Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502723—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by venting arrangements
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0605—Metering of fluids
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/16—Reagents, handling or storing thereof
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0627—Sensor or part of a sensor is integrated
  • B01L2300/0645—Electrodes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0627—Sensor or part of a sensor is integrated
  • B01L2300/0663—Whole sensors
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0874—Three dimensional network
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/18—Means for temperature control
  • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
  • B01L2300/1827—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
  • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
  • G01N33/5438—Electrodes

Definitions

• the present invention generally relates to devices, systems, and methods for evaluating multiple, isolated portions of a fluid sample.
• the devices, systems, and methods described herein can be used for various applications, including, for example, in medical diagnostics for evaluating biological fluid samples, such as for assessing coagulation in a blood sample.
• microfluidics for diagnostics and other applications
• Most current microfluidic cartridges are open systems, where there is open communication between the internal contents of the cartridge and the external environment or a portion of an automated reader, such as a syringe.
• Some of these systems are stated to minimize the chance of contamination to or from the outside environment or reader parts, such as through the use of specialized filters located near the outlet or inlet ports.
• none of these systems eliminates the risk of contamination. Contamination can be dangerous, especially when working with biological or environmental samples that may be harmful upon aerosolization.
• an open system with filters is not suitable for evaluating gas-phase fluid samples, as the gas in such samples may escape through the pores of the filters.
• valves may be “liquid-tight” but not “air-tight,” and thus air may escape through such valves.
• Other cartridges may include a waste or other chamber that is open (e.g., a vented waste chamber) and not closed, in order to reduce the internal pressure in 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 in such cartridges serves as an outlet and opening to the external environment.
• the present invention provides devices, systems, and methods for evaluating a fluid sample.
• the devices can be used, for example, for the in vitro evaluation of a blood sample, to assess a subject’s coagulation status and/or phenotype.
• the devices also can be used to detect one or more analytes in a fluid sample.
• An example device comprises a microfluidic cartridge having a plurality of channels, each channel of the plurality of channels configured to receive a portion of the sample through a common inlet connected to a first end (which may be referred to as the upstream end) of the plurality of channels, and each channel of the plurality of channels leading to a downstream chamber (or series of chambers, etc.) in the channel.
• a 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.) in which some measurement is taken and/or some other analysis occurs.
• a chamber (or well, etc.) where analysis occurs is referred to herein as a testing chamber, sensing chamber, measurement chamber, testing well, sensing well, etc. Downstream of each of the testing chambers 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.
• Embodiments of the present invention also provide a device for evaluating a fluid sample, the device comprising: a microfluidic cartridge having a plurality of channels, which cartridge accepts 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 the inlet), and wherein the plurality of channels comprises at least three channels, such that the fluid sample is split into at least three channels.
• the channels each has a predetermined target volume (e.g., a metered volume), and in further embodiments, the predetermined target volume is dictated by the geometry of each such channel.
• each of the at least two channels may comprise a metering chamber, such that the fluid sample that is in each of the at least two channels fills one or more metering chambers.
• the fill of the metering chamber in each of such channels is based on pressure differentials caused by the size and shape of each of the channels, as described herein.
• At least one of the channels of the plurality of channels comprises a waste chamber at its downstream end.
• the cartridge may be a microfluidic cartridge, chip, or other device, and the plurality of channels may be microfluidic channels.
• the methods can be performed using a device requiring less than about 1 mL, less than about 500 pL, less than about 100 pL, or less than about 50 pL, or about 5 pL or less, of a fluid sample (e.g., in some embodiments, about one drop of fresh whole blood or whole blood anti coagulated with sodium citrate, is sufficient).
• channel width, channel height, or both may be less than 10 mm, less than 6 mm, or less than 2 mm.
• a single channel may have varying dimensions (e.g., width, height, radius, etc.) along a length of the channel (for example, certain regions along a channel’s path may be wider or have a greater radius than other regions); in addition, one or more of the plurality of channels may have different dimensions than the other channels.
• the channels (and the chambers and other regions along the length of each channel) may be of various shapes, such as, for example, equilateral polygons (e.g., hexagon), asymmetrical polygons (e.g., a six-sided polygon with various side lengths), or straight channels (e.g., a rectangular or cylindrical channel).
• the corners and edges where two faces meet may be rounded (e.g., to promote fluid flow through the channel).
• one or more of the channels of the plurality of channels may have the same volume, or the volume may vary from channel to channel.
• one or more of the plurality of channels comprises multiple functional regions (e.g., a metering chamber, a reagent chamber, a mixing chamber, a sensing chamber, etc.) connected in series, all such regions may have the same volume, or volume may vary from region to region (e.g., in a preferred embodiment, a metering chamber may have a volume of about 3 pL, a reagent chamber may have a volume of about I pL, and a sensing chamber may have a volume of about 2 pL).
• the material(s) that form the channels (and thus the material(s) that line the interior surface of the channels, for example) may be the same across one or more channels or may differ from channel to channel.
• the channels’ relative locations to one another also may vary, depending on the embodiment.
• the channels all may be formed within the same substrate and located on the same plane; the channels all may be formed within the same substrate, but may be situated on different (e.g., parallel) planes; the channels may be formed in multiple substrates (e.g., in some embodiments, by layering materials) and located on a single plane; or the channels may be formed in multiple substrates and located on different planes (e.g., as in a multi-planar cartridge with some channels formed within one plane of a substrate and other channels located on another plane, and wherein at least one of the walls of the channels on one or more planes is comprised of a second substrate, such as a layered material, e.g., pressure sensitive adhesive).
• a second substrate such as a layered material, e.g., pressure sensitive adhesive
• the cartridge may contain sensors (e.g., electrode sensors).
• the part of the device that houses the sensors may also form one or more of the walls of one or more of the channels.
• one or more of the walls of one or more of the channels may be made from the sensor itself — e.g., as in a multi-layer PCB (printed circuit board) with one or more exposed electrode tracings.
• an exposed electrode tracing may act as a sensor, or it may serve other purposes, such as, e.g., serving as an integrated heating or cooling unit.
• Sensors may be exposed electrical tracings (e.g., a pair of electrodes, interdigitated electrodes, etc., for measuring impedance) or may be other types of sensors attached to a PCB (e.g., a photodiode, camera lens, spectrophotometer, photonic sensor, piezo sensor, etc.).
• a PCB e.g., a photodiode, camera lens, spectrophotometer, photonic sensor, piezo sensor, etc.
• the PCB may include a stimulant (e.g., a light source, etc.) and a receiver (e.g., a photodiode, etc.).
• the cartridge may include multiple sensors, where the sensors are of the same type (e.g., electrode sensors that measure an electrical property, such as impedance) or of various types.
• a cartridge may comprise one type of sensor on one side and a different type of sensor on the other side; for any sensor, the sensor may be part of or attached to a PCB, as discussed above.
• a multi-layer cartridge may comprise a PCB as a layer, wherein the PCB comprises a sensor or sensors, and the cartridge may further comprise additional sensors within or on one or more other layers.
• Sensor layers can be made from multiple individual or connected PCBs, such as a PCB that is a bottom layer of a cartridge and that communicates with a PCB that is a top layer of a cartridge; such communication may be achieved via a light source and photodiode, etc., where the PCBs are positioned such that they measure activity and/or characteristics (e.g., by intercepting signals) of a portion of a fluid sample in a channel.
• two PCBs can align with each other to form a channel, while each PCB may take different measurements (e.g., one PCB comprises sensors that measure electrical impedance, and a second PCB comprises sensors that take optical measurements, such as for machine learning, artificial intelligence, etc.).
• two PCBs each PCB comprising sensors
• a PCB may comprise electrodes, for example, as sensors to measure a property of the fluid sample and perform testing, or for sensing the position of the fluid (e.g., as fluidic position sensors).
• the cartridges as described herein may comprise fluidic position sensors.
• Fluidic position sensors may be based on electrical impedance using electrodes, for example (and as mentioned above), or may be made of another component attached to the PCB.
• fluidic position sensors may provide active feedback to the external component actuating the cartridge, e.g., to ensure fluidic movement within the cartridge and/or for precise control of fluidic movement.
• Such a feature, depicted, for example, in the embodiments illustrated in the figures, is particularly advantageous, as it allows a single cartridge design to be suitable for testing various types of fluid samples with varying viscosities (e.g., blood samples of varying hematocrits, urine and saliva, etc.).
• a fluidic position sensor may provide feedback to an external control system; such sensor and feedback may be used to detect a fluid’s position within a cartridge and make appropriate adjustments to fluid flow (in certain embodiments, e.g., to confirm that a minimum required fill has been achieved) or to control fluidic movement (in various embodiments, e.g., by applying a particular amount of pressure in response to input received from the fluidic position sensor).
• a fluidic position sensor (which may be a pair of electrode sensors or may be another sensor that measures light, ultrasound, radiofrequency, absorbance, etc.) is used to detect the fill volume of a metering chamber, and once the fill volume of the metering chamber has reached a specific level, the speed or pressure applied to the actuator is modified (e.g., reduced) such that the remainder of the fluid sample that has not yet been split into a channel moves to a channel comprising a waste chamber instead of to a channel comprising a metering chamber.
• the microfluidic cartridge may comprise sensors that measure hematocrit in the sample. Hematocrit information can be used to assess the sample’s viscosity. Sensors that measure hematocrit and provide such hematocrit information to an external control system can be used to control and adjust fluidic movement via sensor feedback.
• certain layering materials may be selected (e.g., glass, either for a whole layer or to make optical windows, to facilitate measurement using optical sensors), or certain coatings may be applied (e.g., to alter surface topology), and/or certain treatments may be used (e.g., treating a region of the cartridge with a chemical, such as to make a surface hydrophilic or hydrophobic, or with a biological material).
• the material(s) of the cartridge body and/or cartridge layers may be selected to facilitate the fluidic dynamics that are best suited for the type of testing to be conducted in the cartridge (e.g., a plastic, such as polycarbonate, is more hydrophobic than glass and may be better suited than glass for certain types of analyses).
• the materials used may affect fluidic movement and the pressure within the system, similar to how viscosity of the fluid or the location of the fluid within the cartridge (e.g., whether the fluid is in the middle of a large chamber or is in a comparatively narrow resistor) can affect fluidic movement.
• a more hydrophobic material lining the surface of the channels can help reduce capillary action and improve accurate metering, especially when the system is under sufficiently low pressure such that capillary action is possible.
• a cartridge may be a reusable cartridge and may comprise features that facilitate cleaning the cartridge prior to reuse.
• the cartridges described herein may be made of a single material or of multiple materials.
• a plurality of channels is formed in a substrate, which can be made of one or more of various materials (e.g., acrylic, polycarbonate, liquid crystal polymer, etc.), and by any one or any combination of methods (e.g., injection molding, machining, laser etching, layering, etc.).
• the cartridge comprises a primary substrate and one or more layers.
• a plurality of flow paths may be formed in the primary substrate such that, in the substrate itself, the plurality of flow paths is open on one side (for instance, on the top side of the flow paths), and one or more cover layers may be attached to the primary substrate to fully enclose the flow paths molded in the substrate and form channels. While various embodiments are discussed with reference to “channels,” other terms (e.g., lanes, compartments, partitions, etc.) can describe spaces that are physically separated from each other and that, in some embodiments, have the same geometry as each other.
• a cartridge has a common inlet connected to the first end of a plurality of channels.
• the common inlet is the cartridge’s only opening.
• a cartridge may have one or more other openings, in addition to a common inlet.
• a cartridge may have a common inlet and a vent port. Regardless of how many openings a cartridge may have, it is an essential feature of such embodiments that each opening be closed after sample input and prior to effecting movement of the fluid sample through the plurality of channels in the cartridge.
• a vent port (if present) may comprise a porous membrane that swells, such that its pores close, when it is wetted by a fluid sample.
• the cartridge may have no internal moving parts.
• a cartridge may comprise a bellows, which can be actuated by an external component so as to push the volume of air contained in the bellows into the cartridge, as a way to control internal pressure and hence fluidic movement.
• the cartridge may have a single moving part, which pressurizes the system and moves the fluid sample through a plurality of channels.
• a cartridge may include, for example, an integrated syringe with a plunger; in such embodiments, an external actuator may move the plunger to control pressure in the system.
• the external component can selectively pressurize or de-pressurize the system in order to move the fluid forward or backward (e.g., for purposes such as mixing fluid, to move fluid to a particular position within a testing chamber, etc.).
• the geometry of each of the plurality of channels may incorporate air springs and resistive regions.
• the cartridge may lack active control elements (other than, e.g., an actuator as described herein), valves, vents, and hydrophobic membranes.
• control elements may be used to allow the closing of the system; such elements include, for example, a porous membrane, which swells so that its pores close upon contact with the fluid sample.
• the cartridge may use forward and backward movement of the fluid sample (such as for mixing of the fluid sample with a reagent).
• fluid flow may be unidirectional.
• a fluid or fluid sample may include, without limitation, a sample of bodily fluid (e.g., blood, plasma, saliva, urine, cerebrospinal fluid (CSF), peritoneal fluid, thoracic fluid (including pleural fluid), or pericardial fluid), a sample of bodily gases (e.g., a sample from exhalation), chemical and/or environmental samples (including, without limitation, a sample of a chemical in liquid or gaseous form), a sample of water, or a sample of air.
• a sample of bodily fluid e.g., blood, plasma, saliva, urine, cerebrospinal fluid (CSF), peritoneal fluid, thoracic fluid (including pleural fluid), or pericardial fluid
• a sample of bodily gases e.g., a sample from exhalation
• chemical and/or environmental samples including, without limitation, a sample of a chemical in liquid or gaseous form
• a sample of water or a sample of air.
• a “blood sample” refers to a whole blood sample or a plasma sample.
• the term plasma includes both platelet-rich-plasma (PRP) and platelet-poor-plasma (PPP).
• PRP platelet-rich-plasma
• PPP platelet-poor-plasma
• the blood sample can be a whole blood sample or a plasma sample. Using whole blood can be particularly useful for certain applications, such as those implemented at the bedside of a patient.
• the fluid sample is a gas and the medium used to pressurize the system and move the sample downstream through channels can be another gas or air or, in some embodiments, may be a liquid.
• the medium used to pressurize the system may be selected so as to minimize reactivity with the sample and permeability to the sample.
• a medium other than air there may be a container (e.g. a pouch, etc.) that holds this medium and that releases the medium upon the start of the cartridge test or with the start of the actuator (e.g., with the start of actuation of the bellows or syringe pump), such as with a perforating needle or other component.
• a chamber may refer to a particular location within a channel, even if the channel’s geometry at that particular location does not change and is the same as the channel’s geometry before and after the chamber.
• the channel’s geometry may change at a chamber, such that, for example, the channel’s volume increases at the location of a chamber.
• test chambers can also describe regions of a channel (or other areas of a cartridge) where some kind of measurement or analysis takes place.
• closed system and variations thereof generally refer to a cartridge that is a dead-end system.
• a closed system uses pressure gradients to cause back pressure in specific places at various points in time to control fluid flow.
• references to numbered features in the figures that include a letter refer only to that specific alphanumerical feature; references to features that do not include a letter (e.g., 140) are intended to refer to all such numbered features (e.g., 140a, 140b, 140c, etc.).
• FIG. 1 depicts the operational principle underlying the closed cartridge air spring design.
• FIG. 2 extends the operational principle depicted in FIG. 1 to a multi-channel microfluidic cartridge, wherein the channels are connected to a common inlet at their upstream ends.
• FIG. 3 models the time-dependent fluidic flow of a Hagen-Poiseuille system like the closed microfluidic air spring cartridge.
• FIGs. 4A-4E depict fluidic flow at various, progressing states of cartridge pressurization.
• FIG. 4A depicts fluid flow at state 0.
• FIG. 4B depicts fluid flow at state 1.
• FIG. 4C depicts fluid flow at state 2.
• FIG. 4D depicts fluid flow at state 3.
• FIG. 4E depicts fluid flow at state 4.
• FIG. 5A and FIG. 5B each depict an embodiment of an air spring cartridge system.
• FIG. 6A depicts an embodiment of an air spring cartridge system that has nine testing chambers and three waste chambers.
• FIG. 6B depicts part of a channel (such as channel a of the embodiment depicted in
• FIG. 6A that includes additional paths (e.g., additional chambers, or a series of additional chambers) and/or resistive features between the metering chamber (130) and the testing chamber (140).
• additional paths e.g., additional chambers, or a series of additional chambers
• resistive features between the metering chamber (130) and the testing chamber (140).
• FIG. 7 depicts the system corresponding to some of the cartridge embodiments described herein (e.g., in FIGs. 10A, 10B, and 11).
• FIG. 8 shows how certain features in a single microfluidic channel (e.g., a single channel of the cartridge shown in FIGs. 10A-10B) are situated within a multi-layer microfluidic cartridge.
• FIG. 9 shows an embodiment in which each of nine channels of a cartridge comprises multiple electrode pairs (each electrode pair is shown as a pair of dots).
• two electrode pairs are part of a feedback mechanism that is used to control actuation of the active element controlling fluid flow.
• another electrode pair (shown as the pair of dots in the center of the nine channels) is located in or near the common channel where the fluid sample resides before it is split into multiple channels; this electrode pair may serve as a sensor, and also may be used as part of the feedback mechanism used to control fluid flow.
• FIG. 10A provides a top view and a bottom view of the multi-layer microfluidic cartridge employing the system depicted schematically in FIG. 7.
• FIG. 10B shows various layers of the same cartridge embodiment depicted in FIGs. 8 and 10 A.
• FIG. 11 shows a top view of an embodiment of the microfluidic cartridge employing the system depicted schematically in FIG. 7.
• FIGs. 12A-12B show embodiments of a cartridge in use, where a fluid sample has been inserted.
• FIG. 12A provides images showing a cartridge in use where the fluid sample is water that has been mixed with dye to facilitate imaging.
• FIG. 12B provides images showing a cartridge in use where the fluid sample is citrated whole blood.
• FIGs. 13A-13F illustrate reagent mixing and demonstrate how the separated portions of a fluid sample remain isolated from each other as the portions flow through their respective channels in a cartridge.
• Dye of different colors was spotted into chambers, with each chamber receiving one or two colors (red, yellow, or red and yellow) (FIG. 13A), and allowed to dry (FIG. 13B).
• FIG. 13C a water fluid sample was introduced into and pushed through an assembled cartridge, and there is complete separation of the fluid sample portions and no mixing of color between the channels.
• FIG. 13D shows how the dry- spotted dye(s) mix with the aliquots of the fluid sample in the mixing chambers, with uniform distribution of the dye(s) within the fluid sample in the mixing chambers.
• FIG. 13E and 13F show images when the water fluid aliquots (after mixing with the dry-spotted red and/or yellow dye) have entered into the testing chambers (which had been dry-spotted with blue dye); in the testing chambers the blue dye does not uniformly disperse into and mix with the fluid sample, demonstrating that in this cartridge embodiment the mixing illustrated in FIG. 13D is necessary to achieve full mixing of the sample with dry-spotted dye(s).
• FIG. 14 shows sacrificial retention of a fluid sample in a closed cartridge system; such sacrificial fluid retention can be used to accommodate evaporation of a fluid sample in certain embodiments.
• the present invention provides a self-contained microfluidic cartridge for use in analyzing a fluid sample.
• a key feature of the disclosed embodiments is that they allow for independent evaluation of multiple, isolated portions of a fluid sample using a single cartridge.
• the microfluidic cartridges described herein combine a parallelized air spring design with stationary features to control flow of a fluid sample inserted into the cartridge; such cartridges provide for the control of movement of the fluid sample through a closed system using only a single active element external to the cartridge (e.g., an actuator that operates a syringe, plunger, bellows, pump, etc.).
• the external active element of the cartridge pushes air and the fluid sample through a closed microfluidic cartridge, without the need for any additional valves or other control elements.
• one compartment of air is upstream of the fluid sample and the other compartment of air is downstream of the fluid sample.
• the upstream air is used to push the fluid sample portions and the air that is downstream of these portions, resulting in movement of the fluid sample downstream through the channels. This process results in compression and increased pressure, as the volumes holding the upstream air, fluid sample, and downstream air are made smaller.
• microfluidic cartridges described herein are dead-end systems (such that their flow paths are closed, with no openings such as vents, etc.) that rely on pressure gradients and back pressure to control fluid flow to and through the desired microfluidic channels (which may comprise one or more chambers) at various points in time.
• the devices, systems, and methods described herein achieve these pressure gradients and back pressure using an air reservoir, air springs, and metering chambers.
• the pressure gradients and back pressure ensure unidirectional fluid flow through the microfluidic cartridge.
• some embodiments may also use resistors (e.g., a tesla valve, a meandering fluid flow path, etc.) to control and/or prevent backflow of a fluid sample.
• each portion of the fluid sample can be metered to a known volume (referred to herein as an aliquot volume or metered volume), such that the volume of fluid sample contained in each of the channels, and in each chamber of a channel (e.g., a reagent chamber, a testing chamber) is of a known volume; b. each portion of the fluid sample contained in each of the channels (and thus in each chamber of a channel (e.g., testing chambers as well as waste chambers)) is isolated from all other portions of the fluid sample that are contained in the other channels (and in any chambers along those other channels); and c. the cartridge operates as a closed system and its contents are not exposed to the outside environment.
• a known volume referred to herein as an aliquot volume or metered volume
• each portion of a fluid sample such as embodiments where it is desirable to expose each portion of a fluid sample to a different condition (for example, to expose the portions to different reagents, or to different concentrations of a reagent or reagents), it is imperative to ensure that each portion is of a known volume (e.g., such that the concentration of an added reagent can be known and controlled).
• the cartridges described herein provide for metering portions of a fluid sample to achieve portions of known volumes (a portion of the fluid sample, once metered to a target volume, is called an aliquot).
• the target volumes (which are also called metered volumes) across the channels may be the same for all of the channels or may differ across channels.
• one or more other portions of the fluid sample may not be metered and may be received into a channel comprising a waste chamber (for example, channels 120j, 120k, and 120Z in FIG. 7). It is also critical to ensure that a chemical or physical reaction in one of the channels (e.g., in one of the channel’s chambers) does not affect any chemical or physical reaction that may occur in any of the other channels (e.g., in chambers of the other channels).
• a closed system to contain the sample within the cartridge and ensure safety for the persons inserting the sample and/or running the analysis, as well as to allow for the safe disposal of the cartridge (which contains the fluid sample) after analysis and use of the cartridge.
• Embodiments of the present invention provide microfluidic cartridges that operate as closed systems that contain a volume of fluid (which can be air or liquid) that is displaced in a controlled fashion to control the movement of an inserted fluid sample.
• the closed cartridges as described herein may rely on a single actuator to control fluid sample movement through the cartridge’s channels, though other mechanisms of controlling movement of the fluid sample may be employed.
• a single, closed actuator e.g., plunger, bellows, etc.
• such cartridges can be made at a lower cost compared to other devices that require additional elements for controlling fluid sample insertion and flow.
• using a cartridge with a closed system may also obviate the need for complex fluid management (e.g., post-analysis procedures, such as washing) external to the cartridge, for example, in the analyzer that obtains measurements from the cartridge.
• complex fluid management e.g., post-analysis procedures, such as washing
• the present invention generally relates to a closed air spring microfluidic cartridge that is useful for analyzing a fluid sample, for example for evaluating physical and/or chemical characteristics of a fluid sample.
• PV nRT
• R the ideal gas constant.
• distal air inside the cartridge must remain unvented during the testing process (in other words, the distal air (which is the air that becomes trapped downstream of the fluid sample, upon insertion of the fluid sample) must not be released from the closed cartridge during use).
• the distal air which is the air that becomes trapped downstream of the fluid sample, upon insertion of the fluid sample
• the system provides feedback to the actuator — e.g., via electrode sensors positioned at various locations in one or more of the channels, where such sensors determine the amount of fluid within the channels (for example, within particular chambers of the channels) — and through a feedback mechanism with the actuator, the actuator can then compensate for the pressure change within the system (e.g., by pushing more or less, and/or by pushing faster or slower).
• the viscosity of the fluid sample also may play a role in pressure changes and the system’s feedback to the actuator.
• the cartridge’s feedback loop thus allows the system to accommodate such variables.
• a channel is any continuous path for fluid flow and may comprise a series of connected fluid-flow paths — for example, a channel may be relatively narrow and then may widen into a chamber, after which the channel narrows again; and a channel may comprise multiple chambers along its path. In the various embodiments described herein, it is immaterial how long the channel is, so long as the distal air is not vented.
• V X P O V O /P X
• a physical property of the fluid sample may change during testing, as the result of changes in temperature or reactions that take place in the sample, for example.
• a change in temperature may affect viscosity and in turn affect the velocity of the fluid sample’s flow, especially in cases of fluids that exhibit nonNewtonian behavior, such as blood, etc.
• the fluid sample’s viscosity may change as the sample moves along the path of a channel with changing dimensions and that thereby exposes the sample to different shear. Further, some testing conditions or reactions in the sample also may affect the pressure within the channel.
• fluidic position sensors may be included within the cartridge, and the cartridge may contain an active feedback mechanism for controlling movement of the sample within the cartridge.
• the fluidic position sensors can communicate with a compensation mechanism providing for an active feedback loop, where the system is pressurized or de-pressurized based on information regarding the fluid sample’s position that is obtained from the position sensors; the pressurization and de-pressurization controls the sample’s movement, such that the sample reaches the desired location within the channel at the desired time and maintains such desired location for a desired time period.
• the active feedback loop involving the position sensors which can result in pressurization or de- pressurization of the channel, provides adequate control of the fluid sample’s movement through and within the channel.
• FIG. 2 shows the manner in which a microfluidic cartridge may receive a fluid sample through a common inlet connected to a first end of a plurality of channels. The sample in the common inlet is then split into multiple channels; certain channels contain an aliquot of the fluid sample, each aliquot having its own channel and each aliquot having a pre-determined volume, while another channel receives any excess fluid sample.
• the volumes of the aliquots may be the same or may differ.
• such different aliquot volumes can be achieved by using different metering well geometries, and/or by manipulating resistor regions in the channels to direct different amounts of the fluid sample to different metering wells.
• the position of an aliquot of the fluid sample within a channel may be determined by the volume of the air distal to (i.e., downstream of) the aliquot.
• Using the volume of the distal air (which is the air downstream of the aliquot) to determine the location of the aliquot within a channel provides a way to determine aliquot location that is independent of channel geometry and other variables (e.g., viscosity of the fluid sample).
• Such method of determining aliquot location, and further of using an air spring design to adjust the location of the aliquot within the channel provide several advantages over conventional microfluidic designs.
• the air spring system as described herein can, for example, tolerate manufacturing defects that may result in non-identical channel geometries (where identical channel geometries are desired). Another advantage is that using an air spring system renders the cartridge suitable for different sample types, including fluid samples that differ in their physical properties (e.g., viscosities).
• the air spring system described herein can accommodate a wide range of fluid properties (such as a wide range of viscosities, densities, compressabilities, etc.). For example, under the same conditions, a fluid of a higher viscosity may take longer than a fluid of lower viscosity to reach a given destination (e.g., the testing chamber) in the cartridge.
• the air spring system can accommodate such viscosity differences, and different rates of fluid movement, by adjusting pressurization within the channel(s) based on the location of the fluid sample within the channels and/or the volume of distal air, and thus a cartridge as described herein can be used for fluid samples that greatly differ in their physical properties (e.g., blood versus saliva).
• the individual aliquots of the sample may acquire different physical properties while in the cartridge (e.g., due to each aliquot’s exposure to different conditions within the cartridge, for example in channels having different reagents, as discussed herein); the air spring system can accommodate such different physical properties of the individual aliquots, by allowing for independent adjustments in each aliquot’s position within the cartridge based on the volume of distal air and using the pressurization/de-pressurization feature discussed above.
• FIG. 3 illustrates time-dependent flow in a Hagan-Poiseuille system like the air spring microfluidic cartridge embodiments described herein.
• AP pressure differential
• ju resistance to fluid flow
• AP U QI U' ⁇ where r is the radius of a cross-section of a cylindrical microchannel.
• Some embodiments may use a rectangular microfluidic channel, in which case resistance (A) is related to u as well as the height (A), width (w), and length (Z) of the microchannel, so that:
• a metering step first can be performed to ensure that each channel contains an aliquot of the fluid sample that has a specific, known volume.
• this metering step further ensures that each aliquot of the fluid sample downstream of the metering is exposed to a known concentration of a reagent (or of multiple reagents).
• the fluid sample will flow into and through each channel.
• the geometry of a channel can provide resistance to flow; such resistance may be localized to a specific region of the channel. Such resistance will create back pressure, and such back pressure may contribute to a pressure differential (the difference between Pi and P2 in FIG. 3).
• Such back pressure thus can serve as a way to slow the flow of the fluid in the channel. Accordingly, selecting certain channel dimensions (such as channel cross section and/or channel length) for the cartridge’s channels is a way to further control the rate of movement of fluid through the channels. Flow will continue, and the fluid will continue to move downstream, provided there is a pressure differential (see, e.g., FIG. 3).
• FIGS. 4A-4E are schematics depicting fluid placement and proximal and distal air, at progressing time-dependent states, in three channels of a single microfluidic cartridge.
• FIG. 4A depicts a cartridge at the time a fluid sample is inserted at a common inlet, and before the sample has split into the three channels and before any metering has occurred.
• FIG. 4B further shows air trapped by the fluid sample in each of the three closed microfluidic channels; this air is distal (downstream) of the fluid sample portions in each channel and is associated with volumes VI 1, V2i, and V3i.
• the pressure associated with VI 1, V2i, and V3i in each of the channels is equal to the pressure Ph, whereas there is a pressure differential in the channel leading to P2i (i.e., Pl 1 7 ⁇ P2i).
• pressure Pl 1 becomes greater than pressure P2i and the fluid sample moves into the chamber associated with P2i.
• 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., the channels associated with VI 1, V2i, and V3i in FIG. 4B; channels with chambers 130a and 130b in FIG. 5A).
• the devices, systems, and methods described herein can be applied to samples from any individual, including mammals (e.g., humans, such as human patients, as well as nonhuman mammals), reptiles, birds, and fish, among others, and can be useful for research and veterinary medicine.
• An individual can be, for example, mature (e.g., adult) or immature (e.g., child, infant, neonate, or pre-term infant).
• the devices, systems, and methods described herein can also be used for environmental samples (e.g., to test for the presence of an analyte in a water sample).
• the devices, systems, and methods described herein can be used in diagnostics and/or prognostics.
• the devices, systems, and methods described herein allow for multiplexed testing of a patient sample in a closed system. Such testing is particularly useful, for instance, in the context of infectious hemorrhagic diseases (e.g., Dengue virus, Marburg virus, Ebola virus, etc.), where a disease-specific cartridge can be used to both diagnose and predict the likelihood of hemorrhagic complications.
• infectious hemorrhagic diseases e.g., Dengue virus, Marburg virus, Ebola virus, etc.
• the devices, systems, and methods described herein can be used to guide therapy of a patient, including, without limitation, in the hospital setting.
• physicians can use a multi-channel microfluidic cartridge that allows for multiplexed, independent testing of an aliquoted fluid sample without cross-talk between the channels (see, e.g., the cartridge embodiments of FIGs. 7, 10A-B) to perform, for example, the assays described in U.S. Application No. 16/046,816 (published as US 2019/0111431) and U.S. Application No. 17/195,615 in order to, for instance, ensure that patients requiring anticoagulation therapy are receiving an appropriate dose of anticoagulant so as to solicit the proper physiological response.
• the devices, systems, and methods described herein likewise can be used in the management of diseases or disorders that result in pathological bleeding or clotting (e.g., COVID-19 caused by the SARS-CoV-2 virus, hemophilia, etc.), including, without limitation, for the clinical and pre-clinical assessment of treatments (e.g., drugs).
• the disclosed devices, systems, and methods also can be used for research and discovery.
• the devices, systems, and methods described herein can be useful for basic drug discovery, for understanding disease or disorder pathophysiology, for pre- clinical assessment of new compounds and biologies, and for monitoring adverse events and off-target effects of experimental treatments.
• the schematic in FIG. 5A depicts a basic closed cartridge system; the system comprises a plurality of channels (120), each channel of the plurality of channels (120a, 120b, and 120j) connected on a first end to a common inlet 100 via a common channel 110, and each channel of the plurality of channels having towards its second end a separate chamber or series of chambers (e.g., 130a, 130b, 160a).
• a separate chamber in each of the channels may be a metering chamber 130 or a waste chamber 160.
• a fluid sample is introduced (e.g., inserted) via common inlet 100.
• an actuator not shown in FIG. 5A
• air upstream of the fluid sample is pushed, causing the sample to move along microfluidic channel 110 before splitting into channels 120a, 120b, and 120j.
• Each microfluidic channel 120a, 120b, and 120j contains at least one resistive element (121a, 121b, and 121j, respectively) that controls the rate of fluid flow in that channel, and thus into each of the chambers in that channel (e.g., chambers 130a and 130b and waste chamber 160a).
• such resistive features may include, for example, the geometric setup of the microfluidic channel (e.g., a change in the channel’s aspect ratio or other change in channel geometry that reduces channel volume, etc.).
• a resistive feature e.g., 121j
• channel 110 may split into more than three microfluidic channels (see, e g , FIGs 6A, 7, 10A)
• 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 (metered volumes of the fluid sample are also called aliquots).
• the resistance of 121a is equal to the resistance of 121b, and 130a and 130b serve as metering chambers that accept the same volume of a fluid sample at the same rate.
• a channel e.g., 110
• the microfluidic cartridges of FIGs. 6A, 7, 10A, and 11 each show nine metering chambers (130a-130i).
• each channel that is used for testing will include a region that is a metering chamber.
• each testing chamber 140a and 140b may contain sensors (e.g., electrode sensors) and/or may facilitate some other method of measurement (e.g., optical detection). Sensors may be contained within a channel or may reside outside of a channel.
• sensors may include any component that allows for measuring or that otherwise measures characteristics of the fluid sample, including, for example, by measuring electrical impedance, measuring electrical capacitance, measuring electrical resistance, assessing flow rate (e.g., by assessing the flow rate of beads that are present in the fluid sample), measuring flow velocity, measuring pressure, measuring viscoelasticity, fluorescence detection (e.g., using fluorescent fibrinogen), measuring turbidity, infrared light detection, infrared spectroscopy, detection using acoustic sensors, detection using photonic sensors, flow cytometry, and methods of visual detection (e.g., for visual clot detection).
• flow rate e.g., by assessing the flow rate of beads that are present in the fluid sample
• measuring flow velocity e.g., by assessing the flow rate of beads that are present in the fluid sample
• measuring pressure e.g., measuring viscoelasticity
• fluorescence detection e.g., using fluorescent fibrinogen
• measuring turbidity e.g., infrared
• one or more testing chambers 140 may also contain a reagent (including, e.g., multiple reagents).
• Reagents may include coagulation factors, calcium, fluorogenic or chromogenic substrates, coagulation activators (e.g., glass, celite, phospholipids, kaolin, etc.), electrochemical reagents (e.g., for thrombin generation, etc.), and platelet activators (e.g., adenosine phosphate, collagen, ristocetin, epinephrine, etc.).
• metering chamber 130 e.g., 130a
• testing chamber 140 e.g., 140a
• at least one of these additional chambers may contain one or more reagents.
• Each testing chamber (or well, etc.) 140 connects to a distal air spring 150 (e.g., 150a) via a chamber (or a series of chambers) 210 (e.g., 210a).
• Each air spring is a closed well of a predetermined volume such that it and the waste chamber(s) (160) control fluidic movement.
• the volume of the air springs can be increased; such increase in volume will decrease the pressure of the system (including the pressure of the system when the aliquots are at their final destination in the testing chambers).
• the volume of the air springs can be decreased; such decrease in volume will increase the pressure of the system (including the pressure of the system when the aliquots are at their final destination in the testing chambers). Similarly, increasing or decreasing the volumes of each waster chamber (160) will affect how much pressure is within the cartridge and the air spring system.
• a cartridge may comprise more than two metering chambers 130a and 130b and/or more than a single waste chamber 160a. Exemplary embodiments of such cartridges are illustrated, by way of example, in FIGs. 6A and 7. It should be understood by those skilled in the art that embodiments of a cartridge comprising a closed air spring system may include features that are not depicted in FIG. 5A (see, e.g., FIGs. 6A, 7), and/or that are not shown in the specific embodiments described herein.
• the fluid sample may be desirable to split in multiple, successive steps. For example, successive or modular splitting may improve metering time and/or fluidic fill across multiple channels. Such improvements may be important in embodiments wherein the fluid sample is split into a large number of portions.
• the fluid sample is split into three separate portions, each portion moving into its own channel, and each of the three separate portions is then subsequently split across three or more metering chambers and one or more waste chambers.
• the embodiment shown in FIG. 5A may include additional features, such as the features of area 113 shown in FIG. 5B.
• the inlet 100 connects to a reservoir 111, and resistor 112 controls and/or prevents backflow of the fluid sample.
• Reservoir 111 may serve, for example, as an additional metering region prior to splitting the fluid sample into multiple metering chambers and one or more waste chambers.
• resistive feature 112 may comprise, for example, a tesla valve, a meandering microfluidic path, and/or a porous membrane. In some embodiments, it may be desirable to combine multiple resistive elements (e.g., multiple resistor regions in parallel or in series).
• the paths connecting each of the chambers in a channel may also include resistive features 201 and/or 211 (see, e.g., FIG. 5B).
• resistance to fluid flow may be achieved via the geometry (including shape and/or dimensions of that shape) of one or more of the paths between chambers (e.g., 200, 210 (including only at certain regions thereof)), such that there is no distinct corresponding resistor separate from the channel itself.
• the fluid sample may be split into at least twelve channels: at least nine channels (120a-i), each channel containing an aliquot of the fluid sample, and three waste channels (120j— /); see, for example, the embodiment depicted in FIG. 6A.
• the fluid sample may split into the at least twelve channels at a single time, or the fluid sample may first split into less than twelve portions, followed by subsequent splittings (e.g., the fluid sample may split twice, three times, four times, etc.).
• the common inlet 100 connects to microfluidic channels 120a-/ directly via common channel 110 (compare, e.g., FIG. 5A).
• the fluid sample splits into three channels, each such channel splitting further into three channels (see the channel labeled 120a) ahead (upstream) of metering chamber 130.
• the fluid sample may flow to a first fluid reservoir 111 and/or resistor 112 before splitting (see, e.g., FIGs. 5B, 7).
• one or more of the channels in a cartridge may further include additional paths (e.g., additional chambers, or a series of additional chambers) and/or resistive features between the metering chamber (130) and the testing chamber (140).
• each of the microfluidic channels (at one more locations along each channel’s path, e.g., 120, 200, 210) may, but need not necessarily, include resistive elements.
• FIG. 7 provides a schematic for an embodiment comprising resistive elements at various locations along channel paths.
• each of the additional chambers and/or resistive features between the metering chamber (130) and the testing chamber (140) in each of the channels of the cartridge may serve a particular purpose or provide a particular function.
• one or more chambers in between a metering chamber (130) and a testing chamber (140) may contain one or more reagents.
• the closed microfluidic cartridge and the various chambers and features included in the channels therein are designed to allow: a. splitting of a fluid sample into multiple channels for analysis (e.g., 120a-i) and at least one waste channel (e.g., 120j); b. isolating each of the portions of the fluid sample in each of the channels for analysis (e.g., 120a-i), and thereby separating such portions from the portions of the fluid sample in each of the waste channels (e.g., 120j— /) and thus in each of the waste chambers (160a-c), and further c.
• each of the portions of the fluid sample in each of the channels for analysis e.g., 120a
• each of the other portions of the fluid sample in each of the other channels for analysis e.g., 120b-i
• d. metering the portion of the fluid sample in each of the channels for analysis such as in chambers 130a-i
• each portion in the chambers downstream of metering e.g., reagent chambers, testing chambers (or wells, etc.
• introducing each of the portions of a known volume called aliquots
• reagents such as in a chamber as depicted by 300
• each aliquot of the fluid sample with the one or more reagents (such as in chamber 310); and g. measuring or otherwise analyzing one or more physical and/or chemical characteristics of each aliquot of the fluid sample (such as in chamber 140), which permits evaluating each aliquot’s reaction in response to the one or more reagents.
• FIGs. 6B and 7 show such a cartridge, which may be used, for example, to detect and/or evaluate blood clotting.
• a cartridge may comprise multiple, stacked layers, and the cartridge’s channels and/or chambers may be made of different materials and/or reside along different planes.
• FIG. 8 shows one such example, where the chambers along the path of one of the microfluidic channels in FIG. 7 (e.g., the channel comprising 120a, 130a, 300a, 310a, 140a, and 150a) are shaped and positioned in three dimensions, such shapes and positions achieving various functions (e.g., items a through g described above).
• the reagent chamber and mixing chamber are sized and positioned relative to one another, as well as to the channel path connecting them, such that the reagent chamber 300 may accommodate up to 1.10 pL of an aliquot of the fluid sample, the mixing chamber 310 may accommodate up to 2.55 pL of an aliquot of the fluid sample, and channel path 400 connecting them may accommodate up to 0.85 pL of an aliquot of the fluid sample.
• the volume of the mixing chamber (and the volume of the fluid sample aliquot in the mixing chamber) may be up to five times, or as little as 1.5 times, larger than the volume of the reagent chamber (and the volume of the fluid sample aliquot in the reagent chamber).
• the ratio of the volume of the fluid sample aliquot in the reagent chamber to the volume of the fluid sample aliquot in the mixing chamber may depend on, e.g., the hydroscopic behavior of the one or more reagents, the properties of the fluid sample, and/or the properties of the interior surface of the reagent chamber (e.g., hydrophobic, hydrophilic, patterned, etc.).
• the configuration depicted in FIG. 8 ensures that the one or more reagents are fully mixed with the aliquot of the fluid sample through a process of power-washing, which takes place in the reagent chamber and which is then followed by mixing in the larger volume mixing chamber 310, which is connected to the testing chamber 140
• this process of power-washing followed by mixing may be particularly desirable, for example, where one or more reagents have been dry-spotted on or within the microfluidic cartridge.
• the efficacy of power-washing is illustrated in FIGs. 13C-13F
• FIG. 13A shows the spotting of droplets with different color dyes and FIG. 13B shows the same droplets once dried (“dry-spotted”) and covered with a polycarbonate sealing layer that is placed on top to completely form the channels.
• the differently-colored dyes are dry-spotted in the reagent chambers, and each reagent chamber contains a red and/or yellow dye (at different concentrations).
• the testing chambers contain spots of blue dye. There are no dye spots in the mixing chambers, which cannot be seen in FIG. 13 A.
• FIGs. 13C and 13D show images as water is flowing through the microfluidic channels in the cartridge.
• FIG. 13C shows a color gradient in the reagent chambers, as water flows through and brings the dried colored spot (the dried dye of red and/or yellow) into solution; the dried dye is used to represent an example reagent such as a coagulation agonist.
• the color dye fully mixes with the fluid sample (which, in this demonstration, is water) when each fluid sample aliquot is in the microchannel’s mixing chamber, as shown in FIG. 13D (which shows dye dissolved uniformly throughout the water within the mixing chambers).
• This demonstration using dye also shows that there is no color mixing or bleed-over between the nine channels through which fluid is flowing in parallel; in other words, there is no cross-talk between the channels, and thus the multiple aliquots can be tested simultaneously.
• FIGs. 13E and 13F show images of the cartridge when each water aliquot with the red and/or yellow dye mixed therein has been pushed farther downstream into each microchannel’s testing chamber containing the blue dye.
• FIGs. 13A-13F demonstrate two important concepts: 1) the dye color(s) used to represent the reagents (the red and/or yellow dyes) in each microchannel is fully mixed with the fluid sample aliquot by the time the aliquot, with the reagent(s), reaches the testing chamber (see FIG. 13D), and 2) the reagent that was dry-spotted directly into the testing chamber (represented in this illustration with the blue spot) does not fully mix with the fluid sample aliquot in each microchannel, as seen by the blue color gradient in each testing chamber (see FIGs. 13E, 13F).
• FIG. 13E, 13F show images of the cartridge when each water aliquot with the red and/or yellow dye mixed therein has been pushed farther downstream into each microchannel’s testing chamber containing the blue dye.
• 13E shows the testing chambers, with each testing chamber having a blue dye color gradient.
• the reagent chamber that contained red dye to represent the reagent if the red dye had fully mixed with the blue spot in the testing chamber, the resulting color in the entire testing chamber should have been purple, but it was not.
• the microchannel with the reagent chamber that contained yellow dye to represent the reagent if the blue spot in the testing chamber had fully mixed with the fluid sample, the resulting color in the entire testing chamber should have been green (a combination of yellow from the reagent in the reagent chamber and blue from the testing chamber reagent), but it was not (see also FIG. 13F).
• a cartridge (such as 60 in FIG. 10B) may comprise an array of components.
• a cartridge may comprise a cartridge body 600 that may include a plunger 601, filter plug 608, support ring 605, and a septum 602.
• the cartridge may further comprise a first pressure sensitive adhesive (PSA) layer 606, a second PSA layer 607, and printed circuit board 800.
• PSA layers 606 and 607 and printed circuit board 800 additionally may be aligned with each other and with the cartridge body 600, and adhered or bonded (e.g., heat sealed, heat welded, laser welded, etc.) to each other and the cartridge body 600.
• the first PSA layer 606 and second PSA layer 607 may have either single-sided or double-sided adhesive.
• a back seal 604 may also be bonded to the bottom of the cartridge body 600 to fashion the microfluidic channels.
• a fluid sample is moved through the microfluidic cartridge by pushing air in the cartridge after insertion of the fluid sample.
• Air can be pushed via an integrated plunger 601 (see, e.g., FIG. 10B).
• an actuator may push on a plunger 601, such that the plunger 601 travels the length (or a portion of the length) of an integrated syringe 700 that moves the fluid sample to the desired locations within the cartridge at specific moments in time.
• a cartridge may alternatively be outfitted with part 901, which includes a bellows 900 that operates to move the fluid sample through the cartridge (see, e.g., FIG. 11).
• Various materials e.g., acrylic, polycarbonate, glass, silicone, etc.
• fabrication methods can be used to make the cartridge body 600.
• one or a combination of existing fabrication methods e.g., injection molding, machining, laser etching, layering (which may include any combination of the foregoing, with or without photoablation and/or sacrificial layers)
• the tests and testing conditions e.g., reagent(s) to be used, temperature, etc.
• the selection of material(s) to use in manufacturing the cartridge body will inform the selection of material(s) to use in manufacturing the cartridge body.
• a cartridge may comprise one or more layers of PSA.
• a PSA layer may allow for creating fluid connections between layers (and different planes) of the cartridge (in such instances, a PSA layer with a particular pattern of openings can be used).
• PSA layers can be mass produced and cut with commercial laser cutters. Small production runs can also be achieved with off-the-shelf laser cutters, depending on the desired resolution of the openings.
• PSA layers may be die cut to create the desired pattern and/or geometry.
• PCB 800 printed circuit board
• such PCB can be rigid or flexible, or may comprise layers with one or more of such layers being flexible and other layer(s) being rigid.
• a flexible PCB made of Kapton® is attached to (e.g., adhered to, laser welded to, etc.) a rigidized layer (such as a rigid PCB).
• a rigid PCB allows for fluorescent measurements.
• PCB 800 also may contain heating elements within the electronics. Including heating elements may be desirable for coagulation testing or polymerase chain reaction (PCR) testing, for example.
• One or more temperature sensors and/or heating elements can be integrated into the PCB, to achieve and/or maintain a desirable temperature in one or more particular regions of the cartridge, such as over the regions of the cartridge containing the fluid sample.
• each testing chamber may have its own temperature sensor and/or heating element.
• localizing individual temperature sensors and/or heating elements to individual regions of a cartridge allows for exposing each aliquot to a different temperature and/or applying different temperatures to different portions of each channel. Temperature measurements also may be used by a testing unit for a feedback temperature control system.
• Cartridges having closed systems wherein each of the aliquots of the fluid sample is isolated, as described herein, may be used to perform multiple tests (e.g., to assess coagulation in each of the aliquots) simultaneously.
• tests may involve assessing activated clotting time, prothrombin time, activated partial thromboplastin time, dilute thrombin time, or thrombin time.
• Such tests may involve coagulation factor activity assays, chromogenic assays (e.g., to assess anti-Factor Xa activity), fluorescence-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 US 2019/0111431) and U.S. Application No. 17/195,615, the contents of each of which are incorporated by reference in their entirety herein.
• electrochemistries of the fluid sample aliquots can be evaluated, such as, for example, for the measurement of one or more electrolytes (e.g., glucose or lactate, etc.) and/or biomarkers (e.g., genetic or protein biomarkers).
• electrolytes e.g., glucose or lactate, etc.
• biomarkers e.g., genetic or protein biomarkers
• enzyme-linked immunosorbent assays can be performed in the microchannels, such as for the detection and quantification of one or more biomarkers and/or antibodies.
• ELISAs can be performed using electrodes that may or may not be functionalized (e.g., directly with antibodies, with nanogold or microgold particles, and/or with carbon nanotubes), or may be performed using agglutination, fluorescence, magnetic beads, chromogenic methods, sensors within or outside of the testing chambers, or any combination thereof.
• Isolating each of the fluid sample aliquots, such that each aliquot moves through a single microchannel makes it possible, in some embodiments, to use a single cartridge to perform multiple platelet activation and inhibition tests, including aggregometry, adenosine triphosphate (ATP) generation (e.g., via luciferase measurements), platelet adhesion (e.g., using imaging, or mechanical and/or electrical sensors), and/or platelet counts (e.g., via imaging and/or with other sensors).
• both coagulation and platelet tests can be performed using a single cartridge.
• PCR can be performed — for example, traditional PCR with thermocycling or isothermal reactions, or newer PCR methods, such as those using clustered regularly interspaced short palindromic repeats (CRISPR).
• CRISPR clustered regularly interspaced short palindromic repeats
• sensors may be incorporated into the testing chambers in order to perform any of the tests described above or that otherwise may be desired.
• sensors include, for example, gas sensors that can be used to detect pathogens, toxins, volatile organic compounds (VOCs), or other hazardous chemical or biological agent.
• VOCs volatile organic compounds
• such sensors may be used to evaluate the chemical composition of the fluid sample within the testing chamber.
• Other embodiments may involve electrode sensors, as discussed above.
• the cartridge may comprise sensors in addition to the sensors used for evaluating the sample aliquot in the testing chamber.
• additional sensors may be placed at points along the fluid path that are not within the testing chamber (e.g., an additional sensor may be located upstream of the testing chamber). Further, additional sensors may be positioned inside the channel or outside of the channel.
• an electrode pair that can be used for evaluating the amount of hematocrit in a blood sample may be included, as shown in FIG. 9.
• a material used to manufacture a cartridge may be transparent in order to allow optical measurements and imaging of the fluid sample aliquots within the channels.
• one or more layers may be transparent or may otherwise allow for optical measurements or imaging.
• Such optical measurements or imaging can take place at one or more locations of the channels, including at locations that are not testing chambers.
• such cartridges may or may not comprise electrode sensors for measuring an electrical property (e.g., electrical impedance) of the sample aliquots.
• testing may require exposing one or more of the sample aliquots to multiple reagents at various times (e.g., by first exposing an aliquot to one reagent, followed by exposure of the aliquot to another reagent) and may also require heating and/or cooling of the fluid sample aliquot(s).
• coagulation testing may require heating the cartridge containing the fluid sample to 37 °C; in further embodiments, testing may require heating the cartridge and/or the portions of the fluid sample to the body temperature of the patient, to approximate physiological conditions.
• the microchannels of a closed microfluidic cartridge facilitate some amount of fluid retention on their surface(s) (described herein as sacrificial fluid retention) to compensate for any evaporation of the sample within the cartridge during testing (see, e.g., FIG. 14, which shows fluid sample (which has been colored with a dark dye) along the interior walls of the microchannels even after the fluid sample aliquot has traveled all the way to the testing chambers).
• the materials that are used in assembling the cartridge components may be chosen or treated so that their surfaces that contact the fluid sample promote a certain amount of fluid sample retention along specific paths of each microfluidic channel (or at least those channels used for testing and comprising testing chambers). For example, if the exposed surface area of the fluid sample when it is in the testing chamber (the area of the fluid sample contacting air or other gas when the fluid sample is in the testing chamber) is smaller than the exposed surface area of the sacrificial fluid retention along the walls of the microchannel, the sacrificial fluid will be preferentially evaporated towards an equilibrated system, with minimal evaporation of the fluid sample and therefore minimal effect on the testing chamber sample volume.
• Channel surfaces may be coated (e.g., with a hydrophilic coating) to promote retention of sacrificial fluid.
• using a PSA layer to form the walls of the microfluidic channels can provide channel surfaces that promote sacrificial fluid retention; for example, for a PSA layer made of polycarbonate, the fluid sample tends to grab onto the PSA as it passes through the channel, resulting in the retention of sacrificial fluid.
• This sacrificial fluid is spread along the PSA lining the sides of the channels, giving the sacrificial fluid a larger surface area of exposure to the air (or other gas) in the cartridge compared to the exposed surface area of the sample in the testing chamber, as the sample in the testing chamber is exposed to the air (or other gas) only on two sides (the upstream and downstream sides, and the surface area of those sides further can be controlled with channel geometry).

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• 2022
  • 2022-08-09 WO PCT/US2022/039851 patent/WO2023018728A1/en not_active Ceased
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