Optical reaction well for assay device

OPTICAL REACTION WELL FOR ASSAY DEVICE

TECHNICAL FIELD

[0001] The present invention relates to the field of microfluidic devices that are capable of performing biological assays. In particular, the present disclosure is directed toward systems, devices, and methods for transmitting a fluid sample from a fluid source into multiple sample chambers configured to accommodate biological assays.

BACKGROUND

[0002] Many existing microfluidic devices are configured to transmit a fluid sample from one location within the microfluidic device, e.g., a common source, to one or more alternative locations within the microfluidic device, e.g., one or more sample chambers. In particular embodiments in which microfluidic devices are configured to transmit a fluid sample from one location within the microfluidic device to a single alternative location within the microfluidic device, existing microfluidic devices may use dead-end filling, in which the fluid sample is transferred into a closed system against an internal pressure of the closed system. Dead-end filling enables precise filling of the single location of the microfluidic device such that overflow of the fluid sample, and thus waste of fluid sample, is minimized. This precision provided by dead-end filling is particularly important in embodiments in which the fluid sample comprises expensive components.

[0003] In alternative embodiments in which microfluidic devices are configured to transmit a fluid sample from one location within the microfluidic device to multiple alternative locations within the microfluidic device, this transfer of the fluid sample is oftentimes performed asynchronously such that one or more of the locations completes filling at different times. Asynchronous completion of filling is problematic in embodiments in which the microfluidic device is used to perform assays, because the reliability of the assay results depends upon the uniformity of the variables that affect the results, such as reaction timing. Furthermore, asynchronous filling of the multiple locations of the microiluidic device may result in imprecise filling of one or more of the multiple locations of microfluidic device such that overflow of the fluid sample, and thus waste of fluid sample, occurs. Not only is this particularly undesirable in embodiments in which components of the fluid sample are expensive, but in embodiments in which the micro fluidic device is used to perform assays, imprecise filling can increase the likelihood of heterogeneity of the fluid sample across the multiple locations, thereby further tarnishing the reliability of the assay results. In addition to these shortcomings of the existing

WO 2019/183608 microfluidic devices described above, many existing microfluidic devices also do not include built-in features to facilitate actuation of assays.

[0004] The novel devices described herein include microfluidic devices that are configured to control transmission of a fluid sample from a common fluid source to multiple sample chambers using dead-end filling, such that the multiple sample chambers are filled concurrently, !he devices described herein enable precise filling of the multiple sample chambers such that overflow of the fluid sample, and thus waste of the fluid sample is minimized. Furthermore, concurrent filling of the multiple sample chambers, as enabled by the devices described herein, increases the likelihood of homogeneity of the fluid sample across the multiple sample chambers, and improves the uniformity of reaction (lining across the multiple sample chambers, thereby improving the reliability of assay results generated by the microfluidic device.

[0005] In certain embodiments, the novel devices described herein also include features io facilitate actuation of assays. For instance, in certain embodiments, one or more of the sample chambers of the novel devices described herein comprise a double tapered chamber that minimizes the trapping of bubbles within the sample chamber during filling with the fluid sample. Minimizing bubble trapping is advantageous during assay actuation because in some embodiments, bubbles interfere with the results of the assay.

SUMMARY

[0006] The present disclosure relates generally to microfluidic devices that transmit a fluid sample from a fluid source into multiple sample chambers configured to accommodate biological iiss3.ys.

[0007] In one aspect, the disclosure provides an apparatus that comprises a common fluid source and a plurality of independent, continuous fluidic pathways connected to the common fluid source. Each independent, continuous fluidic pathway comprises a sample chamber and a. pneumatic compartment. The sample chamber, having a fluid volume, is connected to the common fluid source. The pneumatic compartment, having a. pneumatic volume, is connected to the sample chamber, and thereby is indirectly connected to the common fluid source. Each fluidic pathway of the plurality of independent, continuous fluidic pathways is a closed system excluding the connection between the sample chamber and the common fluid source. In some embodiments, the fluid volume of one fluidic pathway of the apparatus is greater than the fluid volume of another fluidic pathway of the apparatus. To support concurrent delivery of a sample to each sample chamber, the ratio of fluid volume to pneumatic volume is substantially equivalent for each fluidic pathway of the plurality of fluidic pathways.

WO 2019/183608

[0008] In some embodiments of the apparatus, the sample chamber comprises an assay chamber and an entry conduit that connects the common fluid source to the assay chamber. In certain implementations, the assay chamber volume is between 1 pL and 35 pL. Similarly, the pneumatic compartment may comprise an air chamber and a pneumatic conduit that connects the sample chamber to the air chamber. Therefore, each fluidic pathway may comprise an entry conduit, an assay chamber, a pneumatic conduit, and air chamber.

[0009] In some embodiments, the assay chamber comprises a double tapered chamber that in turn comprises a tapered inlet, a tapered outlet, and two curved boundaries. The tapered inlet is in fluidic communication with a terminus of the entry conduit of the fluidic pathway. Similarly, the tapered outlet is in fluidic communication with a terminus of the pneumatic compartment, frequently with a terminus of the pneumatic conduit. The two curved boundaries extend from the tapered inlet to the tapered outlet such that together, the two curved boundaries enclose the volume of the assay chamber. Additionally, the tapered inlet and the tapered outlet are separated by the largest dimension of the assay chamber volume. Furthermore, each curved boundary comprises a midpoint, and a distance between the two curved boundaries decreases as the boundaries curve from the midpoint toward the tapered inlet and from the midpoint toward the tapered outlet.

[0010] In certain embodiments, the assay chamber comprises a first bounding surface formed in a monolithic substrate, and a second bounding surface formed by a plug. The plug comprises a body and a cap. The body of the plug protrudes into the monolithic substrate of the assay chamber at a depth such that the assay chamber volume can be readily changed by altering the depth at which the body of the plug protrudes into the monolithic substrate of the assay chamber. In particular, the cap of the plug forms the second bounding surface of the assay chamber. In further embodiments, a film may form a third bounding surface of the assay chamber such that the first bounding surface, the second bounding surface, and the third bounding surface together enclose the assay chamber volume. In some embodiments, the plug cap comprises an internal cavity configured to hold one or more dried reagents for use in an assay to take place in the assay chamber. Additionally, a magnetic mixing element may be located in the assay chamber to facilitate actuation of an assay in the assay chamber.

[0011] In certain aspects of the disclosed apparatus, one or more films may be adhered to a portion of the apparatus. For example, a first film 12 may be adhered to a surface of at least a portion of the apparatus such that the first film forms one wall of one or more chambers, compartments, or conduits of the apparatus. In certain embodiments discussed in further detail

WO 2019/183608 below, it may be desirable to seal a portion of one or more of the fluidic pathways of the apparatus using heat. Accordingly, in such embodiments, a second film 14, having a higher melting temperature, may be adhered to the first film 12.

[0012] In another, distinct aspect, the disclosure provides a method of simultaneously filling a plurality of sample chambers. The method includes providing an apparatus according to one or more of the embodiments described above. For use in simultaneous filling of the plurality of the sample chambers, the common fluid source of the provided apparatus contains a fluid sample, and each independent, continuous fluidic pathway of the provided apparatus contains a gas, such as, for example, air. After provision of the apparatus, a supply pressure is applied to the fluid sample in the common fluid source, thereby forcing the fluid sample from the common fluid source, into the sample chamber of each fluidic pathway of the apparatus. In certain embodiments, the supply pressure is applied at a constant pressure. In alternative embodiments, the supply pressure is applied from a lower pressure to a higher pressure. In certain aspects, the fluid sample travels to the plurality of sample chambers via the entry conduits against a gravitational force. This transmission of the fluid sample into the sample chamber of each fluidic pathway of the apparatus compresses the gas within the fluidic pathways toward the pneumatic compartments of the fluidic pathways. This in turn causes an increase in the internal pressure in the pneumatic compartments of the fluidic pathways. When the internal pressure in a pneumatic compartment equals the supply pressure, the fluid sample stops flowing from the common fluid source into the fluidic pathway.

[0013] In some embodiments of the disclosed method, at least two sample chambers of the provided apparatus differ in volume. For example, a fluid volume of a sample chamber of a first fluidic pathway of the provided apparatus may be greater than a fluid volume of a sample chamber of a second fluidic pathway of the provided apparatus. Generally, a rate of flow from the common fluid source into each sample chamber of the plurality of sample chambers is proportional to a fluid volume of the sample chamber. Additionally, as noted above, the ratio of fluid volume to pneumatic volume is substantially equivalent for each fluidic pathway of the provided apparatus. Therefore, sample chambers of the provided apparatus, including the differentially sized sample chambers of the first fluidic pathway and the second fluidic pathway, fill at a substantially proportional rate such that the sample chambers fill simultaneously.

[0014] As described above, certain embodiments of the apparatus provided by the disclosed method may comprise one or more sample chambers that in turn comprise a double tapered chamber. In such embodiments in which one or more sample chambers of the provided apparatus

WO 2019/183608 comprise a double tapered chamber, the two curved boundaries of the double tapered chamber slow the rate of fluid advance at the leading front meniscus of the fluid sample, such that when the fluid sample reaches the tapered outlet, the meniscus of the fluid sample is substantially symmetric with respect to the largest dimension of the assay chamber, thereby minimizing the trapping of bubbles within the assay chamber during filling.

[0015] Also as described above, in certain aspects, it may be desirable to seal the fluidic pathways of the apparatus using heat. In such aspects, the method disclosed herein further comprises sealing each fluidic pathway of the plurality of fluidic pathways when the fluid sample stops flowing from the common fluid source into the fluidic pathway. This step of sealing can be performed by heat staking.

[0016] In yet another aspect, the disclosure provides an apparatus for rehydrating a dried reagent that is distinct from the various embodiments of the apparatus described above. In such aspects, the apparatus at issue comprises an assay chamber. The assay chamber comprises a first bounding surface formed in a monolithic substrate and a second bounding surface formed by a plug. The plug comprises a body and a cap. The body of the plug protrudes into the monolithic substrate of the assay chamber at a depth such that the assay chamber volume can be readily changed by altering the depth at which the body of the plug protrudes into the monolithic substrate of the assay chamber. In particular, the cap of the plug forms the second bounding surface of the assay chamber. Together, the first bounding surface and the second bounding surface of the assay chamber enclose a volume of the assay chamber. An internal cavity of the formed in the cap of the plug can hold one or more dried reagents for use in an assay to occur in the assay chamber. The assay chamber contains a magnetic mixing element within the volume of the assay chamber. The magnetic mixing element is capable of gyration within the assay chamber volume.

[0017] In certain embodiments of the apparatus for rehydrating a dried reagent, the assay chamber comprises a third bounding surface of a film. In such embodiments, the first bounding surface, the second bounding surface, and the third bounding surface together enclose the assay chamber volume.

[0018] In yet another distinct aspect, the disclosure provides a method of solubilizing a dried reagent. This method includes providing the apparatus for rehydrating a dried reagent according to one of the embodiments described above. The method further comprises filling the assay chamber with a fluid and inducing gyration of the magnetic mixing element within the assay

WO 2019/183608 chamber of the apparatus by rotating a magnet exterior to the assay chamber, !his gyration of the magnetic mixing element within the assay chamber solubilizes the reagent within the fluid.

[0019] In general, in one embodiment, a plug includes a body with a. bottom surface, a central opening in the body, and a dried reagent on the bottom surface, wherein the body is formed from a material transmissive to excitation wavelengths and emission wavelengths in at least one of a red spectrum, a blue spectrum and a green spectrum.

[0020] This and other embodiments can include one or more of the following features. The dried reagent can be on a portion of the bottom surface wider than a width of the central opening in the body. A width of the central opening can be wider than a portion of the bottom surface containing the dried reagent. The plug can further include a cavity in the bottom surface with the dried reagent within the cavity. The plug can further include a plug thickness between a central opening bottom and the plug body bottom wherein a depth of the cavity is less than 90% of the plug thickness. The plug can further include a plug thickness between a central opening bottom and the plug body bottom wherein a depth of the cavity is less than 70% of the plug thickness. The plug can further include a plug thickness between a central opening bottom and the plug body bottom wherein a depth of the cavity is less than 50% of the plug thickness. The plug can further include an annulus on the plug bottom surface from an outer edge of the plug body to a perimeter of the cavity. The annulus can completely encircle the perimeter of the cavity. The cavity can further include a perimeter in the bottom surface wherein an angle of initiation of the cavity is measured from the perimeter relative to the bottom surface and the angle of initiation is 60 degrees or less. The cavity can be wider than the plug body central opening. The plug body central opening can be wider than the cavity. The plug body bottom surface can further include a bounded area on the plug body bottom surface wherein the dried reagent is within the bounded area. The bounded area on the plug bottom surface can be provided by a feature on the plug body bottom surface. The feature can be raised above the plug bottom surface or recessed into the plug bottom surface. The feature can have a curved cross section or a rectangular cross section. A width of the bounded area can be greater than a width of the body central opening, width of the bounded area can be less than a width of the body central opening, or a width of the bounded area can be about the same as a width of the body central opening. The plug can have a polished or smooth finish facilitating the transmissivity of the excitation wavelengths and the emission wavelengths. The plug can further include a flange on the plug body around the central opening in the plug body. The dried reagent can be selected from the group consisting of nucleic acid synthesis reagents, peptide synthesis reagents, polymer synthesis reagents, nucleic acids, nucleotides, nucleobases, nucleosides, peptides, amino acids, monomers, detection reagents,

WO 2019/183608 catalysts or combinations thereof. The dried reagent can be a continuous film adhering to the plug bottom surface. The dried reagent can be a lyophilized reagent. The dried reagent can include a plurality of droplets adhering to the plug bottom surface.

[0021] In general, in one embodiment, an assay chamber includes a tapered inlet, a tapered outlet״ a plug including a bottom surface and a central opening in the body, wherein the body is formed from a material transmissive to excitation wavelengths and emission wavelengths in at least one of an ultraviolet spectrum, a blue spectrum, a green spectrum and a red spectrum, two curved boundaries, wherein each curved boundary extends from the tapered inlet to the tapered outlet such that together, the two curved boundaries and the plug enclose a volume of the assay chamber, and a shoulder extending from each curved boundary wherein the plug contacts each shoulder such that a boundary of the assay chamber is provided by the two curved boundaries, the shoulders extending from each of the curved boundaries and the plug.

[0022] ^,This and other embodiments can include one or more of the following features. Plug within the assay chamber can have a dried reagent thereon. A cavity on the plug can be positioned between each of the shoulders and the dried reagent is in the cavity. A portion of the curved boundaries or of the shoulders can be shaped to conform to a perimeter of the cavity. The dried reagent on the plug can be positioned between each of the shoulders. A flat portion of the bottom of the plug body can contact the shoulders. A height of each of the shoulders can be used to adjust the volume of the assay chamber. The height of each of the shoulders can be 100 micrometers or more. The height of each of the shoulders can be no greater than a distance of the two curving boundaries from each other at a point of greatest separation. The shoulders can be shaped to maintain an overall curved boundary of the assay chamber from the tapered inlet to the tapered outlet. ’The two curved boundaries and the shoulders can be formed in a monolithic substrate. The assay can further include a film adhered to a surface the monolithic substrate, wherein the film forms one wall of the assay chamber. The assay chamber can have a plug.

[0023] In general, in one embodiment, an apparatus includes a common fluid pathway, and a plurality of independent, continuous fluidic pathways connected to the common fluid pathway, wherein each independent, continuous fluidic pathway includes an assay chamber, and a pneumatic compartment. The assay chamber is connected to the common fluid pathway, the assay chamber having a fluid volume defined in part by a plug having a dried reagent thereon; and the pneumatic compartment, having a pneumatic volume, is connected to the common fluid pathway via the assay chamber. Each fluidic pathway of the plurality of independent, continuous fluidic pathways is a closed system excluding the connection between the assay

WO 2019/183608 chamber and common fluid source. Each assay chamber includes a double tapered chamber, and a shoulder extending from each curved boundary wherein the plug contacts each shoulder such that a boundary of the assay chamber is provided by the two curved boundaries, the shoulders extending from each of the curved boundaries and the plug. The double tapered chamber includes a tapered inlet in fluidic communication with a terminus of the entry conduit of the fluidic pathway, a tapered outlet in fluidic communication with a terminus of the pneumatic compartment, and two curved boundaries, wherein each curved boundary extends from the tapered inlet to the tapered outlet such that together, the two curved boundaries enclose the volume of the assay chamber.

[0024] This and other embodiments can include one or more of the following features. A cavity on the plug can be positioned between each of the shoulders and the dried reagent is in the cavity. A dried reagent on the plug can be positioned between each of the shoulders. A flat portion of the bottom of the plug body can contact the shoulders. A height of each of the shoulders can be used to adjust the volume of the assay chamber. The height of each of the shoulders can be 100 micrometers or more. The shoulders can be shaped to maintain an overall curved boundary of the assay chamber from the tapered inlet to the tapered outlet. The two curved boundaries can be formed in a monolithic substrate. The body of the plug can protrude into the monolithic substrate of the assay chamber at a depth such that the assay chamber volume can be readily changed by altering the depth at which the body of the plug protrudes into the monolithic substrate of the assay chamber. A portion of the curved boundaries or of the shoulders can be shaped to conform to a perimeter of the cavity. The apparatus can further include a first film adhered to a surface of at least a portion of the apparatus. The first film can form one wall of one or more chambers, compartments, or conduits of the apparatus. The apparatus can further include a second film adhered to the first film. The second film can have a higher melting temperature than the first film. The apparatus can further include a heat staked region formed in each of the fluidic pathways using the first film or the second film, !he heat staked region can seal off the common fluid pathway from the assay chamber and the pneumatic chamber. The apparatus can further include a raised platform within each of the plurality of independent, continuous fluidic pathways, the raised platform positioned between an inlet to the assay chamber and the common fluid pathway. The heat staked region can be formed using a portion of the raised platform. The apparatus can have a plug.

[0025] In general, in one embodiment, a method of simultaneously filling a plurality of sample chambers includes: (1) pressurizing a fluid sample within a common fluid pathway; (2) introducing the fluid sample into a plurality of entry conduits from the common fluid pathway;

WO 2019/183608 (3) flowing the fluid sample along each of the entry conduits towards an entry conduit terminus in each of the entry conduits, each entry conduit connected to a sample chamber; (4) flowing the fluid sample along a tapered inlet portion of each sample chamber; (5) flowing the fluid sample adjacent a pair of shoulders and along a plug within each sample chamber; (6) flowing the fluid sample along a tapered outlet portion of each sample chamber towards a pneumatic compartment terminus: and (7) displacing a gas contained within each entry conduit and each sample chamber into a. pneumatic chamber in communication with each pneumatic compartment terminus.

[0026] This and other embodiments can include one or more of the following features. Pressuring the fluid sample step can be performed at a constant pressure. The constant pressure can be one of 5, 10, 20, 40 or 60 psi. The pressurizing the fluid step can further include pressuring the fluid sample at a series of increasing pressure levels. Each increasing level of pressure can be applied for a consistent duration. Each increasing level of pressure can be increased by a constant amount. The pressurizing the fluid sample can apply a series of pressure levels from a. lower pressure level to a higher pressure level. In use, the pneumatic chamber can be above the sample chamber such that the steps of flowing the fluid sample along a tapered outlet portion of the sample chamber towards a pneumatic compartment terminus and displacing a gas contained within each entry conduit can be performed against a gravitational force. In use, the plurality of sample chambers can be oriented such that each pneumatic chamber associated with a specific sample chamber of the plurality of sample chamber is positioned above the sample chamber. Flowing the fluid sample into the sample chamber of each fluidic pathway of the apparatus can compress the gas within the fluidic pathways toward the pneumatic compartments of the fluidic pathways. The method can further include maintaining the pressure reached during the pressurizing a fluid sample step when an internal pressure in each of the pneumatic compartments equals the pressure applied to the common fluid pathway. The method can further include increasing a pressure within each pneumatic compartment during the displacing a gas step, and stopping increasing the pressure when a pressure applied to the common fluid pathway equals the pressure within each pneumatic compartment. The method can further include stopping each of the flowing the sample steps when the internal pressure in each of the pneumatic compartments equals a pressure applied to the common fluid pathway. At least two sample chambers of the plurality of sample chambers can differ in volume. A flowrate from the common fluid pathway into each sample chamber of the plurality of sample chambers can be proportional to a fluid volume of the sample chamber and there are at least two different flowrates. The method can further include simultaneously filling each sample chamber of the plurality of sample chambers. The method can further include flowing the fluid sample along

WO 2019/183608 two diverging curved boundaries within the sample chamber during or after the flowing the fluid sample along the tapered inlet step. The method can further include flowing the fluid sample along two converging curved boundaries within the sample chamber after or during the flowing the fluid sample along the pair of shoulders step. Convergence of the two curved boundaries can slow the rate of fluid advance at a leading front of a meniscus of the fluid sample, such that when the fluid sample reaches the tapered outlet, the meniscus of the fluid sample is substantially symmetric with respect to the largest dimension of the assay chamber, thereby minimizing the trapping of bubbles within the assay chamber during filling. The method can further include positioning a meniscus in each sample chamber adjacent to the pneumatic chamber terminus. The method can further include performing one or more of the steps so as to position one or more bubbles formed within the fluid sample adjacent to a meniscus of the fluid sample within the sample chamber. The meniscus can be proximate to the pneumatic chamber terminus. The method can further include sealing each one of the plurality of entry conduits while performing the step of pressurizing a fluid sample within the common fluid pathway. The method can further include sealing each one of the plurality of entry conduits when the step of flowing the fluid sample along a tapered portion of the sample chamber stops. The method can further include sealing each one of the plurality of entry conduits when the step of flowing the fluid sample along each of the entry conduits from the common fluid pathway stops. The step of sealing can further include heat staking a portion of the entry conduit closed. The method can further include heating a portion of a first film adjacent to each of the entry conduits, melting the first film to seal off the each one of the entry conduits. Idle method can further include simultaneously sealing all entry conduits. The method can further include heating without melting a second film separated from the entry conduit by the first film. The method can further include fusing a portion of the entry conduit to a portion of the first film without melting the second film. After sealing each one of the entry conduits a portion of a first film or a. second film can be fused to a raised platform formed in each one of the entry conduits.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present application is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the subject matter, there are shown in the drawings exemplary embodiments of the subject matter: however, the presently disclosed subject matter is not limited to the specific methods, devices, and systems disclosed. In addition, the drawings are not necessarily drawn to scale. In the drawings:

WO 2019/183608

[0028] FIG. 1 is an illustration of an apparatus for transmitting a fluid sample from a fluid source into multiple sample chambers, in accordance with an embodiment.

[0029] FIG. 2A depicts an apparatus at a time A during simultaneous filling of sample chambers of the apparatus with a fluid sample, in accordance with an embodiment.

[0030] FIG. 2B depicts an apparatus at a time B during simultaneous filling of sample chambers of the apparatus with a fluid sample, in accordance with an embodiment.

[0031] FIG. 2C depicts an apparatus at a time C during simultaneous filling of sample chambers of the apparatus with a fluid sample, in accordance with an embodiment.

[0032] FIG. 2D depicts an apparatus at a time D during simultaneous filling of sample chambers of the apparatus with a fluid sample, in accordance with an embodiment.

[0033] FIG. 2E depicts an apparatus at a time E during simultaneous filling of sample chambers of the apparatus with a fluid sample, in accordance with an embodiment.

[0034] FIG. 2F depicts an apparatus at a time F during simultaneous filling of sample chambers of the apparatus with a fluid sample, in accordance with an embodiment.

[0035] FIG. 2G is a cross section of an inlet conduit having a conduit feature such as a raised platform within a heat stake zone.

[0036] FIG. 2H is a perspective and section view of an apparatus showing a conduit feature in a heat stake zone for the common fluid line. FIG. 21 is a cross section view of the common fluid line of FIG. 2H showing the raised platform within the heat stake zone.

[0037] FIG. 2J is a top down view of an apparatus indicating a heat stake zone along each of the independent fluid conduits. FIG. 2K is a cross section of one of the independent fluid conduits in FIG. 2J showing the conduit feature or raised platform to facilitate heat staking the channel.

[0038] FIG. 3A is an illustration of an independent fluidic pathway, in accordance with an embodiment.

[0039] FIG. 3B is an illustration of an assay chamber, in accordance with an embodiment.

[0040] FIG. 4 is an illustration of a portion of an apparatus for transmitting a fluid sample from a fluid source into multiple sample chambers, in accordance with an embodiment.

[0041] FIG. 5 is a three-dimensional illustration of an assay chamber, in accordance with an embodiment.

WO 2019/183608

[0042] FIG. 6A is a three-dimensional illustration of a plug, in accordance with an embodiment.

[0043] FIG. 6B is a cross-sectional illustration of a plug, in accordance with an embodiment.

[0044] FIG. 6C is an enlarged view of FIG. 6B.

[0045] FIG. 7A is a three-dimensional, cross-sectional illustration of an assay chamber, in accordance with an embodiment. FIG. 7B is an enlarged view of the interior of the assay chamber and mixing bah of FIG. 7A.

[0046] FIG. 7C and 7D are perspective and cross section views of the plug, mixing ball and film used when assembling an embodiment of the assay chamber of FIG. 7A.

[0047] FIG. 8 is a cross section view׳ of a plug having a flat bottom surface.

[0048] FIG. 9A is a section view׳ of the plug in FIG. 8 having a dried reagent having a volume vl along the entire plug bottom surface. FIG. 9B is a bottom up view of the plug of FIG. 9A showing the dried reagent volume vl along the bottom surface.

[0049] FIG. 10A is a section view of the plug in FIG. 8 having a dried reagent having a volume v2 partially covering the plug bottom surface having a width similar to the plug central opening. FIG. 10B is a bottom up view of the plug of FIG. 10 A showing the dried reagent volume v2 along the bottom surface.

[0050] FIG. 11A is a section view of the plug in FIG. 8 having a dried reagent having a volume v3 along the plug bottom surface having a width that is less than the width of the plug central opening. FIG. 1 IB is a bottom up view of the plug of FIG. 11A showing the dried reagent volume v3 along the bottom surface within the wddth of the plug central opening.

[0051] FIGs. 12A and 12B illustrate a feature along the plug bottom surface for retaining a volume of a dried reagent. FIG. 12A illustrates a raised feature having a rectangular־ cross section. FIG. 12B illustrates a recessed feature having a rectangular cross section.

[0052] FIGs. 13A and 13B illustrate a feature along the plug bottom surface for retaining a volume of a dried reagent. FIG. 13 A illustrates a raised feature having a circular cross section. FIG. 12B illustrates a recessed feature having a circular cross section.

[0053] FIG. 14A is a section view of the plug in FIG. 13 A having a dried reagent having a volume vl along the entire plug bottom surface between the raised features. FIG. 14B is a bottom up view׳ of the plug of FIG. 14A showing the dried reagent volume vl along the bottom surface within the boundary of the raised feature.

WO 2019/183608

[0054] FIG. 15A is a section view of the plug in FIG. 13A having a dried reagent having a volume v2 along a portion of the plug bottom surface between the raised features. FIG. 15B is a bottom up view of the plug of FIG. 15A showing the dried reagent volume v2 along the bottom surface within the boundary of the raised feature having about the same width as the plug open central portion.

[0055] FIG. 16A is a section view of the plug in FIG. 13A having a dried reagent having a volume v3 along a portion of the plug bottom surface between the raised features. FIG. 16B is a bottom up view of the plug of FIG. 16A showing the dried reagent volume v3 along the bottom surface within the boundary of the raised feature having a width that is less than the width of the plug open central portion.

[0056] FIG. 17A is a section view of a plug having a cavity as in FIGs. 6A and 6B having a dried reagent having a volume v3. The cavity covers nearly all of the plug bottom surface. FIG. 17B is a bottom up view of the plug of FIG. 17A showing the dried reagent volume vl along the bottom surface within the cavity.

[0057] FIG. 18A is a section view of a plug having a cavity as in FIGs. 6A and 6B having a dried reagent having a volume v2. The cavity covers less than all of the plug bottom surface and is wider than the plug central opening. FIG. 18B is a bottom up view of the plug of FIG. 18A showing the dried reagent volume v2־ along the bottom surface within the cavity.

[0058] FIG. 19A is a section view of a plug having a cavity as in FIGs. 6A and 6B having a dried reagent having a volume v3. The cavity covers less than all of the plug bottom surface and is not as wide as the plug central opening. FIG. 19B is a bottom up view of the plug of FIG. 19A showing the dried reagent volume v3 along the bottom surface within the cavity.

[0059] FIG. 20 is a cross section view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a recessed portion to accommodate the cavity in the plug.

[0060] FIG. 21 is a cross section view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a recessed portion to accommodate the cavity in the plug. The shoulder height shown in FIG. 21 is less than the shoulder height of FIG. 20.

[0061] FIG. 22 is a cross section view of an assay chamber taken through the midpoint of the chamber showing the shoulder supporting the plug and the double tapered sidewalls towards the outlet.

WO 2019/183608

[0062] FIG. 23 is a cross section view of an opening in the optical side of an apparatus sized to receive a plug to be supported by the shoulder. The opening is covered by the first film layer and the second film layer.

[0063] FIG. 24 is a cross section view of an opening in the optical side of an apparatus sized to receive a plug to be supported by the shoulder. The shoulder and tapering sidewall are visible in this view. A plug is shown inserted into the opening but not yet seated against the shoulder.

[0064] FIG. 25 is a perspective view of the optical side of an apparatus showing five plug openings. The plug support rings are shown around each of the openings. The shoulders used to support the plugs are visible within each of the openings.

[0065] FIG. 26A is a cross section view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a shoulder height for engaging the plug to provide a chamber depth. There is a recessed portion in the shoulder to accommodate the cavity in the plug.

[0066] FIG. 26B is a view from the non-opticai side of the plug and shoulder combination in FIG. 26.A. The shoulders support the plug, maintain the tapered sidewall and provide a recess so that the cavity and dried reagents are exposed within the chamber.

[0067] FIG. 27A is a cross section view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a shoulder height for engaging the plug to provide a chamber depth. There is a recessed portion in the shoulder to accommodate the cavity in the plug.

[0068] FIG. 27B is a view from the non-optical side of the plug and shoulder combination in FIG. 27A. The shoulders support the plug, maintain the tapered sidewall and provide a recess so that the cavity and dried reagents are exposed within the chamber.

[0069] FIG. 28A is a cross section view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a shoulder height for engaging the plug to provide a chamber depth. There is a recessed portion in the shoulder to accommodate the cavity in the plug.

[0070] FIG. 28B is a view front the non-optical side of the plug and shoulder combination in FIG. 28A. The shoulders support the plug, maintain the tapered sidewall and provide a recess so that the cavity and dried reagents are exposed within the chamber.

[0071] FIG. 29A is a cross section view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a

WO 2019/183608 shoulder height for engaging the plug to provide a chamber depth. ’There is a recessed portion in the shoulder to accommodate the cavity in the plug.

[0072] FIG. 29B is a view from the non-optical side of the plug and shoulder combination in FIG. 29.A. The shoulders support the plug, maintain the tapered sidewall and provide a recess so that the cavity and dried reagents are exposed within the chamber.

[0073] FIG. 30A is a cross section perspective view of an assay chamber taken through the inlet and the outlet which show's the plug bottom surface supported by a shoulder. The shoulder has a shoulder height for engaging the plug to provide a chamber depth. There is a recessed portion in the shoulder to accommodate the cavity in the plug.

[0074] FIG. 30B is a view from the non-optical side of the plug and shoulder combination in FIG. 30A. The shoulders support the plug, maintain the tapered sidewall and provide a recess so that the cavity and dried reagents are exposed within the chamber.

[0075] FIG. 30C represents the shape and the volume of an assay chamber formed using the plug and shoulder configuration of FIGs. 30A and 30B.

[0076] FIG. 31A is a cross section perspective view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a shoulder height for engaging the plug to provide a chamber depth. There is a recessed portion in the shoulder to accommodate the cavity in the plug.

[0077] FIG. 3 IB is a view from the non-optical side of the plug and shoulder combination in FIG. 31 A. The shoulders support, the plug, maintain the tapered sidewall and provide a recess so that the cavity and dried reagents are exposed within the chamber.

[0078] FIG. 3 IC represents the shape and the volume of an assay chamber formed using the plug and shoulder configuration of FIGs. 31A and 3I B.

[0079] FIG. 32A is a cross section perspective view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a shoulder height for engaging the plug to provide a chamber depth. ’There is a recessed portion in the shoulder to accommodate the cavity in the plug.

[0080] FIG. 32B is a view from the non-optical side of the plug and shoulder combination in FIG. 32A. ’The shoulders support the plug, maintain the tapered sidewall and provide a recess so that the cavity and dried reagents are exposed within the chamber.

WO 2019/183608

[0081] FIG. 32C represents the shape and the volume of an assay chamber formed using the plug and shoulder configuration of FIGs. 32A and 32B.

[0082] FIGs. 32D and 32E are perspective and cross section views of the plug and cavity used in the plug, shoulder and assay chamber shown in FIGs. 32A, 32B and 32C.

[0083] FIG. 33A is a cross section perspective view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a shoulder height for engaging the plug to provide a chamber depth. There is a recessed portion in the shoulder to accommodate the cavity in the plug.

[0084] FIG. 33B is a view from the non-optical side of the plug and shoulder combination in FIG. 33A. The shoulders support the plug, maintain the tapered sidewall and provide a recess so that the cavity and dried reagents are exposed within the chamber.

[0085] FIG. 33C represents the shape and the volume of an assay chamber formed using the plug and shoulder configuration of FIGs. 33A and 33B.

[0086] FIGs. 33D and 33E are perspective and cross section views of the plug and cavity used in the plug, shoulder and assay chamber shown in FIGs. 33A, 33B and 33C.

[0087] FIGs. 34A and 34B are non-optical side and optical side views, respectively, of an apparatus ha ving a mixture of plugs with optical transmissive properties within an optical zone and at least one plug without optical properties in another zone of the apparatus where optical capabilities are not needed.

[0088] FIGs. 35A and 35B are non-optical side and optical side views, respectively, of an apparatus having only plugs with optical transmissive properties.

[0089] FIGs. 36A - 36K are an example sequence of loading a fluid sample into a sample chamber as performed before the exemplary filled chamber states shown in FIGs. 3A and 3B.

[0090] FIG. 37 is an optical side view of an apparatus having five optically transmissive plugs. There results viewed through each of the plugs indicates that three chambers have a detectable emission and two chambers do not have a detectable emission.

DETAILED DESCRIPTION

[0091] Systems, devices, and methods for transmitting a fluid sample from a fluid source into multiple sample chambers, are provided herein. In some embodiments, the devices include a plurality of independent, continuous fluidic pathways, each fluidic pathway comprising a sample chamber connected to a fluid source, and a pneumatic compartment connected to the sample

WO 2019/183608 chamber. The ratio of the volume of the sample chamber to the volume of the pneumatic compartment is substantially equivalent for each fluidic pathway sharing the common fluidic source. In some embodiments, the sample chamber of each fluidic pathway indudes a double tapered chamber, a magnetic mixing element, and/or a plug. In some embodiments, the methods include simultaneously filling the plurality of sample chambers with the fluid sample. In some embodiments, the methods include filling the sample chambers with a fluid sample, and mixing the fluid sample in the sample chambers using a magnetic mixing element held within each sample chamber.

[0092] Before the disclosed embodiments are described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0093] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the present disclosure. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges and are also encompassed within the present disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present disclosure.

[0094] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0095] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which these disclosed embodiments belong. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the disclosed embodiments, representative illustrative methods and materials are now described. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

WO 2019/183608

[0096] It is noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

SYSTEMS

[0097] Included in the disclosure are systems, devices, and methods for transmitting a fluid sample from a fluid source into multiple sample chambers. Systems according to the subject embodiments include a fluid source and a plurality of independent, continuous fluidic pathways, each fluidic pathway comprising a sample chamber connected to the fluid source, and a pneumatic compartment connected to the sample chamber. The fluid source, the sample chamber, and the pneumatic compartment are used in conjunction with one another to transmit a fluid sample from the fluid source into the sample chamber.

[0098] FIG. 1 is an illustration of an apparatus 100 for transmitting a fluid sample from a fluid source into multiple sample chambers, in accordance with an embodiment. The apparatus includes a common fluid source 101 connected to a plurality of independent, continuous fluidic pathways. In alternative embodiments, rather than including the ten fluidic pathways shown in FIG. 1, the apparatus may include any number of fluidic pathways. For example, in some embodiments, the apparatus may include two, five, twelve, or twenty independent, continuous fluidic pathways.

[0099] Each independent, continuous fluidic pathway 110 of the plurality of independent, continuous fluidic pathways comprises a sample chamber and a pneumatic compartment. In some embodiments, each sample chamber comprises an entry conduit 122 and an assay chamber 121. In some further embodiments, each pneumatic compartment comprises a pneumatic conduit 132 and an air chamber 131. Therefore, in such implementations, each fluidic pathway comprises an entry conduit, an assay chamber, a pneumatic conduit, and an air chamber.

[00100] The common fluid source is an inlet, chamber, conduit, or the like capable of supplying a fluid sample to each fluidic pathway of the apparatus at a supply pressure. Ilie common fluid source is connected to, and is in fluidic communication with, each fluidic pathway of the plurality of fluidic pathways. In other implementations, as illustrated in FIG. 1, the common fluid source is connected to, and is in fluidic communication with, the entry conduit of each fluidic pathway. Accordingly, the common fluid source can supply a fluid sample to each fluidic pathway of the apparatus via its respective entry conduit.

[00101] The entry conduit of each fluidic pathway is in turn connected to, and in fluidic communication with, the assay chamber of the fluidic pathway . The assay chamber of the fluidic

WO 2019/183608 pathway is in turn connected to, and in fluidic communication with, the pneumatic conduit of the fluidic pathway. In certain implementations, such as illustrated in FIG. 1, the pneumatic compartment coinprises a pneumatic conduit that is in turn connected to, and in fluidic communication with, the air chamber of the fluidic pathway. In other implementations the pneumatic compartment can be comprised of a single structure directly connected to the sample chamber, or the assay chamber thereof.

[00102] The terms “fluidic connection,” and “fluidic continuity” as used herein, refers to any duct, channel, tube, pipe, or pathway through which a substance, such as a liquid, gas, or solid may pass substantially unrestricted when the pathway is open. When the pathway is closed, the substance is substantially restricted from passing through. Typically, limited diffusion of a substance through the material of a substrate, which may or may not occur depending on the compositions of the substance and materials, does not constitute fluidic communication.

[00103] Due to the continuity of each fluidic pathway, a fluid sample front the common fluid source can travel throughout the fluidic pathway. Specifically, a fluid sample from the common fluid source can travel through the entry conduit, into the assay chamber, through the pneumatic conduit, and into the air chamber of each fluidic pathway. However, in some embodiments it may be desirable to confine the fluid sample to the assay chambers of the apparatus such that the fluid sample is not tost to the pneumatic compartment. Such embodiments are described in greater detail below. In some embodiments, the apparatus is oriented such that the fluid sample travels into the assay chamber via the entry conduit against a gravitational force.

[00104] Excluding the connection between the common fluid source and the entry conduit of each fluidic pathway, each fluidic pathway is a closed system. As used herein, the term “closed system” refers to a system that can exchange heat and energy but not matter, with its surroundings. The term closed system is not intended to exclude limited permeability of gases, such as water vapor or oxygen into the substrate in which the fluidic pathway is formed. In other words, matter contained within a fluidic pathway cannot travel into or out of the fluidic pathway, except via the connection between the common fluid source and the entry conduit of the fluidic pathway. In certain methods of using the apparatus described herein, the fluidic connection between the common fluid source and each of the fluidic pathways is sealed, e.g. by heat staking, during operation of the method.

[00105] By sealing off a connection between the common fluid source and an entry conduit of a fluidic pathway, the fluidic pathway becomes a completely closed system from which matter cannot travel in or out, and for which, devoid of any changing variables, internal pressure within

WO 2019/183608 the fluidic pathway remains constant. One such embodiment of sealing off a connection between the common fluid source and an entry conduit of a fluidic pathway is discussed in greater detail below with regard to FIGS. 2E and 2F.

[00106] As noted above, each fluidic pathway includes a sample chamber and a pneumatic compartment. In turn, each sample chamber includes an entry conduit and an assay chamber, and each pneumatic compartment includes a pneumatic conduit and an air chamber.

[00107] The entry conduit is configured to transport a fluid sample from the common fluid source into the assay chamber of the fluidic pathway. The assay chamber is configured to contain an assay. In some embodiments, an assay chamber may include features to facilitate the assay. For example, in some embodiments discussed in further detail below with regard to FIG. 3B, each assay chamber is configured to minimize formation of bubbles during transmission of the fluid sample into the assay chamber. This feature is advantageous during assay actuation because in some embodiments, bubbles prevent complete filling of the chamber or otherwise interfere with the results of the assay. Additionally, in some embodiments discussed in further detail below with regard to FIGS. 4-7, each assay chamber is configured to include a plug, and/or to contain dried reagents and/or a magnetic mixing element to facilitate assay actuation.

[00108] The sample chamber is fluidically connected to the pneumatic compartment. In those embodiments comprising an entry conduit and an assay chamber, the assay chamber is connected to a pneumatic compartment of the fluidic pathway. As mentioned above, the pneumatic compartment can include a. pneumatic conduit and an air chamber. The pneumatic conduit connects the assay chamber to the air chamber of the fluidic pathway.

[00109] In embodiments in which each assay chamber of an apparatus is configured to contain an assay as described above, it may be beneficial to control a rate of flow of a fluid sample into the assay chambers to ensure that each assay chamber is filled with a precise amount of fluid sample, and to ensure that the composition of the fluid sample is homogenous across all assay chambers of the apparatus. This enables standardization of assays that may take place in the assay chambers, in some embodiments. Accordingly, the pneumatic compartment of each fluidic pathway is configured to control the flow rate of a fluid sample into the assay chamber of the fluidic pathway according to the internal pressure of the pneumatic compartment. The configuration and the function of the pneumatic compartments are discussed in greater detail below.

[00110] Both the sample chamber and the pneumatic compartment of each fluidic pathway have a volume. The sample chamber has a volume hereinafter referred to as the “fluid volume.”

WO 2019/183608 !he fluid volume of a fluidic pathway includes the volume of the entry conduit and the volume of the assay chamber of the fluidic pathway. Similarly, the pneumatic compartment has a volume hereinafter referred to as the “pneumatic volume.” The pneumatic volume of a fluidic pathway includes the volume of the pneumatic conduit and the volume of the air chamber of the fluidic pathway . In order to achieve concurrent filling of each sample chamber, particularly when the volume of the sample chambers vary across the plurality of fluid pathways, the ratio of the fluid volume to the pneumatic volume is the same for each fluidic pathway sharing a common fluid source.

[00111] Prior to the introduction of a fluid sample into each fluidic pathway of an apparatus by a common fluid source, the fluidic pathways contain a gas, such as, e.g., air, at an initial air pressure. Throughout this disclosure air should be interpreted to encompass a mixture of atmospheric gasses or any other gas mixture or pure gas that is compatible with the assays carried out in the apparatus. Furthermore, as one skilled in the art would understand, any gas can be used in place of air in the fluidic pathways of the devices and methods described herein. For example, in some embodiments, air can be substituted with another gas, e.g., an inert gas such as, e.g., nitrogen or argon.

[00112] The initial pressure of air within a fluidic pathway in part determines the internal pressure of the fluidic pathway. When a fluid sample is introduced into the fluidic pathways by the common fluid source, the air within each fluidic pathway is displaced within the fluidic pathway by the advancing fluid sample. Specifically, the advancing fluid sample enters each fluidic pathway via an entry conduit of the fluidic pathway and displaces the air within the fluidic pathway in a direction of the air chamber. As a result of this displacement, the volume that is occupied by the air in the fluidic pathway decreases. As a result of this decreased volume occupied by the air, the pressure of the air, and thus the internal pressure of the fluidic pathway, increases. Specifically, the internal pressure increases proportionally to the decrease in the volume occupied by the air.

[00113] The balance of supply pressure and internal pressure within a fluidic pathway determines a rate of flow of a fluid sample within the fluidic pathway. Specifically, if the internal pressure within the fluidic pathway is less than a pressure at which the fluid sample is supplied to the fluidic pathway, the fluid sample continues to advance within the fluidic pathway, and continues to displace the air contained within the fluidic pathway such that the internal pressure increases. However, the closer in value that the internal pressure is to the supply pressure of the fluid sample, the more pressure the air exerts on the fluid sample within the fluidic pathway, and

WO 2019/183608 the more the internal pressure retards a rate of flow of the fluid sample within the fluidic pathway. Once the internal pressure within the fluidic pathway equals the supply pressure of the fluid sample, the fluid sample stops flowing into the fluidic pathway. Accordingly, by controlling a volume in which air is contained within a fluidic pathway, and thereby controlling the internal pressure of the fluidic pathway, it is possible to control the rate of flow of the fluid sample within the fluidic pathway.

[00114] It may be desirable to control the rate of flow of a fluid sample within a fluidic pathway in a variety of circumstances. Particularly, in certain embodiments, it may be desirable to control a rate of flow of a fluid sample within a fluidic pathway such that the fluid sample is confined to the sample chamber of the fluidic pathway and does not flow into the pneumatic compartment of the fluidic pathway. In other words, it may be desirable to control the rate of flow of a fluid sample within a. fluidic pathway such that the sample chamber (and in some embodiments, the assay chamber in particular) is substantially filled with the fluid sample. As used herein, the term “substantially filled” or “substantially full” means that at least 90% of the fluid volume of the sample chamber contains the fluid sample, and at most 10% of the pneumatic volume of the pneumatic compartment contains the fluid sample. In particular, filling 10% or less of the pneumatic volume of the pneumatic compartment with the fluid sample does not disrupt the operation of the apparatus.

[00115] To substantially fill a sample chamber of a fluidic pathway, the pressure at which the fluid sample is supplied to the sample chamber must equal the internal pressure within the fluidic pathway when the sample chamber is substantially filled with the fluid sample. Furthermore, because the fluid sample is confined to the sample chamber of the fluidic pathway when the sample chamber is substantially filled, the air contained within the fluidic pathway is compressed into the pneumatic compartment of the fluidic pathway. Thus, to substantially fill a. sample chamber of a fluidic pathway, the pressure at which the fluid sample is supplied to the sample chamber must equal the internal pressure contained within the pneumatic compartment of the fluidic pathway when the sample chamber is substantially filled with the fluid sample.

[00116] As mentioned above, internal pressure depends, in part, on the volume of air being displaced from the sample chamber and, in part, on the volume confining the displaced air. Thus when a fluid sample substantially fills the sample chamber of a fluidic pathway, the displaced air is confined within the pneumatic compartment, and the internal pressure depends on the pneumatic volume of the pneumatic compartment and on the fluid volume of the sample chamber.

WO 2019/183608

[00117] Therefore, to achieve a balance between the internal pressure and the supply pressure of the fluid sample when the sample chamber is substantially filled with the fluid sample, the pneumatic volume of the fluidic pathway can be intentionally selected in view of the fluid volume of the sample chamber and the supply pressure. Additionally, as an initial internal pressure within the fluidic pathway and the supply pressure of the fluid sample are also factors in achieving equivalence between the internal pressure contained within the pneumatic volume and the supply pressure of the fluid sample, the ambient internal pressure within the fluidic pathway and the supply pressure of the fluid sample can also be intentionally selected to ensure that the supply pressure of the fluid sample equals the internal pressure contained within the pneumatic compartment when the sample chamber is substantially filled with the fluid sample. This equivalence between the internal pressure and the supply pressure results in a net zero force on the fluid sample contained within the fluidic pathway, thereby stopping the flow of the fluid sample into the fluidic pathway just when the sample chamber is substantially filled.

[00118] In further embodiments, it may be desirable to control the rate of flow of a fluid sample within the multiple fluidic pathways of the apparatus such that the sample chamber of each fluidic pathway fills simultaneously. However, as noted above, in some embodiments, the fluid volume of each fluidic pathway of the apparatus can differ. This variation in fluid volumes means that if a fluid sample flows into each fluidic pathway at the same rate, the sample chambers will not fill simultaneously. Rather, if the fluid sample flows into fluidic pathways of varying fluid volumes at the same rate, the fluidic pathways with smaller fluid volumes will fill with the fluid sample before the fluidic pathways with larger fluid volumes.

[00119] To ensure that the sample chamber of each fluidic pathway fi lls simultaneously regardless of its fluid volume, the fluidic pathways can be configured such that a rate of flow of a fluid sample into each fluidic pathway is proportional to the fluid volume of the fluidic pathway. For example, a first fluidic pathway with a fluid volume that is twice the fluid volume of a second fluidic pathway will be configured such that a rate of flow of a fluid sample into the first fluidic pathway is twice the rate of flow of the fluid sample into the second fluidic pathway. In this way, a sample chamber of the first fluidic pathway and a sample chamber of the second fluidic pathway will fill simultaneously.

[00120] As described above, a rate at which a fluid sample from the common fluid source flows into a fluidic pathway depends on an internal pressure of the fluidic pathway and the supply pressure. Specifical ly, the closer in value that the internal pressure is to the supply pressure, the more the internal pressure retards a rate of flow of the fluid sample within the

WO 2019/183608 fluidic pathway. Furthermore, as described above, the internal pressure of the contained air depends, in part, on a volume in which the air is contained. Specifically, air that is displaced into a portion of a fluidic pathway with a smaller volume will have a greater increase in pressure than a similar volume of air that initially is displaced into a portion of the fluidic pathway with a larger volume.

[00121] Therefore, to configure the fluidic pathways such that a rate of flow of a fluid sample into each fluidic pathway is proportional to the fluid volume of the fluidic pathway, each fluidic pathway can be configured such that the volume in which the air is contained is proportional to the fluid volume of the fluidic pathway. In embodiments in which the sample chamber of the fluidic pathway is the portion of the fluidic pathway being filled with the fluid sample, the volume in which the air is contained is the pneumatic volume of the fluidic pathway. ’Thus in such embodiments, to achieve an inverse proportionality between the internal pressure of a fluidic pathway and the fluid volume of the fluidic pathway, each fluidic pathway is configured such that the pneumatic volume of the fluidic pathway is proportional to the fluid volume of the fluidic pathway.

[00122] Furthermore, to achieve simultaneous filling of the sample chamber of each fluidic pathway of an apparatus, this proportionality between the fluid volume of the fluidic pathway and the pneumatic volume of the fluidic pathway must be the same for all of the fluidic pathways of the apparatus. Specifically, a ratio of the fluid volume to the pneumatic volume of a fluidic pathway must be substantially equivalent for each fluidic pathway of the apparatus. Note that as used herein, “substantially equivalent” means that the ratios of the fluid volume to the pneumatic volume differ by no more than +/- 10%.

[00123] In certain embodiments, the volumes of the entry conduit and the pneumatic conduit are negligible relative to the volumes of the assay chamber and the air chamber, respectively. Specifically, as used herein, “negligible” means that the volume of an entry conduit of a fluidic pathway comprises no more than 10% of the volume of an assay chamber of the fluidic pathway, and similarly, the volume of the pneumatic conduit of a fluidic pathway comprises no more than 10% of the volume of an air chamber of the fluidic pathway. Therefore, in such embodiments, the fluid volume of the sample chamber is comprised, in large part, of the volume of the assay chamber, and in small part, of the volume of the entry conduit. Similarly, in such embodiments, the pneumatic volume of the pneumatic compartment is comprised, in large part, of the volume of the air chamber, and in small part, of the volume of the pneumatic conduit.

WO 2019/183608

[00124] In some embodiments, the fluid volume of one or more fluidic pathways of an apparatus can differ. For example, a. fluid volume of a first fluidic pathway of the apparatus can be greater than a fluid volume of a second fluidic pathway of the apparatus. Thi s difference in fluid volumes across the fluidic pathways of the apparatus can be the result of a difference in volumes of the assay chambers and/or a difference in volumes of the entry conduits of the fluidic pathways.

[00125] In embodiments in which the fluid volume of one or more fluidic pathways of an apparatus differs, the pneumatic volume of the one or more fluidic pathways of the apparatus would also differ for reasons discussed in further detail below. A difference in pneumatic volumes across the fluidic pathways of the apparatus can be the result of a difference in volumes of the air chambers and/or a difference in volumes of the pneumatic conduits of the fluidic pathways.

[00126] In embodiments in which the volumes of the entry conduits and the pneumatic conduits of the fluidic pathways are negligible relative to the volumes of the assay chambers and the air chambers, to enable simultaneous filling of the sample chamber of each fluidic pathway, a ratio of the volume of the assay chamber to the air chamber of a fluidic pathway may be substantially equivalent for each fluidic pathway.

METHODS

[00127] FIGS. 2A-2F depict the apparatus of FIG. 1 at a plurality of sequential time points during simultaneous filling of the sample chambers with a fluid sample from the common fluid source, in accordance with an embodiment. In some embodiments, the apparatus is oriented during filling of the sample chambers such that the fluid sample travels into the sample chambers against a gravitational force. As described above with regard to FIG. 1, the fluid volume of each fluidic pathway of the apparatus may vary. Therefore, to enable simultaneous filling of the sample chamber of each fluidic pathway of the apparatus, a ratio of the fluid volume to the pneumatic volume is substantially equivalent for each fluidic pathway of the apparatus.

[00128] As shown in the legend at the bottom left-hand corner of FIGS. 2A-2F, air is denoted within the fluidic pathways by white space. Contrastingly, the fluid sample is denoted within the common fluid source and the fluidic pathways by black space.

[00129] FIG. 2A depicts the apparatus 200 at a time A. At the time A, the fluidic pathways are filled with air 250. The air contained within the fluidic pathways at the time A is has an initial air pressure that contributes, at least in part, to the internal pressure of the fluidic pathways. At the

WO 2019/183608 time A, the common fluid source 201 supplies the fluid sample 260 to the fluidic pathways at a supply pressure that is greater than the internal pressure within the fluidic pathways. Because the supply pressure of the fluid sample is greater than the internal pressure within the fluidic pathways, the fluid sample advances from the common fluid source toward the entry conduit 222 of each fluidic pathway. As the fluid sample advances within the apparatus, the air contained within the fluidic pathways is displaced by the fluid sample toward the air chambers of the apparatus. This displacement of the air into a smaller volume causes the pressure of the air, and thus the internal pressure, to increase. As the internal pressure increases, the air exerts an increasing amount of pressure on the advancing fluid sample, thereby slowing the rate of flow of the fluid sample.

[00130] FIG. 2B depicts the apparatus 200 at a time B that is subsequent to the time A. At the time B, the common fluid source 201 continues to supply the fluid sample 260 to the fluidic pathways at a supply pressure.

[00131] In some embodiments, throughout the filling of the sample chamber of each fluidic pathway, the supply pressure of the fluid sample is applied at a constant, pressure. In other words, the supply pressure at one time point is equivalent to the supply pressure at all other time points.

[00132] In alternative embodiments, the supply pressure is applied in a ramping fashion, such that the supply pressure increases over time from a lower pressure to a higher pressure. In other words, the supply pressure at a first time point is greater than the supply pressure at a second time point subsequent to the first time point. In such embodiments in which the supply pressure is applied in a ramping fashion, the supply pressure may increase linearly over time from a lower pressure to a higher pressure. In alternative embodiments, the ramping of the supply pressure may follow a parabolic trajectory. In alternative embodiments, the ramping of the supply pressure may follow any alternative trajectory. In embodiments in which the supply pressure is applied in a ramping fashion, this ramping of the supply pressure can dislodge air bubbles that may have formed within a sample chamber during filling, because the increasing supply pressure compresses and detaches the bubbles from their positions within the sample chamber, enabling the bubbles to be released into the pneumatic compartment. In particular embodiments in which the fluid sample travels into the sample chambers against a gravitational force, this orientation of the apparatus aids the detached bubbles in traveling to the top of the sample chamber and into the pneumatic compartment.

[00133] Turning back to FIG. 2B, at the time B, the supply pressure is still greater than the internal pressure within the fluidic pathways. Because the supply pressure of the fluid sample is

WO 2019/183608 still greater than the internal pressure within the fluidic pathways, the fluid sample continues to advance into each fluidic pathway toward the air chamber 231 of the fluidic pathway.

Specifically, as shown in FIG. 2B, the fluid sample has advanced into at least the entry conduit 222 of each fluidic pathway. Furthermore, the fluid sample has advanced into the assay chambers 221 of a portion of the fluid pathways. Accordingly, air is trapped within the fluidic pathways. As the volume of fluid sample supplied to the fluidic pathways via the common fluid source increases, and accordingly as the fluid sample advances within the fluidic pathways towards the air chambers, the air contained within each fluidic pathway is displaced into a smaller volume within the fluidic pathway. Accordingly, the internal pressure of the fluidic pathway increases. As a result of this increase in internal pressure of each fluidic pathway, the air exerts an increasing amount of pressure on the advancing fluid sample in the fluidic pathway, thereby decreasing the rate of flow of the fluid sample lit the fluidic pathway.

[00134] However, the internal pressure within each fluidic pathway does not increase uniformly across all of the fluidic pathways of the apparatus. Rather, an internal pressure of a fluidic pathway depends on the volume into which the air is displaced. Specifically, as the fluid travels through each of the channels, the air upstream of the fluid is compressed. The compressed gas generates a back-pressure that resists the advancing fluid flow. This backpressure is inversely proportional to the ratio of the contained volume over the original volume in accordance with the ideal gas law. For example, fluid in a channel that is connected to a large pneumatic volume would experience a higher back-pressure than a fluid through a channel of the same length that is connected to a smaller pneumatic volume.

[00135] In turn, the velocity of the fluid is proportional to the difference between the pressure applied to the channel, and the back-pressure that comes from the compressed gas in the pneumatic volume and upstream channel. As such, fluid in a channel with a larger pneumatic volume would travel faster than fluid in a channel of the same size with a smaller pneumatic volume.

[00136] Furthermore, as the upstream gas volume is further compressed, the back-pressure increases proportionally. Since the velocity of the fluid is proportional to the pressure difference, the velocity of the fluid gradually decreases as the channels fill. The fluid flow stops when the pressure applied to the fluid is equal to the back-pressure from the compressed pneumatic volumes.

[00137] As described above with regard to FIG. 1, to enable simultaneous filling of the sample chamber of each fluidic pathway, in some embodiments of the apparatus, each fluidic

WO 2019/183608 pathway is configured such that the pneumatic volume of the fluidic pathway is proportional to the fluid volume of the fluidic pathway. For example, a fluidic pathway with a relatively large fluidic volume also has a relatively large pneumatic volume. ^,Therefore, in such embodiments, because a rate of flow of a fluid sample in a fluidic pathway is proportional to the pneumatic volume of the fluidic pathway, the rate of flow of a fluid sample in a fluidic pathway is also proportional to the fluid volume of the fluidic pathway. In other words, fluidic pathways with a larger fluid volume experience a relatively greater rate of flow of a fluid sample than fluidic pathways with a smaller fluid volume. This phenomenon can be seen in FIG. 2B. Specifically, as seen in FIG. 2B, at the time B, the fluidic pathways with larger fluid volumes contain a larger volume of the fluid sample than the fluidic pathways with smaller fluid volumes because the fluidic pathways with larger fluid volumes experience a relatively greater rate of flow of the fluid sample than the fluidic pathways with smaller fluid volumes.

[00138] As further discussed with regard to FIG. 1, to achieve simultaneous filling of the sample chamber of each fluidic pathway of an apparatus, in some embodiments, the proportionality between the fluid volume of the fluidic pathway and the pneumatic volume of a fluidic pathway is the same for all of the fluidic pathways of the apparatus. Specifically, a ratio of the fluid volume to the pneumatic volume of a fluidic pathway is substantially equivalent for each fluidic pathway of the apparatus. Based on this substantial equivalence between the fluidic pathways, the sample chamber of each fluidic pathway fills at a substantially proportional rate, thereby enabling simultaneous filling of the sample chambers. As referred to herein, “substantially proportional” means that the rates at which the sample chambers fill differ by no more than +/- 10%. This phenomenon can also be seen in FIG. 2C. Specifically, as seen in FIG. 2C, at the time C, the same proportion of the sample chamber of each fluidic pathway is filled with the fluid sample. This simultaneous filling of the sample chambers occurs not only at the time C, but throughout the filling of the sample chambers as discussed in more detail below with regard to FIGS. 2C and 2D.

[00139] FIG. 2C depicts the apparatus 200 at a time C that is subsequent to the time B. At the time C, the common fluid source 201 continues to supply the fluid sample 260 to the fluidic pathways at a supply pressure that is greater than the internal pressure within the fluidic pathways. Because the supply pressure of the fluid sample is still greater than the internal pressure within the fluidic pathways, the fluid sample continues to advance into each fluidic pathway toward the air chamber 231 of the fluidic pathway. Specifically, as shown in FIG. 2C, the fluid sample has advanced into the assay chamber 221 of each of the fluidic pathways of the apparatus.

WO 2019/183608

[00140] The air trapped within each fluidic pathway is held at a pressure according to the volume in which the air is contained. Specifically, air contained within a smaller volume has a greater pressure than air contained within a relatively larger volume.

[00141] Furthermore, the air trapped within each fluidic pathway affects a rate of flow of the fluid sample in the fluidic pathway according to the internal pressure of the fluidic pathway that is dependent, at least in part, on the pressure of the trapped air. Specifically, air at a higher pressure decreases a rate of flow of the fluid sample in the fluidic pathway more than air at a relatively lower pressure. Therefore, because air pressure is inversely proportional to the volume in which the air is contained, air contained within a smaller volume decreases a rate of flow of the fluid sample in the fluidic pathway more than air within a larger volume.

[00142] In the embodiment of the apparatus shown in FIG. 2C, each fluidic pathway of the apparatus is configured such that the pneumatic volume of the fluidic pathway is proportional to the fluid volume of the fluidic pathway, and a ratio of the fluid volume to the pneumatic volume of a fluidic pathway is substantially equivalent for each fluidic pathway of the apparatus. As a result, the sample chamber of each fluidic pathway fills at a substantially proportional rate, thereby enabling simultaneous filling of the sample chambers of the apparatus.

[00143] FIG. 2D depicts the apparatus 200 at a time D that is subsequent to the time C. At the time D, the common fluid source 201 continues to supply the fluid sample 260 to the fluidic pathways at a supply pressure. However, at the time D, the internal pressure within each fluidic pathway has increased such that the internal pressure within each fluidic pathway equals the supply pressure. Due to this equivalence between the supply pressure and the internal pressure within each fluidic pathway, the fluid sample ceases to advance within the fluidic pathways.

[00144] In the embodiment shown in FIG. 2D, the fluid sample stops flowing into each fluidic pathway when the fluid sample has substantially filled the assay chamber 221 of the fluidic pathway. As discussed above, to stop flow of the fluid sample into a fluidic pathway when the assay chamber of the fluidic pathway is substantially filled, the internal pressure within the pneumatic volume and the supply pressure of the fluid sample must be equal when the assay chamber is substantially filled with the fluid sample. To accomplish this, the pneumatic volume of the fluidic pathway, an initial air pressure within the fluidic pathway, and the supply pressure of the fluid sample can be intentionally selected. In this way, the fluid sample can be confined to the assay chambers of the apparatus.

[00145] As further shown in FIG. 2D, the assay chamber of each fluidic pathway completes filling at the same time D. In other words, the filling of the sample chambers of FIG. 2D is

WO 2019/183608 simultaneous. As discussed above, this simultaneous filling of the sample chambers is the result of the pneumatic volume of a fluidic pathway being proportional to the fluid volume of the fluidic pathway, and a ratio of the fluid volume to the pneumatic volume of a fluidic pathway being substantially equivalent for each fluidic pathway of the apparatus.

[00146] FIG. 2E depicts the apparatus 200 at a time E that, is subsequent ίο the time D. At the time E, the fluid sample 260 has stopped flowing into the fluidic pathways, and the sample chamber of each fluidic pathway is substantially filled. The level of the fluid sample within each fluidic pathway is maintained by the equilibrium between the supply pressure of the fluid sample and the internal pressure within the pneumatic compartment of the fluidic pathway.

[00147] To maintain this level of the fluid sample within each fluidic pathway without continued application of the supply pressure by the common fluid source 201, a portion of the entry conduit 222 of each fluidic pathway can be sealed. One acceptable method of sealing the entry conduits is heat staking with a heated element 284 such that the fluidic pathway is sealed off from the common fluid source. Note that the supply pressure of the fluid sample is maintained during the heat staking process as shown in FIG. 2E.

[00148] In some embodiments, a first film is adhered to a surface of at least a portion of the apparatus, such that the first film forms one wall of the entry conduit of each fluidic pathway. In one implementation the first film has a similar melting point as the substrate of the apparatus.

[00149] In further embodiments, a second film is adhered to the first film. In such embodiments, the second film has a higher melting point than the first film and the surface of the apparatus such that when heat is applied to the apparatus via the heated element to heat stake the entry conduit of each fluidic pathway, the first film and the surface of the apparatus melt prior to the second film. This higher melting point of the second film prevents the pressurized fluid sample from escaping from the fluidic pathways as the first film and the surface of the apparatus are melted. The result of this heat staking process is a melted first film, which forms a heat stake 203 as depicted in FIG. 2F.

[00150] The sealing process renders each fluidic pathway a completely closed system from which matter cannot travel in or out, and for which, devoid of any changing variables, the internal pressure within each fluidic pathway remains constant.

[00151] In embodiments in which the assay chamber of each fluidic pathway is configured to contain an assay, sealing the entry conduits is beneficial because it isolates the fluidic pathway from the environment such that the assay can be performed in an enclosed and controlled volume without contamination across fluidic pathways or to the environment. Furthermore, the constant -30WO 2019/183608 internal pressure locked into the fluidic pathway by the heat staking minimizes the formation of bubbles within the assay chambers during actuation of the assay.

[00152] FIG. 2F depicts the apparatus 200 at a time F that is subsequent to the time E. At the time F, the heat staking process is complete, and the heat stake 203 is in place such each fluidic pathway is sealed off from the common fluid source 201. As a result of the heal staking, internal pressure within each fluidic pathway remains constant such that the level of the fluid sample is maintained within each fluidic pathway without the assistance of the supply pressure from the common fluid source. Accordingly, as shown in FIG. 2F, the supply pressure from the common fluid source is released. At this stage, the apparatus is prepared for use in one or more assays.

[00153] FIG. 2G is a cross section of a inlet conduit having a conduit feature such as a raised platform within a heat stake zone.

[00154] FIG. 2H is a perspective and section view of an apparatus showing a conduit feature in a heat stake zone for the common fluid line. FIG. 21 is a cross section view of the common fluid line of FIG. 2H showing the raised platform within the heat stake zone.

[00155] FIG. 2J is a top down view of an apparatus indicating a heat stake zone along each of the independent fluid conduits. FIG. 2K is a cross section of one of the independent fluid conduits in FIG. 2J showing the conduit feature or raised platform to facilitate heat staking the channel.

DEVICES

[00156] FIG. 3 A is an illustration of an independent fluidic pathway 310, in accordance with an embodiment. The independent fluidic pathway comprises a sample chamber 320 and a pneumatic compartment 330. The sample chamber comprises an entry conduit 322 and an assay chamber 221. The sample chamber comprises a fluid volume. The pneumatic compartment comprises a pneumatic conduit 332 and an air chamber 331. The pneumatic compartment comprises a pneumatic volume.

[00157] The fluid volume and the pneumatic volume of the fluidic pathway are configured to contain air 350 and/or a fluid sample 360. As shown in FIG. 3A. air is denoted within the fluidic pathway by white space. Contrastingly, the fluid sample is denoted within the fluidic pathway by a crosshatch pattern.

[00158] In the embodiment shown in FIG. 3A, the fluid volume is substantially filled with the fluid sample, and the pneumatic volume is filled with air. However, lit alternative embodiments, the fluid volume and the pneumatic volume may be filled with any ratio of the fluid sample

WO 2019/183608 and/or air. For example, prior to introduction of the fluid sample to the fluidic pathway, the entire fluidic pathway can be filled with air. The process by which a fluidic pathway is filled with the fluid sample is discussed in detail below with regard to FIGS. 2A-2F.

[00159] The entry conduit of the fluidic pathway is configured to transport the fluid sample from a common fluid source into the assay chamber of the fluidic pathway. A portion of the entry conduit that connects the entry conduit to the assay chamber is referred to as an entry conduit terminus 323.

[00160] The assay chamber is configured to contain an assay. In some embodiments, the assay chamber may include features to facilitate the assay. For example, as discussed in further detail below with regard to FIG. 3B, the assay chamber is configured to minimize formation of bubbles during transmission of the fluid sample into the assay chamber.

[00161] The pneumatic conduit connects the assay chamber to the air chamber of the fluidic pathway. A portion of the pneumatic conduit that connects the pneumatic conduit to the assay chamber is referred to as a pneumatic compartment terminus 333.

[00162] As described below with regard to FIG. 1, the pneumatic compartment is configured to control the rate flow of a fluid sample into the assay chamber of the fluidic pathway according to an internal pressure within the pneumatic compartment.

[00163] The independent fluidic pathway is a continuous system. Specifically, the entry conduit is connected to, and in fluidic communication with, the assay chamber. The assay chamber is connected to, and in fluidic communication with, the pneumatic conduit. 'The pneumatic conduit is connected to, and in fluidic communication with, the air chamber.

[00164] As a result of the continuity of the independent fluidic pathway, a fluid sample can travel throughout the fluidic pathway. Specifically a fluid sample can travel through the entry conduit, into the assay chamber, through the pneumatic conduit, and into the air chamber of each fluidic pathway.

[00165] Excluding an opening at one end of the entry conduit, the independent fluidic pathway is a closed system. In other words, matter contained within the fluidic pathway cannot travel into or out of the fluidic pathway, except via the one opening of the entry conduit.

Therefore, by sealing off the one opening of the entry conduit, the fluidic pathway becomes a completely closed system from which matter cannot travel in or out, and for which, devoid of any changing variables, internal pressure within the fluidic pathway remains constant.

WO 2019/183608

[00166] FIG. 3B is an illustration of an assay chamber 321, in accordance with an embodiment. The assay chamber comprises an assay chamber volume. In some embodiments, assay chamber volume is between 1 pL and 35 pL.

[00167] As shown in the legend at the bottom left-hand corner of FIG. 3B, air 350 is denoted within the assay chamber volume by white space. Contrastingly, a fluid sample 360 is denoted within the assay chamber volume by a crosshatch pattern. In the embodiment of the assay chamber depicted in FIG. 3B, the sample chamber is substantially filled with the fluid sample. In other words, at least 90%׳ of the volume of the sample chamber contains the fluid sample, and at most 10% of the pneumatic compartment contains the fluid sample.

[00168] As described above with regard to FIG. 1, in some embodiments, the assay chamber is configured to contain an assay. In such embodiments, the assay chamber may include features to facilitate the assay. For example, in the embodiment of the assay chamber depicted in FIG. 3B, the assay chamber is configured to minimize formation of bubbles during transmission of the fluid sample into the assay chamber. This feature is advantageous during assay actuation because bubbles alter the effective volume of the assay chamber and can interfere with the results of the assay.

[00169] In the embodiment shown in FIG. 3B, to minimize formation of bubbles during transmission of the fluid sample into the assay chamber, the assay chamber comprises a doubled tapered chamber 340. !he role of the double tapered chamber in minimizing bubble formation is discussed in greater detail below. The double tapered chamber comprises a tapered inlet 341, a tapered outlet 342, a first curved boundary 344, and a second curved boundary 345.

[00170] The tapered inlet is an inlet of the double tapered chamber that is configured to receive a fluid sample from an entry conduit. Specifically, the tapered inlet is connected to, and in fluidic communication with, the entry conduit via an entry conduit terminus 323. As noted above, the entry conduit terminus is a portion of the entry conduit that connects the entry conduit to the assay chamber. Therefore, to receive a fluid sample in the double tapered chamber, the fluid sample travels through the entry conduit terminus and into the double tapered chamber via the tapered inlet.

[00171] The tapered outlet is an outlet of the double tapered chamber that is connected to, an in fluidic communication with, a pneumatic compartment via a pneumatic compartment terminus 333. As noted above, the pneumatic compartment terminus is a portion of a pneumatic conduit of the pneumatic compartment that connects the pneumatic compartment to the assay chamber. The

WO 2019/183608 tapered inlet and the tapered outlet are separated by a largest dimension 348 of the assay ch amber vol ume.

[00172] In embodiments in which the double tapered chamber is substantially filled with the fluid sample, as shown in FIG. 3B, air may be contained within the pneumatic compartment, including within the pneumatic compartment terminus. In such embodiments, the tapered outlet can serve to connect the fluid sample located within the double tapered chamber and the air located within the pneumatic compartment, such that the fluid sample can interface with the air. This interface between the fluid sample and the air can be used to control a rate of flow of the fluid sample, as discussed in detail above with regard to FIGS. 1-2D.

[00173] The double tapered chamber compri ses two curved boundaries, the first curved boundary and the second curved boundary. Each curved boundary extends from the tapered inlet to the tapered outlet such that the two curved boundaries enclose the assay chamber volume. Therefore, the only pathways in or out of the double tapered chamber are via the tapered inlet and the tapered outlet, as described above.

[00174] Each curved boundary of the double tapered chamber comprises a midpoint. Specifically, the first curved boundary comprises a first curved boundary midpoint 346 and the second curved boundary comprises a second curved boundary midpoint 347. A distance between the two curved boundaries decreases as the boundaries curve front the midpoint toward the tapered inlet and from the midpoint toward the tapered outlet. In other words, each curved boundary is concave with regard to a center point 343 of the assay chamber volume. In some embodiments, this gradual decrease in the distance between the two curved boundaries as the boundaries curve from their midpoints toward the tapered inlet and the tapered outlet of the assay double tapered chamber, occurs at the same rate towards both the tapered inlet and the tapered outlet, such that curved boundary is symmetric about the midpoint of the curved boundary. In further embodiments, this gradual decrease in the distance between the two curved boundaries as the boundaries curve from their midpoints toward the tapered inlet and the tapered outlet of the assay double tapered chamber, occurs at the same rate towards both the tapered inlet and the tapered outlet for both the first curved boundary and the second curved boundary, such that two curved boundaries are symmetric to one another about the largest dimension of the assay chamber volume.

[00175] As mentioned above, the configuration of the assay chamber as the double tapered chamber shown in FIG. 3B minimizes the formation of bubbles during transmission of the fluid sample into the assay chamber. Specifically, as the fluid sample flows into the double tapered

WO 2019/183608 chamber, the interface between the fluid sample and the air within the double tapered chamber comprises a meniscus 361. The meniscus of the fluid sample includes a leading front 362. The leading front of the meniscus is a portion of the meniscus that leads the advance of the fluid sample within the assay chamber. In embodiments in which the fluid sample substantially fills the assay chamber, such as the embodiment depicted in FIG. 3B, the leading front of the meniscus is a portion of the meniscus that is in closest proximity to the tapered outlet.

[00176] To minimize formation of bubbles during transmission of the fluid sample into the assay chamber, as the fluid sample flows into the double tapered chamber, the two curved boundaries of the double tapered chamber slow the rate of advance of the fluid sample at the leading front of the meniscus of the fluid sample, such that when the fluid sample reaches the tapered outlet, the meniscus of the fluid sample is substantially symmetric with respect to the largest dimension of the assay chamber. As used herein, “substantially symmetric” means that at the point that the leading front of the meniscus of the fluid sample reaches the tapered outlet, the trailing front of the meniscus is has progressed at least half of the distance from the midpoint of the curved boundary to the tapered outlet. Ensuring that the meniscus of the fluid sample is substantially symmetric with respect to the largest dimension of the assay chamber volume by the time the fluid sample reaches the tapered outlet, minimizes the trapping of bubbles within the assay chamber during filling.

[00177] FIG. 4 is an illustration of a portion of an apparatus 400, in accordance with an embodiment. Similar to the embodiments of the apparatuses discussed above with regard to FIGS. 1-3A, the apparatus depicted in FIG. 4 comprises a plurality of fluidic pathways.

Furthermore, each fluidic pathway is a continuous, independent fluidic pathway that includes an entry conduit 422, an assay chamber 421, a pneumatic conduit 432, and an air chamber 431. The common fluid source and each fluidic pathway’s attachment thereto has been cropped from the illustration of FIG. 4.

[00178] As noted above with regard to FIGS. 1 and 3A, excluding a connection between a common fluid source and the entry conduit of a fluidic pathway, each fluidic pathway is a closed system. In some embodiments, to configure a fluidic pathway to be a closed system, the fluidic pathway comprises one or more bounding surfaces. For example, as discussed above with regard to FIGS. 2E and 2F, a bounding surface of each entry conduit of the apparatus may be a first film and/or a second film.

[00179] In the embodiment of the apparatus shown in FIG. 4, the assay chamber of each fluidic pathway comprises two bounding surfaces, such that the assay chamber is an enclosed

WO 2019/183608 system except for a connection of the assay chamber to the entry conduit and a connection of the assay chamber to the pneumatic conduit. Specifically, as shown in FIG. 4, the assay chamber of each fluidic pathway comprises a first bounding surface formed in a monolithic substrate 402, and a second bounding surface formed by a plug cap 472. The plug cap is a portion of a plug (labeled in FIGS. 6A-7) that protrudes into the monolithic substrate at a depth. The plug is placed within an opening of the monolithic substrate. Together, the monolithic substrate and the plug cap form a continuous bounding surface of the assay chamber.

[00180] In some implementations, each assay chamber further comprises a third bounding surface that is formed by a film. In such implementations, together, the monolithic substrate, the plug, and the film enclose the assay chamber volume.

[00181] In certain embodiments, as shown in FIGS. 47־, the plug cap includes a flange 473. The flange comprises a projecting rim of the plug cap that can be welded and/or adhered to a surface of the assay chamber, thereby stabilizing the position of the plug within the opening of the monolithic substrate of the assay chamber such that the plug is not ejected from the opening during pressurization of contents within the assay chamber. The configurations of the monolithic substrate and the plug cap are discussed in further detail below with regard to FIGS. 5-6B.

[00182] FIG. 5 is a three-dimensional illustration of an assay chamber 521, in accordance with an embodiment. As discussed in detail above, the assay chamber is connected to, and is in fluidic communication with, both an entry conduit 522, and a pneumatic conduit 532. .Additionally, the assay chamber is bounded by a monolithic substrate 502 and a plug cap 572.

[00183] In a preferred implementation, the monolithic substrate of the assay chamber is a single structural component. In some embodiments, the monolithic substrate is injection molded. In some embodiments, such as the embodiment depicted in FIG. 5, the monolithic substrate may form a portion or all of a double tapered chamber, as discussed above with regard to FIG. 3B. Specifically, the monolithic substrate may form the two curved boundaries of the double tapered chamber, as discussed above with regard to FIG. 3B.

[00184] As mentioned above with regard to FIG. 4, the plug cap is a component of a plug (labeled in FIGS. 6A-7). As also noted above, in some embodiments, the plug cap includes a flange 573 that can be welded and/or adhered to a surface of the assay chamber to stabilize the position of the plug within the opening of the monolithic substrate of the assay chamber. The plug protrudes into the monolithic substrate at a depth such that the component of the plug that is visible on the exterior of the assay chamber is the plug cap. In embodiments in which the plug cap includes a flange, the flange is also visible on the exterior of the assay chamber as shown in -36WO 2019/183608

FIGS. 4 and 5. In some embodiments, such as embodiments in which the assay chamber is used to contain an assay, the plug is transparent such that the assay within the assay chamber is optically detectable from outside of the assay chamber.

[00185] FIG. 6.A is a three-dimensional illustration of a plug 670, in accordance with an embodiment. The plug comprises a plug cap 672 and a plug body. In some embodiments, such as the embodiment shown in FIG. 6A, the plug cap also includes a flange 673. In further embodiments, such as the embodiment shown in FIG. 6A, the plug cap also includes an internal cavity 674, discussed in further detail below.

[00186] In some embodiments, such as the embodiments shown in FIGS. 4 and 5, the plug comprises a bounding surface of an assay chamber. Specifically, the plug can be placed within an opening of a monolithic substrate of the assay chamber such that the plug cap forms a bounding surface of the assay chamber and the plug body protrudes into the monolithic substrate, and also into the assay chamber, at a depth. In some embodiments, a volume of the assay chamber depends at least in part on the depth at which the plug body protrudes into the monolithic substrate to form a bounding surface of the assay chamber. Specifically, the greater the depth that the plug body protrudes into the assay chamber, the smaller the volume of the assay chamber.

[00187] As noted above, in some embodiments, the plug cap includes the flange shown in FIG. 6.A. The flange can be welded and/or adhered to a surface of the assay chamber to stabilize the position of the plug within the opening of the monolithic substrate of the assay chamber.

[00188] As also noted above, in certain implementations, the plug cap may include the internal cavity, as shown in FIG. 6A. The surface of the cap, including the optional internal cavity, is in fluidic communication with the assay chamber. In certain embodiments, particularly in embodiments which an assay chamber is used to contain an assay, the internal cavity of the plug body may contain one or more dried reagents (labeled in FIG. 7). In such embodiments, the assay chamber can be used to re־hydrate and/or solubilize the one or more dried reagents, as discussed in further detail below with regard to FIG. 7.

[00189] FIG. 6B is a cross-sectional illustration of the plug 670, in accordance with an embodiment.

[00190] FIG. 6C is an enlarged view of the cross section view in FIG. 6B. FIG. 6C is a cross section view of a plug 670. The plug 670 has a bottom surface 676 and a cavity 674 containing dried reagents 75. An annulus 680 is formed in the bottom surface 676 around a perimeter of the cavity 674. The cavity 674 begins with an angle of initiation 686 which is measured from the

WO 2019/183608 plug bottom surface towards the cavity surface. The angle of initiation may vary depending upon the desired about of dried reagents to be held in the cavity 674. The geometry of the cavity and initiation angle 686 may also be selected to minimize impact on the transmissive qualities of the plug 670. The shape of the cavity including the initiation angle may be selected to optimize the ability of a plug to be transmissive to excitation wavelengths and emission wavelengths within a spectrum used with an apparatus. In one exemplary aspect, the excitation and emission wavelengths are in at least one of a red spectrum, a blue spectrum and a green spectrum.

[00191] In various embodiments, an initiation angle 686 is selected to provide a cavity 674 of different depths. In one embodiment, there is a cavity having a depth that is less than is less than 90% of the plug thickness 685. In another embodiment, there is a cavity having a depth that is less than is less than 70% of the plug thickness 685. In still another embodiment, there is a cavity having a depth that is less than is less than 50% of the plug thickness 685. In still other embodiments, the initiation angle 686 is 60 degrees or less. In another embodiment, the initiation angle 686 is 30 degrees or less. In yet another embodiment, the initiation angle 686 is 20 degrees or less. In yet another embodiment, the initiation angle 686 is 10 degrees or less.

[00192] FIG. 7A is a three-dimensional, cross-sectional illustration of an assay chamber 721, in accordance with an embodiment. FIG. 7B is an enlarged view of the interior of the assay chamber and mixing ball of FIG. 7A. FIG. 7C and 7D are perspective and cross section views of the plug, mixing ball and film used when assembling an embodiment of the assay chamber of FIG. 7A.

[00193] As described above, the assay chamber is connected to, and in fluidic communication with, both an entry conduit 722 and a pneumatic conduit 732. Also as described above, the assay chamber is bounded by a monolithic substrate 702 and a plug 770. In particular, the plug is fixed within an opening in the monolithic substrate such that a plug body 771 protrudes into monolithic substrate at a depth, and a plug cap 772 forms a bounding surface of the assay chamber. A volume of the assay chamber depends, in part, on the depth at which the plug body protrudes into the monolithic substrate. In further embodiments, the assay chamber can also be bounded by a film.

[00194] As discussed above, the assay chamber may include features to facilitate an assay. For instance, in some embodiments, the plug is traitsparent such that an interior of the assay chamber is optically detectable from outside of the assay chamber. As noted above, in some embodiments, the plug cap includes a. flange 773. The flange can be welded and/or adhered to a surface of the assay chamber to stabilize the position of the plug within the opening of the

WO 2019/183608 monolithic substrate of the assay chamber. As also noted above, in certain implementations, the plug cap includes an internal cavity 774. In some embodiments, the internal cavity of the plug cap may contain dried reagents 775 for use in an assay. The dried reagents can be re-hydrated and/or solubilized by a fluid sample that enters the assay chamber via the entry conduit as discussed above.

[00195] To decrease the amount of time required to rehydrate and/or solubilize the dried reagents, the interior of the assay chamber may contain a magnetic mixing element 781 that is capable of gyration. Gyration of the magnetic mixing element can aid in mixing contents contained within the assay chamber, and therefore can aid in rehydrating and/or solubilizing the dried reagents. In some embodiments, the magnetic mixing element may be spherical in shape. In alternative embodiments, the magnetic mixing element may comprise any alternative shape.

[00196] To drive gyration of the magnetic mixing element within the assay chamber, an exterior magnet 782 that is capable of rotation may be located exterior to the assay chamber. To drive rotation of the exterior magnet such that the exterior magnet induces gyration of the magnetic mixing element, in some embodiments the exterior magnet may be mechanically coupled to a motor 783 capable of driving rotation of the exterior magnet. By rotating the exterior magnet, gyration of the magnetic mixing element within the assay chamber is induced.

[00197] In embodiments in which the exterior magnet is located above a center of the assay chamber, the exterior magnet may induce balanced spinning of the magnetic mixing element within the assay chamber. Balanced spinning is ineffective in mixing contents. Therefore, to avoid balanced spinning of the magnetic mixing element such that contents contained within the assay chamber are more effectively mixed, in some embodiments, such as the embodiments shown in FIGs. 7A-7D, the exterior magnet is located off-center of the assay chamber. By locating the exterior magnet &off-center of the assay chamber, the magnetic mixing element does not rotate in a perfectly balanced fashion within the center of the assay chamber, but rather moves around the assay chamber in a gyrating motion. This more effectively mixes contents contained within the assay chamber.

[00198] Figures 7A-7D provide multiple views of an assay chamber 721, in accordance with an embodiment. As illustrated in FIG. 7B, the assay chamber is connected to, and in fluidic communication with, both an entry condim 722 and a pneumatic conduit 732. In this implementation, the assay chamber is bounded by a plug 770, a film 712 and a monolithic substrate 702, in which a first curved boundary 744 and second curved boundary 745 are formed. The plug is fixed within an opening in the monolithic substrate such that a plug body 771

WO 2019/183608 protrudes into monolithic substrate at a depth. The bottom surface of the plug 776 having a cavity 774 forms one bounding surface of the assay chamber. The monolithic substrate defines a. second bounding surface and the film defines the final bounding surface of the assay chamber. A volume of the assay chamber depends, in part, on the depth at which the plug body protrudes into the monolithic substrate.

[00199] As discussed above, the assay chamber may include features to facilitate an assay. To decrease the amount of time required to rehydrate and/or solubilize the dried reagents, the interior of the assay chamber may contain a magnetic mixing element 76V that is capable of gyration. Gyration of the magnetic mixing element can aid in mixing contents contained within the assay chamber, and therefore can aid in rehydrating and/or solubilizing the dried reagents. In some embodiments, the magnetic mixing element may be spherical in shape. In alternative embodiments, the magnetic mixing element may comprise any alternative shape.

[00200] Figure 7A is a three-dimensional, cross-sectional illustration of a mixing system according to the invention. To drive gyration of the magnetic mixing element 781 within the assay chamber, an exterior magnet 782 that is capable of rotation may be located exterior to the assay chamber. To drive rotation of the exterior magnet such that the exterior magnet induces gyration of the magnetic mixing element, in some embodiments the exterior magnet may be mechanically coupled to a motor 7£3 capable of driving rotation of the exterior magnet. By rotating the exterior magnet, gyration of the magnetic mixing element within the assay chamber is induced.

[00201] In embodiments in which the exterior magnet is located above a center of the assay chamber, the exterior magnet may induce balanced spinning of the magnetic mixing element within the assay chamber. Balanced spinning is ineffective in mixing contents. Therefore, to avoid balanced spinning of the magnetic mixing element such that contents contained within the assay chamber are more effectively mixed, in some embodiments, such as the embodiments shown in FIG. 7A, the exterior magnet is located off-center of the assay chamber. By locating the exterior magnet off-center of the assay chamber, the magnetic mixing element does not rotate in a perfectly balanced fashion within the center of the assay chamber, but rather moves around the assay chamber in a gyrating motion. This more effectively mixes contents contained within the assay chamber. In an alternative embodiment, the exterior magnet could move in any noncircular route, e.g. side to side, in order to induce movement of the mixing element within the mixing chamber. In yet another implementation, movement of the mixing element can be

WO 2019/183608 induced by a nonmagnetic force, e.g. by vibration or other movement of the assembly containing a mixing chamber.

[00202] FIG. 7C provides a three-dimensional blown-out side-view illustration of a mixing chamber. FIG. 7D illustrates the same blow-out configuration in a cross-sectional view, wherein the crass section is made perpendicular to the direction of fluid flow, i.e. from the inlet to the outlet. The parts of the mixing chamber can be assembled in any order that one of ordinary skill might find useful and convenient for the specific implementation. In one method of assembly, the method coinprises a first step of providing a monolithic substrate having an inlet conduit 722, an outlet conduit, a first curved boundary, a second curved boundary and an opening for a plug 798 formed therein. In a second step a film 712 is attached to the surface of the monolithic substrate that is opposite of the opening for the plug. The film can be attached to the monolithic substrate using any method known in the art, e.g. with adhesive or welding, such as heat staking or laser welding. In a third step, a ball is placed in the mixing chamber bounded by the monolithic substrate 702 and the film 712. In a fourth step, a plus 770 is inserted into the opening in the monolithic substrate to form the final bounding surface of the mixing chamber. The plug can comprise one 01־ more dried reagents placed within a cavity 774 of the plug or otherwise on the bottom surface of the plug. Optionally, the method can further comprise the step of welding the plug to the monolithic substrate. Such welding seals the interface between the monolithic substrate and the plug to eliminate the potential for leaking if or when the mixing chamber is pressurized.

[00203] An alternate assembly method comprises providing a monolithic substrate as described herein, inserting a plug into the opening in the monolithic substrate 795, placing a mixing element in the mixing chamber formed by the substrate and plug, and attaching a film to the substrate. Optionally, the plug can be welded to monolithic substrate at any point after insertion.

[00204] Yet another assembly method comprises providing a plug 770, optionally having a dried reagent thereon, placing a mixing element in the cavity 774 of the plug, inserting the plug into an opening of a monolithic substrate having feature as described herein, and then attaching a film to the substrate on the face opposite to the face in which the plug was inserted.

EXAMPLES

[00205] Several separate experiments were performed to determine operational ranges of the magnetic mixing element described above with regard to FIGs. 7 A.

[00206] Example. 1: Unidirectional mixing

WO 2019/183608

[00207] A first experiment involved rehydrating dried reagents using unidirectional gyration in a device as illustrated in FIG. 7A for 60 seconds at l,030rpm and 2,060rpm. Additional dried reagents were rehydrated using a standard laboratory benchtop protocol for use as a positive control. These rehydrated reagents were used to amplify nucleic acid sequences using LAMP. The performance of the positive control was used as a baseline to which the results of the two device rehydration conditions were compared.

[00208] The results of the first experiment are depicted below in Table 1. Considering the standard deviation of the Time-to-Positive (Tp) and the percentage of reactions that resulted in amplification, the experiment results demonstrate that l,030rpm is insufficient to rehydrate dried reagents when utilizing unidirectional gyration for 60 seconds. Increasing the gyration speed to 2,060rpm decreases the standard deviation and results in 100% amplification, suggesting increased gyration speed is necessary for unidirectional mixing.

!Able 1

<td> Condition</td><td> Av erage 1 p (min)</td><td> Std Dev (min)</td><td> Reactions that .Amplified</td>

<td> Device - 1,030 rpm gyration (n=3)</td><td> 15.0</td><td> 4.04</td><td> 83%</td>

<td> Device - 2,060 rpm gyration (n=3)</td><td> 10.9</td><td> 2.11</td><td> 100%</td>

<td> Positive Controls (n=4)</td><td> 10.0</td><td> 0.29</td><td> 100%</td>

[00209] Example 2: Alternating gyration low speed

[00210] A second experiment - performed in two separate parts - involved alternating the gyration direction every 15 seconds for 60 seconds using a gyration speed of l,030rpm.

Additional dried reagents were rehydrated using a standard laboratory benchtop protocol for use as a positive control. These rehydrated reagents were used to amplify nucleic acid sequences using LAMP. The performance of the positive control was used as a baseline to which the results of the device rehydration condition were compared.

[00211] The results of the second experiment are depicted below in Table 2. In comparison to the results of l,030rpm unidirectional gyration in the Example 1, these results demonstrate that alternating the gyration direction every 15 seconds improves the rehydration performance of the device and permits a lower gyration speed to be employed.

WO 2019/183608

Table 2

<td> Condition</td><td> Average Tp (min)</td><td> Std Dev (min)</td><td> Reactions that Amplified</td>

<td> Device - 1,030 rpm gyration (n=3)</td><td> 9.7</td><td> 0.33</td><td> 100%</td>

<td> Positive Controls (n-6)</td><td> 9.4</td><td> 0.74</td><td> 100%</td>

[00212] Example 3: Alternating gyration medium speed

[00213] A third experiment involved alternating the gyration direction every 15 seconds for 60 seconds using a gyration speed of 2,060rpm. The remainder of the protocol was identical to the second experiment.

[00214] The results of the third experiment are depicted below in Table 3. In contrast to the results of 2,060rpm unidirectional gyration in Example 1, these results demonstrate that alternating the gyration direction every 15 seconds improves the device's rehydration performance even at 2,060rpm by reducing the standard deviation of the rehydrated reagents Time-to-Positive. These results further indicate that the increased shear caused by alternating the gyration direction does not noticeably damage the rehydrated enzyme.

Table 3

<td> Condition</td><td> Average Tp (min)</td><td> Std Dev (min)</td><td> Reactions that Amplified</td>

<td> 2,060 rpm, alternating gyration directions (n=3)</td><td> 9.49</td><td> 0.633</td><td> 100%</td>

<td> Positive Controls (n=4)</td><td> 8.88</td><td> 0.304</td><td> 100%</td>

[00215] The extent of rehydration of dried reagents rehydrated with the device using 2,060rpm alternating gyration for 60 seconds was compared to controls rehydrated using the standard laboratory benchtop protocol. Concentration of the resulting solutions was quantified against a standard curve using a spectrophotometer. The results of the fourth experiment are depicted below in Table 4. These results further demonstrate that the device is capable of matching the rehydration performance of the standard benchtop protocol.

WO 2019/183608

Table 4

<td> Condition</td><td> Percent of 1 x Concentration</td><td> Std Dev</td>

<td> Device (n=4)</td><td> 82.2%</td><td> 2.2%</td>

<td> Controls (11=2)</td><td> 91.0%;</td><td> 1.8%</td>

[00216] Example 4: Extended alternating gyration

[00217] A fourth experiment involved unidirectional gyration at 2,060rpm for 30 seconds and 45 seconds rather than the 60 second duration used by the three proceeding experiments. The remainder of the protocol was identical to that described in Example 2 and Example 3

[00218] The results of the fourth experiment are depicted below in Table 5. These results demonstrate that the device is capable of rehydrating dried reagents to an acceptable degree in fewer than 60 seconds. Incorporating the results of Example 2, the addition of alternating gyration direction has the potential to further strengthen this capability.

Table 5

<td> Sample</td><td> Average T_P</td><td> Std Dev T_P</td>

<td> 45 seconds</td><td> 10.27</td><td> 0.712</td>

<td> 30 seconds</td><td> 10.01</td><td> 0.390</td>

<td> Dry down control (n=4)</td><td> 9.23</td><td> 0.327</td>

[00219] Example 5: No loss of nucleic acid

[00220] To confirm that no nucleic acid is lost by virtue of mixing within a device as described in FIGS. 1-7A, a known concentration of nucleic acid target was loaded into the device without the presence of dried reagents for 60 seconds with 2,060 rpm alternating gyration and no gyration. The concentration of resulting nucleic acid solutions were compared to the original input solution via RT-qPCR.

[00221] The results of the first experiment are depicted below in Table 6, which demonstrate that neither the surface of device itself nor the act of gyrating the mixing bead results in detectable nucleic acid loss or damage.

WO 2019/183608

Table 6

<td></td><td> Average of All Middle Aliquot C_qs</td><td> Standard Deviation of All Middle Aliquot C_qs</td>

<td> 2,060 rpm: alternating</td><td> 25.31</td><td> 0.167</td>

<td> No mixing</td><td> 25.44</td><td> 0.223</td>

<td> Positive control</td><td> 24.97</td><td> 0.611</td>

[00222] Example 6: Heat staking

[00223] One experiment was performed to demonstrate the effectiveness of heat staking the apparatus, as described with regard to FIGS. 2E and 2F.

[00224] A test coupon was constructed, consisting of five wells, each connected to its own pneumatic compartment. Ilie volumes of the wells (including the channels leading into the wells from the common line and the pneumatic conduit leading to the air chamber from the assay chamber) were 5.28 mm’, 7.56 mm13.12 ,’־ mm”, 5.32 mm־’, and 9.96 mm”, respectively. The volumes of the air chambers were 9.24 mm13.22 ,’־ mm³, 22.96 mm³, 9.29 mm³ and 17.43 mm³, respectively. Water was filled into the sample coupon at a ramped pressure of 9.2 and 10 psi. The assay chambers filled evenly, and without significant bubbles caused by the filling process itself. At 9.2 psi, the wells were all substantially filled and at 10 psi, the wells were completely filled and the fluid extended into the pneumatic conduit connecting the wells to the air chambers. Therefore, a pressure in between the two (for instance 9.6 psi) was assumed to be ideal for complete filling.

[00225] After heat staking, the fluidic pathways hold pressure on both sides: air and water (to 10 psig). Furthermore, the fluidic pathways still hold pressure 10 days after heat staking (as observed by doming of pressurized wells and no liquid leakage.)

[00226] Example 7: Amplification and detection

[00227] One experiment was performed to demonstrate that after heat staking the fluidic pathways described with regard to FIGS. 1-7A, heating the fluidic pathways does not induce significant bubble formation within the sample chamber. A test coupon was constructed, consisting of five wells, as described in Example 6.

[00228] Isothermal Amplification Buffer (New England Biolabs) supplemented with MgC12, dNTPs, LAMP primers, F AM-molecular beacon probe, Bst 2 polymerase (New England Biolabs), and RTx Warmstart (reverse transcriptase; New England Biolabs), and 100,000 copies of CT 23S DNA (as template) was filled into the sample coupon with pressure ramping to 13 psi.

WO 2019/183608

The coupon was then heat staked and heated to 64°C. Very small bubbles formed in some assay chambers during the first few minutes at elevated temperature. Over the course of 30 minutes, these tiny bubbles were stable and did not interfere with amplification or image processing. Amplification within each of the five wells was visually detectable within 9-15 minutes of exposure to 64°C.

[00229] FIG. 8 is a cross section view of a plug having a flat bottom surface.

[00230] FIG. 9A is a section view of the plug in FIG. 8 having a dried reagent having a volume vl along the entire plug bottom surface. FIG. 9B is a bottom up view of the plug of FIG. 9A showing the dried reagent volume vl along the bottom surface.

[00231] FIG. 10A is a section view of the plug in FIG. 8 having a dried reagent having a volume v2 partially covering the plug bottom surface having a width similar to the plug central opening. FIG. 10B is a bottom up view׳ of the plug of FIG.10A showing the dried reagent volume v2׳ along the bottom surface.

[00232] FIG. 1 LA is a section view of the plug in FIG. 8 having a dried reagent having a volume v3 along the plug bottom surface having a width that is less than the width of the plug central opening. FIG. 1 IB is a bottom up view of the plug of FIG. 11A showing the dried reagent volume v3־ along the bottom surface within the width of the plug central opening.

[00233] FIGs. 12A and 12B illustrate a feature along the plug bottom surface for retaining a volume of a dried reagent. FIG. 12A illustrates a raised feature having a rectangular cross section. FIG. 12B illustrates a recessed feature having a rectangular cross section.

[00234] FIGs. 13A and 13B illustrate a feature along the plug bottom surface for retaining a volume of a dried reagent. FIG. 13A illustrates a raised feature having a circular cross section. FIG. 12B illustrates a recessed feature having a circular cross section.

[00235] FIG. 14A is a section view of the plug in FIG. 13A having a dried reagent having a volume vl along the entire plug bottom surface between the raised features. FIG. 1413 is a bottom up view of the plug of FIG. 14A showing the dried reagent volume vl along the bottom surface within the boundary of the raised feature.

[00236] FIG. 15A is a section view of the plug in FIG. 13A having a dried reagent having a volume v2 along a portion of the plug bottom surface between the raised features. FIG. 15B is a bottom up view of the plug of FIG. 15A showing the dried reagent volume v2 along the bottom surface within the boundary of the raised feature having about the same width as the plug open central portion.

WO 2019/183608

[00237] FIG. 16A is a section view of the plug in FIG. 13A having a dried reagent having a volume v3 along a portion of the plug bottom surface between the raised features. FIG. 16B is a bottom up view of the plug of FIG. 16A showing the dried reagent volume v3 along the bottom surface within the boundary of the raised feature having a width that is less than the width of the plug open central portion.

[00238] FIG. 17A is a section view of a plug having a cavity as in FIGs. 6A and 6B having a dried reagent having a volume v3. The cavity covers nearly all of the plug bottom surface. FIG. 17B is a bottom up view of the plug of FIG. 17A showing the dried reagent volume vl along the bottom surface within the cavity.

[00239] FIG. 18A is a section view of a plug having a cavity as in FIGs. 6A and 6B having a dried reagent having a volume v2. The cavity covers less than all of the plug bottom surface and is wider than the plug central opening. FIG. 18B is a bottom up view of the plug of FIG. 18A showing the dried reagent volume v2 along the bottom surface within the cavity.

[00240] FIG. 19A is a section view of a plug having a cavity as in FIGs. 6A and 6B having a dried reagent having a volume v3. The cavity covers less than all of the plug bottom surface and is not as wide as the plug central opening. FIG. 19B is a bottom up view of the plug of FIG. 19A showing the dried reagent volume v3 along the bottom surface within the cavity.

[00241] FIG. 20 is a cross section view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a recessed portion to accommodate the cavity in the plug.

[00242] FIG. 21 is a cross section view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a recessed portion to accommodate the cavity in the plug. The shoulder height shown in FIG. 21 is less than the shoulder height of FIG. 20.

[00243] FIG. 22 is a cross section view of an assay chamber taken through the midpoint of the chamber showing the shoulder supporting the plug and the double tapered sidewalls towards the outlet.

[00244] FIG. 23 is a cross section view of an opening in the optical side of an apparatus sized to receive a plug to be supported by the shoulder. The opening is covered by the first film layer and the second film layer.

WO 2019/183608

[00245] FIG. 24 is a cross section view of an opening in the optical side of an apparatus sized to receive a plug to be supported by the shoulder. The shoulder and tapering sidewall are visible in this view. A plug is shown inserted into the opening but not yet seated against the shoulder.

[00246] FIG. 25 is a perspective view of the optical side of an apparatus showing five plug openings. The plug support rings are shown around each of the openings. The shoulders used to support the plugs are visible within each of the openings.

[00247] FIG. 26A is a cross section view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a shoulder height for engaging the plug to provide a chamber depth, !here is a recessed portion in the shoulder to accommodate the cavity in the plug.

[00248] FIG. 26B is a view from the non-optical side of the plug and shoulder combination in FIG. 26A. The shoulders support the plug, maintain the tapered sidewall and provide a recess so that the cavity and dried reagents are exposed within the chamber. The shoulder and plug configuration illustrated in FIGs. 26A and 26B produces a 7.5 μ I well.

[00249] FIG. 27A is a cross section view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a shoulder height for engaging the plug to provide a chamber depth. There is a recessed portion in the shoulder to accommodate the cavity in the plug.

[00250] FIG. 27B is a view from the non-optical side of the plug and shoulder combination in FIG. 27.A. The shoulders support the plug, maintain the tapered sidewall and provide a recess so that the cavity and dried reagents are exposed within the chamber. The shoulder and plug configuration illustrated in FIGs. 27A and 27B produces a 3.9 pl well. The plugs and bore sizes of the plug and shoulder combinations in the illustrative embodiments of FIGs. 26A-27B have the same bore opening of 4.7 mm measured at the fluid end of the body. Advantageously, these examples of illustrate how different shoulder dimensions (such as shoulder height) may be used to provide different volumes.

[00251] FIG. 28A is a cross section view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a shoulder height for engaging the plug to provide a chamber depth. There is a recessed portion in the shoulder to accommodate the cavity in the plug.

[00252] FIG. 28B is a view from the non-optical side of the plug and shoulder combination in

FIG. 28A. The shoulders support the plug, maintain the tapered sidewall and provide a recess so

WO 2019/183608 that the cavity and dried reagents are exposed within the chamber. 'The shoulder and plug configuration illustrated in FIGs. 28 A and 28B produces a 18.5 pl well.

[00253] FIG. 29A is a. cross section view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a shoulder height for engaging the plug to provide a chamber depth. There is a recessed portion in the shoulder to accommodate the cavity in the plug.

[00254] FIG. 29B is a view from the non-optical side of the plug and shoulder combination in FIG. 29A. The shoulders support the plug, maintain the tapered sidewall and provide a recess so that the cavity and dried reagents are exposed within the chamber. The shoulder and plug configuration illustrated in FIGs. 29A and 29B produces a. 13.8 pl well.

[00255] FIG. 30A is a cross section perspective view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a shoulder height for engaging the plug to provide a chamber depth. There is a recessed portion in the shoulder to accommodate the cavity in the plug.

[00256] FIG. 30B is a view from the non-optical side of the plug and shoulder combination in FIG. 30A. The shoulders support the plug, maintain the tapered sidewall and provide a recess so that the cavity and dried reagents are exposed within the chamber. The shoulder and plug configuration illustrated in FIGs. 30A and 30B produces a 13.8 pl well.

[00257] FIG. 30C represents the shape and the volume of an assay chamber formed using the plug and shoulder configuration of FIGs. 30A and 30B.

[00258] As detailed above, Figs 28A, B and FIGs 31A-C provide an 18.5 pl well. Figs. 29A, 29B and FIGs. 30A-30C provide a 13.8 pl well. The plug size in each of these different plug and shoulder height configurations is 5.9 mm. Advantageously, multiple different chamber volumes may be achieved using a common plug size or configuration based on the adjustment of shoulder configuration and dimensions, such as for example, height, in order to provide an apparatus with an assortment of different chamber volumes.

[00259] FIG. 31A is a cross section perspective view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a shoulder height for engaging the plug to provide a chamber depth. There is a recessed portion in the shoulder to accommodate the cavity in the plug.

WO 2019/183608

[00260] FIG. 3 IB is a view from the non-optical side of the plug and shoulder combination in FIG. 31 A. The shoulders support the plug, maintain the tapered sidewall and provide a recess so that the cavity and dried reagents are exposed within the chamber.

[00261] FIG. 31C represents the shape and the volume of an assay chamber formed using the plug and shoulder configuration of FIGs. 31A and 3IB. The shoulder and plug configuration illustrated in FIGs. 31A and 31B produces a 18.5 pl well.

[00262] FIG. 32A is a cross section perspective view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a shoulder height for engaging the plug to provide a chamber depth. There is a recessed portion in the shoulder to accommodate the cavity in the plug.

[00263] FIG. 32B is a view from the non-optical side of the plug and shoulder combination in FIG. 32A. The shoulders support the plug, maintain the tapered sidewall and provide a recess so that the cavity and dried reagents are exposed within the chamber.

[00264] FIG. 32C represents the shape and the volume of an assay chamber formed using the plug and shoulder configuration of FIGs. 32A and 32B.

[00265] FIGs. 32D and 32E are perspective and cross section views of the plug and cavity used in the plug, shoulder and assay chamber shown in FIGs, 32A, 32B and 32C.

[00266] FIG. 33A is a cross section perspective view of an assay chamber taken through the inlet and the outlet which shows the plug bottom surface supported by a shoulder. The shoulder has a shoulder height for engaging the plug to provide a chamber depth. There is a recessed portion in the shoulder to accommodate the cavity in the plug.

[00267] FIG. 33B is a view from the non-optical side of the plug and shoulder combination in FIG. 33A. The shoulders support the plug, maintain the tapered sidcwaH and provide a recess so that the cavity and dried reagents are exposed within the chamber.

[00268] FIG. 33C represents the shape and the volume of an assay chamber formed using the plug and shoulder configuration of FIGs. 33A and 33B.

[00269] FIGs. 33D and 33E are perspective and cross section views of the plug and cavity used in the plug, shoulder and assay chamber shown in FIGs. 33A, 33B and 33C,

[00270] FIGs. 34A and 34B are non-optical side and optical side views, respectively, of an apparatus having a mixture of plugs with optical transmissive properties within an optical zone

WO 2019/183608 and at least one plug without optical properties in another zone of the apparatus where optical capabilities are not needed.

[00271] FIGs. 35A and 35B are non-optical side and optical side views, respectively, of an apparatus having only plugs with optical transmissive properties.

[00272] FIGs. 36A - 36K are an example sequence of loading a fluid sample into a sample chamber as performed before the exemplary filled chamber states shown in FIGs. 3A and 3B.

[00273] The following example depicted in FIGs 36A-36K is a sequence of loading a fluid sample into a sample chamber at 0.2 pL/min. FIGs 36A-36K demonstrate the process prior to arriving at the filled state shown in FIGs 3A and 3B. The sample chamber has a tapered inlet in fluidic communication with a fluid source and a tapered outlet in fluidic communication with a pneumatic compartment. The sample chamber also has a bounding surface formed by an optically transparent plug with dried reagents needed to perform a biological assay. The filling of the sample chamber is viewed on the film side as demonstrated in FIG. 8A and FIG. 9A.

[00274] FIG. 36A shows an assay chamber prior to loading a fluid sample. Each fluidic pathway in the device, including each assay chamber, is filled with air. The irregular circle seen is the edge of the dried reagents 3675 used to perform the biological assay. The area within the irregular circle contains reagents dried on the internal cavity of the plug 3674 whereas the area outside of the irregular circle is reagent free.

[00275] FIG. 36B shows the assay chamber shortly after pressure is applied to the fluid sample, causing it to flow from the common fluid source (not shown), through an entry conduit and then into the sample chamber via the tapered inlet 3641. As the fluid sample flows into the assay chamber, it displaces air present in the chamber, forcing the air towards the tapered outlet and into the pneumatic compartment (not shown). The meniscus 3661 of the fluid sample, which is representative of the liquid to air interface, contacts both sides of the chamber along the tapered inlet portion of the assay chamber. In the view illustrated in FIG. 36B, the meniscus of the fluid sample has advanced beyond the flat annulus of the plug’s bottom surface, contacting the internal cavity 3674 but does not yet reaching the edge of the dried reagents 3675 located in the internal cavity.

[00276] FIG. 36C shows the fluid sample continuing to fill the assay chamber as the meniscus 361 continues to advance across the internal cavity 3674, contacting the edge of the dried reagents. In this view, the center of meniscus 3661 mildly leads ahead of the edges in contact with the assay chamber. This could be attributed to a number of different factors. For example, the dried reagents in the internal cavity 3674 preferentially wet over the reagent free areas of the -51 WO 2019/183608 plug. Alternatively or additionally, the assay chamber increases in depth as the meniscus moves toward the midpoint 3643 of the assay chamber and increases in width, perpendicular to fluid flow, such that the widest and deepest part of the assay chamber is located at midpoint 3643 between the tapered inlet and tapered outlet. Dried reagents gradually separate from the interval cavity 3674 as the leading face of the fluid sample generates mixing of the sample fluid for rehydration and dissolution to begin. Beyond the meniscus 3661, and closest to the pneumatic compartment, t dried reagents on the air side of meniscus have yet to participate in the rehydration. The air in the assay chamber and pneumatic compartment are further compressed, building in pressure.

[00277] FIG. 36D shows the meniscus 3661 further advancing to fill the assay chamber. The movement of the meniscus 3661 is relatively uniform within the sample chamber such that all portions of the leading face advance in a substantially uniform manner as the meniscus approaches the deepest part of the internal cavity 3674 and the widest part of the sample chamber, the width of the chamber defined as perpendicular to overall fluid flow. Dried reagents continue to liftoff the surface of the internal cavity 3674 and mix with the fluid sample. Approximately half of the sample chamber is filled with fluid sample and half remains filled with air.

[00278] FIG. 36E illustrates how the meniscus 3661 can move substantially nonuniformly. The left edge of the meniscus travels at a faster rate compared to the right edge of the meniscus. This, in part, could be attributed to the preferential wetting of the dried reagents versus the reagent free plug surface. In this instance, the edge of the dried reagents is located slightly offcentered towards the left portion of the assay chamber. Having contacted less of the dried reagents 3675, the right edge of the meniscus 3661 lags compared to the left edge. Air bubbles and incomplete filling may arise from the nonuniform movement of the meniscus across the sample chamber. If one edge of the meniscus reaches the outlet prior to the other, the outlet can become filled with liquid, thus trapping air in the assay chamber. Partial filling and entrapment of air bubbles is reduced by the tapered inlet and outlet of the sample chamber, which retard speed at which the meniscus advances as it approaches the outlet. Lastly, rehydration and dissolution of the dried reagents continues to occur.

[00279] FIG. 36F illustrates the fluid sample flowing along the tapered outlet 3642 portion of the assay chamber. When the left edge of the meniscus 3661 slows as it enters the taper outlet, the right edge of the meniscus begins to catch up with the left portion. The meniscus has passed the deepest and widest dimensions of the sample chamber and now flows into the tapered outlet.

WO 2019/183608

As the meniscus flows through tapered outlet, the depth and width of the sample chamber steadily decrease. This configuration causes an increase in the surface retarding forces acting on the leading left portion of the meniscus and provides a more balanced distribution of forces. Dissolution of the dried reagents 3675 is readily apparent. . The dried reagents that first contacted the fluid sample upon loading have had the greatest amount of time suspended in the fluid sample and experience the greatest dissolution. This is shown by streaks directed towards the center of the sample chamber in the direction of fluid flow.

[00280] FIG. 36G shows the fluid sample continuing to flow through the tapered outlet 3642 portion of the assay chamber. The right edge of the meniscus is nearly parallel with the left edge as the entire meniscus 3661 progresses into the taper. The depth and width of the assay chamber continues to decrease as the meniscus approaches the tapered outlet leading to the pneumatic compartment. The majority of the dried reagents are in contact with the fluid sample and continue to dissolve.

[00281] FIG. 36H shows that as the fluid approaches the pneumatic compartment terminus, the movement of the meniscus 3661 is now uniform and the left and right potions advance substantially evenly. The meniscus of the fluid sample has now passed the entirety of the dried reagents such that all areas of the dried reagents are exposed to the fluid sample and are capable of rehydration and dissolution.

[00282] FIG 361 shows the assay chamber as soon as it has reached its filled state. All air that once occupied the fluidic pathway and assay chamber has been pushed to the pneumatic compartment. Partial filling and bubble entrapment have been avoided and rehydration and dissolution continue to occur.

[00283] FIG. 36J shows the sample chamber 20 seconds post filling. The dried reagents continue to dissolve as the amount of time the dried reagents are in contact with the fluid sample increases. Faint streaks remain visible as residual particles of the dried reagent circulate in the assay chamber and continue to dissolve.

[00284] FIG. 36K shows the sample chamber after a total of 40 seconds post filling. Minor amounts of residual dried reagent are faintly noticeable, while the majority of the reagents are now suspended in the fluid sample.

[00285] After filling is completed, one may wish to generate further mixing between the reagents and fluid sample. In one implementation, convective movement of the fluid sample is induced by heat cycling of the filled sample chambers to mix the dried reagents and produce a homogenous solution. A heater in contact with the sample chambers is used to provide the heat -53 WO 2019/183608 cycling. Preferably, the heater is placed on the non-imaging face of the apparatus, which can be sealed with a film. In order to induce convective movement in the chamber, the heater is set to a first temperature for a first interval and then set to a second temperature for a second interval. This cycle of first temperature followed by second temperature can be repeated at least twice, more preferably at least three times. In some implementations, the cycle is repeated five times, or any number of times necessary produce the desired amount of mixing. The first temperature and the second temperature are not the same. Preferably, the first temperature is at least 5°C greater than the second temperature, and more preferably at least 7 °C great and even more preferably at least 10°C greater. In one implementation, the sample chambers are heat cycled between a low temperature of 55°C and high temperature of 65°C. In a preferred implementation, the sample chambers are heat cycled between a low temperature of 60°C and the high temperature of 70°C. According to another implementation, further mixing of the dried reagents and fluid sample is generated by subtly shuttling the sample fluid back and forth across the internal cavity of the plug to create turbulence. This movement of the fluid can be accomplished by using any positive motive force, negative motive force, or a combination of the two.

[00286] In one implementation constant pressure is applied to the fluid sample to flow it through the sample chamber. In an alternative implementation, the sample chamber is filled using a pressure ramp. For example, from 0 to f second the pressure source is set to 0 kPa. At 1 second, the pressure source is set to 60 kPa. After f second, the pressure source is increased 0.44 kPa for every 0.2 second time step until a final pressure of f f 5 kPa is reached after a total of 26 seconds. While this implementation is described in the context of pressure, the filling of the sample chamber can be accomplished using any motive force. In one implementation, a positive motive force is used. In another implementation, a negative motive force is used.

[00287] In one exemplary use of an apparatus embodiment, there is provide a method of simultaneously filling a plurality of sample chambers. The exemplary method includes a step of pressurizing a fluid sample within a common fluid pathway. Next, there is a step of introducing the fluid sample into a plurality of entry conduits from the common fluid pathway. There is next a step of flowing the fluid sample along each of the entry conduits towards an entry conduit terminus in each of the entry conduits, each entry conduit connected to a sample chamber. Thereafter, there is a step of flowing the fluid sample along a tapered inlet portion of each sample chamber and then a step of flowing the fluid sample adjacent a pair of shoulders and along a plug within each sample chamber. The method continues by flowing the fluid sample along a tapered outlet portion of each sample chamber towards a pneumatic compartment

WO 2019/183608 terminus. During the steps above, the flow of the fluid is displacing a gas contained within each entry conduit and each sample chamber into a pneumatic chamber in communication with each pneumatic compartment terminus.

[00288] In one aspect, the method of filling is performed where the pressuring the fluid sample step is performed at a constant pressure. The constant pressure may be one of 5, 10, 20, 40 or 60 psi depending on the embodiment and specific apparatus configuration Optionally, the method steps of pressurizing the fluid step may include pressuring the fluid sample at a series of increasing pressure levels. In one implementation, each increasing level of pressure is applied for a consistent duration. In one implementation, the increasing level of pressure is increased by a constant amount. In one specific implementation, the apparatus is oriented lit use so that the pneumatic chamber is above the sample chamber such that the steps of flowing the fluid sample along a tapered outlet portion of the sample chamber towards a pneumatic compartment terminus and displacing a gas contained within each entry conduit are performed against a gravitational force. Additionally, or optionally, in use during the filling method, the plurality of sample chambers are oriented such that each pneumatic chamber associated with a specific sample chamber of the plurality of sample chamber is positioned above the sample chamber.

[00289] FIG. 37 is an optical side view of an apparatus having five optically transmissive plugs. The results viewed through each of the plugs indicates that three chambers have a detectable signal and two chambers do not. The signal detectable through the plugs can be a fluorescent signal, responsive to excitation light provided to the assay chamber through the optically transmissive plug. Alternatively, the signal can be luminescence resulting from a chemical and/or enzymatic reaction taking place in an assay chamber. In yet another implementation, the signal detectable through the optically transmissive plug may simply be a color change resulting from a colorimetric assay. In order to better visualize color changes, in some implementations a light, e.g. a white light, can be used to illuminate the wells through the plugs.

[00290] As illustrated by this example, the body of the optically transmissive plugs is optionally formed from a material transmissive to excitation wavelengths and emission wavelengths in at least one of a red spectrum, a blue spectrum and a green spectrum.

[00291] When a feature or element is herein referred to as being “on” another feature or element, it can be directly on the other feature or element or intervening features and/or elements may also be present. In contrast, when a feature or element is referred to as being “directly on” another feature or element, there are no intervening features or elements present. It will also be

WO 2019/183608 understood that, when a feature or element is referred to as being “connected”, “attached” or “coupled” to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being “directly connected”, “directly attached” or “directly coupled” to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.

[00292] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “/”.

[00293] Spatially relative terms, such as “under”, “below”, “lower”, “over”, “upper” and the like, may be used herein for ease of description to describe one element or feature’s relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features. Thus, the exemplary term “under” can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms “upwardly”, “downwardly”, “vertical”, “horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[00294] Although the terms “first” and “second” may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below

WO 2019/183608 could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.

[00295] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising” means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term “comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[00296] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “about” or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/- 0.1% of the stated value (or range of values), +/- 1% of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), +/- 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “X” is disclosed the “less than or equal to X” as well as “greater than or equal to X” (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

WO 2019/183608

[00297] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[00298] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

WO 2019/183608

Reference Number List

<td> Item</td><td> Last 2 Digits</td>

<td> apparatus</td><td> 00</td>

<td> common fluid source</td><td> 01</td>

<td> monolithic substrate</td><td> 02</td>

<td> heat stake</td><td> 03</td>

<td> raised platform</td><td> 05</td>

<td> independent fluidic pathway</td><td> 10</td>

<td> first film</td><td> 12</td>

<td> second film</td><td> 14</td>

<td> sample chamber</td><td> 20</td>

<td> assay chamber</td><td> 21</td>

<td> entry conduit</td><td> 22</td>

<td> entry conduit terminus</td><td> 23</td>

<td> pneumatic compartment</td><td> 30</td>

<td> air chamber</td><td> 31</td>

<td> pneumatic conduit</td><td> 32</td>

<td> pneumatic compartment terminus</td><td> 33</td>

<td> double tapered chamber</td><td> 40</td>

<td> tapered inlet</td><td> 41</td>

<td> tapered outlet</td><td> 42</td>

<td> double tapered chamber center point</td><td> 43</td>

<td> first curved boundary</td><td> 44</td>

<td> second curved boundary</td><td> 45</td>

<td> first curved boundary midpoint</td><td> 46</td>

<td> second curved boundary midpoint</td><td> 47</td>

<td> largest dimension</td><td> 48</td>

<td> air</td><td> 50</td>

<td> recess</td><td> 52</td>

<td> fluid sample</td><td> 60</td>

<td> meniscus</td><td> 61</td>

<td> leading front</td><td> 62</td>

<td> Plug</td><td> 70</td>

<td> plug body</td><td> 71</td>

<td> plug cap</td><td> 72</td>

<td> plug cap flange</td><td> 73</td>

<td> plug cap internal cavity</td><td> 74</td>

<td> dried reagents</td><td> 75</td>

<td> plug bottom surface</td><td> 76</td>

<td> central opening</td><td> 77</td>

<td> central opening side wall</td><td> 78</td>

<td> central opening bottom</td><td> 79</td>

<td> flat annulus</td><td> 80</td>

<td> magnetic mixing element</td><td> 81</td>

<td> exterior magnet</td><td> 82</td>

<td> motor</td><td> 83</td>

<td> heated element</td><td> 84</td>

WO 2019/183608

<td> plug thickness</td><td> 85</td>

<td> initiation angle</td><td> 86</td>

<td> raised feature</td><td> 87</td>

<td> recessed feature</td><td> 88</td>

<td> shoulder height</td><td> 92</td>

<td> shoulder</td><td> 95</td>

<td> assay chamber volume</td><td> 96</td>

<td> supporting ring/raised annulus</td><td> 97</td>

<td> opening in monolithic substrate for plug</td><td> 98</td>