Diagnostic cartridge and architecture with common extraction chamber for lysis hybridization, and wash

Abstract

A diagnostic system includes a cartridge and an instrument. The cartridge includes a plurality of zones in fluid communication with each other, including an extraction zone including extraction chambers including magnetic particles, wherein each of the extraction chambers is configured to hold the magnetic particles, and a detection zone including detection chambers, each detection chamber including a heating element, and a waste storage chamber to receive a lysate. The instrument interfaces with the cartridge and includes: a lyse system to interface with at least one of the extraction chambers, and a plurality of magnetic field generators, wherein the plurality of magnetic field generators include: a magnetic field generator to generate a magnetic field to dock the magnetic particles to at least one of the extraction chambers, and a magnetic field generator to capture the magnetic particles to the heating element.

Description

Atty. Dkt. No.: 86357086DIAGNOSTIC CARTRIDGE AND ARCHITECTURE WITH COMMON EXTRACTION CHAMBER FOR LYSIS HYBRIDIZATION, AND WASH BACKGROUND

[0001] A presence of nucleic acids (e.g., DNA and RNA) in a biological sample or specimen may be useful in diagnosing patients. For example, a presence or amount of a certain nucleic acid may indicate infection or other illness or disease. Preparation of the biological sample may allow the biological sample to be amplified so that an increased number of the nucleic acids proportional to the starting amount of nucleic acids can be detected. Amplification of the nucleic acids may be performed through a method such as a polymerase chain reaction. Detection of the nucleic acids may indicate a presence, absence, or amount of the nucleic acid present in the biological sample.SUMMARY

[0002] This disclosure relates to devices and apparatus for detecting or measuring the presence, absence or amount of a nucleic acid of interest (e.g., a target nucleic acid), in a sample containing or suspected of containing the nucleic acid of interest.

[0003] At least one example relates to a diagnostic system. The diagnostic system includes a cartridge including a plurality of zones in fluid communication with each other. The plurality of zones include an extraction zone including one or more extraction chambers including: a plurality of magnetic particles, wherein each of the one or more extraction chambers is configured to hold the plurality of magnetic particles, and a waste storage chamber to receive a lysate, and a detection zone including one or more detection chambers, each detection chamber including one or more heating elements. The diagnostic system includes an instrument to interface with the cartridge, including: a lyse system to interface with at least one of the one or more extraction chambers, and a plurality of magnetic field generators, wherein the plurality of magnetic field generators include: at least one magnetic field generator to generate a magnetic field to dock the plurality of magnetic particles to at least one of the one or more extraction chambers, and at least one magnetic field generator to force the plurality of magnetic particles towards the one or more heating elements.

[0004] Another aspect relates to a method for detecting a presence, absence, or amount of a nucleic acid of interest. The method includes inserting, into a cartridge, a fluid including a biological sample, wherein the cartridge includes a plurality of zones including an extractionAtty. Dkt. No.: 86357086zone including one or more extraction chambers, each extraction chamber including a plurality of magnetic particles, and a detection zone including one or more detection chambers, each detection chamber including one or more heating elements, wherein each zone of the plurality of zones is in fluid communication with each other, and wherein the cartridge further includes one or more reagents. The method further includes lysing the biological sample into at least one of the one or more extraction chambers to release the one or more nucleic acids of interest from the biological sample, wherein lysing the biological sample produces a lysate, hybridizing, into the extraction chamber, the one or more nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles, holding the hybridized one or more magnetic particles on a side of the extraction chamber, washing the hybridized one or more magnetic particles with at least one wash buffer, trapping the hybridized one or more magnetic particles into proximity of the one or more heating elements of the at least one detection chamber, amplifying the one or more nucleic acids of interest in at least one of the plurality of zones, via an amplification reaction, to provide a plurality of the one or more nucleic acids of interest, and detecting a plurality of amplification products indicative of the presence, absence, or amount of the plurality of amplified nucleic acids of interest via an optical unit in communication with the at least one detection chamber of the one or more detection chambers.

[0005] At least one aspect relates to a system. The system includes a cartridge including a plurality of zones in fluid communication with each other, including an extraction zone including one or more extraction chambers including: a plurality of magnetic particles, wherein each of the one or more extraction chambers is configured to hold the plurality of magnetic particles, wherein a biological sample is lysed within at least one of the one or more extraction chambers to release one or more nucleic acids from the biological sample, wherein lysing the biological sample produces a lysate, and wherein the one or more nucleic acids are hybridized to the plurality of magnetic particles, and a waste storage chamber to receive the lysate, a detection zone including one or more detection chambers, each detection chamber including one or more heating elements, wherein a first wash buffer is configured to deliver the hybridized magnetic particles to at least one of the one or more detection chambers from the one or more extraction chambers. The system includes an instrument to interface with the cartridge, including: a lyse system to interface with at least one of the one or more extraction chambers, and a plurality of magnetic field generators, wherein the plurality of magnetic field generators include at least one magnetic field generator to generate a magnetic field to dockAtty. Dkt. No.: 86357086the plurality of magnetic particles to an extraction zone, and at least one magnetic field generator to forward the plurality of magnetic particles toward the one or more heating elements.BRIEF DESCRIPTION OF THE FIGURES

[0006] These and other aspects and features of the present implementations are depicted by way of example in the figures discussed herein. Present implementations can be directed to, but are not limited to, examples depicted in the figures discussed herein. Thus, this disclosure is not limited to any figure or portion thereof depicted or referenced herein, or any aspect described herein with respect to any figures depicted or referenced herein.

[0007] FIG. 1 A depicts a block diagram of a cartridge and an instrument, in accordance with example implementations.

[0008] FIG. IB depicts a block diagram of a cartridge and an instrument, in accordance with example implementations.

[0009] FIG. 2 depicts a method, in accordance with example implementations.

[0010] FIG. 3A depicts a perspective view of a cartridge device, in accordance with example implementations.

[0011] FIG. 3B depicts a perspective view of a cartridge device, in accordance with example implementations.

[0012] FIG. 4 depicts a cartridge architecture, in accordance with example implementations.

[0013] FIG. 5A depicts a first cross sectional view of a detection chamber of the cartridge of FIGS 1 A-4, in accordance with example implementations.

[0014] FIG. 5B depicts a second cross sectional view of a detection chamber of the cartridge of FIGS 1 A-4, in accordance with example implementations.

[0015] FIG. 6 depicts a method, in accordance with example implementations.

[0016] FIG. 7 depicts a method, in accordance with example implementations.

[0017] FIG. 8A depicts a blister device, in accordance with example implementations.Atty. Dkt. No.: 86357086

[0018] FIG. 8B depicts the blister device of FIG. 8 A, in accordance with example implementations.

[0019] FIG. 8C depicts the blister device of FIG. 8 A, in accordance with example implementations.

[0020] FIG. 8D depicts the blister device of FIG. 8 A, in accordance with example implementations.

[0021] FIG. 9A depicts a blister device, in accordance with example implementations.

[0022] FIG. 9B depicts an actuator of the blister device of FIG. 9A, in accordance with example implementations.

[0023] FIG. 10A depicts a system, in accordance with example implementations.

[0024] FIG. 10B depicts a system, in accordance with example implementations.

[0025] FIG. 10C depicts a system, in accordance with example implementations.

[0026] FIG. 11A depicts a cartridge environment, in accordance with example implementations.

[0027] FIG. 11B depicts a cartridge environment, in accordance with example implementations.

[0028] FIG. 12A depicts a cartridge environment in a cross-sectional view, in accordance with example implementations.

[0029] FIG. 12B depicts a cartridge environment in a cross-sectional view, in accordance with example implementations.

[0030] FIG. 12C depicts a cartridge environment in a cross-sectional view, in accordance with example implementations.

[0031] FIG. 12D depicts a cartridge environment in a cross-sectional view, in accordance with example implementations.Atty. Dkt. No.: 86357086

[0032] FIG. 13 A depicts a cartridge environment in plan view, in accordance with example implementations.

[0033] FIG. 13B depicts a cartridge environment in plan view, in accordance with example implementations.

[0034] FIG. 14 depicts a cartridge panel, in accordance with example implementations.

[0035] FIG. 15 depicts a user interface for cartridge environment, in accordance with example implementations.

[0036] It will be recognized that the figures are schematic representations of examples for purposes of illustration. The figures are provided for the purpose of illustrating example implementations with the explicit understanding that the figures will not be used to limit the scope of the meaning of the claims. Thus, the description is not limited to the examples and / or implementations provided in the drawings.DETAILED DESCRIPTION

[0037] Biological samples may contain molecules or particles that are of interest. For example, the molecules may be indicative of disease, illness, genetic abnormalities, etc. Molecular diagnostics may be of use to accurately diagnose potential infections or other diseases. Currently, molecular diagnosing may be performed, at least partially, manually. For example, preparing a sample to be analyzed for diagnostic purposed may be performed manually by a human. Manual sample preparation may be slow. Further, a skilled technician may be required to perform the sample preparation. The preparation may be required to be performed in a certified laboratory meeting certain standards or conditions. This may be costly and timeconsuming.

[0038] Further, molecular detection may be performed by amplifying a molecule of interest (also referred to as a “target” or “target molecule”) in the biological sample and detecting a presence, absence, and / or amount of the molecule of interest. In various examples, the molecule of interest may be a nucleic acid. Specifically, the target molecule may be an oligonucleotide (a “target oligonucleotide”). The target oligonucleotide may be a nucleic acid of interest that is present in and extracted from the biological sample. The target oligonucleotide may be single stranded or double stranded (i.e., before denaturation). The target nucleic acid sequence is theAtty. Dkt. No.: 86357086sequence that is amplified. This specific nucleic acid sequence may characterize the presence of a pathogen (e.g., a virus or bacteria) for which the diagnostic method is being used.

[0039] One method of amplification may be isothermal amplification (e.g., the entire reaction or reaction chamber is heated and cooled to a uniform temperature). Isothermal reactions may be associated with low-plex reactions (e.g., reactions having a limited number of targets or components), high costs for reagents used in the reactions, and less sensitivity and / or specificity compared to other types of amplification reactions, such as polymerase chain reactions (PCR). Further, sample preparation methods, specifically for magnetized particles used for amplification reactions, may include additional steps. For example, a chaotropic salt / alcohol sample preparation method may include air drying alcohol and eluting a target molecule from the magnetic particles. These additional steps may incur additional costs and may increase an amount of time it takes for diagnosing. An extraction-free sample preparation may be limited by a reaction volume, as high costs of reagents may limit volumes that can be used for a given reaction or analysis. A limited reaction volume may subsequently limit an amount of target analytes that can reach the amplification reaction step(s), thus reducing sensitivity of the results.

[0040] The systems and methods described herein provide rapid, low-cost, point-of-care molecular diagnostics. The systems and methods may be convenient to both health care providers running the diagnostics and patients waiting to be diagnosed. The systems and methods described herein utilize pulse-controlled amplification (PCA) to amplify and subsequently detect molecules of interest that may be found in a biological sample. PCA reactions may enable rapid thermocycling with a low power expenditure by using paramagnetic particles to concentrate DNA or other nucleic acid targets into a thin thermocycling zone. Additionally, PCA utilizes polymerase chain reaction (PCR) chemistry to replicate and amplify target molecules (e.g., nucleic acids of interest, such as DNA or RNA). The paramagnetic particles may automate and integrate solid-phase extraction of the sample specimen with PCA and a real-time multi-channel detection, thus reducing both a run time of the diagnostics and a cost of running the diagnostics. Additionally, the diagnostics are performed in a low-cost, disposable cartridge that provides an automated sample-to-answer process. Further, the systems and methods described herein reduce power consumption of instruments used to carry out a multiplex reaction by utilizing low power, yet solid-state and rapid, thermocycling for PCR.Atty. Dkt. No.: 86357086

[0041] In one example, a cartridge can include a plurality of zones. The cartridge may be inserted into an instrument that helps to facilitate amplification and detection of the nucleic acid of interest. Different zones of the plurality of zones of the cartridge may include different components and / or have different functionalities. Each of the plurality of zones and / or components may be fluidly coupled and / or may fluidly communicate with one another. This communication may facilitate the steps of the method (e.g., lysing, hybridization, washing, amplification, detection, etc.) to be performed in or on the cartridge to detect and / or analyze nucleic acids of interest, thus reducing a time to reach a diagnosis. For example, a extraction zone of the plurality of zones may include an input chamber to input a biological sample containing the nucleic acids of interest. A detection zone may include one or more extraction chambers, and a third zone may include one or more detection chambers. Additional zones may include, for example, storage for liquid and / or dry reagents (e.g., a wash buffer, a system composition, lyophilized reagents, etc.). A zone may also be used for reconstitution of a master mix used during amplification. The different zones may be coupled via fluid channels. The cartridge can be inserted into an instrument when the process is ready to begin, and the cartridge may only need to be removed from the instrument upon completion of the processes without any external fluid or reagents being added to the process.

[0042] The cartridge may further include valves (e.g., membrane pinch valves, wax valves, volcano valves, etc.) to permit fluid flow from zone to zone, chamber to chamber, etc. in such a way that fluids and reagents are delivered to the appropriate locations within the cartridge at appropriate times / during appropriate steps of the processes. Further, an instrument interfacing with the cartridge may include a plurality of pumps (e.g., syringe pumps, air cylinder pumps, diaphragm pumps, etc.) to allow fluid flow through the cartridge. Thus, each step that occurs in the process of detecting a molecule of interest, from input of the sample to detection of the amplified molecules, may be performed on the cartridge. Further, all reagents, reactants, and other components used during the processes may be stored in and disposed of on the cartridge. The cartridge used to perform the processes herein may be a single use cartridge. Thus, each component (e.g., reagents) needed for the various reactions and processes may be stored on the cartridge. Wet and / or dry reagents may be stored in blister devices having frangible seals and / or valves that can control flow of the liquids. Further, used or discarded liquids can be transported back to an original storage blister to reduce a need for a waste chamber in the cartridge or removal of waste external to the cartridge.Atty. Dkt. No.: 86357086

[0043] Furthermore, the systems and methods described herein utilize PCA reactions, which may be PCR-like amplifications. The amplification may be able to be performed rapidly (e.g., within 5 to 10 minutes), thus reducing a total time it takes for a sample to be analyzed and a diagnosis to be made. Further, the systems and methods described herein leverage chemistry associated with PCR reactions for a multiplex reaction (e.g., a reaction that facilitates detecting multiple nucleic acids of interest) and real-time detection. Multiplex reactions allow for the ability to detect and amplify multiple nucleic acids of interest. Further, multiplex reactions may allow for detection and amplification of different types of nucleic acids of interest simultaneously (e.g., while the nucleic acids are in the same chamber or different chambers of the cartridge). This may allow for diagnosis of multiple diseases, infections, etc. with one biological sample, thus reducing time, costs, and labor associated with diagnostics.

[0044] As previously stated, real-time detection may reduce analysis times and a time to make a diagnosis based on the analysis results. Further, solid-phase extraction may enhance a sensitivity of the results, though purification and concentration. Solid-phase extraction may also allow more challenging specimen types to be analyzed. Specificity of capture of the target molecules may also be increased through hybridization of the target molecules.

[0045] The systems and methods described herein utilize pulse-controlled amplification to amplify molecules (e.g., nucleic acids) of interest that may be found in a biological sample. The biological sample may be lysed (e.g., with a system composition and / or a control substance) to isolate the molecules of interest from the biological sample. In some examples, the molecules of interest are nucleic acids and may be referred to as “nucleic acids of interest.” The nucleic acids of interest may also be referred to herein as “target oligonucleotides,” each of which may have a unique oligonucleotide sequence. The isolated molecules of interest may be hybridized to magnetic particles having attached capture oligonucleotides. The capture oligonucleotides may refer to the oligonucleotides attached to the particles, each having a nucleic acid sequence that is complementary to the “target oligonucleotide.” A capture oligonucleotide may be an oligonucleotide attached to the magnetic particles having a sequence designed to be complementary to the “target oligonucleotide.” The capture oligonucleotide may be a synthetic single-stranded segments of nucleic acid (DNA or RNA). The sequence of the capture oligonucleotide may be designed specifically to match (i.e., complement) the nucleic acid sequence of the target oligonucleotide. In various examples, the capture oligonucleotides may be further used to isolate and amplify the nucleic acid of interest.Atty. Dkt. No.: 86357086However, it should be understood that various other methods can be used to capture nucleic acids of interest and subsequently isolate and amplify the nucleic acids of interest in proximity to a heating element.

[0046] Hybridization may occur by annealing the molecule of interest with the attached capture oligonucleotides having a complementary sequence. The hybridized molecules of interest and magnetic particles may be amplified to generate an increased or amplified number of molecules of interest via a PCA reaction. The presence, absence, and / or amount of the molecule of interest may be determined using, for example, optical detection.

[0047] In various implementations, a lysate generated during lysing of the biological sample may deliver paramagnetic particles with captured nucleic acid targets (e.g., from the biological sample) to a detection chamber, where a PCA reaction occurs. However, lysate includes human specimens and other chemical components that may interfere with or inhibit PCA if present during a PCA amplification reaction. When lysate delivers the paramagnetic particles with the attached nucleic acid targets to a detection or PCA chamber, residual inhibitors (e.g., human specimens and / or unwanted chemicals) are deposited in the chamber. Further, solid materials in the lysate may be deposited onto heating elements (e.g., foils) within the detection or PCA chambers, even if a liquid lysate is significantly or substantially removed from the chamber. Washing the detection or PCA chamber with wash buffer insufficiently removes the lysate and any solids or precipitates from the lysate such that there are enough undesirable components present in the chamber that performance of the PCA reaction is reduced.

[0048] Accordingly, the systems and methods described herein utilize a wash buffer, rather than a lysate, to transport the magnetic beads with attached nucleic acids of interest from a lyse chamber to a detection or PCA chamber. Use of a wash buffer prevents lysate from reaching the detection or PCA chamber, preventing possible contamination. Hybridization and washing of the magnetic particles and nucleic acids of interest are performed in the same chamber where lysing is performed. Use of a wash buffer, rather than a lysate, to transport the magnetic particles with attached nucleic acids of interest to a detection chamber offers many technical advantages. For example, a reduced (e.g., or zero) amount of lysate carried over from a lyse chamber to a detection / PCA chamber increases the sensitivity of a PCA reaction and associated nucleic acid detection. Additionally, use of a wash or transport buffer facilitates more consistent capture of magnetic particles compared to use of a lysate, due to the increased variability associated with lysates coming from a lysed human sample. Further,Atty. Dkt. No.: 86357086suspension / resuspension of magnetic particles (e.g., via sonication) may produce more uniform magnetic particles and / or fewer aggregates of magnetic particles relative to when lysate is used. Variability of magnetic particle capture onto a foil or heating element of the detection / PCA chamber is also reduced. Additionally, a workflow as described herein more closely mimics a standard laboratory manual workflow.

[0049] Referring now to Fig. 1A, a diagnostic system 100A, comprising a cartridge 100 and an instrument 150 is shown, according to an example. The system 100A may be configured to detect a nucleic acid of interest. In some examples, the cartridge 100 of the diagnostic system may include a series of zones or chambers that may be defined as cavities. The zones / chambers / cavities may be interconnected via a plurality of channels. The series of cavities / chambers can be individually loaded with reagents. The cavities / chambers can be loaded with the same or with different types of reagents. Loading the cavities / chambers with different types of reagents can permit a multi-step reaction. The reagents / reactants can be disposed in the chambers or blister devices, as will be described herein.

[0050] As shown in FIG. 1A, the cartridge 100 includes an extraction zone 104 including a first extraction chamber 114 having reagents 125 and magnetic particles 116, and a detection zone 108 comprising a detection chamber 120 having a heating element 122. The instrument 150 includes a lyse system, a first magnetic field generator 156a, a second magnetic field generator 156b, electrical components 160, and a controller 164.

[0051] As stated above, the cartridge 100 may also include a plurality of zones, shown as an extraction zone 104 and a detection zone 108. Each zone may be or include a plurality of chambers. For example, the extraction zone 104 may include one or more extraction chambers, shown as the extraction chamber 114. Each of the one or more extraction chambers includes a plurality of magnetic particles. Each of the one or more extraction chambers is configured to hold the plurality of magnetic particles. The extraction zone 104 may also include the waste storage chamber 168. The waste storage chamber may receive a lysate. The plurality of zones may include the detection zone 108, which may include one or more detection chambers 120. Each detection chamber 120 includes one or more heating elements. Further, the one or more detection chambers may be or include one or more amplification chambers or one or more pulse-controlled amplification (PCA) chambers.Atty. Dkt. No.: 86357086

[0052] Each zone of the plurality of zones may be in fluid communication with each other (e.g., the other zones of the plurality of zones). It should be understood that the cartridge 100 may include any number of zones. In various examples, each zone is configured to perform or is associated with certain actions. Further, each zone may include one or more chambers or components in addition or alternative to those described herein. For example, the extraction zone 104 may also store a plurality of reagents 125.

[0053] Fluid within the cartridge 100 may move through a plurality of fluidic channels. The channels may be fluidly coupled via a plurality of fluidic junctions. Transport of fluid through the cartridge 100 may be moderated by a plurality of valves. For example, the cartridge 100 may include a plurality of pinch valves, membrane valves, etc. configured to selectively permit and restrict flow through the fluidic channels. For example, when a valve is closed, fluid movement may be restricted. The fluidic channels may permit fluid flow from one of the plurality of zones and / or chambers to another zone and / or chamber. For example, fluid may flow from the extraction zone or an extraction chamber to a detection zone or detection chambers. In various examples, the instrument 150 may include a pump to interface with the cartridge 100, specifically the fluid channels of the cartridge 100, to pump air to move air and the fluid through the channels. In various examples, a pressure differential across each valve opening can be applied to transport fluid in and out of each of the plurality of zones, each of the plurality of chambers, each blister device, etc.

[0054] The cartridge 100 may include a greater or fewer number of zones than what is shown in FIG. 1A. In various examples, different elements of the cartridge 100 may be included in different zones, depending on the configuration of the cartridge 100. For example, as shown in FIG. 1A, the cartridge 100 may include a extraction zone 104 having an extraction chamber 114 and a detection zone 108 having the detection chamber 120. However, in some examples, the extraction zone 104 may also include the wash buffers 110a and 110b.

[0055] In various examples, the extraction chamber 114 may house a lysing of the biological sample to release one or more nucleic acids of interest from the biological sample. The extraction chamber 114 may be referred to as a “lyse chamber,” a “lyse and hybridization chamber,” and / or a “hybridization chamber.” The extraction chamber 114 may be a fluidic structure with an open cavity or void to define a chamber that could be filled with the biological sample comprising the one or more nucleic acids of interest. The extraction chamber 114 may be configured to house a lyse reaction. For example, a biological sample may enter theAtty. Dkt. No.: 86357086extraction chamber 114 and may be lysed, for example by sonication, thermal lysis, thermal sonication, or another lysing method. The lyse reaction may damage the content of the biological sample to release one or more nucleic acids from the biological sample. The lyse reaction performed in the extraction chamber 114 may cause the one or more nucleic acids of interest to be released from the biological sample. The lyse reaction may disrupt, or lyse, cells, and / or tissue samples. That is, the biological sample is lysed within at least one of the one or more extraction chambers 114 to release one or more nucleic acids from the biological sample. Lysing the biological sample produces a lysate.

[0056] As shown in FIG. 1A, the cartridge 100 may include a first wash buffer 110a and a second wash buffer 110b. It should be understood that the wash buffers 110 may be included in a zone (e.g., the extraction zone 104). In various examples, the wash buffers 110 are not included in a zone. The system 100A may include one or more wash buffers 110. In various examples, the cartridge 100 may include a chamber in a extraction zone of a plurality of zones to store the wash buffer 110. The wash buffer 110 may be transported from the chamber or other location to at least one extraction chamber 114 and / or at least one detection chamber 120 to suspend magnetic particles 116 and / or remove undesired elements of the biological sample when the biological sample is in the extraction chamber 114 and / or detection chamber 120. In various examples, the extraction chamber 114 and / or the detection chamber 120 may be washed to remove undesirable elements from the chamber being washed. The wash buffers may be stored in a plurality of blister devices 126.

[0057] In various examples, one or more components or liquid reagents 125 (e.g., the system composition and / or the wash buffers 110) may be stored, housed, or otherwise contained in a blister device 126. Example blister devices are described in greater detail with respect to FIGS.8A-12B. The blister devices 126 may store liquid reagents (e.g., the system composition, the wash buffer 110, liquid reagents used for amplification, etc.). The blister device 126 may allow fluid to be released so that the fluid can reach a destination (e.g., the detection chamber 120). In various examples, the cartridge 100 may be configured such that the system composition and / or the wash buffer 110 may return to the blister device 126 upon completion of use. For example, the wash buffer 110 may be released from a blister device 126 storing the wash buffer 110 and may be delivered to the detection chamber 120 to wash the contents of the detection chamber. After washing is complete, the wash buffer 110 may return to the blister device 126 to be stored.Atty. Dkt. No.: 86357086

[0058] The blister device 126 can be configured to transport its contents in a direction according to a selection of a pumping direction through the blister device 126. In some examples, the blister device 126 is configured to transport contents of the blister device 126 in a direction from a first valve toward a second valve. In some other examples, the blister device 126 is configured to transport contents of the blister device 126 in a direction from the second valve toward the first valve. Blister devices may also be referred to herein as a blisters. The blister device(s) 126, when present in the cartridge 100, may be or be part of the extraction zone and / or the detection zone of the plurality of zones.

[0059] The blister device 126 may comprise a chamber (e.g., a storage cavity) to store a reagent, a first actuator at a first end of the zone, and / or a second actuator at a second end of the zone opposite to the first end of the zone. The blister device 126 can be configured to be hermetically sealed-off from the channel(s) of the cartridge 100 when the diagnostic device / system is in a non-activated state.

[0060] The wash buffers 110 refers to a buffer used for washing magnetic particles 116 and the extraction chamber 114 and / or detection chamber 120. In some examples, the wash buffers 110 can comprise water, a salt, a buffering compound or component (e.g., tris buffer) and / or a surfactant. In some examples, the salt is KC1, MgCh NaCl, etc. In some examples, the wash buffers 110 comprises between 20mM and 45mM salt. In some examples, the wash buffer can comprise between about 0.001% and about 0.1% (e.g., about 0.001%, 0.01%, 0.05%, or 0.1%) surfactant. In some examples, the surfactant is selected from Tween ®20, Tween® 80, Tween® 85, SPAN® 80 or SPAN® 85. In some examples, the surfactant is Tween® 20.

[0061] The system 100A may include a plurality of wash buffers 110. Each wash buffer 110 may have a different composition (e.g., one buffer can be or include salt while another buffer can be or include water). For example, a first wash buffer 110a may have a more aggressive wash chemical relative to a second wash buffer. A more aggressive wash chemical may be a harsher chemical that can remove a greater number of undesired elements in the sample of fluid relative to a less aggressive wash chemical, such as because the more aggressive wash chemical can have a higher chemical concentration, pH level, a higher specificity for removing the undesired elements, etc. Thus, the first wash buffer may be used to wash the extraction chamber 114 and / or detection chamber 120 and the second wash buffer 110b may be used to rinse the detection chamber 120. For example, the first wash buffer may be introduced to a chamber (e.g., the detection chamber 120, the extraction chamber 114, etc.) to wash or remove undesiredAtty. Dkt. No.: 86357086elements of the sample. Undesired elements may be, for example: remaining sample fluid that is not the nucleic acid of interest, contaminants, or other elements or molecules that may interfere with use of the extracted nucleic acid of interest. The second wash buffer may be introduced to the chamber after the first wash buffer has exited the chamber and the wash has been completed. The rinse may remove any remaining elements or contaminants not removed by the first wash buffer.

[0062] In some examples, the wash buffers 110 are used to suspend and / or resuspend the magnetic particles 116 within the extraction chamber 114. Additionally, in some examples, the wash buffers 110 may be used to transport the hybridized magnetic particles 116 from the extraction chamber 114 to the detection chamber 120. For example, a first wash buffer 110a is configured to deliver the hybridized magnetic particles to at least one of the one or more detection chambers 120 from the one or more extraction chambers 114.

[0063] A controller (e.g., the controller 164) of the instrument 150 may cause the wash buffers to enter the chambers 114 and 120. For example, the controller 164 may dispense a second wash buffer into the at least one of the one or more extraction chambers 114. The second wash buffer may wash the hybridized one or more magnetic particles of the plurality of magnetic particles. The controller 164 may also dispense the first wash buffer into the at least one of the one or more extraction chambers 114 to perform a first wash. The first wash may trap the hybridized one or more magnetic particles 116 of the plurality of magnetic parti clesl 16 and suspend the hybridized one or more magnetic particles 116 of the plurality of magnetic particles 116 within the first wash buffer 110a.

[0064] Upon use of the wash buffers 110, the buffers may be transported to a waste storage chamber 168. In some examples, the waste storage chamber 168 stores lysate after lysing and hybridization of the magnetic particles 116 has occurred in the extraction chamber 114. In some examples, a wash buffer may be stored in the waste storage chamber after the buffer has transported the hybridized magnetic particles 116 from the extraction chamber 114 to the detection chamber 120.

[0065] In some examples, the cartridge 100 may also include one or more reagents 125. The reagents 125 may be located in or proximate at least one of the one or more extraction chambers. For example, as shown in FIG. 1A, the one or more reagents 125 are positioned proximate the extraction chamber 114, however, it should be understood that the reagents 125Atty. Dkt. No.: 86357086may be stored in any chamber or zone and / or multiple chambers or zones of the cartridge 100. In various examples, the cartridge 100 may include only one reagent in only one chamber (e.g., the extraction chamber 114). In various other examples, no reagent 125 may be stored in any extraction chamber 114, but may be stored elsewhere in the cartridge 100. For example, the reagents may reside in any extraction or detection chambers, but may be stored elsewhere instead.

[0066] The one or more 125 may be or include liquid reagents 125 and / or dry reagents 125. The reagents 125 may be used in lysing, hybridization, washing, amplification, and / or detection of the nucleic acids of interest. The reagents 125 may also be or include a dry reagent storing an internal positive control (IPC) organism. The reagents 125 may be or include a master mix reagent. The master mix (MM) reagent may be or include a master mix lyophilized (“lyo”) bead. The master mix reagent may be used during amplification for reverse transcription and / or amplification and real-time detection of the nucleic acids of interest. In some examples, magnetic particles 116 may be included with the IPC, such that the magnetic particles 116 are delivered to the extraction chamber 114 prior to lysis, thereby eliminating a need to collect magnetic particles 116 and transport them to the extraction chamber 114 after lysis. The magnetic particles 116 are described in greater detail herein.

[0067] In various examples, liquid reagents 125 may be stored in blister devices (e.g., blister devices 126 of FIGS. 1A and IB) of the cartridge. For example, liquid reagents (e.g., wash buffer, system composition) may be stored in a metal-lined blister device. Blister devices are described in greater detail with respect to FIGS. 8A-9B. The one or more reagents 125 may be located in at least one of the one or more extraction chambers 114 or at least one of the one or more detection chambers 120.

[0068] In various examples, dry reagents 125 may be stored as lyophilized (e.g., freeze-dried) pellets or cakes, air-dried pellets or cakes, and / or sealed with a plastic plug or film. For example, a dry reagent may be an enzyme used for DNA or RNA elongation during amplification. The dry reagent can be or include master mix or a PCR mixture. The dry reagent 125 may be stored as a pellet. Dry reagents may be dissolved in order to be properly utilized. In various examples, a device may push against a piston to push a liquid out of a blister device to a location of a pellet to dissolve the pellet. The dissolved pellet may then be transported to a desired location (e.g., the detection chamber 120).Atty. Dkt. No.: 86357086

[0069] In some examples, the extraction chamber 114 includes a heating system. The extraction chamber 114 may be heated to a specific temperature to release the desired molecule of interest from the biological sample (e.g., the nucleic acid of interest). In some other examples, the heating extraction zone or chamber may be a serpentine channel where a fluid of interest having the biological sample is heated during the fluid passage. A serpentine channel may include a series of U-shaped channels that alternate in direction. A serpentine channel may increase a distance the fluid flowing through the channel travels. This may allow ample time to head the fluid to a target temperature.

[0070] In various examples, the biological sample can be lysed by a different lyse system, such as a lyse system 152, which may be located within the instrument 150. In various examples, the lysate can be lysed by a plurality of lysing techniques, such as a combination of the lyse system 152, mechanical agitation, an external heat source, ultrasonic agitation, impellers, and / or ceramic or glass beads. In various examples, the lyse system 152 may include a sonicator (shown in FIG. IB as sonicator 154) to interface with the one or more extraction chambers of the cartridge 100. The sonicator may be used for lysing. The sonicator 154 will be described in greater detail with respect to FIG. IB. The sonicator may also be referred to as a “sonotrode.”

[0071] The extraction chamber 114 may include a plurality of magnetic particles 116. The plurality of magnetic particles 116 may include one or more capture oligonucleotides that may be complementary to the one or more nucleic acids of interest. The capture oligonucleotides may be complementary to the nucleic acids of interest because the capture oligonucleotides may be configured and / or selected to bind specifically to (e.g., and only to) the one or more nucleic acids of interest. The extraction chamber 114 may house the one or more nucleic acids released from the lysed biological sample. In the extraction chamber 114, the one or more nucleic acids may be hybridized after lysing occurs. The one or more nucleic acids are hybridized to the plurality of magnetic particles 116. For example, in the extraction chamber 114, the one or more nucleic acids of interest may be hybridized with at least one capture oligonucleotide attached to one or more magnetic particles 116 of the plurality of magnetic particles. The one or more nucleic acids are hybridized to the plurality of magnetic particles by hybridizing the one or more nucleic acids of interest with at least one capture oligonucleotide attached to the one or more magnetic particles of the plurality of magnetic particles.

[0072] The term “hybridize” as used herein refers to a process where two substantially complementary nucleic acid strands (at least about 65% complementary over a stretch of atAtty. Dkt. No.: 86357086least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary) anneal to each other under appropriately stringent conditions to form a duplex or heteroduplex through formation of hydrogen bonds between complementary base pairs. For example, hybridization may refer to the formation of a double strand from two single strands, which can each include a nucleic acid and / or a capture oligonucleotide. The capture oligonucleotide may be, for example a DNA or RNA sequence having a complementary sequence to the nucleic acid of interest. Under suitable reaction conditions, the hybridization generally leads to the lowest possible energy state that can be achieved by the combination of the two single strands. In other words, under suitable conditions, the two single strands may bind to each other in such a way that, with respect to the sequences of the two single strands, the greatest possible complementarity (i.e., specificity) is produced.

[0073] In some examples, hybridizations are conducted with probe-length nucleic acid molecules, 15-100 nucleotides in length, or 18-50 nucleotides in length. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, and the thermal melting point I of the formed hybrid. The stringency of hybridization conditions may be estimated and / or adjusted such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not. In some examples, specific hybridization occurs under stringent hybridization conditions. An oligonucleotide or polynucleotide (e.g., a probe or a capture oligonucleotide) that is specific for a target nucleic acid will “hybridize” to the target nucleic acid under suitable conditions.

[0074] As stated, the cartridge 100 may include a plurality of the magnetic particles 116. Specifically, the extraction chamber 114 may include a plurality of the magnetic particles 116. The magnetic particles 116 may also be referred to as “magnetic beads,” “beads,” “paramagnetic beads,” “functionalized beads,” and / or “particles.” As used herein, “magnetic beads” or “magnetic particles” refer to microparticles that have ferromagnetic or paramagnetic properties. For example, the magnetic particles 116 may be or include (strept)avidin with biotinylated oligonucleotides, covalently bound oligonucleotides, polymer beads with embedded iron particles, etc. The size of the microparticles is optionally in a range from approximately 10 nm to approximately 2 mm, optionally in a range from 100 nm to 1 mm, optionally in a range from 500 nm to 50 pm. The shape of the microparticles can be freelyAtty. Dkt. No.: 86357086selected and can, for example, be spherical, cube-shaped, cuboid, or ellipsoidal. The magnetic particles with ferromagnetic properties are optionally formed from at least one of the following materials or contain at least one of the following materials: iron, nickel, cobalt, AlNiCo, SmCo, Nd2Fei4B, NieoFe2o (“Permalloy”), and / or NiFeCo alloys. Optionally, the magnetic particles with paramagnetic properties are made of at least one of the following materials formed or contain at least one of the following materials: alkaline earth metals, alkali metals, and / or rare earths. Alternatively, a magnetic microparticle can be formed from a non-magnetic material such as glass and / or silicate, with magnetic substances being embedded therein. For example, such a microparticle can have a core made of magnetic materials. The magnetic microparticles are optionally provided with at least one coating in order to enable or promote functionalization with nucleic acids, in particular with extraction nucleic acids and / or capture oligonucleotides. Optionally, at least one extraction nucleic acid and / or one capture oligonucleotides and a maximum of 1012extraction nucleic acids and / or capture oligonucleotides are functionalized on a microparticle. Optionally, an areal density of extraction nucleic acids and / or capture oligonucleotides that are functionalized on the surface of a magnetic microparticle is in a range from 0.0001 to 1 per square nanometer. The microparticles can optionally have a coating which enables and / or facilitates functionalization with capture oligonucleotides. For example, the surface of the magnetic microparticles can be at least partially functionalized with streptavidin.

[0075] The magnetic particles 116 may have a nucleic acid attached (e.g., once hybridization occurs). Specifically, the attached nucleic acids may be attached to the magnetic particles 116 by capture oligonucleotides that are attached to the magnetic particles 116. The capture oligonucleotides attached to the magnetic particles 116 are selected to be complementary in part or in full to the one or more nucleic acids of interest. In the extraction chamber 114, the one or more nucleic acids may be hybridized to the capture oligonucleotides attached to the magnetic particles 116. In some examples, the magnetic particles 116 comprise one or more capture oligonucleotides that bind to the one or more nucleic acids of interest (e.g., from the extraction chamber 114). In various examples, the capture oligonucleotides may be the same or different from each other. For example, a first capture oligonucleotides attached to a first magnetic particle 116 may have a first sequence, and a second capture oligonucleotides attached to a second magnetic particle 116 may have a second sequence.

[0076] In various examples, the extraction chamber 114 houses both lysis and hybridization of the magnetic particles with the nucleic acids of interest occurs. During hybridization, the fluidAtty. Dkt. No.: 86357086containing the biological sample may be mixed (e.g., partial mixing, complete mixing, etc.). Mixing the fluid may occur to distribute the magnetic particles 116 throughout the fluid (e.g., the lysate). Mixing may additionally or alternatively occur using one or more of ultrasonic mixing, acoustic mixing, impellers, mechanical agitation, diffusion, and / or channel geometry, such as turns, ridges, and / or mini chambers.

[0077] During hybridization, the fluid may also be heated. The solution may be heated in a variety of ways, such as using an external contact heat source, ultrasonic energy, acoustic energy, and / or infrared radiation (IR).

[0078] The detection zone 108 may be, in various examples, a detection zone including one or more detection chambers 120. In various examples, a different zone may include the one or more detection chambers. The detection chambers 120 may not be a part of or associated with a zone.

[0079] The cartridge 100 may include one or more detection chambers 120. The detection chambers 120 may be configured to amplify and detect the one or more nucleic acids of interest that have been lysed (e.g., from the biological sample) and hybridized (e.g., to the functionalized magnetic particles 116). Thus, in various examples, at least one of the one or more detection chambers 120 is an amplification chamber. The detection chambers 120 may amplify the nucleic acid of interest through, for example, pulse controlled amplification (PC A). PCA is described in greater detail with respect to FIG. 7.

[0080] As used herein, the terms “amplify” or “amplification” with respect to nucleic acid sequences, refer to methods that increase the representation of a population of nucleic acid sequences in a sample. Copies of a particular target nucleic acid sequence generated in vitro in an amplification reaction may be referred to as “amplicons” or “amplification products.” In various examples, amplification products may refer to any products amplified during an amplification reaction. For example, amplification products may include nucleic acids, unquenched fluorophores generated during each replication cycle of the amplification reaction, etc. Amplification may be exponential or linear. A target nucleic acid may be DNA (such as, for example, genomic DNA and complementary DNA (cDNA) or RNA). While the methods described hereinafter relate to amplification using polymerase chain reaction (PCR), numerous other methods such as isothermal methods, rolling circle methods, etc., may be used either in place of, or together with, PCR methods. In at least one of the detection chambers 120, aAtty. Dkt. No.: 86357086lyophilized master mix reagent may be stored. The master mix reagent may refer to a reagent used during an amplification reaction, such as a PCR or PCA reaction.

[0081] In various examples, the amplification reactions may utilize primers to perform amplification of the nucleic acids. Primers are short, artificial, single-stranded segments of nucleic acid (e.g., DNA) that are designed to be complementary to the beginning and / or end of the target sequence that will be amplified. The primer sequences may be shorter than the one of the capture oligonucleotides. For example, a primer may contain about 10 to 25 nucleotides.

[0082] Primers may perform specific functions during amplification. For example, during the amplification / elongation step of the PCR, the primers may bind to both ends of the nucleic acid of interest (e.g., the DNA sequence of interest), thus bookending the sequence of interest that need to be amplified. Enzymes (e.g., DNA polymerase) may then copy the part of the target oligonucleotide sequence that falls between the primers, selectively amplifying the sequence of interest. In some examples, the capture oligonucleotides described above may also be used as primers during amplification. In other examples, the primers may be oligonucleotides different than the capture oligonucleotides. In some examples, the primers may be forward and / or reverse primers. Forward and reverse primers may denote a direction of elongation during the polymerization by the polymerase enzyme. The primers may be used during the amplification / elongation step of the reaction and may comprise part of the master mix composition described herein.

[0083] The detection chamber 120 may include a heating element 122. For example, a detection zone of a plurality of zones may include one or more detection chambers 120 having one or more heating elements 122. Each detection chamber 120 may include a heating element 122. In various examples, at least one of the one or more detection chambers may be an amplification chamber. Further, the heating element of the amplification chamber may be a foil configured to interact with the electrical components 160 of the instrument 150. The electrical components 160 may provide an electrical connection between the instrument 150 and the detection chamber 120, thus providing heat modulation to the detection chamber 120 to amplify the one or more nucleic acids of interest that have been hybridized to the capture oligonucleotides of the plurality of magnetic particles. Specifically, the electrical connection may activate the one or more heating elements 122 of each of the one or more detection chambers 120.Atty. Dkt. No.: 86357086

[0084] The heating element 122 may be positioned or located at a side portion (e.g., only a single side, such as a single wall of the detection chamber 120) of the detection chamber 120. In some examples, the heating element 122 may heat the entirety of the detection zone 108. For example, the detection zone 108 may include a plurality of detection chambers 120, and a single heating element 122 may heat all of the plurality of detection chambers 120. The heating element 122 may include a plurality of layers comprising at least a foil. The heating element 122 may be attached to one or more of an adhesive and / or a heat spreader or conductor. The heating element 122 may be or include, in various examples, a continuous, structured, or shaped metal foil, metal wires, a conductor and / or resistor layer deposited and / or plated, and / or backed by a heat spreader. The heating element 122 may be configured to heat the detection chamber 120 during the PCA process. The heating element 122 may be, for example, a foil to locally heat a portion of the detection chamber 120. For example, a heating element 122 of the amplification chamber (e.g., the detection chamber 120) may be or include a foil to interact with electrical components 160 of the instrument 150 provide an electrical connection to activate the one or more heating elements 122 of each of the one or more detection chambers 120. The heating element 122 may provide heat modulation to amplify the one or more nucleic acids of interest that have been hybridized to the at least one capture oligonucleotide attached to one or more magnetic particles 116 of the plurality of magnetic particles in the extraction chamber 114. The detection chamber 120 is described in greater detail with respect to FIGS.5 A and 5B.

[0085] During the PCA reaction occurring in the detection chamber 120, the heating element 122 may be used to heat a portion of the volume of the detection chamber 120. For example, only a portion of the detection chamber 120 proximate the heating element 122 may be heated. The heating element 122 may be heated by a short electric pulse or other process. Each pulse may be short (e.g., between about 10 and about 1200 microseconds) and each pulse may be delivered quickly in succession. In some examples, the heating time in at least one amplification cycle, in at least 10, at least 20, at least 40, at least 80, or in at least 160 amplification cycles is more than 1 nanosecond, more than 5 nanoseconds, or more than 10 nanoseconds and less than 100 milliseconds, less than 10 milliseconds, less than 1 millisecond, less than 300 microseconds, less than 100 microseconds, less than 50 microseconds, less than 30 microseconds, less than 10 microseconds, less than 5 microseconds, or less than 1.5 microseconds.Atty. Dkt. No.: 86357086

[0086] Due to a resistance of the foil and the rapid pulsing, heat created by the pulses may be local (e.g., the pulses heat only a small portion of the detection chamber 120 surrounding the heating element). In various examples, the pulses may be generated by an energy or power source. When the pulses are ceased or removed, the portion of the detection chamber 120 that has been heated by the pulses may return to the same temperature as the rest of the detection chamber not proximate the heating element 122. As such, denaturation of the nucleic acids or other molecules occurs in a small volume near the heating element 122.

[0087] Denaturation of a nucleic acid may include separate it into its two single strands. For example, the original can be separated from the complement during denaturing. Denaturing may also be referred to as melting. The denaturing of the nucleic acid double strand may be thermally induced. For example, at least a part of the nucleic acid double strand or the whole double strand is exposed to a temperature, described as a denaturing temperature, which causes or at least encourages a separation of the nucleic acid double strands. The denaturing temperature may not be a fixed temperature but may be a temperature interval, within which the temperature during denaturing varies. The denaturing temperature may be selected to be so high that nucleic acid double strands can be separated and / or so low that a DNA polymerase, which may aid in denaturation, is not substantially damaged. In some examples, a denaturation temperature may be between 90 and 100 degrees Celsius. For example, a denaturing temperature may be 95 degrees Celsius.

[0088] This heating process may cause the overall temperature of the detection chamber 120 to be isothermal (e.g., no change, substantially no change, or minimal change in temperature is seen during heating for the PCA reaction), because the heated portion of the detection chamber 120 is small enough that an overall temperature change is not seen by the detection chamber 120. In various implementations, heat flow in and out of the detection chamber 120 may be present. The heating element 122 may cause a slight (e.g., less than 1 degree Celsius) increase in the overall temperature of the detection chamber 120. For example, heat may be unable to be transferred out of the detection chamber 120, and each pulse generated by the heating element 122 may add heat to the chamber. However, the pulse may heat a small enough volume of the detection chamber 120 relative to the total volume of the detection chamber 120 such that the volume heated by the heating element 122 can rapidly return to a setpoint temperature of the detection chamber 120.Atty. Dkt. No.: 86357086

[0089] Referring still to FIG. 1A, the instrument 150 is shown, according to an example implementation. The diagnostic system 100A may include the instrument 150 to interface with (e.g., connect to) the cartridge 100. The cartridge 100 may be inserted into the instrument 150 to perform the processes described herein. In various examples, the cartridge 100 may be a single use cartridge. For example, one cartridge may be used one time to detect the presence of nucleic acids in one biological sample.

[0090] The instrument 150 may include a lyse system 152. The lyse system 152 may interface with at least one of the one or more extraction chambers 114 of the cartridge 100. The lyse system 152 may include a sonicator 154 (shown in FIG. IB). The sonicator 154 may interface with the at least one extraction chamber 114. The lyse system 152 may be utilized when the biological sample enters the extraction chamber 114 of the cartridge 100 to lyse the sample and release the nucleic acids of interest. The lyse system 152 may be coupled to the extraction chamber 114.

[0091] In various examples, the lyse system 152 may be a lyse system comprising sonication (e.g., use of ultrasonic energy), thermal lysis, and / or thermal sonication system. In some examples, the lyse system 152 may include a heating system. In examples where the lyse system 152 includes a heating system, the lyse system 152 may heat the extraction chamber 114 to a target temperature (e.g., between 35 and 100 degrees Celsius), such as by heating the heating element of the extraction chamber 114 to the target temperature, to release the desired molecule of interest (e.g., nucleic acid) from the biological sample. For example, the lyse system 152 may heat the extraction chamber 114 to 95 degrees Celsius.

[0092] In some examples, the lyse system 152 is programmable. Thus, a user may be able to control, set, determine, etc. lysing protocol parameters (e.g., using the controller 164), such as the sample volume, sonication power level, acoustic frequency, and lysing duration. The lyse system 152 may also provide a cooling feature, enabled by a heat exchanging sub-assembly, which may prevent the biological sample from exceeding a maximum set temperature during operation.

[0093] The instrument 150 may include a plurality of magnetic field generators 156a and 156b to generate a magnetic field. The magnetic field may be used to dock the plurality of magnetic particles 116 to at least one zone of the plurality of zones of the cartridge 100. In various examples, the magnetic field generators 156 are magnets (e.g., a permanent magnet). TheAtty. Dkt. No.: 86357086magnetic field generators 156 may be positioned at or proximate the detection chamber 120 and / or the extraction chamber 114. The magnetic field generator may be configured to generate a magnetic field such that the magnetic particles 116 that are hybridized with the nucleic acid of interest and / or the capture oligonucleotides are separated from the lysate solution such that the PCA reaction can occur. The magnetic field generators 156 may be a current carrying conductor or other device that may be powered on or activated such that electric charges being moving to create the magnetic field. In various examples, the instrument 150 may be activated or powered on to activate the magnetic field generator.

[0094] The magnetic field generator 156a may generate a magnetic field to dock the plurality of magnetic particles to at least one of the one or more extraction chambers. For example, after lysis, the lysate may be transferred to the waste storage chamber 168. During transport of the lysate, the magnetic field generator 156a may generate a magnet field that traps the magnetic particles 116 to a side of wall of the extraction chamber 114. The magnetic field generator 156b may force the plurality of magnetic particles towards the one or more heating elements 122, capture the plurality of magnetic particles onto the one or more heating elements 122, cause the plurality of magnetic particles to attach to the heating element 122, cause the plurality of magnetic particles to be located within a target distance of the heating element 122, etc.). For example, the magnetic field generator 156b may generate a magnetic field that docks the magnetic particles 116 to the heating element 122.

[0095] In some examples, the magnetic field generator 156 is configured to generate a variable magnetic field in such a manner that acts on at least a part of the magnetic particles 116 present in the detection chamber and that are linked to the nucleic acid of interest.

[0096] In another example, the variable magnetic field may act on at least a part of the magnetic particles 116 present in the detection chamber 120 in such a manner that the magnetic particles 116 attach to the local heating element 122. Further, the variable magnetic field may be configured to act on the magnetic particles 116 attached to the local heating element 122 in such a manner that they leave the local heating element 122 and are suspended in a reaction solution. This may allow the magnetic microparticles 116 to be optionally attached to and / or repelled from the local heating element 122 multiple times, allowing them to hybridize with additional target nucleic acids in the reaction solution.Atty. Dkt. No.: 86357086

[0097] The magnet or plurality of magnets may be or include a permanent magnet and / or an electromagnet that can be changed in position and / or orientation relative to the reaction container. For example, when using a permanent magnet, the magnetic field may be changed by changing an orientation of the permanent magnet to the extraction chamber 114 and / or detection chamber 120 and / or a distance of the permanent magnet from the extraction chamber 114 and / or detection chamber 120. The direction of the magnetic field can also be changed, for example, by reversing the permanent magnet relative to the reaction container such that, for example, the side of the permanent magnet facing the reaction container changes from the magnetic north pole to the south pole of the permanent magnet or vice versa. When using an electromagnet, for example, the variable magnetic field can be changed by changing the current flow, in such a manner as the current intensity and / or the direction of the current flow. For example, the electromagnet may comprise one or more solenoid coils and optionally a ferromagnetic core. The magnet may be formed on a side of the local heating element 122 facing away from the detection chamber 120. This may offer the advantage that the permanent magnet in this arrangement makes it particularly effective and easy to attract the magnetic microparticles 116 to the local heating element. Alternatively or additionally, the one or more magnets may be changed in position relative to the detection chamber to provide a variable magnetic field in the reaction solution. Alternatively or additionally, several magnets with different polarity can be brought to the detection chamber to achieve a variable magnetic field in the detection chamber.

[0098] The instrument 150 may include electrical components 160. The electrical components 160 may provide an electrical connection between the instrument 150 and one or more zones, chambers, components, etc. of the cartridge 100. For example, the electrical components 160 may provide an electrical connection between the instrument 150 and the cartridge 100 to activate the one or more heating elements 122 of each of the one or more detection chambers 120. An interaction of the detection chamber 120 and the electrical components 160, via an electrical connection, may provide heat modulation to amplify the one or more nucleic acids of interest that have been hybridized to the capture oligonucleotides of the plurality of magnetic particles 116. The electrical components may provide a connection to the heating element 122 in the detection chamber.

[0099] The instrument 150 may include an optical unit 162. The optical unit 162 may be coupled with at least one of the one or more detection chambers. The optical unit 162 may beAtty. Dkt. No.: 86357086configured to detect a presence, absence, amount, etc. of the nucleic acid of interest after amplification has been performed. In various examples, the optical unit 162 is to detect a plurality of amplification products indicative of a presence, absence, or amount of the amplified one or more nucleic acids of interest. The optical unit 162 may detect the nucleic acid via a label bound to the nucleic acid of interest during amplification.

[0100] In various examples, the cartridge 100 may include a transparent window in at least one detection chamber (e.g., the detection chamber 120) of the one or more detection chambers to allow the optical unit 162 to detect a plurality of amplification products indicative of a presence, absence, or amount of the plurality of amplified nucleic acids of interest. In various examples, the detection chamber 120 may include the transparent window. A transparent window may permit light from the optical unit 162 to pass through the cartridge 100 and / or detection chamber 120 so that light can enter the detection chamber 120 and detect the amplification products indicative of a presence, absence, or amount of the plurality of amplified nucleic acids of interest.

[0101] In some examples, detection by the optical unit 162 may be performed in the detection chamber 120 and may be done using a label bound to the nucleic of interest. Specifically, the optical unit 162 may be used for fluorescence sensing. For example fluorophores may be generated during each replication cycle of the amplification reaction. Fluorophores may be used to infer the presence or amount of nucleic acids in the detection chamber 120. In PCA (and / or PCR), fluorophores may be attached to capture oligonucleotides and a quencher molecule prior to amplification. This compound molecule may be included in the master mix reagent. In the proximity of the quencher, the compound molecule may not fluoresce. However, during PCA (and / or PCR), the compound molecule may anneal or attach to a portion of the target nucleic acids. A polymerase enzyme, while constructing a double stranded amplicon, may release the fluorophore to separate the fluorophore from the quencher and allow the fluorophore to fluoresce.

[0102] In some examples, the detection step may include detecting the fluorophore via a sensor located within the optical unit 162. For example, the optical unit 162 and any sensors located within may be positioned in the instrument 150 such that the optical unit 162 and / or the sensors interface with and / or are located proximate the detection chamber 120 when the cartridge 100 is inserted into the instrument 150. In some examples, the sensor may be a fluorescence sensor coupled to the optical unit 162 and configured to detect the fluorescenceAtty. Dkt. No.: 86357086from one or more of the plurality of zones of the cartridge 100 (e.g., the detection zone 108, within the detection chamber 120, etc.).

[0103] In some examples, the optical unit 162 may detect the nucleic acid of interest by detecting an intensity of fluorescent probes in the detection chamber 120. The detection chamber 120 may be configured to transmit fluorescent emission to a sensor within the optical unit 162 located in the instrument. For example, during amplification, fluorophores may be released (e.g., using Taqman probes) into solution during elongation for specific detection of amplified products. The optical unit 162 may detect the fluorophores that may be indicative of the presence, absence, amount, etc. of the nucleic acid of interest.

[0104] In some cases, the diagnostic system 100A may further include a controller 164 (e.g., a computer including a processor and memory configured to control various aspects of the diagnostic system 100A). For example, the instrument 150 may, in some implementations, include the controller 164. The controller 164 may facilitate execution of instructions that cause the processor to perform one or more actions. For example, the memory of the controller 164 may store instructions that cause the processor to perform various actions. For example, the instructions may cause the processors to initiate actions (e.g., actuating valves, etc.) that cause the biological sample and / or nucleic acids of interest to be lysed, hybridized, transported, amplified, etc.

[0105] For example, the controller may include one or more non-transitory computer-readable storage media storing instructions thereon that, when executed by one or more processors, cause the one or more processors to perform operations. For example, the controller may lyse a biological sample into at least one of the extraction chambers 114 to release one or more nucleic acids of interest from the biological sample. As described, lysing the biological sample produces a lysate. The instructions may cause the processors to hybridize, into the at least one extraction chamber 114, the one or more nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles 116.

[0106] The instructions of the controller 164 may also cause the processors dispense a first wash buffer 110a into the at least one of the one or more extraction chambers 114. As described, the first wash buffer may wash the hybridized one or more particles of the plurality of magnetic particles 116. The instructions of the controller 164 may cause the one or more processors to:Atty. Dkt. No.: 86357086dispense a second wash buffer 110b into the at least one of the one or more extraction chambers 114 to perform a second wash. The second wash may trap the hybridized one or more magnetic particles of the plurality of magnetic particles 116 and suspend the hybridized one or more magnetic particles of the plurality of magnetic particles 116 within the second wash buffer 110 (e.g., in the extraction chamber 114). The instructions of the controller 164 may cause the processors to transfer the hybridized one or more magnetic particles 116 to at least one detection chamber 120 of the one or more detection chambers 120 via the second wash buffer 110b. In some examples, the controller 164 instructions cause the one or more processors to lyse the biological sample via the sonotrode (e.g., the sonicator 154).

[0107] Referring now to FIG. IB, a diagnostic system 100B is shown, according to an example implementation. The system 100B includes a cartridge 100 and an instrument 150. The cartridge 100 and the instrument 150 may include the same or similar elements as the elements found in the cartridge and instrument of the system 100A. For example, the extraction chamber 114, and the detection chamber 120 may be the same as those described with respect to FIG.1A.

[0108] As described above with respect to FIG. 1 A, the cartridge 100 of the diagnostic system 100B includes the extraction chamber 114, the detection chamber 120, the first wash buffer 110a, the second wash buffer 110b, the plurality of blister devices 126a-b (individually described as blister device 126, and together described as blister devices 126), and the waste storage chamber 168. The instrument 150 includes the lyse system 152 having a sonicator 154 and the magnetic field generators 156a and 156b.

[0109] The cartridge 100 can include a sample input chamber. The sample input chamber may be a chamber or cavity in which a biological sample can be inserted so that the biological sample enters the cartridge 100 and can be analyzed. For example, the cartridge 100 may be configured to receive a biological sample. The sample input chamber may include a liquid port for receiving liquids. The biological sample may be, for example, saliva, blood, cells, etc. In various examples, the biological sample may include one or more nucleic acids. Of the one or more nucleic acids, the biological sample may include one or more nucleic acids of interest. The nucleic acids of interest may be nucleic acids that the cartridge 100 is being used to amplify and analyze. In various examples, the nucleic acids of interest may not be included in a biological sample. For example, the nucleic acids of interest may be isolated and input into the sample input chamber. The nucleic acids of interest may be, for example, DNA, RNA, mRNA,Atty. Dkt. No.: 86357086etc. The one or more nucleic acids of interest may be the same targets or different targets (e.g., the cartridge 100 can be used to detect and / or analyze one or more of the same type of nucleic acid of interest or different nucleic acids of interest). For example, the diagnostic system 100A of 100B may be configured to amplify multiple nucleic acids of interest to, for example, detect multiple infections, diseases, etc. Amplification and identification of these multiple nucleic acids may be performed simultaneously, thus reducing an amount of time and cost of detecting the presence of multiple nucleic acids. The sample input chamber is shown and described in greater detail with respect to FIGS. 12A-D.

[0110] The cartridge 100 may also include a plurality of zones, shown as a extraction zone 104 and a detection zone 108. Each zone may be or include a plurality of chambers. For example, the extraction zone 104 may include one or more extraction chambers 114 and the detection zone 108 may include one or more detection chambers 120. Each zone of the plurality of zones may be in fluid communication with each other (e.g., the other zones of the plurality of zones). It should be understood that the cartridge 100 may include any number of zones. In various examples, each zone is configured to perform or is associated with certain actions. For example, the extraction zone 104 may be configured to receive and / or house a system composition. Further, the extraction zone 104 may be configured to deliver the system composition to various other components and / or zones of the cartridge 100 to rinse or wash the materials from the respective zones. In various examples, the plurality of zones may include an extraction zone and a detection zone. Further, each zone may include one or more chambers. For example, the plurality of zones may include an extraction zone having one or more extraction chambers and a detection zone having one or more detection chambers.[OHl] The system composition may be a buffer or composition useful for lysis and hybridization of a sample (e.g., of the biological sample). The system composition may also be referred to as a “system buffer.” The system composition may include ingredients (e.g., a buffer) to control a pH of the solution. In some examples, the system composition can comprise water, a salt and / or a surfactant. In some examples, the salt is MgCh, NaCl, KC1, or (NHFESCU In some examples, the system composition comprises between 50mM and IM salt. In some examples, the system composition comprises between about 0.001% and about 0.1% (e.g., about 0.001%, 0.01%, 0.05%, or 0.1%) surfactant. In some examples, the surfactant is selected from Tween® 20 (polysorbate 20), Tween® 80 (polysorbate 80), Tween® 85 (polysorbate 85), SPAN® 80 (sorbitan monooleate) or SPAN® 85 (sorbitane trioleate). In some examples, theAtty. Dkt. No.: 86357086surfactant is Tween® 20. The system composition may also include a buffering component to buffer the pH of the solution. For example, the buffering component may be a tris buffer.

[0112] Each zone may include one or more chambers configured to perform specific actions. As shown in FIG. IB, the extraction zone 104 may include the extraction chamber 114 and the detection zone may include the detection chamber 120. In various examples, each zone may include different types of chambers, different numbers of chambers, different configurations of chambers, etc. In addition to or alternative to chambers, each zone of the plurality of zones may include elements such as blister devices storing solutions (e.g., reagents, buffer solutions, etc.), bubble traps, seals, conduits through which fluid flows from one zone to one or more other zones.

[0113] In various examples, the biological sample may be lysed with the system composition and an internal positive control (IPC). The resulting solution of the biological sample, the system composition, and the IPC may be referred to as a lysate. The IPC may be included in the lysate to control false negative results. During amplification, the IPC may be amplified with the nucleic acids of interest to indicate that the solution being amplified is functional and a negative result (e.g., the nucleic acids of interest are not detected) is reliable. The IPC may be a lyophilized organism or synthetic organism. In various examples, the IPC may be a protein or organism present in a human sample. Further, the extraction chamber 114 may include a plurality of beads (e.g., glass beads, ceramic beads) used to mechanically agitate or lyse the organisms present in the lysate.

[0114] During hybridization, the fluid may also be heated. The solution may be heated in a variety of ways, such as using an external contact heat source, ultrasonic energy, acoustic energy, and / or infrared radiation (IR).

[0115] The detection zone 108 may include one or more detection chambers 120. In various examples, a different zone may include the one or more detection chambers (e.g., the detection zone 108). The detection chambers 120 may not be a part of or associated with a zone.

[0116] The cartridge 100 may include one or more detection chambers 120. The detection chambers 120 may be configured to amplify and detect the one or more nucleic acids of interest that have been lysed (e.g., from the biological sample) and hybridized (e.g., to the functionalized magnetic particles 116). Thus, in various examples, at least one of the one or more detection chambers 120 is an amplification chamber. The detection chambers 120 mayAtty. Dkt. No.: 86357086amplify the nucleic acid of interest through, for example, pulse controlled amplification (PCA). PCA is described in greater detail with respect to FIG. 7.

[0117] Referring still to FIG. IB, the instrument 150 is shown, according to example implementations. The diagnostic system 100A may include the instrument 150 to interface with (e.g., connect to) the cartridge 100. The components of the instrument 150 may be positioned such that specific components are located proximate to specific corresponding components of the cartridge 100 when inserted into the instrument 150. The cartridge 100 may be inserted into the instrument 150 to perform the processes described herein. In various examples, the cartridge 100 may be a single use cartridge. For example, one cartridge may be used one time to detect the presence of nucleic acids in one biological sample.

[0118] The instrument 150 of FIG. IB may include the lyse system 152, the magnetic field generator 156a, the magnetic field generator 156b, and the controller 164 described above with respect to FIG. 1 A.

[0119] As stated above, the lyse system 152 may include a sonicator 154. The sonicator 154 may interface with the at least one extraction chamber 114 (e.g., the extraction chamber 114). The sonicator 154 may deliver ultrasonic waves to the extraction chamber 114 to lyse the fluid present in the chamber. In some examples, the biological sample may be lysed using a sonication system, by using a sonotrode (e.g., the sonicator 154). The lyse system 152 of the instrument 150 may therefore include an ultrasonic transducer or sonicator 154 that transmits ultrasonic energy to the extraction chamber (e.g., the extraction chamber 114) into the biological sample to cause ccll / sporc / tissuc disruption. Efficient transfer of the ultrasonic energy from the sonicator 154 to the sample within the extraction chamber 114 may be dependent, at least in part, upon maintaining the contact between the transducer tip of the sonicator 154 present in the instrument and the cartridge according to a predetermined force.

[0120] Referring now to FIG. 2, a method 200 for detecting a presence, absence, or amount of a nucleic acid of interest is shown, according to an example.

[0121] At process 202, a fluid comprising a biological sample is inserted into a cartridge (e.g., the cartridge 100). In various examples, the biological sample may include the nucleic acid of interest. In various examples, the biological sample may contain another non-nucleic acid molecule of interest. In various examples, the nucleic acid of interest may be a DNA strand and / or an RNA strand. The cartridge may include a plurality of zones. Each zone of the pluralityAtty. Dkt. No.: 86357086of zones may be in fluid communication with each other. For example, the plurality of zones may be or include an extraction zone and / or a detection zone in fluid communication with one another. The extraction zone may include one or more lyse chambers (e.g., extraction chambers 114) and the detection zone may include one or more detection chambers 120. In various examples, each detection chamber 120 may include one or more heating elements 122. The cartridge may further include one or more reagents 125 and a plurality of magnetic particles 116.

[0122] At process 204, the biological sample is lysed into at least one of the one or more extraction chambers (e.g., extraction chamber 114). Lysing the biological sample may release the one or more nucleic acids of interest from the biological sample. Lysing the biological sample produces a lysate. In various examples, the biological sample may be lysed by, for example, sonication (e.g., using the sonicator 154), heating, mechanical agitation, etc. In various examples, the biological sample may be mixed with an IPC and / or a system composition to form the lysate, which may then be lysed to release the nucleic acids of interest from the biological sample. For example, the method can include lysing in the device a biological sample can include the one or more nucleic acids of interest to release the one or more nucleic acids of interest from the biological sample. For example, the biological sample is lysed by performing one or more of sonication, thermal lysis, or thermal sonication.

[0123] At process 206, the one or more nucleic acids of interest are hybridized into the at least one extraction chamber 114 of the one or more extraction chambers 114. The one or more nucleic acids of interest may be hybridized to or with at least one capture oligonucleotide attached to one or more magnetic particles 116 of the plurality of magnetic particles 116. For example, the extraction chamber 114 may include a plurality of magnetic particles 116. The magnetic particles 116 may include attached capture oligonucleotides. The nucleic acids of interest may be hybridized to the capture oligonucleotides.

[0124] At process 208, the hybridized one or more magnetic particles (e.g., the one or more nucleic acids of interest may be hybridized to or with at least one capture oligonucleotide attached to one or more magnetic particles 116) are held on a side of the extraction chamber. For example, the hybridized one or more magnetic particles are held to the side of the extraction chamber via or in an applied magnetic field generated by the magnetic field generator 156a.Atty. Dkt. No.: 86357086

[0125] After lysis at process 204 and / or hybridization at process 206, hybridization, the lysate may be transported to a waste storage chamber (e.g., the waste storage chamber 168).

[0126] At process 210, the hybridized one or more magnetic particles are washed with at least one wash buffer. Washing may be performed by introducing the wash buffer 110 into the at least one detection chamber 120. For example, the wash buffer 110 may be released from a blister device 126 and may be transported to the detection chamber 120. A plurality of wash buffers may be used to wash the detection chamber 120.

[0127] The method 200 may further include suspending the hybridized one or more magnetic particles. The hybridized one or more magnetic particles may be suspended within the extraction chamber 114 via a wash buffer. The method 200 may further include transporting the hybridized one or more magnetic particles to at least one detection chamber of the one or more detection chambers.

[0128] The method 200 may include delivering a plurality of amplification reagents to the at least one detection chamber. In various examples, the plurality of amplification reagents 125 may include, for example, a lyophilized master mix reagent, an enzyme for use in amplification, etc. The amplification reagents 125 may be located in various locations of the cartridge (e.g., in blister devices 126) and may be transported to the detection chamber prior to amplification. In various examples, the amplification reagents may be stored as dry or wet reagents. Dry reagents may be dissolved prior to use.

[0129] The method 200 may include transporting the hybridized one or more magnetic particles to the at least one detection chamber via the at least one wash buffer. The nucleic acids of interest may be transported, for example, via one or more channels within the cartridge 100. The hybridized one or more magnetic particles 116 are delivered to the at least one detection chamber 120 via the at least one wash buffer 110 such that the lysate does not interface with the at least one detection chamber 120.

[0130] At process 212, the hybridized one or more magnetic particles are trapped into proximity of the one or more heating elements (e.g., heating element 122) of the at least one detection chamber (e.g., the detection chamber 120). In various examples, the detection chamber 120 may be locally heated at or around the heating element 122 to perform amplification. As such, the magnetic particles containing the nucleic acids of interest may beAtty. Dkt. No.: 86357086trapped near the heating element 122 so that the nucleic acids of interest can undergo the amplification reaction to be identified by the optical unit 162.

[0131] At process 214, the one or more nucleic acids of interest may be amplified in at least one of the plurality of zones. The one or more nucleic acids of interest may be amplified via an amplification reaction. The amplification reaction may provide a plurality of the one or more nucleic acids of interest.

[0132] In various examples, amplifying the one or more nucleic acids of interest may be performed in the at least one detection chamber 120 by generating a pulse of current via an electrical connection of an instrument 150. The instrument 150 may receive and interact with the cartridge 100. The pulse of current may modulate a temperature proximate the one or more heating elements 122 of the at least one detection chamber 120 to increase a temperature proximate the one or more heating elements of the at least one detection chamber 120 to between 90 and 110 degrees Celsius. For example, the temperature may be increased to 100 degrees Celsius. In various examples, only a portion of the detection chamber may be heated by the pulses. In various examples, amplification may be performed by a PCA reaction. At process 216, a plurality of amplification products indicative of the presence, absence, or amount of the plurality of amplified nucleic acids of interest may be detected. The amplification products may be detected via an optical unit 162 in communication with the at least one detection chamber 120 of the one or more detection chambers 120. For example, during amplification, the plurality of nucleic acids of interest may be tagged, for example, using fluorophores. The optical unit 162 may detect an amount of fluorophores, which may be indicative of a presence, absence, and / or amount of the nucleic acid of interest in the biological sample.

[0133] Referring now to FIG. 3A, a side view of an example cartridge 300A device is shown, in accordance with present implementations. The cartridge 300A may be the same as or similar to the cartridge 100 of FIGS. 1A and IB. The side view of the cartridge 300A may show a side of the cartridge that interfaces with the instrument 150. As shown, the cartridge 300A may include a sample input cover 303, a sample input port 305, an extraction chamber 114, magnetic particles 116, a waste storage chamber 168, an overflow chamber 366, and a detection chamber 120.Atty. Dkt. No.: 86357086

[0134] The sample input port 305 may be a port or cavity coupled with a sample input chamber. The sample input port 305 may receive the biological sample and direct the sample into the sample input chamber.

[0135] The sample input cover 303 may be a cover that interfaces with the sample input port 305. The sample input cover 303 may seal the sample input chamber by preventing liquid from exiting the sample input chamber through the sample input port 305. The sample input chamber may receive a biological sample or other fluid containing one or more nucleic acids of interest. Thus, the cartridge 300 A may include one or more sample fill indicators to indicate a fill level of the sample input chamber. The sample fill indicators may be lines, tick marks, or other visual indicators of a volume of liquid or fluid in the sample input chamber. The sample input chamber may include a transparent material to view a volume of fluid in the sample input chamber. In some examples, the sample input cover 303 may be a cap (e.g., as shown in FIGS 3A and 3B). The cap may be hinged to flip or rotate to open and close, thereby covering and uncovering the sample input port 305. In some examples, the sample input cover 303 may be configured as a slider to slide along an axis to cover and uncover the sample input port 305.

[0136] Referring now to FIG. 3B, a side view of an example cartridge 300B is shown, in accordance with present implementations. The cartridge 300B may be the same as or similar to the cartridge 100 of FIGS. 1A and IB. Further, FIG. 3B may depict a second side of the cartridge 300 A of FIG. 3 A. That is, cartridge 300 A may be the same cartridge as cartridge 300B.

[0137] The cartridge 300B includes a sample input chamber, the system composition stored in a blister device 126a, a first wash buffer 110a stored in a blister device 126b, a second wash buffer 110b stored in a blister device 126c, a PCA buffer 308 stored in a blister device 126d, and the detection chamber 120. The cartridge 300B may also include membrane pinch valves 302, a pump fluidic interconnect 304, and membrane pinch valves 306.

[0138] Membrane pinch valves 302 and 306 may be specific types of valves used to restrict and permit fluid flow through the cartridge. Valves 302 and 306 may also be different types of valves, such as wax valves or volcano valves. The use of valves is described in greater detail with respect to FIG. 4.

[0139] The pump fluidic interconnect 304 may be used to pump air, fluid, etc. through the cartridge 100. A pump maybe, for example, a syringe pump, an air cylinder pump, a diaphragmAtty. Dkt. No.: 86357086pump, etc. The use of a pump in the cartridge 100 is described in greater detail with respect to FIG. 4.

[0140] The PCA buffer 308 may refer to a buffer used to reconstitute a master mix reagent used for reverse transcription and PCA amplification. In some examples PCA buffer 308 comprises water, a salt, a buffer compound (e.g., tris buffer) and optionally a surfactant. In some examples, the salt is MgCh or NaCl. In some examples, the PCA buffer 308 comprises between 0. ImM and 15mM salt. In some examples, the system composition comprises between about 0.001% and about 0.1% (e.g., about 0.001%, 0.01%, 0.05%, or 0.1%) surfactant. In some examples, the surfactant is selected from Tween®20, Tween® 80, Tween® 85, SPAN® 80 or SPAN® 85. In some examples, the surfactant is Tween®-20. In some examples, at least one parameter of the PCA buffer 308 can be adapted to enable hybridization of the target nucleic acid to the functional nucleic acid at a desired complementarity. For example, a concentration of the salt (e.g., MgCh) in the PCA buffer 308 can be increased in order to enable hybridization even with low complementarity, whereas optionally the concentration of the salt (e.g., MgCh) in the PCA buffer 308 can be reduced in order to enable hybridization only from a certain higher degree of complementarity.

[0141] As used herein, a “master mix” (“MM”) or “PCR mixture” refers to a mixture of reagents useful for an amplification reaction (e.g., a PCA reaction, an RT-PCA reaction, a PCR reaction, an RT-PCR reaction, a qPCR reaction). In some examples, the master mix may comprise polymerase, dNTPs, primers (e.g., at least a forward and a reverse primer specific for a target), a probe comprising a detectable label (e.g., a fluorescent probe), and / or a reverse transcriptase. In some examples, the dNTPs comprise a detectable label. In some examples, the master mix is 3X concentration (meaning the master mix comprises 3 times the concentration of each amplification ingredient than needed for the amplification reaction, a 3X master mix is reconstituted (diluted) three folds in a PCA buffer), 5X concentration (meaning the master mix comprises 5 times concentration of each amplification ingredient than needed for the amplification reaction, a 5X master mix is reconstituted (diluted) five folds in a PCA buffer), or 10X concentration (meaning the master mix comprises 10 times the concentration of each amplification ingredient than needed for the amplification reaction, a 10X master mix is reconstituted (diluted) ten folds in a PCA buffer). In some examples, the master mix is lyophilized. In some examples, the master mix is lyophilized in a lyoprotectant, such as trehalose. In some examples, a lyophilized master mix is reconstituted in the PCA buffer 308Atty. Dkt. No.: 86357086as described herein. In various examples, reconstitution of the master mix may be performed by one or more of: reciprocating flow between chambers and / or channels, ultrasonic and / or acoustic mixing, impellers, mechanical agitation, diffusion, and / or channel geometry (e.g., turns, ridges, mini-chambers, etc.).

[0142] FIG. 4 depicts an example diagnostic system architecture, in accordance with present implementations. As illustrated by way of example in FIG. 4, an example diagnostic system architecture 400 can include a cartridge and an instrument. The cartridge may be the same as or similar to the cartridge 100. Further, the instrument may be the same as or similar to the instrument 140.

[0143] The cartridge of the example diagnostic system architecture 400 may include a first vent membrane 404, a plurality of valves 408a-s, a plurality of frangible seals 410a-h, a sample input chamber 412, a sample filter 414, a p-trap 416, an umbrella valve 418, a plurality of bubble traps 420a-d, a system composition blister device 422, wash buffer blister devices 424a and 424b, a plurality of fluidic junctions 426a-d, a sample input (SI) metered section 430, an IPC 431, a lyse chamber 432 (e.g., the extraction chamber 114) having an external ultrasonic horn 434, lysing beads 436, an infrared sensor 437, and a filter for lysing 439, lyophilized functional paramagnetic particles 454, a PCA buffer 440 (stored in a blister device), a PCA buffer metered section 442, a master mix mixing chamber 444 having a master mix lyo bead 446 (e.g., a lyophilized PC master mix), a second vent membrane 448, a waste chamber 468, an overflow chamber 466, and a PCA chamber 456.

[0144] As shown in FIG. 4, the instrument of the diagnostic system architecture 400 may include a pump 402 (e.g., a syringe pump), a plurality of fluid sensors 428a-e, and a plurality of external magnets 458a and 458b. The elements of the instrument may be positioned such that certain components interface with certain components of the cartridge. For example, FIG.4 indicates locations of fluid sensors 428a-e within the instrument relative to the elements within the cartridge. For example, fluid sensors 428a-e may be disposed within the instrument at various locations corresponding to elements located in the cartridge such that the sensors 428a-e sense fluid motion, movement, etc. within the cartridge at the indicated positions. For example, sensor 428a is shown to be located at an inlet of the SI metered section 430. The sensor 428a may not be physically located within the cartridge at the inlet of the SI metered section. Rather, the sensor 428a may be positioned within the instrument such that, upon insertion of the cartridge into the instrument, the position of the sensor 428a aligns with theAtty. Dkt. No.: 86357086inlet of the SI metered section 430. Further, as stated, the instrument the diagnostic system architecture 400 may also include a magnet 458a to interface with the PC A chamber 456 and a magnet 458b to interface with the lyse chamber 432. The magnet 458a may be positioned within the instrument such that, upon insertion of the cartridge into the instrument, the location of the magnet 458a aligns with the position of the PCA chamber 456 within the cartridge. Similarly, the magnet 458b may be positioned within the instrument such that, upon insertion of the cartridge into the instrument, the location of the magnet 458b aligns with the position of lyse chamber 432 within the cartridge

[0145] In various examples, the system composition blister devices 422 and the wash buffer blister devices 424 may be similar to the blister devices 126, respectively. The system composition stored in the blister device 422 may be similar to the system composition described herein and the wash buffer composition stored in the blister devices 424a and 424b may be similar to the wash buffers 110a and 110b. The lyophilized functionalized magnetic particles 454 may be similar to the magnetic particles 116. Further, the PCA buffer 440 may be similar to the PCA buffer 308. The lyse chamber 432 may be similar to the extraction chamber. The PCA chamber 456 may also be similar to the detection chamber 120. The waste chamber 468 may be similar to the waste storage chamber 168.

[0146] The diagnostic system architecture 400 may also include an overflow chamber 466. As will be described below, the overflow chamber 466 may store the wash buffer (e.g., after the magnetic particles are delivered to the PCA chamber 456). The overflow chamber 466 may be used in addition to or alternative to the lyse chamber 432 and / or the wash buffer storage blisters 126a and / or 126b. For example, one or more of the overflow chamber 466, the lyse chamber 432, and / or the wash buffer blister 424a or 424b may store the wash buffer. For example, the wash buffer may be stored in the overflow chamber 466 or return to one or more of the lyse chamber 432 or the original wash buffer blister 424a or 424b upon delivering the magnetic particles 116 to the PCA chamber 456.

[0147] A user may fdl the sample input chamber 412 with a sample liquid. The sample liquid may be a biological sample containing one or more nucleic acids or other molecules of interest. The sample input chamber 412 may be similar to or the same as the sample input chamber described with respect to FIGS. 1A and IB. The user may fdl the sample input chamber 412 via a pipette, an exact volume pipette, a dropper, syringe injection, etc. The sample input chamber 412 may include fdl guides to indicate a fdl level. Upon fdling the sample inputAtty. Dkt. No.: 86357086chamber 412, all 408a-s may be open. An umbrella valve 418 may disable the sample input chamber 412 from filling the sample metering circuit (e.g., the SI metered section 430). In various examples, the umbrella valve 418 may be a type of check valve. For example, the umbrella valve 418 may prevent flow back towards the sample input chamber 412. The umbrella valve 418 may have a sufficient cracking pressure in a forward direction, thereby preventing the fluid in the sample input chamber 412 from reaching the sample metering section 430 from gravity (e.g., a head height pressure).Upon filling the sample input chamber 412, the chamber may be closed. For example, the sample input cover 303 may be closed.

[0148] Upon filling the sample input chamber 412, the sample may be pressurized through the valves 408a. Pressure may be vented through normally open valves 408k, 408n and / or 408o and the second vent membrane 448. For example, pressure may be vented through valve 408k. Thus, the sample may be pushed through the umbrella valve 418, the bubble trap 420a, and into the sample metering channels (e.g., the SI metered section 430). Fluid flow may be monitored as the liquid moves past an inlet of the SI metered section 430. In various examples, a first fluid flow sensor 428a may be positioned within the instrument such that the sensor aligns with and senses fluid at an inlet of the SI metered section 430. It should be understood that the positions of the fluid flow sensors 428 may be positioned to correspond to any locations of the cartridge 100. For example, a second fluid flow sensor 428b may also be positioned within the instrument such that the sensor aligns with and senses fluid at an outlet of the SI metered section 430. The sensors 428a and 428b may monitor the fluid flow until the liquid reaches a sample metering outlet sensor. Responsive to the liquid reaching the outlet sensor, flow may be stopped. For example, valves may close to prevent movement of the liquid.

[0149] The fluid flow sensors may detect if a cartridge channel or chamber has liquid or air present. The fluid flow sensors may track progress of liquid slugs, meter liquid by triggering the halt of flow to control volume of fluid (e.g., sensors 428b, 428c, and 428e), help in reciprocating mixing by triggering when a flow should be reversed (e.g., sensor 428e), and / or help locate the reconstituted master mix reagent in the PCA chamber (e.g., sensors 428c and 428e).

[0150] In various examples, sensors from the instrument 150 may be optical and / or capacitive. Optical sensors may be or include LEDs and photodiodes to detect changes in contrast, color, reflection, etc. in a microfluidic channel or chamber. Capacitive sensors may be or includeAtty. Dkt. No.: 86357086capacitor plates to detect change in dielectric between liquid and air in a microfluidic channel or chamber.

[0151] Responsive to metering the sample, frangible seals 410a and 410b may be opened. Opening the frangible seals 410a and 410b may enable fluid flow into and out of the system composition blister device 422. The cartridge may then be pressurized through the first vent membrane 404 and pinch valve 408b, thus purging the sample from the SI metered section 430. Further, the sample may reconstitute an IPC 431 (e.g., a lyophilized IPC particle), and the sample may fill the lyse chamber 432. Pressure may be vented through one or more of pinch valves 408m, 408n, and / or 408g and / or the second vent membrane 448 to allow the fluid flow. For example, pressure may be vented through the pinch valve 408g to fill the lyse chamber 432 with the sample and the IPC 431.

[0152] In various examples, the system composition fluid may flow through the SI metered section 430 as the blister device 422 empties. In various examples, it may be beneficial to fully empty the blister device 422 for improved performance. Air may then be pushed through the blister device 422 (e.g., via syringe pump 402) to fully empty the blister device 422 and purge any remaining system composition fluid from the SI metered section 430 into the lyse chamber 432. Metering sensors (e.g., sensors 428a and 428b) may manage a flow rate through the SI metered section 430 and minimize an amount of air pushed into the lyse chamber 432 from the system composition blister device 422.

[0153] Responsive to emptying the system composition blister device 422, the cartridge 100 may be pressurized through the first vent membrane 404 and the pinch valve 408f. The lyse chamber 432 may then be pressurized to ensure contact between a lyse chamber film and a lyse chamber heater. In various examples, all valves may be closed to isolate the lyse chamber 432 during lysis. In various examples, lysis may be an ultrasonic lysis. As such, the ultrasonic horn 434 may be pulsed, and a temperature of the lyse chamber 432 may be controller via the horn 434, the heater, and / or a temperature sensor. The ultrasonic pulses may lyse the fluid and mix the sample, the system composition, and the IPC 431. Lysing beads 436 may be used to aid the lysing process.

[0154] Responsive to lysing the sample, the cartridge 100 may be pressurized through the first vent membrane 404 and the pinch valve 408f. Further the valves 408p, 408m and / or 408n may be opened. This may allow the lysate (e.g., the sample, the system composition, and the IPCAtty. Dkt. No.: 86357086lyo material mixture) to be delivered from the lyse chamber 432 past the magnetic particles 454 and a location corresponding to the sensor 428d, and into the waste chamber 468. The entrance of the lysate and magnetic particles 454 into the waste chamber 468 may cause the magnetic bead lyo particle 454 (e.g., a lyophilized particle comprising or including magnetic particles, such as magnetic particles 116, that is to be reconstituted in order for the magnetic particles to be utilized) to be reconstituted. In various examples, all of the lysate is delivered from the lyse chamber 432. Flow may be stopped responsive to a determination that a trailing meniscus of the lysate is detected by a waste chamber inlet sensor (e.g., the fluid sensor 428d). The waste chamber 468 may be kept full of fluid to minimize air generation in the chamber. When the magnetic bead lyo particle 454 has been reconstituted (e.g., such that the magnetic bead lyo particle 454 is or includes reconstituted magnetic particles, referred to as magnetic particles 454), the lysate and magnetic particles 454 are transported back into the lyse chamber 432. The movement between the lyse chamber 432 and the waste chamber 468 may be repeated (e.g., until the magnetic bead lyo particle 454 is reconstituted).

[0155] Flow of the lysate back to the lyse chamber 432 may occur via use of a bidirectional syringe pump (e.g., the syringe pump 402). Valves 408m, 4081, and 408f may be opened and the syringe pump 402 is pulled, thereby creating a negative pressure to cause the lysate to flow back into the lyse chamber 432.. In various examples, the waste chamber inlet sensor 428d may detect when all of the lysate has been purged from the waste chamber 468.

[0156] The magnetic bead lyo particle 454 may be included with the IPC 431. As such, the magnetic particles 454 may enter the lyse chamber 432 when the IPC 431 is delivered to the lyse chamber. During such occurrences, the lysate may not exit the lyse chamber 432 and enter the waste chamber 468 to reconstitute the magnetic bead lyo particle 454.

[0157] Responsive to the lysate reentering the lyse chamber 432 (or, when the magnetic bead lyo particle 454 is included with the IPC 431, responsive to lysis occurring), hybridization may occur. For example, the lysate may be mixed in the lyse chamber 432 using the ultrasonic horn 434. In order to minimize air generation, the IR sensor 437 may indirectly monitor fluid flow to ensure neither the lyse chamber 432 maintains an adequate fluid level. For example, the IR sensor 437 may monitor a temperature of the lyse chamber 432 (e.g., as part of a closed feedback loop), and temperature changes detected by the IR sensor 437 may correspond to changes in fluid level of the lyse chamber 432. Ultrasonic pulsing by the ultrasonic horn 434 may allow mixing of the magnetic particles 454 into the lysate such that the magnetic particlesAtty. Dkt. No.: 86357086454 are hybridized with the nucleic acids of interest. A heating rate of the fluid may be increased by mixing the fluid with itself. Mixing the fluid with itself may improve a heating rate because the heat source may be applied on one side of the liquid volume, reducing an amount of time to conduct the heat compared to when the liquid is in a static state. The region may include controlled heating during hybridization to promote capture of the sample target RNA to the paramagnetic particles. For example, the zone 460 may be heated (e.g., by an external heat source).

[0158] After hybridization and mixing of the lysate, the magnetic particles 454 are trapped to a side or wall of the lyse chamber 432. For example, the external magnet 458b is actuated to interface with the lyse chamber 432 and apply a magnetic field that causes the magnetic particles 454 to be held or trapped to the wall of the lyse chamber 432.

[0159] While the hybridized magnetic particles 454 are trapped to the side of the lyse chamber 432, the lysate (e.g., including the system composition and the IPC 431) is pushed out of the lyse chamber 432 and into the waste chamber 468. The lysate is pushed into the waste chamber 468 by pressurizing through the first vent membrane 404 and the pinch valve 408f. Further the valves 4081, 408p, 408m and / or 408n may be opened. This may allow the lysate (e.g., the system composition and the IPC lyo material mixture) to be delivered from the lyse chamber 432 past the location corresponding to the sensor 428d, and into the waste chamber 468. Transporting the lysate past the valve 4081 may prevent the lysate from moving past a manifold that interacts with the PCA chamber 456.

[0160] In various examples, a plurality of wash buffers may be enabled for use in the cartridge 100. For example, two wash buffers may be used. In various examples, the first wash buffer may be a more aggressive chemistry that may be more inhibitory to a final PCA reaction relative to the second wash buffer. For example, the first wash buffer may include water, 0.017M ofMgCh, 0.1 IM of Tris-HCL for a pH of 8.0, and 0.056% of Tween® 20. In various examples, NaCl or KC1 may be utilized rather than MgCh.

[0161] The first wash buffer may enter the lyse chamber 432. The first wash buffer may be enabled by opening the frangible seals 410c and / or 41 Od of the wash buffer blister device 424a. The first wash buffer may then be moved from the blister device 424a through the lyse chamber 432 by pressurizing through the valves 408c and 408d. Valves 408q, 408p, and 408g may be open to allow fluid flow. The volume of the first wash buffer may be predefined. For example,Atty. Dkt. No.: 86357086an amount of wash buffer stored in the wash buffer blister 424a is a predetermined, known amount, and the entirety of the contents of the wash buffer blister 424a is to be released and transported throughout the cartridge 400 such that a known amount of wash buffer is used. In some implementations, the volume of the first wash buffer may be controlled using the IR sensor 437. For example, the IR sensor 437 may sense a temperature change within the lyse chamber 432 that is associated with a fluid volume change within the lyse chamber 432. In various examples, the blister device 424a may not be fully emptied in order to minimize air passing downstream through a common channel to the lyse chamber 432.

[0162] Optionally, the first wash buffer may be returned to the wash buffer blister device 424a by pressurizing valve 408f and opening valves 408q, 408p, 408c, and 408e to allow fluid flow back into the wash buffer blister device 424a. In other implementations, the first wash buffer may be stored in the waste chamber 468. The second wash buffer may then enter the lyse chamber to perform a second wash. In various examples, the second wash buffer may be a less aggressive chemistry that may be less inhibitory to a final PCA reaction relative to the first wash buffer. For example, the second wash buffer may include 0.01 M Tris buffer composition, 0.1 M NaCl, and 0.0015 M KC1, with a pH 8.0 at 25 degrees Celsius and 0.01% Tween® 20 when dissolved in one liter of deionized water. In some cases, the second wash buffer may include the same or a similar composition to the first wash buffer (e.g., water, 0.017M of MgCh, 0.1 IM of Tris-HCL for a pH of 8.0, and 0.056% of Tween® 20).

[0163] The second wash buffer may be enabled by opening the frangible seals 410e and / or 410f of the wash buffer blister device 424b. The second wash buffer may then be moved from the blister device 424b through the lyse chamber 432 by pressurizing through the valve 408d. Valves 408r, 408p, and 408g may be open to allow fluid flow. The volume of the second wash buffer may be predefined. For example, an amount of wash buffer stored in the wash buffer blister 424b is a predetermined, known amount, and the entirety of the contents of the wash buffer blister 424b is to be released and transported throughout the cartridge 400 such that a known amount of wash buffer is used. In some implementations, the volume of the second wash buffer may be controlled using the IR sensor 437. For example, the IR sensor 437 may sense a temperature change within the lyse chamber 432 that is associated with a fluid volume change within the lyse chamber 432. In various examples, the blister device 424b may not be fully emptied in order to minimize air passing downstream through a common channel to the lyse chamber 432.Atty. Dkt. No.: 86357086

[0164] While the first wash buffer (or the second wash buffer, when a second wash is performed) is in the lyse chamber 432, the magnet 458b may be retracted, thereby releasing the hybridized magnetic particles 454 from the wall or side of the lyse chamber 432. In some examples, the magnet 458b may not be retracted and the hybridized magnetic particles 454 may remain held to the wall of the lyse chamber 432 for the duration of the wash. The ultrasonic horn 434 may be used to resuspend the magnetic particles 454 in the first (or second) wash buffer. When the hybridized magnetic particles 454 are transported from the lyse chamber 432 to the PCA chamber 456 via a wash buffer, the magnetic 458b is retracted to permit transport of the hybridized magnetic particles 454.

[0165] The hybridized magnetic particles 454 suspended in either the first or second wash buffer is transported from the lyse chamber 432 to the PCA chamber 456. The fluid may flow from the lyse chamber 432 to the PCA chamber 456 by opening valve 408p such that fluid flows into the PCA chamber 456. The sensor 428c may be used to monitor fluid flow into the PCA chamber. When in the PCA chamber 456, the magnetic particles 454 may be captured onto a heating element (e.g., an SS foil, the heating element 122, etc.). The magnet 458a may interface with the PCA chamber 456 to pull the paramagnetic particles 454 to a PCA foil (e.g., a heating element 122), thus separating the particles from the wash buffer. This may be referred to as a solid phase extraction. The magnet 458a may be static or movable (e.g., such that the magnet is actuated to interface with the PCA chamber 456 when the magnetic particles enter the PCA chamber 456).

[0166] When the magnetic particles are captured onto the heating element, the wash buffer is moved from the PCA chamber 456 to one of the wash buffer blister device 424, the waste chamber 468, or the overflow chamber 466. When the wash buffer is returned to the wash buffer blister device 424, flow may be reversed by pressurizing valve 408o and opening valves 408q, 408c, and 408e (when the wash buffer is the first wash buffer) or valves 408r and 408e (when the wash buffer is the second wash buffer) to allow fluid flow. The wash buffer may be returned to its blister device 424 until the end of the fluid flow is detected (e.g., by any sensor 428). When the wash buffer is moved to the overflow chamber 466, the valve 408n is opened and the wash buffer is moved. Valves 408o, 408p, 4081, and 408m may be opened to move the wash buffer from the overflow chamber 466 to the waste chamber 468.

[0167] In various examples, the PCA buffer 440 may be metered (e.g., at the PCA buffer metered section 442). Valves 410g and 41 Oh are opened to allow the PCA buffer 440 to exitAtty. Dkt. No.: 86357086the blister device and move to the PCA buffer metered section 442. Flow may be moved out by sucking flow at the valve 408k and opening the valves 408h and 408i to allow flow. A bubble trap 420d may be used to catch air from the PCA buffer 440 that moves downstream. The PCA buffer 440 may continue to fill the PCA buffer metered section 442 until the PCA buffer 440 is sensed by the PCA buffer metering sensor 428e.

[0168] After the PCA buffer flows past a location aligned with the sensor 428e, the valve 408i is closed and the valve 408j is opened to allow pressurized air to separate remaining PCA buffer from a controlled metered PCA buffer volume. The metered volume may be sucked into the master mix (MM) mixing chamber 444, and the master mix lyo bead 446 may be reconstituted. Flow may be stopped responsive to a trailing meniscus of the PCA buffer is sensed by the PCA buffer metering sensor (e.g., the sensor 428e).

[0169] The master mix (MM) lyo bead 446 may be mixed well into the PCA buffer. The MM lyo bead 446 may be mixed into the PCA buffer by recirculating flow between the MM metering section and the MM mixing chamber 444. Flow may be recirculated by alternating positive and negative pump pressure with valve 408k and valve 408j open. Once mixing is complete, the master mix is pulled into the metering channels and out of the mixing chamber 444. Reconstitution and mixing of the master mix lyo bead 446 may be performed in parallel with hybridization described above. In some examples, reconstitution and mixing of the master mix lyo bead 446 is performed during capture of the magnetic particles in the lyse chamber 432.

[0170] Responsive to the master mix being fully mixed, the master mix may be loaded into the PCA chamber 456. The master mix may enter the PCA chamber 456 by applying a vacuum to valve 408o, with valves 408s and 408j open to allow fluid flow. The master mix may enter the PCA well until a trailing liquid meniscus of the master mix arrives at a location corresponding to the PCA inlet sensor 428c.

[0171] Responsive to the master mix arriving at the PCA chamber 456, PCA may be performed. The PCA chamber 456 may be pressurized via the valve 408o to reduce an impact of air bubble growth during PCA temperature cycling. After pressurization, the chamber may be isolated from the pump by closing all valves of the cartridge 100. An isothermal temperature in the PCA chamber 456 may be controlled via a plurality of heaters on each side of the PCAAtty. Dkt. No.: 86357086chamber 456. In various examples, electrical pulsing of the PCA foil (e.g., the heating element 122) may create temperature pulses for the PCA reaction.

[0172] Referring now to FIG. 5A, a cross-sectional view of the detection chamber 120 is shown, according to an example. The detection chamber 120 may include a plurality of temperature regulators 502 surrounding the detection chamber 120, a plastic layer 506, an adhesive 508, and a heat spreader 510. In various examples, a detection chamber well 504 may be a cavity formed between the plastic layer 506 and the heating element 122. The detection chamber well 504 may be configured to house the nucleic acids of interest for amplification and detection. The heating element 122 may be coupled to an energy source, which is coupled to the controller 164 and the electrical components 160. In various examples, the electrical components 160, the controller 164, and / or the energy source may be located in the instrument 150. In various examples, the detection chamber 120 may also be referred to as an amplification chamber, a reaction chamber, a PCA chamber, a PCA reaction chamber, etc. In various examples, the cartridge 100 may include a plurality of detection chambers and / or other types of chambers. For example, the cartridge 100 may include an amplification chamber and a detection chamber.

[0173] The detection chamber 120 may be configured to house an amplification reaction. Amplification may be performed using a nucleic acid amplification method selected from one or more of: pulse-controlled amplification (PCA), reverse transcriptase pulse controlled amplification (RT-PCA), polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR) or real-time polymerase chain reaction (qPCR). In some examples, the step of amplifying the nucleic acid of interest is done by pulse-controlled amplification (PCA).

[0174] PCA reactions may be utilized to amplify the molecule of interest. Specifically, in said PCA reactions, only a portion of the chamber in which the reaction occurs may be heated for amplification, as opposed to the entirety of the reaction chamber. This may facilitate optimization of temperature control and provide for a more efficient amplification. Compared to polymerase chain reactions (PCR) for amplification, PCA reaction cycles may have a decreased duration, thus decreasing an amount of time for amplification and detection to occur.

[0175] In various examples, during the PCA reaction, a small volume of the fluid in the detection chamber well 504 may be temporarily heated (e.g., thermocycled). For example, 99%Atty. Dkt. No.: 86357086of the fluid volume may be unheated, and 1% of the fluid volume may be temporarily heated during the PCA reaction. For example, 99% of the fluid volume may remain at an isothermal temperature (e.g., between 60 and 70 degrees Celsius), while the 1% of the fluid volume being heated may temporarily heat to between 90 and 110 degrees Celsius. For example, the isothermal volume may remain at 65 degrees Celsius, while the heated fluid volume may be temporarily heated to 100 degrees Celsius (and return to 65 degrees Celsius when electrical pulses are not being delivered to temporarily heat the heating element 122).

[0176] As shown in FIG. 5 A, line 511 indicates a border of a denaturation zone. For example, under the line 511, the magnetic particles 116 are shown. During the PCA reaction, nucleic acids of interest may be attached to the magnetic particles 116 and may generally stay within the area outlined by the line 511. Thus, in various examples, the denaturation zone may be a volume of the detection chamber well 504 (e.g., about 15 micrometer thick) where the detection chamber 120 is locally heated to perform the PCA reaction. As described herein, denaturation may describe the separation of a nucleic acid into its two single strands. Denaturation may allow for amplification of the nucleic acids of interest, as each single strand may be used to replicate another strand, increasing the number of nucleic acid strands. Further, the denaturation zone of the detection chamber 120 may be the portion of the chamber that is heated, while the temperature of the remainder of the detection chamber 120 is unchanged. For example, the heating element 122 may deliver pulses and generate heat so that the temperature of the denaturation zone increases. As described herein, the pulses may be configured such that the generated heat dissipates quickly and does not cause a temperature change to the detection chamber well 504 as a whole (e.g., the overall chamber is isothermal but the denaturation zone experiences a temperature increase).

[0177] In some examples, more than one nucleic acid of interest may be amplified by thermocycling. Thermocycling may be performed when the magnetic particles 116 are functionalized with different capture oligonucleotides designed to be linked to different nucleic acid of interest.

[0178] The temperature regulators 502 may be or include heatsinks. The temperature regulators 502 may be heating and / or cooling elements configured to bring a temperature of the system to an isothermal temperature above room temperature. For example, the temperature regulators 502 may raise an isothermal temperature of the detection chamber 120 to between 60 and 70 degrees Celsius. For example, the isothermal temperature of the detection chamberAtty. Dkt. No.: 86357086may be raised to 65 degrees Celsius. As the heating element 122 is delivered an electrical pulse to locally heat a portion of the detection chamber 120, the temperature regulators 502 may remove heat from the detection chamber 120 to prevent a temperature of the overall detection chamber 120 from raising beyond the isothermal temperature. In various examples, a temperature sensor may be located on or proximate the detection chamber 120. The temperature sensor may monitor the temperature of the detection chamber in various locations. For example, a temperature sensor may monitor a temperature at or near the heating element 122 and / or a temperature away from the heating element 122 (e.g., a location at which the temperature should remain constant or relatively constant). The temperature data may be sent to the controller 164. Responsive to receiving the temperature data, the controller 164 may activate or otherwise control the pulses delivered by the energy source.

[0179] The plastic layer 506 may be a first layer of the detection chamber 120. In various examples the plastic layer 506 may be a few hundred micrometers thick (e.g., around 200 micrometers thick). The plastic may be or include a base of the cartridge 100. For example, the plastic layer 506 may be the same material that the base of the cartridge 100 is made out of. For example, the plastic layer 506 may also be referred to as the cartridge base layer 506. The cartridge may be made of a dielectric material. In some examples, the walls of the cartridge may comprise a polymer material, such as (but not limited to) a cyclic olefin copolymer (COC) material. In some examples, the polymer material may comprise polyethylene, polypropylene, polycarbonate, polymethylmethacrylate (PMMA), and the like.

[0180] The heating element 122 may be coupled to or adjacent to the denaturation zone (e.g., marked by the line 511). The heating element 122 may be a resistive local heating element. Particularly, the heating element 122 may be a foil (e.g., a metal foil). Heating of the heating element 122 may be achieved by means of short electrical pulses with which the local heating element(s) 122 are energized. For example, the energy source may be coupled to the heating element 122. The controller 164 may control the energy source to generate pulses to the heating element 122 to heat the heating element 122, and, subsequently, a portion of the detection chamber well 504.

[0181] In some examples, generating pulses of energy may be performed such that only the immediate vicinity of the heating element 122 is heated locally for a short time. Heating of the heating element 122 may allow denaturation of the nucleic acid molecules in the reactionAtty. Dkt. No.: 86357086volume, while the bulk of the reaction volume (i.e., the reaction solution) may remain at a base temperature at which elongation and / or hybridization can take place.

[0182] When a current flows through the heating element 122 the heating element 122 may begin to heat up at the beginning of the heating pulse.

[0183] In various examples, the heating element 122 may be around ten micrometers thick. To realize the lowest possible heat capacity, the heating element 122 may have a thickness of less than 100 micrometers in at least one dimension (e.g., less than 50 micrometers, less than 30 micrometers). The thickness of the heating element 122 may be sufficiently low to provide sufficient electrical resistance or impedance. For example, a thinner heating element 122 may have an increased resistance and, consequently, allow for greater Joule heating. In order to make the heating element 122 not too fragile, the material thickness in each dimension may be at least 100 nm, at least 1 micrometers and / or 5 micrometers or 10 micrometers. In some examples, the heating element 122 may comprise a thickness of about 10 to about 50 microns, a thickness of about 15 to about 40 microns, or a thickness of about 20 to about 30 microns, and in some examples, a thickness of about 25 microns.

[0184] In some examples, the heating element 122 may be formed of a metallic foil. For example, the heating element 122 may be formed of ferromagnetic materials such as steel, stainless steels, nickel, and / or highly conductive non-ferrous metals, such as brass and / or copper. In some examples, the heating element 122 may comprise a material such as: stainless steel, brass, titanium, tantalum, tungsten, aluminum, copper, platinum, gold, silver, zinc, indium tin oxide (ITO), and combinations thereof. In some such examples, the first layer material is a stainless-steel material. Alternatively or additionally, the heating element 122 may be at least partially formed of very hard materials, such as tungsten, which may allow very thin designs of the local heating element 122. In addition, the heating element 122 may have a very high thermal conductivity.

[0185] In some examples, the heating element 122 forms at least a part of a container wall of the reaction container. This may allow for direct contact between the heating element 122 and the sample fluid or reaction solution to be established in a simple manner.

[0186] In various examples, the heating element 122 may be a first layer of a plurality of layers comprising the detection chamber 120. The first layer (e.g., the heating element 122, also referred to herein as a “heating element” or a “foil”) may comprise an electrically activatableAtty. Dkt. No.: 86357086heating element that may be to generate heat within the detection chamber 120. In some examples, the heating element 122 may comprise an electrically conductive material. Upon application of an electrical signal to induce heating, the electrically conductive material (e.g., the heating element 122) may generate power (P) depending on its resistivity (R) and current (I) where P=I2x R. Accordingly, in some instances, the heating element 122 also may sometimes be referred to as being an electrically resistive sheet. In various examples, the heating element 122 may be aPCA foil. For example, the heating element 122 may be stainless steel.

[0187] In various examples, the foil (e.g., the heating element 122 or the first layer) may be wider than the PCA chamber or detection chamber 120. This may allow electrical probes of the instrument 150 (e.g., the energy source) to contact the detection chamber 120, specifically the heating element 122. This may allow for the generation of the electrical pulses for PCA.

[0188] In some examples, a wall of the detection chamber 120 may further comprise a second sheet or layer. The second sheet may be the adhesive 508 or a different second layer. The second sheet may act to electrically isolate the first layer from a third layer (e.g., heat spreader 510. In some examples, the adhesive 508 may comprise a thickness of about 10 microns to about 200 microns. In some examples, the adhesive 508 comprises an adhesive layer, such as a pressure sensitive adhesive (PSA) layer. In some examples the adhesive 508 may comprise a PSA layer having a thickness up to 200 microns. In some examples, the adhesive 508 may be a heat spreader pressure sensitive adhesive. In some examples, the adhesive 508 may comprise a material including both thermosetting and thermoplastic properties. In some such examples, the material of the adhesive 508 may comprise acrylic adhesive materials. In some of these examples, the adhesive 508 may comprise a thermal bonding adhesive, such as but not limited to: a Pyralux®-based material from DuPont de Nemours, Inc. of Wilmington, Delaware; and a FastelFilm material obtainable from Fastel Adhesives and Substrate Products via www.fasteladhesives.com; and the like.

[0189] In some examples, the wall of the detection chamber comprises a third layer. The third layer may be a heat spreader 510. The heat spreader 510 may be located in the detection chamber 120. In various examples, the heat spreader 510 may directly contact a cartridgecontact heater in the instrument 150. For example, the cartridge-contact heater may interface with the heat spreader 510 to heat each of the one or more detection chambers. That is, the heat spreader 510 may be or include a thermally conductive elastomer plate or a metal plate andAtty. Dkt. No.: 86357086may be to transfer heat from the cartridge-contact heater to the detection chamber 120. Specifically, the heat spreader 510 can be or include one or more of a thermally conductive elastomer plate or a metal plate operable as a thermal conduit for transferring heat between the cartridge-contact heater and the detection chamber 120.

[0190] In some examples, the heat spreader 510 comprises a thermally conductive metal sheet. In some examples, the heat spreader 510 may comprise a material such as: aluminum, copper, brass, and combinations thereof or other thermally conductive materials. In some other examples, the heat spreader 510 comprises aluminum. A metal heat spreader 510 may, due to its rigidity, provide worse thermal contact relative to an elastomer. However, a metal heat spreader 510 may rapidly spread heat flow laterally throughout the portion of the detection chamber 120 that is heated during PCA.

[0191] In various examples, the heat spreader 510 may be an elastomer. For example, the heat spreader 510 may be a fiberglass reinforced silicone film. Specifically, the heat spreader 510 may be a thermally conductive elastomer. An elastomer heat spreader 510 may provide better thermal contact compared to a metal heat spreader due to a lower thermal contact impedance, but may not actually “spread” heat through the detection chamber 120.

[0192] The heat spreader 510 may comprise a thickness of about 150 microns to about 500 microns, and in some examples a thickness of about 250 to about 400 microns. The thickness provides a mechanical stiffness sufficient to resist or prevent deformation of the heating element 122.

[0193] In various examples, the adhesive 508 and / or the heat spreader 510 may be optional. Thus, in various examples, the one or more heating elements of the detection chamber 120 includes only the heating element 122 (e.g., a foil).

[0194] Referring now to FIG. 5B, a system 500B including the detection chamber 120 is shown, according to an example. The system 500B may be the same as or similar to 500A. Further, components of the system 500B may be the same as or similar to the components of the system 500A. For example, the system 500B shows a detection chamber 120 having the detection chamber well 504, the cartridge base (or plastic layer) 506, the heating element 122, the adhesive 508, and the heat spreader 510. In various examples, the system 500B may include a second adhesive 512.Atty. Dkt. No.: 86357086

[0195] For example, in various examples, the second adhesive 512 may be a foil PSA layer. The foil PSA layer may be a three layer element. For example, the foil PSA layer may include an adhesive layer, a backer layer, and another adhesive layer.

[0196] The system 500B of FIG. 5B further shows an optical film 514 attached to, coupled to, or otherwise affixed to the detection chamber 120. In various examples, the optical film 514 may allow detection of the amplified nucleic acids of interest. For example, the optical film 514 may have high transparency and / or low haze. This may allow the optical unit 162 of the instrument 150 to detect a presence, absence, and / or amount of a nucleic acid of interest. For example, during the PCA reaction, the nucleic acids may be tagged with a fluorophore. The optical unit 162 may utilize, for example, a sensor within the detection chamber 120 to detect fluorescence corresponding to an amount of the nucleic acid of interest.

[0197] Referring now to FIG. 6, a method 600 for detecting the presence, absence, amount, etc. of a nucleic acid of interest, according to some examples. Generally, a user may input an amount of sample (e.g., such as swab specimen eluted in a transport media) into the cartridge 100. The cartridge 100 may meter a proper amount of the sample and system composition, and internal positive control (IPC) may be added. The target organisms in the sample and IPC are lysed. Nucleic acids from target organisms and IPC may hybridize (e.g., bind) to paramagnetic particles. Paramagnetic particles with bound capture oligonucleotides may be held onto a side (e.g., wall) of the extraction chamber. The extraction chamber and the held paramagnetic particles are washed with a wash buffer and suspended in the buffer. A second wash buffer may also be used. The hybridized paramagnetic particles are transferred to the detection chamber 120 (e.g., a PCA chamber). The hybridized paramagnetic particles are then captured onto a pulse heater in the detection chamber 120 with a magnetic field. The detection chamber 120 and captured magnetic particles 116 are washed with stored wash buffer 110. PCA buffer 308 may be metered and used to reconstitute Master Mix reagent used for PCA. Reconstituted Master Mix reagent may be transferred into the detection chamber 120 and covers the magnetic particles trapped on the heating element. PCA (e.g., RT-PCA) is performed with real-time multi-channel detection. Results of PCA and detection of target nucleic acids are reported to the user.

[0198] At block 602, a specimen sample may be collected. The specimen sample may be a biological sample collected from, for example, a human that contains a molecule of interest. For example, the biological sample may include one or more nucleic acids of interest. AAtty. Dkt. No.: 86357086molecule of interest may be a molecule to be amplified and detected. The molecule of interest may be used for various purposes, such as diagnosing the person that the specimen sample belongs to. In various examples, the specimen sample may be collected by a nasal swab or other retrieval device. Further, the specimen sample may be collected from the person and eluted in a commercial transport medium, such as Copan UTM ®, to be input into the cartridge 100. At block 604, the sample may be input into the cartridge 100. For example, the sample may be input into the sample input chamber. The sample volume of the specimen may be greater than a predefined value (e.g., between 275 and 325 microliters). For example, the sample volume may be greater than 300 microliters. At block 606, the volume of the specimen inserted into the cartridge may be metered so that the sample volume used in the detection process is at or around the predefined value. For example, 320 microliters of the specimen may be collected, and the volume may be metered to 300 microliters to be inserted into the cartridge. Metering a sample may include, for example, utilizing the sample as stored in the cartridge 100 or as delivered by a user, a defined volume between a liquid sensor and a cut-off junction, and / or a defined volume between an overflow valve and a cut-off junction.

[0199] At block 608, the system composition or buffer may be stored. For example, the system composition may be stored in a blister device attached to the cartridge and / or a sealed chamber integrated into the cartridge body. The system composition may be used for lysis and / or hybridization. For example, the system composition may be stored in a blister device and transported to one or more extraction chambers 114 for use in lysing the biological sample and / or hybridizing the nucleic acids of interest of the lysed biological sample.

[0200] In various examples, the system composition may comprise water, salt, a buffering compound, and / or surfactants. The system composition may include relatively high concentrations of salts. Types of salts found in the system composition may include, for example, KC1, MgCh and / or NaCl. In various examples, the surfactant may be Tween® 20. Further, the cartridge 100 may contain a certain volume of the system composition. For example, the cartridge 100 may include between 300 and 800 microliters of the system composition. For example, the cartridge 100 may include 500 microliters of the system composition. At block 610, the volume of the system composition may be metered so that the system composition volume used in the lysing and / or hybridization processes is at or around the predefined value. For example, 465 microliters of the specimen may be collected, and theAtty. Dkt. No.: 86357086volume may be metered to 450 microliters to be transported to and / or used in the extraction chamber 114.

[0201] At block 612, the wash buffer (e.g., wash buffer 110, first wash buffer 110a and / or second wash buffer 110b) may be stored. For example, the wash buffers 110 may be stored in blister devices attached to the cartridge and / or a sealed chamber integrated into the cartridge body. The wash buffers 110 may be used for washing magnetic particles (e.g., magnetic particles 116) and / or the extraction chamber 114. For example, the wash buffer 110 may be stored in a blister device and transported to one or more extraction chambers 114 for use in washing the extraction chamber to prepare for transport of hybridized magnetic particles 116 to the detection chamber 120, amplification, and detection of the nucleic acids of interest of the biological sample.

[0202] In various examples, the wash buffers 110 may be used to suspend the magnetic particles 116 within the extraction chamber 114. Further, a wash buffer (e.g., a second wash buffer) may be used to transport the magnetic particles 116 from the extraction chamber 114 to the detection chamber 120.

[0203] In various examples, the wash buffers 110 may comprise water, salt, a buffering compound, and / or surfactants. The wash buffers 110 may include moderately high concentrations of salts (e.g., relative to the salt concentrations in the system composition). Types of salts found in the wash buffer 110 may include, for example MgCh and / or NaCl. In various examples, the surfactant may be Tween® 20. The wash buffer 110 may also include KC1. Further, the cartridge 100 may contain a certain volume of the wash buffer 110. For example, the cartridge 100 may include between 100 and 400 microliters of the wash buffer 110. For example, the cartridge 100 may include 250 microliters of the wash buffer 110. In various examples, the cartridge 100 may include a plurality of wash buffers 110, each having a different composition and / or different uses. For example, a first wash buffer 110a may include a stronger washing agent, and a second wash buffer 110b may be milder. For example, the first wash buffer 110 may include 0.05 M tris buffer, 0.15 M NaCl, 0.0025 M KC1, and 0.05% Tween® 20, while the second wash buffer 110 may include 0.025 M tris buffer, 0.05 M NaCl, 0.0015 M KC1, and 0.01% Tween® 20. The first wash buffer 110a may be used to wash the extraction chamber 114, and the second wash buffer may be used to rinse the extraction chamber 114.Atty. Dkt. No.: 86357086

[0204] At block 614, the PCA buffer (e.g., PCA buffer 308) may be stored. For example, the PCA buffer 308 may be stored in a blister device attached to the cartridge and / or a sealed chamber integrated into the cartridge body. The PCA buffer 308 may be used to reconstitute a master mix reagent used for reverse transcription and PCA amplification (e.g., the amplification process). For example, the PCA buffer 308 may be stored in a blister device and transported to one or more detection chambers 120 (e.g., a PCA chamber) for use preparing for amplification of the nucleic acids of interest.

[0205] In various examples, the PCA buffer 308 may comprise water and / or salt. The PCA buffer 308 may include small concentrations of salts (e.g., relative to the salt concentrations in the system composition and / or the wash buffer 110). Types of salts found in the wash buffer 110 may include, for example MgCh. In various examples, the PCA buffer 308 may include a surfactant (e.g., Tween® 20) and / or a buffering compound. Further, the cartridge 100 may contain a certain volume of the PCA buffer 308. For example, the cartridge 100 may include between 100 and 400 microliters of the PCA buffer 308. For example, the cartridge 100 may include 200 microliters of the PCA buffer 308.

[0206] At block 616, the volume of the PCA buffer 308 may be metered so that the PCA buffer volume used in the reconstitution process is at or around the predefined value. For example, 235 microliters of the specimen may be collected, and the volume may be metered to 60 microliters to be transported to and / or used in the detection chamber 120 Metering the PCA buffer 308 may control a concentration of the subsequently reconstituted master mix reagent, which may ensure proper amplification of the nucleic acids of interest. In various examples, the PCA buffer 308 may be metered by pumping the PCA buffer 308 through a defined volume between a liquid sensor and a fluidic T-junction bisecting the PCA buffer. Air may then be pumped into the T-junction such that a defined volume of the PCA buffer 308 is further transported.

[0207] At block 618, the biological sample may be lysed, stored with internal positive control (IPC), and mixed with system composition. For example, the metered biological sample may be transported to the extraction chamber 114. The system composition and the IPC may be added to the extraction chamber 114 prior to, concurrent with, and / or subsequent to addition of the biological sample to the extraction chamber 114. The IPC may be a lyophilized organism or synthetic organism. In various examples, the IPC may be a protein and / or organism present in a human sample. At block 618, the sample, the system composition, and the IPC may beAtty. Dkt. No.: 86357086mixed using sonication. For example, the lyse system 152 may be coupled with the extraction chamber 114. The sonicator 154 may perform sonication to mix the sample, buffer, and IPC.

[0208] In various examples, the extraction chamber 114 may be heated by an external heater and / or the sonicator 154. Sonication may induce cavitation upon organisms (e.g., the IPC). In various examples, small beads or particles (e.g., ceramic beads, glass beads) may be included in the extraction chamber 114. The beads or particles may be agitated to mechanically lyse the solution. In various examples, lysis may result from one or more of heat, cavitation, and / or the lysing beads.

[0209] At block 620, potential targeted nucleic acids in the solution lysed at block 618 may be hybridized to stored functionalized paramagnetic particles (e.g., magnetic particles 116). The solution lysed in the extraction chamber 114 may be referred to as a “lysate.” In some examples, in the extraction chamber 114, the lysate may reconstitute lyophilized paramagnetic particles. The lyophilized paramagnetic particles may be functionalized with capture oligonucleotides that may be designed to capture RNA and / or DNA strands from target organisms. The oligonucleotides may be utilized as a half of a primer pair during amplification (e.g., during the PCA amplification reaction). In some examples, the paramagnetic particles 116 may be included in the extraction chamber 114 during lysis at block 618. In other examples, the paramagnetic particles 116 may be added to the extraction chamber 114 after lysis (e.g., after block 618 and before block 620).

[0210] During hybridization, the magnetic particles 116 may be mixed and agitated with the lysate. Mixing and agitation may optimize a capture efficiency of the target nucleic acids. In various examples, hybridization may occur between 55 and 65 degrees Celsius. For example, hybridization may occur at 62 degrees Celsius. In various examples, hybridization may occur with a high salt content (e.g., MgCh or NaCl). Mixing may occur, for example, by reciprocating flow between two chambers such that vortices form at an entrance of each chamber to mix the lysate with the magnetic particles 116.

[0211] After hybridization, the hybridized magnetic particles 116 may be held to a side or wall of the extraction chamber 114. The hybridized magnetic particles 116 may be held to the side of the extraction chamber 114 in an applied magnetic field generated by the magnetic field generator 156. While the hybridized magnetic particles 116 are trapped, the lysate may be disposed or transported to waste (e.g., to the waste storage chamber 168). The lysate may beAtty. Dkt. No.: 86357086disposed such that the lysate does not contact any components or surface of the cartridge 100 that may subsequently be in contact with the master mix reagent.

[0212] At block 622, the extraction chamber 114 is washed using the wash buffer (e.g., the wash buffer 110). Specifically, washing may occur with a first wash buffer 110a. The first wash buffer 110a may resuspend the hybridized paramagnetic particles 116 in the extraction chamber 114. Resuspension may be performed, for example, via sonication (e.g., by the sonicator 154). Washing the extraction chamber 114 may also remove undesirable components in the extraction chamber 114 and / or on the magnetic particles 116 that may interfere with amplification of the nucleic acids of interest. In some examples, a second wash buffer 110b is used to perform a second washing of the extraction chamber 114 (e.g., after the first wash using the first wash buffer 110a). The second wash may occur by again trapping the hybridized magnetic particles 116 to a side or wall of the extraction chamber 114 and exchanging fluids (e.g., the lysate) in the extraction chamber 114. The wash buffers 110 may be pumped through the extraction chamber 114 to wash the chamber. Upon washing, the wash buffer 110 may return to the blister device storing the wash buffer. For example, the first wash buffer 110a may return to a blister device that originally stored the wash buffer. In some examples, the second wash buffer 110b may not return to the blister device, as the wash buffer may be used to transport the hybridized magnetic particles 116 to the detection chamber 120. In various examples, a volume, time, flow rate, and / or flow directionality may be adjusted based on needs of the amplification and / or assay.

[0213] At block 624, the magnetic particles 116 are trapped onto a heating element (e.g., the heating element 122) in the detection chamber 120 (e.g., a PCA chamber). That is, responsive to a washing of the extraction chamber 114 with the first or second wash buffer 110, the hybridized magnetic particles 116 may be suspended in the wash buffer 110 and are transported to the detection chamber 120. The wash buffer including the hybridized magnetic particles 116 may be delivered to the detection chamber 120. Further, in various examples, a flow of the wash buffer may be continuous, in discrete steps, or any combination thereof. In the detection chamber 120, the magnetic particles 116 are trapped onto the heating element. An external permanent magnet (e.g., generated by the magnetic field generator 156) may create a magnetic field. The generated magnetic field may attract the paramagnetic particles onto a surface of the heating element 122. In various examples, the magnetic particles may be distributed uniformly across the heating element 122. The heating element 122 may be or include, in variousAtty. Dkt. No.: 86357086examples, a continuous, structured, or shaped metal foil, metal wires, a conductor and / or resistor layer deposited and / or plated, and / or backed by a heat spreader. In various examples, in order for the PCA process to occur, the paramagnetic particles with the captured nucleic acids may be concentrated at a thermocycling zone of the heating element.

[0214] At block 626, a PCA master mix reagent may be reconstituted. A master mix may be used for reverse transcription, PCA amplification, and / or real-time fluorescence detection. The master mix may be lyophilized (e.g., freeze dried). The PCA master mix reagent may be stored, for example, in a lyophilized pellet or cake, an air-dried pellet or cake, and / or sealed with a plastic plug or film.

[0215] In various examples, the PCA master mix reagent may include a plurality of active ingredients, such as: reverse transcriptase, polymerase, and dNTPs. For each reaction target, the master mix reagent may include a set of primers (e.g., one half of a primer pair used in PCR) and a fluorescent probe. In various examples, lyophilization excipients may include sugars, such as, for example, trehalose.

[0216] In various examples, a metered amount of the master mix may be used to reconstitute the master mix reagent. The master mix reagent may be lyophilized. The PCA buffer 308 may be mixed with the master mix by reciprocally pumping between a storage chamber storing the original master mix reagent and a channel leading to the chamber.

[0217] At block 628, the detection chamber 120 is loaded with the reconstituted master mix reagent. In order for amplification to be performed, the master mix reagent may be located in the detection chamber 120 where temperature incubation and / or thermocycling occurs. In various examples, air bubbles may interfere with optical detection of the nucleic acid of interest. Thus, the system may remove any air bubbles in the detection chamber 120 after the master mix reagent is loaded. Air bubbles may be managed, for example, by air bubble traps with stagnant chamber geometries, hydrophobic vent membranes over a channel or chamber, columns, posts, filters, elongated vertical chambers for buoyant bubble collection, etc. In various examples, air may be minimized to reduce movement when the solution is heated. Specifically, air on at least one end of the reaction solution may be minimized. For example, a valve may be positioned on one or more entrances and / or exits to the detection chamber 120 to remove air from the detection chamber 120.Atty. Dkt. No.: 86357086

[0218] At block 630, amplification is performed. In various examples, amplification may be or include a PCA reaction, as will be described in greater detail with respect to FIG. 7. Upon amplification, a real-time multi-channel detection may be performed to detect the nucleic acids of interest. For example, an optical unit may detect an absence, presence, and / or amount of the nucleic acid of interest. In various examples, detection may be performed using optical fluorescence and / or electrochemical detection with functionalized surfaces.

[0219] At block 634, the results of the amplification may be analyzed and presented. For example, the results may be analyzed to determine a presence, absence amount, etc. of the nucleic acid of interest. The results may be displayed, for example, via a user interface.

[0220] Referring now to FIG. 7, a method 700 for pulse controlled amplification (PCA) is shown, according to an example implementation. PCA may be performed to amplify the one or more nucleic acids of interest to be able to be detected. In various examples, PCA may be performed in one or more components of the cartridge 100, such as the extraction chamber 114 and / or the detection chamber 120. In various examples, a method other than PCA may be utilized to amplify the one or more nucleic acids of interest. For example, PCR may be used to amplify the nucleic acids.

[0221] At block 702, the lysate containing the nucleic acid of interest is hybridized to capture oligonucleotides. Hybridization may occur in the extraction chamber 114. After hybridization, the nucleotides of interest and the capture oligonucleotides may be transported to the detection chamber 120. The magnetic particles 116 with the capture oligonucleotides are shown in FIG.7 as magnetic particles with capture oligonucleotides 714. In the detection chamber, the magnetic particles may be docked to the heating element 122, shown in FIG. 7 at element 716. For example, the magnetic field generator 156 may generate a magnetic field such that the magnetic particles 116 dock to the heating element 122.

[0222] At block 704, the capture oligonucleotides attached to the docked magnetic particles may undergo elongation to increase a length of the strand or strands of the capture oligonucleotides. For example, if the capture oligonucleotide is RNA, the capture oligonucleotide may undergo reverse transcription. If the capture oligonucleotide is DNA, the capture oligonucleotide may undergo elongation. Transcription or reverse transcription and / or elongation may occur when the detection chamber 120 is at an annealing or elongationAtty. Dkt. No.: 86357086temperature (e.g., between 50 and 80 degrees Celsius). For example, the annealing or elongation temperature may be 72 degrees Celsius.

[0223] At block 706, an electrical pulse 718 may be delivered through the heating element 122. The electrical pulse may generate heat such that a heating zone having the local reaction liquid is heated to a predefined temperature value The predefined temperature value may be, for example, within a range of around 90 to 105 degrees Celsius. For example, the liquid may be heated to 100 degrees Celsius. The predefined temperature value may be a denaturation or melting temperature at which the DNA or RNA denatures. The local reaction liquid may be a portion of the total volume of liquid in the detection chamber 120. For example, the local reaction liquid may be between less than 1% and 5% of the total liquid volume in the reaction chamber. In various examples, the local reaction liquid may be liquid surrounding the magnetic particles 116, the capture oligonucleotides, the nucleic acids of interest, etc. that undergo a reaction / amplification. During heating of the heating element 122 and the local reaction liquid, the capture oligonucleotides attached to the magnetic particles 116 may denature. As a result, the target oligonucleotides may become free in solution. In various examples, the heating zone may quickly return to an annealing or elongation temperature value from the denaturation temperature value. For example, due to a heat capacity of the detection chamber 120, the locally heated area or volume of the detection chamber 120 may rapidly decrease. During pulse delivery (e.g., while the heating element 122 heats a portion of the reaction chamber), temperature changes may occur at a rate greater than 10.000 degrees Celsius per second. For example, the temperature may change at 11.000 degrees Celsius per second.

[0224] At block 708, primers 720 may bind to single strand oligonucleotides on the magnetic particles (e.g., the primers anneal to the denatured oligonucleotides). The primers 720 are short, single-stranded segments of nucleic acid (e.g., DNA) that are designed to be complementary to the beginning and / or end of the target sequence that will be amplified (e.g., the nucleic acid of interest). In some examples, the primers 720 may be forward and / or reverse primers (e.g., denoting a direction of elongation during the polymerization by the polymerase enzyme). In some examples, the primers may be forward and / or reverse primers. Forward and reverse primers may denote a direction of elongation during the polymerization by the polymerase enzyme. The primers 720 are used during the amplification and / or elongation steps of the reaction and may be part of the master mix composition. In some examples, the primers 720 are complementary to the target oligonucleotide.Atty. Dkt. No.: 86357086

[0225] During the amplification / elongation step of the PCR, the primers 720 may bind to the nucleic acid of interest (e.g., the DNA sequence of interest) on each end of the sequence of interest that is to be amplified (e.g., the target nucleic acid is “bookended” by the primers). As will be described herein, enzymes (e.g., DNA polymerase 722) may copy the part of the target oligonucleotide sequence that falls between the primers, selectively amplifying the sequence of interest.

[0226] In various examples, the capture oligonucleotide may also be used as a primer. For example, if the capture oligonucleotide is being used as a reverse primer (e.g., relative directionality during elongation by the polymerase), then forward primers may be free in solution and may bind to the single strand oligonucleotide that are captured (by the capture oligonucleotide that also shares the function of the reverse primer). In other words, the annealing of two primers (as being described here) may first occur. One of the two primers may not be free in solution because it is also the capture oligonucleotide. As such, a free amplicon (e.g., a copy of the target nucleic acids) anneals itself to the capture oligonucleotide that also doubles as the reverse primer. The forward primer may be free in solution to also bind to the amplicon. The binding of the amplicon to the capture oligonucleotide may be the same as or similar to hybridization described above. Further, the reaction may have “forward” and “reverse” primers switch places, where the capture oligonucleotide functions as the forward primer and the free primer in solution is the reverse primer.

[0227] At block 710, the strand of the primers and the oligonucleotides may be elongated. For example, polymerase 722 may elongate the strand. Elongation of the single strand into a double strand may occur between forward and reverse primers attached to the double strand in the annealing step. Thus, elongation may occur between the forward primer and the capture oligonucleotide (which also functions as the reverse primer). During elongation, fluorophores may be released into the solution for detection of the amplified nucleic acids of interest. For example, Taqman probes may be used to release fluorophores into the solution. The optical unit 162 may detect the fluorophores to determine a corresponding value, absence, presence, etc. of the amplified nucleic acid of interest.

[0228] The processes described with respect to blocks 706-710 may be repeated. For example, after the strand has been elongated at block 710, another electrical pulse may occur at block 708. Thus, more oligonucleotides are again denatured, and the process may repeat to generateAtty. Dkt. No.: 86357086a plurality of the nucleic acids of interest, for example, a certain number of times. The number of times may be a predetermined number or may depend upon a number of amplified strands.

[0229] In various examples, a fraction of the released oligonucleotides may be recaptured by capture oligonucleotides on a functionalized magnetic particle. These oligonucleotides may be used for the cyclic amplification reaction, thus causing exponential replication of the target nucleotides.

[0230] FIG. 8A depicts an example blister device 800, in accordance with present implementations. As illustrated by way of example in FIG. 8, the example blister device 800 can include at least a first rupturing member 810 (also referred to herein as a “piercing member”) to open or close a valve 812a (also referred to herein as a “one-time open valve” or a “valve seal”) of the blister device 800 (e.g., a portion of the lidding foil 860 adjacent with the rupturing member), a metal coated polymer film 820, a reagent storage cavity 830, a second rupturing member 840 to open or close valve 812b of blister device 800 (e.g., a portion of the lidding foil 860 adjacent the rupturing member), a first fluid input-output area 850, the lidding foil 860 at least partially corresponding to the reagent storage cavity 830, and a second fluid input-output area 870. One or more zones as discussed herein can comprise or include one or more blister devices similar to the blister device 800. This technical solution is not limited to blister devices discussed by way of example, and is not limited to exclude blister devices at any zone. The blister device 800 may be used to store liquid reagents in the cartridge 100. For example, the system composition and / or the wash buffer 110 may be stored in a blister device 800. The blister device 800 may be attached, coupled, or otherwise affixed to the cartridge 100. The blister device 800 can provide a technical improvement to achieve bidirectional flow between and through zones of a cartridge as discussed herein.

[0231] As shown in FIG. 8 A, the blister device 800 comprises rupturing members 810 and 840 that can be actuated to open the valve 812. The rupturing members 810 and 840 may be integrated into the metal coated polymer film 820. In various examples, the rupturing members 810 and 840 may be used to rupture one-time open valves (e.g., valves 812). One-time open valves 812 may minimize water loss out of the storage cavity 830 through the closed or sealed rupturing members 810 and 840. The rupturing members may allow a common pump (e.g., the syringe pump 402 of FIG. 4) to deliver the contents of the blister device 800 to the microfluidic network of the cartridge 100. For example, the fluid entering and exiting the blister device 800 may enter and / or exit one or both of the one-time open valves 812. Further, the valves 812 mayAtty. Dkt. No.: 86357086allow the reagents stored in the blister device 800 to return to the blister device 800 after use. For example, a blister device 800 may store a wash buffer. The wash buffer may exit the blister device 800 (e.g., through a valve 812) for use in the hybridization or detection chamber to wash the nucleic acids of interest. After washing is complete, the wash buffer may reenter the blister device 800 through the valves 812a and / or 812b. Storing used liquid reagents in the original blister device 800 may reduce the use of additional waste chambers in or on the cartridge 100, which may reduce a cost of manufacturing the cartridge 100 and / or may reduce a size of the cartridge 100. As such, the blister device 800 may additionally function as a waste chamber to store a used or exhausted liquid reagent.

[0232] As described above, the blister device 800 may include one or more one-time open valves 812. The opening of the valves 812 may allow a pump of the cartridge to deliver the reagents 125 stored in a chamber or cavity 830 of the blister device to one or more locations of the cartridge. The valves may also allow the reagents to return to the chamber after use of the reagents. As will be described with respect to FIG. 12, the rupturing members 810 and 840 may interface with an actuator of the instrument 150 to modulate a compression of the one or more rupturing members .

[0233] The blister device 800 may be sealed off from channels and chambers of the cartridge 100 by the rupturing members 810 and 840 and / or the unruptured valves 812. The rupturing members 810 and 840 may cover a first port (e.g., the valve 812a and / or the first fluid inputoutput area 850) when the rupturing member is in a non-ruptured state. The blister device 800 may be in fluid communication with the channels and the chambers of the cartridge through the first port and / or via the first fluid input-output area 850 when the rupturing member is in a ruptured state.

[0234] The blister device 800 may comprise a second port (e.g., the valve seal 812b and / or an area underneath the rupturing member 840 and / or the second input output area 870) and may be sealed-off from the channels and the chambers of the cartridge by the second rupturing member. The blister device 800 may be in fluid communication with the channels and the chambers of the cartridge through the second port and / or via the second fluid input-output area 870 when said the rupturing member is in a ruptured state.

[0235] The blister device 800 may enclose a storage chamber when the storage chamber is configured in hermetically sealed-off relation from the channels and / or when the cartridge ofAtty. Dkt. No.: 86357086the present disclosure is in a non-activated state. The storage chamber is configured for open communication with at least one of the plurality of zones and / or detection chambers such that liquid may flow freely between channels. For example, the blister device 800 may, when ruptured, allow fluid to flow freely between the blister device 800 and the extraction chamber 114.

[0236] In some examples, a first channel can be sealed-off from the blister device 800 by the valve 812a covering a first port when the first valve 812a is in a non-ruptured state. The first channel can be in fluid communication with the blister device 800 through the first port when the first valve 812a is in a ruptured state.

[0237] In some examples, a second channel can be sealed-off from the blister device 800 by the valve seal 812b covering the second port when the second valve 812b is in a non-ruptured state. The second channel can be in fluid communication with the chamber through the second port when the second valve 812b is in a ruptured state.

[0238] The first and second valve seals 812a and 812b may be ruptured by the first and second rupturing members 810 and 840, respectively, to bring the first and second channels into fluid communication with the storage chamber through the first and second ports of the blister device 800. Upon selectively piercing, by the first and second rupturing members 810 and 840, at least one of the first and second valve seals 812a and 812b of the blister device 800 and forming the opening therein, the desired reagent can be introduced through a corresponding one of the channels having an opened port.

[0239] FIGS. 8B, 8C, and 8D depict a detailed view of the rupturing members 810 and / or 840 of the blister device 800 and the valves 812 of FIG. 8A, according to some examples. A chamber between the rupturing members 810 and / or 840 and the valve seals 812a and 812b, respectively, may include first and second fluid input-output areas 850 and 870. In various examples, the rupturing members 810 and / or 840 may be actuated, compressed, or otherwise manipulated to open a valve 812 (also referred to as a “one-time open valve” or a “valve seal”) and permit fluid flow. However, over-travel of the rupturing members 810 and 840 may cause a pressure restriction when liquid attempts to move through the blister device 800 and / in and out of the blister device 800. FIG. 8B depicts the valve 812 of the blister device 800 prior to actuation or travel of the rupturing member 810. The rupturing member 810 and the first inputoutput area 850 are shown. However, the elements of FIGS. 8B, 8C, and 8D may be the sameAtty. Dkt. No.: 86357086or similar for rupturing member 840 and area 870. As shown in FIG. 8B, the rupturing member 810 may be uncompressed, and the valve 812a may be unbroken. The fluid contained in the blister device may be stored in a chamber of the blister device and may enter and exit the blister device from the input output area 850 via a channel 814. FIG. 8C shows the blister device 800 when the rupturing member 810 is not over traveled (e.g., the rupturing member 810 is only partially compressed). This may permit fluid to flow upon actuation of the rupturing member 810. FIG. 8D depicts the valve 812a (e.g., valve seal) of the blister device 800 after actuation, specifically after over-travel of the rupturing member 810. As shown in FIG. 8D, flow may be restricted due to over-travel of the rupturing member 810. For example, in FIG. 8D, the rupturing member 810 has over traveled to reduce a size of the input / output area 850, thus restricting fluid flow. As such, fluid may have difficulty traveling through the blister device 800 and to and from other components of the cartridge 100 via the channel 814.

[0240] FIG. 9A depicts an example blister system 900, in accordance with present implementations. As illustrated by way of example in FIG. 9A, an example blister system 900 can include at least a blister device 902 and one or more actuators 910 having stand offs 920 to control actuation depth, a contact geometry 930 to open a frangible seal of the blister device 902, an actuator 960, and an actuation direction 970. The blister device 902 may include frangible seals 940 (also referred to as a valve, e.g., the valve 812) and a reagent storage cavity 950. In various examples, the blister device 902 may be located in or on the cartridge 100. The actuator 910 may be located in or on the instrument 150. The actuator 910 (and / or 960) may be positioned in the instrument 150 such that the actuator aligns with the seals 940 of the blister device 902 to allow the actuator to break one or more seals of the blister device 902. The blister device 800 can correspond at least partially in one or more of structure and operation to the blister device 902, and can include or couple to one or more of the actuator 910, the stand offs 920, or the actuator 960 by one or more of the contact geometry 930 and the frangible seals 940 to operate the actuators 910 and 960 in the actuation direction 970. For example, the actuation direction 970 can correspond to a first direction of movement of the actuators 910 or 960 toward the frangible seals 940 according to a piercing operation of one or more rupturing members (e.g., the rupturing members 810 and 840) to open one or more of the frangible seals 940. For example, the actuation direction 970 can correspond to a second direction of movement of the actuators 910 or 960 away from the frangible seals 940 subsequent to a piercing operation to open one or more of the frangible seals 940. Various examples can comprise a blister device in accordance with this disclosure, such as (but not limited to) theAtty. Dkt. No.: 86357086examples of the blister devices depicted in FIGS. 8A-9B. The blister system 900 may be part of other devices (e.g., other cartridges and systems) that interface with other instruments and systems.

[0241] As discussed with respect to FIGS. 8A- 8D, over-travel of the piercing elements (e.g., rupturing members) of the blister device 902 may cause flow restriction. The actuator 910 may prevent over constriction. As shown in FIGS. 9 A and 9B, the actuator 910 may include stand offs 920 and a contact geometry. The stand offs 920 and the contact geometry 930 may prevent over travel. For example, the stand offs 920 may extend further or be longer than the contact geometry. Thus, the stand offs 920 may prevent the contact geometry 930 from contacting the seals 940 at too great a depth, thus causing flow restriction.

[0242] FIG. 10A depicts an example system, in accordance with present implementations. As illustrated by way of example in FIG. 10 A, an example system 1000 A can include at least a diagnostic cartridge 1010, a diagnostic instrument 1020, and a dropper 1030 A. The diagnostic cartridge may be the same as or similar to the cartridge 100, and the diagnostic instrument 1020 may be the same as or similar to the instrument 150. Specifically, as will be described in greater detail herein, the cartridge may include a sample input chamber comprising a liquid port. For example, a biological sample may be inserted into the cartridge via the sample input chamber. The cartridge may include a liquid port via which liquid samples may be received.

[0243] The diagnostic cartridge 1010 can receive and interact with a sample fluid by one or more components thereof. For example, the diagnostic cartridge 1010 can include one or more portions corresponding to zones, chambers, channels, reservoirs, containers, or any combination thereof. Specifically, the diagnostic cartridge 1010 may include a plurality of zones. The plurality of zones may include an extraction zone having one or more extraction chambers and a detection zone having one or more detection chambers. Each zone of the plurality of zones may be in fluid communication with each other.

[0244] The diagnostic cartridge 1010 can include one or more contents corresponding to chemicals, biochemicals, liquids, solutions, powders, materials, or any combination thereof, that can be present at or transferable between one or more of portions of the diagnostic cartridge 1010. The diagnostic cartridge 1010 can include one or more components that can interact with one or more corresponding components of the diagnostic instrument 1020. For example, the diagnostic cartridge 1010 can include a heating element located in each detection chamber ofAtty. Dkt. No.: 86357086the one or more detection chambers that can be placed in contact with or proximate to a heating element of the diagnostic instrument 1020, to heat content of a portion of the diagnostic cartridge 1010. The portion of the cartridge 1010 may be heated via an electrical connection to activate the one or more heating elements of each of the one or more detection chambers of the diagnostic cartridge 1010. The diagnostic cartridge 1010 can have a structure corresponding to a rectangular shape of a size corresponding to a palm. The diagnostic cartridge 1010 can have a height greater than a width of the diagnostic cartridge 1010, and a depth greater than a height of the diagnostic cartridge 1010.

[0245] For example, the diagnostic cartridge 1010 can be at least partially insertable into the diagnostic instrument 1020. That is, the cartridge 1010 may interface with the instrument. The instrument 1020 can include a sensor configured to detect the state of the cartridge or its contents. For example, the instrument 1020 can detect whether the cartridge 1010 is fully inserted into the diagnostic instrument 1020 by an optical sensor or the like. For example, the instrument 1020 can detect whether one or more portions of the cartridge are filled or ready for performance of a diagnostic test, by one or more sensors configured to detect fill level of one or more portions.

[0246] The diagnostic cartridge 1010 can couple with the dropper 1030 A via a sample input chamber disposed at a surface of the diagnostic cartridge 1010. The sample input chamber may include a liquid port to receive a liquid, such as the biological sample. The sample input chamber can be disposed at a top surface of the diagnostic cartridge 1010, when the diagnostic cartridge 1010 is oriented to be inserted into the diagnostic instrument 1020. For example, the cartridge is configured to couple to the instrument to provide a user interface presenting at least one of an indication of an amount of the sample fluid in the cartridge or a status of the diagnostic test performed by the cartridge. For example, the cartridge has a physical interface configured to couple the cartridge to a holding device configured to hold the cartridge upright during injection of the sample fluid.

[0247] The diagnostic instrument 1020 can interact with the diagnostic cartridge 1010 and can generate one or more indications corresponding to one or more interactions with the diagnostic cartridge 1010. The diagnostic instrument 1020 can include one or more electrical and electronic components to initiate, control, and / or stop one or more interactions, and to generate one or more indications. For example, the diagnostic instrument 1020 can include one or more sensor devices, motors, actuators, processors, displays, or any combination thereof. TheAtty. Dkt. No.: 86357086diagnostic instrument 1020 can include a receptacle having a width corresponding to a width of the diagnostic cartridge 1010, and a height corresponding to a height of the diagnostic cartridge 1010. The diagnostic instrument 1020 can include one or more logical or electronic devices including but not limited to integrated circuits, logic gates, flip flops, gate arrays, programmable gate arrays, and the like. One or more electrical, electronic, or like devices, or components associated with the diagnostic instrument 1020 can also be associated with, integrated with, integrable with, replaced by, supplemented by, complemented by, or the like, a system processor or any component thereof. For example, an interaction can correspond to one or more chemical, biochemical, electrical, or electrochemical reactions corresponding to identification of one or more chemicals or biochemicals. One or more interactions can result in one or more changes in electrical, optical, chemical, or other characteristics and / or properties inside the cartridge. Such characteristics and / or properties can be detected with one or more sensors to, for example, identify a component (e.g., a molecule or microorganism), to determine a state of the component, to detect the component’s presence, and / or to determine the component’s quantity.

[0248] The diagnostic instrument 1020 can include a system processor that can execute one or more instructions associated with the system 1000, according to any of the depictions 1000A-C of the system 1000. The system processor can include an electronic processor, an integrated circuit, or the like including one or more of digital logic, analog logic, digital sensors, analog sensors, communication buses, volatile memory, nonvolatile memory, and the like. The system processor can include, but is not limited to, at least one microcontroller unit (MCU), microprocessor unit (MPU), central processing unit (CPU), graphics processing unit (GPU), physics processing unit (PPU), embedded controller (EC), or the like. The system processor can include a memory operable to store or storing one or more instructions for operating components of the system processor and operating components operably coupled to the system processor. The one or more instructions can include at least one of firmware, software, hardware, operating systems, embedded operating systems, and the like. The system processor or the diagnostic instrument 1020 generally can include at least one communication bus controller to effect communication between the system processor and the other elements of the system 1000.

[0249] The dropper 1030 A can store contents and expel contents to the diagnostic cartridge 1010. For example, the dropper 1030A can have a cylindrical shape including an inlet at a firstAtty. Dkt. No.: 86357086end and an outlet at a second end. The dropper 1030 A can include a reservoir corresponding to the cylindrical shape. For example, the dropper 1030A can have a reservoir with a volume corresponding to a medical syringe. The inlet of the dropper 1030 can correspond to a cap that can be opened to allow filling of the reservoir with contents. The outlet of the dropper 1030 A can correspond to a nozzle. For example, the nozzle of the dropper 1030A can have a shape that can be mated with the sample input chamber of the diagnostic cartridge 1010. The dropper 1030A can be in a state disconnected from or not mated with the diagnostic cartridge 1010. For example, the dropper 1030A can be in a state prior to or subsequent to filling of the diagnostic cartridge 1010 with the content of the dropper 1030 A.

[0250] FIG. 10B depicts an example system, in accordance with present implementations. As illustrated by way of example in FIG. 10B, an example system 1000B can include at least a dropper 1030B operated by a user 1040 according to a direction 1050. The dropper 1030B can correspond at least partially in one or more of structure and operation to the dropper 1030 A. For example, the dropper 1030B can be in a state corresponding to filling of the diagnostic cartridge 1010 with the content of the dropper 1030 A. The user 1040 can correspond to an individual operating one or more of the dropper 1030B, the diagnostic cartridge 1010, and the diagnostic instrument 1020. For example, the user 1040 can orient the dropper 1030B to align the sample input chamber of the diagnostic cartridge 1010 to face the outlet of the dropper 1030B. For example, the user 1040 can move one or more of the diagnostic cartridge 1010 and the dropper 1030B in the direction 1050 to mate the inlet of the diagnostic cartridge 1010 with the outlet of the dropper 1030B.

[0251] FIG. 10C depicts an example system, in accordance with present implementations. As illustrated by way of example in FIG. 10C, an example system 1000C can include at least a display device 1060. The display device 1060 can present one or more indications of one or more biochemical characteristics associated with a sample fluid, and can include an electronic display. An electronic display can include, for example, a liquid crystal display (LCD), a lightemitting diode (LED) display, an organic light-emitting diode (OLED) display, or the like. The display device 1060 can receive, for example, capacitive or resistive touch input. The display device 1060 can be housed at least partially within the diagnostic instrument 1020. The display device 1060 can present one or more indications via one or more user interfaces that can include one or more graphical presentations and graphical control affordances. For example, a control affordance can include a portion of a user interface configured to detect user input. ExampleAtty. Dkt. No.: 86357086I / O components thus include, without limitation, a touchscreen display, a keypad or keyboard, biometric sensors such as a fingerprint scanners, buttons, switches, computer mice, microphones (e.g., for voice inputs such as test commands (e.g., “begin test”), passcodes (e.g., passcode known to authorized user), and / or voice recognition (e.g., analysis of voice signature of a user to record identity and / or compare voice signature for authentication or identity confirmation), speakers (e.g., for status updates such as “test in progress” or “test complete” or for speaking instructions for how to use an instrument or cartridge such as “insert cartridge further”), and / or other input / output devices.

[0252] In various examples, the diagnostic cartridge 1010 may include one or more extraction chambers (e.g., extraction chambers 114), one or more detection chambers (e.g., detection chamber 120 one or more reagents (e.g., reagents 125), and a plurality of magnetic particles (e.g., magnetic particles 116).

[0253] At least one of the one or more extraction chambers is a hybridization chamber to hybridize the one or more nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles. Further, at least one of the one or more detection chambers is an amplification chamber and one or more heating elements of the amplification chamber is a foil to interact with an electrical connection of an instrument to provide heat modulation to amplify the one or more nucleic acids of interest that have been hybridized to the at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles.

[0254] The diagnostic cartridge may include a transparent window in at least one detection chamber of the one or more detection chambers to allow an optical unit in communication with the at least one detection chamber to detect a plurality of amplification products indicative of a presence, absence, or amount of the plurality of amplified nucleic acids of interest.

[0255] The one or more extraction chambers may be used to lyse the biological sample to release one or more nucleic acids of interest from the biological sample. In various examples, one of the one or more extraction chambers may be a hybridization chamber to hybridize the one or more nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles.

[0256] Each of the one or more detection chambers may include a heating element at side portion of the detection chamber. The heating element may include a plurality of layers. ForAtty. Dkt. No.: 86357086example, a layer of the heating element may be a foil. In various examples, the plurality of layers may further include a heat spreading element and / or an adhesive.

[0257] At least one of the one or more detection chambers may be an amplification chamber configured to amplify the nucleic acids of interest. One or more heating elements of the amplification chamber may be a foil to interact with an electrical connection of an instrument to provide heat modulation to amplify the one or more nucleic acids of interest that have been hybridized (e.g., in the extraction chambers 114) to the at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles.

[0258] The cartridge may further include one or more blister devices. Each blister device may store reagents (e.g., the wash composition). Each of the one or more blister devices may include one or more one-time open valves to allow a pump of the cartridge to deliver the reagents stored in a chamber of the blister device to one or more locations of the cartridge. The valves may also allow the reagents to return to the chamber after use of the reagents. The one or more onetime open valves may interface with an actuator of an instrument configured to modulate travel of a piercing element to open a valve or seal.

[0259] FIG. 11A depicts an example cartridge environment, in accordance with present implementations. As illustrated by way of example in FIG. 11 A, an example cartridge environment 1100 A can include at least a lower fill indication member 1110, an upper fill indication member 1112, a face 1120, a side portion 1130, and a view window 1140A. The cartridge environment 1100 A can correspond to a first state of a portion 1100 of the system 1000 according to one or more of systems 1000A-C as illustrated by way of example in FIG.10A-C.

[0260] The lower fill indication member 1110 can correspond to a physical component of the diagnostic cartridge 1010. The lower fill indication member 1110 can be located at a position that indicates a minimum fill level of a liquid with respect to the view window 1140A. For example, the lower fill indication member 1110 can indicate whether an amount of sample fluid from the dropper 1030B meets or exceeds a first vertical level in the view window 1140 A with respect to a particular diagnostic test. For example, the lower fill indication member 1110 can be formed or placed at a location over the view window 1140 A along a vertical direction of the view window 1140A to indicate a minimum amount of fluid corresponding to a particular diagnostic test. For example, the lower fill indication member 1110 can be formed as aAtty. Dkt. No.: 86357086component of the diagnostic cartridge 1010 at a first vertical position to indicate a minimum amount of fluid corresponding to a COVID-19 diagnostic test. For example, the lower fill indication member 1110 can be formed as a component of the diagnostic cartridge 1010 at a second vertical position to indicate a minimum amount of fluid corresponding to a seasonal flu diagnostic test. For example, the cartridge 1010 can include the predetermined position corresponding to a minimum amount of the sample fluid needed for performance of the diagnostic test.

[0261] The upper fill indication member 1112 can correspond to a physical component of the diagnostic cartridge 1010 distinct from the lower fill indication member 1110. The upper fill indication member 1112 can be located at a position that indicates a maximum fill level of a liquid with respect to the view window 1140 A. For example, the upper fill indication member 1112 can indicate whether an amount of sample fluid from the dropper 1030B meets or exceeds a second vertical level in the view window 1140 A with respect to a particular diagnostic test. For example, the upper fill indication member 1112 can be formed or placed at a location over the view window 1140A along a vertical direction of the view window 1140A to indicate a maximum amount of fluid corresponding to a particular diagnostic test. For example, the upper fill indication member 1112 can be formed as a component of the diagnostic cartridge 1010 at a third vertical position to indicate a maximum amount of fluid corresponding to a CO VID-19 diagnostic test. For example, the upper fill indication member 1112 can be formed as a component of the diagnostic cartridge 1010 at a fourth vertical position to indicate a maximum amount of fluid corresponding to a seasonal flu diagnostic test.

[0262] The face 1120 can at least partially frame view window 1140 A. The face 1120 can at least partially integrate with or connect with one or more of the lower fill indication member 1110 and the upper fill indication member 1112. For example, the face 1120 can be formed to include a portion of a component of the diagnostic cartridge 1010 defining an opening at least partially surrounding the view window 1140 A. For example, the face 1120 can be formed with one or more of the lower fill indication member 1110 and the upper fill indication member 1112. For example, the face 1120, the lower fill indication member 1110, and the upper fill indication member 1112 can be integrally formed of a single solid piece. For example, the single solid piece can include or be a stiff polymer or plastic. The face 1120 can be oriented according to a face plane that intersects a top plane corresponding to the top surface of the diagnostic cartridge 1010 and a front plane corresponding to the front surface of the diagnosticAtty. Dkt. No.: 86357086cartridge 1010. For example, the face plane can be oriented at a 45 degree angle with respect to one or more of the top plane and the front plane. Thus, the face 1120 can provide a technical improvement to increase visibility of a view window 1140A during a filling process of the diagnostic cartridge 1010.

[0263] The side portion 1130 can correspond to a component of the diagnostic cartridge 1010 covering at least a portion of the diagnostic cartridge 1010. For example, the side portion 1130 can be integrally formed with one or more of the lower fill indication member 1110, the upper fill indication member 1112, and the face 1120. For example, the face 1120, the lower fill indication member 1110, and the upper fill indication member 1112 can be integrally formed of a single solid piece with the side portion 1130.

[0264] The view window 1140A can correspond to at least a portion of a reservoir of the diagnostic cartridge 1010 that aligns with an opening in the face 1120. For example, the reservoir of the diagnostic cartridge 1010 can be disposed along the face plane to render visible at least a portion of side wall through which a fill level of a liquid in the reservoir. For example, at least the portion of the reservoir aligned with the opening of the face 1120 can include or be a transparent or translucent material. The view window 1140 A can correspond to a state of a view window 1140 having no fluid visible therethrough. For example, the view window 1140A can frame a reservoir of the diagnostic cartridge 1010 absent any fluid or containing an amount of fluid below a threshold of visibility in the view window 1140A.

[0265] FIG. 11B depicts an example cartridge environment, in accordance with present implementations. As illustrated by way of example in FIG. 11B, an example cartridge environment 1100B can include at least a view window 1140B, and a fluid 1150 visible from view window 1140B.

[0266] The view window 1140B can correspond at least partially in one or more of structure and operation to the view window 1140B. The view window 1140B can correspond to a state of a view window 1140 having fluid 1150 visible therethrough. For example, the view window 1140B can frame a reservoir of the diagnostic cartridge 1010 containing an amount of liquid visible in the view window 1140A. For example, the fluid 1150 visible from view window 1140B can be at a level above the lower fill indication member 1110 and below the upper fill indication member 1112. Thus, the lower fill indication member 1110 and the upper fill indication member 1112 can provide a technical improvement to increase visibility of fluidAtty. Dkt. No.: 863570861150 deposited to the diagnostic cartridge 1010 during depositing of the fluid 1150 by the user 1040.

[0267] FIG. 12A depicts an example cartridge environment in a cross-sectional view, in accordance with present implementations. As illustrated by way of example in FIG. 12 A, an example cartridge environment 1200 A in a cross-sectional view can include at least an upper portion 1210, a lower portion 1212, a reservoir 1220, a fluid 1230 A in the dropper 1030B, a fluid 1232A in the reservoir 1220, and an air gap 1234A between the dropper 1030B and the reservoir 1220. The cartridge environment 1200A can correspond to a first filling state including the diagnostic cartridge 1010 and the dropper 1030B. For example, the first filling state can correspond to a start of a transfer of fluid 1230 A from the dropper 1030B to the diagnostic cartridge 1010.

[0268] The upper portion 1210 can correspond to a portion of the reservoir 1220 structured to couple with or mate with the outlet of the dropper 1030B. For example, the upper portion 1210 can define an opening extending from a top surface of the diagnostic cartridge 1010 to the reservoir 1220. For example, the upper portion 1210 can define an opening having a cross section corresponding to a cross section of the outlet of the dropper 1030B. For example, the opening of the upper portion 1210 can have a circular cross section corresponding to a circular cross section of the dropper 1030B.

[0269] The lower portion 1212 can correspond to a portion of the reservoir 1220 structured to transport and store fluid. For example, the lower portion 1212 can extend in a direction corresponding to the face plane. For example, the lower portion 1212 can be integrally formed with the upper portion 1210 at an angle corresponding to an angle between the face plane and either the top plane or the front plane, or both. The position of the lower portion 1212 at the angle can result in the lower portion having a sloped surface 1214. The sloped surface 1214 of the lower portion 1212 can be oriented to allow transport of the fluid 1232A without or mitigating dispersion of the fluid 1232A in the reservoir 1220. For example, the sloped surface 1214 can be oriented to prevent “breakage” of the fluid and to transport fluid in one body or a minimum number of bodies according to a surface tension of the fluid. For example, the sloped surface 1214 can be formed or oriented to achieve a technical improvement to prevent or minimize formation of separate droplets of the fluid 1232A. For example, the sloped surface 1214 can be formed or oriented to achieve a technical improvement to prevent or minimize formation of bubbles in the fluid 1232A. For example, the sloped surface 1214 can have anAtty. Dkt. No.: 86357086angle of 45 degrees with respect to the top plane of the diagnostic cartridge 1010, to mitigate dispersion of a fluid 1232A having a surface tension property corresponding, for example, to at least one of a liquid sample or a liquid sample mixed or otherwise combined with a transport medium. For example, a transport medium can correspond to or include a commercial transport medium, such as Copan UTM ®.

[0270] The reservoir 1220 can include both the upper portion 1210 and the lower portion 1212. For example, the reservoir 1220 can have a cylindrical shape enclosing a cavity that abuts the face 1120 and the view window 1240 A. For example, the reservoir 1220 can be attached with a body of the diagnostic cartridge 1010 and can include or be a transparent or translucent material. The reservoir 1220 can have one or more dimensions to achieve a technical improvement to eliminate or mitigate dispersion of the fluid 1232A in the reservoir 1220. For example, the reservoir 1220 can be formed with one or more of a particular inner depth 1222 and a particular outer depth 1224 corresponding to a surface tension property corresponding to at least one of a liquid sample or a liquid sample mixed or otherwise combined with a transport medium. For example, the inner depth 1222 can be between about 10 millimeters (mm) and about 20 mm, and can be about 15 mm. For example, the outer depth 1224 can be about 4 mm greater than the inner depth 1222. For example, the inner depth can correspond to a horizontal direction from a front of the reservoir 1220 at the view window 1140 A to a rear of the reservoir 1220 at a surface opposite to the front of the reservoir in the horizontal direction. For example, the horizontal direction can be parallel to the top plane of the diagnostic cartridge 1010. For example, the reservoir can have a depth ranging from about 5 mm to about 10 mm, and a width ranging from about 5 mm to about 10 mm. For example, the reservoir can correspond to a chamber. For example, the chamber can include a first surface coupled to the opening, and a second surface that is sloped with respect to the first surface such that the sample fluid flows down the second surface when the cartridge is upright. For example, the system can include a reservoir that has a depth between about 5 mm and about 10 mm and a width between about 5 mm and about 10 mm.

[0271] The fluid 1230 A in the dropper 1030B can be transported between the dropper 1030B mated with the reservoir 1220. For example, the fluid 1230A can be expelled from the outlet of the dropper 1030B into the upper portion 1210 of the reservoir 1220 and onto the sloped surface 1214 of the lower portion 1212 of the reservoir 1220. The outlet of the dropper 1030B can be positioned at a distance from the sloped surface 1214 corresponding to a distance toAtty. Dkt. No.: 86357086mitigate or eliminate dispersion of the fluid 1232A in the reservoir 1220. The fluid can flow into the reservoir and fill the reservoir 1220 according to a fill line, with minimal or no dispersion that may render the fill level of the fluid 1232A difficult or impossible to detect visually. The air gap 1234A between the dropper 1030B and the reservoir 1220 can provide an outlet for air to escape the reservoir 1220 as it is replaced by the fluid 1232A. Thus, the air gap 1234A can provide the technical improvement of mitigating or preventing dispersion of the fluid 1232A by providing a pathway for air to flow that prevents or mitigates pressurization of or foaming of the fluid 1232A.

[0272] FIG. 12B depicts an example cartridge environment in a cross-sectional view, in accordance with present implementations. As illustrated by way of example in FIG. 12B, an example cartridge environment 1200B in a cross-sectional view can include at least a fluid 1230A in the dropper 1030B, a fluid 1232B in the reservoir 1220, and an air gap 1234B between the dropper 1030B and the reservoir 1220. The cartridge environment 1200B can correspond to a second filling state including the diagnostic cartridge 1010 and the dropper 1030B. For example, the first filling state can correspond to a continuation of a transfer of fluid from the dropper 1030B to the diagnostic cartridge 1010, subsequent to the first filling state.

[0273] For example, the dropper 1030B can be removed from the inlet of the upper portion 1210 of the reservoir 1220 at any point during a filling process of the reservoir. However, the dropper 1030B does not need to be removed from the inlet of the upper portion 1210 to complete a filling process. The fluid 1230B can correspond to an amount of fluid less than an amount of the fluid 1230A. The fluid 1232B can correspond to an amount of fluid greater than an amount of the fluid 1232A. For example, the fluid 1232B can continue to flow into the reservoir 1220 with an absence of formation of bubbles or droplets. The air gap 1234B can be increased to the size of the inlet of the upper portion 1210 of the reservoir 1220 upon removal of the dropper 1030B from the inlet of the upper portion 1210.

[0274] FIG. 12C depicts an example cartridge environment in a cross-sectional view, in accordance with present implementations. As illustrated by way of example in FIG. 12C, an example cartridge environment 1200C in a cross-sectional view can include at least a fluid 1230C in the dropper 1030B, and a fluid 1232C in the reservoir 1220. The cartridge environment 1200C can correspond to a third filling state including the diagnostic cartridge 1010 and the dropper 1030B. For example, the third filling state can correspond to a continuation of a transfer of fluid from the dropper 1030B to the diagnostic cartridge 1010,Atty. Dkt. No.: 86357086subsequent to the second filling state. The fluid 1230C can correspond to an amount of fluid less than an amount of the fluid 1230A. The fluid 1232C can correspond to an amount of fluid greater than an amount of the fluid 1232B. For example, the fluid 1232C can continue to flow into the reservoir 1220 with an absence of formation of bubbles or droplets.

[0275] FIG. 12D depicts an example cartridge environment in a cross-sectional view, in accordance with present implementations. As illustrated by way of example in FIG. 12D, an example cartridge environment 1200D in a cross-sectional view can include at least a fluid 1230D in the dropper 1030B, and a fluid 1232D in the reservoir 1220. The cartridge environment 1200D can correspond to a fourth filling state including the diagnostic cartridge 1010 and the dropper 1030B. For example, the fourth filling state can correspond to a continuation of a transfer of fluid from the dropper 1030B to the diagnostic cartridge 1010, subsequent to the third filling state. The fluid 1230D can correspond to an amount of fluid less than an amount of the fluid 1230C, or an absence of fluid in the dropper 1030B. The fluid 1232D can correspond to an amount of fluid greater than an amount of the fluid 1232C. For example, the fluid 1232D can complete a flow into the reservoir 1220 with an absence of formation of bubbles or droplets. For example, a level of the fluid 1232 can be visible through the view window 1140B according to a level corresponding to the fill level of the reservoir 1220.

[0276] FIG. 13 A depicts an example cartridge environment in plan view, in accordance with present implementations. As illustrated by way of example in FIG. 13 A, an example cartridge environment 1300 A in plan view can include at least a cartridge inlet 1310A, and an inlet cover 1320A. The cartridge inlet 1310A can correspond at least partially in one or more of structure and operation to the inlet of the upper portion 1210 of the reservoir 1220. The cartridge inlet 1310A can correspond to an open state that allows the outlet of the dropper 1030 A to mate with the cartridge inlet 1310A. In some examples, the inlet cover 1320A can slide from an open position to a closed position (e.g., similar to or the same as the sample input cover 303b). In some examples, the inlet cover can be a cap that can close or open over the cartridge inlet 1310A (e.g., similar to or the same as the sample input cap 303a). For example, the inlet cover 1320A can be at a position corresponding to the open state that allows the outlet of the dropper 1030A to mate with the cartridge inlet 1310A. For example, the cartridge can include a lid moveable to cover and seal the opening, the lid configured to permit the diagnostic test to proceed when covering the opening.Atty. Dkt. No.: 86357086

[0277] FIG. 13B depicts an example cartridge environment in plan view, in accordance with present implementations. As illustrated by way of example in FIG. 13B, an example cartridge environment 13006 in plan view can include at least a cartridge inlet 1310B, and an inlet cover 1320B. The cartridge inlet 1310B can correspond at least partially in one or more of structure and operation to the cartridge inlet 1310A. The cartridge inlet 1310B can correspond to a closed state that prevents or blocks the outlet of the dropper 1030 A from mating with the cartridge inlet 1310A, and prevents or blocks egress of fluid in the reservoir 1220. The inlet cover 1320B can correspond to a closed position, subsequent to a filling operation of the reservoir 1220. For example, the inlet cover 1320B can be at a position corresponding to the closed state that prevents or blocks the outlet of the dropper 1030 A from mating with the cartridge inlet 1310B.

[0278] This technical solution can provide at least a technical improvement to mitigate or eliminate contamination into an environment external to the cartridge 1010, by mitigating or eliminating dispersion of a sample fluid into an environment exterior to the cartridge 1010 via the cartridge inlet 1310. For example, the cartridge 1010 can transition between cartridge environments 1300A-B by movement of a cartridge at a predetermined velocity or within a range of predetermined velocities. For example, the inlet cover 1320A can move via a spring having a tension sufficiently low as to prevent a “snap” of the inlet cover 1320A into the depicted position of FIG. 13B. The “snap” can correspond to a speed of traversal of the inlet cover 1320 sufficient to cause ejection of at least a portion sample flid from the reservoir 1220 via the cartridge inlet 1310. For example, a speed of traversal can be greater than or equal to 0.5 seconds, but is not limited thereto. For example, the speed of traversal can be based on a spring coupling the inlet cover 1320A to the upper portion 1210 of the reservoir 1220.

[0279] For example, the technical solution can include the technical improvement to mitigate contamination into an environment external to the cartridge 1010, by a combination of structures of the cartridge 1010, to achieve the technical improvement of substantial mitigation, up to complete elimination, of dispersion of a sample fluid into an environment exterior to the cartridge 1010 via the cartridge inlet 1310. For example, the cartridge inlet 1310, corresponding to the upper portion 1210 of the reservoir 1220, can be structured to minimize dispersion by a structure to allow a tip of the dropper 1030 to be fully inserted into the upper portion 1210, with an allowance restricted to the air gap 1234A. Further, the inlet cover 1320 can be structured to mitigate or eliminate contact with sample fluid that has potentially been dispersed onto the top surface of the cartridge 1010. For example, the inlet cover 1320 can be structuredAtty. Dkt. No.: 86357086to have a shape that covers the cartridge inlet 1310 including the cartridge inlet 1310 and a portion of the top surface of the cartridge 1010 within a predetermined distance of the cartridge inlet 1310. For example, the predetermined distance can correspond to a square or rectangular region of the top surface located surrounding the cartridge inlet 1310, as illustrated by way of example in Fig. 13 A.

[0280] FIG. 14 depicts an example cartridge panel, in accordance with present implementations. As illustrated by way of example in FIG. 14, an example cartridge panel 1400 can include at least a front portion 1420, and a top portion 1430. The panel 1400 can correspond to a single piece integrally formed to include the lower fill indication member 1110, the upper fill indication member 1112, the face 1120, the side portion 1130, to define the view window 1140. The face 1120 can include the lower fill indication member 1110, the upper fill indication member 1112, the face 1120, and the view window 1140. The top portion 1430 can include the cartridge inlet 1310, and the inlet cover 1320. For example, the cartridge inlet 1310 can be integrally formed with the top portion 1430, and the inlet cover 1320 can be attachable to the top portion 1430 to moveably slide at least from the open position to the closed position along the top portion 1430. The panel 1400 can achieve a technical improvement to provide a customizable cartridge indication via members 1110 and 1112 that can be formed to correspond to a particular diagnostic test, and can be attached to a diagnostic cartridge 1010 to customize the diagnostic cartridge 1010 to the diagnostic test and to customize the members 1110 and 1112 for the diagnostic test. This technical solution is not limited to the cartridge panel 1400. For example, at least one of the cartridge inlet 1310 or the inlet cover 1320 can be integrally formed with, integrated with, attached with, or coupled with the reservoir 1220.

[0281] FIG. 15 depicts an example user interface for cartridge environment, in accordance with present implementations. As illustrated by way of example in FIG. 15, an example user interface 1500 for a cartridge environment can include at least an environment presentation 1510, a diagnostic presentation 1520, and a reservoir presentation 1530. The user interface 1500 can be presented on or by the display device 1060. For example, the system can include a device configured to securely couple with the cartridge and provide a user interface. The user interface 1500 can be configured to present an indication of a status of the diagnostic test performed by the cartridge.

[0282] The environment presentation 1510 can present a visual representation of one or more of the diagnostic cartridge 1010 and the diagnostic instrument 1020. For example, theAtty. Dkt. No.: 86357086environment presentation 1510 can present, at a first portion of the user interface, an indication of an arrangement of the diagnostic cartridge 1010 with respect to the diagnostic instrument 1020. For example, the arrangement can correspond to an attachment or mating of the diagnostic cartridge 1010 with the diagnostic instrument 1020 by at least partially inserting the diagnostic cartridge in the diagnostic instrument 1020.

[0283] The diagnostic presentation 1520 can present a visual indication of a diagnostic test corresponding to the diagnostic cartridge 1010 or a diagnostic test corresponding to the diagnostic cartridge 1010 or the panel 1400. For example, the diagnostic presentation 1520 can present, at a second portion of the user interface, an indication of identifiers of the diagnostic test. For example, the identifiers can include an identification of one or more aspects of the test, the patient, the cartridge, or any combination thereof.

[0284] The reservoir presentation 1530 can present a visual indication of a fill level corresponding to a diagnostic test or the panel 1400 with respect to one or more of the member 1110 and 1112. For example, the reservoir presentation 1530 can include one or more members 1110 and 1112 present according to the member 1110 and 1112 on the panel 1400. For example, the reservoir presentation 1530 can present a visual indication including both the members 1110 and 1112, according to a panel 1400 that includes both the members 1110 and 1112. For example, the reservoir presentation 1530 can present a visual indication including only the member 1110, according to a panel 1400 that includes only the member 1110. For example, the reservoir presentation 1530 can present a visual indication including only the member 1112, according to a panel 1400 that includes only the member 1112. For example, the reservoir presentation 1530 can prompt the user to perform a filling operation according to a diagnostic test to level indicated by one or more of the members formed according to the diagnostic test. For example, the device can include where the one or more prompts correspond to injection of the sample fluid into the cartridge. For example, the device can include where the one or more prompts correspond to performance of the diagnostic test.

[0285] Aspects of this technical solutions disclosed herein may include a cartridge such as, in various examples, a cartridge consistent with the above disclosure. The cartridge may receive sample fluids to be tested. The cartridge may include components with structures to mitigate erroneous indications of amounts of sample fluid, during deposition of the sample fluid. A diagnostic testing architecture can include a single-use or limited-use cartridge that includes one or more contents that can interact with a sample fluid to perform a particular medicalAtty. Dkt. No.: 86357086diagnostic by triggering one or more physical reactions with at least a portion of a sample fluid deposited at the cartridge. For example, contents can correspond to chemicals, liquids, or solids having particular chemical, biological, electrical, or mechanical properties, or any combination thereof. The diagnostic testing architecture can include a diagnostic testing device (e.g., an instrument) to detect the results or effects of one or more interactions with the sample fluid at the cartridge, and can determine and output one or more results corresponding to the results or effects of the one or more interactions. Thus, this technical solution can include one or more components including but not limited to one or more of a cartridge configured according to a particular diagnostic test, a dropper operable to deposit a sample fluid to the cartridge or a component thereof, and a diagnostic instrument to exchange signals with the cartridge and generate or output an indication corresponding to receipt of the sample fluid and / or to an interaction between the sample fluid and one or more portions of the cartridge.

[0286] At least one aspect is directed to one or more components that can be combined or assembled, for example, to form a cartridge configured to perform a particular diagnostic. For example, a cartridge can include one or more zones that can include one or more contents as discussed herein, according to a diagnostic to be performed. For example, a diagnostic can correspond to a medical diagnostic. For example, a medical diagnostic can include one or more tests for the presence of indicators of a molecule, microorganism, disease, or condition. In an example, a cartridge may comprise one or more diagnostic tests to detect indicators of one or more viruses (or components thereof) that can cause one or more diseases such as COVID-19, influenza, etc. The cartridge can include a body including one or more zones having one or more contents, and a panel portion including one or more indicators corresponding to a particular diagnostic. For example, a body having a reservoir integrated therewith can couple with a panel having a view window that can be aligned with a portion of the reservoir, in accordance with an attachment of the body with the panel. The panel can include one or more members that can be positioned at one or more locations relative to the reservoir to indicate various fill levels of the reservoir. The fill levels may indicate amounts of sample fluid suitable for performing a particular diagnostic. For example, the panel can be configured to include one or more of a minimum fill indicator and / or a maximum fill indicator that can align with the reservoir of the body and that can be positioned based on how much of a sample fluid is to be injected into the cartridge for the diagnostic. Thus, the panel can provide a technical solution to customize one or more indicators corresponding to a particular diagnostic.Atty. Dkt. No.: 86357086

[0287] At least one aspect is directed to a cartridge that can include a reservoir and an inlet structured to receive sample fluid, and to minimize or eliminate dispersion of the fluid in a manner that can result in an incorrect indication of sample fluid in the view window corresponding to the reservoir. For example, a user can input a sample fluid into a cartridge via an inlet located at an upper portion of the cartridge while concurrently viewing the view window and a portion of the reservoir aligned with the view window. The reservoir can include a transparent or translucent material to permit viewing of the sample fluid within the reservoir from the view window. The user can also view, at the view window, one or more members indicating at least one of a minimum fill level or a maximum fill level of the reservoir. The user can deposit sample fluid into the reservoir via the inlet while viewing the view window and the members to ensure that a fill level of the sample fluid in the reservoir appears above or below the members. This technical solution can accurately indicate a fill level of a reservoir during the deposit of the sample fluid, at least by a structure of the reservoir to minimize or eliminate dispersion of the sample fluid that may cause a fill level to appear dispersed or unclear. For example, formation of bubbles during depositing of a sample fluid can result in a layer of foam that may cause the volume of a fluid in the reservoir to appear incorrectly greater than its actual volume. Thus, this technical solution can provide at least a technical improvement to eliminate dispersion of a fluid during deposition to decrease risk of incorrect fluid sample deposit by a user to a cartridge, and thus reduce or eliminate waste of cartridge devices, biological specimens, or any combination thereof.

[0288] The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” “having,” “containing,” “involving,” “characterized by,” “characterized in that,” and variations thereof herein, is meant to encompass the items listed thereafter, equivalents thereof, and additional items, as well as alternate implementations consisting of the items listed thereafter exclusively. In one implementation, the systems and methods described herein consist of one, each combination of more than one, or all of the described elements, acts, or components.

[0289] References to “or” may be construed as inclusive so that any terms described using “or” may indicate any of a single, more than one, and all of the described terms. References to at least one of a conjunctive list of terms may be construed as an inclusive OR to indicate any of a single, more than one, and all of the described terms. For example, a reference to “at least one of ‘A’ and ‘B’” can include only ‘A,’ only ‘B,’ as well as both ‘A’ and ‘B.’ Such referencesAtty. Dkt. No.: 86357086used in conjunction with "comprising" or other open terminology can include additional items. References to “is” or “are” may be construed as nonlimiting to the implementation or action referenced in connection with that term. The terms “is” or “are” or any tense or derivative thereof, are interchangeable and synonymous with "can be" as used herein, unless stated otherwise herein.

[0290] Directional indicators depicted herein are example directions to facilitate understanding of the examples discussed herein and are not limited to the directional indicators depicted herein. Any directional indicator depicted herein can be modified to the reverse direction or can be modified to include both the depicted direction and a direction reverse to the depicted direction, unless stated otherwise herein. While operations are depicted in the drawings in a particular order, such operations are not required to be performed in the particular order shown or in sequential order, and all illustrated operations are not required to be performed. Actions described herein can be performed in a different order. Where technical features in the drawings, detailed description or any claim are followed by reference signs, the reference signs have been included to increase the intelligibility of the drawings, detailed description, and claims. Accordingly, neither the reference signs nor their absence have any limiting effect on the scope of any clam elements.

[0291] The various ranges provided herein include the stated range and any value or sub-range within the stated range. Furthermore, when “about” is utilized to describe a value or percentage this includes, refers to, and / or encompasses variations (up to + / - ten %) from the stated value or percentage. In describing and claiming the examples disclosed herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0292] Although specific examples have been illustrated and described herein, a variety of alternate and / or equivalent implementations may be substituted for the specific examples shown and described without departing from the scope of the present disclosure. This application is intended to cover any adaptations or variations of the specific examples discussed herein. For example, the method 200 may include additional operations not explicitly recited or may exclude certain recited operations in some examples. Various examples of the cartridge 100 or the instrument 150 may include additional components not explicitly recited, may exclude certain recited components, or may include the recited components in different relative positions than shown in the examples described above. Therefore, it is intended that the scope of this disclosure be limited only by the claims and the equivalents thereof.

Claims

Atty. Dkt. No.: 86357086WHAT IS CLAIMED IS:

1. A diagnostic system comprising:a cartridge comprising a plurality of zones in fluid communication with each other, comprising:an extraction zone comprising one or more extraction chambers comprising:a plurality of magnetic particles, wherein each of the one or more extraction chambers is configured to hold the plurality of magnetic particles; anda waste storage chamber to receive a lysate; anda detection zone comprising one or more detection chambers, each detection chamber comprising one or more heating elements; andan instrument to interface with the cartridge, comprising:a lyse system to interface with at least one of the one or more extraction chambers; anda plurality of magnetic field generators, wherein the plurality of magnetic field generators comprise:at least one magnetic field generator to generate a magnetic field to dock the plurality of magnetic particles to at least one of the one or more extraction chambers; andat least one magnetic field generator to force the plurality of magnetic particles towards the one or more heating elements.

2. The diagnostic system of claim 1, further comprising:a controller comprising one or more non-transitory computer-readable storage media storing instructions thereon that, when executed by one or more processors, cause the one or more processors to:lyse a biological sample into at least one of the extraction chambers to release one or more nucleic acids of interest from the biological sample, wherein lysing the biological sample produces a lysate; andhybridize, into the at least one extraction chamber, the one or more nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles.Atty. Dkt. No.: 863570863. The diagnostic system of claim 2, wherein the instructions further cause the one or more processors to:dispense a first wash buffer into the at least one of the one or more extraction chambers, the first wash buffer to wash the hybridized one or more particles of the plurality of magnetic particles.

4. The diagnostic system of claim 3, wherein the instructions further cause the one or more processors to:dispense a second wash buffer into the at least one of the one or more extraction chambers to perform a second wash, the second wash to trap the hybridized one or more magnetic particles of the plurality of magnetic particles and suspend the hybridized one or more magnetic particles of the plurality of magnetic particles within the second wash buffer; andtransfer the hybridized one or more magnetic particles to at least one detection chamber of the one or more detection chambers via the second wash buffer.

5. The diagnostic system of claim 2, wherein the lyse system comprises a sonotrode, and wherein instructions cause the one or more processors to lyse the biological sample via the sonotrode.

6. The diagnostic system of claim 1, wherein the one or more detection chambers comprise one or more amplification chambers or one or more pulse-controlled amplification (PCA) chambers.

7. A method for detecting a presence, absence, or amount of a nucleic acid of interest, the method comprising:inserting, into a cartridge, a fluid comprising a biological sample, wherein the cartridge comprises a plurality of zones comprising an extraction zone comprising one or more extraction chambers, each extraction chamber comprising a plurality of magnetic particles, and a detection zone comprising one or more detection chambers, each detection chamber comprising one or more heating elements, wherein each zone of the plurality of zones is in fluid communication with each other, and wherein the cartridge further comprises one or more reagents;Atty. Dkt. No.: 86357086lysing the biological sample into at least one of the one or more extraction chambers to release the one or more nucleic acids of interest from the biological sample, wherein lysing the biological sample produces a lysate;hybridizing, into the extraction chamber, the one or more nucleic acids of interest with at least one capture oligonucleotide attached to one or more magnetic particles of the plurality of magnetic particles;holding the hybridized one or more magnetic particles on a side of the extraction chamber;washing the hybridized one or more magnetic particles with at least one wash buffer; trapping the hybridized one or more magnetic particles into proximity of the one or more heating elements of the at least one detection chamber;amplifying the one or more nucleic acids of interest in at least one of the plurality of zones, via an amplification reaction, to provide a plurality of the one or more nucleic acids of interest; anddetecting a plurality of amplification products indicative of the presence, absence, or amount of the plurality of amplified nucleic acids of interest via an optical unit in communication with the at least one detection chamber of the one or more detection chambers.

8. The method of claim 7, further comprising:suspending the hybridized one or more magnetic particles;transporting the hybridized one or more magnetic particles to at least one detection chamber of the one or more detection chambers; anddelivering a plurality of amplification reagents to the at least one detection chamber.

9. The method of claim 8, wherein lysing the biological sample is performed via sonication.

10. The method of claim 7, wherein the hybridized one or more magnetic particles are held on the side of the extraction chamber in an applied magnetic field.

11. The method of claim 7, further comprising transporting the lysate to a waste storage chamber.

12. The method of claim 8, wherein hybridized one or more magnetic particles are transported to the at least one detection chamber via the at least one wash buffer.Atty. Dkt. No.: 8635708613. The method of claim 7, wherein amplifying the one or more nucleic acids of interest is performed in the at least one detection chamber by generating a pulse of current via an electrical connection of an instrument to receive and interact with the cartridge.

14. The method of claim 13, wherein the pulse of current modulates a temperature proximate the one or more heating elements of the at least one detection chamber to increase a temperature proximate the one or more heating elements of the at least one detection chamber to between 90 and 110 degrees Celsius.

15. The method of claim 7, wherein the hybridized one or more magnetic particles are delivered to the at least one detection chamber via the at least one wash buffer such that the lysate does not interface with the at least one detection chamber.

16. A system comprising:a cartridge, comprising:a plurality of zones in fluid communication with each other, comprising:an extraction zone comprising:one or more extraction chambers comprising:a plurality of magnetic particles, wherein each of the one or more extraction chambers is configured to hold the plurality of magnetic particles;wherein a biological sample is lysed within at least one of the one or more extraction chambers to release one or more nucleic acids from the biological sample, wherein lysing the biological sample produces a lysate, and wherein the one or more nucleic acids are hybridized to the plurality of magnetic particles; anda waste storage chamber to receive the lysate; and a detection zone comprising one or more detection chambers, each detection chamber comprising one or more heating elements;wherein a first wash buffer is configured to deliver the hybridized magnetic particles to at least one of the one or more detection chambers from the one or more extraction chambers; andan instrument to interface with the cartridge, comprising:Atty. Dkt. No.: 86357086a lyse system to interface with at least one of the one or more extraction chambers; anda plurality of magnetic field generators, wherein the plurality of magnetic field generators comprise:at least one magnetic field generator to generate a magnetic field to dock the plurality of magnetic particles to an extraction zone; and at least one magnetic field generator to force the plurality of magnetic particles towards the one or more heating elements.

17. The system of claim 16, wherein the one or more nucleic acids are hybridized to the plurality of magnetic particles by hybridizing the one or more nucleic acids of interest with at least one capture oligonucleotide attached to the one or more magnetic particles of the plurality of magnetic particles.

18. The system of claim 17, further comprising a controller comprising one or more non-transitory computer-readable media storing instructions thereon that, when executed by one or more processors, cause the one or more processors to:dispense a second wash buffer into the at least one of the one or more extraction chambers, the second wash buffer to wash the hybridized one or more magnetic particles of the plurality of magnetic particles.

19. The system of claim 18, wherein the instructions further cause the one or more processors to:dispense the first wash buffer into the at least one of the one or more extraction chambers to perform a first wash, the first wash to trap the hybridized one or more magnetic particles of the plurality of magnetic particles and suspend the hybridized one or more magnetic particles of the plurality of magnetic particles within the first wash buffer.

20. The system of claim 16, wherein the one or more detection chambers comprise one or more amplification chambers or one or more pulse-controlled amplification (PCA) chambers.

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