Preamplification pulse-controlled amplification

Abstract

A method includes inserting, into a cartridge, a fluid comprising a biological sample, lysing the biological sample into an extraction chamber to release nucleic acids, delivering amplification reagents to a first detection chamber, amplifying the nucleic acids in the first detection chamber for a first number of cycles, dividing the nucleic acids obtained via the amplification reaction into a plurality of aliquots, delivering each of the aliquots to a different second detection chamber, performing a plurality of second amplifications, each second amplification performed for one of the aliquots each second amplification performed for a second number of cycles, and detecting a plurality of amplification products indicative of the presence, absence, or amount of the plurality of amplified nucleic acids, wherein a different amplification product is detected in each aliquot of the plurality of aliquots.

Description

Atty. Dkt. No.: 86357096PREAMPLIFICATION PULSE-CONTROLLED AMPLIFICATION 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 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 comprising a biological sample. The cartridge includes a plurality of zones, including an extraction zone having one or more extraction chambers and a detection zone having one or more detection chambers. Each detection chamber includes one or more heating elements. Each zone of the plurality of zones is in fluid communication with each other. The cartridge further includes one or more reagents and a plurality of magnetic particles. 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, delivering a plurality of amplification reagents to a first detection chamber of the at least one detection chamber, amplifying the one or more nucleic acids of interest in the first detection chamber for a first number of cycles, via a first amplification reaction, to provide a plurality of the one or more nucleic acids of interest, dividing the plurality of the one or more nucleic acids obtained via the first amplification reaction into a plurality of aliquots, delivering each of the plurality of aliquots to a different second detection chamber of the at least one detection chamber, each aliquot of the plurality of aliquots associated with a different nucleic acid of interest to be detected, performing a plurality of second amplification reactions, each second amplification reaction performed for one of the plurality of aliquots in the second detection to which the aliquot was delivered, each second 14937-8761-0640.1Atty. Dkt. No.: 86357096amplification performed for a second number of cycles, the second number of cycles greater than the first number of cycles, and detecting a plurality of amplification products indicative of the presence, absence, or amount of the plurality of amplified nucleic acids of interest. A different amplification product is detected in each aliquot of the plurality of aliquots.

[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 comprising a biological sample. The cartridge includes a plurality of zones, including an extraction zone having a lyse chamber having a plurality of magnetic particles and a detection zone including one or more detection chambers, each detection chamber including one or more heating elements. Each zone of the plurality of zones is in fluid communication with each other. The cartridge further includes one or more reagents. The method further includes lysing the biological sample into at least one of the lyse chamber to release the one or more nucleic acids of interest from the biological sample, hybridizing, into the lyse 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 magnetic particles on a side of the lyse chamber, wherein the plurality of magnetic particles are hybridized to the one or more nucleic acids of interest, washing the hybridized plurality of magnetic particles at least one wash buffer, suspending the hybridized plurality of magnetic particles with a first wash buffer, transporting the one or more nucleic acids of interest with the at least one capture oligonucleotide attached to the one or more magnetic particles to at least one detection chamber of the one or more detection chambers, delivering a plurality of amplification reagents to a first detection chamber of the at least one detection chamber, amplifying the one or more nucleic acids of interest in the first detection chamber for a first number of cycles, via a first amplification reaction, to provide a plurality of the one or more nucleic acids of interest, dividing the plurality of the one or more nucleic acids obtained via the first amplification reaction into a plurality of aliquots, delivering each of the plurality of aliquots to a different second detection chamber of the at least one detection chamber, each aliquot of the plurality of aliquots associated with a different nucleic acid of interest to be detected, performing a plurality of second amplification reactions, each second amplification reaction performed for one of the plurality of aliquots, each second amplification performed for a second number of cycles, the second number of cycles greater than the first number of cycles, and detecting a plurality of amplification products indicative of the presence, absence, or amount of the plurality of24937-8761-0640.1Atty. Dkt. No.: 86357096amplified nucleic acids of interest. A different amplification product is detected in each aliquot of the plurality of aliquots.

[0005] Another aspect relates to a diagnostic system. The diagnostic system includes: a cartridge comprising a plurality of zones in fluid communication with each other. The cartridge includes an extraction zone including: one or more extraction chambers including: a plurality of magnetic particles. The one or more extraction chambers are configured to hold the plurality of magnetic particles. The extraction zone includes a waste storage chamber configured to receive a lysate from the one or more extraction chambers. The cartridge includes a detection zone including: a plurality of mixing chambers including one or more different reagents, a plurality of detection chambers, and a heating element configured to heat the plurality of detection chambers.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. 2A depicts a method of preamplification, in accordance with example implementations.

[0010] FIG. 2B depicts a method of preamplification, in accordance with example implementations.

[0011] FIG. 3 depicts a method of preamplification, in accordance with example implementations.

[0012] FIG. 4A depicts a first cartridge architecture, in accordance with example implementations.34937-8761-0640.1Atty. Dkt. No.: 86357096

[0013] FIG. 4B depicts a second cartridge architecture, in accordance with example implementations.

[0014] FIG. 4C depicts a third cartridge architecture, in accordance with example implementations.

[0015] FIG. 5A depicts a first cross sectional view of a detection chamber of the cartridge of FIGS 1 A-4C, in accordance with example implementations.

[0016] FIG. 5B depicts a second cross sectional view of a detection chamber of the cartridge of FIGS 1A-4C, in accordance with example implementations.

[0017] FIG. 6 depicts a method for performing preamplification pulse-controlled amplification followed by a full amplification, in accordance with example implementations.

[0018] FIG. 7 depicts a sequence of pulse-controlled amplification, in accordance with example implementations.

[0019] FIG. 8 depicts a plurality of types of pulse-controlled amplification, in accordance with example implementations.

[0020] FIG. 9A depicts a blister device, in accordance with example implementations.

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

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

[0023] FIG. 9D depicts the blister device of FIG. 9A, in accordance with example implementations.

[0024] FIG. 10A depicts a blister device, in accordance with example implementations.

[0025] FIG. 10B depicts an actuator of the blister device of FIG. 10A, in accordance with example implementations.

[0026] FIG. 11 A depicts a system, in accordance with example implementations.44937-8761-0640.1Atty. Dkt. No.: 86357096

[0027] FIG. 1 IB depicts a system, in accordance with example implementations.

[0028] FIG. 11C depicts a system, in accordance with example implementations.

[0029] FIG. 12A depicts a cartridge environment, in accordance with example implementations.

[0030] FIG. 12B depicts a cartridge environment, in accordance with example implementations.

[0031] FIG. 13 A depicts a cartridge environment in a cross-sectional view, in accordance with example implementations.

[0032] FIG. 13B depicts a cartridge environment in a cross-sectional view, in accordance with example implementations.

[0033] FIG. 13C depicts a cartridge environment in a cross-sectional view, in accordance with example implementations.

[0034] FIG. 13D depicts a cartridge environment in a cross-sectional view, in accordance with example implementations.

[0035] FIG. 14A depicts a cartridge environment in plan view, in accordance with example implementations.

[0036] FIG. 14B depicts a cartridge environment in plan view, in accordance with example implementations.

[0037] FIG. 15 depicts a cartridge panel, in accordance with example implementations.

[0038] FIG. 16 depicts a user interface for cartridge environment, in accordance with example implementations.

[0039] 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.54937-8761-0640.1Atty. Dkt. No.: 86357096DETAILED DESCRIPTION

[0040] 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.

[0041] 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 the sequence 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.

[0042] 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 target64937-8761-0640.1Atty. Dkt. No.: 86357096analytes that can reach the amplification reaction step(s), thus reducing sensitivity of the results.

[0043] 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.

[0044] 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 first zone of the plurality of zones may include an input chamber to input a biological sample containing the nucleic acids of interest. A second 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 can74937-8761-0640.1Atty. Dkt. No.: 86357096be 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.

[0045] 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 storage chamber in the cartridge or removal of waste external to the cartridge.

[0046] 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.

[0047] 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 may84937-8761-0640.1Atty. Dkt. No.: 86357096also 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.

[0048] 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. However, 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.

[0049] 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.

[0050] High-plex PCA amplification (e.g., without preamplification and dilution / division of the biological sample) may be difficult to perform successfully. For example, off-target amplification, uneven amplification, etc., may prevent analytes of interest from being sufficiently amplified such that all analytes of interest can be successfully detected. Additionally, division of the biological sample into aliquots without preamplification may reduce sensitivity during detection. For example, dividing the biological sample into three94937-8761-0640.1Atty. Dkt. No.: 86357096aliquots prior to detection may reduce a detection limit by three times, making detection of an analyte difficult. Detection may be performed using high Stoke-shift fluorescent dyes or temperature-differentiated fluorescent probes, which may address limitations of optical fluorescence detection. However, these methods may not address challenges posed by high-plex PCA amplification. Further, running sample preparations multiple times, either with a larger amount of the biological sample or using a shared lysed sample, can address the challenges posed by high-plex PCA amplification. However, these methods can increase a size, complexity, and cost of the workflow. As such, pre-amplifying the biological sample prior to division of the sample and subsequent second amplifications can address the challenges associated with high-plex PCA amplification (e.g., off-target or uneven amplification) while maintaining sufficient sensitivity to detect the analytes of interest in the sample.

[0051] Specifically, the systems and methods described herein provide detection of a plurality of analytes (e.g., nucleic acids of interest) in a multiplex or high-plex reaction. A high-plex reaction may be a type of multiplex reaction in which a large (e.g., above a certain threshold value) number of analytes are analyzed from a single biological sample. A large number of analytes may be, for example, eight or more analytes. To enable analysis of a large number of analytes, the systems and methods described herein utilize a plurality (e.g., two or more) of sequential PCA amplifications. A first amplification (also referred to as a “preamplification”) may partially amplify all analytes present in a biological sample. The biological sample containing the partially amplified products may then be diluted and divided into a plurality of aliquots. A second amplification reaction may be performed on each of the plurality of aliquots (e.g., in parallel). The second amplification reactions may be PCA amplifications. Each amplified aliquot may be analyzed such that a different analyte is detected in each aliquot.

[0052] As described above, performing a preamplification reaction prior to a PCA reaction may provide several technical advantages. For example, in high-plex PCA reactions (specifically bead PCA reactions, as the preamplification reaction may be), replicating the nucleic acids of interest on an order of 104times may be easier (e.g., faster, cheaper, etc.) than replicating on an order of 109times. Further, standard (e.g., six channel, six target) optical detection may be able to be used, since each aliquot may have six or fewer targets to be detected. Further, a multi (e.g., six-) plex amplification and detection may still be performed in during preamplification and / or the second amplifications. Additionally, as stated above, preamplification may enhance a limit of detection of lower-plex PCA reactions. Additionally,104937-8761-0640.1Atty. Dkt. No.: 86357096in some implementations, the detection chambers may share a common heating element (e.g., a shared foil). This may reduce costs associated with including additional reach on / detecti on chambers and / or heating elements.

[0053] Referring now to Fig. 1A, a diagnostic system 100A, comprising a cartridge 100, is shown, according to an example embodiment. The system 100 A 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.

[0054] As shown in FIG. 1A, the cartridge 100 includes an extraction zone 104 comprising a first extraction chamber 114a having reagents 125 and a second extraction chamber 114b having magnetic particles 116 and reagents 125, and a detection zone 108 comprising a first detection chamber 120a having a heating element 122 and a second detection chamber 120b having the heating element 122. As shown, the first and second detection chambers 120 may have separate heating elements 122. In some examples, the first and second detection chambers 120 may share a common heating element 122. For example, a single heating element 122 may heat the entire detection zone 108.

[0055] In some examples, the extraction zone 104 may be a first zone 104 and the detection zone 108 may be a second zone 108. Each zone may be or include a plurality of chambers. For example, the first zone 104 may include extraction chambers 114a and / or 114b and the second zone 108 may include the detection chamber 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 first zone 104 may be an extraction zone, and the second zone 108 may be a detection zone. Further, each zone may include one or more chambers. For example, the first zone 104 may be an extraction zone including one or more extraction chambers 114, and the second zone 108 may be a detection zone including one or more detection chambers 120.114937-8761-0640.1Atty. Dkt. No.: 86357096

[0056] 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. Membrane pinch valves and may be specific types of valves used to restrict and permit fluid flow through the cartridge. Valves and 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 FIGS. 4A-4C.

[0057] 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, an instrument may be coupled to the cartridge 100 and 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. A pump fluidic interconnect may be used to pump air, fluid, etc. through the cartridge 100. A pump may be, for example, a syringe pump, an air cylinder pump, a diaphragm pump, etc. The use of a pump in the cartridge 100 is described in greater detail with respect to FIGS. 4A-4C. 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.

[0058] The cartridge 100 may include a greater or fewer number of zones and / or chambers than what is shown in FIG. 1 A. For example, as is shown and will be described in FIG. IB, the cartridge 100 may include the extraction zone 104 and the detection zone 108, as well as a first wash buffer 110a and a second wash buffer 110b. 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, the cartridge 100 may include a first zone 104 that is an extraction zone having extraction chambers 114 and a second zone 108 that is a detection zone having the detection chamber 120.

[0059] In various examples, at least one of the plurality of extraction chambers 114 may be an extraction chamber 114a. The extraction chambers 114a may house a lysing of the biological sample to release one or more nucleic acids of interest from the biological sample. The extraction chamber 114a may be a fluidic structure with an open cavity or void to define a124937-8761-0640.1Atty. Dkt. No.: 86357096chamber that could be filled with the biological sample comprising the one or more nucleic acids of interest. The extraction chamber 114a may be configured to house a lyse reaction. For example, a biological sample may enter the extraction chamber 114a 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 114a 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.

[0060] The cartridge 100 may also include one or more reagents 125. The reagents 125 may be located in at least one of the one or more extraction chambers. For example, as shown in FIG. 1 A, the extraction chambers 114 include one or more reagents 125, however, it should be understood that the reagents 125 may 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 detection chamber 120). In various other examples, no reagent 125 may be stored in any extraction chamber 114a or extraction chamber 114b (also called a hybridization chamber herein), 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.

[0061] 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.

[0062] In various examples, liquid reagents 125 may be stored in blister devices (e.g., blister devices 900 described with respect to FIG. 9) 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. 9A-10B. 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 120.134937-8761-0640.1Atty. Dkt. No.: 86357096

[0063] 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).

[0064] In some examples, the reagents 125 may include a PC A buffer. The PC A buffer may refer to a buffer used to reconstitute a master mix reagent used for reverse transcription and PCA amplification. In some examples PCA buffer comprises water, a salt, a buffer compound (e.g., tris buffer) and optionally a surfactant. In some examples, the salt is MgCh, KC1, or NaCl. . In some examples, the PCA buffer comprises between O.lmM 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 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 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 can be reduced in order to enable hybridization only from a certain higher degree of complementarity.

[0065] 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 the144937-8761-0640.1Atty. Dkt. No.: 86357096amplification 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 as described herein. In various embodiments, 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.).

[0066] In some examples, the extraction chamber 114a includes a heating system. The extraction chamber 114a 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.

[0067] In various examples, the biological sample can be lysed by a different lyse system (e.g., the lyse system 152 of FIG. IB), which may be located within an instrument (e.g., the instrument 150 of FIG. IB) coupled to the cartridge 100. In various examples, the lysate can be lysed by a plurality of lysing techniques, such as a combination of the lyse system, 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.”

[0068] In various examples, at least one of the plurality of extraction chambers 114 may be an extraction chamber 114b. For example, in various embodiments, the extraction zone (e.g., the first zone 104) of the plurality of zones of the cartridge 100 includes at least two extraction chambers 114. As such, at least one of the two (or one or more) extraction chambers 114 may be an extraction chamber 114b. The extraction chamber 114b may include a plurality of154937-8761-0640.1Atty. Dkt. No.: 86357096magnetic 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 114b may receive, from the extraction chamber 114a, the one or more nucleic acids released from the lysed biological sample. In the extraction chamber 114b, the one or more nucleic acids may be hybridized. For example, in the extraction chamber 114b, 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.

[0069] In various examples, the cartridge 100 may include a plurality of hybridization chambers 114b. For example, the cartridge 100 may include two hybridization chambers 114b. The lysate may be moved from the first hybridization chamber to the second hybridization chamber to fully mix and hybridize the nucleic acids of interest. In some examples, the cartridge 100 may include a single extraction chamber 114a, in which both lysing and hybridization are performed.

[0070] 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 at least 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.

[0071] 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) is164937-8761-0640.1Atty. Dkt. No.: 86357096influenced 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.

[0072] The cartridge 100 may include a plurality of the magnetic particles 116. Specifically, the extraction chamber 114b may include a plurality of the magnetic particles 116. The magnetic particles 116 may also be referred to as “magnetic beads,” “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 freely selected 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, formed of, and / 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 which174937-8761-0640.1Atty. Dkt. No.: 86357096enables and / or facilitates functionalization with capture oligonucleotides. For example, the surface of the magnetic microparticles, also called herein paramagnetic beads, paramagnetic particles, and magnetic particles, can be at least partially functionalized with streptavidin.

[0073] The magnetic particles 116 may have a nucleic acid attached. 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 structure of a nucleic acid attached to a magnetic particle by a capture oligonucleotide may be referred to as a “hybridized magnetic particle” or a “hybridized nucleic acid.” 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 114b, the one or more nucleic acids from the extraction chamber 114a 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 114a). In various examples, the capture oligonucleotides may be the same or different from each other. For example, a first capture oligonucleotide attached to a first magnetic particle 116 may have a first sequence, and a second capture oligonucleotide attached to a second magnetic particle 116 may have a second sequence.

[0074] In various examples, the cartridge 100 includes a plurality of hybridization chambers (e.g., the extraction chamber 114b). During hybridization, the fluid containing the biological sample may move between the plurality of hybridization chambers to allow for mixing (e.g., partial mixing, complete mixing, etc.) of the solution or fluid during hybridization. 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.

[0075] 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).

[0076] 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. The detection chambers 120 may not be a part of or associated with a zone.184937-8761-0640.1Atty. Dkt. No.: 86357096

[0077] 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 embodiments, 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). Types of PCA reactions may include, for example, bead PCA reactions, free PCA reactions, and hybrid PCA reactions. Such reactions are described in greater detail with respect to FIGS.7 and 8.

[0078] Specifically, the nucleic acids of interest may undergo a plurality of amplification reactions in a plurality of detection chambers 120. For example, as will be described herein, the nucleic acids of interest may undergo a first preamplification reaction and a second amplification reaction. A preamplification reaction may follow the same steps or processes as a “normal” amplification reaction. However, in a preamplification reaction, the reaction may be performed for a smaller number of cycles relative to a “normal” amplification reaction, thereby producing fewer amplification products in a preamplification reaction relative to an amplification reaction. For example, in a preamplification reaction, the reaction may be performed for a number of cycles such that an amplification factor of the reaction is on the order of between 103and 104, while an amplification reaction may be performed for a number of cycles such that an amplification factor of the reaction is on the order of 109+. The number of cycles performed for a preamplification reaction may be, for example between 20 and 50 cycles. The number of cycles performed for an amplification reaction (e.g., a standard or non-preamplification reaction) may be, for example, 50 to 200+ cycles. In some examples, the number of preamplification cycles may be a percentage of a number of total amplification cycles or non-preamplification cycles. For example, the number of preamplification cycles may be 10% to 35% of the total number of amplification cycles.

[0079] As used herein, the terms “amplify,” “amplification,” and / or “preamplification” 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 may194937-8761-0640.1Atty. Dkt. No.: 86357096include 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, a lyophilized 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.

[0080] 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.

[0081] 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.

[0082] 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 various electrical components of the instrument. The electrical204937-8761-0640.1Atty. Dkt. No.: 86357096components may provide an electrical connection between the instrument 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.

[0083] 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. 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 114b. The detection chamber 120 is described in greater detail with respect to FIGS.5 A and 5B.

[0084] During the PCA reach on(s) 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 than214937-8761-0640.1Atty. Dkt. No.: 8635709630 microseconds, less than 10 microseconds, less than 5 microseconds, or less than 1.5 microseconds.

[0085] 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. 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.

[0086] 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.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 PC A 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 the224937-8761-0640.1Atty. Dkt. No.: 86357096volume heated by the heating element 122 can rapidly return to a setpoint temperature of the detection chamber 120.

[0087] Referring now to FIG. IB, a diagnostic system 100B is shown, according to an example embodiment. The system 100B includes the 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 chambers 114a and 114b, and the detection chambers 120a and 120b may be the same as those described with respect to FIG. 1A.

[0088] In addition to the elements described above with respect to FIG. 1 A, the cartridge 100 of the diagnostic system 100B includes a first wash buffer 110a and a second wash buffer 110b. Additionally, the extraction zone 104 may include a waste storage chamber 168. As shown in FIG. IB, the extraction zone 104 may include the extraction chamber 114a that is configured as a lyse chamber or lysis chamber and includes the magnetic particles 116. The detection zone 108 may include a first detection chamber 120a and a second detection chamber 120b, each including a heating element 122. In some examples, the detection chambers 120a and 120b may share a common heating element 122. The detection zone 108 may further include a first mixing chamber 123a and a second mixing chamber 123b, each including reagents 125. The instrument 150 includes a lyse system 152 having a sonicator 154, a first magnetic field generator 156a, and a second magnetic field generator 156b.

[0089] The cartridge 100 can also 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 sample input chamber may include a sample input cap that interfaces with a liquid port of the cartridge 100. The sample input cap may seal the sample input chamber by preventing liquid from exiting the sample input chamber through the liquid port. The sample input chamber may receive a biological sample or other fluid containing one or more nucleic acids of interest. Thus, the cartridge 100 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 the234937-8761-0640.1Atty. Dkt. No.: 86357096sample input chamber. The sample input cover may be configured as a slider to slide along an axis to cover and uncover a liquid port of the sample input chamber.

[0090] 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, etc. 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. 13A-D.

[0091] The cartridge 100 may also include a plurality of zones, shown as the extraction zone 104 and the detection zone 108. Each zone may be or include a plurality of chambers. For example, the first zone 104 may include one or more extraction chambers or lyse chambers 114a and one or more waste storage chambers 168. The detection zone 108 may include one or more detection chambers 120 and one or more mixing chambers 123. 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.244937-8761-0640.1Atty. Dkt. No.: 86357096

[0092] 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 (NH^SC . 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, the surfactant 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.

[0093] In some examples, the extraction zone 104 may include the system composition. It should be understood that the system composition may be included in a zone different than the extraction zone 104. In various examples, the system composition is not included in a zone. The system 100 A may include one or more system compositions.

[0094] As shown in FIG. IB, the cartridge 100 may include a plurality of wash buffers 110. It should be understood that the wash buffers 110 may or may not be included in a zone. For example, as shown in FIG. IB, the wash buffers 110 are not included in a zone. However, in some examples, one or both of the wash buffers 110 may be included in the extraction zone 104. In various examples, the cartridge 100 may include a chamber in a first zone of a plurality of zones to store the wash buffer 110. The wash buffer 110 may be transported from the chamber to at least one extraction chamber 114a to remove undesired elements of the biological sample when the biological sample is in the detection chamber 120. In various embodiments, the extraction chamber 114a, the extraction chamber 114b, and / or the detection chamber 120 may be washed to remove undesirable elements from the chamber being washed.

[0095] The wash buffers 110 refer to buffers used for washing magnetic particles 116 and the extraction chamber 114a. In some examples, the wash buffer 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 buffer 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, the254937-8761-0640.1Atty. Dkt. No.: 86357096surfactant is selected from Tween ®20, Tween® 80, Tween® 85, SPAN® 80 or SPAN® 85. In some examples, the surfactant is Tween® 20.

[0096] The system 100B 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 110a may be used to wash the extraction chamber 114a and the second wash buffer 110b may be used to rinse the extraction chamber 114a. For example, the first wash buffer 110a may be introduced to a chamber (e.g., the detection chamber 120, the extraction chamber 114a, etc.) to wash or remove undesired elements 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 110b may be introduced to the chamber after the first wash buffer 110a has exited the chamber (e.g., has been transported to the waste storage chamber 168) and the wash has been completed. The rinse may remove any remaining elements or contaminants not removed by the first wash buffer.

[0097] 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. Example blister devices are described in greater detail with respect to FIGS. 9A-10B. The blister devices may store liquid reagents (e.g., the system composition, the wash buffers 110, liquid reagents used for amplification, etc.). The blister device 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 buffers 110 may return to the blister device upon completion of use. For example, the wash buffers 110 may be released from a blister device storing the wash buffers 110 and may be delivered to the extraction chamber 114a to wash the contents of the lyse chamber. After washing is complete, the wash buffer 110 may return to the blister device to be stored.264937-8761-0640.1Atty. Dkt. No.: 86357096

[0098] In some examples, the wash buffer 110 may be transported to the waste storage chamber 168. For example, upon washing the extraction chamber 114a, a lysate may be transported to the waste storage chamber 168. For example, upon hybridization, a magnetic field generator 156 may generate a magnetic field that traps the hybridized magnetic particles 116 to a side or wall of the extraction chamber 114a. The remaining contents of the extraction chamber 114a (e.g., the lysate, the wash buffer 110, the reagents 125, etc.) may be transported to the waste storage chamber 168.

[0099] The blister device can be configured to transport its contents in a direction according to a selection of a pumping direction through the blister device. In some examples, the blister device is configured to transport contents of the blister device in a direction from a first valve toward a second valve. In some other examples, the blister device is configured to transport contents of the blister device 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), when present in the cartridge 100, may be or be part of the first zone and / or the second zone of the plurality of zones.

[0100] The blister device 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 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.

[0101] 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 114a may include a plurality of beads (e.g., glass beads, ceramic beads) used to mechanically agitate or lyse the organisms present in the lysate.274937-8761-0640.1Atty. Dkt. No.: 86357096

[0102] 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).

[0103] 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 embodiments, 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 FIGS. 7 and 8.

[0104] The detection zone 108 may also include a plurality of mixing chambers 123. Specifically, a shown, the detection zone 108 includes a first mixing chamber 123a and a second mixing chamber 123b. Each of the mixing chambers 123 may be hybridization chambers. For example, upon washing the lysate in the extraction chamber 114a, the hybridized magnetic particles 116 may be transported to at least one of the mixing chambers 123. In each of the mixing chambers 123, hybridization may occur in a manner described herein with respect to hybridization chambers (e.g., the extraction chamber 114b of FIG. 1 A). The reagents 125 in each of the mixing chambers 123 may be used during hybridization.

[0105] Referring still to FIG. IB, the instrument 150 is shown, according to example embodiments. The diagnostic system 100B 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.

[0106] 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. The sonicator 154 may interface with the at least one extraction chamber 114 (e.g., the extraction chamber 114a). The lyse system 152 may be utilized when the biological sample enters the extraction chamber 114a of the cartridge 100 to284937-8761-0640.1Atty. Dkt. No.: 86357096lyse the sample and release the nucleic acids of interest. The lyse system 152 may be coupled to the extraction chamber 114a.

[0107] 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 114a to a target temperature (e.g., between 35 and 100 degrees Celsius), such as by heating the heating element of the extraction chamber 114a 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 114a to 95 degrees Celsius.

[0108] 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 a controller), 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.

[0109] 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 114a). The sonicator 154 may deliver ultrasonic waves to the extraction chamber 114a 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 114a) into the biological sample to cause cell / spore / tissue 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.

[0110] The instrument 150 may include a plurality of magnetic field generators 156 (e.g., a first magnetic field generator 156a and a second magnetic field generator 156b) to generate a magnetic field. The magnetic field generators 156 may be movable (e.g., may be movable within the instrument 150) to interface with a plurality of locations of the cartridge 100. The294937-8761-0640.1Atty. Dkt. No.: 86357096magnetic 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 generator 156 is a magnet (e.g., a permanent magnet). The magnetic field generator 156 may be positioned at or proximate the extraction chamber 114a and / or the detection chamber(s) 120. 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 generator 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 embodiments, the instrument 150 may be activated or powered on to activate the magnetic field generator.[OHl] In some examples, the magnetic field generator 156a may be configured to generate a magnetic field to dock the magnetic particles 116 to the extraction chamber 114a. Further, the second magnetic field generator 156b may be configured to 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 distance of the heating element 122, etc.

[0112] 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.

[0113] 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.

[0114] 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 reaction304937-8761-0640.1Atty. Dkt. No.: 86357096container. For example, when using a permanent magnet, the magnetic field may be changed by changing an orientation of the permanent magnet to the detection chamber 120 and / or a distance of the permanent magnet from the 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.

[0115] Referring now to FIG. 2A, a method 200 for detecting a presence, absence, or amount of a nucleic acid of interest is shown, according to an example embodiment.

[0116] 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 plurality of 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 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. The plurality of magnetic particles 116 may be or include streptavidin-coated paramagnetic beads.314937-8761-0640.1Atty. Dkt. No.: 86357096

[0117] In some examples, the method for detecting the presence, absence, or amount of a nucleic acid of interest includes a plurality of magnetic particles 116 that are streptavidin-coated paramagnetic beads. The plurality of magnetic particles include a plurality of groups of magnetic particles. Each group can include a subset of the plurality of magnetic particles. Each subset of magnetic particles is of a different type, and each magnetic particle of the subset is attached to a single capture oligonucleotide. As referred to herein “different type” may refer to magnetic particles that are different due to different sequences of the attached capture oligonucleotides.

[0118] In some other examples, the plurality of magnetic particle includes a plurality of groups of magnetic particles, and each group includes a subset of the plurality of magnetic particles. Each subset of magnetic particles is of a different type, and each magnetic particle of the subset of magnetic particles is attached to a single type of capture oligonucleotide. In other words, in a first option, the magnetic particles form subsets or groups of particles, and each individual particle that is part of a subset is attached to the same capture oligonucleotides or type of capture oligonucleotides (e.g., all having the same nucleic acid sequences).. Such magnetic particles are capable of capturing only one type of nucleic acid of interest. In order to have multiplexing capabilities, the use of different subsets of beads, each having specific different capture oligonucleotides attached, may be used.

[0119] In other examples still, each magnetic particle of the plurality of magnetic particles 116 is of a different type than another magnetic particle of the plurality of magnetic particles 116. Further, each magnetic particle is attached to different type of capture oligonucleotide. In other words, in a second option, the magnetic particles are attached to multiple different capture nucleotides or types of capture oligonucleotides (e.g., each having a different sequence), meaning that each individual magnetic particle is capable of capturing a different nucleic acid of interest. In order to have multiplexing functionality (e.g., the functionality of capturing and hybridizing different nucleic acids of interest) only one group of magnetic particles would be used.

[0120] In some examples, rather than each magnetic particle having a single capture oligonucleotide targeting a single nucleotide sequence or nucleic acid of interest, each magnetic particle may have multiple or all unique capture oligonucleotides attached, thereby targeting all of the different target sequences of interest. For example, when there are six target sequences to be amplified and detected, rather than utilizing six different types of functionalized magnetic324937-8761-0640.1Atty. Dkt. No.: 86357096particles, each with a unique capture oligonucleotide for each target sequence, the system may include a single type of functionalized magnetic particle with six unique capture oligonucleotides attached (e.g., one for each target sequence). In some examples where six target sequences are present, the system may include two types of magnetic particles 116, a first type of magnetic particle having four capture oligonucleotide types attached, and a second type of magnetic particle having two capture oligonucleotide types attached. Other implementations or combinations may exist (e.g., two types of magnetic beads, each with three types of capture oligonucleotides attached, seven target sequences and three type of magnetic particles where two types have three capture oligonucleotides attached and one type has one capture oligonucleotide attached, etc.).

[0121] At process 204, the biological sample is lysed into at least one of the one or more extraction chambers (e.g., extraction chambers 114a). Lysing the biological sample may release the one or more nucleic acids of interest from the biological sample. 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 a 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 including 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 one or more of sonication, thermal lysis, or thermal sonication.

[0122] The method 200 may include hybridizing the one or more nucleic acids of interest. The one or more nucleic acids of interest are hybridized into the at least one extraction chamber 114a 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 114b 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.

[0123] The method 200 may further include transporting the one or more nucleic acids of interest with the at least one capture oligonucleotide attached to the one or more magnetic particles 116 to at least one detection chamber 120 of the one or more detection chambers 120. The nucleic acids of interest may be transported, for example, via the second wash buffer 110b.334937-8761-0640.1Atty. Dkt. No.: 86357096

[0124] The method 200 may further include trapping the one or more magnetic particles 116 into close proximity of the one or more heating elements 122 of the at least one detection chamber 120. For example, the magnetic particles 116 may be trapped within a threshold distance of the one or more heating elements 122 of the at least one 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 be trapped near the heating element 122 so that the nucleic acids of interest can undergo the amplification reaction to be identified by an optical unit.

[0125] The method 200 may further include washing, in the extraction chamber 114, the one or more nucleic acids of interest hybridized with the at least one capture oligonucleotide attached to the one or more magnetic particles. The hybridized one or more nucleic acids of interest may be washed with a first wash buffer 110a. The method 200 may further include washing the one or more nucleic acids of interest hybridized with the at least one capture oligonucleotide attached to the one or more magnetic particles with a second wash buffer 110b. Washing with the second wash buffer may suspend the one or more magnetic particles 116 within the second wash buffer 110b. Washing the hybridized magnetic particles 116 may remove or wash undesired elements from the extraction chamber(s) 114. Washing may be performed by introducing the wash buffer 110a and / or 110b into the at least one extraction chamber 114. For example, the wash buffer 110 may be released from a blister device 126 (as shown in FIGS. 3A-3G and described with respect to FIGS. 1 and 9A-10B) and may be transported to the extraction chamber(s) 114.

[0126] At process 206, a plurality of amplification reagents 125 may be delivered to a first detection chamber 120 of the at least one detection chamber 120. 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.

[0127] At process 208, the one or more nucleic acids of interest are amplified in the first detection chamber 120 via a first amplification reaction. Specifically, the first amplification reaction may be a bead-based pulse-controlled amplification (PCA) reaction that utilizes the plurality of magnetic particles 116. The first amplification reaction may be a preamplification.344937-8761-0640.1Atty. Dkt. No.: 86357096That is, the one or more nucleic acids of interest may be amplified for a first number of cycles. The first number of cycles may be a limited number (e.g., on the order of 104cycles) relative toa number of cycles performed for a non-preamplification reaction. The first amplification reaction may provide a plurality of the one or more nucleic acids of interest.

[0128] At process 210, the plurality of the one or more nucleic acids of interest obtained via the first amplification reaction are divided into a plurality of aliquots.

[0129] At process 212, each of the plurality of aliquots is delivered to a different second detection chamber 120 of the at least one detection chamber 120. Each aliquot of the plurality of aliquots may be associated with a different nucleic acid of interest to be detected.

[0130] At process 214, a plurality of second amplification reactions are performed. Each second amplification reaction is performed for one of the plurality of aliquots in the second detection chamber to which the aliquot was delivered. Each of the plurality of second amplification reactions is one of a free PCA reaction that does not utilize the plurality of magnetic particles or a hybrid PCA reaction that utilizes the plurality of magnetic particles and a plurality of biotinylated primers.

[0131] Each second amplification reaction is performed for a second number of cycles. The second number of cycles may be greater than the first number of cycles. For example, the first number of cycles may be on the order of 104cycles, and the second number of cycles may be on the order of 109cycles. Further, each of the plurality of second amplification reactions are performed in parallel.

[0132] 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. A different amplification product is detected in each aliquot of the plurality of aliquots. The amplification products may be detected via an optical unit 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 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. 2B, a method 220 is shown for performing a preamplification reaction to detect one or more nucleic acids of interest, according to an example embodiment.354937-8761-0640.1Atty. Dkt. No.: 86357096

[0134] At process 222, 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 plurality of 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 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. The plurality of magnetic particles 116 may be or include streptavidin paramagnetic beads.

[0135] In some examples, the plurality of magnetic particles include a plurality of groups of magnetic particles, each group including a subset of the plurality of magnetic particles. Each subset of magnetic particles is of a different type, and each magnetic particle of the subset is attached to a single capture oligonucleotide. Additionally or alternatively, each group of the plurality of magnetic particles is to attach to a different type of capture oligonucleotide. Said capture oligonucleotides are able to hybridize with a specific nucleic acid of interest. In some examples, each magnetic particle of the plurality of magnetic particles is of a different type than another magnetic particle of the plurality of magnetic particles, and each magnetic particle of the plurality of magnetic particles is attached to a different type of capture oligonucleotide.

[0136] For instance, in some examples, each of the magnetic particles 116 may be of the same type, and each of the magnetic particles 116 may attach to a different capture oligonucleotide or type of capture oligonucleotide (e.g., each capture oligonucleotide is attached to the same bead or type of bead). This may cause each magnetic particle and attached capture oligonucleotide to hybridize with a different type of nucleic acid of interest (e.g., because each type of capture oligonucleotide has a specific sequence that matches or is complementary to the sequence of a specific nucleic acid of interest).

[0137] In other examples, each group of magnetic particles 116 may be of a different type, and each magnetic particle of the group of magnetic particles 116 (e.g., and / or each magnetic particle within a group) may attach to the same capture oligonucleotide or type of capture oligonucleotide. Different groups of magnetic particles 116 may attach to different capture364937-8761-0640.1Atty. Dkt. No.: 86357096oligonucleotides in this way. For example, there may be a number (e.g., five) of different groups of magnetic particles, each group having a number (e.g., 100) of magnetic particles. All of the magnetic particles within a group may attach or link to the same type of capture oligonucleotide. This may cause each magnetic particle and attached capture oligonucleotide within a group to hybridize with the same type of nucleic acid of interest. Each group of magnetic particles may attach to a different type of capture oligonucleotide such that the magnetic particles in a first group hybridize with different nucleic acids of interest than the magnetic particles in a second group.

[0138] At process 224, the biological sample is lysed into at least one of the one or more extraction chambers (e.g., extraction chambers 114a). Lysing the biological sample may release the one or more nucleic acids of interest from the biological sample. 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 a 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 including 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 one or more of sonication, thermal lysis, or thermal sonication. In some examples, lysing the biological sample produces a lysate.

[0139] At process 226, the one or more nucleic acids of interest are hybridized. The one or more nucleic acids of interest are hybridized into the at least one extraction chamber 114a 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 114b 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.

[0140] At process 228, the hybridized magnetic particles are held to a side of the extraction chamber 114a. The hybridized magnetic particles 116 may be held to a side of the extraction chamber 114a via a magnetic field generated by the external magnetic field generator 156. In some examples, the lysate may be transported to a waste storage chamber after the hybridized magnetic particles are held to the side of the lyse chamber.374937-8761-0640.1Atty. Dkt. No.: 86357096

[0141] At process 230, in the extraction chamber 114, the one or more nucleic acids of interest hybridized with the at least one capture oligonucleotide attached to the one or more magnetic particles are washed. The hybridized one or more nucleic acids of interest may be washed with a first wash buffer 110a. The method 200 may further include washing the one or more nucleic acids of interest hybridized with the at least one capture oligonucleotide attached to the one or more magnetic particles with a second wash buffer 110b. Washing with the second wash buffer may suspend the one or more magnetic particles 116 within the second wash buffer 110b. Washing the hybridized magnetic particles 116 may remove or wash undesired elements from the extraction chamber(s) 114. Washing may be performed by introducing the wash buffer 110a and / or 110b into the at least one extraction chamber 114. For example, the wash buffer 110 may be released from a blister device 126 (as described with respect to FIGS. 1 and 9A-10B) and may be transported to the extraction chamber(s) 114.

[0142] At process 232, the hybridized plurality of magnetic particles 116 are suspended in the extraction chamber 114a using the first wash buffer 110a. Suspending the hybridized magnetic particles 116 may include sonicating the hybridized plurality of magnetic particles via a sonotrode.

[0143] At process 234, the one or more nucleic acids of interest with the at least one capture oligonucleotide attached to the one or more magnetic particles 116 are transported to at least one detection chamber 120 of the one or more detection chambers 120. The nucleic acids of interest may be transported, for example, via the second wash buffer 110b.

[0144] At process 236, a plurality of amplification reagents 125 may be delivered to a first detection chamber 120 of the at least one detection chamber 120. 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.

[0145] At process 238, the one or more nucleic acids of interest are amplified in the first detection chamber 120 via a first amplification reaction. Specifically, the first amplification reaction may be a bead-based pulse-controlled amplification (PCA) reaction that utilizes the plurality of magnetic particles 116. The first amplification reaction may be a preamplification.384937-8761-0640.1Atty. Dkt. No.: 86357096That is, the one or more nucleic acids of interest may be amplified for a first number of cycles. The first number of cycles may be a limited number (e.g., on the order of 104cycles) relative toa number of cycles performed for a non-preamplification reaction. The first amplification reaction may provide a plurality of the one or more nucleic acids of interest.

[0146] At process 240, the plurality of the one or more nucleic acids of interest obtained via the first amplification reaction are divided into a plurality of aliquots.

[0147] At process 242, each of the plurality of aliquots is delivered to a different second detection chamber 120 of the at least one detection chamber 120. Each aliquot of the plurality of aliquots may be associated with a different nucleic acid of interest to be detected.

[0148] At process 244, a plurality of second amplification reactions are performed. Each second amplification reaction is performed for one of the plurality of aliquots in the second detection chamber to which the aliquot was delivered. Each of the plurality of second amplification reactions is one of a free PCA reaction that does not utilize the plurality of magnetic particles or a hybrid PCA reaction that utilizes the plurality of magnetic particles and a plurality of biotinylated primers.

[0149] Each second amplification reaction is performed for a second number of cycles. The second number of cycles may be greater than the first number of cycles. For example, the first number of cycles may be on the order of 104cycles, and the second number of cycles may be on the order of 109cycles. Further, each of the plurality of second amplification reactions are performed in parallel.

[0150] At process 246, a plurality of amplification products indicative of the presence, absence, or amount of the plurality of amplified nucleic acids of interest may be detected. A different amplification product is detected in each aliquot of the plurality of aliquots. The amplification products may be detected via an optical unit 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 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.

[0151] Referring now to FIG. 3, a method 300 for detecting the presence, absence, or amount of a nucleic acid of interest is shown, according to an example embodiment. At process 302, a394937-8761-0640.1Atty. Dkt. No.: 86357096solid phase extraction is performed. The solid phase extraction may be performed by hybridizing the nucleic acids of interest on the plurality of magnetic particles 116 (e.g., using a capture oligonucleotide).

[0152] At process 304, a preamplification reaction is performed, as described above with respect to FIGS. 2A and 2B and below with respect to FIGS. 4A-4C. During preamplification, each target present in the sample may be amplified by 103to 104times (e.g., as opposed to 109times to reach a level of detectability of the targets). No detection of the preamplification products may be performed.

[0153] At process 306, amplicons (e.g., preamplification products) are eluted off of the magnetic particles 116. Products used in PC A reactions may be diluted, and the resulting fluid is aliquoted.

[0154] At process 308, a master mix is reconstituted. A number of master mixes that are reconstituted may correspond to a number of aliquots. For example, when the diluted PCA products (with the preamplification products) are aliquoted into three aliquots, three master mixes may be reconstituted.

[0155] At process 310, PCA amplification reactions are performed. The PCA reactions may be hybrid or free PCA reactions. In some examples, the amplification reactions may be performed with real-time fluorescent detection of the amplification products indicative of a presence, absence, or amount of the nucleic acids of interest.

[0156] FIGS. 4A-4C depict example diagnostic system architectures, in accordance with present implementations. As illustrated by way of example in FIGS. 4A-4C, example diagnostic system architectures 400A-400C 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 150. FIGS. 4A-4C illustrate different embodiments of the same cartridge 100.

[0157] The cartridge of the example diagnostic system architecture 400 may include a first vent membrane 404, a plurality of valves 408a-w, a plurality of frangible seals 410a-h, a liquid sample input chamber 412, a sample filter 414, a p-trap 416, an umbrella valve 418, a plurality of bubble traps 420a-e, a system composition blister device 422, wash buffer blister devices 424a and 424b, a plurality of junctions 426a-n, a sample input (SI) metered section 430, an IPC404937-8761-0640.1Atty. Dkt. No.: 86357096lyo particle 431, a lyse chamber 432 (e.g., the extraction chamber 114a) having an external ultrasonic horn 434, lysing beads 436, and an infrared sensor 437, a magnetic particle 438, a PC A buffer 440 (stored in a blister device), a PC A buffer metered section 442, a first master mix mixing chamber 444 having a first master mix 446 (e.g., a lyophilized PC master mix), a second vent membrane 448, a plurality of second master mix mixing chambers 450a-c, each having a second master mix 454a-c, respectively, a metered diluted PCA product chamber 452, a plurality of PCA chambers 456a-c, a plurality of air springs 464a-c, each coupled to a respective PCA chamber 456a-c, a PCA mixing chamber 466, a waste storage chamber 468, and a preamplification PCA chamber 470. In some implementations, the lysis chamber 432 interfaces with an external heat zone 460, the plurality of second mixing chambers 450a-c interface with a second external heat zone 453, and the plurality of PCA chambers 456a-c and the preamplification PCA chamber 470 interface with a third external heat zone 462.

[0158] As shown in FIGS. 4A-4C, the instrument of the diagnostic system architecture 400 may include a pump 402 (e.g., a syringe pump), a plurality of fluid sensors 428a-i, and a plurality of external magnets 458a-b. The elements of the instrument may be positioned such that certain components interface with certain components of the cartridge. For example, FIGS.4A-C indicate locations of fluid sensors 428a-i within the instrument relative to the elements within the cartridge. For example, fluid sensors 428a-i may be disposed within the instrument at various locations corresponding to elements located in the cartridge such that the sensors 428a-i 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 the inlet of the SI metered section 430. Further, as stated, the instrument the diagnostic system architecture 400 may also include a plurality of external magnets 458a-b. The external magnet 458a may interface with the preamplification PCA chamber 470. The external magnet 458b may interface with the lysis chamber 432. The external magnets 458 may be positioned within the instrument such that, upon insertion of the cartridge into the instrument, the locations of the magnet 458a-b align with the positions of the preamplification PCA chamber 470 and the lysis chamber 432, respectively, within the cartridge.414937-8761-0640.1Atty. Dkt. No.: 86357096

[0159] In various examples, the system composition blister device 422 and the wash buffer blister devices 424 may be similar to the blister devices 126a and 126b, respectively. The system composition stored in the blister device 422 may be similar to the system composition and the wash buffer composition stored in the blister devices 424a and 424b may be similar to the wash buffer 110. The lyse chamber 432 may be similar to the extraction chamber 114a and the second MM mixing chambers 450a-c may be similar to the extraction chambers 114b. The PCA chambers 456 may also be similar to the detection chamber 120.

[0160] The following description refers to processed performed in any of the cartridges 400A, 400B, and / or 400C. A user may fill the liquid 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 FIG. 1. The user may fill the sample input chamber 412 via a pipette, an exact volume pipette, a dropper, syringe injection, etc. The sample input chamber 412 may include fill guides to indicate a fill level. Upon filling the sample input chamber 412, all valves 408a-w 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 liquid 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 liquid sample input chamber 412, the chamber may be closed. For example, the sample input cover 303 may be closed.

[0161] 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, 408h, 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 locations424937-8761-0640.1Atty. Dkt. No.: 86357096of 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.

[0162] 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 428f), help in reciprocating mixing by triggering when a flow should be reversed (e.g., sensors 428d, 428e, and 428f), and / or help locate the reconstituted master mix reagent in the PCA chamber (e.g., sensors 428c and 428f).

[0163] 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 include capacitor plates to detect change in dielectric between liquid and air in a microfluidic channel or chamber.

[0164] 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 lyo particle 431, and the sample may fill the lyse chamber 432. Pressure may be vented through one or more of pinch valves 408n, 408h, 408o, and / or 408k and / or the second vent membrane 448 to allow the fluid flow. For example, pressure may be vented through the pinch valve 408k.

[0165] In various examples, the system composition fluid (e.g., system composition ) 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) may434937-8761-0640.1Atty. Dkt. No.: 86357096manage 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.

[0166] 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 408d. 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 lyo particle 431.

[0167] Optionally, responsive to lysing the sample with the IPC lyo particle 431 and the system composition, the resulting lysate may be transported from the lyse chamber 432 to a location where the MB lyo particle 438 is stored. The fluid sensor 428d may monitor transport of the fluid to determine when the lysate has entered the storage location of the MB lyo particle 438. The lysate may be transported from the lyse chamber 432 to the storage location of the MB lyo particle 438 by opening the valves 408d, 408p, and / or 408h. The lysate may then be moved back to the lyse chamber 432. Movement of the lysate between the lyse chamber 432 and the storage location of the MB lyo particle 438 may mix the lysate and ensure that all of the MB lyo particle 438 is collected and mixed with the lysate.

[0168] In some examples, the MB lyo particle 438 may be included with the IPC lyo particle 431. In such examples, the lysate may not be moved to a location of the MB lyo particle 438 and may instead remain in the lyse chamber 432.

[0169] In some cases, upon lysing, hybridization may occur in the lysis chamber 432. Hybridization may occur through incubation at a hybridization temperature (e.g., a temperature capable of causing the nucleic acids of interest to hybridize with the magnetic particles) for a certain period of time (e.g., 1 to 5 minutes). The lysate may be cooled to the hybridization temperature. Cooling may be accelerated via, for example, external connective cooling and / or addition of system buffer (having a lower temperature than the lysate) to the lyse chamber 432.

[0170] As described herein, hybridization may occur in the lyse chamber 432. However, it should be understood that hybridization may occur in a chamber that is not the lyse chamber 432. For example, in some cases, the diagnostic system architecture 400 may include one or 444937-8761-0640.1Atty. Dkt. No.: 86357096more additional hybridization chambers, and the lysate may be transported from the lyse chamber 432 to the hybridization chamber to allow hybridization to occur.

[0171] Responsive to hybridization, the external magnet 458b may apply a magnetic field to the lysis chamber 432. This may cause the magnetic particles hybridized to the nucleic acids of interest in the lysis chamber 432 to be held to the side (e.g., a wall) of the lysis chamber 432. The valves 408h, 408p, 408o, and / or 408n may be opened. The lysate (e.g., the system composition and the IPC lyo material mixture) without the hybridized paramagnetic particles may be transported from the lysis chamber 432 to the waste storage chamber 468.

[0172] 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 of MgCh, 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.The first wash buffer may be enabled by opening the frangible seals 410c and / or 410d of the wash buffer blister device 424a. The first wash buffer may then be moved from the blister device 424a through the PCA chambers 456 by pressurizing through the valves 408c and 408i. Valves 408q, 408n, and 408o may be open to allow fluid flow. In embodiments where hybridization occurs in the lysis chamber 432, the wash buffer may flow from the blister 424a to the lysis chamber 432 responsive to the opening of the frangible seals 410c and 410d. The volume of the first wash buffer may be controlled using the sensors 428d and 428e positioned within the instrument such that the sensor aligns with and senses fluid in the hybridization chambers. After passing a controlled volume of the first wash buffer to the lysis chamber 432, and after washing the lysis chamber 432 with the first wash buffer, flow may be reversed by pressurizing valve 408g and 408h and opening valves 408q, 408i, and 408j to allow fluid flow. The first wash buffer may be returned to the blister device 424a until the end of the fluid flow is detected (e.g., by any sensor 428). Responsive to the detection of the end of the fluid flow, additional air and / or liquid may be purged to the lyse chamber 432.

[0173] 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 liter454937-8761-0640.1Atty. Dkt. No.: 86357096of 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). The second wash buffer may be enabled by opening the frangible seals 410e and 410f of the wash buffer blister device 424b. The second wash buffer may then be moved from the blister device to the lysis chamber 432. In embodiments where hybridization occurs in the lysis chamber 432 (e.g., as shown in FIGS. 4A-4C), the wash buffer may flow from the blister 424a to the lysis chamber 432 responsive to the opening of the seals 410c and 410d. The volume of the second wash buffer may be controlled using the sensor 428d positioned within the instrument such that the sensor aligns with and senses fluid in the hybridization chambers. After passing a controlled volume of the second wash buffer the lysis chamber 432, flow may be reversed by pressurizing through the opened valves 408g and 408h and opening valves 408r and 408j to allow fluid flow back to the blister device 424b. The second wash buffer may be returned to the blister device 424b until the end of the fluid flow is detected (e.g., by any sensor 428). Responsive to the detection of the end of the fluid flow, additional air and / or liquid may be purged to the lyse chamber 432.

[0174] In various examples, the PCA buffer 440 may be metered (e.g., at the PCA buffer metered section 442). The PCA buffer blister valves 410g and 41 Oh may be opened. Flow may then move out of the PCA buffer 440. Flow may be moved out by sucking flow at the valve 408f and opening the valves 408e and 4081 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 428f.

[0175] After the PCA buffer flows past a location aligned with the sensor 428f, the valve 408e is closed and the valve 408m 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 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 428f). In the MM mixing chamber 444, a preamplification master mix (e.g., a preamplification master mix lyo bead) may be mixed.

[0176] The master mix (MM) lyo material may be mixed well into the PCA buffer. The MM lyo material 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 and464937-8761-0640.1Atty. Dkt. No.: 86357096negative pump pressure with valve 408f and valve 408m open. Once mixing is complete, the master mix is pulled into the metering channels and out of the mixing chamber 444.

[0177] Responsive to the master mix being fully mixed, the master mix may enter the preamplification PCA chamber 470 by applying a vacuum to valve 408h, with valves 408s and 408m open to allow fluid flow. The master mix may enter the preamplification PCA chamber 470 until a trailing liquid meniscus of the master mix arrives at a location corresponding to the inlet sensor 428c.

[0178] Responsive to the master mix arriving at the preamplification PCA chamber 470, a first PCA reaction may be performed. The first PCA reaction may be a preamplification reaction. That is, the first amplification reaction may be performed for a limited number of cycles relative to a number of cycles performed in second amplification reactions. The preamplification PCA chamber 470 may be pressurized via the valve 408h 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 preamplification PCA chamber 470 may be controlled via a plurality of heaters on each side of the preamplification PCA chamber 470. In various examples, electrical pulsing of the PCA foil (e.g., the heating element 122) may create temperature pulses for the first PCA reaction. In various embodiments, the first PCA reaction may be a bead PCA reaction.

[0179] After the preamplification reaction, the products of the first PCA reaction are eluted, and the preamplification PCA chamber 470 is heated. In some examples, the preamplification PCA chamber 470 may be heated via the heating element 122. For example, the heating element 122 is a pulse heating element that may be or include a metal foil that directly contacts the amplification reagents (e.g., the preamplification PCA chamber 470). In some examples, the preamplification PCA chamber 470 is heated using a bulk heater / cooler that is external to the cartridge (e.g., is part of the instrument 150) but is positioned within the instrument such that the bulk heater / cooler interfaces or contacts the preamplification PCA chamber 470 and / or the PCA chambers 456. As shown in FIGS. 4A-4C, the bulk heater / cooler may generate heat for the external heat zone 462. In various examples, heating may occur by applying energy to (e.g., heating) only the heating element 122 or applying energy to the heating element 122 and the external bulk heater / cooler (e.g., to heat zone 462). Heating the heating element 122 may cause the external heat zone 462 to heat, , thereby heating the preamplification PCA chamber 470. The heating element 122 may be heated to, for example 95 degrees Celsius for a474937-8761-0640.1Atty. Dkt. No.: 86357096predetermined length of time (e.g., X seconds). Upon completion of the first amplification reaction, the preamplification PCA chamber 470 is depressurized, and the PCA products are pumped into the PCA mixing chamber 466.

[0180] Remaining PCA buffer 440 (e.g., PCA buffer not used in master mix reconstitution) from the PCA buffer blister is transported to a location of the cartridge that interfaces with a location of the sensor 428e. Dilution may then occur by performing a reciprocating mix of the PCA buffer 440 with the PCA products in the PCA mixing chamber 466. The diluted, mixed PCA product is then metered, and the metered diluted PCA product 452 is transported to a cartridge location that interfaces with the sensor 428e.

[0181] The metered diluted PCA product is pumped to locations of the cartridge that interface with sensors 428g, 428h, and 428i. The sensors 428g, 428h, and 428i may be used to monitor fluid flow and location and trigger the closing of the valves 408u, 408v, and 408w. Closing of the valves 408u, 408v, and 408w may halt flow of the metered diluted PCA products. Flow of the metered diluted PCA products to the mixing chambers 450 may be split based on a number of lyophilized MM reagents and reactions. In embodiments where free PCA reactions are performed as the second amplification reactions, no magnetic particles may be present in the MM mixing chambers 450 or the PCA chambers 456. In embodiments where hybrid PCA reactions are performed as the second amplification reactions, such as those described with respect to FIGS. 4B and 4C, the lyophilized MM reagents 454 may be or include streptavidin-coated magnetic particles, which may enhance the hybrid PCA reaction. Further a plurality of the primers (e.g., half of the primers) may be biotinylated.

[0182] The metered diluted PCA products are pumped to the MM mixing chambers 450a-c, either in parallel or in series. As stated, the number of MM mixing chambers utilized may be based on a number of aliquots that the PCA products are to be divided into (e.g., based on a number of targets to be detected). The MM mixing chambers 450a-c may be configured to mix master mixes used during the second amplification reactions (e.g., non-preamplification reactions). The diluted PCA products may be reciprocatingly mixed with the lyo MMs 454. Each of the chambers 450 may mix the diluted PCA products in parallel. For example, the portion of the diluted PCA products pumped into the MM mixing chamber 450a are mixed with the lyo MM 454a while the portion of the diluted PCA products pumped into the MM mixing chamber 450b are mixed with the lyo MM 454b. The sensors 428g, 428h, and 428i may monitor flow and control flow reversal. In embodiments where hybrid PCA reactions are484937-8761-0640.1Atty. Dkt. No.: 86357096performed as the second amplification reactions, such as in FIGS. 4B and 4C, the lyo MM (e.g., the magnetic particles) may also be resuspended in the mixed solution. Further, FIG. 4C depicts an embodiment where an enhanced hybrid PCA reaction is performed as the second amplification reaction. In such embodiments, a mixing area, indicated by the external heat zone 462, may be heated to an optimum hybridization temperature. After a sufficient incubation time for hybridization, the reconstituted lyo MM 454 (that includes the magnetic particles) may be heated (e.g., to 90 degrees Celsius). In some cases, such hybridization may be optional (e.g., due to a slow hybridization reaction rate). Heating the reconstituted lyo MM 454 may facilitate hot-starting polymerase, which may reduce off-target amplification.

[0183] The valves 408u, 408v, and 408w are opened, and the fluid in each of the chambers 450 is pumped into a respective PCA chamber 456. The fluid may be pumped from each of the MM mixing chambers 450 to the respective PCA chambers 456 in series or in parallel. In embodiments where hybrid PCA reactions are performed as the second amplification reactions, such as in FIGS. 4B and 4C, the magnetic particles suspended in the lyo MM 454 are captured onto a foil (e.g., the heating element 122). Second PCA reactions may be performed in each of the PCA chambers 456 in parallel. Specifically, the second PCA reactions may be free PCA reactions (as in FIG. 4A) or hybrid PCA reactions (as in FIGS. 4B and 4C). In some examples, as shown in FIG. 4C, bead-PCA reactions may be performed as the second amplification reactions. Real-time optical detection may be performed for each of the reactions. Upon completion of the second PCA reactions, the system is depressurized.

[0184] Referring now to FIG. 5A, a cross-sectional view of the detection chamber 120 is shown, according to an example embodiment. 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 a controller and the electrical components. In various examples, the electrical components, the controller, 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 types494937-8761-0640.1Atty. Dkt. No.: 86357096of chambers. For example, the cartridge 100 may include an amplification chamber and a detection chamber.

[0185] 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).

[0186] 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.

[0187] 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% of 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).

[0188] 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 may504937-8761-0640.1Atty. Dkt. No.: 86357096allow 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).

[0189] 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.

[0190] 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 chamber may 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 embodiments, 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 a controller. Responsive to receiving the temperature data, the controller may activate or otherwise control the pulses delivered by the energy source.

[0191] 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, the514937-8761-0640.1Atty. Dkt. No.: 86357096plastic 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.

[0192] 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 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.

[0193] 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 reaction volume, 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.

[0194] 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.

[0195] 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,524937-8761-0640.1Atty. Dkt. No.: 86357096a 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.

[0196] 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.

[0197] 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.

[0198] 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 activatable heating 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 a PC A foil. For example, the heating element 122 may be stainless steel.

[0199] In various examples, the foil (e.g., the heating element 122 or the first layer) may be wider than the PCA 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.534937-8761-0640.1Atty. Dkt. No.: 86357096

[0200] 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 layer 508 may comprise a PSA layer having a thickness up to 200 microns. In some examples, the adhesive layer 508 may be a heat spreader pressure sensitive adhesive. In some examples, the adhesive layer 508 may comprise a material including both thermosetting and thermoplastic properties. In some such examples, the material of the adhesive layer 508 may comprise acrylic adhesive materials. In some of these examples, the adhesive layer 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.

[0201] 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 and may 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.

[0202] 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.544937-8761-0640.1Atty. Dkt. No.: 86357096

[0203] 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.

[0204] The heat spreader 510may 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.

[0205] 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).

[0206] Referring now to FIG. 5B, a system 500B including the detection chamber 120 is shown, according to an example embodiment. 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.

[0207] 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.

[0208] 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 an optical unit 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 may utilize, for example, a sensor within the detection chamber 120 to detect fluorescence corresponding to an amount of the nucleic acid of interest.554937-8761-0640.1Atty. Dkt. No.: 86357096

[0209] Referring now to FIG. 6, a method 600 for detecting the presence, absence, amount, etc. of at least one nucleic acid of interest, according to some embodiments. 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 sample may include a plurality of nucleic acids of interest. 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 (e.g., analytes, nucleic acids of interest, etc.) 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 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. PC A buffer may be metered and used to reconstitute Master Mix reagent used for PCA. Reconstituted Master Mix reagent may be loaded into the detection chamber 120 with trapped magnetic particles. A first PCA (e.g., RT-PCA) is performed to preamplify the target organisms. The preamplified sample is diluted and divided into a plurality of aliquots. PCA is performed on each aliquot with real-time fluorescent detection, where a different target organism is detected in each aliquot. Results of PCA and detection of target nucleic acids are reported to the user.

[0210] 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 a plurality (e.g., two or more) nucleic acids of interest. A molecule 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 100564937-8761-0640.1Atty. Dkt. No.: 86357096or 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.

[0211] At block 608, the system composition (e.g., system composition ) 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 (e.g., one or both of the extraction chamber 114a and the extraction chamber 114b) for use in lysing the biological sample and / or hybridizing the nucleic acids of interest of the lysed biological sample.

[0212] 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 the volume may be metered to 450 microliters to be transported to and / or used in the extraction chamber 114a and / or the extraction chamber 114b.

[0213] At block 612, the wash buffer (e.g., wash buffer 110) may be stored. For example, the wash buffer 110 may be stored in a blister device attached to the cartridge and / or a sealed chamber integrated into the cartridge body. The wash buffer 110 may be used for washing magnetic particles (e.g., magnetic particles 116) and / or the extraction chamber(s) 114a and / or 114b. For example, the wash buffer 110 may be stored in a blister device and transported to one or more extraction chambers 114a or 114b (e.g., a lyse chamber) for use in washing the extraction chamber (and / or the magnetic particles 116) to prepare for subsequent amplification and detection of the nucleic acids of interest of the biological sample.

[0214] In various examples, the wash buffer 110 may comprise water, salt, a buffering compound, and / or surfactants. The wash buffer 110 may include moderately high574937-8761-0640.1Atty. Dkt. No.: 86357096concentrations 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 may include a stronger washing agent, and a second wash buffer 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 may be used to wash the extraction chamber 114a and / or the magnetic particles 116, and the second wash buffer may be used to rinse the extraction chamber 114 and / or the magnetic particles 116.

[0215] At block 614, the PC A buffer may be stored. For example, the PC A buffer may be stored in a blister device attached to the cartridge and / or a sealed chamber integrated into the cartridge body. The PCA buffer 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 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. The PCA buffer may also be delivered to the preamplified sample. For example, PCA buffer that remains after using a portion of the PCA buffer to reconstitute the master mix reagent may be to the extraction chamber 114a where the preamplified products are stored. The PCA buffer may be mixed with the preamplified products.

[0216] In various examples, the PCA buffer may comprise water and / or salt. The PCA buffer 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 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. For example, the cartridge 100 may include between 100 and 400 microliters of the PCA buffer. For example, the cartridge 100 may include 200 microliters of the PCA buffer.584937-8761-0640.1Atty. Dkt. No.: 86357096

[0217] At block 616, the volume of the PC A buffer 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 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 may be metered by pumping the PCA buffer 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 is further transported.

[0218] At block 618, 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. Reconstitution of the master mix reagent may, in some examples, be performed in parallel with hybridization and / or capture of the magnetic particles 116 in the extraction chamber 114 (e.g., block 620).

[0219] 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.

[0220] At block 620, 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 114a. The system composition and the IPC may be added to the extraction chamber 114a prior to, concurrent with, and / or subsequent to addition of the biological sample to the extraction chamber 114a. 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 be mixed using sonication. For example, the lyse system 152 may be coupled with the extraction chamber 114a. The sonicator 154 may perform sonication to add heat to the extraction chamber 114a, mix the contents of the extraction chamber 114a (e.g., the sample, the buffer, and the IPC), and / or induce cavitation.594937-8761-0640.1Atty. Dkt. No.: 86357096

[0221] In various examples, the extraction chamber 114a 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 114a. 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.

[0222] At block 622, potential targeted nucleic acids in the solution lysed at block 620 may be hybridized to stored functionalized paramagnetic particles (e.g., magnetic particles 116). The functionalized magnetic particles 116 may be included during lysis at block 620 or added to the extraction chamber 114a after lysing the sample, the buffer, and the IPC at block 620. The solution lysed in the extraction chamber 114a may be referred to as a “lysate.” In some examples, the lysate may be transported to the extraction chamber 114b. In the extraction chamber 114b, 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).

[0223] During hybridization, the magnetic particles 116 may be mixed and agitated with the lysate. A greater number of magnetic particles 116 may be utilized to perform hybridization in a high-plex reaction relative to hybridization in low-plex reactions. The use greater number of magnetic particles 116 may increase time efficiency of hybridization. The nucleic acids from the target organisms and the IPC may hybridize or bind to the magnetic particles 116. 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 (e.g., a first extraction chamber 114b and a second extraction chamber 114b such that vortices form at an entrance of each chamber to mix the lysate with the magnetic particles 116.

[0224] During or after hybridization, the magnetic particles 116 may be held to the side of the extraction chamber. An applied magnetic field (e.g., the magnetic field generator 156a or 156b) may cause the magnetic particles 116 to be held to the side (e.g., a wall) of the extraction604937-8761-0640.1Atty. Dkt. No.: 86357096chamber 114. Upon lysing the sample, the buffer, and the IPC at block 612, the resulting lysate is disposed to waste storage. For example, the lysate may be transferred to the waste storage chamber 168. In some examples, the lysate may be stored in the blister device used to store the system composition . The lysate may be stored in locations of the cartridge 100 (e.g., the extraction chamber(s) 114, the waste storage chamber 168, etc.) such that the lysate does not contact any surface of the cartridge 100 that may subsequently be in contact with the master mix reagent. As such, the lysate may be stored and transported throughout the cartridge 100 such that the lysate does not contact any valves, channels, chambers, etc. that the master mix reagent contacts. For example, the lysate may not be transported to the detection chamber 120.

[0225] At block 624, one or both of the extraction chambers 114a and 114b and the magnetic particles 116 are washed using the wash buffer (e.g., the wash buffer 110). Washing the extraction chamber(s) 114 and / or the hybridized magnetic particles 116 may remove undesirable components in the extraction chamber(s) 114 and / or on the magnetic particles 116 that may interfere with amplification of the nucleic acids of interest. The wash buffer 110 may be pumped through the extraction chamber(s) 114 to wash the chamber(s). Upon washing, the wash buffer 110 may return to the blister device storing the wash buffer. In various examples, a volume, time, flow rate, and / or flow directionality may be adjusted based on needs of the amplification and / or assay.

[0226] Upon washing the contents of the extraction chamber(s) 114, the magnetic particles 116 may be resuspended in the wash buffer 110. In various embodiments, resuspension of the magnetic particles 116 may be performed via sonication (e.g., using the sonicator 154).

[0227] In various embodiments, a second wash may be formed with a second wash buffer (e.g., a first wash is performed as described above with a first wash buffer 110a, and the second wash is performed with a wash buffer 110b). The second wash may be performed similar to the first wash, but with a different wash buffer. The second wash may trap the magnetic particles 116 and exchange fluids in the extraction chamber(s) 114.

[0228] At block 626, the magnetic particles 116 (e.g., that are bound with the target oligonucleotides) are trapped onto a heating element (e.g., the heating element 122) in the detection chamber 120 (e.g., a PCA chamber). The heating element 122 may be or include, in various examples, a continuous, structured, or shaped metal foil, a pulse heating element, metal wires, a conductor and / or resistor layer deposited and / or plated, and / or backed by a heat614937-8761-0640.1Atty. Dkt. No.: 86357096spreader. In various examples, in order for the PC A process to occur, the paramagnetic particles with the captured nucleic acids may be concentrated at a thermocycling zone of the heating element. Thus, after hybridization, the hybridized magnetic particles may be transported to the detection chamber 120, where the particles are “trapped” or otherwise attached to the heating element 122.

[0229] The hybridized magnetic particles may be delivered to the detection chamber 120. The hybridized magnetic particles 116 may be trapped or attached to the heating element 122 via a magnetic field (e.g., generated by the magnetic field generator 156). An external permanent magnet (e.g., 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. Further, in various examples, a flow of the hybridized magnetic particles may be continuous, in discrete steps, or any combination thereof.

[0230] At block 618, as stated above, the PCA master mix reagent is reconstituted. 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 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.

[0231] 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. The reconstituted master mix reagent may cover the trapped magnetic particles 116 on the heating element 122. 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 send 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 120.624937-8761-0640.1Atty. Dkt. No.: 86357096

[0232] At block 630, a first amplification is performed. The first amplification may be a preamplification. The preamplification may be or include an RT-PCA or bead-based PC A, as will be described in greater detail with respect to FIGS. 7 and 8. Preamplification may be performed for a limited number of cycles (e.g., below a threshold number of cycles). For example, preamplification may be performed for a number of cycles (e.g., 20) and / or for a specific amplification factor (e.g., on the order of 104) when typical (e.g., non-preamplification) amplification is performed for a greater number of cycles (e.g., at least 50) and / or for a specific amplification factor (e.g., on an order of 109). In some examples, preamplification may be performed for a minimum number of cycles (e.g., at least 20 cycles) and / or until a minimum amplification factor is achieved (e.g., on an order of 103to 104). In some examples, the number of preamplification cycles may be a percentage of a number of total amplification cycles or non-preamplification cycles. For example, the number of preamplification cycles may be 10% to 35% of the total number of full (e.g., non-preamplification) amplification cycles. Preamplification of the hybridized magnetic particles 116 in the detection chamber 120 may produce a preamplificate. Trapped magnetic particles 116 may be heated using the heating element 122 to elute amplicons off of the particles 116.

[0233] As described herein, preamplification may allow for easier amplification and detection of analytes in high-plex reactions. . For example, it may be easier to amplify a large number (e.g., 18) analytes of interest to a lower amplification factor (e.g., on an order of 104) and / or for a fewer number of cycles than to a greater amplification factor (e.g., an order of 109) and / or for a greater number of cycles. First performing a preamplification by a limited number of cycles before performing a second amplification using a typical number of cycles (e.g., a greater number of cycles than during preamplification) may reduce challenges resulting from off-target amplification (e.g., including primer-dimers), competing reactions, exhausting reaction components, the presence of inhibitors, etc. Further, amplification efficiency may be greatest during the early amplification cycles (e.g., the number of cycles performed during preamplification).

[0234] During preamplification, no detection of the analytes or nucleic acids of interest may be performed. By not detecting the analytes in the preamplificate, the challenges associated with detecting a large number (e.g., greater than six) targets with a multi-channel detection system. Additionally, during preamplification, a larger area of the heating element 122 may be utilized to accommodate the greater amount of magnetic particles 116 used in a high-plex634937-8761-0640.1Atty. Dkt. No.: 86357096hybridization (e.g., as in block 622). In addition to a greater area of the heating element 122, longer heating pulses and / or longer cooling times per cycle may be used during the preamplification. Longer heating pulses may extend a denaturation time, thereby enabling longer amplicons used in nested and / or semi-nested primer designs.

[0235] At block 632, the preamplificate is diluted. For example, as described above, PCA buffer that is not used to reconstitute the master mix reagent may be added to the products of the preamplification (e.g., the preamplificate). The PCA buffer is mixed with the preamplificate. Upon dilution, the diluted preamplificate is divided or split into a plurality of aliquots. For example, the diluted preamplificate may be divided into 3, 4, 5, 6, etc., aliquots. Dilution may increase a volume of fluid to allow for a sufficient fluid volume of each aliquot, thereby enabling second amplification reactions to be performed on each aliquot. Dilution may also dilute out any off-target amplification products and / or primers resulting from preamplification.

[0236] In some examples, to dilute the preamplificate, the amplicons (e.g., amplification products, preamplified nucleic acids of interest, etc.) may be eluted from the magnetic particles in a preamplification detection chamber 120. The preamplificate may be diluted with the PCA buffer, and the preamplificate is divided into a plurality of aliquots. In other embodiments, the preamplificate (e.g., the original master mix used during preamplification) may be discarded. Fresh (e.g., unused) PCA buffer may be transported into the preamplification PCA chamber (e.g., a first PCA chamber). The amplicons (e.g., replicated targets) are eluted from the magnetic particles 116 into the fresh PCA buffer and the solution including the fresh PCA buffer is divided into a plurality of aliquots.

[0237] At block 634, each aliquot reconstitutes a master mix reagent. For example, when the preamplificate has been divided into three aliquots, three master mix reagents may be reconstituted (e.g., one by each aliquot). Each reconstitution may be performed in parallel (e.g., at the same time), and delivery of the reconstituted master mixes to each of the second detection chambers 120 may be performed in parallel. Each master mix may target a smaller set of nucleic acids relative to the master mix reagent constituted at block 618. Each reconstituted master mix reagent may include bare streptavidin paramagnetic beads that may be used for a subsequent amplification (e.g., a hybrid PCA).644937-8761-0640.1Atty. Dkt. No.: 86357096

[0238] As stated above, primes for second amplification reactions may be nested and / or seminested relative to the primers used during the first amplification (e.g., the preamplification). The use of nested and / or semi-nested primers may improve overall sensitivity and / or specificity of the PCA reactions and detection. In some examples, the primers may be biotinylated. Further, in embodiments in which the second amplification reactions are hybrid PCA reactions, streptavidin-coated paramagnetic beads may be included in the master mix.

[0239] Further, during reconstitution at block 634, the master mix may be heated (e.g., using the heating element 122). Heating the master mix may activate hot-start polymerases prior to the second amplification reactions rather than at the start of the second amplification reactions.

[0240] At block 636, each master mix reagent reconstituted at block 634 is transported to and loaded into an associated second detection chamber 120. For example, the preamplification performed at block 630 may be performed in a first detection chamber 120. Each aliquot and associated reconstituted master mix reagent is transported to a respective second detection chamber 120. The second detection chambers 120 may be PCA chambers.

[0241] At block 638, second amplifications are performed on each of the aliquots. The second amplifications may be or include PCA reactions. Specifically, the second amplifications may be free PCA reactions and / or hybrid PCA reactions. Each of the second amplification reactions performed on each of the aliquots may be performed in parallel (e.g., at the same time or substantially the same time) using the same heating element 122 (e.g., the same foil). The second amplifications may be performed using real-time detection. That is, as each amplification occurs or after each amplification reaction has occurred, the presence, absence, or amount of the nucleic acid of interest is detected. As stated herein, a different nucleic acid (e.g., or analyte) of interest may be detected in each aliquot.

[0242] As stated, the second amplification reactions may be hybrid PCA and / or free PCA reactions. Free PCA reactions may have a worse associated limit of detection compared to bead PCA. However, the preamplification reaction may be a bead PCA reaction or any other type of preamplification reaction, thereby overcoming the challenges associated with the lower limit of detection of the free PCA (e.g., because preamplification increases a number of copies of the nucleic acids or targets of interest to a value above a limit of detection associated with free PCA). Further, the use of a free PCA reaction may streamline workflow. For example, without use of a free PCA reaction, the preamplificate may be reconcentrated onto a second set of654937-8761-0640.1Atty. Dkt. No.: 86357096magnetic particles 116 to facilitate second bead PCA reactions. The lack of magnetic particles 116 in free PCA may eliminate signal noise (e.g., during detection) that may be associated with the random distribution of magnetic particles 116 on the heating element 122. Additionally, due to the aliquoting of the diluted preamplificate, each free PCA reaction and associated detection may be limited to a certain number of analytes below a threshold value (e.g., six or fewer analytes). In some examples, when free PCA reactions are performed, the lyophilized master mix reagents may be stored outside each detection chamber 120 (e.g., each PCA chamber).

[0243] The second amplification reactions may be hybrid PCA reactions. For example, when one or more primers are biotinylated and streptavidin-coated magnetic particles are included in the detection chamber 120, hybrid PCA reactions may be performed. Hybrid PCA reactions may enhance a limit of detection relative to the limit of detection associated with free PCA reactions. In some examples, when hybrid PCA reactions are performed, a streptavidin magnetic particle may be included with the lyophilized master mix reagents. Additionally, biotinylated primers may naturally bind to streptavidin-coated magnetic particles (e.g., via biotin-avidin binding, if the primers are not already attached prior to lyophilization). The biotinylated primers bound to the streptavidin-coated magnetic particles may help concentrate newly generated amplicons within a thermocycling zone by capturing the newly generated amplicons to the magnetic particles (e.g., due to the biotinylated capture primers on the magnetic particles). . A hybrid PCA reaction ay then be performed without explicit prior hybridization. The hybrid PCA reactions performed for each aliquot may be performed in parallel. Additionally, the lyophilized magnetic particles and master mix components may be stored outside of each detection chamber 120 (e.g., each PCA chamber). In some examples, the lyophilized magnetic particles and master mix components may be stored within each detection chamber 120 (e.g., each PCA chamber).

[0244] The 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. The optical unit may include a plurality of channels, each channel to detect a different nucleic acid (or analyte) of interest in each aliquot.

[0245] At block 640, the results of the second amplifications may be analyzed and presented or reported (e.g., to a user). For example, the results may be analyzed to determine a presence,664937-8761-0640.1Atty. Dkt. No.: 86357096absence amount, etc. of the nucleic acid of interest. The results may be displayed, for example, via a user interface.

[0246] Referring now to FIG. 7, a method 700 for pulse controlled amplification (PCA) is shown, according to an example embodiment. 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 114b 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.

[0247] At block 702, the lysate containing the nucleic acid of interest is hybridized to capture oligonucleotides. Hybridization may occur in the extraction chamber 114b. 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.

[0248] 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 elongation temperature (e.g., between 50 and 80 degrees Celsius). For example, the annealing or elongation temperature may be 72 degrees Celsius.

[0249] At block 706, an electrical pulse 718 maybe 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 a674937-8761-0640.1Atty. Dkt. No.: 86357096portion 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.

[0250] 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.

[0251] 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.

[0252] 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., relative684937-8761-0640.1Atty. Dkt. No.: 86357096directionality 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.

[0253] 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 may detect the fluorophores to determine a corresponding value, absence, presence, etc. of the amplified nucleic acid of interest.

[0254] 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 generate a 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.

[0255] 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.694937-8761-0640.1Atty. Dkt. No.: 86357096

[0256] Referring now to FIG. 8, a plurality of types of PC A reactions are shown, according to example embodiments. Specifically, bead PCA 802, free PCA 804, and hybrid PCA 806 are shown, according to some examples.

[0257] Each type of PCA reaction 802, 804, and 806 may be performed in a detection chamber 120. During a PCA reaction, each detection chamber 120 may include an isothermal zone 808, a pulsed thermocycling zone 810, and a foil 814.

[0258] A bead PCA reaction 802 may include processed similar to those described in FIG. 7. As shown in FIG. 8, bead PCA 802 may further include, in the pulsed thermocycling zone 810, a plurality of paramagnetic beads 812 hybridized with captured target nucleic acids. A plurality of master mix components 816 may be mixed into the isothermal zone 808 of the detection chamber 120. Due to the hybridized paramagnetic beads 812 located in the thermocycling zone 810, the target nucleic acids 818 may be captured on the magnetic particles and may be located within the thermocycling zone 810.

[0259] A free PCA reaction 804 may include similar steps as those performed in a bead PCA reaction. For example, a free PCA reaction 804 may be performed with similar spatial and / or temporal temperature profiles as that associated with bead PCA reactions 802. As shown in FIG. 8, free PCA 804 includes, similar to bead PCA 802, a pulsed thermocycling zone 810. However, the thermocycling zone 810 may not include paramagnetic beads with captured target nucleic acids 812. Due to a lack of magnetic beads in the detection chamber 120, the target nucleic acids 818 may not be concentrated in or near the thermocycling zone 810 and may instead be dispersed throughout the detection chamber 120 (e.g., within the isothermal zone 808 and the thermocycling zone 810). Additionally, in free PCA reactions 804, the foil 814 may generate pulses that are longer (e.g., have a greater duration) relative to other PCA reactions. Longer pulses may expand the thermocycling zone 810. Further, free PCA reactions 804 may have shorter cycles relative to bead PCA reactions 802.

[0260] A hybrid PCA reaction 806 may include performing both a PCR reaction and a PCA reaction. As with free PCA reactions 804, hybrid PCA reactions 806 may utilize similar spatial and temporal temperature profiles as those for bead PCA reactions 802. Additionally, hybrid PCA reactions 806 may be performed with streptavidin-coated paramagnetic beads. The use of such paramagnetic beads may enhance or increase a concentration of target nucleic acids 818 to the foil 814 before and / or during amplification. This may be due to the biotinylated primers704937-8761-0640.1Atty. Dkt. No.: 86357096820 (e.g., capture oligonucleotides) on or coupled with the streptavidin-coated paramagnetic beads, thereby omitting an explicit hybridization or capture process performed prior to amplification. Hybrid PCA reactions 806 may also include primers 820. Specifically, the primers 820 may be biotinylated primers that are initially (e.g., before the PCA reaction occurs and / or at the start of the PCA reaction) suspended in a free master mix solution.

[0261] FIG. 9A depicts an example blister device 900, in accordance with present implementations. As illustrated by way of example in FIG. 9, the example blister device 900 can include at least a first rupturing member 910 (also referred to herein as a “piercing member”) to open or close a valve seal 912a (also referred to herein as a “one-time open valve”) of the blister device 900 (e.g., a portion of the lidding foil 960 adjacent with the rupturing member), a metal coated polymer film 920, a reagent storage cavity 930, a second rupturing member 940 to open or close valve seal 912b of blister device 900 (e.g., a portion of the lidding foil 960 adjacent the rupturing member), a first fluid input-output area 950, the lidding foil 960 at least partially corresponding to the reagent storage cavity 930, and a second fluid inputoutput area 970. One or more zones as discussed herein can comprise or include one or more blister devices similar to the blister device 900. 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 900 may be used to store liquid reagents in the cartridge 100. For example, the system composition and / or the wash buffer 90 may be stored in a blister device 900. The blister device 900 may be attached, coupled, or otherwise affixed to the cartridge 100. The blister device 900 can provide a technical improvement to achieve bidirectional flow between and through zones of a cartridge as discussed herein.

[0262] As shown in FIG. 9A, the blister device 900 comprises rupturing members 910 and 940 that can be actuated to open the valve seals 912. The rupturing members 910 and 940 may be integrated into the metal coated polymer film 920. In various examples, the rupturing members 910 and 940 may be used to rupture one-time open valves (e.g., valve seals 912). One-time open valves 912 may minimize water loss out of the storage cavity 930 through the closed or sealed rupturing members 910 and 940. 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 900 to the microfluidic network of the cartridge 100. For example, the fluid entering and exiting the blister device 900 may enter and / or exit one or both of the one-time open valves 912. Further, the valves 912 may allow the reagents stored in the blister device 900 to return to the blister device714937-8761-0640.1Atty. Dkt. No.: 86357096900 after use. For example, a blister device 900 may store a wash buffer. The wash buffer may exit the blister device 900 (e.g., through a valve 912) 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 900 through the valves 912a and / or 912b. Storing used liquid reagents in the original blister device 900 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 900 may additionally function as a waste chamber to store a used or exhausted liquid reagent.

[0263] As described above, the blister device 900 may include one or more one-time open valves 912. The opening of the valves 912 may allow a pump of the cartridge to deliver the reagents 125 stored in a chamber or cavity 930 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 910 and 940 may interface with an actuator of the instrument 150 to modulate a compression of the one or more rupturing members .

[0264] The blister device 900 may be sealed off from channels and chambers of the cartridge 100 by the rupturing members 910 and 940 and / or the unruptured valves 912. The rupturing members 910 and 940 may cover a first port (e.g., the valve 912a and / or the first fluid inputoutput area 950) when the rupturing member is in a non-ruptured state. The blister device 900 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 950 when the rupturing member is in a ruptured state.

[0265] The blister device 900 may comprise a second port (e.g., the valve 912b and / or an area underneath the rupturing member 940 and / or the second input output area 970) and may be sealed-off from the channels and the chambers of the cartridge by the second rupturing member. The blister device 900 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 970 when said the rupturing member is in a ruptured state.

[0266] The blister device 900 may enclose a storage chamber when the storage chamber is configured in hermetically sealed-off relation from the channels and / or when the cartridge of the present disclosure is in a non-activated state. The storage chamber is configured for open724937-8761-0640.1Atty. Dkt. No.: 86357096communication 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 900 may, when ruptured, allow fluid to flow freely between the blister device 900 and the extraction chamber 114b.

[0267] In some examples, a first channel can be sealed-off from the blister device 900 by the valve seal 912a covering a first port when the first valve seal 912a is in a non-ruptured state. The first channel can be in fluid communication with the blister device 900 through the first port when the first valve seal 912a is in a ruptured state.

[0268] In some examples, a second channel can be sealed-off from the blister device 900 by the valve seal 912b covering the second port when the second valve seal 912b is in a nonruptured state. The second channel can be in fluid communication with the chamber through the second port when the second valve seal 912b is in a ruptured state.

[0269] The first and second valve seals 912a and 912b may be ruptured by the first and second rupturing members 910 and 940, 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 900. Upon selectively piercing, by the first and second rupturing members 910 and 940, at least one of the first and second valve seals 912a and 912b of the blister device 900 and forming the opening therein, the desired reagent can be introduced through a corresponding one of the channels having an opened port.

[0270] FIGS. 9B, 9C, and 9D depict a detailed view of the rupturing members 910 and / or 940 of the blister device 900 and the valve seals 912 of FIG. 9A, according to some embodiments. A chamber between the rupturing members 910 and / or 940 and the valve seals 912a and 912b, respectively, may include first and second fluid input-output areas 950 and 970. In various examples, the rupturing members 910 and / or 940 may be actuated, compressed, or otherwise manipulated to open a valve seal 912 (also referred to as a “one-time open valve” or a “valve”) and permit fluid flow. However, over-travel of the rupturing members 910 and 940 may cause a pressure restriction when liquid attempts to move through the blister device 900 and / in and out of the blister device 900. FIG. 9B depicts the valve seal 912 of the blister device 900 prior to actuation or travel of the rupturing member 910. The rupturing member 910 and the first input-output area 950 are shown. However, the elements of FIGS. 9B, 9C, and 9D may be the same or similar for rupturing member 940 and area 970. As shown in FIG. 9B, the rupturing734937-8761-0640.1Atty. Dkt. No.: 86357096member 910 may be uncompressed, and the valve seal 912a 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 950 via a channel 914. FIG. 9C shows the blister device 900 when the rupturing member 910 is not over traveled (e.g., the rupturing member 910 is only partially compressed). This may permit fluid to flow upon actuation of the rupturing member 910. FIG. 9D depicts the valve seal 912a of the blister device 900 after actuation, specifically after over-travel of the rupturing member 910. As shown in FIG. 9D, flow may be restricted due to over-travel of the rupturing member 910. For example, in FIG.9D, the rupturing member 910 has over traveled to reduce a size of the cavity 950, thus restricting fluid flow. As such, fluid may have difficulty traveling through the blister device 900 and to and from other components of the cartridge 100 via the channel 914.

[0271] FIG. 10A depicts an example blister system 1000, in accordance with present implementations. As illustrated by way of example in FIG. 10A, an example blister system 1000 can include at least a blister device 1002 and one or more actuators 1010 having stand offs 1020 to control actuation depth, a contact geometry 1030 to open a frangible seal of the blister device 1002, an actuator 0, and an actuation direction 1070. The blister device 1002 may include frangible seals 1040 (also referred to as a valve, e.g., the valve 912) and a reagent storage cavity 1050. In various examples, the blister device 1002 may be located in or on the cartridge 100. The actuator 1010 may be located in or on the instrument 150. The actuator 1010 (and / or 0) may be positioned in the instrument 150 such that the actuator aligns with the seals 1040 of the blister device 1002 to allow the actuator to break one or more seals of the blister device 1002. The blister device 900 can correspond at least partially in one or more of structure and operation to the blister device 1002, and can include or couple to one or more of the actuator 1010, the stand offs 1020, or the actuator 0 by one or more of the contact geometry 1030 and the frangible seals 1040 to operate the actuators 1010 and 0 in the actuation direction 1070. For example, the actuation direction 1070 can correspond to a first direction of movement of the actuators 1010 or 0 toward the frangible seals 1040 according to a piercing operation of one or more rupturing members (e.g., the rupturing members 910 and 940) to open one or more of the frangible seals 1040. For example, the actuation direction 1070 can correspond to a second direction of movement of the actuators 1010 or 0 away from the frangible seals 1040 subsequent to a piercing operation to open one or more of the frangible seals 1040. Various examples can comprise a blister device in accordance with this disclosure, such as (but not limited to) the embodiments of the blister devices depicted in FIGS. 9A-10B.744937-8761-0640.1Atty. Dkt. No.: 86357096The blister system 1000 may be part of other devices (e.g., other cartridges and systems) that interface with other instruments and systems.

[0272] As discussed with respect to FIGS. 9A- 9D, over-travel of the piercing elements (e.g., rupturing members) of the blister device 1002 may cause flow restriction. The actuator 1010 may prevent over constriction. As shown in FIGS. 10A and 10B, the actuator 1010 may include stand offs 1020 and a contact geometry. The stand offs 1020 and the contact geometry 1030 may prevent over travel. For example, the stand offs 1020 may extend further or be longer than the contact geometry. Thus, the stand offs 1020 may prevent the contact geometry 1030 from contacting the seals 1040 at too great a depth, thus causing flow restriction.

[0273] FIG. 11 A depicts an example system, in accordance with present implementations. As illustrated by way of example in FIG. 11 A, an example system 1100 A can include at least a diagnostic cartridge 1110, a diagnostic instrument 1120, and a dropper 1130A. The diagnostic cartridge may be the same as or similar to the cartridge 100, and the diagnostic instrument 1120 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.

[0274] The diagnostic cartridge 1110 can receive and interact with a sample fluid by one or more components thereof. For example, the diagnostic cartridge 1110 can include one or more portions corresponding to zones, chambers, channels, reservoirs, containers, or any combination thereof. Specifically, the diagnostic cartridge 1110 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.

[0275] The diagnostic cartridge 1110 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 1110. The diagnostic cartridge 1110 can include one or more components that can interact with one or more corresponding components of the diagnostic instrument 1120. For example, the diagnostic cartridge 1110 can include a heating element located in each detection chamber of the one or more detection chambers that can be placed in contact with or proximate to a heating754937-8761-0640.1Atty. Dkt. No.: 86357096element of the diagnostic instrument 1120, to heat content of a portion of the diagnostic cartridge 1110. The portion of the cartridge 1110 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 1110. The diagnostic cartridge 1110 can have a structure corresponding to a rectangular shape of a size corresponding to a palm. The diagnostic cartridge 1110 can have a height greater than a width of the diagnostic cartridge 1110, and a depth greater than a height of the diagnostic cartridge 1110.

[0276] For example, the diagnostic cartridge 1110 can be at least partially insertable into the diagnostic instrument 1120. That is, the cartridge 1110 may interface with the instrument. The instrument 1120 can include a sensor configured to detect the state of the cartridge or its contents. For example, the instrument 1120 can detect whether the cartridge 1110 is fully inserted into the diagnostic instrument 1120 by an optical sensor or the like. For example, the instrument 1120 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.

[0277] The diagnostic cartridge 1110 can couple with the dropper 1130A via a sample input chamber disposed at a surface of the diagnostic cartridge 1110. 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 1110, when the diagnostic cartridge 1110 is oriented to be inserted into the diagnostic instrument 1120. 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.

[0278] The diagnostic instrument 1120 can interact with the diagnostic cartridge 1110 and can generate one or more indications corresponding to one or more interactions with the diagnostic cartridge 1110. The diagnostic instrument 1120 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 1120 can include one or more sensor devices, motors, actuators, processors, displays, or any combination thereof. The diagnostic instrument 1120 can include a receptacle having a width corresponding to a width764937-8761-0640.1Atty. Dkt. No.: 86357096of the diagnostic cartridge 1110, and a height corresponding to a height of the diagnostic cartridge 1110. The diagnostic instrument 1120 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 1120 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.

[0279] The diagnostic instrument 1120 can include a system processor that can execute one or more instructions associated with the system 1100, according to any of the depictions 1100A-C of the system 1100. 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 1120 generally can include at least one communication bus controller to effect communication between the system processor and the other elements of the system 1100.

[0280] The dropper 1130A can store contents and expel contents to the diagnostic cartridge 1110. For example, the dropper 1130A can have a cylindrical shape including an inlet at a first end and an outlet at a second end. The dropper I I 30 A can include a reservoir corresponding to774937-8761-0640.1Atty. Dkt. No.: 86357096the cylindrical shape. For example, the dropper 1130A can have a reservoir with a volume corresponding to a medical syringe. The inlet of the dropper 1130 can correspond to a cap that can be opened to allow filling of the reservoir with contents. The outlet of the dropper 1130A can correspond to a nozzle. For example, the nozzle of the dropper 1130A can have a shape that can be mated with the sample input chamber of the diagnostic cartridge 1110. The dropper 1130A can be in a state disconnected from or not mated with the diagnostic cartridge 1110. For example, the dropper 1130A can be in a state prior to or subsequent to filling of the diagnostic cartridge 1110 with the content of the dropper 1130A.

[0281] FIG. 1 IB depicts an example system, in accordance with present implementations. As illustrated by way of example in FIG. 11B, an example system 1100B can include at least a dropper 1130B operated by a user 1140 according to a direction 1150. The dropper 1130B can correspond at least partially in one or more of structure and operation to the dropper 1130A. For example, the dropper 1130B can be in a state corresponding to filling of the diagnostic cartridge 1110 with the content of the dropper 1130A. The user 1140 can correspond to an individual operating one or more of the dropper 1130B, the diagnostic cartridge 1110, and the diagnostic instrument 1120. For example, the user 1140 can orient the dropper 1130B to align the sample input chamber of the diagnostic cartridge 1110 to face the outlet of the dropper 1130B. For example, the user 1140 can move one or more of the diagnostic cartridge 1110 and the dropper 1130B in the direction 1150 to mate the inlet of the diagnostic cartridge 1110 with the outlet of the dropper 1130B.

[0282] FIG. 11C depicts an example system, in accordance with present implementations. As illustrated by way of example in FIG. 11C, an example system 1100C can include at least a display device 1160. The display device 1160 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 1160 can receive, for example, capacitive or resistive touch input. The display device 1160 can be housed at least partially within the diagnostic instrument 1120. The display device 1160 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. Example I / O components thus include, without limitation, a touchscreen display, a keypad or keyboard,784937-8761-0640.1Atty. Dkt. No.: 86357096biometric 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.

[0283] In various examples, the diagnostic cartridge 1110 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).

[0284] 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.

[0285] 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.

[0286] 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.

[0287] 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. For794937-8761-0640.1Atty. Dkt. No.: 86357096example, 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.

[0288] 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.

[0289] 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.

[0290] FIG. 12A depicts an example cartridge environment, in accordance with present implementations. As illustrated by way of example in FIG. 12 A, an example cartridge environment 1200 A can include at least a lower fill indication member 1210, an upper fill indication member 1212, a face 1220, a side portion 1230, and a view window 1240 A. The cartridge environment 1200 A can correspond to a first state of a portion 1200 of the system 1000 according to one or more of systems 1000A-C as illustrated by way of example in FIG.10A-C.

[0291] The lower fill indication member 1210 can correspond to a physical component of the diagnostic cartridge 1110. The lower fill indication member 1210 can be located at a position that indicates a minimum fill level of a liquid with respect to the view window 1240 A. For example, the lower fill indication member 1210 can indicate whether an amount of sample fluid from the dropper 1130B meets or exceeds a first vertical level in the view window 1240 A with respect to a particular diagnostic test. For example, the lower fill indication member 1210 can be formed or placed at a location over the view window 1240A along a vertical direction of the view window 1240 A to indicate a minimum amount of fluid corresponding to a particular diagnostic test. For example, the lower fill indication member 1210 can be formed as a804937-8761-0640.1Atty. Dkt. No.: 86357096component of the diagnostic cartridge 1110 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 1210 can be formed as a component of the diagnostic cartridge 1110 at a second vertical position to indicate a minimum amount of fluid corresponding to a seasonal flu diagnostic test. For example, the cartridge 1110 can include the predetermined position corresponding to a minimum amount of the sample fluid needed for performance of the diagnostic test.

[0292] The upper fill indication member 1212 can correspond to a physical component of the diagnostic cartridge 1110 distinct from the lower fill indication member 1210. The upper fill indication member 1212 can be located at a position that indicates a maximum fill level of a liquid with respect to the view window 1240 A. For example, the upper fill indication member 1212 can indicate whether an amount of sample fluid from the dropper 1130B meets or exceeds a second vertical level in the view window 1240 A with respect to a particular diagnostic test. For example, the upper fill indication member 1212 can be formed or placed at a location over the view window 1240 A along a vertical direction of the view window 1240 A to indicate a maximum amount of fluid corresponding to a particular diagnostic test. For example, the upper fill indication member 1212 can be formed as a component of the diagnostic cartridge 1110 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 1212 can be formed as a component of the diagnostic cartridge 1110 at a fourth vertical position to indicate a maximum amount of fluid corresponding to a seasonal flu diagnostic test.

[0293] The face 1220 can at least partially frame view window 1240 A. The face 1220 can at least partially integrate with or connect with one or more of the lower fill indication member 1210 and the upper fill indication member 1212. For example, the face 1220 can be formed to include a portion of a component of the diagnostic cartridge 1110 defining an opening at least partially surrounding the view window 1240 A. For example, the face 1220 can be formed with one or more of the lower fill indication member 1210 and the upper fill indication member 1212. For example, the face 1220, the lower fill indication member 1210, and the upper fill indication member 1212 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 1220 can be oriented according to a face plane that intersects a top plane corresponding to the top surface of the diagnostic cartridge 1110 and a front plane corresponding to the front surface of the diagnostic814937-8761-0640.1Atty. Dkt. No.: 86357096cartridge 1110. 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 1220 can provide a technical improvement to increase visibility of a view window 1240A during a filling process of the diagnostic cartridge 1110.

[0294] The side portion 1230 can correspond to a component of the diagnostic cartridge 1110 covering at least a portion of the diagnostic cartridge 1110. For example, the side portion 1230 can be integrally formed with one or more of the lower fill indication member 1210, the upper fill indication member 1212, and the face 1220. For example, the face 1220, the lower fill indication member 1210, and the upper fill indication member 1212 can be integrally formed of a single solid piece with the side portion 1230.

[0295] The view window 1240 A can correspond to at least a portion of a reservoir of the diagnostic cartridge 1110 that aligns with an opening in the face 1220. For example, the reservoir of the diagnostic cartridge 1110 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 1220 can include or be a transparent or translucent material. The view window 1240A can correspond to a state of a view window 1240A having no fluid visible therethrough. For example, the view window 1240A can frame a reservoir of the diagnostic cartridge 1110 absent any fluid or containing an amount of fluid below a threshold of visibility in the view window 1240A.

[0296] FIG. 12B depicts an example cartridge environment, in accordance with present implementations. As illustrated by way of example in FIG. 12B, an example cartridge environment 1200B can include at least a view window 1240B, and a fluid 1250 visible from view window 1240B.

[0297] The view window 1240B can correspond at least partially in one or more of structure and operation to the view window 1240B. The view window 1240B can correspond to a state of a view window 1240 having fluid 1250 visible therethrough. For example, the view window 1240B can frame a reservoir of the diagnostic cartridge 1110 containing an amount of liquid visible in the view window 1240A. For example, the fluid 1250 visible from view window 1240B can be at a level above the lower fill indication member 1210 and below the upper fill indication member 1212. Thus, the lower fill indication member 1210 and the upper fill indication member 1212 can provide a technical improvement to increase visibility of fluid824937-8761-0640.1Atty. Dkt. No.: 863570961250 deposited to the diagnostic cartridge 1110 during depositing of the fluid 1250 by the user 1240.

[0298] FIG. 13 A depicts an example cartridge environment in a cross-sectional view, in accordance with present implementations. As illustrated by way of example in FIG. 13 A, an example cartridge environment 1300 A in a cross-sectional view can include at least an upper portion 1310, a lower portion 1312, a reservoir 1320, a fluid 1330A in the dropper 1130B, a fluid 1332A in the reservoir 1320, and an air gap 1334A between the dropper 1130B and the reservoir 1320. The cartridge environment 1300A can correspond to a first filling state including the diagnostic cartridge 1110 and the dropper 1130B. For example, the first filling state can correspond to a start of a transfer of fluid 1330A from the dropper 1130B to the diagnostic cartridge 1110.

[0299] The upper portion 1310 can correspond to a portion of the reservoir 1320 structured to couple with or mate with the outlet of the dropper 1130B. For example, the upper portion 1310 can define an opening extending from a top surface of the diagnostic cartridge 1110 to the reservoir 1320. For example, the upper portion 1310 can define an opening having a cross section corresponding to a cross section of the outlet of the dropper 1130B. For example, the opening of the upper portion 1310 can have a circular cross section corresponding to a circular cross section of the dropper 1130B.

[0300] The lower portion 1312 can correspond to a portion of the reservoir 1320 structured to transport and store fluid. For example, the lower portion 1312 can extend in a direction corresponding to the face plane. For example, the lower portion 1312 can be integrally formed with the upper portion 1310 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 1312 at the angle can result in the lower portion having a sloped surface 1314. The sloped surface 1314 of the lower portion 1312 can be oriented to allow transport of the fluid 1332A without or mitigating dispersion of the fluid 1332A in the reservoir 1320. For example, the sloped surface 1314 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 1314 can be formed or oriented to achieve a technical improvement to prevent or minimize formation of separate droplets of the fluid 1332A. For example, the sloped surface 1314 can be formed or oriented to achieve a technical improvement to prevent or minimize formation of bubbles in the fluid 1332A. For example, the sloped surface 1314 can have an834937-8761-0640.1Atty. Dkt. No.: 86357096angle of 45 degrees with respect to the top plane of the diagnostic cartridge 1110, to mitigate dispersion of a fluid 1332A 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 ®.

[0301] The reservoir 1320 can include both the upper portion 1310 and the lower portion 1312. For example, the reservoir 1320 can have a cylindrical shape enclosing a cavity that abuts the face 1220 and the view window 1240A. For example, the reservoir 1320 can be attached with a body of the diagnostic cartridge 1110 and can include or be a transparent or translucent material. The reservoir 1320 can have one or more dimensions to achieve a technical improvement to eliminate or mitigate dispersion of the fluid 1332A in the reservoir 1320. For example, the reservoir 1320 can be formed with one or more of a particular inner depth 1322 and a particular outer depth 1324 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 1322 can be between about 10 millimeters (mm) and about 20 mm, and can be about 15 mm. For example, the outer depth 1324 can be about 4 mm greater than the inner depth 1322. For example, the inner depth can correspond to a horizontal direction from a front of the reservoir 1320 at the view window 1240A to a rear of the reservoir 1320 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 1110. 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.

[0302] The fluid 1330A in the dropper 1130B can be transported between the dropper 1130B mated with the reservoir 1320. For example, the fluid 1330A can be expelled from the outlet of the dropper 1130B into the upper portion 1310 of the reservoir 1320 and onto the sloped surface 1314 of the lower portion 1312 of the reservoir 1320. The outlet of the dropper 1130B can be positioned at a distance from the sloped surface 1314 corresponding to a distance to844937-8761-0640.1Atty. Dkt. No.: 86357096mitigate or eliminate dispersion of the fluid 1332A in the reservoir 1320. The fluid can flow into the reservoir and fill the reservoir 1320 according to a fill line, with minimal or no dispersion that may render the fill level of the fluid 1332A difficult or impossible to detect visually. The air gap 1334A between the dropper 1130B and the reservoir 1320 can provide an outlet for air to escape the reservoir 1320 as it is replaced by the fluid 1332A. Thus, the air gap 1334A can provide the technical improvement of mitigating or preventing dispersion of the fluid 1332A by providing a pathway for air to flow that prevents or mitigates pressurization of or foaming of the fluid 1332A.

[0303] FIG. 13B depicts an example cartridge environment in a cross-sectional view, in accordance with present implementations. As illustrated by way of example in FIG. 13B, an example cartridge environment 13006 in a cross-sectional view can include at least a fluid 1330A in the dropper 1130B, a fluid 1332B in the reservoir 1320, and an air gap 1334B between the dropper 1130B and the reservoir 1320. The cartridge environment 1300B can correspond to a second filling state including the diagnostic cartridge 1110 and the dropper 1130B. For example, the first filling state can correspond to a continuation of a transfer of fluid from the dropper 1130B to the diagnostic cartridge 1110, subsequent to the first filling state.

[0304] For example, the dropper 1130B can be removed from the inlet of the upper portion 1310 of the reservoir 1320 at any point during a filling process of the reservoir. However, the dropper 1130B does not need to be removed from the inlet of the upper portion 1310 to complete a filling process. The fluid 1330B can correspond to an amount of fluid less than an amount of the fluid 1330A. The fluid 1332B can correspond to an amount of fluid greater than an amount of the fluid 1332A. For example, the fluid 1332B can continue to flow into the reservoir 1320 with an absence of formation of bubbles or droplets. The air gap 1334B can be increased to the size of the inlet of the upper portion 1310 of the reservoir 1320 upon removal of the dropper 1130B from the inlet of the upper portion 1310.

[0305] FIG. 13C depicts an example cartridge environment in a cross-sectional view, in accordance with present implementations. As illustrated by way of example in FIG. 13C, an example cartridge environment 1300C in a cross-sectional view can include at least a fluid 1330C in the dropper 1130B, and a fluid 1332C in the reservoir 1320. The cartridge environment 1300C can correspond to a third filling state including the diagnostic cartridge 1110 and the dropper 1130B. For example, the third filling state can correspond to a continuation of a transfer of fluid from the dropper 1130B to the diagnostic cartridge 1110,854937-8761-0640.1Atty. Dkt. No.: 86357096subsequent to the second filling state. The fluid 1330C can correspond to an amount of fluid less than an amount of the fluid 1330A. The fluid 1332C can correspond to an amount of fluid greater than an amount of the fluid 1332B. For example, the fluid 1332C can continue to flow into the reservoir 1320 with an absence of formation of bubbles or droplets.

[0306] FIG. 13D depicts an example cartridge environment in a cross-sectional view, in accordance with present implementations. As illustrated by way of example in FIG. 13D, an example cartridge environment 1300D in a cross-sectional view can include at least a fluid 1330D in the dropper 1130B, and a fluid 1332D in the reservoir 1320. The cartridge environment 1300D can correspond to a fourth filling state including the diagnostic cartridge 1110 and the dropper 1130B. For example, the fourth filling state can correspond to a continuation of a transfer of fluid from the dropper 1130B to the diagnostic cartridge 1110, subsequent to the third filling state. The fluid 1330D can correspond to an amount of fluid less than an amount of the fluid 1330C, or an absence of fluid in the dropper 1130B. The fluid 1332D can correspond to an amount of fluid greater than an amount of the fluid 1332C. For example, the fluid 1332D can complete a flow into the reservoir 1320 with an absence of formation of bubbles or droplets. For example, a level of the fluid 1332 can be visible through the view window 1240B according to a level corresponding to the fill level of the reservoir 1320.

[0307] FIG. 14A depicts an example cartridge environment in plan view, in accordance with present implementations. As illustrated by way of example in FIG. 14A, an example cartridge environment 1400 A in plan view can include at least a cartridge inlet 1410A, and an inlet cover 1420 A. The cartridge inlet 1410A can correspond at least partially in one or more of structure and operation to the inlet of the upper portion 1310 of the reservoir 1320. The cartridge inlet 1410A can correspond to an open state that allows the outlet of the dropper 1130A to mate with the cartridge inlet 1410A. In some examples, the inlet cover 1420 A can slide from an open position to a closed position. In some examples, the inlet cover can be a cap that can close or open over the cartridge inlet 1410A. For example, the inlet cover 1420A can be at a position corresponding to the open state that allows the outlet of the dropper 1130A to mate with the cartridge inlet 1410A. 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.864937-8761-0640.1Atty. Dkt. No.: 86357096

[0308] FIG. 14B depicts an example cartridge environment in plan view, in accordance with present implementations. As illustrated by way of example in FIG. 14B, an example cartridge environment 1400B in plan view can include at least a cartridge inlet 141 OB, and an inlet cover 1420B. The cartridge inlet 141 OB can correspond at least partially in one or more of structure and operation to the cartridge inlet 1410A. The cartridge inlet 141 OB can correspond to a closed state that prevents or blocks the outlet of the dropper 1130A from mating with the cartridge inlet 1410A, and prevents or blocks egress of fluid in the reservoir 1320. The inlet cover 1420B can correspond to a closed position, subsequent to a filling operation of the reservoir 1320. For example, the inlet cover 1420B can be at a position corresponding to the closed state that prevents or blocks the outlet of the dropper 1130A from mating with the cartridge inlet 141 OB.

[0309] This technical solution can provide at least a technical improvement to mitigate or eliminate contamination into an environment external to the cartridge 1110, by mitigating or eliminating dispersion of a sample fluid into an environment exterior to the cartridge 1110 via the cartridge inlet 1410. For example, the cartridge 1110 can transition between cartridge environments 1400A-B by movement of a cartridge at a predetermined velocity or within a range of predetermined velocities. For example, the inlet cover 1420A can move via a spring having a tension sufficiently low as to prevent a “snap” of the inlet cover 1420A into the depicted position of FIG. 14B. The “snap” can correspond to a speed of traversal of the inlet cover 1420 sufficient to cause ejection of at least a portion sample flid from the reservoir 1320 via the cartridge inlet 1410. 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 1420A to the upper portion 1310 of the reservoir 1320.

[0310] For example, the technical solution can include the technical improvement to mitigate contamination into an environment external to the cartridge 1110, by a combination of structures of the cartridge 1110, 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 1110 via the cartridge inlet 1410. For example, the cartridge inlet 1410, corresponding to the upper portion 1310 of the reservoir 1320, can be structured to minimize dispersion by a structure to allow a tip of the dropper 1130 to be fully inserted into the upper portion 1310, with an allowance restricted to the air gap 1334A. Further, the inlet cover 1420 can be structured to mitigate or eliminate contact with sample fluid that has potentially been dispersed onto the top surface of the cartridge 1110. For example, the inlet cover 1420 can be structured874937-8761-0640.1Atty. Dkt. No.: 86357096to have a shape that covers the cartridge inlet 1410 including the cartridge inlet 1410 and a portion of the top surface of the cartridge 1110 within a predetermined distance of the cartridge inlet 1410. For example, the predetermined distance can correspond to a square or rectangular region of the top surface located surrounding the cartridge inlet 1410, as illustrated by way of example in Fig. 14A.

[0311] FIG. 15 depicts an example cartridge panel, in accordance with present implementations. As illustrated by way of example in FIG. 15, an example cartridge panel 1500 can include at least a front portion 1520, and a top portion 1530. The panel 1500 can correspond to a single piece integrally formed to include the lower fill indication member 1210, the upper fill indication member 1212, the face 1220, the side portion 1230, to define the view window 1240. The face 1220 can include the lower fill indication member 1210, the upper fill indication member 1212, the face 1220, and the view window 1240. The top portion 1530 can include the cartridge inlet 1410, and the inlet cover 1420. For example, the cartridge inlet 1410 can be integrally formed with the top portion 1530, and the inlet cover 1420 can be attachable to the top portion 1530 to moveably slide at least from the open position to the closed position along the top portion 1530. The panel 1500 can achieve a technical improvement to provide a customizable cartridge indication via members 1210 and 1212 that can be formed to correspond to a particular diagnostic test, and can be attached to a diagnostic cartridge 1110 to customize the diagnostic cartridge 1110 to the diagnostic test and to customize the members 1210 and 1212 for the diagnostic test. This technical solution is not limited to the cartridge panel 1500. For example, at least one of the cartridge inlet 1410 or the inlet cover 1420 can be integrally formed with, integrated with, attached with, or coupled with the reservoir 1320.

[0312] FIG. 16 depicts an example user interface for cartridge environment, in accordance with present implementations. As illustrated by way of example in FIG. 16, an example user interface 1600 for a cartridge environment can include at least an environment presentation 1610, a diagnostic presentation 1620, and a reservoir presentation 1630. The user interface 1600 can be presented on or by the display device 1160. For example, the system can include a device configured to securely couple with the cartridge and provide a user interface. The user interface 1600 can be configured to present an indication of a status of the diagnostic test performed by the cartridge.

[0313] The environment presentation 1610 can present a visual representation of one or more of the diagnostic cartridge 1110 and the diagnostic instrument 1120. For example, the884937-8761-0640.1Atty. Dkt. No.: 86357096environment presentation 1610 can present, at a first portion of the user interface, an indication of an arrangement of the diagnostic cartridge 1110 with respect to the diagnostic instrument 1120. For example, the arrangement can correspond to an attachment or mating of the diagnostic cartridge 1110 with the diagnostic instrument 1120 by at least partially inserting the diagnostic cartridge in the diagnostic instrument 1120.

[0314] The diagnostic presentation 1620 can present a visual indication of a diagnostic test corresponding to the diagnostic cartridge 1110 or a diagnostic test corresponding to the diagnostic cartridge 1110 or the panel 1500. For example, the diagnostic presentation 1620 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.

[0315] The reservoir presentation 1630 can present a visual indication of a fill level corresponding to a diagnostic test or the panel 1500 with respect to one or more of the member 1210 and 1212. For example, the reservoir presentation 1630 can include one or more members 1210 and 1212 present according to the member 1210 and 1212 on the panel 1500. For example, the reservoir presentation 1630 can present a visual indication including both the members 1210 and 1212, according to a panel 1500 that includes both the members 1210 and 1212. For example, the reservoir presentation 1630 can present a visual indication including only the member 1210, according to a panel 1500 that includes only the member 1210. For example, the reservoir presentation 1630 can present a visual indication including only the member 1212, according to a panel 1500 that includes only the member 1212. For example, the reservoir presentation 1630 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.

[0316] 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 medical894937-8761-0640.1Atty. Dkt. No.: 86357096diagnostic 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.

[0317] 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.904937-8761-0640.1Atty. Dkt. No.: 86357096

[0318] 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.

[0319] 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.

[0320] 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 references914937-8761-0640.1Atty. Dkt. No.: 86357096used 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.

[0321] 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.

[0322] 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.

[0323] 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.924937-8761-0640.1

Claims

Atty. Dkt. No.: 86357096WHAT IS CLAIMED IS:

1. 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 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 and a plurality of magnetic particles;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;delivering a plurality of amplification reagents to a first detection chamber of the at least one detection chamber;amplifying the one or more nucleic acids of interest in the first detection chamber for a first number of cycles, via a first amplification reaction, to provide a plurality of the one or more nucleic acids of interest;dividing the plurality of the one or more nucleic acids obtained via the first amplification reaction into a plurality of aliquots;delivering each of the plurality of aliquots to a different second detection chamber of the at least one detection chamber, each aliquot of the plurality of aliquots associated with a different nucleic acid of interest to be detected;performing a plurality of second amplification reactions, each second amplification reaction performed for one of the plurality of aliquots in the second detection to which the aliquot was delivered, each second amplification performed for a second number of cycles, the second number of cycles greater than the first number of cycles; anddetecting a plurality of amplification products indicative of the presence, absence, or amount of the plurality of amplified nucleic acids of interest, wherein a different amplification product is detected in each aliquot of the plurality of aliquots.

2. The method of claim 1, wherein the plurality of magnetic particles comprise streptavidin-coated paramagnetic beads, and wherein one of:the plurality of magnetic particles comprise a plurality of groups of magnetic particles, each group comprising a subset of the plurality of magnetic particles, wherein each subset of 934937-8761-0640.1Atty. Dkt. No.: 86357096magnetic particles is of a different type, and wherein each magnetic particle of the subset is attached to a single capture oligonucleotide; oreach magnetic particle of the plurality of magnetic particles is of a different type than another magnetic particle of the plurality of magnetic particles, and each magnetic particle is attached to a different type of capture oligonucleotide.

3. The method of claim 1, wherein the first amplification reaction is a bead-based pulse-controlled amplification (PCA) reaction that utilizes the plurality of magnetic particles; and each of the plurality of second amplification reactions is one of a free PCA reaction that does not utilize the plurality of magnetic particles or a hybrid PCA reaction that utilizes the plurality of magnetic particles and a plurality of biotinylated primers.

4. The method of claim 1, wherein the plurality of second amplification reactions are performed in parallel.

5. The method of claim 1, further comprising:hybridizing, into the at least one extraction chamber of the one or more extraction chambers, 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.

6. The method of claim 5, further comprising:washing the one or more nucleic acids of interest hybridized with the at least one capture oligonucleotide attached to the one or more magnetic particles with a first wash buffer.

7. The method of claim 5, further comprising:washing the one or more nucleic acids of interest hybridized with the at least one capture oligonucleotide attached to the one or more magnetic particles with a second wash buffer, the washing with the second wash buffer to suspend the one or more magnetic particles within the second wash buffer.

8. The method of claim 7, further comprising:transporting the one or more nucleic acids of interest hybridized with the at least one capture oligonucleotide attached to the one or more magnetic particles to at least one detection chamber of the one or more detection chambers via the second wash buffer; and944937-8761-0640.1Atty. Dkt. No.: 86357096trapping the one or more magnetic particles within a threshold distance of the one or more heating elements of the at least one detection chamber.

9. 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 a lyse 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;lysing the biological sample into at least one of the lyse chamber to release the one or more nucleic acids of interest from the biological sample;hybridizing, into the lyse 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 plurality of magnetic particles on a side of the lyse chamber, wherein the plurality of magnetic particles are hybridized to the one or more nucleic acids of interest; washing the hybridized plurality of magnetic particles with at least one wash buffer; suspending the hybridized plurality of magnetic particles with a first wash buffer; transporting the one or more nucleic acids of interest with the at least one capture oligonucleotide attached to the one or more magnetic particles to at least one detection chamber of the one or more detection chambers;delivering a plurality of amplification reagents to a first detection chamber of the at least one detection chamber;amplifying the one or more nucleic acids of interest in the first detection chamber for a first number of cycles, via a first amplification reaction, to provide a plurality of the one or more nucleic acids of interest;dividing the plurality of the one or more nucleic acids obtained via the first amplification reaction into a plurality of aliquots;delivering each of the plurality of aliquots to a different second detection chamber of the at least one detection chamber, each aliquot of the plurality of aliquots associated with a different nucleic acid of interest to be detected;954937-8761-0640.1Atty. Dkt. No.: 86357096performing a plurality of second amplification reactions, each second amplification reaction performed for one of the plurality of aliquots, each second amplification performed for a second number of cycles, the second number of cycles greater than the first number of cycles; anddetecting a plurality of amplification products indicative of the presence, absence, or amount of the plurality of amplified nucleic acids of interest, wherein a different amplification product is detected in each aliquot of the plurality of aliquots.

10. The method of claim 9, wherein each of the first and second amplification reactions are one of a free pulse-controlled amplification (PCA) reaction or a hybrid PCA reaction.

11. The method of claim 9, wherein the plurality of magnetic particles are held to the side of the lyse chamber via an applied magnetic field.

12. The method of claim 9, wherein lysing the biological sample produces a lysate, and wherein the method further comprises transporting the lysate to a waste storage chamber.

13. The method of claim 9, wherein suspending the hybridized plurality of magnetic particles comprises sonicating the hybridized plurality of magnetic particles via a sonotrode.

14. The method of claim 5, further comprising:washing the one or more nucleic acids of interest hybridized with the at least one capture oligonucleotide attached to the one or more magnetic particles with a second wash buffer, washing with the second wash buffer to suspend the one or more magnetic particles within the second wash buffer.

15. 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 the one or more extraction chambers are configured to hold the plurality of magnetic particles;964937-8761-0640.1Atty. Dkt. No.: 86357096a waste storage chamber configured to receive a lysate from the one or more extraction chambers;a detection zone comprising:a plurality of mixing chambers comprising one or more different reagents;a plurality of detection chambers; anda heating element configured to heat the plurality of detection chambers.

16. The system of claim 15, further comprising:an instrument to interface with the cartridge, comprising:a lyse system; andat least one of: a plurality of magnetic field generators or a plurality of movable magnetic field generators to interact with the cartridge at one or more detection chambers of the plurality of detection chambers.

17. The system of claim 16, wherein the lyse system further comprises a sonotrode to interface with at least one of the one or more extraction chambers.

18. The system of claim 15, wherein:the plurality of magnetic particles comprise one or more of: oligonucleotides capable of capturing different nucleic acids of interest or different types of magnetic particles, each type of magnetic particle having a different oligonucleotide capable of capturing a specific nucleic acid of interest,wherein a first magnetic field generator is configured to generate a magnetic field to dock the plurality of magnetic particles to at least one of the one or more extraction chambers; andwherein a second magnetic field generator is configured to force the plurality of magnetic particles towards the one or more heating elements.

19. The system of claim 15, wherein the one or more different reagents are master mix reagents capable of detecting different nucleic acids of interest.

20. The system of claim 15, wherein the one or more extraction chambers is a lyse chamber, and wherein the plurality of detection chambers comprise a plurality of amplification chambers,974937-8761-0640.1Atty. Dkt. No.: 86357096the one or more extraction chambers communicably coupled with the plurality of detection chambers via a plurality of fluid channels.984937-8761-0640.1

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