrate the positioning of the slider and lancing element in the pre-venting position shown in FIG. 10E. As shown in FIG. 10F, when the engager 380 of the slider 322 engages the engagement zone 209 of the sample collection device 200 in the pre-venting position, the piercing elements 381, 382, ​​and 383 have not yet been biased downward by the slider 322 toward their respective reservoirs. In such a pre-venting position, the tracks 384, 385, and 386 may not yet contact the contact lancing elements 381, 382, ​​and 383, respectively. As shown in FIG. 10G, in the pre-venting position, the piercing element 381 is positioned upward but has not yet pierced the sealing material 320, which seals the fluid within the sample preparation reservoir 317. In FIG. 10G, the track 384 of the slider 322 has not yet contacted the lancing element 381 of the lancing device 323.

[0266] 10H, the sample collection device 200 is moved further distally within the input tunnel 301 from a pre-mixing and pre-venting position toward a mixing and venting position. As the collector pushes the sample collection device 200 distally, the shuttle 324 is moved partially within the sample preparation reservoir 317. The shuttle 324 may be moved within the cartridge, for example, by the application of a force above 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 such that one or more reagent balls within the shuttle 324 and the collected sample 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 such that fluid from the sample preparation reservoir 317 does not leak beyond the second end 371 into the input tunnel 301. Additionally, as distal movement of the sample collection reservoir 200 creates a transition from the pre-mixing position to the mixing position, the second end 371 of the shuttle continuously fluidly seals the fluid in the sample preparation reservoir 317 from the input tunnel 301.

[0267] As the collector pushes the sample collection device 200 distally, the seal piercer 321 may also be moved from a pre-venting position toward a venting position. The seal piercer 321 may be moved within the cartridge, for example, by the application of a force above a threshold force by the sample collection device 200 (e.g., at the engagement zone 209) on the seal piercer 321 (e.g., at the engager 380 of the slider 322). As the sample collection device 200 is moved distally, the seal piercer 321 punctures the seal material across the sample preparation reservoir 317, the irrigant reservoir 318, and / or the substrate reservoir 319, venting the fluid within the reservoirs. In response to insertion of the sample collection device 200 into the input tunnel 301, the seal piercer 321 may be moved within the input tunnel 301 in a first direction, e.g., generally laterally in a direction generally parallel to the movement of the sample collection device 200, and in a second (different) direction, e.g., downward in a generally vertical manner toward the individual reservoirs, 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] 10I, as the sample collection device 200 moves the slider 322 distally in a direction generally parallel to the movement of the sample collection device 200, the slider 322 may contact the contact lancing device 323 and move the lancing device 323 in a different direction, for example, 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 contact the puncturing elements 381, 382, ​​and 383 of the lancing device 323, respectively. The distal movement of the slider 322 may cause the lancing device 323 to puncture the seal material and vent the reservoir.

[0269] 10J illustrates the piercing element 381 piercing the seal material 320 over 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 into the piercing position.

[0270] 10K shows shuttle 324 in the mixing position and the seal piercer in the venting position. In the mixing position, first end 366 of shuttle 324, one or more reagent ball compartments of the shuttle storing one or more reagent balls, and / or one or more sample compartments storing the sample to be analyzed may be disposed within sample preparation reservoir 317. As described above, shuttle 324 (e.g., at second end 371) and sample collection device 200 (e.g., via tip 204 inserted into opening 372 and distal seal zone 208) may also fluidly seal sample preparation reservoir 317 in the mixing position such that the collected sample, reagent balls, and fluids within sample preparation reservoir 317 are sealed within the reservoir. In this manner, the collected sample, reagent balls, and fluids may be mixed within sample preparation reservoir 317.

[0271] Advantageously, insertion of the sample collection device 200 vents the sample preparation reservoir 317, the detergent reservoir 318, and / or the substrate reservoir 319, a configuration that ensures that the reservoirs remain fluidly sealed prior to insertion of the sample collection device 200 and facilitates drainage of the reservoirs into the analysis channel when the outlets of the individual reservoirs allow fluid to flow therethrough.

[0272] In the mixing position, the seal piercer 321 may move away from the punctured hole, opening the punctured hole and facilitating venting. As shown in FIG. 10K, the engagement zone 209 of the sample collection device 200 may be moved distally beyond the engager 380 such that the engager 380 disengages from the engagement zone 209 of the sample collection device 200 in the venting position. Additionally, in 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. In this manner, the proximal seal zone 207 provides additional structure to minimize or eliminate, 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. In the mixing position, the locking member 387 may be biased inward within the input tunnel 301 such that a locking end of the locking member 387 engages the sample collection device 200. The locking end may lock into the engagement zone 209. The locking end may be a protrusion sized to fit within a groove in the engagement zone 209, as shown. The locking member 387 may also define a portion of the input channel 301, as shown, and may be coupled to the cartridge housing at its end opposite the locking end. Advantageously, locking the sample collection device 200 within the input tunnel 301 (e.g., vertically and / or axially) aids in sealing the sample preparation reservoir 317 over time, such that once locked, the sample collection device 200 cannot be retracted, as the user cannot inadvertently pull the sample collection device 200 out of the cartridge device 300 once testing has begun, promoting safe disposal and testing consistency.

[0274] 10L and 10M further depict the sample collection device 200 (FIG. 10L) and internal component 316 (FIG. 10M) in a mixing position within the cartridge device 300 for clarity.

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

[0276] The ultrasonic generator element 327 may be configured to emit acoustic waves, moving fluid in the sample preparation reservoir 317 between the reagent bowl compartment 367 and the sample compartment 370 in a wave pattern and mixing the fluid in the sample preparation reservoir 317. Such mixing may create a fluid mixture of the sample, fluid from the reservoir, and dissolved reagent bowls. The acoustic emissions from the ultrasonic generator element 327 may heat the fluid in the sample preparation reservoir 317 and mix the contents of the sample preparation reservoir 317 at the macro- and micro-level for amplification, such as isothermal amplification. The reagent bowls may contain, for example, polymerase, primers, and signaling agents for isothermal amplification. The shuttle 324 may have a compartment divider 368, which may be a flange configured to divide the reagent bowl compartment 367 and the sample compartment 370. Fluid flowing around the compartment divider 368 may promote the formation of the wave pattern. The compartment divider 368 may have a slot configured to allow fluid to flow through the compartment divider 368 via the slot during mixing. The ultrasonic generator element 327 may form part of a wall, e.g., the bottom wall, of the sample preparation reservoir 317 and may be positioned off-center of the sample preparation reservoir 317 to promote mixing of the fluid within the sample preparation reservoir 317. For example, the ultrasonic generator element 327 may be positioned off-center with respect to a central axis of the sample preparation reservoir 317 that extends perpendicular to a longitudinal axis extending through the center of the cartridge's input tunnel. Such off-center positioning of the ultrasonic generator element 327 also promotes improved mixing. The ultrasonic generator element 327 may be electrically coupled to a printed circuit board via one or more spring contacts, as described in detail below.

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

[0278] After contacting contact switch 389 and puncturing the seal material, seal piercer 321' may move such that post 388 no longer depresses contact switch 389 and piercer 323' moves from puncturing a hole in the seal material to vent the individual reservoirs. Insertion of the sample collection device into the input tunnel may cause the seal piercer to puncture the seal material before the shuttle begins movement from the pre-mixing position to the mixing position or during movement from the pre-mixing position to the mixing position.

[0279] 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. Beneficially, the spring contact 392 absorbs the movement of the ultrasonic generator element 327' such that the circuit board 331' vibrates minimally in a suitable manner when the ultrasonic generator element 327' is activated, for example, in response to a signal transmitted by a reader device, and the spring contact 392 allows for ease and repeatability of assembly compared to solder, which can adversely affect the ultrasonic generator element 327'.

[0280] As will be readily understood by one of ordinary skill in the art, although FIGS. 11A and 11D do not depict a shuttle and / or reagent bowls within the input tunnel, such features may be included in these embodiments.

[0281] 12A-12E, alternative configurations for collecting and analyzing fluid samples are described. Cartridge device 300'' may be constructed similarly to cartridge device 300 described above, except that cartridge device 300'' may be modified for enhanced collection of relatively large volumes of fluid. Additionally, sample collection device 200'' may be constructed similarly to sample collection device 200 described above, except that sample collection device 200'' may have a modified collection area for enhanced collection of relatively large volumes of fluid. For example, sample collection device 200'' and 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 portion 201'' modified for enhanced collection of relatively large volumes of fluid (e.g., approximately 10-100 microliters), which may include a distal seal zone 208'', a wicking portion 211, an intermediate seal zone 212, and a shroud 213. The distal portion 201'' of the sample collection device 200'' is adapted to be exposed to a sample, preferably a liquid sample, absorb at least a portion of the sample, and be compressed to expel the collected sample from the distal portion 201'' into the cartridge device 300'' for analysis of the expelled sample. The wicking portion 211 is configured to wick and absorb the sample and may be formed from a wicking material. The wicking 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 sealing members, e.g., O-rings, configured to create a fluid-tight seal that reduces or prevents fluid absorbed onto the wicking portion 211 from migrating proximally from within the shroud 213 beyond the intermediate seal zone 212. The wicking portion 211 may be at least partially disposed within the shroud 213 and slide within a lumen of the shroud 213. The wicking portion 211 may be configured to be transparent when exposed to fluid, such that as an increasing amount of sample fluid is collected, an increasing amount of the wicking portion 211 becomes transparent. The sample collection device 200'' may include a sample collection indicator 214 configured to visually alert a collector based on the volume of sample fluid collected. In one embodiment, the sample collection indicator 214 visually alerts a collector that at least a predetermined volume of sample fluid has been collected, optionally as the volume of collected sample increases. For example, the sample collection indicator 214 may change color when a predetermined volume of sample or more has been collected. As another example, an increasing amount of sample collection indicator 214 may become visible as an increasing volume of sample is collected.For example, sample collection indicator 214 may be a colored thread embedded within wicking portion 211 that becomes gradually visually exposed as increasing volumes of collected fluid sample cause the surrounding wicking material to become more transparent so that a collector can monitor the progress of fluid collection and determine when a sufficient volume of sample has been collected. In one embodiment, sample collection indicator 214 includes a transparent area on shroud 213. Additionally or alternatively, sample collection indicator 214 may change color as the volume of collected sample increases.

[0283] Shuttle 324″ may include a first end 366″, a reagent bowl compartment 367″, a compartment divider 368″, and a sample compartment 370″ that are constructed similarly to those individual components described above. Preferably, compartment divider 368″, like compartment divider 368′, does not have a slot, such that compartment divider 368″ fluidly seals reagent bowl compartment 367″ from sample compartment 370″ in the pre-mixing position. Second end 371″ of shuttle 324″ includes distal flange 393 and proximal flange 394 and may be modified to have cavity 395. Additionally, unlike opening 372 of shuttle 324″, opening 372″ is configured to allow the ejected sample flow to be compressed from sample collection device 200″ into sample compartment 370″, rather than being part of sample collection device 200″. Second end 371" (e.g., at distal flange 393) may be configured to fluidly seal sample compartment 370" in both the pre-mixing position and the mixing position, and continuously during transitions therebetween. Distal flange 393 may include one or more sealing members, e.g., O-rings, configured to create a fluid-tight seal that reduces or prevents fluid from flowing proximally from sample compartment 370". Additionally, compartment divider 368" may be configured to fluidly seal sample compartment 370" in the pre-mixing position. Compartment divider 368" may include one or more sealing members, e.g., O-rings, configured to create a fluid-tight seal that reduces or prevents fluid from flowing distally from 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 a sample, for example, from the inner cheek, throat, mouth, nasal cavity, ear, urine, blood, plasma, saliva, etc. The wicking portion 211 of the sample collection device 200'' is designed to retain a portion of the sample and enable analysis of the presence, absence, and / or amount of one or more target analytes in the sample using the cartridge device 300'' and a reader device. The cartridge device 300'' may be electrically coupled to the reader device 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 aperture 302″ that defines the opening of the input tunnel 301″. In the pre-mixing position, the shuttle 324″ is disposed within the input tunnel 301″, and the first end 366″ of the shuttle 324″ forms a wall of the sample preparation reservoir 317″, fluidly sealing the fluid within the sample preparation reservoir 317″. In this pre-mixing position, the reagent ball 375″ housed by the shuttle 324″ is within the input tunnel 301″ and is not exposed to the fluid within the sample preparation reservoir 317″.

[0286] 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 distal seal zone 208″ may contact the second end 371″ (e.g., at the cavity 395 and / or the proximal flange 394) as shown. The cavity 395 may be sized slightly larger than the outer surface of the wicking portion 211 so that the distal end of the wicking portion 211 fits snugly within the cavity 395. The distal seal zone 208″ may be configured to fluidically seal the sample collection device 200″ to the shuttle 324″ such that fluid expelled from the sample collection device 200″ progresses 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 in 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 expel the collected fluid sample, for example, through an opening 372″ in the second end 371″ and into a sample compartment 370″ of the shuttle 324″. As the sample collection device 200″ is moved distally, the wicking portion 211 may be compressed to expel the collected sample, and the distal end of the wicking portion 211 may remain substantially in a fixed position during compression. The intermediate seal zone 212 and / or the shroud 213 may move distally during such compression in proportion to the movement of the handle of the sample collection device 200″. Preferably, as the wicking portion 211 is compressed and sample is expelled therefrom, the expelled sample proceeds through the opening 372″ into the sample compartment 370″. During 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 a wall of the sample preparation reservoir 317'', sealing the fluid within the sample preparation reservoir 317''. The cartridge device 300'' may include a proximal shoulder 396 configured to hold the shuttle 324'' in a pre-mixing position during compression of the wicking portion 211. The proximal shoulder 396 may engage the proximal flange 394 and hold the shuttle 324'' in place.

[0288] A maximum predetermined volume of sample may be configured to be retained in sample compartment 370″. Cartridge device 300″ may include an overflow compartment 397 and an overflow lumen 398. Overflow compartment 397 and overflow lumen 398 may be part of the cartridge housing or internal components within the cartridge. If the amount of sample introduced into sample compartment 370″ exceeds a predetermined volume, e.g., greater than about 20 μl, the excess sample may proceed to overflow compartment 397, which is fluidly connected to sample compartment 370″ via overflow lumen 398, for example. Limiting the volume of sample in sample compartment 370″ may improve the precision, accuracy, and / or consistency of the analysis because, in the mixing position, a maximum predetermined volume of sample is inserted into sample preparation reservoir 317. Overflow compartment 397 may otherwise be sealed to prevent or reduce leakage of excess sample in overflow compartment 397.

[0289] FIG. 12D shows the sample collection device 200″ and cartridge device 300″ in the mixing position, and FIG. 12E shows an enlarged view of a portion of FIG. 12D for clarity. As a collector pushes the sample collection device 200″ distally from the pre-mixing position to the mixing position, the shuttle 324″ is moved partially within the sample preparation reservoir 317″. Application of a force above a threshold force by the sample collection device 200″ (at the distal seal portion 208″ and / or the distal end of the wicking material 211) to the shuttle 324″ (e.g., preferably at the second end 371″ within the cavity 395) may move the shuttle 324″ from the pre-mixing position to the mixing position, where the ejected sample in the sample compartment 370″ and the reagent ball 375″ are mixed in the fluid of the sample preparation reservoir 317″. For example, the threshold force may be the force required to push the shuttle 324″ distally past the proximal step 396. As the shuttle 324″ moves distally, the first end 366″ may unseal such that one or more reagent balls in the shuttle 324″ and the sample collected in 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″ to prevent fluid from the sample preparation reservoir 317″ from leaking beyond the second end 371″ and / or the intermediate seal zone 212 into the input tunnel 301″. Additionally, as distal movement of the sample collection device 200″ creates 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 shoulder 399 configured to hold the shuttle 324'' in the mixing position and prevent further distal movement. The distal shoulder 399 may engage the proximal flange 394 to hold the shuttle 324'' in place.

[0290] In the mixing position, first end 366″ of shuttle 324″, one or more reagent ball compartments of the shuttle storing one or more reagent balls, and / or one or more sample compartments storing the sample to be analyzed may be disposed within sample preparation reservoir 317″. As described above, shuttle 324″ (e.g., at second end 371″) and sample collection device 200″ (e.g., via distal seal zone 208″ inserted into cavity 395) may also fluidly seal sample preparation reservoir 317″ in the mixing position, such that the collected sample, reagent balls, and fluids within sample preparation reservoir 317″ are sealed within the reservoir. In this way, the collected sample, reagent balls, and fluids may be mixed within 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''. In the mixing position, the locking member 387'' may be biased inward within the input tunnel 301'' such that a locking end of the locking member 387'' engages the sample collection device 200''. The locking end may lock into an engagement zone of the sample collection device 200''. The locking end may be a protrusion that is sized to fit within a groove on the shaft of the sample collection device 200'' or a groove on the shroud 213, as shown. The locking member 387'' may also define a portion of the input channel 301'', as shown, 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., vertically and / or axially) promotes sealing of the sample preparation reservoir 317'' over time, such that once locked, the sample collection device 200'' cannot be retracted, as the user cannot inadvertently pull the sample collection device 200'' out of the cartridge device 300'' once testing has begun, promoting safe disposal and testing consistency.

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

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

[0294] In FIG. 13A, an exploded view of cartridge device 300''' is shown. The cartridge device 300''' may include a sample preparation reservoir 317''', a cleaning agent reservoir 318''', a substrate reservoir 319''', an input tunnel component 326''', an overflow compartment 397''', and / or internal components 316''', which may include posts 610 and 612, 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 component 327''', an absorbent 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 components may be disposed within 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 another housing. In the case of multiple housings, such separate housings may be configured to mate with each other.

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

[0296] Posts 610 and 612 are configured to couple to seal piercer 321''' and allow seal piercer 321''' to be moved from a pre-vention position to a venting position. Posts 610 and 612 may be integrally formed with internal component 316''' or may be separate pieces.

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

[0298] Collet 618 is preferably disposed within input tunnel 301'" between opening 302'" and sample preparation reservoir 317'". Collet 618 may also be disposed at least partially proximal to shuttle 324'" within input tunnel 301'". For example, in the pre-mixing position, an end, e.g., the second end of the shuttle, may be disposed within collet 618. Collet 618 may also be configured to hold shuttle 324'" in the pre-mixing position and decouple it from shuttle 324'" during insertion of a sample collection device into input tunnel 301'". In this manner, collet 618 may hold shuttle 324'" in the pre-mixing state until a force applied from the sample collection device decouples collet 618 from shuttle 324'", such that collet 618 remains in place within input tunnel 301'". Collet 618 has a lumen sized to allow insertion of a distal portion of a sample collection device therethrough. Collet 618 may have a generally tubular shape, as illustrated in FIG. 13A . Collet 618 may also be configured to activate contact switch 389′″. For example, collet 618 may activate contact switch 389′″ in response to a force applied by a sample collection device on collet 618 during insertion of the sample collection device into input tunnel 301′″.

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

[0300] 13B, the sample collection device 200''' may be constructed similarly to the sample collection device 200'' described above, but 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''' illustratively includes an engagement zone 209''' having multiple grooves and protrusions distal to and spaced apart from the proximal seal zone 207'''. The multiple grooves and protrusions are configured for engagement with one or more components of a cartridge device due to the 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 sequentially engage grooves of the multiple grooves in 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 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 configured to activate a contact switch.

[0301] Similar to sample collection device 200'', sample collection device 200''' may include a distal portion 201''' modified for improved collection of relatively large volumes of fluid (e.g., approximately 10-100 microliters, preferably approximately 20 microliters), which may include a distal seal zone 208''', a wicking portion 211''', an intermediate seal zone, and / or a shroud 213'''. Sample collection device 200''' may also include a proximal portion 202''', a shaft 203''' extending between distal portion 201'''' and proximal portion 202'''', a handle 206''', a proximal seal zone 207'''', and / or an engagement zone 209''' having a shoulder 220, similar to similarly primed reference numbers previously described. Sample collection device 200''' is configured for full or partial insertion into cartridge device 300''' after sample collection. The sample collection device 200''' and cartridge device 300''' may be useful, among other things, 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''' so that the shoulder 220 contacts and displaces a seal piercer, vents one or more reservoirs within the cartridge device 300''', and / or contacts a collet and activates a contact switch, while the shoulder of the distal seal zone 208''' may be sized to move distally through the input tunnel without displacing a seal piercer and / or activating a contact switch.

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

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

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

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

[0306] Referring to FIG. 13FF, sample collection device 200''''''' may be constructed similarly to sample collection device 200'''''' shown in FIG. 13P, except that tip 204''''''' of sample collection device 200''''''' includes a ring 224 rather than a tube. Distal portion 201''''''' including tip 204'''''''' is configured to be exposed to the sample such that up to a predetermined volume of the sample (e.g., 10 microliters, preferably less than about 2 microliters) is disposed within the groove formed by ring 224 for analysis. Collection of a predetermined volume of sample is expected to promote accuracy of analyte analysis because a substantially known amount of sample will be analyzed. Tip 204'''''''' may have a rounded end as shown and may be configured to collect sample from any desired area or location, although tip 204''''''' may be particularly useful when collecting blood samples. 13GG, 13HH, 13II, 13JJ, 13KK, and 13LL are back, side, front, back, side, and front views, respectively, of sample collection device 200''''''''.

[0307] 13MM, sample collection device 200'''''''' may be constructed similarly to sample collection device 200'''''' shown in FIG. 13P, except that tip 204'''''''' of sample collection device 200'''''''' is not a tube but includes first ring 226 and second ring 228. Distal portion 201'''''''''', including tip 204'''''''', is configured to be exposed to sample such that up to a predetermined volume of sample (e.g., 10 microliters, preferably less than about 5 microliters) is disposed within the groove formed by first ring 226 and second ring 228 for analysis. Collection of a predetermined volume of sample is expected to facilitate accuracy of analyte analysis, as a substantially known amount of sample will be analyzed. Tip 204'''''''' may have a rounded end, as shown, and may be configured to collect a sample from any desired area or location, although tip 204'''''''' may be particularly useful when collecting a blood sample. Figures 13NN, 13OO, 13PP, 13QQ, 13RR, and 13SS are back, side, front, back, side, and front views, respectively, of sample collection device 200''''''''.

[0308] 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, e.g., saliva, blood, plasma, or urine, such as 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 and 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, in an upper surface of the collet 618. The slot 626 is sized to receive a portion of the seal piercer therethrough. For example, an engager of the seal piercer may extend through the slot into the input tunnel to allow 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 the 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 the pre-mixing position. Illustratively, the collet 618 includes a first locking arm 628 and a second locking arm 630 on opposite 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 place within the input tunnel during compression of the distal portion and expel sample fluid into the shuttle. Each locking arm may include a ramp and a protrusion, shown in FIG. 14B as ramp 632 and protrusion 634 for locking arm 630. Protrusion 634 may be coupled to the shuttle and hold the shuttle in place in the 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 decouple 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, decoupling the first and second locking arms 628 and 630 from the shuttle. For example, a shoulder of the sample collection device may contact a ramp on the locking arm and deflect the protrusion outward as the sample collection device moves distally along the ramp within the input tunnel. The ramp is shaped such that the protrusion decouples from the proximal flange of the shuttle, unlocking the shuttle and allowing it to move from a pre-mixing position to a mixing position where it is partially disposed within the sample preparation reservoir.

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

[0311] 14C and 14D, an alternative collet for use in the cartridge is described. Collet 618′ may be specifically designed for relatively small sample collections, e.g., nasal, ear, or blood samples, when the sample does not need to be compressed from the distal portion of the sample collection device. As can be seen by comparing FIGS. 14C and 14D with FIGS. 14A and 14B, collet 618′ is similar to collet 618, except that collet 618′ does not have locking arms. In the pre-mixing position, collet 618′ has one or more protrusions 637 disposed at its distal end 622′ and configured to contact the proximal end of the shuttle. For example, one or more protrusions 637 may contact a sealing member, e.g., an O-ring, at the second end 371, 371′ of shuttle 324, 324′ to retain the sealing member in place. The one or more protrusions may have a lead-in angle configured to guide a distal portion of the sample collection device into the opening in the second end of the shuttle.

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

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

[0314] The seal piercer 321''' is configured to puncture the seal material 320''' and vent fluids in the sample preparation reservoir 317''', the detergent reservoir 318''', and / or the substrate reservoir 319'''. Preferably, the seal piercer 321''' is configured to sequentially pierce the seal material 320''' across the reservoirs during piercing, reducing resistance on the sample collection device. The seal piercer 321''' may be configured to be contacted by a distal portion, e.g., at a shoulder, of the sample collection device within the input tunnel 301''' and move within the housing 304''' to puncture the seal material 320''' and vent fluids in the sample preparation reservoir 317''', the detergent reservoir 318''', and / or the substrate reservoir 319''' in response to a force applied by the sample collection device. Illustratively, the seal piercer 321''' is a single piece. The seal piercer 321''' is disposed within the housing 304''' and may be partially disposed within the input tunnel 301'''. For example, the engager 380''' of the seal piercer 321''' may be disposed within the input tunnel 301''', for example, through the slot 626 of the collet 618. The seal piercer 321''' has one or more piercing elements with edges sharp enough to cut open the seal material 320'''. Illustratively, the seal piercer 321''' has a first piercing element 381''' having a piercing end positioned adjacent the sample preparation reservoir 317''', a second piercing element 382''' having a piercing end positioned adjacent the irrigant reservoir 318''', and a third piercing element 383''' having a piercing end positioned adjacent the substrate reservoir 319'''.

[0315] The seal piercer 321''' may also include slots 638 and 640 configured to receive portions of the posts 610 and 612, respectively. Thus, the seal piercer 321''' may move within the housing 304''' while portions of the posts 610 and 612 remain within the slots 638 and 640. The cartridge 300''' may also include one or more ramps configured to deflect the one or more piercing elements toward and pierce the seal material 320'''. The one or more ramps may be directly coupled to the housing 304''' of the cartridge 300'''. The peaks of the one or more ramps may be positioned such that when the sample collection device is fully inserted into the input tunnel 301''', the one or more piercing elements advance past the peaks to facilitate venting the reservoir. Illustratively, cartridge 300'" has a first ramp 642 positioned adjacent to sample preparation reservoir 317'", a second ramp 644 positioned adjacent to irrigant reservoir 318'", and a third ramp 646 positioned adjacent to substrate reservoir 319'". The distance between each ramp and each piercing element in the pre-venting position may vary so that the reservoirs are pierced sequentially. Each ramp may have a central notch to fit over a portion of seal piercer 321'" adjacent the piercing element in the venting and mixing position, as shown in FIG. 15B.

[0316] As the sample collection device 200''' is moved distally through the input tunnel 301''', the seal piercer 321''' preferably does not move within the cartridge housing and remains in a pre-vent position until the sample collection device 200''' fixedly engages the seal piercer 321'''. The seal piercer 321''' may fixedly engage the sample collection device 200''' once the sample collection device 200''' is fully inserted into the input tunnel 301''', for example, by temporarily or permanently coupling an engager 380''' of the seal piercer 321''' to the sample collection device 200''' at shoulder 220.

[0317] As the collector pushes the sample collection device 200''' distally, the seal piercer 321''' is moved from the pre-vent position toward the vent position. The seal piercer 321''' may be moved within the cartridge by, for example, the application of a force above a threshold force by the sample collection device (e.g., at the shoulder 220, which may be at the distal end of the shroud 213''' and / or the distal end of the engagement zone 209''') on the seal piercer 321''' 200''' (e.g., at the engager 380'''). As the sample collection device 200''' is moved distally, the piercing element first pierces the seal material above the reservoir at the shortest distance from the ramp. For example, as the sample collection device 200''' is moved distally, the seal piercer 321''' moves generally parallel to the movement of the sample collection device 200''' until the second piercing element 382''' contacts the second ramp 644, which deflects the second piercing element 382''' downward, piercing the seal material 320''' across the irrigant reservoir 318'''. As the seal piercer 321''' continues to move distally, the second piercing element 382''' moves over the peak of the second ramp 644 and moves upward from the pierced hole above the irrigant reservoir 318''' and the third piercing element 383''' contacts the third ramp 646 which deflects the third piercing element 383''' downward, piercing the seal material 320''' across the substrate reservoir 319'''. As the seal piercer 321''' continues to move distally, the third piercing element 383''' moves over the peak of the third ramp 646 and moves upward from the punctured hole above the substrate reservoir 319''' and the first piercing element 381''' contacts the first ramp 642, which deflects the first piercing element 381''' downward, piercing the seal material 320''' over the sample preparation reservoir 317'''. As the seal piercer 321''' continues to move distally, the first piercing element 381''' moves over the peak of the first ramp 642 and moves upward from the punctured hole above the sample preparation reservoir 317'''.As will be appreciated by one skilled in the art, the order of puncturing may be varied for sequential puncturing.

[0318] Advantageously, the configuration in which insertion of the sample collection device 200''' sequentially vents the sample preparation reservoir 317''', the detergent reservoir 318''', and / or the substrate reservoir 319''' ensures that the reservoirs remain fluidly sealed prior to insertion of the sample collection device 200, reduces resistance forces during insertion of the sample collection device, and facilitates drainage of the reservoirs into the analysis channel when the individual reservoir outlets allow fluid to flow therethrough.

[0319] 16A-16J, a configuration for collecting and analyzing samples using cartridge 300'" is described. By way of example, sample collection device 200'" is inserted into cartridge 300'", however, it should be understood that because cartridge 300'" is designed for general-purpose use for different types of samples and different types of indications, any sample collection device described herein may be used, and certain internal components may be substituted, such as shuttles, reagent bowls, and / or collets, based on the application. Sample collection device 200'" is as described above with respect to FIG. 13B. Sample collection device 200'" illustratively includes engagement zone 209'" having multiple grooves and protrusions distal to and spaced from a sealing zone in the proximal portion of sample collection device 200'". The plurality of grooves and protrusions are configured for engagement with one or more components of the cartridge device 300'" due to the non-retractability of the sample collection device 200'" during partial and full insertion into the cartridge 300'". The engagement zone 209'" may facilitate secure engagement between the sample collection device 200'" and the cartridge device, e.g., via locking member 387'", such that when the sample collection device 200'" is partially inserted a predetermined distance into the input tunnel of the cartridge, e.g., when the distal-most groove of the engagement zone 209'" engages locking member 387'", the sample collection device 200'" becomes irreversibly mated with the cartridge. The sample collection device 200'" remains irreversibly mated with the cartridge as insertion continues toward the fully inserted position. For example, the locking member 387''' may engage grooves of the plurality of grooves in the engagement zone 209''' successively in a distal-to-proximal direction as the sample collection device 200''' is moved distally within the input tunnel 301'''. The locking member 387''' may engage the proximal-most groove of the plurality of grooves in the engagement zone 209''' when the sample collection device 200''' i...

Claims

1. A sample analysis cartridge, comprising: an input tunnel extending from the opening, the input tunnel configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to a sample containing the target nucleic acid; a reagent bowl containing reagents for amplifying the target nucleic acid by an amplification reaction; a reservoir configured to hold a fluid, the reservoir further configured to receive the sample from the sample collection device and to receive the reagent ball such that the sample mixes with an effective amount of the reagent for amplification of the target nucleic acid within the fluid of the reservoir to produce an amplicon comprising a signal transduction substance; an analysis channel configured to receive the fluid containing the amplicons from the reservoir; a sensor disposed at least partially within the analysis channel such that the amplicons indicative of at least one of the presence, absence, or amount of the target nucleic acid in the sample are localized across the sensor, and the sensor senses a signal indicative of at least one of the presence, absence, or amount of the target nucleic acid in the sample based on a reaction between the signal transduction entity and a substrate of the amplicons localized across the sensor; A sample analysis cartridge comprising:

2. A sample analysis cartridge as described in claim 1, wherein the reagent ball contains a signal transduction substance for detecting amplification of the target nucleic acid, a primer for amplifying the target nucleic acid, and a polymerase.

3. A sample analysis cartridge as described in claim 2, wherein the reagent ball further contains one or more affinity molecules covalently or non-covalently bound to solid particles for detection of the target nucleic acid.

4. The reagent ball is disposed within the input tunnel, and the reagent ball is not exposed to the fluid in the reservoir in a pre-mixing position; The sample analysis cartridge of claim 1 , wherein the reagent ball is configured to move into the reservoir for mixing with the fluid in the reservoir at a mixing position.

5. A sample analysis cartridge as described in claim 1, further comprising a shuttle defining a first compartment and a second compartment, the first and second compartments configured to be positioned within the reservoir in a mixing position.

6. A sample analysis cartridge as described in claim 5, wherein the shuttle is configured to move within the input tunnel so that the first and second compartments are not exposed to the fluid in the reservoir in a pre-mixing position.

7. A sample analysis cartridge as described in claim 5, further comprising a piezoelectric transducer configured to emit energy into the reservoir to move the fluid in the reservoir between the first and second compartments and mix the reagent, the sample, and the fluid within the reservoir.

8. A sample analysis cartridge as described in claim 5, wherein the first compartment includes a reagent ball compartment configured to store the reagent ball, and the second compartment includes a sample compartment configured to receive the sample from the sample collection device.

9. A sample analysis cartridge as described in claim 1, further comprising a temperature sensor configured to sense a temperature indicative of the temperature of the fluid in the reservoir.

10. A sample analysis cartridge as described in claim 9, wherein the temperature sensor is located on a printed circuit board positioned adjacent to the piezoelectric transducer.

11. A sample analysis cartridge as described in claim 10, wherein the piezoelectric transducer is configured to emit energy into the reservoir to move the fluid in the reservoir and mix the reagent, the sample, and the fluid in the reservoir, and to emit the energy in a modified manner based on the sensed temperature.

12. A sample analysis cartridge as described in claim 11, wherein the energy emitted by the piezoelectric transducer is configured to cause the amplification reaction to cure on the target nucleic acid.

13. The sample analysis cartridge of claim 1, further comprising a contact switch configured to indicate insertion of the sample collection device into the input tunnel.

14. A sample analysis cartridge as described in claim 1, wherein the reagent ball further includes magnetic particles configured to be magnetically held across a target working electrode of the sensor.

15. A sample analysis cartridge as described in claim 1, further comprising a substrate reservoir configured to hold a substrate solution containing the substrate, the substrate reservoir having an outlet configured to allow the substrate solution to pass therethrough and into the analysis channel up to at least the portion of the sensor so that the substrate reacts with a signal transduction substance localized across the sensor.

16. A sample analysis cartridge comprising: an input tunnel extending from the opening, the input tunnel configured to allow insertion of a sample collection device having a distal portion adapted to be exposed to a sample containing the target nucleic acid; a reservoir configured to hold a fluid, the reservoir further configured to receive the sample from the sample collection device such that the sample mixes with an effective amount of reagents for amplification of the target nucleic acid within the fluid of the reservoir to produce an amplicon comprising a signal transduction agent; a shuttle defining a first compartment and a second compartment, the first and second compartments configured to be disposed within the reservoir in a mixing position; an analysis channel configured to receive the fluid containing the amplicons from the reservoir; a sensor disposed at least partially within the analysis channel such that the amplicons indicative of at least one of the presence, absence, or amount of the target nucleic acid in the sample are localized across the sensor, and the sensor senses a signal indicative of at least one of the presence, absence, or amount of the target nucleic acid in the sample based on a reaction between the signal transduction entity and a substrate of the amplicons localized across the sensor; A sample analysis cartridge comprising:

17. The sample analysis cartridge of claim 16, further comprising a reagent bowl containing the reagents for amplifying the target nucleic acid by an amplification reaction.

18. A sample analysis cartridge as described in claim 17, wherein the reagent ball contains a signal transduction substance for detecting amplification of the target nucleic acid, a primer for amplifying the target nucleic acid, and a polymerase.

19. A sample analysis cartridge as described in claim 18, wherein the reagent ball further contains one or more affinity molecules covalently or non-covalently bound to solid particles for detection of the target nucleic acid.

20. The reagent ball is disposed within the input tunnel, and the reagent ball is not exposed to the fluid in the reservoir in a pre-mixing position; 20. The sample analysis cartridge of claim 17, wherein the reagent ball is configured to move into the reservoir for mixing with the fluid in the reservoir at a mixing position.

21. A sample analysis cartridge as described in claim 16, wherein the shuttle is configured to move within the input tunnel so that the first and second compartments are not exposed to the fluid in the reservoir in a pre-mixing position.

22. The sample analysis cartridge of claim 16, further comprising a piezoelectric transducer configured to emit energy into the reservoir to move the fluid in the reservoir between the first and second compartments and mix the reagent, the sample, and the fluid within the reservoir.

23. A sample analysis cartridge as described in claim 16, wherein the first compartment includes a reagent ball compartment configured to store a reagent ball containing the reagents for amplifying the target nucleic acid by an amplification reaction, and the second compartment includes a sample compartment configured to receive the sample from the sample collection device.

24. The sample analysis cartridge of claim 16, further comprising a temperature sensor configured to sense a temperature indicative of the temperature of the fluid in the reservoir.

25. A sample analysis cartridge as described in claim 24, wherein the temperature sensor is located on a printed circuit board positioned adjacent to the piezoelectric transducer.

26. A sample analysis cartridge as described in claim 25, wherein the piezoelectric transducer is configured to emit energy into the reservoir to move the fluid in the reservoir and mix the reagent, the sample, and the fluid in the reservoir, and to emit the energy in a modified manner based on the sensed temperature.

27. ​​A sample analysis cartridge as described in claim 26, wherein the energy emitted by the piezoelectric transducer is configured to cause an amplification reaction to harden on the target nucleic acid.

28. The sample analysis cartridge of claim 16, further comprising a contact switch configured to indicate insertion of the sample collection device into the input tunnel.

29. A sample analysis cartridge as described in claim 16, further comprising a reagent ball containing the reagents for amplifying the target nucleic acid by an amplification reaction, the reagent ball further comprising magnetic particles configured to be magnetically held across the target working electrode of the sensor.

30. A sample analysis cartridge as described in claim 16, further comprising a substrate reservoir configured to hold a substrate solution containing the substrate, the substrate reservoir having an outlet configured to allow the substrate solution to pass therethrough and into the analysis channel up to at least the portion of the sensor so that the substrate reacts with a signal transduction substance localized across the sensor.

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