Methods for operating microfluidic devices

Abstract

Methods of preparing a microfluidic device for operation are presented. In some embodiments, the interface network is pressurized with a liquid to purge at least a portion of the gas present in the interface network while another portion of the gas remains trapped in at least one region of the interface network and is at least partially perfused to the sample chamber. The sample chamber can be pressurized with a liquid to purge at least a portion of the gas present in the sample chamber while another portion of the gas remains trapped in at least one region of the sample chamber and is at least partially perfused to the assay chamber. The assay chamber can be pressurized with a liquid to purge at least a portion of the gas present in the assay chamber into the containment network, and the containment network can be pressurized to at least partially purge the containment network.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 416,175, filed October 14, 2022, and U.S. Provisional Patent Application No. 63 / 417,894, filed October 20, 2022, the contents of both of which are incorporated herein in their entireties.

[0002] Technical Field

[0002] The present disclosure relates generally to the operation of microfluidic devices, and more particularly to methods of preparing microfluidic devices for use. [Background technology]

[0003] background

[0003] Microfluidic devices are essential for performing many experiments in biological applications, and therefore it is essential to devise efficient and rapid ways to perform experiments using microfluidic devices. Summary of the Invention [Means for solving the problem]

[0004] overview Microfluidic devices allow thousands of reactions to be performed on a handheld device, or chip. However, preparing such devices for the introduction of samples and reagents can be difficult because surface tension and other properties of fluids (liquids and gases) that are of little importance on larger scales become dominant on the microfluidic scale.

[0005]

[0005] Removing air from a microfluidic device is particularly important in preparing the device for use.

[0006] Surprisingly, the method of the present teachings has been found to not only reduce the time it takes to prepare a microfluidic device by more than 50%, but also dramatically (by two orders of magnitude) reduce the number of measurements affected by air bubbles. For details, see the case below where errors (false positives and false negatives combined) were reduced from over 740 using conventional methods to just 4 using one embodiment of the present teachings.

[0007]

[0007] Microfluidic devices typically have four general features: a network of channels for delivering sample to the reaction area, a network of channels for delivering reagents (e.g., assays) to the reaction area, a means for preventing the sample and reagents from mixing / interacting until desired, and a means for isolating the two reaction areas from each other.

[0008]

[0008] In the present application, the means for preventing the sample and reagents from mixing / interacting until desired comprises a network of microfluidic channels to which pressure can be applied to actuate valves, and the means for isolating the two reaction regions from each other comprises a network of microfluidic flow paths to which pressure can be applied to actuate valves.

[0009]

[0009] In one aspect, a method is disclosed for preparing a microfluidic device for operation, formed from a gas-perfusable material having multiple separate fluid networks at a distance that allows them to communicate with each other (i.e., a distance that allows gas to perfuse between two adjacent networks), wherein the networks comprise elements that can trap air during filling, the method comprising the following steps: (a) pressurizing a first network to a pressure greater than the pressure associated with the second network by introducing a liquid into the first network to purge a portion of a first gas from the first network with another portion of the first gas remaining trapped in at least one region of the first network and at least partially perfused into the second network; and (b) purging a portion of a second gas from the second network with another portion of the second gas remaining trapped in at least one region of the second network. (c) introducing a second liquid into a second network in fluid communication with the chamber in which the measurement is performed to pressurize the second gas in the second network to a pressure greater than the pressure associated with the third network, to purge the second gas while the third gas remains trapped in at least one region of the third network and is at least partially perfused into the third network; (d) introducing a fourth liquid into the fourth network to purge the third gas from the fourth network.

[0010]

[0010] The first, second, and third gases may be the same gas, e.g., air, or any two of the gases may be different. Furthermore, the same or different liquids may be employed to pressurize the network.

[0011]

[0011] The networks may be connectable through closable interfaces / valves. In some embodiments, such interfaces / valves may be formed from an elastomeric material.

[0012]

[0012] In some embodiments, the maximum thickness of perfusable material between at least one of the networks and an adjacent network is less than the minimum thickness of perfusable material extending from at least one of the networks to the edge of the body of the microfluidic device.

[0013] Any of the first, second, and third liquids may be aqueous or organic liquids. By way of example and not limitation, the liquids may be water, alcohol, and / or oil, among others.

[0014]

[0014] Furthermore, any of the gases to be pressurized may be any of air, nitrogen, oxygen, carbon dioxide, argon, among others.

[0015] In some embodiments, a microfluidic device may include chambers, such as sample chambers and assay chambers, for receiving samples and liquid assays (e.g., reagents needed to perform a particular assay). In some such embodiments, separate networks may control fluid communication between the chambers.

[0016]

[0016] In some embodiments, the volume of trapped gas in the purged network may be greater than the volume of trapped gas, if any, in the subsequently purged network.

[0017] In some embodiments, at least one of the networks may be at least partially filled with liquid before receiving trapped gas by perfusion from an adjacent network that has been purged by pressure. By way of example, trapped gas received by an at least partially filled network may flow as bubbles through the liquid of that network. In some embodiments, gas received by the at least partially filled network may push liquid (or at least a portion thereof) out of the network.

[0018]

[0018] In some embodiments, the pressure associated with any of the above pressurized networks can be equal to, greater than, or less than atmospheric pressure.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings are not necessarily to scale or exhaustive. Rather, emphasis has generally been placed on illustrating the principles of the embodiments described herein. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments consistent with the present disclosure. Together with the description, the drawings serve to explain the principles of the present disclosure.

[0020]

[0020] The drawings are as follows: [Brief explanation of the drawings]

[0021] [Figure 1A] 1 illustrates a schematic representation of a microfluidic device implemented in some embodiments of the present teachings. [Figure 1B] 1 illustrates a schematic representation of a microfluidic device implemented in some embodiments of the present teachings. [Figure 2A] 1 illustrates a schematic representation of a microfluidic device implemented in some embodiments of the present teachings. [Figure 2B] 1 illustrates a schematic representation of a microfluidic device implemented in some embodiments of the present teachings. [Figure 3]

[0022] 1 illustrates an example of various steps in the operation of a microfluidic device and the time savings that can be achieved when utilizing embodiments of the present teachings. [Figure 4]

[0023] 1A-1C illustrate schematically the trapping of air in a chamber of a microfluidic device and the purging of air from the chamber. [Figure 5A]

[0024] 1 shows an example of a conventional method for purging various components of a microfluidic device. [Figure 5B]

[0024] An example of a conventional method for purging various components of a microfluidic device is shown. [Figure 5C]

[0024] An example of a conventional method for purging various components of a microfluidic device is shown. [Figure 6A]

[0025] 1 illustrates an example method for preparing a microfluidic device according to an embodiment of the present teachings. [Figure 6B]

[0025] An example method for preparing a microfluidic device according to an embodiment of the present teachings is provided. [Figure 6C]

[0025] An example method for preparing a microfluidic device according to an embodiment of the present teachings is provided. [Figure 7A]

[0026] 1 shows pressure and temperature profiles of a microfluidic device in accordance with an embodiment of the present teachings. [Figure 7B]

[0026] Figure 1 shows pressure and temperature profiles of a microfluidic device according to an embodiment of the present teachings. [Figure 7C]

[0026] Figure 1 shows pressure and temperature profiles of a microfluidic device according to an embodiment of the present teachings. [Figure 8]

[0027] 10 shows an example of the improvement in reducing errors caused by air bubbles that can result when using a microfluidic device prepared in accordance with an embodiment of the present technology. [Figure 9]

[0028] FIG. 1 shows a reference workflow for gene expression (GE) and genotyping (GT) runs performed on a Standard BioTools 96.96 IFC microfluidic device, with example experiments applying a conventional (reference) loading method and an example experiment applying a loading method according to an embodiment of the present technology (high speed). [Figure 10]

[0029] An example of multiple GT and GE runs over an 8 hour operating shift using a loading method according to an embodiment of the present technology (high speed) is shown. [Figure 11]

[0030] 1 shows average instrument run times across various microfluidic devices and application types from a validation study using the Standard BioTools X9 Genomics System. [Figure 12]

[0031] 1 shows an experimental comparison of ΔΔCt values ​​from the same Standard BioTools 96.96 IFC microfluidic device run using human tissue cDNA samples and panel A of the Advanta IO gene expression panel using a conventional (reference) loading method and a loading method according to an embodiment of the present technology (fast). [Figure 13]

[0032] 1 shows experimental results of a gene expression uniformity study performed on a Standard BioTools 96.96 IFC microfluidic device using a conventional (reference) loading method and a loading method according to an embodiment of the present technology (fast). [Figure 14A]

[0033] 1 shows TaqMan genotyping performance of the Standard BioTools 96.96 IFC microfluidic device in experiments using conventional (reference) loading methods. [Figure 14B]

[0034] 1 shows TaqMan genotyping performance of a Standard BioTools 96.96 IFC microfluidic device in an experiment using a loading method according to an embodiment of the present technology (high speed). [Figure 15A]

[0035] 1 includes a schematic representation of a study evaluating the effect of various levels of bubble formation in a Standard BioTools 96.96 IFC microfluidic device using a loading method according to an embodiment of the present technology (fast). [Figure 15B]

[0035] Photographs of a study evaluating the effect of different levels of bubble formation in a Standard BioTools 96.96 IFC microfluidic device using a loading method according to an embodiment of the present technology (fast). DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description

[0036] 1A, 1B, 2A, and 2B schematically illustrate a microfluidic device 100 comprising two sets of wells 120 and 140 for samples and assays, respectively. Sample analysis may be performed by delivering sample and assay liquids from the sample and assay wells through a network of microfluidic channels to a sample chamber 240 and an assay chamber 230, respectively, formed in a fluid manifold 160, allowing the two liquids to mix by diffusion, and performing sample analysis. It should be understood that FIGS. 1A, 1B, 2A, and 2B are not necessarily drawn to scale, but rather emphasis is placed on illustrating the present teachings.

[0023]

[0037] 2A and 2B show schematic views of a portion of a fluid manifold of a microfluidic device; while FIG. 2A shows a single unit 200 in three dimensions, it should be understood that this unit may be repeated, e.g., shown are 96 x 96 repetitions of 9,216 single units in such a manifold; in this illustration, the microfluidic device has 96 sample inputs 120 and 96 reagent inputs 140. FIG. 2B is a schematic cross-sectional view of various microfluidic structures formed in a perfusable material 250, with the numbers in square brackets indicating the relative distances through the perfusable material between the various structures. It should be understood that the structures shown in FIGS. 2A and 2B are only a small portion of the structures in the fluid manifold; for example, channels 210 and 220 are only a portion of the network of which they are a part.

[0024]

[0038] In this particular example, the sample chambers 240 in the fluid manifold are taller (i.e., have a greater height) than the assay chambers 230. As used herein, the term "tall" refers to the dimension of the chamber perpendicular to the underside of the device. Here, "tall" refers to the dimension perpendicular to the surface of the device. As shown in Figures 2A and 2B, the sample chambers 240 and assay chambers 230 exist in pairs, with Figure 2A showing a single pair 230, 240 and Figure 2B showing two adjacent pairs 230a, 240a and 230b, 240b.

[0025]

[0039] Fluid manifold 160 (also referred to herein as a "chip manifold") also provides fluid connections and control between the sample and assay chambers.

[0026]

[0040] More particularly, the fluid manifold comprises a network of channels for providing fluid communication between the liquid in the sample chamber and the liquid in the assay chamber.

[0027]

[0041] Additionally, the fluid manifold includes an interface network including a plurality of microfluidic channels (control channels) 210 that can control fluid communication between the sample chambers 240 and the assay chambers 230; that is, the interface network is used to prevent the sample and reagents from mixing / interacting until desired. In particular, the interface network can be used in a closed state to isolate the assay chamber from the sample chamber. In an open state, the interface network can allow communication between the assay chamber and the sample chamber. In other words, when the control channel of the interface network is closed, it can act as a valve that prevents the passage of fluid between the sample chamber and the assay chamber until a reaction is intended to be initiated, at which point the interface valve is changed from the closed state to the open state to allow fluid communication between the sample chamber and its mating assay chamber, for example, by diffusion of sample and assay liquids.

[0028]

[0042] In addition, the fluid manifold may comprise a containment network including a plurality of control channels 220 that, when in a closed state, may be used to isolate each sample chamber and assay chamber pair from fluid communication with other such pairs, e.g., to isolate two reaction regions from each other, where a reaction region is a combined sample chamber and assay chamber pair when the associated interface network control channel is in an open state.

[0029]

[0043] More specifically, the control channel 220 of the containment network can act as a valve that, when closed, inhibits the passage of liquid from a first pair of sample well-assay wells (e.g., 230a, 240a) to a second (and optionally adjacent) pair of sample well-assay wells (e.g., 230b, 240b), thereby isolating the reaction in each pair from the other pairs.

[0030]

[0044] In this example, the valve is made from a thin, flexible layer of polydimethylsiloxane (PDMS) to allow the valve to be actuated into an open or closed state by back pressure, and the perfusable material 250 in this example also comprises PDMS.

[0031]

[0045] Further details regarding the above-mentioned exemplary microfluidic devices can be obtained by reference to U.S. Pat. Nos. 8,220,487, 8,163,492, and 9,643,178, each of which is incorporated herein by reference in its entirety.

[0032]

[0046] FIG. 3 is a diagram of various steps in a typical operation of a microfluidic device 100, illustrating an example of the time savings that can be achieved when using various embodiments of the present teachings to prepare a microfluidic device for operation versus traditional device preparation methods. In FIG. 3, a human icon represents a step in the workflow where manual intervention occurs. The numbers in the bar graphs represent time in minutes and do not include time spent on manual intervention. A traditional workflow 310 is shown, which uses substantially separate and sequential steps of priming, loading, and mixing (e.g., all priming steps are completed before all loading steps) in preparation for sample and assay interaction (here, PCR (polymerase chain reaction)) for subsequent analysis. This traditional workflow is compared to one embodiment of the present invention 320, in which the priming and loading steps are interwoven (e.g., loading steps may be performed before all priming steps are completed, or vice versa). A significant time savings in preparing a microfluidic device for an experiment is demonstrated, with the priming, loading, and mixing time reduced from 154 minutes to 58 minutes.

[0033]

[0047] In some embodiments, in addition to changing the order in which the microfluidic device is purged, increasing the applied pressure relative to conventional methods can also be beneficial in shortening the time required to prepare the microfluidic device for use. For example, in some conventional methods, the assay chamber is pressurized and the sample chamber is depressurized before closing the containment valve. In contrast, in various embodiments of the present invention, both the assay chamber and the sample chamber are pressurized, e.g., to substantially the same pressure, before closing the containment valve.

[0034]

[0048] Note that the time savings shown in Figure 3 are achieved even though the manual intervention between priming and loading, and between loading and mixing in conventional approaches, is eliminated.

[0035]

[0049] In this example, the priming step involves purging the control channels of the microfluidic device (e.g., elements 210, 220 in FIGS. 2A, 2B) by pressurizing them with liquid. By way of example, the microfluidic device may include an interface network (e.g., element 210 in FIGS. 2A, 2B) including control channels for controlling fluid communication between sample chambers and assay chambers, and a containment network (e.g., element 220 in FIGS. 2A, 2B) for controlling fluid communication between adjacent pairs of sample and assay chambers that are in fluid communication during the mixing phase.

[0036]

[0050] The loading step refers to delivering sample liquid and assay liquid to the sample chamber and assay chamber pair in preparation for a mixing step in which the sample liquid and assay liquid in the sample chamber and assay chamber pair are mixed to perform sample analysis such as PCR (e.g., reacting the sample with assay reagents).

[0037]

[0051] When preparing a microfluidic device to introduce samples and reagents for subsequent mixing and analysis, it is important to ensure that air is purged from the microfluidic device before starting sample analysis, as air bubbles trapped in the sample and / or assay chambers can adversely affect the accuracy of the analysis (e.g., resulting in false positives or false negatives; data from such reaction areas cannot be used; the reaction may need to be repeated with new samples and reagents, thereby wasting sample and reagents).

[0038]

[0052] The present invention in various aspects and embodiments can reduce the time required to prepare a microfluidic device (e.g., as shown in FIG. 3 and further described below) relative to conventional methods, as well as reduce the generation of air bubbles.

[0039]

[0053] In this example, removing air present in a tall chamber, i.e., the sample chamber in this example, is more difficult than removing air present in a short chamber, i.e., the assay chamber in this example. Such air entrapment in a chamber is shown schematically in Figure 4 for structures formed in perfusable material 450.

[0040]

[0054] However, it should be understood that the methods of the present invention are not limited to tall and short geometrical differences, but can be applied to geometries that are significantly different in volume, e.g., large versus small, since at the microfluidic scale, surface tension forces can dominate over buoyancy-driven gas movement.

[0041]

[0055] As can be seen in Figure 4, air trapped in a short chamber can be much more easily purged through liquid backpressure or by passing the air through the illustrated exhaust port. However, for tall chambers, such as the sample chamber in this example, air can become trapped and be forced into the perfusable material 450 under pressure. For example, this may be due to the fact that the only channel available to remove air trapped at the top of a tall chamber is the bulk body 450 of the microfluidic device (e.g., PDMS), rather than through a thin layer connected to an exhaust port that is available earlier in the purge sequence, i.e., before all channels and chambers are filled with liquid.

[0042]

[0056] Additionally, purging significant amounts of air through the body of a microfluidic device can result in air dissolving in the body (e.g., in the PDMS), which can lead to the formation of air bubbles in the chambers as the trapped air returns to the chambers, a problem exacerbated the higher the pressures used.

[0043]

[0057] Thus, in conventional methods, chambers where the sample-reagent reaction is measured (e.g., via fluorescence) are purged of air last, since these areas are most susceptible to air bubbles that may adversely affect the analysis (i.e., are most likely to produce errors or erroneous results). In the exemplary microfluidic device used for illustration purposes here, the area where the measurement is performed is the sample chamber (e.g., element 240 in Figures 2A and 2B).

[0044]

[0058] The sample chamber is the chamber in which the measurement takes place and is the larger chamber, although it should be understood that in various embodiments the method is generally applicable to configurations in which the measurement chamber is not the sample chamber.

[0045]

[0059] According to some embodiments of the present teachings, the order of conventional operations of a microfluidic device is altered in a manner that counterintuitively allows for more efficient purging of the device, for example, by purging the larger chamber where measurements are taken (the sample chamber in these examples) last, but much earlier in the device preparation process.

[0046]

[0060] Surprisingly, the method of the present teachings was found to not only reduce the time it takes to prepare a microfluidic device by over 50%, but also dramatically (by two orders of magnitude) reduce the number of measurements affected by air bubbles - see in detail the case below where errors (false positives and false negatives combined) were reduced from over 740 using conventional methods to just 4 using one embodiment of the present teachings.

[0047]

[0061] 5A, 5B, and 5C show an example of a conventional method for purging various components of a microfluidic device, in which the control channels of the interface network (A) and the control channels of the containment network (B) are first purged, e.g., via pressurization of those channels with a liquid. The assay chamber (C) and the sample chamber (D) are then purged by introducing a liquid assay (i.e., reagents necessary to perform the assay) and sample into the assay chamber and the sample chamber, respectively.

[0048]

[0062] Each trace in Figure 5B is a line pattern encoding the structure shown in Figure 5A, where the long-dashed trace is the pressure as a function of time in the interface network (A), the short-dashed trace is the pressure as a function of time in the containment network (B), the solid trace is the pressure as a function of time in the assay chamber (C), and the dotted trace is the pressure as a function of time in the sample chamber (D), and Figure 5C shows the sequence schematically with arrows indicating the purging of air from the structure.

[0049]

[0063] In such conventional methods of purging microfluidic devices, purged air dissolved in the bulk PDMS can return to the sample or assay chamber, and it was therefore thought necessary to purge the sample chamber (D) last to prevent air from predominating through the perfusable material (bulk PDMS) and back into the sample chamber.

[0050]

[0064] In contrast, in various embodiments of the present teachings, rather than sequentially or simultaneously filling all control networks (A and B) before purging the sample and assay chambers (C and D), the containment network (B) is not purged until the assay chamber (C) and sample chamber (D) have been purged.

[0051]

[0065] 6A, 6B, and 6C illustrate an example of a method for preparing a microfluidic device according to various embodiments of the present invention. In various embodiments, the sequence of operations includes first purging the interface network (A), then purging the sample chamber (D), followed by purging the assay chamber (C), and finally purging the containment network (B), where purging is provided, for example, via pressurization of the respective channels with a liquid.

[0052]

[0066] Each trace in Figure 6B is a line pattern encoding the structure shown in Figure 6A, where the long-dashed trace is the pressure as a function of time in the interface network (A), the short-dashed trace is the pressure as a function of time in the containment network (B), the solid trace is the pressure as a function of time in the assay chamber (C), and the dotted trace is the pressure as a function of time in the sample chamber (D), and Figure 6C shows the sequence schematically with arrows indicating the purging of air from the structure.

[0053]

[0067] In this example, to purge some of the air (or other gas) present in interface network (A), the control channel of interface network (A) is first pressurized via the introduction of liquid to a pressure greater than the pressure associated with sample chamber (D) and assay chamber (C) and containment network (B). Due to the pressure differential, air trapped in the interface network (or at least a portion thereof) is received by assay chamber (C) and sample chamber (D) and the containment network via perfusion through the thin layer of PDMS.

[0054]

[0068] The sample chamber (D) is then filled with the liquid sample, thereby pressurizing the chamber and thus purging the chamber of any air present therein. The pressure differential between the sample chamber (D) and the assay chamber (C) facilitates perfusion of the air trapped in the sample chamber (D) into the assay chamber (C) and the containment network (B).

[0055]

[0069] The assay chamber (C) is then filled with a liquid (e.g., an assay reagent), which pressurizes the assay chamber (C), thus purging the chamber of any air present therein. The pressure differential between the assay chamber (C) and the containment chamber (B) facilitates perfusion of any air trapped in the assay chamber into the containment network (B).

[0056]

[0070] Finally, the confinement network (B) is pressurized by introducing liquid into the channels of the confinement network (B), thereby purging the air within the confinement network (B) via perfusion to the bulk and exhaust ports (not shown) of the microfluidic body to which the confinement network (B) is connected.

[0057]

[0071] Following purging of the microfluidic device, the control channels of the interface network are transitioned to an open state to allow fluid communication between the sample chambers and the assay chambers, and the control channels of the containment network are transitioned from an open state to a closed state to isolate the fluidly communicating sample and assay chamber pairs. Fluid communication between the paired sample and assay chambers results in mixing of the sample and assay fluids by diffusion.

[0058]

[0072] It should be understood that the examples shown in Figures 5A-5C and 6A-6C were performed using microfluidic devices having the same structure (specifically, Standard BioTools 96.96 IFC microfluidic devices). Additionally, it should be noted that the "filling A and B" steps in Figures 5A-5C were performed at 25°C, and the liquid (oil) used in the "filling A" step was heated to approximately 70°C to reduce fluid viscosity. However, the benefits of reduced preparation time and reduced air bubbles were also observed for embodiments in which the liquid in the "filling A" step was not so heated.

[0059]

[0073] In some embodiments, modulation of the temperature of the microfluidic device may be employed to facilitate device preparation. For example, the microfluidic device may be heated to reduce the viscosity of certain liquids in the control channels of the interface network and containment network, facilitating the closing and opening of these channels. Additionally, heating of the microfluidic device may be employed to promote mixing of the sample liquid and the assay liquid. Illustrative examples of pressure and temperature profiles according to various embodiments are shown in Figures 7A-7C, where the temperature profile corresponds to the shaded area and the pressure profile corresponds to the trace.

[0060]

[0074] The schematic flow path 700 is provided for ease of reference in aligning the pressure traces encoded with a line pattern, where the long-dashed trace is pressure as a function of time in the interface network (A), the short-dashed trace is pressure as a function of time in the containment network (B), the solid trace is pressure as a function of time in the assay chamber (C), and the dotted trace is pressure as a function of time in the sample chamber (D).

[0061]

[0075] The "thermal mixing" region in Figures 7A-7C refers to the time allotted for mixing to occur between the sample and assay chambers by diffusion. During this time, the interface between the sample and assay chambers is open, so the sample and assay are in fluid communication. To increase the rate of diffusion and reduce the time required to reach equilibrium between the sample and assay chambers, the temperature is often increased, a step also performed in conventional methods.

[0062]

[0076] Figure 7A shows an embodiment in which the setup time was reduced to approximately 57 minutes using a Standard BioTools 96.96 IFC microfluidic device with 24 sample wells (e.g., item 120 in Figure 1) and 24 assay wells (e.g., item 140 in Figure 1) empty. The numbers in the fields of the graph in Figure 7A provide more detailed times for the various elements therein.

[0063]

[0077] Figure 7B shows an embodiment of a Standard BioTools 96.96 IFC microfluidic device using 24 sample wells (e.g., item 120 in Figure 1) and 24 assay wells (e.g., item 140 in Figure 1) as empty, reducing setup time to approximately 49 minutes.

[0064]

[0078] Figure 7C shows an embodiment in which the setup time was reduced to approximately 42 minutes using a Standard BioTools 96.96 IFC microfluidic device with all sample wells (e.g., item 120 in Figure 1) and assay wells (e.g., item 140 in Figure 1) filled with liquid, i.e., all filled with sample or assay reagents, respectively.

[0065]

[0079] Methods according to various embodiments of the present invention may provide several advantages, such as significantly reducing device setup time as well as reducing the presence of air bubbles in the sample and / or assay chambers during analysis, which may increase the accuracy of sample analysis by, for example, reducing false positive and false negative results, as well as reducing sample and reagent waste.

[0066]

[0080] FIG. 8 shows an example of the improvement in reducing errors due to air bubbles that may result when using a microfluidic device prepared according to an embodiment of the present invention, such as the microfluidic device 100 described above prepared according to an embodiment of the present invention for gene expression analysis. FIG. 8 compares the occurrence of false negatives and false positives in gene expression analysis using a Standard BioTools 96.96 IFC microfluidic device with 24 sample wells (e.g., item 120 in FIG. 1 ) and 24 assay wells (e.g., item 140 in FIG. 1 ) empty. False negatives and false positives can generally be attributed to air bubbles. With the new method shown in FIG. 8 , it is believed that errors may not be due to air bubbles, but rather may be due to potential leaks between sample chambers.

[0067]

[0081] Using the conventional preparation method (Prior Art. Method), 522 false positives and 224 false negatives were observed, for a total of 746 errors. In comparison, using a preparation method substantially in accordance with the preparation method shown in Figure 7A, only 4 false positives and no false negatives were observed. Errors due to air bubbles were reduced by a factor of 186 (or more than two orders of magnitude).

[0068]

[0082] Various aspects of the present teachings can be further understood with reference to the following discussion of experimental results.

[0069]

[0083] Experimental results

[0084] As can be seen in consideration of the experimental results below, the methods disclosed herein provide loading methods for integrated fluidic circuits (IFCs), particularly the Standard BioTools 96.96 Genotyping (GT) / Gene Expression (GE) IFC microfluidic device, that improve the turnaround time of applications run on the IFC. Furthermore, as described in detail below, the experimental loading methods described in this section do not modify the IFC design, require additional consumables and labor, or adversely affect data quality.

[0070]

[0085] The conventional loading method for an IFC microfluidic device includes the following steps. Step 1: Simultaneously load (prime) the interface control line and the containment control line. Step 2: Load the sample and assay simultaneously (loading / mixing).

[0071]

[0086] In an experimental rapid loading method according to various embodiments of the present teachings, the filling / loading sequence of the IFC microfluidic device was reordered, as described in the detailed description above, to improve air evacuation from the chip.

[0072]

[0087] In this example, heat was applied to reduce the viscosity of the fluid, which allowed for faster filling. Samples and assays were loaded at room temperature (cold exposure). Higher pressures were employed compared to conventional loading methods to reduce the effect of air bubbles in the liquid network. More specifically, in contrast to conventional loading methods, the experimental fast loading method utilized in this example includes the following steps: Step 1: Load the interface control lines. Step 2: Load the sample. Step 3: Load the assay. Step 4: Loading the containment control line. The experimental fast loading method did not employ any separate priming step: the accumulator was loaded with control line fluid at the same time as the sample and assay were pipetted into the inlets.

[0073]

[0088] The experimental rapid loading method was performed using the same existing sample assay reagents, volumes, and preparation procedures as those used in the Standard BioTools 96.96 IFC microfluidic device. The sample and assay mixture preparation in the experimental rapid loading method was the same as the existing sample and assay mixture preparation in the Standard BioTools 96.96 IFC microfluidic device. Additionally, the IFC used in the experimental rapid loading method was the same (same barcode) as the existing Standard BioTools 96.96 IFC.

[0074]

[0089] FIG. 9 shows a reference workflow for gene expression (GE) and genotyping (GT) runs performed on a Standard BioTools 96.96 IFC microfluidic device, with an example experiment using a conventional (reference) loading method and an example experiment using a loading method according to an embodiment of the present technology (fast). As can be seen from the illustration in FIG. 9 , the GE and GT runs using the present fast loading method were each 1.9 hours shorter than the GE and GT runs using the reference loading method. Thus, the GE run using the present fast loading method was 56% shorter than the GE run using the reference loading method, and the GT run using the present fast loading method was 52% shorter than the GT run using the reference loading method. Thus, the example experiment in FIG. 9 shows that data can be obtained using the present fast loading method in approximately half the time required to obtain data using the reference script, although in other embodiments, loading times may be even shorter.

[0075]

[0090] Additionally, FIG. 9 shows that the present fast loading method eliminates some time-dependent user intervention (e.g., between priming and loading / mixing operations), thereby allowing laboratory staff to focus on other tasks.

[0076]

[0091] Figure 10 shows an example of multiple GT and GE runs in an 8-hour operating shift using a loading method according to an embodiment of the present technology (high speed). As shown in Figure 10, the high speed loading method can support up to four GT runs or six GE runs in an 8-hour operating shift, while the reference loading method can only support up to two GT runs or two GE runs in an 8-hour operating shift. Thus, the high speed loading method can increase the GT run capacity per day by up to 200% (192 more samples per day) and the GE run capacity per day by up to 300% (384 more samples per day) compared to the reference loading method.

[0077]

[0092] Figure 11 shows the average instrument run times for various microfluidic devices and application types from a validation study using the Standard BioTools X9 Genomics System. As can be seen from Figure 11, the run times for GE, SNP-type GT, and TaqMan GT in the rapid loading method according to the present teachings are significantly (approximately 50% or more) shorter than the corresponding run times in the reference script when performed on a Standard BioTools 96.96 IFC microfluidic device.

[0078]

[0093] Figure 12 shows an experimental comparison of ΔΔCt values ​​from the same Standard BioTools 96.96 IFC microfluidic device run using a human tissue cDNA sample and panel A of the Advanta IO gene expression panel using a conventional (reference) loading method and a loading method according to an embodiment of the present technology (fast). In this example, ΔΔCt values ​​were calculated from three replicates of each sample, with five reference targets included for normalization. The correlation, slope, and intercept of the comparative plot in Figure 12 can be seen to demonstrate that the reference and fast loading methods provide roughly identical gene expression results.

[0079]

[0094] Figure 13 shows experimental results of a gene expression uniformity study performed on a Standard BioTools 96.96 IFC microfluidic device using a conventional (reference) loading method and a loading method according to an embodiment of the present technology (fast). In the study in Figure 13, a single sample and a single assay were loaded across the entire IFC.

[0080]

[0095] Ideally, in a gene expression uniformity study, all chambers of an IFC would show the same Ct result, and the Ct standard deviation across all chambers could be used as a measure of Ct uniformity across the IFC. As shown in Figure 13, in the uniformity study performed here, the fast loading method exhibited an overall lower Ct standard deviation (mean = 0.067, n = 18) than the reference loading method (mean = 0.090, n = 18). This difference was statistically significant, with a p-value of 7.3e-09 for a t-test on the difference in means. Thus, as shown in Figure 13, the fast loading method resulted in better uniformity across the IFC compared to the reference loading method.

[0081]

[0096] Figure 14A shows TaqMan genotyping performance of a Standard BioTools 96.96 IFC microfluidic device in an experiment using a conventional (reference) loading method. Figure 14B shows TaqMan genotyping performance of a Standard BioTools 96.96 IFC microfluidic device in an experiment using a loading method according to an embodiment of the present technology (fast). Figures 14A and 14B show that the fast and reference loading methods resulted in similar genotyping cluster distributions.

[0082]

[0097] In another example, Table 1 below shows a comparison of known genotyping results in IFCs run using the reference script and the fast script, respectively, using TaqMan GT assays. In this example, identical IFCs were run using 92 samples (+4 NTC) versus 96 assays. Known genotypes for all assayed SNPs were obtained from publicly available sequencing and / or array data. Each IFC result was compared against the known genotype for each sample / assay to determine the concordance of each loading method with the known genotype.

[0083]

[0098] [Table 1]

[0084]

[0099] As shown in Table 1 above, both loading methods yielded similar concordance rates (>99%) on TaqMan GT.

[0085]

[0100] 15A and 15B include schematics and photographs, respectively, of a study evaluating the impact of various levels (e.g., size and location) of bubble formation in a Standard BioTools 96.96 IFC microfluidic device using a loading method according to an embodiment of the present technology (fast). More specifically, the study in FIGS. 15A and 15B evaluated the types of problematic bubble formation that can occur in the reagent chamber / inlet by injecting various amounts of air into the reagent inlet. When no air (0 μL) was injected into the reagent inlet, no bubbles formed in the reagent chamber, and both the reference loading method and the fast loading method of an embodiment of the present invention filled the device well. When less than 1 μL of air was injected into the reagent inlet and bubbles formed at the bottom of the reagent chamber, the reference loading method did not fill the device well, while the fast loading method filled the device well. When more than 1 μL of air was injected into the reagent inlet and bubbles formed only at the top of the reagent chamber, both the reference loading method and the fast loading method filled the device well. However, when more than 1 μL of air was injected into the reagent inlet, causing bubbles to form at the top and bottom of the reagent chamber, the reference loading method did not fill the device well, while the fast loading method did fill the device well.

[0086]

[0101] Thus, the reference loading method filled the device wells only when no bubbles were formed or when bubbles were formed only at the top of the reagent chamber, whereas the fast loading method of one embodiment of the present invention filled the device wells under all bubble formation conditions examined.

[0087]

[0102] In summary, experiments performed on the fast-loading method according to the present teachings confirmed that the fast-loading method offers several advantages over the reference loading method. More specifically, compared to the reference loading method, the fast-loading method exhibited faster overall run times as a result of a) the reduced time required for the instrument to load the sample and assay into the reaction chamber and b) the reduced time required to mix the sample and assay on the IFC. Additionally, the fast-loading method eliminated the need for a separate priming step, thereby eliminating user interaction between the priming step and the loading / mixing step of the reference loading method and reducing hands-on time for laboratory personnel. The fast-loading method also became less susceptible to bubbling at the reagent inlet. Furthermore, the fast-loading method was compatible with all current 96.96 IFCs and provided the above-mentioned advantages without modifying existing thermal cycling and detection steps.

[0088]

[0103] The methods of the present embodiments, as described above, are applicable to a wide variety of microfluidic devices. Furthermore, the methods of preparing a microfluidic device for use according to the present teachings can be implemented in software using known techniques, as shown in the present teachings.

[0089]

[0104] Those skilled in the art will appreciate that various modifications can be made to the above-described embodiments without departing from the scope of the present teachings.

Claims

1. 1. A method of preparing a microfluidic device for operation, comprising: The microfluidic device is formed of a perfusable material having a plurality of separate fluid networks at a communicable distance, the networks including elements that trap air during filling; The method comprises: (a) introducing a first liquid into a first network to pressurize the first gas in the first network to a pressure greater than a pressure associated with the second network to purge a portion of the first gas from the first network while another portion of the first gas remains trapped in at least one region of the first network and is at least partially perfused into a second network; (b) introducing a second liquid into a second network in fluid communication with a chamber in which measurements are taken to pressurize the second gas in the second network to a pressure greater than the pressure associated with the third network to purge a portion of the second gas from the second network while another portion of the second gas remains trapped in at least one region of the second network and is at least partially perfused into a third network; (c) introducing a third liquid into the third network to pressurize the third gas in the third network to a pressure greater than the pressure associated with the fourth network to purge a portion of the third gas from the third network while another portion of the third gas remains trapped in at least one region of the third network and is at least partially perfused into a fourth network; (d) introducing a fourth liquid into the fourth network to purge the third gas from the fourth network; The method of claim 1, wherein the first and second subroutines are executed sequentially.

2. The method of claim 1 further comprising heating the first liquid.

3. The method of claim 1 , wherein the first, second, and third gases are the same.

4. The method of claim 1 , wherein the network is connectable through a closable interface / valve.

5. 10. The method of claim 1, wherein a maximum thickness of the perfusable material between at least one of the networks and an adjacent network is less than a minimum thickness of the perfusable material extending from at least one of the networks to an edge of a body of the microfluidic device.

6. The method of claim 1 , wherein any of the first, second, and third liquids is any of an aqueous liquid and an organic liquid.

7. The method of claim 6 , wherein any of the first, second, and third liquids comprises any of water, alcohol, and oil.

8. The method of claim 1 , wherein any of the first, second, and third liquids comprises any of air, nitrogen, oxygen, carbon dioxide, and argon.

9. The method of claim 1 , wherein the element comprises a chamber.

10. 10. The method of claim 1, wherein the volume of the trapped gas in any of the steps is greater than the volume of the trapped gas, if any, in a subsequent step.

11. 10. The method of claim 1, wherein any of the networks is at least partially filled with liquid before receiving trapped gas from an adjacent network.

12. 12. The method of claim 11, wherein the trapped gas received by the at least partially filled network flows as bubbles through the liquid partially filling the network.

13. The method of claim 11 , wherein the gas received by the at least partially filled network forces the liquid partially filling the network out of the network.

14. The method of claim 1 , wherein the pressure associated with any of the pressurized networks is equal to, greater than, or less than atmospheric pressure.

15. 1. A method of preparing a microfluidic device for operation, comprising: the microfluidic device having a plurality of assay chambers, a plurality of sample chambers, and a microfluidic manifold configured to provide selective coupling between the assay chambers and the sample chambers, the microfluidic manifold comprising a network of interface channels between the assay chambers and the sample chambers, and a network of containment channels for isolating each pair of the assay chambers and the sample chambers from adjacent pairs; The method comprises: (1) pressurizing the interface network with a liquid to a pressure greater than the pressure associated with the sample chamber to purge at least a portion of the gas present in the interface network while another portion of the gas remains trapped in at least one region of the interface network and is at least partially perfused into the sample chamber; (2) pressurizing the sample chamber with a liquid to a pressure greater than the pressure associated with the assay chamber to purge at least a portion of the gas present in the sample chamber while another portion of the gas remains trapped in at least one region of the sample chamber and is at least partially perfused into the assay chamber; (3) pressurizing the assay chamber with a liquid to a pressure greater than the pressure associated with the containment network to purge at least a portion of the gas present in the assay chamber into the containment network; (4) pressurizing the containment network to at least partially purge the containment network; The method of claim 1, wherein the first and second subroutines are executed sequentially.

16. 16. The method of claim 15, wherein the sample chamber is where measurements are made after reaction and / or mixing of the sample with the assay.

17. 16. The method of claim 15, wherein step (2) causes the sample chamber to be purged at least partially through the containment network.

18. 16. The method of claim 15, wherein step (3) evacuates the assay chamber at least partially through the containment network.

19. The method of claim 15 , wherein the sample chamber is taller than the assay chamber.

20. 16. The method of claim 15, further comprising transitioning the interface network from a closed state to an open state to allow mixing of the assay and sample in the assay chamber and the sample chamber.

21. 21. The method of claim 20, further comprising increasing the temperature of the microfluidic device to promote the mixing of the assay and the sample by diffusion.

22. 16. The method of claim 15, wherein performing the sequential steps substantially reduces the probability of bubble formation in the sample chamber and the assay chamber relative to performing steps (1), (2), (3), and (4) of claim 14 in the order (1), (4), (3), and (2).

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