WO2017123855A1 - Dispositifs microfluidiques d'échantillonnage et leurs utilisations - Google Patents

Dispositifs microfluidiques d'échantillonnage et leurs utilisations Download PDF

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Publication number
WO2017123855A1
WO2017123855A1 PCT/US2017/013316 US2017013316W WO2017123855A1 WO 2017123855 A1 WO2017123855 A1 WO 2017123855A1 US 2017013316 W US2017013316 W US 2017013316W WO 2017123855 A1 WO2017123855 A1 WO 2017123855A1
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WIPO (PCT)
Prior art keywords
fluid
sample
path
microfluidic
assay
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PCT/US2017/013316
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English (en)
Inventor
Richard Novak
Jefferson PUERTA
Olivier Henry
Youngjae CHOE
Jonathan SABATÉ DEL RIO
Donald E. Ingber
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President And Fellows Of Harvard College
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Publication of WO2017123855A1 publication Critical patent/WO2017123855A1/fr

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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
    • B01L3/502738—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 integrated valves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1095—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices for supplying the samples to flow-through analysers
    • G01N35/1097—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices for supplying the samples to flow-through analysers characterised by the valves
    • 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
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • 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
    • 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
    • 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
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0655—Valves, specific forms thereof with moving parts pinch valves

Definitions

  • microfluidic sample devices and systems comprising the same for sampling of a fluid in a microfluidic device without disrupting flow of the fluid in the microfluidic device.
  • the microfluidic sample devices and systems described herein can be used for performing an assay on a sample from a cell-culture microfluidic device (e.g., an organ-on-a-chip device) while maintaining flow of cell culture medium therein.
  • microfluidic device or system that is capable of performing automated analytical assays (e.g., immunoassays) using various biosensors and taking samples from microfluidic organ-on-a-chip devices without disrupting the flow of fluid to cell culture in the devices.
  • automated analytical assays e.g., immunoassays
  • microfluidic sample devices and/or systems comprising the same for sampling of a fluid in a microfluidic device while maintaining a substantially continuous flow of the fluid in the microfluidic device.
  • the microfluidic sample devices and/or systems can be configured to conduct one or more assays on a sample fluid from cell-culture microfluidic devices while maintaining a substantially continuous flow of a fluid in the cell-culture microfluidic devices.
  • the inventors have developed a microfluidic sample device comprising a sample fluid path, a main assay path, and a membrane valve located between the sample fluid path and the main assay path.
  • the microfluidic sample device can be used to couple a cell- culture microfluidic device to a downstream fluid-receiving device (e.g., a reservoir or another cell-culture microfluidic device).
  • a downstream fluid-receiving device e.g., a reservoir or another cell-culture microfluidic device.
  • the sample fluid path of a microfluidic sample device can allow for continuous flow of a cell culture medium from a cell-culture microfluidic device (e.g., an organ-on-a-chip device) to a downstream fluid- receiving device (e.g., a reservoir or another cell-culture microfluidic device) while an aliquot of a fluid sample from the cell-culture microfluidic device enters into the main assay path for an assay to be performed via control of the opening and closing of the membrane valve.
  • a cell-culture microfluidic device e.g., an organ-on-a-chip device
  • a downstream fluid- receiving device e.g.
  • microfluidic sample devices and systems comprising the same for sampling of a fluid in a device and/or performing an assay on a sample fluid from the device without disrupting normal operation of the device, as well as uses thereof.
  • a microfluidic sample device comprises (a) a sample fluid path having a sample-fluid inlet for receiving the sample fluid and a sample-fluid outlet for allowing the fluid to exit the microfluidic sample device; (b) a main assay path that is coupled to an assay module for performing an assay; and (c) a membrane valve located between the sample fluid path and the main assay path and being operable between a first valve state and a second valve state.
  • sample fluid flows through the sample fluid path and exits the sample-fluid outlet in the first valve state, or the sample fluid flows from the sample fluid path into the main assay path to permit an assay performed on the sample fluid in the second valve state.
  • microfluidic sample device can be used to periodically perform an assay on a sample fluid from a device without disrupting normal operation of the device.
  • the microfluidic sample device can further comprise a fluid waste path located at an output port of the assay module.
  • the fluid waste path can comprise a microfluidic pump for pulling the sample fluid or wash fluid through the main assay path and the assay module.
  • the fluid waste path can comprise a fluid outlet port that allows a sample fluid or a wash fluid to exit the microfluidic sample device.
  • a microfluidic sample device for periodically performing an assay on a sample fluid.
  • the microfluidic sample device comprises: (a) a sample fluid path having a sample-fluid inlet for receiving the sample fluid and a sample- fluid outlet for allowing the fluid to exit the microfluidic sample device; (b) a main assay path that periodically receives the fluid from the sample fluid path for performing the assay; (c) a fluid waste path for removing the fluid from the microfluidic sample device after the assay is performed on the fluid; and (d) a microfluidic pump located within the fluid waste path for pulling a wash fluid through the main assay path after the assay is performed such that the main assay path is cleansed before subsequently receiving a second sample fluid for another assay.
  • the fluid waste path can comprise a fluid outlet port that allows the sample fluid to exit the microfluidic sample device.
  • the microfluidic sample device can further comprise a membrane valve located between the sample fluid path and the main assay path.
  • the membrane valve can be operated between a first valve state and a second valve state. In the first valve state, the sample fluid flows through the sample fluid path and exits the sample- fluid outlet. In the second valve state, at least a portion of the sample fluid flows from the sample fluid path into the main assay path to permit the assay.
  • the membrane valve can comprise: (i) an actuation structure comprising a membrane actuation mechanism; and (ii) an elastomeric membrane located between the actuation structure and a fluidic structure comprising a portion of the sample fluid path and a portion of the main assay path such that the elastomeric membrane is oriented transverse to the sample fluid path portion and the main assay path portion.
  • the elastomeric membrane can be actuated between the first valve state and the second valve state.
  • the membrane can be actuated by various art-recognized actuation mechanisms, e.g., but not limited to pneumatically-operated actuators, mechanically-operated actuators, and/or electrically-operated actuators.
  • the membrane can be actuated pneumatically to open or close a fluidic conduit between a portion of the sample fluid and a portion of the main assay path.
  • the membrane actuation mechanism can comprise a pneumatic conduit.
  • Application of an air pressure to the pneumatic conduit can actuate the membrane to prevent a sample fluid in the sample fluid path from entering the main assay path.
  • the sample fluid continues to flow through the sample fluid path and exits the sample fluid outlet.
  • Application of vacuum to the pneumatic conduit can actuate the membrane to permit at least a portion of the sample fluid to flow from the sample fluid path into the main assay path.
  • one end portion of the main assay path can comprise a wash inlet for clearing the main assay path with a wash fluid after the sample fluid flows into the assay module, while the other end portion of the main assay path can be coupled to an assay module.
  • the sample fluid in the main assay path can be subjected to at least one or more assays.
  • the microfluidic sample device can further comprise at least one reagent input conduit that controllably releases a reagent into the main assay path.
  • at least one reagent can be controllably released through the corresponding reagent input conduit into a sample fluid while the sample fluid moves along the main assay path toward an assay module.
  • at least one reagent can be controllably released through the corresponding reagent input conduit into the main assay path after a sample fluid has moved along the main assay path and entered an assay module.
  • the added reagent(s) can be directed to the assay module.
  • at least one reagent can be controllably released through the corresponding reagent input conduit into the main assay path and be directed to the assay module via the main assay path before a sample fluid enters the main assay path.
  • no pump is placed in a fluidic path between the reagent input conduits and the assay module.
  • microfluidic sample devices described herein can be integrated into an instrument, e.g., for diagnostic applications, and/or can be used to interconnect at least one device to another as well as to collect samples and/or even to perform assays at the interconnection. Accordingly, systems comprising one or more microfluidic sample devices as described herein are also within the scope of various aspects described herein.
  • One aspect relates to a microfluidic system comprising one or more microfluidic sample devices as described herein.
  • the microfluidic system comprises (a) at least one cell-culture microfluidic device having a chamber including a surface to which the cells are attached, the chamber comprising a fluid inlet for receiving a fluid that passes across the cells and a fluid outlet for exiting the fluid from the cell-culture microfluidic device; (b) at least one downstream fluid- receiving device that receives the fluid from the cell-culture microfluidic device; and (c) at least one microfluidic sample device located between the cell-culture microfluidic device and the downstream fluid-receiving device.
  • the microfluidic sample device used in the microfluidic system can be any embodiment described herein.
  • the microfluidic sample device can comprise a sample fluid path with a sample-fluid inlet for receiving a fluid from the fluid outlet of the cell-culture microfluidic device and a sample-fluid outlet for allowing the fluid to pass to the downstream fluid-receiving device.
  • the main assay path of the microfluidic sample device can be coupled to an assay module for performing a desirable assay.
  • a membrane valve can be located between the sample fluid path and the main assay path, and the membrane valve can be operable (i) in a first valve state to allow the fluid to pass to the downstream fluid- receiving device via the sample-fluid outlet; and (ii) in a second state to allow the fluid to enter into the main assay path.
  • an assay can be performed on the fluid received by the main assay path while maintaining a substantially continuous flow of a fluid between the cell- culture microfluidic device and the downstream fluid-receiving device. This can provide capability to periodically conduct an assay on an effluent from a cell-culture microfluidic device, e.g., to monitor cell culture condition or to monitor cell response over a period of time.
  • the downstream fluid-receiving device is generally a container or a device located downstream of the cell-culture microfluidic device that can hold or store a fluid.
  • the downstream fluid-receiving device can be a fluid reservoir.
  • the downstream fluid-receiving device can be a second cell-culture microfluidic device with a second set of cells cultured therein.
  • the microfluidic sample device can be integral to a cartridge that holds the cell-culture microfluidic device.
  • the cell-culture microfluidic device can be any microfluidic device that can be used for cell culture.
  • the cell-culture microfluidic device can comprise a first chamber, a second chamber, and a membrane located between the first and second chambers. The membrane can comprise at least one type of cells thereon.
  • the cell-culture microfluidic device can comprise an organ-on-a-chip device.
  • the microfluidic system can further comprise an assay module coupled to the main assay path of the microfluidic sample device.
  • An assay module comprises at least one or more sensors for detecting or measuring one or more target analytes.
  • the microfluidic system can comprise a sample multiplexer located between the main assay path and the assay module.
  • one fluidic input can be converted to a plurality of (e.g., at least two or more) fluidic channels.
  • the microfluidic system can comprise a sample demultiplexer located between the assay module and the fluid waste path.
  • a plurality of (e.g., at least two or more) fluidic outputs after sensor measurements can be converted to a single channel, e.g., fluidly connected to a fluid waste path.
  • a method comprises (a) allowing a sample fluid exiting from the microfluidic device to move along a sample fluid path within a microfluidic sample device according to one embodiment described herein; (b) actuating a membrane valve of the microfluidic sample device to a valve state to allow at least an aliquot of the sample fluid to flow from the sample fluid path to the main assay path, while the remaining of the sample fluid can continue to flow and exits the sample-fluid outlet, thereby sampling the fluid periodically while maintaining a substantially continuous flow of the fluid through the microfluidic device in the microfluidic system; and (c) actuating the membrane valve, after the main assay path receives the aliquot, to a different valve state to prevent any additional sample fluid to flow from the sample fluid to the main assay path, but to exit
  • the methods described herein can be applied to microfluidic applications where sampling of a fluid in a microfluidic device is desirable for an assay and a substantially continuous flow of a fluid is desired or preferred to be maintained in the microfluidic device.
  • the microfluidic device can comprise a cell culture device.
  • the cell- culture microfluidic device can be any microfluidic device that can be used for cell culture.
  • the cell-culture microfluidic device can comprise a first chamber, a second chamber, and a membrane located between the first and second chambers. The membrane can comprise at least one type of cells thereon.
  • the cell- culture microfluidic device can comprise an organ-on-a-chip device.
  • the sample-fluid outlet of the sample fluid path can be coupled to a reservoir or a second microfluidic device (e.g., an organ-on-a-chip).
  • a second microfluidic device e.g., an organ-on-a-chip
  • the method can further comprising allowing at least one reagent controllably released from one or more reagent input conduits of the microfluidic sample device to move along the main assay path toward an assay module.
  • the reagents can be introduced into the main assay path and contacted the aliquot received by the main assay path.
  • a reagent include, but are not limited to a nucleic acid extraction agent, a fixation agent, a staining agent, an analyte- specific antibody, a detectable label, or any combinations thereof.
  • Appropriate reagents can be introduced to an assay module configured for specific assay(s).
  • nucleic acid when a nucleic acid extraction reagent is added to the aliquot in the main assay path, nucleic acid can be extracted from cell(s) present in the aliquot prior to entering the assay module, e.g., for a nucleic acid assay such as polymerase reaction for measurement of RNA and/or sequencing.
  • a fixation agent, a staining agent, an analyte-specific antibody, and/or a detectable label are added to the main assay path, cell staining and/or labeling an analyte can be performed on the aliquot.
  • FIG. 1 is a schematic diagram showing a microfluidic system or a cell culture interrogator system according to one embodiment described herein.
  • the system comprises multiple organ-on-a-chip microfluidic devices fluidly connected to each other via a microfluidic sample device (also referred to as "an immunoassay chip” in one embodiment).
  • the system can be used for periodically conducting an assay on a fluid exposed to cells cultured in an organ-on-a-chip microfluidic device.
  • FIG. 2A is a first schematic diagram illustrating the cross-section of a membrane valve and how a membrane valve works in a microfluidic sample device according to one embodiment described herein.
  • FIG. 2B is a second schematic diagram illustrating the cross-section of the membrane valve of FIG. 2A and how the membrane valve works in the microfluidic sample device.
  • FIG. 3 is a schematic diagram illustrating a top view of a portion of a microfluidic sample device according to one embodiment described herein fluidly connecting an organ- on-a-chip microfluidic device to an assay module.
  • FIG. 4 is a schematic diagram illustrating a top view of one embodiment of an assay module.
  • the assay module can comprise one or more sensors for detecting a target molecule, an optional multiplexer, and an optional demultiplexer.
  • FIG. 5 is a schematic diagram illustrating a top view of a microfluidic sample device according to one embodiment described herein.
  • FIG. 6A illustrates a first step in a schematic diagram showing operation of a microfluidic pump according to one embodiment described herein.
  • FIG. 6B illustrates a second step in the operation of the microfluidic pump represented in the schematic diagram of FIG. 6 A.
  • FIG. 6C illustrates a third step in the operation of the microfluidic pump represented in the schematic diagram of FIG. 6 A.
  • FIG. 6D illustrates a fourth step in the operation of the microfluidic pump represented in the schematic diagram of FIG. 6 A.
  • FIG. 6E illustrates a fifth step in the operation of the microfluidic pump represented in the schematic diagram of FIG. 6 A.
  • FIG. 6F illustrates a sixth step in the operation of the microfluidic pump represented in the schematic diagram of FIG. 6 A.
  • FIG. 7 is a schematic diagram showing a top view of a portion of a microfluidic sample device according to one embodiment described herein.
  • the microfluidic sample device comprises a sample fluid path, a main assay path, and a membrane valve located between the sample fluid path and the main assay path.
  • FIG. 8 is a schematic diagram showing a top view of an example multiplexer microfluidic device.
  • the multiplexer microfluidic device converts one fluidic input into multiple fluidic channels.
  • FIG. 9 is a photograph showing an example pneumatic control box.
  • FIG. 10 is an image showing a top half of an example Lab VIEW user interface.
  • FIG. 11 is an image showing a bottom half of an example Lab VIEW user interface.
  • FIG. 12 is a schematic diagram illustrating a top view of a microfluidic sample device according to one embodiment described herein. For simplified illustrations purposes only, details of membrane valves and microfluidic pumps are not shown in the figure.
  • FIG. 13 is a schematic diagram illustrating a top view of a microfluidic sample device according to another embodiment described herein.
  • the microfluidic sample device comprises an entire stack having a fluidic layer and an agent reservoir layer (e.g., a well template).
  • the fluidic layer is disposed on the agent reservoir layer.
  • the agent reservoir layer can comprise a plurality of chambers or wells each for holding an agent or reagent.
  • the chambers and wells can be arranged in an ordered array or in any configuration.
  • the fluidic layer has a similar configuration or design as shown in FIG. 12. For simplified illustrations purposes only, details of membrane valves and microfluidic pumps are not shown in the figure.
  • FIG. 14 is a schematic diagram illustrating a top view of a microfluidic sample device of FIG. 12 or FIG. 13.
  • the microfluidic sample device comprises a fluidic layer and a pneumatic layer.
  • the fluidic layer and pneumatic layer can form a microfluidic cartridge for providing fluidic connection and transfer.
  • the two layers must be distinct layers using traditional fabrication methods (e.g., hot embossing, casting, injection molding, machining, etc.).
  • the two layers are separated by an elastic membrane that provides fluid actuation as a result of deflection using vaccum or pressure.
  • the orientation does not typicall affect function, in most cases.
  • the entire device can be made from a single piece of one or more materials deposited in the fabrication process.
  • This approach results in a monolithic device with the same functional regions (e.g., pneumatic vs. fluidic) as the multi-part device illustrated in the schematic diagram of FIG. 14.
  • the fluidic layer can have a similar configuration or design as shown in FIG. 12.
  • the pneumatic layer comprises membrane valves, microfluidic pumps, and corresponding pneumatic conduits and inlets.
  • FIG. 15 is a schematic diagram illustrating a magnified top view of a portion of a microfluidic sample device of FIG. 12 or FIG. 13 comprising fluidic connections to an assay module.
  • the portion of the microfluidic device can comprise a plurality of waste fluid paths and corresponding fluid outlets or ports of the main assay paths.
  • the fluid outlets or ports of the main assay paths can be fluidly connected to corresponding input ports of an assay module
  • FIG. 16 is a schematic diagram illustrating a top view of a microfluidic analyzer according to one embodiment described herein.
  • the microfluidic analyzer can comprise a microfluidic sample device and an assay module.
  • the microfluidic sample device can comprise an agent reservoir layer, and a microfluidic cartridge in controllable fluidic connection with the agent reservoir layer.
  • the microfluidic cartridge can comprise a fluidic layer and a pneumatic layer.
  • the assay module can be overlaid on top of the microfluidic cartridge.
  • the assay module (e.g., sensing element) can be mounted onto the cartridge. This enables the microfluidic cartridge to provide programmable fluid/reagent delivery to a sensor while the sensor is able to collect information on the target analytes.
  • the sensor can be glued, mounted with adhesive or compression, soldered, etc.
  • microfluidic sample devices and/or systems comprising the same, which can be used to sample a fluid in a microfluidic device while maintaining a substantially continuous flow of the fluid in the microfluidic device.
  • the microfluidic sample devices and/or systems can be configured to conduct one or more assays on a sample fluid from one or more microfluidic devices (e.g., cell-culture microfluidic device(s)) while maintaining a substantially continuous flow of a fluid in the microfluidic device(s) (e.g., cell-culture microfluidic device(s)).
  • the inventors have developed a microfluidic sample device comprising a sample fluid path, a main assay path, and a membrane valve located between the sample fluid path and the main assay path.
  • a microfluidic sample device e.g., a cell-culture microfluidic device
  • a downstream fluid-receiving device e.g., a reservoir or another cell- culture microfluidic device.
  • a sample fluid path within a microfluidic sample device can allow for continuous flow of a fluid (e.g., a cell culture medium) from a microfluidic device (e.g., a cell-culture microfluidic device such as an organ-on-a-chip device) to a downstream fluid-receiving device (e.g., a reservoir or another cell-culture microfluidic device) while at least an aliquot of a sample fluid from the microfluidic device enters into a main assay path within the microfluidic sample device for an assay to be performed via control of the opening and closing of a membrane valve located between the sample fluid path and the main assay path.
  • a fluid e.g., a cell culture medium
  • a microfluidic device e.g., a cell-culture microfluidic device such as an organ-on-a-chip device
  • a downstream fluid-receiving device e.g., a reservoir or another cell-culture microfluidic device
  • microfluidic device e.g., a cell-culture microfluidic device
  • a fluid e.g., a cell culture medium
  • embodiments of various aspects described herein relate to microfluidic sample devices and systems comprising the same for sampling of a fluid in a device and/or performing an assay on a sample fluid from the device without disrupting normal operation of the device, as well as uses thereof.
  • a microfluidic sample device for sampling of a fluid and optionally performing an assay on a sample fluid.
  • a microfluidic sample device comprises (a) a sample fluid path having a sample-fluid inlet for receiving the sample fluid and a sample-fluid outlet for allowing the fluid to exit the microfluidic sample device; (b) a main assay path that is coupled to an assay module for performing an assay; and (c) a membrane valve located between the sample fluid path and the main assay path. The membrane valve is operable between a first valve state and a second valve state.
  • the first valve state is when the sample fluid flows through the sample fluid path and exits the sample- fluid outlet; and the second valve state is when at least an aliquot of the sample fluid flows from the sample fluid path into the main assay path to permit an assay performed on the aliquot.
  • Such microfluidic sample device can be used to periodically perform an assay on a sample fluid from a device without disrupting normal operation of the device.
  • the microfluidic sample device 300 comprises a main body 302, and a sample fluid path 304 and a main assay path 306 disposed in the main body.
  • a membrane valve 308 is located between the sample fluid path 304 and the main assay path 306.
  • the sample fluid path 304 is configured to receive a sample fluid from a microfluidic device 310 (e.g., a cell- culture microfluidic device such as an organ-on-a-chip device) and at least direct at least a portion of the sample fluid to a downstream fluid-receiving device.
  • a microfluidic device 310 e.g., a cell- culture microfluidic device such as an organ-on-a-chip device
  • the main assay path 306 is configured to at least receive an aliquot of the sample fluid received by the sample fluid path 304, a wash fluid, and/or at least one or more reagents, and is further configured to be coupled to a sample multiplexer within an assay module 312 (e.g., an immunoassay chip) for performing an assay.
  • the membrane valve 308 is configured to control fluid flow between the sample fluid path 304 and the main assay path 306.
  • the main body 302 can be made of any thermoplastic and/or thermocurable polymeric or glass materials, or any materials that are compatible with cell culture reagents and assay reagents.
  • Exemplary polymeric materials that can be used in the main body include, but are not limited to, polyurethanes, rubber, molded plastic, polymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLONTM), polyvinylchloride (PVC), polydimethylsiloxane (PDMS), polysulfone, and a combination of two or more thereof.
  • PMMA polymethylmethacrylate
  • TEFLONTM polytetrafluoroethylene
  • PVC polyvinylchloride
  • PDMS polydimethylsiloxane
  • polysulfone polysulfone
  • the sample fluid path 304 has a sample fluid inlet 314 for receiving a sample fluid and a sample fluid outlet 316 for allowing at least a portion of the sample fluid received by the sample fluid path to exit the microfluidic sample device.
  • the membrane valve 308 is actuated to permit fluid flow from the sample fluid path 304 to the main assay path 306, at least an aliquot of a sample fluid received by the sample fluid path 304 can enter the main assay path 308 and the remaining of the sample fluid in the sample fluid path 304 can continue to flow through the sample fluid path 304 and exit the sample fluid outlet 316, if the flow rate of the sample fluid at the sample fluid inlet 314 is greater than the flow rate through the sample fluid path 304.
  • This can allow one to sample at flow rates independent from perfusion rates in a microfluidic device that provides the sample fluid via the sample fluid inlet 314.
  • the sample fluid path 304 is a channel, a conduit, or a duct defining a passageway through and along which a fluid flows, passes or moves between the sample fluid inlet 314 and the sample fluid outlet 316.
  • the sample fluid path 304 can be of any tortuosity or of any path pattern.
  • the sample fluid path 304 can be designed to form a curved pathway, e.g., to maintain the compactness of the microfluidic sample device.
  • FIGs. 3 and 7 show one embodiment of the sample fluid path that can be disposed in the microfluidic sample devices described herein.
  • the sample fluid path 304 can comprise a U-shaped pathway.
  • the sample fluid path 304 can also form a linear pathway, a curved pathway, an irregular-shaped pathway, or a pathway of any other shape.
  • FIG. 3 or FIG. 7 illustrates a microfluidic sample device 300 with a single sample fluid path 304, it should not be construed as limiting.
  • the microfluidic sample device 300 can comprise a plurality of sample fluid paths 304, e.g., at least two or more, including, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more sample fluid paths 304.
  • Multiple sample fluid paths in a microfluidic sample device can allow for performing assays using samples collected from different microfluidic devices 310 (e.g., cell-culture microfluidic devices, organ-on-a-chip devices).
  • each of the sample fluid paths 304 can have a sample fluid inlet 314 independently and fluidly connected to a microfluidic device 310 (e.g., a cell-culture microfluidic device), a sample fluid outlet 316 independently and fluidly connected to a downstream fluid-receiving device, and a membrane valve 308 located between the sample fluid path 304 and a main assay path 306.
  • a microfluidic device 310 e.g., a cell-culture microfluidic device
  • sample fluid outlet 316 independently and fluidly connected to a downstream fluid-receiving device
  • a membrane valve 308 located between the sample fluid path 304 and a main assay path 306.
  • the plurality of sample fluid paths 304 can share the same main assay path 306.
  • the plurality of sample fluid paths 304 can have their individual main assay paths 306.
  • the terms “fluidly communicates” and “fluidly connects” between two or more components e.g., a sample fluid inlet 314 and a microfluidic device 310; or a sample fluid outlet 316 and a downstream fluid-receiving device; or a sample fluid path 304 and a main assay path 306 fluidly connected by a membrane valve) or equivalent thereof means that a fluid (e.g., gas or liquid) can flow from one component (e.g., a microfluidic device 310, a sample fluid outlet 316, or a sample fluid path 304) to another (e.g., a sample fluid inlet 314, a downstream fluid-receiving device, or a main assay path 306, respectively) but does not exclude an intermediate component between the two recited components which are in fluid communication.
  • a fluid e.g., gas or liquid
  • the two components e.g., a sample fluid inlet 314 and a microfluidic device 310; or a sample fluid outlet 316 and a downstream fluid-receiving device
  • the two components are integral to each other.
  • the two components e.g., a sample fluid inlet 314 and a microfluidic device 310; or a sample fluid outlet 316 and a downstream fluid-receiving device; or a sample fluid path 304 and a main assay path 306 fluidly connected by a membrane valve
  • an intermediate component e.g., a sample fluid inlet 314 and a microfluidic device 310; or a sample fluid outlet 316 and a downstream fluid-receiving device; or a sample fluid path 304 and a main assay path 306 fluidly connected by a membrane valve
  • a sample fluid inlet 314 and a microfluidic device 310, or a sample fluid outlet 316 and a downstream fluid-receiving device can be fluidly connected by a fluidic channel and/or tubing.
  • a sample fluid path 304 and a main assay path 306 are fluidly connected by a membrane valve 308 which can control the flow of a fluid between the sample fluid path 304 and the main assay path 306.
  • the sample fluid path 304 comprising a sample-fluid inlet 314 and a sample-fluid outlet 316 can allow for continuous flow of culture medium from a cell-culture microfluidic device (e.g., an organ-on-a-chip device) to a downstream fluid-receiving device (e.g., a reservoir or another cell-culture microfluidic device) while at least an aliquot of a sample fluid from a microfluidic device 300 (e.g., a cell-culture microfluidic device) enters into the main assay path 306 for an assay to be performed through control of the membrane valve 308 located between the sample fluid path 304 and the main assay path 306.
  • a cell-culture microfluidic device e.g., an organ-on-a-chip device
  • a downstream fluid-receiving device e.g., a reservoir or another cell-culture microfluidic device
  • a sample fluid from a microfluidic device 300 e.g.,
  • the length of the sample fluid path 304 can be of any dimension. However, a shorter sample fluid path can minimize dead volume, and/or allow more control over sampling time point and/or less mixing with a smaller cross-section. Similarly, while the width of the sample fluid path 304 can be of any dimension, reducing the width of the sample fluid path can minimize dead volume, and/or allow more control over sampling time point and/or less mixing with a smaller cross-section.
  • the cross-section of the sample fluid path 304 can be of any dimension, which can vary, e.g., with a sampling flow rate.
  • the cross-section of the sample fluid path 304 can have a dimension ranging from about 20 micrometers (" ⁇ ") to about 1000 ⁇ , from about 50 ⁇ to about 1000 ⁇ , from about 100 ⁇ to about 750 ⁇ , or from about 200 ⁇ to about 500 ⁇ .
  • the cross-section of the sample fluid path 304 can have a dimension of about 200 ⁇ .
  • a sample fluid path with a smaller cross-section can avoid or minimize dead volume issues.
  • the cross-section of the sample fluid path 304 can be of any shape, e.g., a circle, an ellipse, a triangle, a square, a rectangle, a polygon or any irregular shape.
  • the sample fluid path 304 can have a circular cross-section.
  • the sample fluid path 304 can have a square cross-section.
  • the main assay path 306 can have a first end portion adapted to fluidly connect to a fluid inlet or port 318 (e.g., for wash buffer), and a second end portion comprising a fluid outlet or port 320 adapted to fluidly connect to an assay module 312.
  • a fluid inlet or port 318 e.g., for wash buffer
  • a fluid outlet or port 320 adapted to fluidly connect to an assay module 312.
  • the fluid inlet or port 318 can be located at one end of the main assay path 306, and/or the fluid outlet or port 320 can be located at another end of the main assay path 306.
  • the fluid inlet or port 318 can be located in close proximity to one end of the main assay path 306, and/or the fluid outlet or port 320 can be located in close proximity to another end of the main assay path 306.
  • the fluid inlet or port 318 of the main assay path 306 can be adapted for receiving a wash buffer.
  • the main assay path 308 can comprise a wash inlet 318 in the first end portion (e.g., at the first end or in close proximity to the first end) for receiving a wash buffer to clean or rinse the main assay path 308 after a sample fluid exits the main assay path 308 and enters an assay module 312.
  • the main assay path 306 is a channel, a conduit, or a duct defining a passageway through and along which a fluid flows, passes or moves between at least one fluid inlet or port 318 and at least one fluid outlet or port 320.
  • the main assay path 306 can be of any path pattern.
  • the main assay path 306 can be designed to form a linear pathway between a fluid inlet or port 318 and a fluid outlet or port 320 as shown in FIG. 3.
  • the main assay path 306 can comprise a linear pathway with branching at one or both end portions, where each branching can comprise a fluid inlet 318 or a fluid outlet 320.
  • the main assay path 306 can also form a curved pathway or any other shape.
  • a curved main assay path can reduce footprint on the microfluidic sample devices described herein.
  • the length of the main assay path 306 can be of any dimension, which can vary with a number of factors including, e.g., the number of reagent input conduits 322, which will be described below, spacing between the reagent input conduits along the main assay path, the number of sample fluid paths, the number of inputs along the main assay path 306, and/or the number of membrane valves 326 located along the main assay path 306.
  • the main assay path can range from about 10 millimeters to about 100 millimeters.
  • the cross-section of the main assay path 306 can be of any dimension, which can vary, e.g., with a flow rate desired in the main assay path 306.
  • the cross-section of the main assay path 306 can have a dimension ranging from about 20 ⁇ to about 1000 ⁇ , from about 50 ⁇ to about 1000 ⁇ , from about 100 ⁇ to about 750 ⁇ , or from about 200 ⁇ to about 500 ⁇ .
  • the cross-section of the main assay path 306 can be of any shape, e.g., a circle, an ellipse, a triangle, a square, a rectangle, a polygon or any irregular shape.
  • the main assay path 306 can have a circular cross- section.
  • the main assay path 306 can have a square cross-section.
  • the main assay path 306 can be used to deliver different types of fluids.
  • the main assay path 306 can direct at least an aliquot of a sample fluid from the sample fluid path 304 to an assay module 312 to perform an assay on the aliquot.
  • the main assay path 306 can direct at least one or more reagents received from one or more reagent input conduits 322 (as described below) to an assay module 312, thereby providing assay reagent(s) that are required to perform an assay.
  • the main assay path 306 can provide a passageway or a chamber/space for an aliquot of a sample fluid to contact and/or mix with one or more reagents, prior to moving to an assay module 312.
  • the microfluidic sample device 300 also comprises a membrane valve 308 located between a sample fluid path 304 and a main assay path 306.
  • the membrane valve 308 is configured to operate between a first valve state to prevent a sample fluid received by the sample fluid path 304 from entering the main assay path 306, and a second valve state to allow at least an aliquot of the sample fluid to flow from the sample fluid path 304 into the main assay path 306, which is then directed to an assay module 312 to perform an assay.
  • Examples of a membrane valve that can be used to control fluid flow between the sample fluid path 304 and the main assay path 306 include, but are not limited to, pinch valves, pneumatic valves, mechanical valves, solenoid valves, thermoresponsive valves, electroresponsive valves, magnetic valves, hydraulic valves, and a combination of two or more thereof.
  • the membrane valve 308 can comprise: (i) an actuation structure 307 comprising a membrane actuation mechanism; and (ii) an elastomeric membrane 308M located between the actuation structure 307 and a fluidic structure 309 comprising a portion of the sample fluid path 304 and a portion of the main assay path 306 such that the elastomeric membrane 308M is oriented transverse to the sample fluid path portion and the main assay path portion.
  • the elastomeric membrane can be actuated between the first valve state and the second valve state as described above.
  • the membrane can be actuated by various art-recognized actuation mechanisms, e.g., but not limited to pneumatically-operated actuators, mechanically-operated actuators, and/or electrically-operated actuators.
  • the membrane can be actuated pneumatically to open or close a fluidic conduit between a portion of the sample fluid and a portion of the main assay path.
  • the membrane actuation mechanism can comprise a pneumatic conduit 324. Application of an air pressure to the pneumatic conduit can actuate the membrane valve 308 to prevent a sample fluid in the sample fluid path 304 from entering the main assay path 306.
  • a pneumatic conduit 324 is a channel, a conduit, or a duct defining a passageway through and along which air flows, passes or moves between at least one pneumatic inlet 328 and a corresponding membrane valve 308.
  • the pneumatic conduit 324 can be of any path pattern.
  • the pneumatic conduit 324 can be designed to form a linear pathway between a pneumatic inlet 328 and a corresponding membrane valve 308 as shown in FIG. 5.
  • the pneumatic conduit 324 can also form a curved pathway or any other shape.
  • the length of the pneumatic conduit 324 can be of any dimension, which can vary with a number of factors including, e.g., air pressure, vacuum, and/or membrane thickness. In general, a shorter pneumatic conduit has a smaller dead volume, which in turn makes the corresponding membrane valves more responsive to air or gas pressure. In some embodiments, the length of the pneumatic conduit 324 can be optimized, e.g., for resistance volume.
  • the length of the pneumatic conduit can be designed to accommodate air or gas pressure, and/or flow rate of gas (e.g., air)/vacuum source and/or speed at which a user desires to drive,
  • the length of the pneumatic conduit 324 can designed based on design constraint s) of any components (e.g., pumps) of the devices or systems described herein.
  • the cross-section of the pneumatic conduit 324 can be of any dimension, which can vary, e.g., with an air or gas flow rate desired in the pneumatic conduit 324. Generally, the air or gas flow rate within the pneumatic conduit 324 should be higher than the sum of the total dead volume and valve volume for a given amount of time, and can also depend on actuation frequency of the corresponding membrane valve. In some embodiments, the cross- section of the pneumatic conduit 324 can have a dimension ranging from about 20 ⁇ to about 1000 ⁇ , about 50 ⁇ to about 1000 ⁇ , from about 100 ⁇ to about 750 ⁇ , or from about 200 ⁇ to about 500 ⁇ .
  • the cross-section of the pneumatic conduit 324 can have a dimension of about 200 ⁇ .
  • a smaller cross-section of the pneumatic conduit 324 can reduce or avoid dead volume issues.
  • the cross-section of the pneumatic conduit 324 can be of any shape, e.g., a circle, an ellipse, a triangle, a square, a rectangle, a polygon or any irregular shape.
  • the pneumatic conduit 324 can have a circular cross-section.
  • the pneumatic conduit 324 can have a square cross-section.
  • FIGs. 2A and 2B illustrate a cross-section of a membrane valve according to one embodiment described herein.
  • the membrane valve 308 can be formed by placing a membrane 308M against a ridge 308R separating two fluidic channels (e.g., a sample fluid path 304 and a main assay path 306 of a microfluidic sample device 300 described herein) and uses the membrane 308M to create a fluidic pathway between the two fluidic channels (e.g., a sample fluid path 304 and a main assay path 306), which is controllably opened or closed by introducing vacuum or air pressure, respectively, to the valve 308. Vacuum or air pressure can be introduced to the membrane 308M through a pneumatic conduit 324.
  • the air pressure forces the membrane 308M to deflect toward the ridge 308R, pushing down against the two fluidic channels (e.g., a sample fluid path 304 and a main assay path 306), which in turn blocks a fluid flow between the two fluid channels (e.g., a sample fluid path 304 and a main assay path 306).
  • the two fluidic channels e.g., a sample fluid path 304 and a main assay path 306
  • the membrane 308M When vacuum is introduced to the valve 308, the membrane 308M is pulled away from the ridge 308R or is pulled away from the two fluidic channels (e.g., a sample fluid path 304 and a main assay path 306), thus providing a fluidic pathway between the two fluidic channels (e.g., a sample fluid path 304 and a main assay path 306).
  • the two fluidic channels e.g., a sample fluid path 304 and a main assay path 306
  • the membrane 308M can be made of any flexible polymeric material(s).
  • An appropriate flexible polymeric material(s) can allow the membrane 308 to form a reversible seal with the surface of the ridge 308R when it is pushed by the air pressure to deflect toward the ridge 308R.
  • the membrane 308M can comprise or essentially consist of, or consist of styrene-ethylene/butylene-styrene (SEBS).
  • SEBS styrene-ethylene/butylene-styrene
  • polystyrene can be used for a pneumatic layer, which is located in a top half of the device, to provide a permanend bond necessary for pressure or vacuum actuation of the membrane 308M without delamination.
  • the bottom portion of the device illustrated in FIGs. 2A and 2B is optionally a material (e.g., polycarbonate) that prevents the membrane 308M from sticking to the fluidic channel discontinuity, which would preven the valve from opening. Accordingly, the membrane 308M can be bonded to the polystyrene with heat bonding, while the other side can be polycarbonate, which will not bond as well to SEBS, to provide differential bonding across the membrane.
  • a material e.g., polycarbonate
  • an aliquot of a sample fluid received by the main assay path can be subjected to at least one or more assays.
  • the microfluidic sample device 300 can further comprise at least one reagent input conduit 322 that controllably releases a reagent into the main assay path 306.
  • the reagent input conduits 322 are located downstream of the membrane valve 308 that controls fluid flow between the sample fluid path 304 and the main assay path 306. The reagent(s) released into the main assay path 3-6 can be directed to an assay module 312.
  • At least one reagent can be controllably released through the corresponding reagent input conduit 322 into an aliquot of a sample fluid while the aliquot moves along the main assay path 322 toward an assay module 322. In some embodiments, at least one reagent can be controllably released through the corresponding reagent input conduit 322 into the main assay path 306 after an aliquot of a sample fluid has moved along the main assay path 306 and entered an assay module 312.
  • At least one reagent can be controllably released through the corresponding reagent input conduit 322 into the main assay path 306 and be directed to the assay module 312 via the main assay path 306 before an aliquot of a sample fluid enters the main assay path 306.
  • the microfluidic sample device 300 can further comprise at least one or more (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more) reagent input conduits 322.
  • the number of the reagent conduits 322 can vary with the number of required different reagents for one or more assays to be performed in the assay module 312.
  • Each of the reagent input conduits 322 is adapted to controllably release a reagent into a fluid as the fluid moves along the main assay path 306 toward the assay module 312. As shown in FIG.
  • the reagent input conduits 322 can each be configured to have a membrane valve 326 located between the reagent input conduit 322 and the main assay path 306.
  • the membrane valve 326 is operable between a first reagent valve state and a second reagent valve state.
  • the first reagent valve state closes the fluidic pathway between the reagent input conduit 322 and the main assay path 306 to inhibit the main assay path 306 from receiving a reagent received by the reagent input conduit 322, while the second reagent valve state opens the fluidic pathway between the reagent input conduit 322 and the main assay path 306 to permit the main assay path 306 to receive a reagent received by the reagent input conduit 322.
  • the membrane valve 326 used to control fluid flow between the reagent input conduit 322 and the main assay path 306 can be of the same type, or of a different type, of the membrane valve 308 used to control fluid flow between the sample fluid path 304 and the main assay path 306.
  • the membrane valve 326 located between the reagent input conduit 322 and the main assay path 306 can be controlled by pneumatic inputs as described for the membrane valve 308 located between the sample fluid path 304 and the main assay path 306 (e.g., as shown in FIG. 5) to switch between the first reagent valve state and the second reagent valve state.
  • a reagent input conduit 322 is a channel, a path, or a duct defining a passageway through and along which a reagent (for performing an assay) flows, passes or moves between at least one reagent inlet 3221 and a corresponding membrane valve 326.
  • the reagent input conduit 322 can be of any path pattern.
  • the reagent input conduit 322 can be designed to form a linear pathway between a reagent inlet 3221 and a corresponding membrane valve 326 as shown in FIG. 5.
  • the reagent input conduit 322 can also form a curved pathway or any other shape.
  • the length of the reagent input conduit 322 can be of any dimension, which can vary with a number of factors including, e.g., arrangement of the reagent input conduits for ease of access, e.g., by a robotic arm.
  • the cross-section of the reagent input conduit 322 can be of any dimension, which can vary, e.g., with a reagent flow rate desired in the reagent input conduit 322.
  • the cross-section of the reagent input conduit 322 can have a dimension ranging from about 50 ⁇ to about 1000 ⁇ , from about 100 ⁇ to about 750 ⁇ , or from about 200 ⁇ to about 500 ⁇ .
  • the cross-section of the reagent input conduit 322 can be of any shape, e.g., a circle, an ellipse, a triangle, a square, a rectangle, a polygon or any irregular shape.
  • the reagent input conduit 322 can have a circular cross-section.
  • the reagent input conduit 322 can have a square cross-section.
  • FIG. 5 illustrates that the reagent input conduits 322 are placed substantially perpendicular to the main assay path 306, the reagent input conduits 322 can also be placed at an angle relative to the main assay path 306. This can maintain the compactness of the microfluidic sample device while maintaining a desired length of a reagent input conduit.
  • the reagent input conduits 322 can be placed at an angle between 45 degrees and 135 degrees, relative to the main assay path 306.
  • FIGs. 3 and 5 illustrate one or another embodiment of the microfluidic sample devices described herein or a portion thereof and are not construed to be limited to the specific design and layout of different components of the microfluidic sample devices as shown therein. Modifications within one of skill in the art are also within the scope of various embodiments described herein. For example, instead of having all the reagent input conduits on the left side and the pneumatic conduits on the right side, relative to the direction of fluid flow in the main assay path, as shown in FIG. 5, one can switch their placements and have them the opposite.
  • the microfluidic sample device 300 further comprises a membrane valve 330 located between the fluid inlet or port 318 of the main assay path 306 and the membrane valve 308 for fluid control between the sample fluid path 304 and the main assay path 308.
  • the membrane valve 330 can be actuated to direct a fluid (e.g., a wash buffer) to flow along the main assay path 306 in another valve state.
  • valve states e.g., opening or closing
  • the membrane valves By controlling the valve states (e.g., opening or closing) of the membrane valves within the microfluidic sample devices described herein, one can control when a sample is taken as well as controlling the inputs of reagents for running an assay.
  • the use of the membrane valves to control fluid control can reduce or minimize disruption in fluid flow in a microfluidic device (e.g., a cell-culture microfluidic device) while a sample of an effluent from the microfluidic device is taken.
  • a microfluidic device e.g., a cell-culture microfluidic device
  • Such advantages can also be leveraged in experiments in which multiple (e.g., at least two or more) microfluidic devices (e.g., a cell culture microfluidic device) are being interconnected by at least one or more microfluidic sample devices described herein.
  • two microfluidic devices e.g., two cell culture microfluidic devices
  • multiple microfluidic devices e.g., cell-culture microfluidic devices
  • the aliquot sample can move along the main assay path 306 and be directed to an assay module 312 with or without reagent(s) added.
  • the main assay path 306 has a second end portion comprising a fluid outlet or port 320 that is adapted to fluidly connect to an assay module 312.
  • the term "an aliquot of a sample fluid,” or “an aliquot sample,” or an equivalent thereof refers to a fluid that is received by a main assay path 306 from a sample fluid path 304.
  • the source of the fluid received by the sample fluid path 304 can be derived from or collected from an effluent or output of a microfluidic device (e.g., a cell- culture microfluidic device) fluidly connected upstream of a microfluidic sample device described herein.
  • a microfluidic device e.g., a cell- culture microfluidic device
  • An assay module 312 is a structural module configured to perform one or more analytical assay(s) on one or more samples.
  • module does not imply that the components or functionality described as part of the module are all configured in a common package or unit. Indeed, any or all of the various components of a module, whether a sensor or other components such as a sample multiplexer or a sample demultiplexer, can be combined in a single package/unit or separately maintained.
  • assay refers to a procedure or a method by which a property or characteristic of at least one component (e.g., cells or supernatant) in a sample and/or a target analyte is detected and/or measured.
  • Assay is a short hand commonly used term for biological assay and is a type of in vitro experiment. Assays can be conducted to measure effects of a test agent on living organisms (e.g., cells, tissues, and/or embryos) cultured in a cell-culture microfluidic device such as an organ-on a-chip. Assays can be qualitative or quantitative.
  • the assay module 312 comprises a sample multiplexer microfluidic device (also referred to as “a sample multiplexer”) 334, one or more sensors 332, and a sample demultiplexer microfluidic device (also referred to as "a sample demultiplexer”) 336.
  • a sample multiplexer microfluidic device also referred to as "a sample multiplexer”
  • sensors 332 one or more sensors 332
  • a sample demultiplexer microfluidic device also referred to as "a sample demultiplexer”
  • a sample demultiplexer microfluidic device also referred to as "a sample demultiplexer”
  • an assay module 312 does not require a sample multiplexer microfluidic device 334 or sample demultiplexer microfluidic device 336.
  • At least one or more component(s) of an assay module 312 can be integrated into or separated from the microfluidic sample device 300.
  • a sample multiplexer 334 and/or a sample demultiplexer 336 can be integrated into or separated from a microfluidic sample device 300 described herein.
  • at least one or more sensors 332 can be integrated into or separated from a microfluidic sample device described herein.
  • a sample multiplexer 334 is a microfluidic device configured to split a single fluidic input into a plurality of (e.g., at least 2 or more, including, e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more) separate fluidic channels.
  • a sample multiplexer can allow a user to control the number of samples to run in a single assay.
  • the samples can be derived or collected from the same or different source microfluidic device(s) (e.g., same cell-culture microfluidic device or different cell-culture microfluidic devices). For example, as shown in FIG.
  • a sample multiplexer can split a single fluidic input into 9 separate fluidic channels since a sensor, in this embodiment, can do up to 8 samples.
  • Each fluidic channel can comprise a membrane valve such that the fluidic channels can be open and close independent of each other by controlling different combinations of valve opening and closing.
  • An extra fluidic channel of the sample multiplexer can be fluidly connected to a waste reservoir, e.g., in case where air bubbles are present within the microfluidic sample device and/or assay module, the air bubbles can be flushed out before they enter a sensing area of the assay module.
  • the membrane valves used in the sample multiplexer can be of the same type or of different types, as used to control flow between the sample fluid path 304 and the main assay path 306, and/or between the reagent input conduits 322 and the main assay path 306.
  • one or more microfluidic resistors can be implemented in a sample multiplexer. This can allow substantially even fluid flow in all fluidic channels when a fluid is desired to flow in all of the fluidic channels.
  • Fluidic resistors are known in the art and can be adapted to be implemented in a sample multiplexer (see, e.g., DOI: 10.1039/B806140H, 10.1063/1.2363931, 10.1039/C2LC20799K).
  • the term "sensor” refers to a device for measuring or detecting a qualitative or quantitative physical parameter indicative of a property of a sample and converting it into a signal which can be read or detected by a user and/or by an instrument.
  • Signal can include, but are not limited to sound (e.g., ultrasound), light (e.g., infrared waves, microwaves, radio waves, ultraviolet waves, fluorescent light, visible light), electrical current or voltage, color, and a combination of two or more thereof.
  • a sensor is a device comprising one or more reactive means being adapted to detect one or more target analytes or molecules such as microorganisms or related (bio-) molecules (e.g., a pH sensor, an enzyme sensor, organelle sensor, tissue sensor, microorganism sensor, immunosensor, and chemical or electrochemical sensor), additionally having the capability to provide a signal for detection.
  • target analytes or molecules such as microorganisms or related (bio-) molecules
  • bio- e.g., a pH sensor, an enzyme sensor, organelle sensor, tissue sensor, microorganism sensor, immunosensor, and chemical or electrochemical sensor
  • reactive is defined as having the capability to selectively interact with such target analytes or molecules.
  • biosensors There are generally two categories of sensors: biosensors and chemical/electro- chemical sensors.
  • biosensors function by providing a means of specifically binding, and therefore detecting, a target biologically active analyte.
  • the biosensor is highly selective, even when presented with a mixture of many chemical and biological entities, such as present in cell culture sample.
  • Electrochemical and chemical sensors which rely on chemically reactive means, generally do not have either the high selectivity or the amplification properties of biosensors but are highly reliable, inexpensive, and often very well established.
  • a sensor can comprise an electrode.
  • An electrode can be used to measure electric impedance of a sample.
  • a sensor can comprise a glass sensing layer with gold interdigitated electrodes.
  • a sensor can comprise sensing surface functionalized with an antibody specific for a target analyte.
  • a sensor can comprise a sheet of glass with a gold layer that is functionalized with a biotinylated thiol self-assembled monolayer, followed by streptavidin, and later, biotinylated capture antibody for specifically binding a target analyte.
  • microfluidic sample devices described herein can be used with any sensor type that requires fluid connections or fluid contact and multiple reagents.
  • sensors that can be used in an assay module include, but are not limited to electrical sensors, mass-sensitive sensors, optical sensors, thermal sensors, magnetic sensors, biochemical sensors, and a combination of two or more thereof.
  • the sensors can be regenerated.
  • the microfluidic sample devices can be used for continuous monitoring of a target analyte in an effluent from a cell-culture microfluidic device.
  • sensors that can be regenerated include antibody- and/or nucleic acid-based sensors, thermal or heat sensors, electrical sensors, light sensitive sensors, and a combination of two or more thereof.
  • a release agent can be flowed across the sensor to release any bound target analyte therefrom without significantly deactivating or denaturing the capture agent (e.g., antibody or nucleic acid).
  • a specific wavelength of light can be used change conformation of a bound target analyte and thus release the bound target analyte from the sensor.
  • the sensors can be fabricated via standard photolithography and optionally gold deposition processes known in the art.
  • the microfluidic sample device described herein and/or the assay module can have a plurality of (e.g., at least two) sensors or sensor spots.
  • the microfluidic sample device described herein can have at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at last ten, at least 20, at least 30, at least 40, at least 50, at least 60 or more sensors or sensor spots.
  • the microfluidic sample device described herein can have up to 64 sensor spots.
  • Each sensor or sensor spot on the microfluidic sample device described herein can be adapted to perform at least one single assay or multiple assays (e.g., serial assays for the same target analyte(s)).
  • the microfluidic sample device described herein can be used to perform a single assay or multiplexing (e.g., multiple assays) depending on the needs of a user.
  • capture agents e.g., but not limited to capture antibodies
  • target analytes e.g., but not limited to target proteins
  • a detectable agent e.g., but not limited to secondary antibodies optionally labeled with a detectable label
  • a detectable label can be flowed into the assay module (or over the sensors), providing a detectable label for subsequent detection (e.g., but not limited to imaging such as epifluorescence imaging) and/or target analysis such as quantification.
  • a sensor can comprise a sheet of glass with an thermally evaporated gold layer that can be functionalized with a surface activating agent to permit attachment of capture agent(s) to the surface of the gold layer.
  • the gold layer can be functionalized with a biotinylated thiol self-assembled monolayer, followed by streptavidin, which can then interact with a biotinylated capture agent.
  • the assay module can comprise a digital RT-PCR for measurement of RNA molecules in a sample fluid.
  • the assay assay module can comprise mass spectroscopy and/or Raman microscopy.
  • the assayed sample can be directed to a waste reservoir for disposal, e.g., by fluidly connect the output of the assay module to a waste outlet 342.
  • the waste outlet 342 can be adapted to fluidly connect to a waste reservoir.
  • each of the assayed samples can be individually directed to a waste outlet 342 and a corresponding waste reservoir.
  • multiple assayed samples can be directed into a sample demultiplexer, which is fluidly connected to a waste outlet 342 and a waste reservoir for disposal.
  • a sample demultiplexer is a microfluidic device configured to convert at least two or more (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more) fluidic inputs into a single fluidic channel.
  • an assay module 312 can also comprises a sample demultiplexer that is adapted to fluidly connect to the outlet of the sensors, wherein the demultiplexer converts 9 fluidic outputs from the sensors into a single fluidic channel fluidly connected to a waste reservoir for disposal.
  • the number of fluidic outputs in the sample demultiplexer can be based on the number of fluidic channels created from a single fluidic input in a corresponding sample multiplexer.
  • the corresponding downstream multiplexer can be designed accordingly, e.g., to convert more than 9 fluidic outputs into one single fluidic channel.
  • the microfluidic sample device 300 can further comprise a fluid waste path 338 for removing a fluid from the microfluidic sample device 300, e.g., after an assay is performed on the fluid, or after a wash buffer is introduced to cleanse the main assay path 306.
  • the fluid waste path 338 is placed downstream of fluidic connections to an assay module 312.
  • the fluid waste path 338 can be placed downstream of an assay module output-receiving port 340.
  • the fluid waste path 338 can comprise a waste outlet 342 that allows a sample fluid or a wash fluid to exit the microfluidic sample device 300.
  • the fluid waste path 338 can comprise a fluid handling module.
  • the fluid handling module is designed to control fluid flow rate and/or direction within the microfluidic sample device during operation.
  • the fluid handling module can comprise a pump system 344.
  • the pump system 344 within the fluid waste path 338 can be configured to drive a sample to be assayed through the main assay path 306 and/or the assay module 312 toward a waste outlet 342 for the assayed sample to exit the microfluidic sample device.
  • Pump systems for control of fluid delivery are known in the art and can be adapted in the system described herein.
  • Examples of a pump system include, but are not limited to, a vacuum-driven system, a pressure-driven system, a peristaltic pump, a pneumatic pump, a mechanical pump, an acoustofluidic pump, an electrofluidic pump, and a combination of two or more thereof.
  • the microfluidic sample device 300 can comprise at least one or more microfluidic pumps 344.
  • the microfluidic pump(s) 344 can be located within a fluid waste path 338 for pulling a fluid through the main assay path 306 and/or the assay module 312 when needed, e.g., pulling a sample aliquot to enter an assay module 312 for an assay to be performed, or pulling an assayed sample out of the assay module 312 for waste disposal, or pulling a wash buffer through the main assay path 306 after an assay is performed such that the main assay path 306 is cleansed before subsequently receiving a second sample fluid for another assay.
  • the microfluidic pump 344 can be controlled using air pressure and vacuum.
  • FIGs. 6A-6F illustrate a microfluidic pump that can be used in the microfluidic sample device described herein, and also operation of the microfluidic pump to produce a full pump stroke.
  • a microfluidic pump can comprise at least three membrane valves (344A, 344B, 344C) connected in series.
  • a first membrane valve (referred to as “diaphragm valve” in FIGs. 6A-6F) 344B is placed at the center of the microfluidic pump 344 and provides the driving force for the pump.
  • a set of two second membrane valves (referred to as "check valve” in FIGs.
  • 344A, 344C are each placed on either side of the first membrane valve 344B.
  • a full pump stroke cycle can be generated and also be repeated to provide pulses of flow. Controlling the speed in which pump stroke cycles run, can control the resulting flow rate of the pump. This can provide a compact system that can be integrated into a microfluidic sample device as described herein.
  • the membrane valves 344A, 344B, 344C of the microfluidic pump 344 can be controlled by pneumatic inputs.
  • the membrane valves 344A, 344B, 344C of the microfluidic pump 344 can be fluidly connected to pneumatic conduits 324 such that each membrane valve is actuated to a desired valve state (e.g., allowing or blocking a fluid to pass through) by introducing air pressure or vacuum into the pneumatic conduit.
  • the pneumatic inputs can be controlled by a series of solenoid valves which can switch from applying air to applying vacuum.
  • the solenoid valves can be controlled by a controller, e.g., a microcontroller, and connected to a computer comprising a specific algorithm (e.g., a Lab VIEW program and interface) to control the timing of the opening and closing of the valves.
  • the algorithm can allow a user to input the number of pump strokes and/or desired volume of each input (e.g., sample volume, wash buffer, and/or reagents for an assay to be performed) so that automation of an assay can be performed on the microfluidic sample devices described herein.
  • the user can also have an option to manually control each membrane valve if it is desirable to manually run an assay.
  • the user can also control the microfluidic pump to change the flow rate of the system.
  • no pump is placed in a fluidic path between the reagent input conduits and the assay module.
  • a microfluidic pump 344 downstream of the assay module 312 this can reduce the likelihood of air bubbles formed with the microfluidic sample device during operation.
  • another aspect described herein is a microfluidic sample device for periodically performing an assay on a sample fluid with minimal air bubble formation within the microfluidic sample device during operation.
  • the microfluidic sample device comprises: (a) a sample fluid path having a sample-fluid inlet for receiving the sample fluid and a sample-fluid outlet for allowing the fluid to exit the microfluidic sample device; (b) a main assay path that periodically receives the fluid from the sample fluid path for performing the assay; (c) a fluid waste path for removing the fluid from the microfluidic sample device after the assay is performed on the fluid; and (d) a microfluidic pump located within the fluid waste path for pulling a wash fluid through the main assay path after the assay is performed such that the main assay path is cleansed before subsequently receiving a second sample fluid for another assay.
  • the fluid waste path can comprise a fluid outlet port that allows the sample fluid to exit the microfluidic sample device.
  • a surface of any fluid paths in contact with a fluid and/or reagent can be modified for reducing non-specific binding of an entity (e.g., a test agent or a target analyte) in a fluid to the surface of the fluid paths.
  • an entity e.g., a test agent or a target analyte
  • a fluid path e.g., sample fluid path, main assay path, reagent input conduit(s)
  • a fluid path e.g., sample fluid path, main assay path, reagent input conduit(s)
  • a surfactant e.g., PLURONIC® 127
  • a blocking protein such as bovine serum albumin
  • Additional surfactant that can be used to reduce the adhesive force between the surface of the fluid paths (e.g., sample fluid path, main assay path, reagent input conduit(s)) and non-specific binding of an entity (e.g., a test agent or a target analyte) in a fluid include, but are not limited to, hydrophilic (especially amphipathic) polymers and polymeric surface-acting agents; non-ionic agents such as polyhydric alcohol-type surfactants, e.g., fatty acid esters of glycerol, pentaerythritol, sorbitol, sorbitan, and more hydrophilic agents made by their alkoxylation, including polysorbates (TWEEN®); polyethylene glycol-type surfactants such as PLURONIC surfactants (e.g., poloxamers), polyethylene glycol (PEG), methoxypolyethylene glycol (MPEG), polyacrylic acid, polyglycosides, soluble polysaccharides,
  • a skilled artisan will readily be able to determine appropriate methods and/or reagents for use to reduce non-specific binding of an entity (e.g., a test agent or a target analyte) in a fluid to the surface of the fluid paths (e.g., sample fluid path, main assay path, reagent input conduit(s)) based on the substrate material of the microfluidic sample devices and/or types of entities to be blocked.
  • entity e.g., a test agent or a target analyte
  • the fluid paths e.g., sample fluid path, main assay path, reagent input conduit(s)
  • FIGs. 12-16 are illustrative examples of parallel device designs and are not construed to be limiting. Modifications to the devices and systems within one of skill in the art are also within the scope of various aspects described herein. For example, the layout, configuration, and/placement of the main assay paths, pneumatic conduits, pneumatic inlets, and/or reagent input conduits within the microfluidic sample devices and microfluidic systems can be modified to suit the size of the device.
  • FIG. 12 illustrates one embodiment of a fluidic layer of a microfluidic sample device comprising multiple main assay paths.
  • a fluidic layer 1200 of a microfluidic sample device can comprise a plurality of (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or more) main assay paths 1206. Accordingly, such microfluidic sample device with multiple main assay paths is also referred to as a "microfluidic parallel sample device.” Microfluidic parallel sample devices can be used to perform multiple different assays (e.g., each for a different target analyte) using a single sample, or to peform duplicate assays on a single sample. As shown in FIG.
  • each main assay path 1206 of the microfluidic parallel sample device 1200 comprises similar components of a main assay path described herein.
  • Each main assay path 1206 can have a first end portion adapted to fluidly connect to a fluid inlet or port 1219, and a second end portion comprising a fluid outlet or port 1220 adapted to fluidly connect to an assay module input port.
  • Each main assay path 1206 can be fluidly connected to its corresponding set (comprising a plurality of, e.g., at least two or more) of reagent input conduits 1222 and fluid waste path 1238.
  • the reagent input conduit 1222 is a channel, a path, or a duct defining a passageway through and along which a reagent (for performing an assay) flows, passes, or moves between at least one reagent inlet and a corresponding membrane valve.
  • the fluid waste path 1238 can comprise an assay module output-receiving port 1240 at first end portion and a waste outlet 1242 at a second portion. For simplified illustrations purposes only, details of membrane valves and microfluidic pumps are not shown in FIG. 12.
  • the fluid waste path can comprise a fluid handling module, e.g., but not limited to a microfluidic pump.
  • Membrane valves can be located between a reagent input conduit 1222 and a main assay path 1206.
  • a sample fluid path is a direct input through one of the condutis 1222.
  • FIG. 14 shows an assembly of a fluidic layer and a pneumatic layer in an exemplary microfluidic sample device.
  • the fluidic layer and pneumatic layer can form a microfluidic cartridge for providing fluidic connection and transfer.
  • the fluidic layer similar to the one in FIG. 12, can comprise a plurality of (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or more) main assay paths 1206 fluidly connected to their corresponding sets of reagent input conduits 1222 and fluid waste paths 1238.
  • the pneumatic layer can comprise membrane valves 1226 located between reagent input conduits 1222 and main assay paths 1206, membrane valves 1230 located between the fluid inlets 1218 and the first reagent input conduit 1222-1, and microfluidic pumps 1244 located within their corresponding fluid waste paths 1238.
  • Each of the membrane valves 1226, 1230 and/or microfluidic pumps 1244 is fluidly connected to its corresponding pneumatic conduit 1244.
  • a subset of the pneumatic conduits 1244 can be fluidly connected to each other and can thus share a single pneumatic inlet 1228, for example, as shown in FIG. 14.
  • each pneumatic conduit 1244 can have its corresponding pneumatic inlet 1228.
  • the reagent input conduits 1222 are fluidly connected to one or more reagent sources.
  • reagent input inlets 1222 of the fluidic layer 1200 are fluidly connected to corresponding chambers 1302 disposed in an agent reservoir layer 1300.
  • the agent reservoir layer 1300 can comprise a plurality of (e.g., at least 2, at least 5, at least 10, at least 20, at least 30, or more, depending on, e.g., the number of reagent input inlets 12221 and fluid inlets 1218) chambers or wells 1302 each for holding an agent or reagent.
  • the chambers and wells 1302 can be disposed in the agent reservoir layer 1300 in an ordered array, for example, as shown in FIG. 13, or in any configuration to suit the need of a user.
  • the fluidic layer 1200 is disposed on the agent reservoir layer 1300 such that each of the fluid inlet 1218 and reagent input inlets 12221 are aligned with a corresponding chamber or well 1302.
  • FIG. 15 shows a magnified top view of a portion of a microfluidic sample device of FIG. 14 comprising a fluidic layer and a pneumatic layer as described above, where the portion comprises fluidic connections to an assay module.
  • the portion of the microfluidic device can comprise a plurality of waste fluid paths 1238 and corresponding fluid outlets or ports 1220 of the main assay paths.
  • the fluid outlets or ports 1220 of the main assay paths can be fluidly connected to corresponding input ports of an assay module. As shown in FIG.
  • the vertical spacing between the fluid outlet or port 1220 (that is adapted to fluidly connected to an assay module input port) and the corresponding assay module output- receiving port 1240 can be of any dimension, e.g., depending on the size of the device, and/or relative positions of the ports 1220, 1240.
  • the vertical spacing between the fluid outlet or port 1220 (that is adapted to fluidly connected to an assay module input port) and the corresponding assay module output-receiving port 1240 can range from about 1 millimeter ("mm") to about 5 mm. The spacing depends on the position of the assay module/sensor and any relevant flow cells or other fluidic channles.
  • the spacing can be greater than 5 mm, possibly 10 mm or 20 mm, based on the sensor layout.
  • the vertical spacing between the fluid outlet or port 1220 (that is adapted to fluidly connected to an assay module input port) and the corresponding assay module output- receiving port 1240 can be about 20 mm.
  • the horizontal spacing between a fluid outlet or port 1220-1 and an adjacent fluid outlet or port 1220-2, or between an assay module output- receiving port 1240-1 and an adjacent assay module output-receiving port 1240-2 can be of any dimension, e.g., depending on the size of the device, and/or relative positions of the ports. In some embodiments, the horizontal spacing can range from about 1 mm to about 5 mm.
  • the horizontal spacing also referred to as the receiving port spacing, is determined based on the reservoir spacing. For example, when using standard plate format wells as shown, the horizontal spacing must align with standard well plate formats. Alternatively, the horizontal spacing skips the wells. In some embodiments, the horizontal spacing can be about 2.5 mm.
  • the microfluidic sample devices and/or microfluidic cartridges described herein can be fluidly connected to an assay module.
  • the assay module can comprise one or a plurality of (e.g., at least two or more) sensors described herein.
  • the microfluidic sample devices and/or microfluidic cartridges described herein can be fluidly connected to an assay module to form a microfluidic analyzer.
  • FIG. 16 shows a top view of a microfluidic analyzer according to one embodiment described herein.
  • the microfluidic analzyer can comprise any embodiment of a microfluidic sample device described herein and an assay module. As shown in FIG.
  • the microfluidic sample device can comprise an agent reservoir layer 1300 and a microfluidic cartridge in controllable fluidic connection with the agent reservoir layer.
  • the microfluidic cartridge can comprise a fluidic layer 1200 and a pneumatic layer as described above.
  • the assay module 1600 can be overlaid on top of the microfluidic sample device or cartridge.
  • the assay module or sensor can be, for example, glued or otherwise attached to the cartridge to provide analytical capability. In another example, the sensor can be integrated into the cartridge.
  • the fluid outlet(s) 1220 of the main assay path(s) 1206 disposed in the fluidic layer 1200 can be fluidly connected to corresponding input port(s) of an assay module 1600; and the assay module output fluid(s) can be directed to the fluid waste path(s) 1238 disposed in the fluidic layer 1200 via assay module output-receiving port(s) 1240 located at the upstream portion of the fluid waste path(s) 1238.
  • Exemplary systems comprising a microfluidic sample device as described herein
  • microfluidic sample devices described herein can be integrated into an instrument, e.g., for diagnostic applications, and/or can be used to interconnect at least one device to another as well as to collect samples and/or even to perform assays at the interconnection. Accordingly, systems comprising one or more microfluidic sample devices as described herein are also within the scope of various aspects described herein.
  • One aspect relates to a microfluidic system comprising one or more microfluidic sample devices as described herein.
  • the microfluidic system comprises (a) at least one cell-culture microfluidic device having a chamber including a surface to which the cells are attached, the chamber comprising a fluid inlet for receiving a fluid that passes across the cells and a fluid outlet for exiting the fluid from the cell-culture microfluidic device; (b) at least one downstream fluid- receiving device that receives the fluid from the cell-culture microfluidic device; and (c) at least one microfluidic sample device located between the cell-culture microfluidic device and the downstream fluid-receiving device.
  • the microfluidic sample device 300 has a sample fluid path 304 with a sample-fluid outlet 316 that allows a fluid received by the sample fluid path to exit the microfluidic sample device 300
  • a user can use the microfluidic sample device 300 to couple a cell-culture microfluidic device 310 (e.g., an organ-on-a-chip device) to a downstream fluid-receiving device 346, e.g., but not limited to a reservoir or another cell- culture microfluidic device (e.g., an organ-on-a-chip device) to form a microfluidic system 100 or a cell culture interrogator system.
  • a cell-culture microfluidic device 310 e.g., an organ-on-a-chip device
  • a downstream fluid-receiving device 346 e.g., but not limited to a reservoir or another cell- culture microfluidic device (e.g., an organ-on-a-chip device) to form a microflu
  • the membrane valve located between the sample fluid path and the main assay path of the microfluidic sample device can be actuated to open, allowing an aliquot of a fluid flowing from the cell-culture microfluidic device to enter into the main assay path, while the rest of the fluid can flow through the sample-fluid outlet of the sample fluid path.
  • the membrane valve of the microfluidic sample device can close so that a fluid from the cell- culture microfluidic device can continuously flow to a downstream fluid receiving device as in a normal operation, via the sample fluid path.
  • the user does not have to physically interact with a cell-culture microfluidic device while sampling for an assay.
  • the microfluidic sample devices described herein permit an assay to perform on a sample while maintaining a substantially continuous flow of a fluid between a cell culture microfluidic device and a downstream fluid-receiving device, the microfluidic sample devices provide capability of automation of experiments involving at least one or more cell-culture microfluidic device.
  • a microfluidic system 100 comprises (a) a first cell-culture microfluidic device 310; (b) a second cell-culture microfluidic device 346 (e.g., a downstream fluid-receiving device); (c) a third cell-culture microfluidic device 348; (d) a first microfluidic sample device 300-1 as described herein located between the first cell-culture microfluidic device 310 and the second cell-culture microfluidic device 346; and (e) a second microfluidic sample device 300-2 as described herein located between the second cell-culture microfluidic device 346 and the third cell- culture microfluidic device 348.
  • the combination of cell-culture microfluidic devices and microfluidic sample devices described herein can be used to culture cells of different tissue types in different devices and rerouting the culture medium, e.g., using the fluid handling module, such as comprising a microfluidic pump, of the microfluidic sample devices described herein.
  • the fluid handling module such as comprising a microfluidic pump
  • the microfluidic sample device 300 used in the microfluidic system 100 can be any embodiment described herein.
  • the microfluidic sample device 300 can comprise a sample fluid path 304 with a sample-fluid inlet 314 for receiving a fluid from the fluid outlet of the cell-culture microfluidic device 310 and a sample-fluid outlet 316 for allowing the fluid to pass to the downstream fluid-receiving device 346.
  • the main assay path 306 of the microfluidic sample device 300 can be coupled to an assay module 312 for performing a desirable assay.
  • a membrane valve 308 can be located between the sample fluid path and the main assay path, and the membrane valve 308 can be operable (i) in a first valve state to allow the fluid to pass to the downstream fluid-receiving device via the sample-fluid outlet; and (ii) in a second state to allow the fluid to enter into the main assay path.
  • an assay can be performed on the fluid received by the main assay path while maintaining a substantially continuous flow of a fluid between the cell-culture microfluidic device and the downstream fluid-receiving device. This can provide capability to periodically conduct an assay on an effluent from a cell-culture microfluidic device, e.g., to monitor cell culture condition or to monitor cell response over a period of time.
  • the downstream fluid-receiving device 346 is generally a container or a device located downstream of the cell-culture microfluidic device that can hold or store a fluid.
  • the downstream fluid-receiving device 346 can be a fluid reservoir.
  • the downstream fluid-receiving device 346 can be a second cell-culture microfluidic device with a second set of cells cultured therein.
  • the microfluidic sample device 300 can be integral to a cartridge that holds the cell-culture microfluidic device. In some embodiments, the microfluidic sample device 300 can be integral to a cartridge as described in the International Patent Application No. WO 2015/013332, the content of which is incorporated herein by reference in its entirety.
  • the cell-culture microfluidic device can be any microfluidic device that can be used for cell culture.
  • the cell-culture microfluidic device can comprise a first chamber, a second chamber, and a membrane located between the first and second chambers.
  • the membrane can comprise at least one type of cells thereon.
  • the cell-culture microfluidic device can comprise an organ-on-a-chip device. Examples of various organ-on-a-chip devices, e.g., as described in International Patent Application Nos: WO 2010/009307, WO 2012/118799, WO 2013/086486, WO 2013/086502, and in U.S. Patent No.
  • the membrane can be porous (e.g., permeable or selectively permeable), non-porous (e.g., non-permeable), rigid, flexible, elastic, or any combination thereof.
  • the membrane can be porous, e.g., allowing exchange/transport of fluids (e.g., gas and/or liquids), passage of molecules such as nutrients, cytokines and/or chemokines, cell transmigration, or any combinations thereof.
  • the membrane can be non-porous.
  • a first surface of the membrane facing the first channel comprises a first type of cells adhered thereon.
  • a second surface of the membrane facing the second channel can comprise a second type of cells adhered thereon.
  • Cells can be established cell lines or derived from a subject. Cells can be derived from a tissue of different organs, including, e.g., intestine, liver, kidney, lung, heart, brain, reproductive organs (e.g., testis or ovaries), eye, skin, and any combinations thereof. Cells can be differentiated cells or stem cells.
  • the microfluidic system 100 can further comprise an assay module 312 as described herein fluidly coupled to the main assay path 306 of the microfluidic sample device 300.
  • An assay module can comprise at least one or more sensors for detecting or measuring one or more target analytes.
  • the microfluidic system 100 can comprise a sample multiplexer 334 located between the main assay path 306 and sensors 332 of the assay module 312.
  • one fluidic input can be converted to a plurality of (e.g., at least two or more) fluidic channels.
  • a sample fluid received in the main assay path 306 can be divided into a plurality of (e.g., at least two or more) even aliquots using the sample multiplexer 334 before passing to the assay module 312.
  • the assay module 312 can comprise a plurality of different analyte-specific sensors 332, and each fluidic channel of the sample multiplexer 332 comprising an aliquot of the sample fluid can be fluidly coupled to an analyte-specific sensor. Thus, a multiplexing assay can be performed on the sample fluid received in the main assay path.
  • the microfluidic sample device comprises a plurality of (e.g., at least two or more) sample fluid paths and corresponding membrane valves that interact with the main assay path
  • a first sample fluid moving from a first sample fluid path into the main assay path can enter a first fluidic channel of the sample multiplexer.
  • a second sample fluid can subsequently move from a second sample fluid path into the main assay path and move toward a second fluidic channel of the sample multiplexer.
  • a wash buffer can be introduced into the main assay path via a wash inlet to remove any residue of the first sample fluid.
  • the first and the second sample fluid paths can be fluidly connected to different sample source (e.g., different chambers of a cell-culture microfluidic device, or different cell-culture microfluidic devices).
  • sample source e.g., different chambers of a cell-culture microfluidic device, or different cell-culture microfluidic devices.
  • a plurality of different samples can be assayed in series or in parallel.
  • a single analyte-specific sensor can be used to assay different samples when one sample is introduced to the sensor at a time.
  • each fluidic channel of the sample multiplexer can be fluidly connected to a corresponding sensor, which allows parallel sensing of different samples.
  • the sample multiplexer can be placed downstream or upstream of one or more sensors.
  • a sample multiplexer can be used where a single sample is assayed for multiple target analytes.
  • a sample multiplexer can be used where multiple samples are assayed for a single target analyte.
  • One of skill in the art can determine appropriate placement of a sample multiplexer with respect to sensor(s) for various assay applications.
  • the microfluidic system 100 can comprise a sample demultiplexer 336 located between sensor(s) 332 of the assay module 312 and the fluid waste path 338.
  • a plurality of (e.g., at least two or more) fluidic outputs after sensor measurements can be converted to a single channel, e.g., fluidly connected to a fluid waste path.
  • a method comprises (a) allowing a sample fluid exiting from the microfluidic device to move along a sample fluid path within a microfluidic sample device according to one embodiment described herein; (b) actuating a membrane valve of the microfluidic sample device to a valve state to allow at least an aliquot of the sample fluid to flow from the sample fluid path to the main assay path, while the remaining of the sample fluid can continue to flow and exits the sample-fluid outlet, thereby sampling the fluid periodically while maintaining a substantially continuous flow of the fluid through the microfluidic device in the microfluidic system; and (c) actuating the membrane valve, after the main assay path receives the aliquot, to a different valve state to prevent any additional sample fluid to flow from the sample fluid to the main assay path, but to exit
  • sample fluid received in the sample fluid path enters the main assay path, when the membrane valve of the microfluidic sample device is actuated to permit fluid flow between the sample fluid path and the main assay path.
  • the perfusion rate is substantially equal to or greater than the sampling flow rate, while an aliquot of the sample fluid enters the main assay path, the remaining of the sample fluid continues to flow through the sample fluid path as if the membrane valve is closed and exits the sample-fluid outlet.
  • the perfusion rate can be substantially the same as the sampling flow rate if the remaining of the sample fluid (upon an aliquot of the sample fluid entering the main assay path) is intended to introduce into another microfluidic device.
  • the perfusion rate can be greater than the sampling flow rate, e.g., by 1000-fold, if the remaining of the sample fluid (upon an aliquot of the sample fluid entering the main assay path) is intended to go to waste.
  • the perfusion rate is lower than or about the same as the sampling flow rate, substantially all of the sample fluid received by the sample fluid path enters the main assay path.
  • the sampling flow rate can vary to suit the need of an application and/or depend on limits of the pump and/or device.
  • the sampling flow rate can vary from microliter(s) per minute to milliter(s) per minute.
  • the sampling flow rate can vary from about 0 microliter per minute - about 500 microliters per minute.
  • the sampling flow rate can vary from about 1 microliter per minute - about 500 microliters per minute.
  • the sampling flow rate can vary from about 5 microliter per minute - about 100 microliters per minute.
  • the sampling flow rate can be up to a milliter per minute or more.
  • the methods described herein can be applied to microfluidic applications where sampling of a fluid in a microfluidic device is desirable for an assay and a substantially continuous flow of a fluid is desired or preferred to be maintained in the microfluidic device.
  • the microfluidic device can comprise a cell culture device.
  • the cell- culture microfluidic device can be any microfluidic device that can be used for cell culture.
  • the cell-culture microfluidic device can comprise a first chamber, a second chamber, and a membrane located between the first and second chambers. The membrane can comprise at least one type of cells thereon.
  • the cell- culture microfluidic device can comprise an organ-on-a-chip device.
  • the sample-fluid outlet of the sample fluid path can be coupled to a reservoir or a second microfluidic device (e.g., an organ-on-a-chip).
  • a second microfluidic device e.g., an organ-on-a-chip
  • the method can further comprising allowing at least one reagent controllably released from one or more reagent input conduits of the microfluidic sample device to move along the main assay path toward an assay module.
  • the reagents can be introduced into the main assay path and contacted the aliquot received by the main assay path.
  • a reagent include, but are not limited to a nucleic acid extraction agent, a fixation agent, a staining agent, an analyte- specific antibody, a detectable label, or any combinations thereof.
  • Appropriate reagents can be introduced to an assay module configured for specific assay(s).
  • nucleic acid when a nucleic acid extraction reagent is added to the aliquot in the main assay path, nucleic acid can be extracted from cell(s) present in the aliquot prior to entering the assay module, e.g., for a nucleic acid assay such as polymerase reaction for measurement of RNA and/or sequencing.
  • a fixation agent, a staining agent, an analyte-specific antibody, and/or a detectable label are added to the main assay path, cell staining and/or labeling an analyte can be performed on the aliquot.
  • the microfluidic sample devices described herein can be fluidly connected to a single or multiple cell-culture microfluidic devices (e.g., with a split-flow valve) where cells are cultured therein.
  • cell staining reagent(s) can be introduced through the reagent input conduit(s) 322 of the microfluidic sample device 300, and can be directed to the downstream cell-culture microfluidice device for fixing and/or staining cells cultured therein.
  • sample preparation for staining can be performed in situ on the cell- culture microfluidic device.
  • the microfluidic sample devices and systems comprising the same can be used to run a plurality of assays in parallel or in series.
  • the microfluidic sample devices and systems described herein can be used to run at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or more assays in parallel or in series.
  • Each assay can be designed to detect and/or measure the same or different target analyte(s).
  • the microfluidic sample devices and systems can be used to perform multiplexing, to measure multiple target analytes, and/or to collect and measure one or more target analytes periodically (e.g., at multiple time points) from a sample source (e.g., in a microfluidic device).
  • a sample source e.g., in a microfluidic device.
  • the multiple main assay paths can be placed separately in individual microfluidic sample devices or all in a single microfluidic device.
  • an assay can be performed repeatedly on a single sensor.
  • a plurality of sample fluids can be directed to the same sensor serially or sequentially (e.g., at different time points) such that multiple assays for the same target analyte(s) can be performed on a single sensor.
  • a microfluidic system comprises multiple cell-culture microfluidic devices (e.g., organ-on-a-chip devices) and cells in an upstream cell-culture microfluidic device are treated with one or more test agent or a library of test agents
  • the microfluidic sample devices and systems can be used to detect responses of the treated cells and cells in downstream cell- culture microfluidic devices.
  • the microfluidic sample devices and systems described herein can be used to integrate multiple organ-on-a-chip devices and/or to perform a compound screen in one or more organ-on-a-chip devices or other flow-through culture systems.
  • a sample aliquot received in the main assay path comprises a cell or a plurality of cells
  • the microfluidic device sample devices and systems described herein can be used for flowing cell sampling/processing.
  • the microfluidic device sample devices and systems described herein can be used for non-adherent cell (e.g., but not limited to immune cells) sampling/processing.
  • the microfluidic device sample devices and systems described herein can be used for rare cell (e.g., but not limited to circulating tumor cells and fetal cells) sampling/processing.
  • a fluid outlet 320 of the main assay path 306 can be fluidly connected to a cytometer.
  • magnetic particles e.g., magnetic nanoparticles
  • a sample fluid residing in the main assay path 306 can be captured by the magnetic particles (e.g., magnetic nanoparticles) and the cell-bound magnetic particles can then be separated from the sample fluid by a magnetic separation method.
  • cells in a sample fluid can be directed to an assay module comprising sensor(s) coated with one or more target cell capture agents such that target cells bind to the target cell capture agents.
  • any embodiments of the devices described herein can be made of any material that is compatible with a fluid to be in contact with the device(s).
  • Exemplary materials that can be used to fabricate different embodiments of the microfluidic sample devices described herein can include, but are not limited to, glass, co-polymer, polymer or any combinations thereof.
  • Exemplary polymers include, but are not limited to, polyurethanes, rubber, molded plastic, polymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLONTM), polyvinylchloride (PVC), polydimethylsiloxane (PDMS), and polysulfone.
  • PMMA polymethylmethacrylate
  • TEFLONTM polytetrafluoroethylene
  • PVC polyvinylchloride
  • PDMS polydimethylsiloxane
  • the methods used in fabrication of any embodiments of the microfluidic sample devices described herein can vary with the materials used, and include embossing, soft lithography methods, microassembly, bulk micromachining methods, surface micro- machining methods, standard lithographic methods, wet etching, reactive ion etching, plasma etching, laser etching, stereolithography and laser chemical three-dimensional writing methods, solid-object printing, machining, modular assembly methods, replica molding methods, injection molding methods, hot molding methods, laser ablation methods, combinations of methods, and other methods known in the art.
  • the microfluidic sample devices described herein can be formed by replica molding, for example, in which a replica of the selected material conforms to the shape of a master or a mold and replicates the features of the master or the mold.
  • the replica can be further sealed to a surface to enclose at least one fluidic path or channel described herein.
  • the microfluidic sample devices described herein can be formed by machining or micromachining.
  • micromachining as used herein can encompass bulk micromachining or surface micromachining as recognized in the art.
  • bulk micromachining defines microstructures such as fluidic elements (e.g., fluidic channels) by selectively etching inside a substrate.
  • surface micromachining creates microstructures such as fluidic elements (e.g., fluidic channels) from a top surface of a substrate material.
  • the microfluidic sample devices described herein can be formed by solid-object printing.
  • the solid-object printing can take a three-dimensional (3D) computer-aided design file to make a series of cross-sectional slices. Each slice can then be printed on top of one another to create the 3D solid object.

Abstract

Cette invention concerne des dispositifs microfluidiques d'échantillonnage et des systèmes comprenant ceux-ci, pour effectuer une ou plusieurs analyses d'un échantillon de fluide à partir de dispositifs microfluidiques de culture cellulaire tout en maintenant un écoulement sensiblement continu d'un fluide dans les dispositifs microfluidiques de culture cellulaire. L'invention concerne en outre des procédés d'utilisation desdits dispositifs. Selon certains modes de réalisation, lesdits dispositifs microfluidiques d'échantillonnage peuvent être utilisés pour interconnecter des dispositifs microfluidiques de type organe sur puce et pour effectuer des analyses au niveau de l'interconnexion.
PCT/US2017/013316 2016-01-13 2017-01-13 Dispositifs microfluidiques d'échantillonnage et leurs utilisations WO2017123855A1 (fr)

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WO2020065040A1 (fr) * 2018-09-27 2020-04-02 Ge Healthcare Bio-Sciences Ab Système et procédé pour un produit pharmaceutique
WO2020086999A1 (fr) * 2018-10-25 2020-04-30 Savran Technologies, Inc. Systèmes et procédés de capture de particules
WO2021064019A1 (fr) * 2019-10-01 2021-04-08 Cytiva Sweden Ab Système de bioprocédé
WO2021138088A1 (fr) * 2019-12-30 2021-07-08 Illumina, Inc. Systèmes et procédés d'actionnement pour une utilisation avec des cellules d'écoulement

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US20140356849A1 (en) * 2011-12-09 2014-12-04 Vanderbilt University Integrated organ-on-chip systems and applications of the same
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* Cited by examiner, † Cited by third party
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WO2020065040A1 (fr) * 2018-09-27 2020-04-02 Ge Healthcare Bio-Sciences Ab Système et procédé pour un produit pharmaceutique
WO2020086999A1 (fr) * 2018-10-25 2020-04-30 Savran Technologies, Inc. Systèmes et procédés de capture de particules
WO2021064019A1 (fr) * 2019-10-01 2021-04-08 Cytiva Sweden Ab Système de bioprocédé
WO2021138088A1 (fr) * 2019-12-30 2021-07-08 Illumina, Inc. Systèmes et procédés d'actionnement pour une utilisation avec des cellules d'écoulement
CN115038524A (zh) * 2019-12-30 2022-09-09 伊鲁米那有限公司 与流通池一起使用的致动系统和方法
EP4084906A4 (fr) * 2019-12-30 2024-01-03 Illumina Inc Systèmes et procédés d'actionnement pour une utilisation avec des cellules d'écoulement

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