CLOSED MICROFLUIDIC CARTRIDGE FOR EVALUATING MULTIPLE DISTINCT PORTIONS OF A FLUID SAMPLE - Patent application

Abstract

The present invention relates to a microfluidic cartridge that operates as a closed system. The microfluidic cartridges described herein allow for the independent evaluation of multiple separated portions of a fluid sample (eg, a blood sample, etc.). In certain embodiments, each partition has a predetermined volume. The present invention also relates to a method for simultaneously assessing multiple separated portions of a fluid sample using a single cartridge.

Description

[Technical field]

[0001] The present invention relates generally to devices, systems, and methods for evaluating multiple separated portions of a fluid sample. The devices, systems, and methods described herein may be used in a variety of applications, including medical diagnostics, for evaluating biological fluid samples, such as, for example, assessing coagulation in a blood sample. [Background technology]

[0002] The use of microfluidics for diagnostic and other applications is becoming increasingly common. Most current microfluidic cartridges are open systems, with open communication between the internal contents of the cartridge and parts of the external environment or automated reader, such as a syringe. Some of these systems are conditioned to minimize the possibility of contamination to or from the outside environment or parts of the reader, such as through the use of dedicated filters placed near the outlet or inlet ports. However, none of these systems eliminate the risk of contamination. Contamination can be dangerous, especially when dealing with biological or environmental samples that may be harmful if aerosolized. Furthermore, open systems with filters are not suitable for evaluating gas-phase fluid samples, since gases in such samples can escape through the pores of the filter. Similarly, in systems that use valves to control fluid flow, air can escape from such valves, since such valves are "liquid-tight" but not "air-tight". Other cartridges may also include waste chambers or other chambers that are not closed but are open (e.g., vented waste chambers) to reduce internal pressure within the cartridge. See, e.g., Miyazaki et al., Processes 8:1360 (2020); Ahrberg et al., Lab Chip 16:3866-84 (2016); Al-Faqueri et al., Sensors 15:4658-76 (2015). The waste chamber of such a cartridge serves as an exit and opening to the external environment. Summary of the Invention [Problem to be solved by the invention]

[0003] There is an urgent need in the art for a device that becomes a closed system once a sample is inserted (e.g., to contain dangerous pathogens or other dangerous agents in a single-use testing cartridge). There is also a need in the art for devices, systems, and methods for evaluating multiple separate portions of a fluid sample such that multiple portions of a single fluid sample can be evaluated independently and simultaneously using a single device. There is a particular need for devices that use stationary features in the device to allow for the division of a fluid sample into multiple separate portions (e.g., using microfluidic channel geometries) and allow each portion to be exposed to one or more reagents independently of the other portions (e.g., to avoid crosstalk between portions and to avoid the propagation of any chemical or physical reactions or cues from one portion of the sample to another), and that are of reduced cost and / or complexity. The present invention addresses these needs. [Means for solving the problem]

[0004] The present invention provides devices, systems, and methods for evaluating a fluid sample. The device can be used for in vitro evaluation of a blood sample, for example, to evaluate a coagulation state and / or phenotype of a subject. The device can also be used to detect one or more analytes in a fluid sample. An exemplary device includes a microfluidic cartridge having a plurality of channels, each of which is configured to receive a portion of a sample through a common inlet connected to a first end (which may also be referred to as an upstream end) of the plurality of channels, each of which opens into a downstream chamber (or series of chambers, etc.) of the channel. The downstream chamber may be a metering chamber or a waste chamber. Downstream of each of the metering chambers may be another chamber (or series of chambers, etc.) where some measurement is performed and / or some other analysis occurs. The chamber (or well, etc.) where the analysis occurs is referred to herein as a test chamber, detection chamber, measurement chamber, test well, detection well, etc. Downstream of each test chamber is an air spring or other closed chamber. The aforementioned chambers may be connected by channel paths of various shapes and lengths, as further described herein.

[0005] An embodiment of the present invention also provides an apparatus for evaluating a fluid sample, the apparatus having a microfluidic cartridge having a plurality of channels, the cartridge receiving a fluid sample through a common inlet connected to a first end of the plurality of channels (such that each of the channels connects to an inlet), the plurality of channels having at least three channels such that the fluid sample is divided into at least three channels. In some embodiments, at least two of the channels each have a predetermined target volume (e.g., a metered volume), and in further embodiments, the predetermined target volume is defined by the shape of each such channel. In certain embodiments, each of the at least two channels has a metering chamber such that the fluid sample in each of the at least two channels fills one or more metering chambers. In some aspects, the filling of the metering chambers in each such channel is based on a pressure differential caused by the size and shape of each of the channels, as described herein. At least one of the plurality of channels has a waste chamber at its downstream end.

[0006] In embodiments of the device, system, and method of the present invention, the cartridge may be a microfluidic cartridge, chip, or other device, and the multiple channels may be microfluidic channels. In some embodiments, the method may be performed using a device that requires less than about 1 mL, less than about 500 μL, less than about 100 μL, or less than about 50 μL, or less than about 5 μL of fluid sample (e.g., in some embodiments, about one drop of fresh whole blood or whole blood anticoagulated with sodium citrate is sufficient). In some embodiments, the channel width, channel height, or both may be less than 10 mm, less than 6 mm, or less than 2 mm. In some embodiments, a single channel may have various dimensions (e.g., width, height, radius, etc.) along the length of the channel (e.g., certain regions along the path of the channel are wider or have a larger radius than other regions), and further, one or more channels may have different dimensions than other channels. The channels (and chambers and other regions along the length of each channel) may be of various shapes, such as, for example, equilateral polygons (e.g., hexagons), asymmetric polygons (e.g., hexagons with various side lengths), or rectilinear channels (e.g., rectangular or cylindrical channels). Furthermore, whatever shape is used, corners and edges where two faces intersect may be rounded (e.g., to facilitate fluid flow through the channel). In certain embodiments, it may be desirable to use a variety of different channel shapes. In some embodiments, one or more channels (e.g., all of the channels) of the plurality of channels may have the same volume, or the volumes may vary from channel to channel. Similarly, in embodiments where one or more of the plurality of channels has multiple functional regions (e.g., metering chambers, reagent chambers, mixing chambers, detection chambers, etc.), such regions connected in series may all have the same volume, or the volumes may vary from region to region (e.g., in a preferred embodiment, the metering chambers may have a volume of about 3 μL, the reagent chambers may have a volume of about 1 μL, and the detection chambers may have a volume of about 2 μL).Additionally, the material forming the channels (and thus, for example, the material lining the inner surface of the channels) may be the same across one or more channels or may vary from channel to channel. The relative positions of the channels may also vary depending on the embodiment. For example, the channels may all be formed in the same substrate and disposed in the same plane, the channels may all be formed in the same substrate but located in different (e.g., parallel) planes, the channels may be formed in multiple substrates (e.g., by stacking materials in some embodiments) and disposed in a single plane, or the channels may be formed in multiple substrates and disposed in different planes (e.g., at least one of the walls of the channels on one or more planes is composed of a layered material, e.g., a second substrate, such as a pressure sensitive adhesive, such as in a multi-planar cartridge with some channels formed in one plane of a substrate and other channels disposed in another plane).

[0007] In various embodiments of the devices, systems, and methods of the present invention, the cartridge may include a sensor (e.g., an electrode sensor). In some embodiments, the portion of the device that houses the sensor may form one or more walls of one or more channels. Alternatively, in certain embodiments, one or more walls of one or more channels may be made from the sensor itself, such as, for example, a multi-layer PCB (printed circuit board) with one or more exposed electrode traces. In embodiments in which a multi-layer PCB with exposed electrode traces is used, the exposed electrode traces may act as the sensor or may serve other purposes, such as, for example, functioning as an integrated heater or cooling unit. The sensor may be an exposed electrical trace (e.g., a pair of electrodes for impedance measurements, interdigital electrodes, etc.) or other types of sensors attached to the PCB (e.g., a photodiode, a camera lens, a spectrophotometer, a photonic sensor, a piezoelectric sensor, etc.). In some embodiments in which a PCB is used, the PCB may include a stimulant (e.g., a light source, etc.) and a receiver (e.g., a photodiode, etc.).

[0008] In some embodiments, the cartridge may include multiple sensors, the sensors being of the same type (e.g., electrode sensors measuring electrical properties such as impedance) or of different types. For example, the cartridge may have one type of sensor on one side and a different type of sensor on the other side, and for either sensor, the sensor may be part of or attached to the PCB, as described above. In certain embodiments, a multi-layer cartridge may have a PCB as a layer, the PCB having one or more sensors, and the cartridge may further have additional sensors in or on one or more other layers. The sensor layer may be made of multiple separate or connected PCBs, such as a PCB that is the bottom layer of the cartridge and communicates with a PCB that is the top layer of the cartridge, such communication may be achieved via a light source and photodiode, etc., and the PCB is arranged to measure (e.g., by intercepting a signal) the activity and / or properties of a portion of the fluid sample in the channel. In some embodiments, two PCBs can be aligned with each other to form a channel, but each PCB can perform a different measurement (e.g., one PCB has a sensor that measures electrical impedance and a second PCB has a sensor that performs an optical measurement, machine learning, artificial intelligence, etc.). In certain aspects, the two PCBs can measure different properties within the same channel (at the same location of the channel, or in different areas of the same channel) independently of each other, although each PCB has a sensor, or may measure different properties within different channels and / or different portions of the cartridge (e.g., through the use of two PCBs, each PCB has a sensor, but electrical impedance can be measured in one channel on one side of the cartridge and optical sensing can be performed in the other channel on the other side of the cartridge).

[0009] In some embodiments, the PCB may include electrodes, for example, as sensors for measuring properties of a fluid sample and performing tests, or for sensing the position of the fluid (eg, as a fluid position sensor).

[0010] In some embodiments, the cartridges described herein may have a fluid position sensor. The fluid position sensor may be based on electrical impedance, for example using electrodes (as described above), or may be made of other components attached to the PCB. In various embodiments, the fluid position sensor may provide active feedback to an external component operating the cartridge, for example, to ensure precise control of fluid movement and / or fluid movement within the cartridge. Such a feature, illustrated, for example, in the embodiments described in the figures, is particularly advantageous as it allows a single cartridge design to be suitable for testing various types of fluid samples with different viscosities (e.g., blood samples with different hematocrits, urine, saliva, etc.). Thus, in some embodiments, the fluid position sensor may provide feedback to an external control system, and such sensor and feedback may be used to detect the position of the fluid within the cartridge and make appropriate adjustments to the fluid flow (in certain embodiments, for example, to ensure that a minimum required fill volume has been achieved) or control the movement of the fluid (in various embodiments, for example, by applying a certain amount of pressure in response to input received from the fluid position sensor). For example, in some embodiments, a fluid position sensor (which may be a pair of electrode sensors or other sensors measuring light, ultrasound, radio frequency, absorbance, etc.) is used to detect the fill of the metering chamber, and when the fill of the metering chamber reaches a certain level, the velocity or pressure applied to the actuator is altered (e.g., reduced) so that the remainder of the fluid sample that has not yet been split into channels moves to the channel with the waste chamber instead of the channel with the metering chamber.

[0011] Additionally, in some embodiments where the microfluidic cartridge is used to evaluate a blood sample, the microfluidic cartridge may have a sensor that measures the hematocrit in the sample. The hematocrit information may be used to evaluate the viscosity of the sample. A sensor that measures the hematocrit and provides such hematocrit information to an external control system may be used to control and regulate the movement of the fluid via sensor feedback.

[0012] In certain embodiments, it may be beneficial to select certain features and / or materials to optimize test conditions in certain regions of the cartridge. For example, certain laminate materials may be selected (e.g., glass, either for the entire layer or to create an optical window to facilitate measurements with an optical sensor), certain coatings may be applied (e.g., to change the surface topology), or certain treatments may be used (e.g., to treat regions of the cartridge with chemicals, such as to make the surface hydrophilic or hydrophobic, or with biological materials).

[0013] Similarly, the materials of the cartridge body and / or cartridge layers may be selected to promote fluid dynamics that are best suited for the type of test to be performed within the cartridge (e.g., plastics such as polycarbonate are more hydrophobic than glass and may be more suitable than glass for certain types of analysis). The materials used may affect the movement of the fluid and the pressure within the system (e.g., whether the fluid is in the middle of a large chamber or in a relatively narrow resistor), just as the viscosity of the fluid or the position of the fluid within the cartridge may affect the movement of the fluid. For example, a more hydrophobic material lining the surfaces of the channels may help inhibit capillary action and improve accurate metering, especially when the system is under sufficiently low pressure that capillary action may occur. Additionally, in some areas of the cartridge, it may be desirable to line the surfaces of the channels with a material that facilitates retention of sacrificial fluids, as described further herein.

[0014] The present invention also provides microfluidic cartridges that are useful as single-use, disposable cartridges. In other embodiments, the cartridges may be reusable cartridges and may have features that facilitate cleaning of the cartridge before reuse. As noted above, the cartridges described herein may be made of a single material or may be made of multiple materials.

[0015] In some embodiments, the channels are formed in a substrate by one or any combination of a number of methods (e.g., injection molding, machining, laser etching, lamination, etc.), which may be made of one or more of a variety of materials (e.g., acrylic, polycarbonate, liquid crystal polymer, etc.). In some embodiments, the cartridge has a primary substrate and one or more layers. For example, in certain embodiments, the channels may be formed in the primary substrate such that the channels are open on one side (e.g., above the channels) in the substrate itself, and one or more cover layers may be attached to the primary substrate to completely surround the channels molded in the substrate to form the channels. Although various embodiments are described with reference to "channels," other terms (e.g., lanes, compartments, partitions, etc.) can describe spaces that are physically separated from one another and, in some embodiments, have the same shape as one another.

[0016] In certain embodiments of the devices, systems, and methods of the present invention, the cartridge has a common inlet connected to a first end of the multiple channels. In some embodiments, the common inlet is the only opening of the cartridge. In other embodiments, the cartridge may have one or more other openings in addition to the common inlet. For example, the cartridge may have a common inlet and a vent. Regardless of how many openings the cartridge has, it is an essential feature of such embodiments that each opening is closed after sample input and before movement of the fluid sample through the multiple channels in the cartridge is performed. For example, the vent (if present) may have a porous membrane that expands to close the pores when wetted with the fluid sample.

[0017] In some embodiments, the cartridge may have no internal moving parts, for example, the cartridge may have a bellows that may be actuated by an external component to force a volume of air contained in the bellows into the cartridge as a way of controlling the internal pressure and therefore the movement of fluid.

[0018] In some aspects, the cartridge may have a single moving part that pressurizes the system to move the fluid sample through multiple channels. The cartridge may include, for example, an integrated syringe with a plunger, and in such an embodiment, an external actuator may move the plunger to control the pressure in the system. In all of these embodiments, an external component may selectively pressurize or depressurize the system to move fluids forward or backward (e.g., to move fluids to specific locations within a test chamber for purposes of mixing fluids, etc.).

[0019] In embodiments of the device, system, and method of the present invention, the shape of each of the plurality of channels may incorporate air springs and resistance areas.

[0020] In various embodiments of the devices, systems, and methods of the present invention, the cartridge may be devoid of active control elements (eg, other than the actuators described herein), valves, vents, and hydrophobic membranes.

[0021] In certain embodiments, internal (i.e., contained within the cartridge) control elements may be used to enable closure of the system, including, for example, a porous membrane that expands upon contact with a fluid sample to close its pores.

[0022] In embodiments of the devices, systems, and methods of the present invention, the cartridge may use forward and reverse flow of the fluid sample (such as for mixing the fluid sample with a reagent), while in other embodiments, the fluid flow may be unidirectional.

[0023] The fluid or fluid sample may include, but is not limited to, a sample of a bodily fluid (e.g., blood, plasma, saliva, urine, cerebrospinal fluid (CSF), peritoneal fluid, pleural fluid (including pleural fluid), or pericardial fluid), a sample of a bodily gas (e.g., a sample from exhaled breath), a chemical sample and / or an environmental sample (including, but not limited to, a sample of a chemical in liquid or gaseous form), a water sample, or an air sample.

[0024] As used herein, a "blood sample" refers to a whole blood sample or a plasma sample, unless otherwise stated. The term plasma includes both platelet rich plasma (PRP) and platelet poor plasma (PPP). In any of the devices, systems, and methods described herein, the blood sample may be a whole blood sample or a plasma sample. The use of whole blood may be particularly useful for certain applications, such as those performed at the patient's bedside.

[0025] In some embodiments, the fluid sample is a gas, and the medium used to pressurize the system and move the sample downstream through the channels may be another gas or air, or in some embodiments, a liquid. The medium used to pressurize the system may be selected to minimize reactivity with and permeability to the sample. In embodiments where a medium other than air is used, there may be a container (e.g., a pouch, etc.) that holds this medium and releases the medium, for example, by a piercing needle or other component, at the start of the cartridge test or upon actuation of the actuator (e.g., upon initiation of actuation of a bellows or syringe pump).

[0026] The term "chamber" and variations thereof are used interchangeably herein with the term "well" and variations thereof and are non-limiting terms. Such chambers are located along the path of the microchannel. In some embodiments, a chamber may refer to a particular location within a channel, even if the shape of the channel at that particular location does not change and is the same as the shape of the channel before and after the chamber. In other embodiments, the shape of the channel may change at the chamber, for example, such that the volume of the channel increases at the location of the chamber. Additionally, while various embodiments are described with reference to "measurement chambers," "sensing chambers," or "test chambers," other terms (e.g., test wells, sensing wells, etc.) may describe areas of the channel (or other areas of the cartridge) where some measurement or analysis takes place.

[0027] The term "closed system" and its variations generally refer to a cartridge that is a dead-end system.

[0028] A closed system uses pressure gradients to create back pressure at specific locations at different times to control fluid flow. As used herein, unless otherwise noted, a reference to a numbered feature in a figure that includes a letter (e.g., 140a) is intended to refer to that particular alphanumeric feature only, and a reference to a feature that does not include a letter (e.g., 140) is intended to refer to all such numbered features (e.g., 140a, 140b, 140c, etc.). [Brief description of the drawings]

[0029] [Figure 1] FIG. 1 illustrates the operating principle underlying the closed cartridge air spring design. [Diagram 2] FIG. 2 extends the operating principle illustrated in FIG. 1 to a multi-channel microfluidic cartridge, where the channels are connected at their upstream ends to a common inlet. [Diagram 3]Figure 3 models the time-dependent fluid flow in a Hagen-Poiseuille system such as a closed microfluidic air spring cartridge. [Figure 4A] Figures 4A-4E illustrate fluid flow at various stages of cartridge pressurization: Figure 4A illustrates fluid flow in state 0; [Figure 4B] Figures 4A-4E illustrate fluid flow at various stages of cartridge pressurization: Figure 4B illustrates fluid flow in state 1; [Figure 4C] Figures 4A-4E illustrate fluid flow at various stages of cartridge pressurization: Figure 4C illustrates fluid flow in state 2; [Figure 4D] 4A-4E illustrate fluid flow at various stages of cartridge pressurization. FIG. 4D illustrates fluid flow at state 3. [Figure 4E] 4A-4E illustrate fluid flow at various stages of cartridge pressurization: FIG. 4E illustrates fluid flow at state 4; [Figure 5A] 5A and 5B each illustrate one embodiment of an air spring cartridge system. [Figure 5B] 5A and 5B each illustrate one embodiment of an air spring cartridge system. [Figure 6A] FIG. 6A illustrates one embodiment of an air spring cartridge system having nine test chambers and three waste chambers. [Figure 6B] FIG. 6B illustrates a portion of a channel (such as channel a in the embodiment illustrated in FIG. 6A) that includes an additional pathway (e.g., an additional chamber, or a series of additional chambers) and / or resistive features between the metering chamber (130) and the test chamber (140). [Figure 7] FIG. 7 illustrates a system corresponding to some of the cartridge embodiments described herein (FIGS. 10A, 10B, and 11). [Figure 8]FIG. 8 illustrates how certain features in a single microfluidic channel (eg, the single channel of the cartridge shown in FIGS. 10A-10B) are arranged within a multi-layer microfluidic cartridge. [Figure 9] 9 shows an embodiment in which each of the nine channels of the cartridge has multiple electrode pairs (each electrode pair shown as a pair of dots). In a particular channel, two electrode pairs are part of a feedback mechanism used to control the actuation of an active element that controls the fluid flow. Additionally, another electrode pair (shown as a pair of dots in the middle of the nine channels) is located in or near the common channel where the fluid sample resides before being split into multiple channels, and this electrode pair may function as a sensor and may be used as part of the feedback mechanism used to control the fluid flow. [Figure 10A] FIG. 10A provides top and bottom views of a multi-layer microfluidic cartridge employing the system illustrated generally in FIG. [Figure 10B] FIG. 10B shows the same various layers of the cartridge embodiment shown in FIGS. 8 and 10A. [Figure 11] FIG. 11 shows a top view of one embodiment of a microfluidic cartridge employing the system illustrated generally in FIG. [Figure 12A] 12A-12B show an embodiment of a cartridge in use with a fluid sample inserted: Figure 12A provides an image showing the cartridge in use, where the fluid sample is water mixed with a dye to facilitate imaging. [Figure 12B] Figures 12A-12B show an embodiment of a cartridge in use with a fluid sample inserted, with Figure 12B providing an image showing the cartridge in use where the fluid sample is citrated whole blood. [Figure 13A]13A-13F show the mixing of reagents and how compartmentalized portions of the fluid sample remain isolated from one another as they flow through their respective channels in the cartridge. Different colored dyes are spotted into the chambers, with each chamber receiving one or two colors (red, yellow, or red and yellow) (FIG. 13A) and allowed to dry (FIG. 13B). [Figure 13B] 13A-13F show the mixing of reagents and how compartmentalized portions of the fluid sample remain isolated from one another as they flow through their respective channels in the cartridge. Different colored dyes are spotted into the chambers, with each chamber receiving one or two colors (red, yellow, or red and yellow) (FIG. 13A) and allowed to dry (FIG. 13B). [Figure 13C] Figures 13A-13F show the mixing of reagents and how the separate portions of the fluid sample remain isolated from one another as they flow through their respective channels in the cartridge. As shown in Figure 13C, a water fluid sample is introduced and forced into the assembled cartridge, yet there is complete separation of the fluid sample portions and no mixing of colors between channels. [Figure 13D] Figures 13A-13F show the mixing of reagents and how the compartmentalized portions of the fluid sample remain isolated from one another as they flow through their respective channels in the cartridge. Figure 13D shows how the dried spotted dye mixes with an aliquot of the fluid sample in the mixing chamber, resulting in an even distribution of the dye within the fluid sample in the mixing chamber. [Figure 13E]Figures 13A-13F show the mixing of reagents and how the compartmentalized portions of the fluid sample remain isolated from one another as they flow through their respective channels in the cartridge. Figures 13E and 13F show images of a fluid aliquot of water (after mixing with dry spotted red and / or yellow dyes) as it enters the test chamber (dry spotted with blue dye), where the blue dye is uniformly dispersed and unmixed in the fluid sample, demonstrating that in this cartridge embodiment, the mixing shown in Figure 13D is necessary to achieve complete mixing of the sample and dry spotted dye. [Figure 13F] Figures 13A-13F show the mixing of reagents and how the compartmentalized portions of the fluid sample remain isolated from one another as they flow through their respective channels in the cartridge. Figures 13E and 13F show images of a fluid aliquot of water (after mixing with dry spotted red and / or yellow dyes) as it enters the test chamber (dry spotted with blue dye), where the blue dye is uniformly dispersed and unmixed in the fluid sample, demonstrating that in this cartridge embodiment, the mixing shown in Figure 13D is necessary to achieve complete mixing of the sample and dry spotted dye. [Figure 14] FIG. 14 illustrates sacrificial retention of a fluid sample in a closed cartridge system, which may be used in certain embodiments to accommodate evaporation of the fluid sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The features and advantages of the present invention will be more readily understood by those skilled in the art upon reading the following detailed description. It is to be understood that certain features of the present invention described above and below in the context of separate embodiments may be combined to form a single embodiment. Conversely, various features of the present invention that are described for brevity in the context of a single embodiment may also be combined to form subcombinations thereof. Furthermore, it is expressly understood that the drawings and specific embodiments of the present invention described herein are illustrated for illustrative purposes, and that the descriptions and drawings are for illustrative purposes only, and that the specific embodiments are not intended to define the limitations of the present invention.

[0031] The present invention provides a self-contained microfluidic cartridge for use in the analysis of a fluid sample. A primary feature of the disclosed embodiments is that it allows for the independent evaluation of multiple partitioned portions of a fluid sample using a single cartridge. More specifically, the microfluidic cartridges described herein combine a parallelized air spring design with stationary features to control the flow of a fluid sample inserted into the cartridge, and such cartridges provide control of the movement of the fluid sample through a closed system using only a single active element (e.g., an actuator operating a syringe, plunger, bellows, pump, etc.) external to the cartridge. The active element external to the cartridge pushes the air and the fluid sample through the closed microfluidic cartridge without the need for any additional valves or other control elements.

[0032] As described below, the closed cartridge system described herein has two compartments of air, one compartment of air upstream of the fluid sample and the other compartment of air downstream of the fluid sample. The upstream air is used to push the fluid sample portion and the downstream air, resulting in the movement of the fluid sample downstream through the channel. This process causes the volume holding the upstream air, the fluid sample, and the downstream air to become smaller, resulting in compression and an increase in pressure.

[0033] The microfluidic cartridges described herein are dead-end systems (i.e., closed flow paths and no openings such as vents) that rely on pressure gradients and back pressure to control the flow of fluid into and through desired microfluidic channels (which may consist of one or more chambers) at various times. In some embodiments, the devices, systems, and methods described herein achieve these pressure gradients and back pressures using air reservoirs, air springs, and metering chambers. In some aspects, the pressure gradients and back pressure ensure unidirectional fluid flow through the microfluidic cartridge. Additionally, in some embodiments, resistors (e.g., Tesla valves, serpentine fluid flow paths, etc.) may be used to control and / or prevent backflow of the fluid sample.

[0034] The devices, systems, and methods described herein offer numerous advantages over existing microfluidic cartridges in that they provide the following features: a. each portion of the fluid sample can be metered into a known volume (referred to herein as an aliquot volume or metered volume) such that the volume of the fluid sample contained in each of the channels and in each chamber of the channel (e.g., reagent chamber, test chamber) is a known volume; b. each portion of the fluid sample contained in each of the channels (and thus each chamber of the channel (e.g., the test chamber, as well as the waste chamber)) is separate from all other portions of the fluid sample contained in other channels (and any chambers along other channels); c. The cartridge operates as a closed system and its contents are never exposed to the outside environment.

[0035] In some embodiments, such as those where it is desirable to expose each portion of the fluid sample to different conditions (e.g., to different reagents, or to different concentrations of a reagent or reagents), it is essential to ensure that each portion is of a known volume (e.g., so that the concentration of the reagent to be added can be known and controlled). Thus, the cartridges described herein provide for metering a portion of the fluid sample to achieve a portion of known volume (a portion of the fluid sample, once metered to a target volume, is called an aliquot). The target volume (also called the metered volume) across the channels may be the same for all channels or may be different across the channels. In addition to the portion of the fluid sample that is metered, one or more other portions of the fluid sample may not be metered and may be received in a channel having a waste chamber (e.g., channels 120j, 120k, 120l in FIG. 7). It is also important to ensure that a chemical or physical reaction in one of the channels (e.g., in one of the chambers of the channel) does not affect a chemical or physical reaction that may occur in any other channel (e.g., in the chambers of the other channel).

[0036] In some embodiments, particularly those in which hazardous and / or infectious fluid samples are analyzed, it is desirable to have a closed system that contains the sample within the cartridge to ensure the safety of the person inserting the sample and / or performing the analysis, and also allows for the safe disposal of the cartridge (containing the fluid sample) following the analysis and use of the cartridge.

[0037] An embodiment of the present invention provides a microfluidic cartridge that operates as a closed system containing a volume of fluid (which may be air or liquid) that is displaced in a controlled manner to control the movement of an inserted fluid sample. The closed cartridges described herein may rely on a single actuator to control the movement of the fluid sample through the channels of the cartridge, although other mechanisms for controlling the movement of the fluid sample may be employed. For cartridges that rely on a single closed actuator (plunger, bellows, etc.), such cartridges can be manufactured at a lower cost than other devices that require additional elements to control the insertion and flow of the fluid sample without requiring other active control elements or valves, vents, hydrophobic membranes, etc. This cost savings is of great benefit, for example, for point-of-care or home analysis (e.g., point-of-care diagnostics), or where the test is likely to be used in the field (e.g., water quality testing), in harsh conditions (e.g., battlefields, zero gravity, etc.), or at other points of need (e.g., infectious disease testing in remote locations overseas). The use of a single closed actuator may also facilitate easier assembly and manufacturability of the cartridge due to fewer parts required to actuate the cartridge. Similarly, the use of a single closed actuator may increase the robustness of the cartridge (e.g., the cartridge may have fewer moving and / or fragile parts), particularly when such cartridges are used as diagnostic instruments.

[0038] In some embodiments, the use of a cartridge with a closed system can also obviate the need for complex fluid management (e.g., post-analysis procedures such as washing) outside the cartridge, e.g., in an analytical device that obtains measurements from the cartridge.

[0039] Working Principle The present invention relates generally to a closed air spring microfluidic cartridge that is useful for analyzing a fluid sample, for example, evaluating the physical and / or chemical properties of the fluid sample.

[0040] The operation of a closed microfluidic cartridge can be explained by reference to the ideal gas law, which describes the behavior of a gas with respect to pressure (P), volume (V), temperature (T), and amount of gas (n), as follows: PV=nRT where R is the ideal gas constant. For a closed microfluidic cartridge to function properly, distal air inside the cartridge must remain unreleased during the test process (in other words, distal air (that is, air that would be trapped downstream of the fluid sample upon insertion of the fluid sample) must not be released from the closed cartridge during use). In some embodiments, n, T, and R are held constant throughout the test process, and at any instant throughout the process, there will be a direct and proportional relationship between P and V. In other embodiments, where T is not held constant, the system provides feedback to the actuators - for example, via electrode sensors placed at various positions in one or more channels, such sensors determining the amount of fluid in the channels (e.g., in a particular chamber of a channel) - and via a feedback mechanism with the actuators, the actuators can compensate for pressure changes in the system (e.g., by pushing more or less, and / or by pushing faster or slower). In some embodiments, the viscosity of the fluid sample may also play a role in the pressure changes and the feedback of the system to the actuators. Thus, the feedback loop of the cartridge (see, e.g., FIG. 9) allows the system to adapt to such changes.

[0041] To illustrate the operation of the air spring system, the following description focuses on the movement of fluid through a single channel. In such a single channel example, a fluid sample is introduced into the microfluidic cartridge at a first end of the microfluidic channel, and the fluid sample travels through the channel, which is closed at a second end of the system (i.e., the downstream, distal end of the channel). A channel is any continuous path for fluid flow and may have a series of connected fluid flow paths, for example, a channel may be relatively narrow and then widen into a chamber, then the channel may narrow again, and the channel may have multiple chambers along its path. In various embodiments described herein, it does not matter how long the channel is, as long as no distal air is released. With such a condition, the initial amount of air inside the cartridge is a known amount, but does not change once the fluid sample is introduced or at any time thereafter during the test, as the air is trapped inside the cartridge by the fluid sample, and is left in the cartridge as the test progresses, with the air trapped by the fluid sample at one end and trapped by the blocked distal end of the channel at the other end. In some embodiments where the temperature is held constant, the pressure and volume of air trapped distal to the fluid sample at the start of the process (t=0) can be used to predict and control the position of the fluid sample at any time later in the process (t=x, x>0) as follows: P0V0=P x V x V x =P0V0 / P x For example, pressure P x By increasing the volume V x This principle, which is the basis of the operation of air springs, is illustrated in FIG.

[0042] In some cases, the physical properties of the fluid sample (e.g., the viscosity of the sample) may change during testing, for example, as a result of temperature changes or reactions occurring in the sample. Changes in temperature affect the viscosity, which in turn may affect the rate of flow of the fluid sample, especially for fluids that exhibit non-Newtonian behavior, such as blood. Furthermore, for non-Newtonian fluids, the viscosity of the fluid sample may change as the sample moves along the path of a channel that changes dimensions, thereby exposing the sample to different shear. Furthermore, some test conditions or reactions in the sample may also affect the pressure in the channel. For example, an increase in temperature may lead to evaporation of a portion of the fluid sample in the channel and / or to expansion of the fluid (if the fluid occupies more volume). In some cases, the compressibility of the sample may affect the movement of the sample through the pressurized channel. To build a robust closed system suitable for multiple sample types and conditions, a fluid position sensor may be included in the cartridge, and the cartridge may include an active feedback mechanism to control the movement of the sample within the cartridge. In such cases, the fluid position sensor may be in communication with a compensation mechanism that provides an active feedback loop, where the system is pressurized or depressurized based on information about the position of the fluid sample obtained from the position sensor, and the pressurization and depressurization controls the movement of the sample such that the sample reaches a desired position in the channel at a desired time and remains at such desired position for a desired time. The active feedback loop involving the position sensor results in pressurization or depressurization of the channel to provide appropriate control of the fluid sample through and within the channel.

[0043] The steps described above for a single channel can be applied to multiple channels to direct a fluid sample through multiple channels of a microfluidic cartridge using a single air source. For example, FIG. 2 illustrates an embodiment in which a microfluidic cartridge may receive a fluid sample through a common inlet connected to a first end of multiple channels. The sample at the common inlet is then split into multiple channels, with certain channels containing aliquots of the fluid sample, each aliquot having a respective channel, each aliquot having a predetermined volume, while other channels receive any excess fluid sample. The aliquot volumes may be the same or different. For example, in embodiments in which the aliquot volumes are different, such different aliquot volumes may be achieved by using different metering well shapes and / or by manipulating resistive regions within the channels to direct different amounts of the fluid sample to different metering wells.

[0044] Furthermore, at any instant in time, for any channel, the position of the aliquot of the fluid sample within the channel may be determined by the volume of air distal to the aliquot (i.e., downstream of the aliquot). Using the volume of distal air (which is the air downstream of the aliquot) to determine the position of the aliquot within the channel provides a method of determining the aliquot position that is independent of the shape of the channel and other variables (e.g., the viscosity of the fluid sample). The method of using an air spring design to determine the aliquot position and further adjust the aliquot position within the channel provides several advantages over conventional microfluidic designs. For example, if the volume of distal air trapped inside the cartridge is large enough (if the pressure within the system is low enough to allow the fluid sample to move relatively easily, thereby providing improved control over the movement of the fluid sample portions), small variations between or across channels that would otherwise cause channel resistances to differ and therefore result in inaccurate test results can be accommodated by using the actuation of the air springs of the system. The air spring system described herein can tolerate, for example, manufacturing imperfections that may result in non-identical channel geometries (where identical channel geometries are desired). Another advantage is that using an air spring system makes the cartridge suitable for different sample types, including fluid samples with different physical properties (e.g., viscosity). Compatibility across a wide range of different sample types is important, for example, for blood diagnostics, since blood samples of different hematocrits move differently through a microfluidic cartridge due to different viscosities.

[0045] Thus, unlike conventional systems, the air spring system described herein can accommodate a wide range of fluid properties (such as a wide range of viscosities, densities, compressibility, etc.). For example, under the same conditions, a more viscous fluid may take longer to reach a predetermined destination (e.g., a test chamber) within the cartridge than a less viscous fluid. The air spring system can accommodate such differences in viscosity and speed of fluid movement by adjusting pressurization within the channel based on the position of the fluid sample within the channel and / or the volume of distal air, and thus the cartridges described herein can be used with fluid samples that have significantly different physical properties (e.g., blood vs. saliva). Similarly, for a single fluid sample, individual aliquots of the sample may acquire different physical properties while within the cartridge (e.g., by exposing each aliquot to different conditions within the cartridge, e.g., channels with different reagents, as described herein), and the air spring system can accommodate such different physical properties of the individual aliquots by enabling independent adjustment of the position of each aliquot within the cartridge based on the volume of distal air and using the pressurization / depressurization features described above.

[0046] 3 shows the time-dependent flow in a Hagan-Poiseuille system, such as the embodiment of the air-spring microfluidic cartridge described herein. To ensure proper metering and therefore a known and determinable volume for each of the channels used in the test, the relationship between the pressure difference (P), volumetric flow rate (Q), and resistance to fluid flow (μ) is given by: ΔP=8μLQ / πr 4 where r is the cross-sectional radius of the cylindrical microchannel. In some embodiments, the resistance (R) is related to μ as well as the height (h), width (w), and length (L) of the microchannel as follows: R = 12 μL / h 2 w To ensure consistent measurements across multiple channels of a microfluidic cartridge and therefore accurate analysis, a metering step is first performed to ensure that each channel contains an aliquot of the fluid sample having a specific known volume. In some embodiments, this metering step further ensures that each aliquot of the fluid sample downstream of the metering is exposed to a known concentration of the reagent (or reagents). By applying pressure, the fluid sample flows into and through each channel. Furthermore, the shape of the channel may provide resistance to the flow, and such resistance may be localized to a specific region of the channel. Such resistance creates a back pressure, and such back pressure may contribute to a pressure difference (the difference between P1 and P2 in FIG. 3). Such back pressure may act as a way to slow down the flow of the fluid in the channel. Thus, selecting specific channel dimensions (such as channel cross-section and / or channel length) for the channels of the cartridge is a way to further control the speed of the movement of the fluid through the channel. The flow continues, and if there is a pressure difference, the fluid continues to move downstream (see, for example, FIG. 3).

[0047] 4A-4E are schematic diagrams illustrating the placement of fluids and proximal and distal air in time-dependent states in three channels of a single microfluidic cartridge. FIG. 4A illustrates the cartridge when a fluid sample is inserted into the common inlet, before the sample splits into the three channels and before metering occurs. In particular, FIG. 4A illustrates the cartridge in state 0 (t=0), where all pressures inside the cartridge are in equilibrium and there are no pressure differences. Then, according to the ideal gas law, splitting and fluid flow are subsequently accomplished, applying pressure to increase P1. For example, FIG. 4B illustrates the same cartridge in state 1 (t=1), where the fluid sample is pushed to a known or predetermined position using an air spring. FIG. 4B further illustrates the air trapped by the fluid sample in the three closed microfluidic channels, which is distal (downstream) of the fluid sample portion of each channel and associated with volumes V11, V21, and V31. The pressure associated with V11, V21, and V31 in each of the channels is equal to pressure P11, but there is a pressure difference in the channel leading to P21 (i.e., P11 ≠ P21). Over time (when pressure is applied to the inlet), pressure P11 becomes greater than pressure P21, and the fluid sample moves to the chamber associated with P21. Clearly, the total volume of the fluid sample inserted into the microfluidic cartridge (e.g., via inlet 100 in FIG. 5A) must be greater than the sum of all metered aliquots across each of the channels used for testing (e.g., channels associated with V11, V21, and V31 in FIG. 4B, channels associated with chambers 130a and 130b in FIG. 5A). FIG. 4C illustrates the cartridge where the pressure is increased to a predetermined state (designated state 2, t=2), filling each of the three channels with the desired fluid volume. The volume of fluid in each of the three channels is then increased, resulting in the air in each of the three channels being compressed, V12, V22, and V32 respectively decreasing and additional fluid being allowed to enter each channel until the desired fluid volume is achieved.At any instant when no fluid is moving, the pressure in each channel associated with distal volume V1, V2, or V3 is equal to pressure P1 and pressure P2 (as the system drives toward pressure equilibrium). When P1 rises and becomes greater than P2 (P1 is equal to the pressure associated with each of V1, V2, and V3), the fluid sample is expelled into the chamber associated with P2. After the desired metered volume of fluid in each channel is achieved and excess fluid sample is in the chamber associated with P2, the air associated with P1 and any air upstream (proximal to the fluid) of the aliquot of fluid sample is expelled to V1. x , V2 x , and V3 x is pushed towards the channel associated with V1 x , V2 x , or V3 x This point in the process (t=x, x>2) is shown in Figure 4D, ensuring isolation of each aliquot in the channel associated with the air. Air continues to push the aliquots through the channels until each aliquot reaches a measurement and / or sensing location (see Figure 4E).

[0048] The devices, systems, and methods described herein may be applicable to samples from any individual, including mammals (e.g., humans, such as human patients, as well as non-human mammals), reptiles, birds, and fish, among others, and may be useful in research and veterinary medicine. The individual may be, for example, mature (e.g., adults), immature (e.g., children, infants, newborns, or premature babies). The devices, systems, and methods described herein may also be used with environmental samples (e.g., to test for the presence of an analyte in a water sample).

[0049] The devices, systems, and methods described herein can be used for diagnosis and / or prognosis. For example, the devices, systems, and methods described herein allow for multiplexed testing of patient samples in a closed system. Such testing is particularly useful, for example, in the case of infectious hemorrhagic diseases (e.g., Dengue virus, Marburg virus, Ebola virus, etc.), where disease-specific cartridges can be used to both diagnose and predict the likelihood of hemorrhagic complications.

[0050] The devices, systems, and methods described herein may be used to guide patient treatment, including, but not limited to, in a hospital. For example, a physician may use a multi-channel microfluidic cartridge that allows multiplexed, independent testing of dispensed fluid samples without crosstalk between channels (see, e.g., cartridge embodiments in Figures 7, 10A-B) to perform the analyses described in U.S. Application No. 16 / 046,816 (published as US2019 / 0111431) and U.S. Application No. 17 / 195,615, for example, to ensure that a patient requiring anticoagulant therapy is receiving an appropriate dose of anticoagulant to elicit an appropriate physiological response.

[0051] The devices, systems, and methods described herein may also be used in the management, including but not limited to, clinical and preclinical evaluation of treatments (e.g., drugs), of diseases or conditions that result in pathological bleeding or clotting (e.g., COVID-19 caused by the SARS-CoV-2 virus, hemophilia, etc.).

[0052] The disclosed devices, systems, and methods can also be used in research and discovery. For example, the devices, systems, and methods described herein are useful for basic drug discovery, understanding the pathophysiology of diseases and illnesses, preclinical evaluation of novel compounds and biologics, and monitoring adverse events and off-target effects of experimental therapies.

[0053] Working Example 5A, 5B, 6A, 6B, and 7 show various embodiments of a closed microfluidic cartridge having an air spring system.

[0054] The schematic of Figure 5A illustrates a basic closed cartridge system having a plurality of channels (120), each of which (120a, 120b, and 120j) is connected at a first end to a common inlet 100 via a common channel 110, and each of which has a separate chamber or series of chambers (e.g., 130a, 130b, 160a) toward its second end. In some embodiments, each separate chamber of a channel may be a metering chamber 130 or a waste chamber 160.

[0055] In a cartridge employing such a design, a fluid sample is introduced (e.g., inserted) through a common inlet 100. Upon actuation of an actuator (not shown in FIG. 5A ) (see, e.g., feature 700 in FIG. 10B , feature 900 in FIG. 11 ), air is pushed upstream of the fluid sample, causing the sample to move along microfluidic channel 110 before splitting into channels 120a, 120b, and 120j. Each microfluidic channel 120a, 120b, and 120j includes at least one resistive element (121a, 121b, and 121j, respectively) that controls the rate of fluid flow within that channel, and thus the rate of fluid flow into each of the chambers within that channel (e.g., chambers 130a, 130b, and waste chamber 160a). In some aspects, such resistive features may include, for example, the geometry of the microfluidic channel (e.g., a change in the aspect ratio of the channel, or other change in the channel shape that reduces the channel volume, etc.). In some embodiments, the resistive features (e.g., 121j) may be, but need not be, physically separate from the channel (e.g., the resistance provided by 121j may be achieved, for example, via the shape of channel 120j, in which case the resistive features would not be separate from the channel). In some embodiments, channel 110 may split into three or more microfluidic channels (see, e.g., FIGS. 6A, 7, 10A).

[0056] In various embodiments of the devices, systems, and methods described herein, portions of the fluid sample may be metered after the fluid sample is split across multiple channels, such that the metered portions have equal fluid volumes (the metered volumes of the fluid sample are also referred to as aliquots). In one example, the resistance of 121a is equal to the resistance of 121b, and 130a and 130b function as metering chambers that receive equal amounts of the fluid sample at the same rate. It should be understood that in some embodiments, there may be more metering chambers than the two metering chambers illustrated in FIG. 5A when a channel (e.g., 110) branches into three or more microfluidic channels (see, e.g., FIGS. 6A, 7, 10A). For example, the microfluidic cartridges of FIGS. 6A, 7, 10A each show nine metering chambers (130a-130i). Generally, each channel used for testing includes an area that is a metering chamber.

[0057] In the cartridge embodiment shown in FIG. 5A, downstream of the metering chamber 130 is a test chamber 140 (also referred to as a sensing chamber or measurement chamber). In such an embodiment, each test chamber 140a and 140b may include a sensor (e.g., an electrode sensor) and / or facilitate some other measurement method (e.g., optical detection). The sensor may be included in the channel or may be external to the channel. As described herein, the sensor may include any component capable of measuring or otherwise measuring a property of the fluid sample, including, for example, measuring electrical impedance, measuring electrical volume, measuring electrical resistance, evaluating flow (e.g., evaluating the flow rate of beads present in the fluid sample), measuring flow rate, measuring pressure, measuring viscoelasticity, fluorescence detection (e.g., using fluorescent fibrinogen), measuring turbidity, infrared light detection, infrared spectroscopy, detection using an acoustic sensor, detection using a photonic sensor, flow cytometry, and methods of visual detection (e.g., visual clot detection).

[0058] In some embodiments, one or more test chambers 140 may also include a reagent (e.g., including multiple reagents) that may include clotting factors, calcium, fluorogenic or chromogenic substrates, clotting activators (e.g., glass, celite, phospholipids, kaolin, etc.), electrochemical reagents (e.g., for thrombin generation, etc.), and platelet activators (e.g., adenosine phosphates, collagen, ristocetin, epinephrine, etc.).

[0059] 6B, in some embodiments, between metering chamber 130 (e.g., 130a) and test chamber 140 (e.g., 140a) is a chamber (or series of chambers) for each of the channels used for testing. In such embodiments, at least one of these additional chambers may contain one or more reagents.

[0060] Each test chamber (or well, etc.) 140 (e.g., 140a) connects to a distal air spring 150 (e.g., 150a) via a chamber (or series of chambers) 210 (e.g., 210a). Each air spring is a closed well with a predetermined volume that, together with a waste chamber (160), controls the movement of fluid. In the design of the cartridge embodiments described herein, the volume of the air spring can be increased, and such an increase in volume reduces the system pressure (including the system pressure when the aliquot is at its final destination in the test chamber). Alternatively, the volume of the air spring can be decreased, and such a decrease in volume increases the system pressure (including the system pressure when the aliquot is at its final destination in the test chamber). Similarly, increasing or decreasing the volume of each waste chamber (160) affects how much pressure is in the cartridge and air spring system.

[0061] In various aspects, the cartridge may have two or more metering chambers 130a and 130b and / or one or more waste chambers 160a. Exemplary embodiments of such cartridges are shown, by way of example, in Figures 6A and 7. It should be understood by those skilled in the art that cartridge embodiments having a closed air spring system may include features not shown in Figure 5A (see, e.g., Figures 6A, 7) and / or not shown in certain embodiments described herein.

[0062] In some embodiments, it may be desirable to split the fluid sample in multiple successive steps. For example, sequential or modular splitting may improve metering time and / or fluid loading across multiple channels. Such improvements may be important in embodiments in which the fluid sample is split into multiple portions. In some embodiments, the fluid sample is split into three separate portions, each portion travels to its own channel, and each of the three separate portions is then split across three or more metering chambers and one or more waste chambers.

[0063] In certain aspects, the embodiment shown in FIG. 5A may include additional features, such as the features of region 113 shown in FIG. 5B. In such an embodiment, inlet 100 connects to reservoir 111, and resistor 112 controls and / or prevents backflow of the fluid sample. Reservoir 111 may function as an additional metering region, for example, before splitting the fluid sample into multiple metering chambers and one or more waste chambers. In some embodiments, resistive feature 112 may include, for example, a Tesla valve, a serpentine microfluidic path, and / or a porous membrane. In some embodiments, it may be desirable to combine multiple resistive elements (e.g., multiple resistive regions in parallel or series). Furthermore, in some embodiments, the paths connecting each of the chambers in the channel (e.g., from 130 to 140 via 200, from 140 to 150 via 210) may also include resistive features 201 and / or 211 (see, for example, FIG. 5B). In some aspects, resistance to fluid flow may be achieved via one or more geometries (including the shape and / or dimensions of that shape) of the pathway between the chambers (e.g., 200, 210 (including only certain regions)), such that there is no separate corresponding resistor apart from the channel itself.

[0064] In certain embodiments of the cartridge described herein, the fluid sample may be split into at least 12 channels, at least 9 channels (120a-i) each containing an aliquot of the fluid sample, and 3 waste channels (120j-l), see, for example, the embodiment illustrated in FIG. 6A. The fluid sample may be split into at least 12 channels at a time, or the fluid sample may be first split into less than 12 portions followed by subsequent splitting (e.g., the fluid sample may be split 2, 3, 4, etc. times). For example, in the embodiment of FIG. 6A, the common inlet 100 connects directly to the microfluidic channels 120a-l via a common channel 110 (compare, for example, FIG. 5A). In other embodiments, such as the embodiment of FIG. 10A, the fluid sample branches into 3 channels, each such channel further branches into 3 channels (see channels labeled 120a) before (upstream) the metering chamber 130.

[0065] In various embodiments, the fluid sample may flow to the first fluid reservoir 111 and / or resistor 112 prior to splitting (see, e.g., FIGS. 5B, 7). As shown in FIG. 6B, in some embodiments, one or more of the channels in the cartridge may further include an additional path (e.g., an additional chamber, or a series of additional chambers) and / or resistive features between the metering chamber (130) and the test chamber (140). In some aspects, each of the microfluidic channels (at one or more locations along the path of each channel, e.g., 120, 200, 210) may, but need not, include a resistive element. FIG. 7 provides a schematic diagram of an embodiment having resistive elements at various locations along the channel path.

[0066] In certain embodiments in which the cartridge has additional chambers and / or resistive features between the metering chamber (130) and the test chamber (140), each of the additional chambers and / or resistive features between the metering chamber (130) and the test chamber (140) in each of the channels of the cartridge (e.g., the cartridge illustrated in FIG. 7) may serve a particular purpose or provide a particular function. For example, as described above, one or more chambers between the metering chamber (130) and the test chamber (140) may contain one or more reagents.

[0067] In certain embodiments (e.g., those shown in Figures 6B and 7), the various chambers and features contained in the closed microfluidic cartridge and the channels therein are: a. splitting a fluid sample into a plurality of analytical channels (e.g., 120a-i) and at least one waste channel (e.g., 120j); b. isolating each portion of the fluid sample in each of the analytical channels (e.g., 120a-i), thereby isolating such portion from the portion of the fluid sample in each of the waste channels (e.g., 120j-l), and thus in each of the waste chambers (160a-c); c. separating each of the portions of the fluid sample in each of the analytical channels (e.g., 120a) from each of the other portions of the fluid sample in each of the other analytical channels (e.g., 120b-i); d. Metering a portion of the fluid sample in each of the analytical channels (such as chambers 130a-i), thereby ensuring that each portion in a chamber downstream of the metering (e.g., a reagent chamber, a test chamber (or well), etc.) is of a known volume (such volumes across the channels may or may not be equal to one another); e. introducing each of the portions (called aliquots) of known volume into one or more reagents (such as in a chamber illustrated by 300); f. mixing each aliquot of the fluid sample with one or more reagents (e.g., in chamber 310); and g. measuring or otherwise analyzing one or more physical and / or chemical properties of each aliquot of the fluid sample (e.g., in chamber 140) to enable assessment of the response of each aliquot to one or more reagents; It is intended to be so. 6B and 7 show such a cartridge, which may be used, for example, to detect and / or assess blood clotting.

[0068] In some embodiments, the cartridge may have multiple stacked layers, and the channels and / or chambers of the cartridge may be made of different materials and / or lie along different planes. Figure 8 shows one such example, where the chambers along one path of the microfluidic channel of Figure 7 (e.g., the channel having 120a, 130a, 300a, 310a, 140a, and 150a) are three-dimensionally shaped and positioned to achieve various functions (e.g., items a through g above). In a preferred embodiment, the reagent chambers and mixing chambers are sized and positioned relative to each other and to the channels connecting them, such that the reagent chamber 300 may accommodate an aliquot of a fluid sample up to 1.10 μL, the mixing chamber 310 may accommodate an aliquot of a fluid sample up to 2.55 μL, and the channel path 400 connecting them accommodates an aliquot of a fluid sample up to 0.85 μL. In some embodiments, the volume of the mixing chamber (and the volume of the fluid sample aliquot within the mixing chamber) may be up to 5 times larger or 1.5 times smaller than the volume of the reagent chamber (and the volume of the fluid sample aliquot within the reagent chamber). In various aspects, the ratio of the volume of the fluid sample aliquot within the reagent chamber to the volume of the fluid sample aliquot within the mixing chamber may depend, for example, on the hydrophilic behavior of one or more reagents, the properties of the fluid sample, and / or the properties of the interior surface of the reagent chamber (e.g., hydrophobicity, hydrophilicity, patterning, etc.).

[0069] In certain embodiments, the configuration illustrated in FIG. 8 ensures that one or more reagents are thoroughly mixed with an aliquot of the fluid sample through a power wash process performed in a reagent chamber and then mixed in a larger volume mixing chamber 310 connected to the test chamber 140.

[0070] In some embodiments, this step of power washing (high pressure washing) followed by mixing may be particularly desirable, for example, when one or more reagents have been dry spotted onto or within the microfluidic cartridge. The effect of power washing is shown in Figures 13C-13F.

[0071] FIG. 13A shows the spotting of droplets with different color dyes, and FIG. 13B shows the same droplets once dried ("dried spots") and covered with a polycarbonate sealing layer placed on top to completely form the channels. The different color dyes are dry spotted into the reagent chambers, each containing red and / or yellow dye (at different concentrations). The test chamber contains a spot of blue dye. Not visible in FIG. 13A, there is no dye spot in the mixing chamber. FIGS. 13C and 13D show images of water flowing through the microfluidic channels in the cartridge. FIG. 13C shows the color gradient in the reagent chambers as the water flows and brings the dried colored spots (red and / or yellow dried dyes) into solution, where the dried dyes are used to show examples of reagents such as clotting agonists. As shown in FIG. 13D (which shows the dyes dissolved homogeneously in the water in the mixing chamber), when each fluid sample aliquot is in the mixing chamber of the microchannel, the color dyes mix thoroughly with the fluid sample (water in this demonstration). This demonstration with dyes also shows that there is no color mixing or bleed-over (leakage) between the nine channels with parallel fluid flow, in other words, there is no cross-interference between the channels, and therefore multiple aliquots can be tested simultaneously. Figures 13E and 13F show images of the cartridge as water aliquots mixed with red and / or yellow dyes are pushed further downstream into the test chambers of each microchannel containing the blue dye.

[0072] Figures 13A-13F illustrate two important concepts: 1) the dye color used to represent the reagent (red and / or yellow dye) in each microchannel is completely mixed with the fluid sample aliquot by the time the aliquot with the reagent reaches the test chamber (see Figure 13D), and 2) the reagent (represented by the blue spot in this figure) dry spotted directly onto the test chamber is not completely mixed with the fluid sample aliquot in each microchannel, as seen by the gradient of blue color in each test chamber (see Figures 13E, 13F). Figure 13E shows the test chambers, each of which has a color gradient of blue dye. In the microchannel with the reagent chamber containing the red dye representing the reagent, if the red dye had completely mixed with the blue spot in the test chamber, the overall color of the test chamber should have been purple, but this did not happen. Similarly, in a microchannel with a reagent chamber containing a yellow dye representing a reagent, if the blue spot in the test chamber mixed completely with the fluid sample, the color of the entire test chamber should be green (a combination of the yellow from the reagent in the reagent chamber and the blue from the reagent in the test chamber), but this did not happen (see also FIG. 13F). Instead, only a relatively small portion of the test chamber turned green, while the majority of the test chamber retained the dye color from the reagent chamber. This figure shows that power washing in the reagent chamber followed by mixing in the mixing chamber provides that the aliquot and reagent are well mixed by the time the aliquot reaches the test chamber.

[0073] In some embodiments, a cartridge (such as 60 in FIG. 10B) may have an array of components. For example, the cartridge may have a cartridge body 600, which may include a plunger 601, a filter plug 608, a support ring 605, and a septum 602. The cartridge may further have a first pressure sensitive adhesive (PSA) layer 606, a second PSA layer 607, and a printed circuit board 800. The PSA layers 606 and 607 and the printed circuit board 800 may further be aligned with each other and with the cartridge body 600, and may be attached or bonded (e.g., heat sealed, thermally welded, laser welded, etc.) to each other and to the cartridge body 600. The first PSA layer 606 and the second PSA layer 607 may have either a single-sided adhesive or a double-sided adhesive. In some embodiments, a back seal 604 may also be bonded to the bottom of the cartridge body 600 to form a microfluidic channel.

[0074] In some embodiments, after insertion of the fluid sample, the fluid sample is moved through the microfluidic cartridge by pushing air within the cartridge. The air may be pushed via an integrated plunger 601 (see, e.g., FIG. 10B). For example, an actuator may push the plunger 601, causing it to move the entire length (or a portion of the length) of the integrated syringe 700, moving the fluid sample to a desired location within the cartridge at a particular moment. In some embodiments, the cartridge may alternatively be equipped with a portion 901 that includes a bellows 900 that is actuated to move the fluid sample through the cartridge (see, e.g., FIG. 11).

[0075] A variety of materials (e.g., acrylic, polycarbonate, glass, silicon, etc.) and manufacturing methods may be used to create the cartridge body 600. For example, one or a combination of existing manufacturing methods (e.g., injection molding, machining, laser etching, lamination (with or without photoablation and / or sacrificial layers, which may include any combination of the foregoing)) may be used. In some embodiments, the tests and test conditions used (e.g., reagents used, temperature, etc.) inform the selection of materials used to manufacture the cartridge body.

[0076] In various aspects, the cartridge may have one or more layers of PSA. In some embodiments, the PSA layer may allow for fluid connections to be formed between layers (and different planes) of the cartridge (in such cases, a PSA layer with a specific aperture pattern may be used). The PSA layer can be mass produced and cut with a commercial laser cutter. Depending on the desired resolution of the apertures, small volume production is also possible with a commercial laser cutter. Furthermore, depending on the pattern and shape (or geometry) of the apertures, the PSA layer can be punched to create the desired pattern and / or geometry.

[0077] In embodiments where the cartridge has a printed circuit board (PCB), such as PCB 800 shown in the embodiment of FIG. 10B, such PCB may be rigid or flexible, or may have layers where one or more of such layers are flexible and other layers are rigid. In an exemplary embodiment, a flexible Kapton® PCB is attached (e.g., glued, laser welded, etc.) to a rigid layer (such as a rigid PCB). In some embodiments, the flexible PCB allows for fluorescence measurements. PCB 800 may also include a heater element within the electronics. For example, including a heater element may be desirable for coagulation testing or polymerase chain reaction (PCR) testing. One or more temperature sensors and / or heater elements may be incorporated into the PCB to achieve and / or maintain a desired temperature over one or more specific regions of the cartridge, such as the region of the cartridge that contains the fluid sample. In some embodiments, each test chamber may have its own temperature sensor and / or heater element. In further embodiments, individual temperature sensors and / or heater elements may be localized to individual regions of the cartridge, allowing each aliquot to be exposed to different temperatures and / or different temperatures to be applied to different portions of each channel. Temperature measurements may also be used by the test unit for a feedback temperature control system.

[0078] A cartridge having a closed system, in which each aliquot of a fluid sample is compartmentalized, may be used to simultaneously perform multiple tests (e.g., to assess clotting in each of the aliquots) as described herein. In embodiments including clotting tests, such tests may include assessing activated clotting time, prothrombin time, activated partial thromboplastin time, dilute thrombin time, or thrombin time. Such tests may include clotting factor activity assays, chromogenic assays (e.g., to assess anti-factor Xa activity), fluorescent-based assays (e.g., fluorescent fibrinogen-based assays), electrochemical-based assays (e.g., thrombin generation), and any of the functional assays disclosed in U.S. Application No. 16 / 046,816 (published as US2019 / 0111431) and U.S. Application No. 17 / 195,615, the contents of each of which are incorporated herein by reference in their entirety.

[0079] In some embodiments, the electrochemical properties of a fluid sample aliquot may be evaluated, for example, for the measurement of one or more electrolytes (such as glucose or lactate) and / or biomarkers (genetic or protein biomarkers). Such analyses may be accomplished by the addition of special reagents, the use of electrode coatings, and / or functionalization of electrodes.

[0080] In some embodiments, an enzyme-linked immunosorbent assay (ELISA) may be performed within the microchannel, such as for detection and quantification of one or more biomarkers and / or antibodies. ELISA may be performed using electrodes that may or may not be functionalized (e.g., directly with antibodies, with nano- or micro-gold particles, and / or with carbon nanotubes), or may be performed using agglutination, fluorescence, magnetic beads, chromogenic methods, sensors inside or outside the test chamber, or any combination thereof.

[0081] Separating each of the fluid sample aliquots so that each aliquot moves through a single microchannel allows, in some embodiments, a single cartridge to be used to perform multiple platelet activation and inhibition tests, including agglutination measurements, adenosine triphosphate (ATP) generation (e.g., via luciferase measurements), platelet adhesion (e.g., using imaging, or mechanical and / or electrical sensors), and / or platelet count (e.g., via imaging and / or other sensors). In further embodiments, both coagulation tests and platelet tests can be performed using a single cartridge.

[0082] In some embodiments, PCR can be performed - for example, traditional PCR using temperature cycling or isothermal reactions, or newer PCR methods such as those using clustered regularly interspaced short palindromic repeats (CRISPR).

[0083] Various types of sensors may be incorporated into the test chamber to perform any of the tests described above or otherwise desired. Such sensors include, for example, gas sensors that can be used to detect pathogens, toxins, volatile organic compounds (VOCs), or other harmful chemical or biological substances. In some embodiments, such sensors may be used to evaluate the chemical composition of the fluid sample in the test chamber. Other embodiments may include electrode sensors as described above. Additionally, in some embodiments, the cartridge may have sensors in addition to the sensor used to evaluate the sample aliquot in the test chamber. Such additional sensors may be located at points along the fluid path that are not within the test chamber (e.g., additional sensors may be located upstream of the test chamber). Additionally, additional sensors may be located inside the channel or outside the channel. For example, in the microfluidic device of FIG. 10A, an electrode pair may be included that may be used to evaluate the hematocrit amount in a blood sample, as shown in FIG. 9.

[0084] In some embodiments, materials used to manufacture the cartridge may be transparent to allow optical measurement and imaging of the fluid sample aliquots within the channels. For example, in a multi-layer cartridge, one or more layers may be transparent or otherwise allow optical measurement or imaging. Such optical measurement or imaging may be performed at one or more locations of the channel, including locations that are not test chambers. Additionally, such cartridges may or may not have electrode sensors for measuring electrical properties (e.g., electrical impedance) of the sample aliquots.

[0085] In some embodiments, a test may require exposing one or more sample aliquots to multiple reagents at various times (e.g., by first exposing an aliquot to one reagent and then exposing the aliquot to another reagent) and may require heating and / or cooling of the fluid sample aliquots. For example, a coagulation test may require heating a cartridge containing a fluid sample to 37° C., and in further embodiments, a test may require heating the cartridge and / or a portion of the fluid sample to the patient's body temperature to approximate physiological conditions.

[0086] In various embodiments, it may be desirable for the microchannels of a closed microfluidic cartridge to promote some fluid retention on their surfaces (described herein as sacrificial fluid retention) to compensate for evaporation of the sample within the cartridge during testing, especially when the testing process involves multiple successive exposure conditions and / or may take longer than 5 minutes (e.g., up to 60 minutes) to complete (see, e.g., FIG. 14 , which shows the fluid sample (stained with a dark dye) along the inner wall of the microchannel even after the fluid sample aliquot has traveled to the test chamber). In some cases, considering the air (or other gas) in contact with the fluid sample (proximal or upstream, distal or downstream), and the temperature within the cartridge, evaporation of the fluid sample may occur within each microchannel until the system reaches equilibrium. In devices using micro- or nano-droplets for testing, evaporation of the sample may be an issue. Typically, these systems incorporate a "sacrificial" droplet at a specific point in the device, with the purpose that such a "sacrificial" droplet, rather than the sample, evaporates, so that the sample is maintained in a liquid state at a constant volume. In certain embodiments of the invention, as shown in FIG. 14, the materials used in assembling the cartridge components may be selected or treated such that their surfaces in contact with the fluid sample promote retention of a constant amount of the fluid sample along a specific path of each microfluidic channel (or at least the channel that is used for testing and has a test chamber). For example, if the exposed surface area of ​​the fluid sample when it is in the test chamber (the area of ​​the fluid sample that contacts air or other gas when it is in the test chamber) is smaller than the exposed surface area of ​​the sacrificial fluid retention along the walls of the microchannel, the sacrificial fluid will preferentially evaporate towards the equilibrated system, minimizing evaporation of the fluid sample and thus minimizing the impact on the sample volume in the test chamber. The channel surfaces may be coated (e.g., with a hydrophilic coating) to promote retention of the sacrificial fluid.In certain embodiments, the use of a PSA layer to form the walls of a microfluidic channel can provide a channel surface that promotes retention of the sacrificial fluid, for example a PSA layer made of polycarbonate, which tends to trap the fluid sample as it passes through the channel, thereby retaining the sacrificial fluid. This sacrificial fluid spreads along the PSA lining the sides of the channel, and the sample in the test chamber is exposed to air (or other gas) on only two sides (upstream and downstream, the surface areas of which can be further controlled by the channel geometry), resulting in a larger surface area of ​​the sacrificial fluid exposed to air (or other gas) in the cartridge compared to the exposed surface area of ​​the sample in the test chamber.

[0087] While the present invention has been particularly shown and described with reference to specific embodiments thereof, it will be understood by those skilled in the art in light of this disclosure that various changes in form and detail may be made therein without departing from the scope of the invention, as encompassed, for example, by the appended claims.

Claims

1. 1. A microfluidic cartridge comprising: an inlet configured to receive a fluid sample; three or more channels having upstream ends connected to the inlet; It has At least two of the channels are test channels, each test channel having a metering chamber and a test chamber; at least one of the channels is a waste channel having a waste chamber; The microfluidic cartridge is a closed system such that air cannot be released from the channel once a fluid sample is received at the inlet.

2. Each test channel further includes a reagent chamber. The microfluidic cartridge of claim 1 .

3. The microfluidic cartridge further comprises two or more pairs of electrode sensors.

3. The microfluidic cartridge of claim 1 or 2.

4. each pair of electrode sensors is positioned to measure an electrical property of the fluid sample; The microfluidic cartridge of claim 3 .

5. the microfluidic cartridge further comprises two or more fluid position sensors; 3. The microfluidic cartridge of claim 1 or 2.

6. Each fluid position sensor is a pair of electrode sensors. The microfluidic cartridge of claim 5 .

7. each fluid position sensor configured to detect the amount of fluid sample in one of the test channels; The microfluidic cartridge of claim 5 .

8. the microfluidic cartridge having at least five test channels; 3. The microfluidic cartridge of claim 1 or 2.

9. The microfluidic cartridge having at least five test channels. The microfluidic cartridge of claim 3 .

10. The microfluidic cartridge having at least five test channels. The microfluidic cartridge of claim 4 .

11. The microfluidic cartridge having at least five test channels. The microfluidic cartridge of claim 5 .

12. A method for evaluating a fluid sample, comprising inserting the fluid sample into the inlet of the microfluidic cartridge of claim 3.

13. A method for evaluating a fluid sample, comprising inserting the fluid sample into the inlet of the microfluidic cartridge of claim 4.

14. A method for evaluating a fluid sample, comprising inserting the fluid sample into the inlet of the microfluidic cartridge described in claim 5.

15. A method for evaluating a fluid sample, comprising inserting the fluid sample into the inlet of the microfluidic cartridge of claim 7.

16. The method comprises dividing the fluid sample into three or more of the channels connected at their upstream ends to the inlet, such that the fluid sample is divided into three or more portions, having at least two portions divided into the test channel and at least one portion divided into the waste channel. The method of claim 13.

17. and measuring a specific volume of each divided portion into one of the test channels, the measuring being performed in the measuring chamber of each test channel.

17. The method of claim 16.

18. all portions divided into the test channel are metered to the same volume; 18. The method of claim 17.

19. The method comprises dividing the fluid sample into three or more of the channels connected to the inlet at their upstream ends, such that the fluid sample is divided into three or more portions, having at least two portions divided into the test channel and at least one portion divided into the waste channel.

15. The method of claim 14.

20. The method further comprises weighing each divided portion of one of the test channels to a specific volume, the weighing being performed in the weighing chamber of each test channel.

20. The method of claim 19.

21. All portions divided into the test channel are metered to the same volume.

21. The method of claim 20.

22. The method comprises dividing the fluid sample into three or more of the channels connected at their upstream ends to the inlet, such that the fluid sample is divided into three or more portions, having at least two portions divided into the test channel and at least one portion divided into the waste channel.

16. The method of claim 15.

23. The method further comprises metering each divided portion of one of the test channels to a specific volume, the metering being performed in the metering chamber of each test channel.

23. The method of claim 22.

24. All portions divided into the test channel are weighed to the same volume.

24. The method of claim 23.

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