WO2004069983A9 - Architecture de microréacteur et procédés associés - Google Patents

Architecture de microréacteur et procédés associés

Info

Publication number
WO2004069983A9
WO2004069983A9 PCT/US2003/025943 US0325943W WO2004069983A9 WO 2004069983 A9 WO2004069983 A9 WO 2004069983A9 US 0325943 W US0325943 W US 0325943W WO 2004069983 A9 WO2004069983 A9 WO 2004069983A9
Authority
WO
WIPO (PCT)
Prior art keywords
chip
reaction site
membrane
less
predetermined reaction
Prior art date
Application number
PCT/US2003/025943
Other languages
English (en)
Other versions
WO2004069983A2 (fr
WO2004069983A3 (fr
Inventor
Seth T Rodgers
Peter A Russo
Howard B Schreyer
Sean J Leblanc
Andrey J Zarur
Xinyu Li
Original Assignee
Bioprocessors Corp
Seth T Rodgers
Peter A Russo
Howard B Schreyer
Sean J Leblanc
Andrey J Zarur
Xinyu Li
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US10/457,015 external-priority patent/US20040058407A1/en
Priority claimed from US10/457,049 external-priority patent/US20040058437A1/en
Application filed by Bioprocessors Corp, Seth T Rodgers, Peter A Russo, Howard B Schreyer, Sean J Leblanc, Andrey J Zarur, Xinyu Li filed Critical Bioprocessors Corp
Priority to CA002496017A priority Critical patent/CA2496017A1/fr
Priority to AU2003303303A priority patent/AU2003303303A1/en
Priority to EP03815293A priority patent/EP1567637A2/fr
Priority to JP2005515706A priority patent/JP2006521786A/ja
Priority to US10/664,067 priority patent/US20050032204A1/en
Publication of WO2004069983A2 publication Critical patent/WO2004069983A2/fr
Publication of WO2004069983A9 publication Critical patent/WO2004069983A9/fr
Publication of WO2004069983A3 publication Critical patent/WO2004069983A3/fr

Links

Classifications

    • 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
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH

Definitions

  • the present invention generally relates to chemical, biological, and/or biochemical reactor chips and other reaction systems such as microreactor systems.
  • the subject matter of this invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.
  • the invention is an apparatus.
  • the apparatus can include a precursor able to react to form a gaseous agent able to substantially alter the pH of a substance within the predetermined reaction site, where the chip is arranged to allow gaseous non-liquid transport of the agent to the predetermined reaction site.
  • the apparatus includes a pH- altering agent dispensing unit integrally connected to the chip in fluid communication with the predetermined reaction site.
  • the invention in accordance with another embodiment, includes a source of gas integrally connected to the chip.
  • the invention includes a laser waveguide in optical communication with a surface defining the predetermined reaction site.
  • the apparatus includes a sensor integrally connected to the chip, where the sensor is able to determine an environmental factor associated with the predetermined reaction site.
  • the environmental factor is at least one of pH, a concentration of a dissolved gas, molarity, osmolarity, glucose concentration, glutamine concentration, pyruvate concentration, apatite concentration, color, turbidity, viscosity, a concentration of an amino acid, a concentration of a vitamin, a concentration of a hormone, serum concentration, a concentration of an ion, shear rate, and degree of agitation.
  • the apparatus may also include an actuator integrally connected to the chip, where the actuator is able to alter the environmental factor.
  • the apparatus may also include an actuator integrally connected to the chip able to alter at least one of the temperature, the pressure, and the environmental factor.
  • the apparatus may include a sensor able to determine an environmental factor associated with at least one of the predetermined reaction sites.
  • the environmental factor may be at least one of the C0 2 concentration, glucose concentration, glutamine concentration, pyruvate concentration, apatite concentration, serum concentration, a concentration of a vitamin, a concentration of an amino acid, and a concentration of a hormone.
  • the apparatus includes a chip comprising a predetermined reaction site having an inlet, an outlet, and a volume of less than about 1 ml.
  • the apparatus in still another set of embodiments, includes a substantially liquid-tight chip comprising a predetermined reaction site having a volume of less than about 1 ml, where the predetermined reaction site is constructed and arranged to maintain at least one living cell at the predetermined reaction site.
  • the apparatus in one set of embodiments, is defined, at least in part, by a chip produced by a process including the step of fastening two components to produce a portion of the chip defining a predetermined reaction site having a volume of less than about 1 ml, where the predetermined reaction site is constructed and arranged to maintain at least one living cell at the predetermined reaction site.
  • the apparatus in another set of embodiments, includes a chip comprising a predetermined reaction site having a volume of less than about 1 ml, where the predetermined reaction site constructed and arranged to maintain at least one living cell at the predetermined reaction site, and the predetermined reaction site has a nonzero evaporation rate of less than about 100 microliters/day.
  • the apparatus is defined, at least in part, by a chip comprising a first predetermined reaction site having a volume of less than about 1 ml and a second predetermined reaction site, where the chip defines a pathway fluidly connecting the first predetermined reaction site and the second predetermined reaction site, and where the pathway crosses a membrane.
  • the apparatus in one set of embodiment, includes a reaction site having a first portion and a second portion separated by a membrane, and at least a first and a second channel in fluidic communication with the second portion of the reaction site.
  • the invention is a method in another aspect.
  • the method includes producing a gas in a chip comprising a predetermined reaction site having a volume of less than about 1 ml by directing a laser at at least a portion of the chip.
  • the invention in a method of producing a chip comprising a predetermined reaction site having a volume of less than 1 ml, includes attaching a first component of the chip to a second component of the chip with or without auxiliary adhesive to produce a portion of the chip that defines the predetermined reaction site.
  • the method in yet another set of embodiments, includes an act of providing a substrate having a surface into which is fabricated a plurality of reaction sites, where at least one reaction site has a volume less than about 2 ml and is divided by a substantially cell impermeable membrane into at least a cell culture portion containing cells and a reservoir portion not containing cells, where the reservoir portion is fluidly connected to at least a first and a second channel fabricated into the surface of the substrate.
  • the method also includes acts of introducing at least one test compound into at least one of the plurality of reaction sites, and monitoring the effect of the test compound on cells located within the cell culture portion.
  • the present invention is directed to a method of making one or more of the embodiments described herein, for example, a chip or other reaction system, such as a microreactor system.
  • the present invention is directed to a method of using one or more of the embodiments described herein, for example, a chip or other reaction system, such as a microreactor system.
  • the present invention is directed to a method of promoting one or more of the embodiments described herein, for example, a chip or other reaction system, such as a microreactor system.
  • the present invention is directed to a method of making a chip and/or a reactor system, e.g., as described in any of the embodiments herein.
  • the present invention is directed to a method of using a chip and/or a reactor system, e.g., as described in any of the embodiments herein, for example, example.
  • the present invention is directed to a method of promoting a chip and/or a reactor system, e.g., as described in any of the embodiments herein.
  • Fig. 1 illustrates one embodiment of the invention
  • Fig. 2 illustrates an example of a microfluidic chip for use with the invention including mixing, heating/dispersion, reaction, and separation units, in expanded view
  • FIG. 3A-3C illustrate various stackable arrangements of chips of the invention
  • Figs. 4A-4C illustrate various energy directors for use with the invention in certain embodiments
  • Figs 5 A and 5B illustrate a device according to one embodiment of the invention, having multiple layers
  • Fig. 6 is a block diagram of an example of a control system of the invention
  • Figs. 7A and 7B illustrate a device according to another embodiment of the invention having a dispensing unit
  • Figs. 8 A and 8B illustrate a device according to another embodiment of the invention where a laser is used to produce a response
  • Figs. 9 A and 9B are cross sectional views of certain embodiments of the present invention
  • Figs. 10A - 10D illustrates certain membranes of the invention in fluid communication with various reaction sites.
  • FIG. 18A and 18B illustrate expanded views of portions of various chips according to another embodiment of the invention
  • Fig. 19 illustrates an expanded view of a portion of a chip according to yet another embodiment of the invention
  • Fig. 20 is a graph illustrating oxygen permeability for an embodiment of the invention as used in a bacterial culture
  • Fig. 21 is a graph illustrating oxygen permeability for an embodiment of the invention as used in a mammalian cell culture
  • Fig. 22 illustrates another embodiment of the invention having a waveguide
  • Fig. 23 is a graph of intensity (in relative units) versus relative concentration, in an embodiment of the invention
  • Fig. 24 is a graph of optical density at 480 nm versus time in an experiment using an embodiment of the invention
  • FIG. 25 illustrates a solid substrate having a reaction site and channels, in accordance with one embodiment of the invention
  • Figs. 26A-26E illustrate various views of the embodiment illustrated in Fig. 25
  • Figs. 27A and 27B illustrate microfabricated bioreactors in accordance with various embodiments of the invention.
  • the present invention generally relates to chemical, biological, and/or biochemical reactor chips and other reaction systems such as microreactor systems, as well as systems and methods for constructing and using such devices.
  • a chip or other reaction system may be constructed so as to promote cell growth within it.
  • the chips or other reaction systems of the invention include one or more reaction sites. The reaction sites can be very small, for example, with a volume of less than about 1 ml.
  • a chip is able to detect, measure and/or control an environmental factor such as the temperature, pressure, CO 2 concentration, O 2 concentration, relative humidity, pH, etc. associated with one or more reaction sites, by using one or more sensors, actuators, processors, and/or control systems.
  • the present invention is directed to materials and systems having humidity and/or gas control, for example, for use with a chip. Such materials may have high oxygen permeability and/or low water vapor permeability.
  • the present invention in still another aspect, generally relates to light- interacting components suitable for use in chips and other reactor systems. These components may include waveguides, optical fibers, light sources, photodetectors, optical elements, and the like. Referring now to Fig.
  • port 9 can be a "self-sealing" port, addressable by a needle (as described more fully below) when at least one side of port 9 is covered by a layer (not shown) of material which, when a needle is inserted through the material and withdrawn, forms a seal generally impermeable to species such as fluids introduced into or removed from the chip via the port.
  • Ports 15 can be defined by voids in layer 2, and can be used to facilitate fluidic connection between and among various layers of a chip and/or an environment external to the chip.
  • layer 2 forms part of a multi-layer chip including multiple reaction sites in different layers
  • another layer may be provided on one side of layer 2 (optionally separated by an intermediate layer or layers) including one set of reaction sites or conduits, and another layer may be provided on the opposite side of layer 2, similarly separated by intermediate layers if desirable, and ports 15 may define passages or routes for fluidic connection between reaction sites and/or conduits of chip layers on opposite sides of layer 2.
  • Ports 15 also may connect to channels communicating with a chamber aligned with a chamber defining reaction site 4, separated from the reaction site by a membrane, e.g. semipermeable membrane.
  • each reaction site 4 along with the associated fluidic connections (e.g., channels 6 and 8, ports 9 and ports 15), together define a reactor 14, as indicated by dotted lines.
  • layer 2 contains six such reactors, each reactor having substantially the same configuration.
  • a reactor may include more than one reaction site, channels, ports, etc.
  • a chip layer may have reactors that do not substantially have the same configuration. Additionally shown in Fig.
  • Device 1 is a series of devices 16 which can be used to secure layer 2 to other layers of a chip and/or to assure alignment of layer 2 with other layers and/or other systems to which the chip is desirably coupled.
  • Devices 16 can define screws, posts, indentations (i.e., that match corresponding protrusions of other layers or devices), or the like.
  • Those of ordinary skill in the art are aware of a variety of suitable techniques for securing layers to other layers and/or chips of the invention to other components or systems using devices such as these.
  • a variety of definitions are now provided which will aid in understanding of the invention. Following, and interspersed with these definitions, is further disclosure, including descriptions of figures, that will fully describe the invention.
  • determining generally refers to the measurement and/or analysis of a substance (e.g., within a reaction site), for example, quantitatively or qualitatively, or the detection of the presence or absence of the substance. “Determining” may also refer to the measurement and/or analysis of an interaction between two or more substances, for example, quantitatively or qualitatively, or by detecting the presence or absence of the interaction.
  • Examples of techniques suitable for use in the invention include, but are not limited to, gravimetric analysis, calorimetry, pressure or temperature measurement, spectroscopy such as infrared, absorption, fluorescence, UV/visible, FTIR ("Fourier Transform Infrared Spectroscopy"), or Raman; gravimetric techniques; ellipsometry; piezoelectric measurements; immunoassays; electrochemical measurements; optical measurements such as optical density measurements; circular dichroism; light scattering measurements such as quasielectric light scattering; polarimetry; refractometry; or turbidity measurements, including nephelometry.
  • a "chip,” as used herein, is an integral article that includes one or more reactors.
  • “Integral article” means a single piece of material, or assembly of components integrally connected with each other.
  • integrally connected when referring to two or more objects, means objects that do not become separated from each other during the course of normal use, e.g., cannot be separated manually; separation requires at least the use of tools, and/or by causing damage to at least one of the components, for example, by breaking, peeling, etc. (separating components fastened together via adhesives, tools, etc.).
  • a chip can be connected to or inserted into a larger framework defining an overall reaction system, for example, a high-throughput system.
  • the system can be defined primarily by other chips, chassis, cartridges, cassettes, and/or by a larger machine or set of conduits or channels, sources of reactants, cell types, and/or nutrients, inlets, outlets, sensors, actuators, and/or controllers.
  • the chip can be a generally flat or planar article (i.e., having one dimension that is relatively small compared to the other dimensions); however, in some cases, the chip can be a non-planar article, for example, the chip may have a cubical shape, a curved surface, a solid or block shape, etc.
  • a "membrane" is a three-dimensional material having any shape such that one of the dimensions is substantially smaller than the other dimensions.
  • the membrane may be generally flexible or non-rigid.
  • a membrane may be a rectangular or circular material with a length and width on the order of millimeters, centimeters, or more, and a thickness of less than a millimeter, and in some cases, less than 100 microns, less than 10 microns, or less than 1 micron or less.
  • the membrane may define a portion of a reaction site and/or a reactor, or the membrane may be used to divide a reaction site into two or more portions, which may have volumes or dimensions which are substantially the same or different.
  • membranes may be semipermeable membranes, which those of ordinary skill in the art will recognize to be membranes permeable with respect to at least one species, but not readily permeable with respect to at least one other species.
  • a semipermeable membrane may allow oxygen to permeate across it, but not allow water vapor to do so, or allows water vapor to permeate it, but at a permeability that is at least an order of magnitude less.
  • a semipermeable membrane may be selected to allow water to permeate across it, but not certain ions.
  • the membrane may be permeable to cations and substantially impermeable to anions, or permeable to anions and substantially impermeable to cations (e.g., cation exchange membranes and anion exchange membranes).
  • the membrane may be substantially impermeable to molecules having a molecular weight greater than about 1 kilodalton, 10 kilodaltons, or 100 kilodaltons or more.
  • the membrane may be impermeable to cells, but be chosen to be permeable to varied selected substances; for example, the membrane may be permeable to nutrients, proteins and other molecules produced by the cells, waste products, or the like. In other cases, the membrane may be gas impermeable.
  • a reaction site may be divided into a first cell culture portion and a second cell culture portion flanking a first reservoir portion and two additional reservoir portions, one of which is separated by a membrane from the first cell culture portion and the other of which is separated by a membrane from the second cell culture portion.
  • first cell culture portion and second cell culture portion flanking a first reservoir portion and two additional reservoir portions, one of which is separated by a membrane from the first cell culture portion and the other of which is separated by a membrane from the second cell culture portion.
  • a substantially transparent material for example, a membrane
  • a material that allows electromagnetic radiation to be transmitted through the material without significant scattering such that the intensity of electromagnetic radiation transmitted through the material is sufficient to allow the radiation to interact with a substance on the other side of the material, such as a chemical, biochemical, or biological reaction, or a cell.
  • the material is substantially transparent to incident electromagnetic radiation ranging between the infrared and ultraviolet ranges (including visible light) and, in particular, between wavelengths of about 400 - 410 nm and about 1,000 nm.
  • the material is at least partially transparent to electromagnetic radiation within the above- mentioned wavelength range to the extent necessary to promote and/or monitor a physical, chemical, biochemical, and/or biological reaction occurring within a reaction site, for example as previously described.
  • the material may be transparent to electromagnetic radiation within the above-mentioned wavelength range to the extent necessary to monitor, observe, stimulate and/or control a cell within the reaction site.
  • reactors examples include chemical or biological reactors and cell culturing devices, as well as the reactors described in International Patent Application Serial No. PCT/US01/07679, published on September 20, 2001 as WO 01/68257, incorporated herein by reference.
  • Reactors can include one or more reaction sites or chambers.
  • the reactor may be used for any chemical, biochemical, and/or biological purpose, for example, cell growth, pharmaceutical production, chemical synthesis, hazardous chemical production, drug screening, materials screening, drug development, chemical remediation of warfare reagents, or the like.
  • the reactor may be used to facilitate very small scale culmre of cells or tissues.
  • a reactor of the invention comprises a matrix or substrate of a few millimeters to centimeters in size, containing channels with dimensions on the order of, e.g., tens or hundreds of micrometers.
  • Reagents of interest may be allowed to flow through these channels, for example to a reaction site, or between different reaction sites, and the reagents may be mixed or reacted in some fashion. The products of such reactions can be recovered, separated, and treated within the system in certain cases.
  • a "reaction site” is defined as a site within a reactor that is constructed and arranged to produce a physical, chemical, biochemical, and/or biological reaction during use of the reactor.
  • More than one reaction site may be present within a reactor or a chip in some cases, for example, At least one reaction site, at least two reaction sites, at least three reaction sites, at least four reaction sites, at least 5 reaction sites, at least 7 reaction sites, at least 10 reaction sites, at least 15 reaction sites, at least 20 reaction sites, at least 30 reaction sites, at least 40 reaction sites, at least 50 reaction sites, at least 100 reaction sites, at least 500 reaction sites, or at least 1,000 reaction sites or more may be present within a reactor or a chip.
  • the reaction site may be defined as a region where a reaction is allowed to occur; for example, the reactor may be constructed and arranged to cause a reaction within a channel, one or more chambers, at the intersection of two or more channels, etc.
  • the reaction may be, for example, a mixing or a separation process, a reaction between two or more chemicals, a light-activated or a light-inhibited reaction, a biological process, and the like.
  • the reaction may involve an interaction with light that does not lead to a chemical change, for example, a photon of light may be absorbed by a substance associated with the reaction site and converted into heat energy or re-emitted as fluorescence.
  • the reaction site may also include one or more cells and/or tissues.
  • the reaction site may be defined as a region surrounding a location where cells are to be placed within the reactor, for example, a cytophilic region within the reactor.
  • the reaction site containing cells may include a region containing a gas (e.g., a "gas head space"), for example, if the reaction site is not completely filled with a liquid.
  • a gas e.g., a "gas head space”
  • the gas head space in some cases, may be partially separated from the reaction site, through use of a gas-permeable or semi-permeable membrane.
  • the gas head space may include various sensors for monitoring temperature, and/or other reaction conditions. Many embodiments and arrangements of the invention are described with reference to a chip, or to a reactor, and those of ordinary skill in the art will recognize that the invention can apply to either or both.
  • a channel arrangement may be described in the context of one, but it will be recognized that the arrangement can apply in the context of the other (or, typically, both: a reactor which is part of a chip). It is to be understood that all descriptions herein that are given in the context of a reactor or chip apply to the other, unless inconsistent with the description of the arrangement in the context of the definitions of "chip” and "reactor" herein.
  • the reaction site may be defined by geometrical considerations.
  • the reaction site may be defined as a chamber in a reactor, a channel, an intersection of two or more channels, or other location defined in some fashion (e.g., formed or etched within a substrate that can define a reactor and/or chip).
  • reaction site may be artificially created, for example, by the intersection or union of two or more fluids (e.g., within one or several channels), or by constraining a fluid on a surface, for example, using bumps or ridges on the surface to constrain fluid flow.
  • the reaction site may be defined through electrical, magnetic, and/or optical systems.
  • a reaction site may be defined as the intersection between a beam of light and a fluid channel. The volume of the reaction site can be very small in certain embodiments.
  • the reaction site may have a volume of less than one liter, less than about 100 ml, les than about 10 ml, less than about 5 ml, less than about 3 ml, less than about 2 ml, less than about 1 ml, less than about 500 microliters, less than about 300 microliters, less than about 200 microliters, less than about 100 microliters, less than about 50 microliters, less than about 30 microliters, less than about 20 microliters or less than about 10 microliters in various embodiments.
  • the reaction site may also have a volume of less than about 5 microliters, or less than about 1 microliter in certain cases.
  • the reaction site may have any convenient size and/or shape.
  • the reaction site may have a dimension that is 500 microns deep or less, 200 microns deep or less, or 100 microns deep or less.
  • cells can be present at the reaction site.
  • Sensor(s) associated with the chip or reactor in certain cases, may be able to determine the number of cells, the density of cells, the status or health of the cell, the cell type, the physiology of the cells, etc.
  • the reactor can also maintain or control one or more environmental factors associated with the reaction site, for example, in such a way as to support a chemical reaction or a living cell.
  • a sensor may be connected to an actuator and/or a microprocessor able to produce an appropriate change in an environmental factor within the reaction site.
  • the actuator may be connected to an external pump, the actuator may cause the release of a substance from a reservoir, or the actuator may produce sonic or electromagnetic energy to heat the reaction site, or selectively kill a type of cell susceptible to that energy.
  • the reactor can include one or more than one reaction site, and one or more than one sensor, actuator, processor, and/or control system associated with the reaction site(s). It is to be understood that any reaction site or a sensor technique disclosed herein can be provided in combination with any combination of other reaction sites and sensors.
  • the channel can include characteristics that facilitate control over fluid transport, e.g., structural characteristics (e.g., an elongated indentation), physical/chemical characteristics (e.g., hydrophobicity vs. hydrophilicity) and/or other characteristics that can exert a force (e.g., a containing force) on a fluid when within the channel.
  • the fluid within the channel may partially or completely fill the channel.
  • the fluid may be held or confined within the channel or a portion of the channel in some fashion, for example, using surface tension (i.e., such that the fluid is held within the channel within a meniscus, such as a concave or convex meniscus).
  • the channel may have any suitable cross-sectional shape that allows for fluid transport, for example, a square channel, a circular channel, a rounded channel, a rectangular channel (e.g., having any aspect ratio), a triangular channel, an irregular channel, etc.
  • the channel may be of any size within the reactor or chip.
  • the channel may have a largest dimension perpendicular to a direction of fluid flow within the channel of less than about 1000 micrometers in some cases, less than about 500 micrometers in other cases, less than about 400 micrometers in other cases, less than about 300 micrometers in other cases, less than about 200 micrometers in still other cases, less than about 100 micrometers in still other cases, or less than about 50 or 25 micrometers in still other cases.
  • the dimensions of the channel may be chosen such that fluid is able to freely flow through the channel, for example, if the fluid contains cells.
  • the dimensions of the channel may also be chosen in certain cases, for example, to allow a certain volumetric or linear flowrate of fluid within the channel.
  • the depth of other largest dimension perpendicular to a direction of fluid flow may be similar to that of a reaction site to which the channel is in fluid communication with.
  • the number of channels, the shape or geometry of the channels, and the placement of channels within the chip can be determined by those of ordinary skill in the art.
  • Chips of the invention may also include a plurality of inlets and/or outlets that can receive and/or output any of a variety of reactants, products, and/or fluids, for example, directed towards one or more reactors and/or reaction sites.
  • the inlets and/or outlets may allow the aseptic transfer of compounds. At least a portion of the plurality of inlets and/or outlets may be in fluid communication with one or more reaction sites within the chip.
  • the inlets and/or outlets may also contain one or more sensors and/or actuators, as further described below.
  • the chip may have any number of inlets and/or outlets from one to tens of hundreds that can be in fluid communication with one or more reactors and/or reaction sites.
  • each reactor may have around 25 inlets and/or outlets, in other cases around 50 inlets and/or outlets, in still other cases around 75 inlets and/or outlets, or around 100 or more inlets and/or outlets in still other cases.
  • the inlets and/or outlets of the chip, directed to one or more reactors and/or reaction sites may include inlets and/or outlets for a fluid such as a gas or a liquid, for example, for a waste stream, a reactant stream, a product stream, an inert stream, etc.
  • the chip may be constructed and arranged such that fluids entering or leaving reactors and/or reaction sites do not substantially disturb reactions that may be occurring therein.
  • fluids may enter and/or leave a reaction site without affecting the rate of reaction in a chemical, biochemical, and/or biological reaction occurring within the reaction site, or without disturbing and/or disrupting cells that may be present within the reaction site.
  • inlet and/or outlet gases may include, but are not limited to, C0 2 , CO, oxygen, hydrogen, NO, N0 2 , water vapor, nitrogen, ammonia, acetic acid, etc.
  • an inlet and/or outlet fluid may include liquids and/or other substances contained therein, for example, water, saline, cells, cell culture medium, blood or other bodily fluids, antibodies, pH buffers, solvents, hormones, carbohydrates, nutrients, growth factors, therapeutic agents (or suspected therapeutic agents), antifoaming agents (e.g., to prevent production of foam and bubbles), proteins, antibodies, and the like.
  • the inlet and/or outlet fluid may also include a metabolite in some cases.
  • a "metabolite,” as used herein, is any molecule that can be metabolized by a cell.
  • a metabolite may be or include an energy source such as a carbohydrate or a sugar, for example, glucose, fructose, galactose, starch, corn syrup, and the like.
  • Other example metabolites include hormones, enzymes, proteins, signaling peptides, amino acids, etc.
  • the inlets and/or outlets may be formed within the chip by any suitable technique known to those of ordinary skill in the art, for example, by holes or apertures that are punched, drilled, molded, milled, etc. within the chip or within a portion of the chip, such as a substrate layer. In some cases, the inlets and/or outlets may be lined, for example, with an elastomeric material.
  • the inlets and/or outlets may be constructed using self-sealing materials that may be re-usable in some cases.
  • an inlet and/or outlet may be constructed out of a material that allows the inlet and/or outlet to be liquid-tight (i.e., the inlet and/or outlet will not allow a liquid to pass therethrough without the application of an external driving force, but may admit the insertion of a needle or other mechanical device able to penetrate the material under certain conditions).
  • the material upon removal of the needle or other mechanical device, the material may be able to regain its liquid-tight properties (i.e., a "self-sealing" material).
  • Non-limiting examples of self- sealing materials suitable for use with the invention include, for example, polymers such as polydimethylsiloxane ("PDMS"), natural rubber, HDPE, or silicone materials such as Formulations RTV 108, RTV 615, or RTV 118 (General Electric, New York, NY).
  • the chip of the present invention may include very small elements, for example, sub-millimeter or microfluidic elements.
  • the chip may include at least one reaction site having a cross sectional dimension of no greater than, for example, 100 mm, 80 mm, 50 mm, or 10 mm.
  • the reaction site may have a maximum cross section no greater than, for example, 100 mm, 80 mm, 50 mm, or 10 mm.
  • the "cross section” refers to a distance measured between two opposed boundaries of the reaction site
  • the "maximum cross section” refers to the largest distance between two opposed boundaries that may be measured.
  • a cross section or a maximum cross section of a reaction site may be less than 5 mm, less than 2 mm, less than 1 mm, less than 500 micrometers, less than 300 micrometers, less than 100 micrometers, less than 10 micrometers, or less than 1 micrometer or smaller.
  • a "microfluidic chip” is a chip comprising at least one fluidic element having a sub-millimeter cross section, i.e., having a cross section that is less than 1 mm.
  • a reaction site may have a generally rectangular shape, with a length of 80 mm, a width of 10 mm, and a depth of 5 mm. While one reaction site may be able to hold and/or react a small volume of fluid as described herein, the technology associated with the invention also allows for scalability and parallelization. With regard to throughput, an array of many reactors and/or reaction sites within a chip, or within a plurality of chips, can be built in parallel to generate larger capacities.
  • a plurality of chips may be operated in parallel, for example, through the use of robotics, for example which can monitor or control the chips automatically.
  • an advantage may be obtained by maintaining production capacity at the small scale of reactions typically performed in the laboratory, with scale-up via parallelization. It is a feature of the invention that many reaction sites may be arranged in parallel within a reactor of a chip and/or within a plurality of chips.
  • reaction sites can be constructed to operate in parallel, or in other cases at least about 7, about 10, about 30, about 50, about 100, about 200, about 500, about 1,000, about 5,000, about 10,000, about 50,000, or even about 100,000 or more reaction sites can be constructed to operate in parallel, for example, in a high-throughput system.
  • the number of reaction sites may be selected so as to produce a certain quantity of a species or product, or so as to be able to process a certain amount of reactant.
  • the parallelization of the chips and/or reactors may allow many compounds to be screened simultaneously, or many different growth conditions and or cell lines to be tested and/or screened simultaneously.
  • any embodiment described herein can be used in conjunction with a collection chamber connectable ultimately to an outlet of one or more reactors and/or reaction sites of a chip.
  • the collection chamber may have a volume of greater than 10 milliliters or 100 milliliters in some cases.
  • the collection chamber in other cases, may have a volume of greater than 100 liters or 500 liters, or greater than 1 liter, 2 liters, 5 liters, or 10 liters.
  • the reactors and/or reaction sites are arranged in parallel within one or more chips, e.g., a plurality of reactors and/or reaction sites may be able to deliver a product to a collection chamber.
  • the reaction site(s) and/or the channels in fluidic communication with the reaction site(s) are free of active mixing elements.
  • the reactor of the chip can be constructed in such a way as to cause turbulence in the fluids provided through the inlets and/or outlets, thereby mixing and/or delivering a mixture of the fluids, preferably without active mixing, where mixing is desired.
  • the reactor and/or reaction site(s) may include a plurality of obstructions in the flow path of the fluid that causes fluid flowing through the flow path to mix, for example, as shown in mixing unit 42 in Fig. 2.
  • These obstructions can be of essentially any geometrical arrangement for example, a series of pillars.
  • active mixing elements is meant to define mixing elements such as blades, stirrers, or the Iike, which are movable relative to the reaction site(s) and/or channels themselves, that is, movable relative to portion(s) of the reactor defining a reaction site a or a channel.
  • Chips of the invention can be constructed and arranged such that they are able to be stacked in a predetermined, pre-aligned relationship with other, similar chips, such that the chips are all oriented in a predetermined way (e.g., all oriented in the same way) when stacked together.
  • the chip often can be constructed and arranged such that at least a portion of the chip, such as a reaction site, is in fluidic communication with one or more of the other chips and/or reaction sites within other chips. This arrangement may find use in parallelization of chips, as discussed herein.
  • the chip is constructed and arranged such that the chip is able to be stably connected to a microplate, for example, as defined in the 2002 SPS/ANSI proposed standard (e.g., a microplate having dimensions of roughly 127.76 ⁇ 0.50 mm by 85.48 ⁇ 0.50 mm).
  • stably connected refers to systems in which two components are connected such that a specific motion or force is necessary to disconnect the two components from each other, i.e., the two components cannot be dislodged by random vibration or displacement (e.g., accidentally lightly bumping a component).
  • microplate is also sometimes referred to as a "microtiter” plate, a "microwell” plate, or other similar terms known to the art.
  • the microplate may include any number of wells.
  • the microplate may be a six-well microplate, a 24-well microplate, a 96-well microplate, a 384-well microplate, or a 1,536-well microplate.
  • the wells may be of any suitable shape, for example, cylindrical or rectangular.
  • the microplate may also have other numbers of wells and/or other well geometries or configurations, for instance, in certain specialized applications.
  • Figs. 3A - 3C illustrate one set of embodiments of the invention in which one or more reaction sites may be positioned in association with a chip such that, when the chip is stably connected to other chips and/or microplates, one or more reaction sites of the chip are positioned or aligned to be in chemical, biological, or biochemical communication with, or chemically, biologically, or biochemically connectable with one or more reaction sites of the other chip(s) and/or one or more wells of the microplate(s).
  • “Alignment,” in this context, can mean complete alignment, such that the entire area of the side of a reaction site adjacent another reaction site or well completely overlaps the other reaction site or well, and vice versa, or at least a portion of the reaction site can overlap at least a portion of an adjacent reaction site or well.
  • “Chemically, biologically, or biochemically connectable” means that the reaction site is in fluid communication with another reaction site or well (i.e., fluid is free to flow from one to the other); or is fluidly connectable to the other site or well (e.g., the two are separated from each other by a wall or other component that can be punctured or ruptured, or a valve in a conduit connecting the two can be opened); or the reaction site and other site or well are arranged such that at least some chemical, biological, or biochemical species can migrate from one to the other, e.g., across a semipermeable membrane.
  • a chip may have six reaction sites that are constructed and arranged to be aligned with the six wells of a 6-well microplate when the chip is stably connected with the microplate (e.g., positioned on top of the microplate), a chip having 96 reaction sites may be constructed and arranged such that the 96 wells are constructed and arranged to be aligned with the 96 wells of a 96-well microplate when the chip is stably connected with the microplate, etc.
  • Detection of the environmental condition may occur, for example, by means of a sensor which may be positioned within the reaction site, or positioned proximate the reaction site, i.e., positioned such that the sensor is in communication with the reaction site in some manner. In some cases, such detection may occur in real-time.
  • the sensor may be, for example, a pH sensor, an optional sensor, an oxygen sensor, a sensor able to detect the concentration of a substance, or the like. Other examples of sensors are further described below.
  • the sensor may be embedded and integrally connected with the chip (e.g., within a component defining at least a portion of the reaction site a channel in fluidic communication with the reaction site, etc.), or separate from the chip in some cases (e.g., within sensing communication).
  • a reactor and/or reaction site within a chip may be coupled to a light delivery and/or other light interacting component(s).
  • the light-interacting component may include a detection system where light (e.g., having a predetermined wavelength) arising from a dye, a fluorescent molecule, etc., may be detected and/or measured.
  • the sensor can include a colorimetric detection system in some cases, which may be external to the chip, or microfabricated into the chip in certain cases.
  • the colorimetric detection system can be external to the chip, but optically coupled to the reaction site, for example, using fiber optics or other light-interacting components that may be embedded in the chip (e.g., such as those described below).
  • a flow of agent may occur along serpentine path 281.
  • a chemical agent generated elsewhere within the chip may be allowed to interact with the reaction site(s) to control the environmental factor(s) therein, or one or more fluidic pathways may be created (e.g., opened) within the chip that allows an agent stored within the chip or external the chip to come into contact with the reaction site . or otherwise affect the reaction site.
  • the agent may be any agent able to alter and/or control one or more environmental factors within the reaction site.
  • the agent may be a non-pH-neutral composition or a pH-altering agent as previously described. As an example, in Fig.
  • the base or alkaline may have a pH of at least about 7, at least about 8, at least about 9, at least about 1 1, or at least about 12 pH units.
  • a "non-neutral” or a “non-pH-neutral” composition is a composition that is either acidic or basic (i.e., the composition has a pH that is either greater than or less than 7, preferably by a significant amount, such as by at least 1 or 2 pH units).
  • the non-pH-neutral composition may be a solid, a liquid, or a gas in some cases.
  • laser beam 232 may optionally pass through one or more other layers and or components of chip 205 before reaching compartment 235 (for example, if those layers and/or components are substantially transparent).
  • the agent-producing precursor(s) 237 may produce agent 238 in this example.
  • Agent 238 may be, in this example, a gas such as a pH-altering gas, for example, ammonium, acetic acid, CO, CO 2 , 0 2 , N 2 , HCl, etc.
  • Non-limiting examples of such materials include quartz, black glass, silicon, black sand, carbon black, and the like.
  • the additional compounds may be substantially unreactive, unable to form a transportable agent (i.e., transportable through a layer or a component of the chip), or the additional compounds may not significantly interfere with the production of the agent or with control of an environmental factor associated with the reaction site.
  • the agent in certain embodiments, may be produced in a reaction that is activated at a certain temperature, such as in a thermal decomposition or degradation reaction.
  • formic acid may be produced by the thermal decomposition of sodium formate, potassium formate, calcium formate, lithium formate, magnesium formate, etc.
  • the pH-altering agent may not be an acid or a base, but be in a form that can be converted into an acid or a base within the chip or within a reaction site.
  • the pH-altering agent may react with water to form an acid or a base within the chip or reaction site.
  • a gas such as C0 2 may react with water to produce carbonic acid, e.g.:
  • Chips of the invention may include one or more fluid pathways for delivery of species or removal of species from a reaction site.
  • a fluidic pathway can be created in situ (after construction of the chip, during chip setup and/or during use of the chip) by permeabilizing or damaging a component separating the compartment from the reaction site (e.g., as in a wall or a membrane), or separates the compartment from a fluidic pathway in fluid communication with the reaction site.
  • the fluidic pathway or other means for fluidic communication may be created by permeabilizing and/or damaging (reversibly or irreversibly) a component that separates the compartment containing the agent (and/or agent precursor(s)) from fluidic communication with the reaction site, or separates the compartment from a channel or other fluidic pathway in fluid communication with the reaction site, thus creating a fluidic connection between the compartment and the reaction site.
  • the component may be permeabilized by heating the component to increase the permeability of the chemical agent or by causing the component to melt or vaporize.
  • the permeability of the component may be enhanced by one, two, or three or more orders of magnitude.
  • the permeabilization of the component may be reversible or at least partially reversible, for example, by decreasing the temperature, or introducing a non-permeabilizing agent.
  • the component in some cases, may also be damaged or otherwise altered or permeabilized through a reaction, for example, a chemical or electrochemical reaction, to produce a fluidic connection with the reaction site.
  • the component may include a metal, such as gold, silver or copper, that can be electrolyzed upon the application of a suitable electrical current.
  • the component may be chemically etched, for example, with a reactive species.
  • the component as discussed above may be mechanically altered and/or damaged, for example, by piercing the component with a microneedle to create a fluidic pathway between the compartment and the reaction site.
  • the microneedle or other mechanical device may originate from within the chip, or externally.
  • the component may be altered on a reversible basis, for example, the component may be self-sealing and/or comprised an elastomeric substance that can be resealed.
  • the component may also be damaged without the use of mechanical forces or chemicals in some cases. For example, energy may be applied to the surface to damage it.
  • the component may be ablated, for example, using heat or light.
  • the component may include a material able to enhance the creation of the fluidic pathway in some embodiments of the invention.
  • the enhancing material may facilitate the absorption of light or other forms of energy, or increase the chemical reaction or transport rate.
  • the component may comprise a material that is able to absorb incident electromagnetic radiation, i.e., a darkened or "black" material, such as quartz, black glass, silicon, black sand, carbon black, and the like.
  • the component may include a catalyst, an enzyme, or a permeation enhancer.
  • the humidity control material may include a membrane or a thin film selected to control the passage of gases and/or water vapor therethrough.
  • the humidity controller is a membrane or a thin film having a desired permeability to one or more gases.
  • the membrane or thin film may be positioned anywhere in the chip where it is able to affect one or more reaction sites in some fashion.
  • the membrane or thin film may be positioned such that it defines the surface of one or more reaction sites.
  • the humidity control material may also include a support layer.
  • a support layer may comprise any material or materials that provides desired support.
  • the support layer may include one of the layers that may otherwise be included in the humidity control material for permeability, such as polydimethylsiloxane or polyfluoroorganic materials, or the support layer may comprise a different material, such as glass (for example, PYREX® glass by Corning Glass of Corning, NY; or indium/tin-coated glass), latex, silicon, or the like.
  • the support layer may be positioned anywhere within the humidity control material, for example, as an outer layer or an intermediate layer, and may be positioned to help protect one or more delicate layers.
  • the use of a support layer may allow a large portion, or nearly all of a reaction site, reactor, or chip to be constructed of the humidity control material.
  • the support layer does not significantly impact the permeability of the humidity control material, or the change in permeability may be accounted for in the design of the humidity control material.
  • the membrane may include a protection layer.
  • the protection layer may be positioned as any component of the humidity control material, for example, as a surface layer, or interposed between a sensitive portion of the humidity control material and the material or environment that may adversely affect it.
  • the protection layer may be positioned on an inner surface of the humidity control material, particularly where the harmful material is within the chip, or on the outer surface of the humidity control material, particularly where the harmful material is outside the chip.
  • the protection layer may also be positioned between other layers, so long as it is able to perform is protective function.
  • the protection layer does not significantly impact the permeability of the humidity control material, or the change in permeability may be accounted for in the design of the humidity control material.
  • the humidity control material is selected to have a certain permeability and/or a certain permeance.
  • the "permeability" of a material is given its ordinary meaning as used in the art, i.e., an intrinsic property that generally describes the ability of a gas to pass through the material.
  • the "permeance" of a material is the actual rate of gas transport through a sample of a material, i.e., an extrinsic property. The permeance of a sample of material is affected by factors such as the area or thickness of the material, the pressure differential across the material, etc.
  • reaction is cell culture, for example to maintain a cell culture, to increase the number of available cells or cell types, or to produce a desirable cellular product.
  • the humidity control material may allow sufficient oxygen to enter by diffusion therethrough to support cell growth.
  • the humidity control material may also be largely impermeable to microorganisms and other cells, for example to prevent contamination.
  • the material has low toxicity.
  • cell culturing may take place over varying lengths of time, depending on the cells being cultured and other factors known to those of ordinary skill in the art.
  • the design of the chip and the nature of the humidity control material may be adapted to the culture time.
  • the chip or humidity control material may be designed to allow it to withstand the time needed for the culture and is preferably designed to be able to be reused many times.
  • cell cultures may be performed in 24 hours, 48 hours, 1 week, 2 weeks, 4 weeks, 6 weeks, 3 months, 1 year, continuously, or any other time required for a specific cell culture.
  • the humidity control material is selected to have a permeability and/or a permeance to one or more gases that corresponds to a range acceptable for culturing certain cells.
  • the humidity control material may have a permeability and/or permeance to oxygen high enough, and/or a permeability and/or permeance to water vapor low enough, to allow cell culturing.
  • permeability ranges of a humidity control material for use in the invention include a permeability to oxygen greater than about 100 (cm 3 s ⁇ p mm/m 2 atm day), and a permeability to water vapor less than about 6xl0 "6 (cm 3 s ⁇ p mm/m 2 atm day).
  • STP refers to "standard temperature and pressure,” referring to a temperature of 273.15K (0 °C) and a pressure of about 10 5 Pa (1 atm).
  • oxygen permeability and water vapor permeability listed herein can be used.
  • an example of a suitable range of oxygen permeability is provided by a membrane having a permeability to oxygen permeability greater than about lxl 0 3 (cm 3 s ⁇ p mm/m 2 atm day) and/or a permeability to water vapor is less than about 6x10 (cm 3 s ⁇ p mm/m 2 atm day).
  • a membrane of the invention having a permeability to oxygen greater than about 100 (cm 3 S ⁇ p mm/m 2 atm day) and a permeability to water vapor lower than about lxl O 5 (cm 3 S ⁇ p mm/m 2 atm day).
  • a permeability to oxygen greater than about 100 (cm 3 S ⁇ p mm/m 2 atm day) and a permeability to water vapor lower than about lxl O 5 (cm 3 S ⁇ p mm/m 2 atm day).
  • humidity control materials having a permeability to oxygen and water vapor, in certain cases, it is desired that the material have very high oxygen permeability and very low permeability to water vapor, e.g., as is indicated in Fig. 12 by "goal" region 80.
  • the material may have an oxygen permeability of greater than about 1000 (cm 3 s ⁇ p micrometer/m 2 day atm), in some cases greater than about 10,000 (cm 3 s ⁇ p micrometer/m 2 day atm), and in some cases greater than about 100,000 (cm 3 s ⁇ p micrometer/m 2 day atm), and/or a permeability to water vapor less than about 1000 (g micrometer/m 2 day), in some cases less than about 100 (g micrometer/m 2 day), and in some cases less than about 10 (g micrometer/m 2 day).
  • an oxygen permeability of greater than about 1000 (cm 3 s ⁇ p micrometer/m 2 day atm), in some cases greater than about 10,000 (cm 3 s ⁇ p micrometer/m 2 day atm), and in some cases greater than about 100,000 (cm 3 s ⁇ p micrometer/m 2 day atm), and/or a permeability to water vapor less than about 1000 (g micrometer/m 2 day), in some cases less than about 100 (g micrometer/m
  • the results of materials such as high density polyethylene (“HDPE”), polyethylene terephthalate (“PET”), polypropylene (“PP”), or poly(4-methylpentene-l) (“PMP”) are shown, and these may be suitable for use with the invention, as further described below.
  • HDPE high density polyethylene
  • PET polyethylene terephthalate
  • PP polypropylene
  • PMP poly(4-methylpentene-l)
  • the humidity control material does not promote cell adhesion, but may include a cell adhesion layer (or a cell adhesion layer can be provided on the material) that may be any of a wide variety of hydrophilic, cytophilic, and/or biophilic materials.
  • Examples of materials that may be suitable for a cell adhesion layer on a humidity control material include, but are not limited to, polyfluoroorganic materials, polyester, PDMS, polycarbonate, polystyrene, and aluminum oxide.
  • the humidity control material may include a layer coated with a material that promotes cell adhesion, for example, using an RGD peptide sequence.
  • it may be desired to modify the surface of a cell adhesion layer for example, by attachment, binding, soaking or other treatments.
  • Example molecules that promote cell adhesion include, but are not limited to, fibronectin, laminin, albumin or collagen.
  • the cell adhesion layer may be positioned as an inner layer or a surface layer of the membrane, or may abut an interior of the chip.
  • the cell adhesion layer does not significantly impact the permeability or permeance of the humidity control material, or the change in permeability or permeance may be accounted for in the design of the humidity control material.
  • Some of the materials used to form the humidity control material, and, in some cases, some of the layers thereof, may be selected based on the gas permeabilities of the materials, for example, as previously described. Those of ordinary skill in the art will know of methods of determining the gas permeability of a material.
  • a sample of a material having a known exposed area and thickness may be placed between two chambers, and a gas (or a liquid) may be placed in one chamber.
  • the experimental time it takes for the gas (or liquid) to diffuse across the material to the other chamber and detected in a suitable fashion may then be related to the gas (or liquid) permeability of the material.
  • the humidity control material may include a polymer (e.g., a single polymer type, a co-polymer, a polymer blend, a polymer derivative, etc.).
  • polyfluoroorganic materials such as polytetra
  • the polymer may be poly(4-methylpentene-l) ("PMP"):
  • the polymer may be poly(4- methylhexene-1 ), poly(4-methylheptene-l) poIy(4-methyloctene-l), etc.
  • the polymer may be poly(l -trimethlsilyl-l-propyne) ("PTMSP"): which, in some cases, may have a permeability coefficient for oxygen of about 5J8xl0 5 (cm 3 S ⁇ p mm/m 2 day atm).
  • PTMSP poly(l -trimethlsilyl-l-propyne)
  • copolymer of these and/or other polymers may be used in the humidity control material.
  • the first and second layers may also each include a mixture of materials in some embodiments.
  • one layer may include at least 50% by weight of one material with the balance comprising one or more other materials.
  • each layer consists essentially of a single material.
  • the area and thickness of the humidity control material, or a layer or portion thereof may be used to select a desired degree of permeance and/or permeability.
  • a more water vapor-permeable material may be made thicker, or its area may be reduced, in order to reduce the amount of water vapor that reaches or leaves the area or region where humidity control is desired.
  • the material may be designed such that it is between about 10 micrometers and 2 mm thick. Within this range, the relative thickness of layers within multiple layers or portions of the material may vary. For example, a relatively thick layer of a polyfluoroorganic material and a relatively thin layer of vinylidene chloride may be useful in particular embodiments.
  • a few micrometers of polytetrafluoroethylene may be deposited or coated onto a layer of polydimethylsiloxane, or a few micrometers of HDPE could be co- molded with PDMS.
  • the polymer (or mixture of polymers) used in the humidity control material may be sufficiently hydrophobic such that the polymer is able to retain water (i.e., water vapor is not able to readily transport through the polymer).
  • the permeability of water through a hydrophobic polymer may be less than about 1000 (g micrometer/m 2 day), 900 (g micrometer/m 2 day), 800 (g micrometer/m 2 day), 600 (g micrometer/m 2 day) or less, as previously described.
  • the polymer(s) used in the humidity control material may have a molecular structure open enough to readily allow the transport of oxygen therethrough.
  • the molecular structure may allow transport of oxygen across the polymer of greater than about 1000 (cm 3 s ⁇ p micrometer/m 2 day atm) or more, as previously described.
  • the polymer is sufficiently branched such that the polymer is unable to form a structure under ambient conditions (e.g., a tightly crystalline structure) that limits the transport of oxygen therethrough, for instance, to less than about 1000 (cm 3 s ⁇ p micrometer/m 2 day atm) or 500 (cm 3 s ⁇ p micrometer/m 2 day atm).
  • the polymer may include a bulky group that prevent the polymer from readily forming a structure under ambient conditions that limits the transport of oxygen therethrough.
  • a "bulky group" on a polymer, as used herein, is a moiety sufficiently large that the polymer is unable to form a crystalline structure under ambient conditions that limits the transport of oxygen therethrough to less than about 1000 (cm 3 s ⁇ p micrometer/m 2 day) or 500 (cm 3 s ⁇ p micrometer/m 2 day).
  • the bulky group may be, for instance, part of the backbone of the polymer or a side chain.
  • Non-limiting examples of bulky side groups include groups containing cyclopentyl moieties, isopropyl moieties, cyclohexyl moieties, phenyl moieties, isobutyl moieties, tert-butyl moieties, cycloheptyl moieties, trimethylsilyl or other trialkylsilyl moieties etc.
  • the polymer may have a structure:
  • each R independently comprises at least one atom
  • Bk is a bulky group.
  • R may be a hydrogen or an alkyl group.
  • the polymer may have several or all of the above-described features.
  • the polymer may be a polymer blend or a copolymer that has sufficient hydrophobicity such that the polymer is able to retain water yet have a molecular structure open enough to allow sufficient oxygen permeability therethrough.
  • the present invention achieves a permeability goal by combining two layers or portions of material. This can be achieved, for example, by including a first, more permeable layer, and a second, less permeable layer; multiple layers may also be used in other embodiments.
  • the humidity control material may be formed out of the same or different materials polymers.
  • the humidity control material may include a first layer including at least about 55% by weight of a first polymer or co-polymer and a second layer comprising no more than about 45% by weight of the first polymer or co-polymer.
  • the humidity control material may include a first layer including at least about 60%, about 70%, or about 80% by weight of a first polymer or co-polymer and a second layer comprising no more than about 40%, about 30%, or about 20% by weight of the first polymer or copolymer.
  • the first polymer may comprise about 100% of the first layer and essentially none of the second layer. In some cases, at least a portion of the first layer may be co-polymerized with the second layer.
  • the humidity control material of the present invention is constructed as a membrane including two or more layers, the two or more layers may be joined in any manner that provides sufficient strength to the membranes. In some cases, the two or more layers may be sufficiently self-supporting and it may not be necessary to join the layers, meaning a space could be left therebetween if desired. In other embodiments, additional layers may be used to support the membrane.
  • examples of acceptable means of joining the layers include laminating the layers together, at least partially intermixing the layers, and co-polymerizing the layers together.
  • the resin that will form each layer may be partially or totally intermixed before the membrane is formed.
  • liquid pre-polymers may be mixed and then a curing agent added, or two partially cured layers can be connected with a curing agent between them, curing the layers together.
  • the humidity control material of the present invention allows light to pass through it.
  • the material may be used where light is important, for example, to facilitate a reaction such as a photocatalyzed reaction, to promote cell or plant growth, to cause a biochemical change to occur, or the like.
  • the material may also allow observation of a region, such as a reactor or reaction site, that is protected by the humidity control material, or is located behind a humidity-controlled region.
  • the humidity control material is translucent, and, in some cases, it is at least substantially transparent.
  • the chip can include a variety of other components.
  • the chip may include components such as a light source, a flowmeter (e.g., for measuring fluid flow of a gas or a liquid), a circuit such as an integrated circuit, a reservoir (e.g., for a solution), a micromechanical or a MEMS ("microelectromechanical system") component, a microvalve, a micropump, or the like, for example, as further described below.
  • the components may be fabricated on the chip using techniques such as those used in standard microfabrication, similar to those used to create semiconductors (See Madou Fundamentals of Microfabrication, CRC Press, Boca Raton, FL 1997; and Maluf, An Introduction of Micromechanical Systems Engineering, Artech House Boston, MA 2000).
  • At least one, two, three or more components are integrally connected to the chip. In certain embodiments, all of the components are integrally connected to the chip.
  • Other examples of components suitable for use with the invention include pylon-like obstructions placed in the flow path of a stream to enhance mixing within the chip, reactor and/or reaction site, or heating, separation, and/or dispersion units within the chip, reactor and/or reaction site.
  • the heating unit may be a miniaturized, traditional heat exchanger.
  • the present invention may include a membrane, such as a membrane that may control humidity (e.g., as previously described) and/or be substantially transparent.
  • a membrane may be positioned anywhere in the a reactor within a chip.
  • the membrane is positioned such that it defines the surface of one or more reaction sites and/or divides a reaction site into two or more portions, which portions may have the same or different dimensions.
  • membrane 410 which may be a humidity controller and/or be substantially transparent, defines a surface of reaction site 41 1.
  • Fig. 10B membrane 410 defines the surface of reaction site 41 1 and a surface of reaction site 412.
  • the membrane can be positioned such that it is in fluidic communication with one or more reaction sites of the chip.
  • the membrane may be positioned such that a pathway fluidly connecting a first reaction site with a second reaction site crosses the membrane.
  • the membrane can be positioned such that it is in fluidic communication with one or more reaction sites of the chip.
  • the membrane may be positioned such that a pathway fluidly connecting a first reaction site with a second reaction site crosses the membrane.
  • membrane 410 does not define surfaces of reaction sites 411 or 412, but is positioned such that at least one pathway fluidly connecting reaction site 411 with reaction site 412 crosses membrane 410.
  • the membrane may be a porous membrane having, for example, a number-average pore size of greater than about 0.03 micrometers and less than about 5 micrometers.
  • the pore size of the membrane may be less than about 4 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1.5 micrometers, less than about 1.0 micrometers, less than about 0.75 micrometers, less than about 0.6 micrometers, less than about 0.5 micrometers, less than about 0.4 micrometers, less than about 0.3 micrometers, less than about 0.1 micrometers, less than about 0.07 micrometers, and in other embodiments, less than about 0.05 micrometers.
  • the pores are also greater than 0.03 micrometers or greater than 0.08 micrometers.
  • the membrane may be chosen to prevent the passage of certain cells there through (e.g., bacterial cells, yeast cells, mammalian cells, etc.). For example, a membrane with a pore size of about 0.2 micrometers may prevent the passage of bacteria cells, and a membrane with a pore size of a bout 1 micrometer may prevent the passage of mammalian cells.
  • a membrane may be chosen to prevent or permit the passage of certain molecules, e.g., micromolecules, having a certain size and/or charge, i.e., a charge and/or size selective membrane.
  • the membrane may be or include polymers or other materials such as polyethylene terephthalate (PET), polysulfone, polycarbonate, acrylics such as polymethyl methacrylate, polyethylene, polypropylene, regenerated cellulose, nitrocellulose, aluminum oxide, glass, fiberglass, and the like.
  • PET polyethylene terephthalate
  • acrylics such as polymethyl methacrylate, polyethylene, polypropylene, regenerated cellulose, nitrocellulose, aluminum oxide, glass, fiberglass, and the like.
  • the membrane may also be substantially transparent, e.g., as previously described.
  • the membrane is a substantially transparent polyethylene terephthalate membrane having a pore size of 2 micrometers or less, for example, a ROTRAC® capillary membrane made by Oxyphen U.S.A., Inc. (New York, NY).
  • a chip of the invention may include a structure adapted to facilitate the reactions or interactions that are intended to take place therein (e.g., within a reaction site).
  • the chip may include structure(s) able to improve or promote cell growth.
  • a surface of a reaction site may be a surface able to promote cell binding or adhesion, or the reactor and/or reaction site within.
  • the chip may include a structure that includes a cell adhesion layer, which may include any of a wide variety of hydrophilic, cytophilic, and/or biophilic materials.
  • the surface may be ionized, coated (e.g., with a support material) and/or micropatterned with any of a wide variety of hydrophilic, cytophilic, and/or biophilic materials, for example, materials having exposed carboxylic acid, alcohol, and/or amino groups.
  • hydrophilic, cytophilic, and/or biophilic materials for example, materials having exposed carboxylic acid, alcohol, and/or amino groups.
  • materials that may be suitable for a cell adhesion layer include, but are not limited to, polyfluoroorganic materials, polyester, PDMS, polycarbonate, polystyrene, and aluminum oxide.
  • the structure may include a layer coated with a material that promotes cell adhesion, for example, an RGD peptide sequence, or the structure may be treated in such a way that it is able to promote cell adhesion, for example, the surface may be treated such that the surface becomes relatively more hydrophilic, cytophilic, and/or biophilic.
  • it may be desired to modify the surface of a cell adhesion layer, for instance with materials that promote cell adhesion, for example, by attachment, binding, soaking or other treatments.
  • Example materials that promote cell adhesion include, but are not limited to, fibronectin, laminin, albumin or collagen.
  • the surface may be formed out of a hydrophobic, cytophobic, and/or biophobic material, or the surface may be treated in some fashion to make it more hydrophobic, cytophobic, and/or biophobic, for example, by using aliphatic hydrocarbons and/or fluorocarbons.
  • the chip may include a "light-interacting component," i.e., a component that interacts with light, for example, by producing light, reacting to light, causing a change in a property of light, directing light, altering light, etc.
  • a "light-interacting component” is a component that interacts with light in some fashion related to chip and/or reactor function, for example, by producing light, reacting to light, causing a change in a property of light, directing light, altering light, etc., in a manner that affects a sample within a chip or reactor and/or determines something about the sample (the presence of the sample, a characteristic of the sample, etc.).
  • the component produces light, such as in a light-emitting diode (“LED”) or a laser.
  • the light-interacting component may be a component that is sensitive to light or responds to light, such as a photodetector or a photovoltaic cell.
  • the light-interacting component may manipulate or alter light in some fashion, for example, by focusing or collimating light, or causing light to diverge, such as in a lens, or spectrally dispersing light, such as in a diffraction grating or a prism.
  • the light-interacting component may be able to transmit or redirect the direction of light in some fashion, such as along a bent path or around a corner, for example, as in a waveguide or mirror.
  • the light-interacting component may alter a property of light incident on the component, such as the degree of polarization or the frequency, for example, as in a polarizer or an interferometer. Other devices, or combinations of devices, are also possible.
  • the term "light-interacting component” does not encompass components or devices that passively transmit light without significant modification, alteration, or redirection, such as air, or a plane of glass or plastic (e.g., a "window”).
  • the term “light-interacting component” also does not generally encompass components that passively absorb essentially all incident light without a response, such as would be found in an opaque material.
  • a light-interacting component may be positioned anywhere on or within the reactor.
  • the light-interacting component may be placed within or adjacent to a reaction site.
  • the light-interacting component is integrally connected with the reaction site, for example, as a wall or a surface of the reaction site.
  • the light-interacting component may be positioned elsewhere in, or integrally connected to, the chip, such that at least a portion of light entering the light- interacting component is in optical communication with the reaction site.
  • optical communication generally refers to any pathway that provides for the transport of electromagnetic radiation, such as visible light.
  • Optical communication includes direct, "line-of-sight" communication.
  • Optical communication may also be facilitated, for example, by the use of optical devices such as lenses, filters, optical fiber, waveguides, diffraction gratings, mirrors, beamsplitters, prisms, and the like.
  • the waveguide may be formed out of a silicon-based material, for example, glass, ion-implanted glass, quartz, silicon, silicon oxide, silicon nitride, silicon carbide, polysilicon, coated glass, conductive glass, indium-tin-oxide glass and the like.
  • the waveguide may comprise other transparent or translucent organic or inorganic materials.
  • the waveguide may comprise a polymer including, but not limited to, polyacrylate, polymethacrylate, polycarbonate, polystyrene, polypropylene, polyethylene, polyimide, polyvinylidene fluoride, an ion-exchanged polymer, and fluorinated derivatives of the above.
  • the waveguide may have more than one central material or more than one surrounding material.
  • both the central and surrounding materials forming the waveguide may each be a glass.
  • a waveguide may be formed out of a polymer and a silicon-based material, such that the material with the lower index of refraction surrounds the material with the higher index of refraction.
  • the waveguide may be constructed out of a single material surrounded by, for example, air or a portion of the chip having a higher index of refraction than the waveguide, thus resulting in a condition where total internal reflection may occur at the waveguide/air or waveguide/chip interface.
  • the waveguide may be constructed by any suitable technique known to those of ordinary skill in the art, for example, by milling, grinding, or machining (e.g., by cutting or etching a channel into a chip substrate, then depositing material into the channel, optionally using a sealant).
  • the waveguide may also be formed, for example, by depositing layers of materials during the chip fabrication process. The deposited material, in some cases, can have a higher index of refraction than the surrounding reactor substrate, thus forming a general core-cladding structure, as previously described.
  • the waveguide may also be constructed by laser etching of materials forming the chip, such as glass or plastic, in such a way as to manipulate/alter the refractive index, relative to the surrounding material.
  • the light source may produce a single wavelength or a substantially monochromatic wavelength, or a wide range of wavelengths, as previously described.
  • the source of light in certain embodiments, may also be generated in a chemical reaction or a biological process, such as a chemical reaction that produces photons, for example, a reaction involving GFP ("green fluorescence protein") or luciferase, or through fluorescence or phosphorescence.
  • a chemical reaction that produces photons
  • GFP green fluorescence protein
  • luciferase luciferase
  • incident electrons, electrical current, friction, heat, chemical or biological reactions may be applied to generate light, for example, within a sample located within a reaction site, or from a reaction center located within the chip in optical communication with the reaction site.
  • the light-interacting component may include a filter, for example, a low pass filter, a high pass filter, a notch filter, a spatial filter, a wavelength-selecting filter, or the like.
  • the filter may be able to, for example, substantially reduce or eliminate a portion of the incident light.
  • the filter may eliminate or substantially reduce light having a wavelength below about 350 nm or greater than about 1000 nm.
  • the filter may be able to reduce noise within the incident light, or increase the signal-to-noise ratio of the incident light.
  • the filter may be able to polarize the incident light, for example, linearly or circularly.
  • the light-interacting component may include an optical element in optical communication with the reaction site.
  • an "optical element” refers to any element or device able to alter the pathway of light entering or exiting the optical element, for example, by focusing or collimating the light, or causing the light to diverge.
  • the optical element may focus the incident light to a single point or a small region, or the optical element may collimate or redirect divergent beams of light to form a parallel or converging beams of light.
  • focus generally refers to the ability to cause rays of light to converge to a point or a small region.
  • the term "collimate” generally refers to the ability to increase the convergence of rays of light, not necessarily to a point or a small region, for example, such that the beam focuses at an infinite distance.
  • diverging beams of light may be collimated into parallel beams of light.
  • the optical element may disperse or cause light to diverge, for example, as in a diverging lens.
  • the optical element may be, for example, a beamsplitter, an optical coating (e.g., a dichroic, an antireflective, or a reflective coating), an optical grating, a diffraction grating, or the like.
  • the optical element may be a lens.
  • the lens may be any lens, such as a converging or a diverging lens.
  • the lens may be, for example, a meniscus, a plano-convex lens, a plano-concave lens, a double convex lens, a double concave lens, a Fresnel lens, a spherical lens, an aspheric lens, a binary lens, or the like.
  • the optical element may also be a mirror, such as a planar mirror, a curved mirror, a parabolic mirror, or the like. In other embodiments, the optical element may cause light to disperse, for example, as in a diffraction grating or a prism.
  • a material having a different index of refraction may be used.
  • the optical element may be a material having a different index of refraction than the waveguide.
  • the index of refraction of the optical element will be about the same as or more than the index of refraction of the waveguide.
  • a material having a graded index of refraction (a "GRIN" material) may be used as an optical element.
  • the GRIN material may minimize the amount of divergence inherent in light reaching the GRIN material.
  • a material of unifo ⁇ n thickness can be made to act as a lens by varying its refractive index along a cross section of the element.
  • the GRIN material may redirect divergent rays of light into a parallel arrangement.
  • the GRIN material does not necessarily have a uniform thickness, and a combination of the graded index of refraction of the material and the shape of the material may be used to focus or collimate the light.
  • the light-interacting component may include a component that is able to convert light to electricity, such as a photosensor or photodetector, a photomultiplier, a photocell, a photodiode such as an avalanche photodiode, a photodiode array, a CCD chip ("charge-coupled device") or the like.
  • the component may be used, in some cases, to determine the state or condition of a substance within a reaction site, for example, through emission (including fluorescence or phosphorescence), absorbance, scattering, optical density, polarization measurements, or other measurements, including using the human eye.
  • the light-interacting component may be used for imaging purposes, for example, to image a portion of a cell or other material located at or near the reaction site, or to determine whether a cell has adhered to a surface.
  • the light-interacting component may be used to produce electricity.
  • a photocell may be integrally fabricated within the chip using one or more layers comprising semiconductor materials.
  • light may be directed to the reaction site, for example, to activate or inhibit a chemical reaction.
  • a reaction may require the use of light for activation, or a light-sensitive enzyme may be inhibited by applying light to the enzyme.
  • light directed to the reaction site may be used as a probe or a signal source.
  • the light may be delivered in a controlled manner to the reaction site in certain embodiments, for example, so that the light reaching the reaction site has a specific wavelength, polarization, or intensity.
  • a portion of the light arising from the reaction site may be detected and analyzed.
  • the light arising from the reaction site may be reflected or refracted light, for example, light directed to the reaction as previously described, or the light may be produced through physical means, for example, through fluorescence or phosphorescence.
  • the light may be generated within the reaction site, as previously described.
  • Light from the reaction site may be analyzed using any suitable analytical technique, for example, infrared spectroscopy, FTIR ("Fourier Transform Infrared Spectroscopy"), Raman spectroscopy, absorption spectroscopy, fluorescence spectroscopy, optical density, circular dichroism, light scattering, polarimetry, refractometry, turbidity measurements, quasielectric light scattering, or any other suitable techniques.
  • imaging of the reaction site may be performed, for example using optical imaging, or infrared imaging.
  • a reactor and/or a reaction site within a chip may be constructed and arranged to maintain an environment that promotes the growth of one or more types of living cells, for example, simultaneously.
  • the reaction site may be provided with fluid flow, oxygen, nutrient distribution, etc., conditions that are similar to those found in living tissue, for example, tissue that the cells originate from.
  • the chip may be able to provide conditions that are closer to in vivo than those provided by batch culture systems.
  • the cells may be any cell or cell type, for instance a prokaryotic cell or a eukaryotic cell.
  • the cell may be a bacterium or other single-cell organism, a plant cell, an insect cell, a fungi cell or an animal cell. If the cell is a single-cell organism, then the cell may be, for example, a protozoan, a trypanosome, an amoeba, a yeast cell, algae, etc.
  • the cell may be, for example, an invertebrate cell (e.g., a cell from a fruit fly), a fish cell (e.g., a zebrafish cell), an amphibian cell (e.g., a frog cell), a reptile cell, a bird cell, or a mammalian cell such as a primate cell, a bovine cell, a horse cell, a porcine cell, a goat cell, a dog cell, a cat cell, or a cell from a rodent such as a rat or a mouse.
  • the cell is from a multicellular organism, the cell may be from any part of the organism.
  • the cell may be a cardiac cell, a fibroblast, a keratinocyte, a heptaocyte, a chondracyte, a neural cell, a osteocyte, a muscle cell, a blood cell, an endothelial cell, an immune cell (e.g., a T-cell, a B-cell, a macrophage, a neutrophil, a basophil, a mast cell, an eosinophil), a stem cell, etc.
  • the cell may be a genetically engineered cell.
  • the cell may be a Chinese hamster ovarian ("CHO") cell or a 3T3 cell.
  • more than one cell type may be used simultaneously, for example, fibroblasts and hepatocytes.
  • cell monolayers, tissue cultures or cellular constructs e.g., cells located on a non-living scaffold), and the like may also be used in the reaction site. The precise environmental conditions necessary in the reaction site for a specific cell type or types may be determined by those of ordinary skill in the art.
  • the cells may produce chemical or biological compounds of therapeutic and/or diagnostic interest, for instance, in nanogram, microgram, milligram or gram or higher quantities.
  • the cells may be able to produce products such as monoclonal antibodies, proteins such as recombinant proteins, amino acids, hormones, vitamins, drug or pharmaceuticals, other therapeutic molecules, artificial chemicals, polymers, tracers such as GFP ("green fluorescent protein") or luciferase, etc.
  • the cells may be used for drug discovery and/or drug developmental purposes.
  • the cells may be exposed to an agent suspected of interacting with the cells.
  • agents include a carcinogenic or mutagenic compound, a synthetic compound, a hormone or hormone analog, a vitamin, a tracer, a drug or a pharmaceutical, a virus, a prion, a bacteria, etc.
  • the invention may be used in automating cell culture to enable high-throughput processing of monoclonal antibodies and/or other compounds of interest.
  • the invention may be used to screen cells, cell types, cell growth conditions, or the like, for example, to determine self viability, self production rates, etc.
  • the invention may be used in high through put screening techniques.
  • the invention may be used to assess the effect of one or more selected compounds on cell growth, normal or abnormal biological function of a cell or cell type, expression of a protein or other agent produced by the cell, or the like.
  • the invention may also be used to investigate the effects of various environmental factors on cell growth, cell biological function, production of a cell product, etc.
  • a reactor and/or a reaction site within a chip may be constructed and arranged to prevent, facilitate, and/or determine a chemical or a biochemical reaction with the living cells within the reaction site (for example, to determine the effect, if any, of an agent such as a drug, a hormone, a vitamin, an antibiotic, an enzyme, an antibody, a protein, a carbohydrate, etc. on a living cell).
  • an agent such as a drug, a hormone, a vitamin, an antibiotic, an enzyme, an antibody, a protein, a carbohydrate, etc. on a living cell.
  • agents suspected of being able to interact with a cell may be added to a reactor and/or a reaction site containing the cell, and the response of the cell to the agent(s) may be determined, using the systems and methods of the invention.
  • the cells may be sensitive to light.
  • the cell may be a plant cell that responds to a light stimulus or is photosynthetic.
  • the light may be used to grow cells, such as mammalian cells sensitive to light, or plant cells.
  • the cell may be a bacterium that is attracted to or is repelled by light.
  • the cell may be an animal cell having a light receptor or other light-signaling response, for example, a rod cell or a cone cell.
  • the cell may be a genetically engineered cell having a light receptor or another light- sensitive molecule, for example, one that decomposes or forms reactive entities upon exposure to light, or stimulates a biological process to occur.
  • the cell may be insensitive to light; light applied to the chip may be used for analysis of the cells, for example, detection, imaging, counting, morphological analysis, or spectroscopic analysis. In still other cases, the light may be used to kill the cells, for example, directly, or by inducing an apoptotic reaction.
  • the chip may be constructed and arranged such that cells within the chip can be maintained in a metabolically active state, for example, such that the cells are able to grow and divide.
  • the chip may be constructed such that one or more additional surfaces can be added to the reaction site, for example, as in a series of plates, or the chip may be constructed such that the cells are able to divide while remaining attached to a substrate.
  • the chip may be constructed such that cells may be harvested or removed from the chip, for example, through an outlet of the chip, or by removal of a surface from the reaction site, optionally without substantially disturbing other cells present within the chip.
  • the chip may be able to maintain the cells in a metabolically active state for any suitable length of time, for example, 1 day, 1 week, 30 days, 60 days, 90 days, 1 year, or indefinitely in some cases.
  • the present invention provides any of the above-mentioned chips packaged in kits, optionally including instructions for use of the chips. That is, the kit can include a description of use of the chip, for example, for use with a microplate, or an apparatus adapted to handle microplates.
  • instructions can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the invention. Instructions also can include any oral or electronic instructions provided in any manner such that a user of the chip will clearly recognize that the instructions are to be associated with the chip. Additionally, the kit may include other components depending on the specific application, for example, containers, adapters, syringes, needles, replacement parts, etc. As used herein, "promoted” includes all methods of doing business including methods of education, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with the invention.
  • a 0.2 micrometer pore size membrane (Osmonics, Minnetonka, MN) was also attached to the top side of the first layer by means of the pressure-sensitive silicone adhesive.
  • a second chip layer (including chamber top) having associated fluidic channels, ports, chambers, other reaction sites, etc. therein was cast in a mold using PDMS. This second layer was fashioned to be alignable with the first chip layer. The second layer was aligned with the chambers in the first chip layer and attached by means of the pressure- sensitive silicone adhesive, forming a completed chip.
  • the LED light was placed in optical communication with a 600 micron diameter optical fiber (P600-2, Ocean Optics) by a lens (74-UV, Ocean Optics), then directed to the chip.
  • the emitted light was collected by a 25.4 mm f-1 lens (Thorlabs, Newton, NJ) and optically communicated to another 600 micron fiber which, in turn, was in optical communication with a computer-controlled spectrophotometer (USB- 2000F, Ocean Optics).
  • the emission intensity reported in both cases was measured at 560 nm. Sample results from these experiments are shown in Fig. 13 where the ratio of intensities was plotted versus the solution pH.
  • Example 3 This example illustrates the preparation of a chip in accordance with an embodiment of the invention.
  • a chip layer having associated fluidic channels, ports, chambers, etc.
  • PDMS polydimethylsiloxane
  • Sylgard 184 Dow Corning, Midland, MI
  • the PDMS layer was cured at 90 °C for 20 minutes.
  • the PDMS layer was attached to a bottom plate by means of a pressure sensitive silicone adhesive layer (Dielectric Polymers, Holyoke, MA).
  • the bottom plate was made of acrylic or polycarbonate and was machined from sheet stock or injection molded.
  • the layers were bonded by compressing the layers in a hydraulic press (Carver, Wabash, IN), forming the completed chip.
  • Example 4 This example illustrates the control of the pH within a reaction site of a chip, according to another embodiment of the invention. Multiple chips similar to the one described in Example 3 were prepared using PDMS, having a geometry similar to the embodiment illustrated in Fig. 5. Each chip included three predetermined reaction site defined by a chamber within the chip. The chamber depth (distance from the surface of the chip) was 500 microns. Three chambers of one chip were each filled with a solution of 50 micromolar chlorophenol red dye (Sigma-Aldrich, Milwaukee, WI).
  • Cholorphenol red is known to undergo a color change from yellow to purple as the solution gets more basic (i.e., as the pH of the solution increases). This color change can be monitored by measuring the absorbance of the solution at a wavelength of 574 nm.
  • the pH of the reaction sites within the chips was determined optically.
  • the light source tungsten halogen; LH-1 ; Ocean Optics
  • LH-1 Ocean Optics
  • P100-2 Ocean Optics
  • 74-UV Ocean Optics
  • the transmitted light 315 now at least partially attenuated by the turbidity of sample 325 within reaction site 320, was collected with another collimating lens/fiber assembly (not shown) which transmitted it to a computer-controlled spectrophotometer 330 (USB-2000; Ocean Optics)
  • a small amount (about 20 microliters) of ammonia solution Sigma-Aldrich, Milwaukee, WI was placed on top of the chip, generally proximate reaction site 320.
  • the light absorbance at 574 nm of the reaction site was monitored over the course of two hours. Three concentrations of ammonia were used, as shown in Fig.
  • Example 5 This example illustrates an embodiment of the invention as used to adjust the pH within a predetermined reaction site while avoiding any liquid contact therein.
  • a microreactor was constructed out of polydimethylsiloxane (PDMS).
  • This particular device had a footprint of 127.77 mm by 85.48 mm, generally the same size as a 96 microwell plate.
  • This particular device was assembled by combining the various layers of materials, membranes, and barrier/interface layers to form a stacked composite structure having a 200 microliter chamber, as described in Example 1.
  • the pH of the chamber was monitored using a pH-altering agent, chlorophenol red, within the cell culture chamber.
  • the emission spectra of the chamber was recorded every 10 seconds for about 90 minutes.
  • a drop of ammonia (20 microliters, 4.0 M) was placed on a thin layer of PDMS covering the chamber.
  • the ammonia gas was allowed to diffuse as a gas across the PDMS to enter the chamber, thus illustrating gaseous non-liquid transport of an agent to the predetermined reaction site.
  • a plot of the optical density of the chamber with respect to time of this experiment is shown in Fig. 16, for wavelengths of 480 nm, 574 nm, and 700 nm.
  • a wavelength of 480 nm is indicative of the agent chlorophenol red, with higher optical density values indicating more alkaline conditions.
  • Example 6 This example illustrates the ratiometric determination of the pH within a reaction site of a chip according to an embodiment of the invention.
  • a chip was prepared using methods similar to those in Example 1.
  • a pH sensor for the chip was constructed by immobilizing a fluorescent, pH-sensitive dye in a gel. The gel was prepared as follows. A stock solution of 15 ml tetraethoxysilane (TEOS) and 20 ml ethanol (both from Sigma-Aldrich, Milwaukee, WI) was prepared and kept sealed until use.
  • TEOS tetraethoxysilane
  • ml ethanol both from Sigma-Aldrich, Milwaukee, WI
  • the gel was placed in fluidic contact within the reaction site. Solutions having known pH values were added into the reaction site. The fluorescence of the gel in contract with the reaction site, indicative of the pH within the reaction site, was monitored using a ratiometric fluorescent procedure. In this procedure, the fluorescent response of the pH- sensitive dye at two different wavelengths (510 and 480 nm) in response to the pH was determined using a commercially-available UV-visible spectrometer. By using solutions having different known pH's within the reaction site, the ratio of the response at 510 nm and the response of 480 nm was shown to be proportional to the pH of the solution, thus demonstrating ratiometric determination of the pH within a reaction site.
  • Example 7 control of the pH within a reaction site of a chip was demonstrated according to one embodiment of the invention.
  • a chip similar to the one described in Example 1 was attached to a control system.
  • a computer was used to record the pH values determined using the ratiometric procedure described above, and, using a control algorithm, the computer was able to determine whether control action to adjust the pH within the reaction site was necessary.
  • a fluidic connection was established between the chip and an external pumping system by opening a valve that connected the chip to the external pumping system.
  • Example 8 In this example, control of the pH within the reaction site was demonstrated in accordance with another embodiment of the invention. A chip similar to the one described in Example 1 was attached to a control system. A fluorescent, pH-sensitive dye was immobilized in a gel in accordance with Example 6, and a computer was connected to the chip, similar to the method described in Example 7.
  • an acid e.g., ammonium hydroxide
  • a base e.g., acetic acid
  • Example 9 This example illustrates various chips of the invention formed from multiple layers of dissimilar materials. A variety of adhesives were used to fix the interface layers to the rigid cell culture or sealing layers depending on the materials involved. One adhesive used for bonding PDMS to polycarbonate was a two-part urethane epoxy mixed with un-cured PDMS.
  • the adhesive process used to bond rigid polycarbonate layers to each other was either sonic welding or a heated press.
  • the reaction site was designed to be about 200 microns thick and had a volume of roughly 20 microliters.
  • a chip 280 having reaction site 240 was fabricated.
  • a polycarbonate layer 244 was attached to PDMS layer 242.
  • a gap within PDMS layer 242 defined reaction site 240 when the chip was assembled, as shown in Fig. 17A.
  • PDMS layer 242 was attached to polycarbonate layer 244 using the above-described two- part urethane epoxy mixed with un-cured PDMS.
  • a similar chip is illustrated in Fig. 17B.
  • reaction site 240 was defined by layer 245, which was a thin, rigid layer of polycarbonate. Between layers 242 and 245 was a gas-permeable film 246 (BIOFOIL® made by VivaScience). Layers 244, 245, 246 and 242 of chip 80 were joined using the above-described adhesive processes.
  • Example 10 This example illustrates various chips of the invention formed from multiple layers of dissimilar materials. A variety of adhesives were used to fix the interface layers to the rigid cell culture or sealing layers depending on the materials involved. One adhesive used for bonding PDMS to polycarbonate was a two-part urethane epoxy mixed with un-cured PDMS. The adhesive process used to bond rigid polycarbonate layers to each other was either sonic welding or a heated press.
  • the reaction site was designed to be about 200 microns thick and had a volume of roughly 20 microliters.
  • the fabrication of the chips illustrated in Figs. 18A and 18B were similar to those described in Example 9, including the adhesion methods.
  • the reservoir layer 248 was fashioned from polycarbonate and was positioned between gas-permeable film 246 (BIOFOIL®) and polycarbonate layer 244.
  • Reservoir layer 248 has a gap (i.e., a hole or a partially hollowed out space) that defines reaction site 50, which was a reservoir in this example.
  • the reaction site 240 was defined by a gap interface layer 242.
  • polycarbonate layer 248 was used to define reaction site 250.
  • Example 11 This example illustrates the fabrication of an embodiment of the invention without using adhesive materials.
  • the reaction site was designed to be about 200 microns thick and had a volume of roughly 20 microliters.
  • the layout of this example, illustrated in Fig. 19, is similar to that illustrated in Fig. 18B of Example 10, except that an additional compression layer 252 was used to mechanically hold the other layers in place. No adhesive materials were used in this example. Instead, screws 253 extending from polycarbonate layer 252 through the other layers of the chip were secured to layer 244 to fabricate chip 280.
  • Example 12 In this example, an embodiment of the present invention is illustrated as used in a chip sealed by a membrane having a permeability to oxygen high enough to allow culture of living cells.
  • the amount of oxygen required in this example is a function of the number of cells present and the oxygen requirements for the cells' metabolism. This is illustrated in the equations 2-4 below.
  • 26C is a plan view of upper silicone sheet 615 showing the upper reservoir portion 630 of the chamber along with its associated channels 650, both of which end at an upper portion port 665 that provides access through the upper silicone sheet 615 to the upper portion channels.
  • the wall 695 of upper reservoir portion 630 lacks abrupt transitions and corners in this example. This facilitates complete mixing and dispersion of material introduced into the upper reservoir portion 630.
  • passages 670 in the upper silicone sheets 615 are aligned with the lower portion ports the lower silicone sheet, allowing access to the lower portion channels through the upper silicone sheet.
  • Fig. 26D is a cross-section of upper silicone sheet 615 along B-B' in Fig. 26C.
  • passage 670 provides an opening through the upper silicone sheet 615. This opening is aligned with one of the lower portion ports when the upper silicone sheet and the lower silicone sheet are joined to form a complete chamber.
  • Upper portion port 665 is molded into upper silicone sheet 615 and provides access to the upper portion channels.
  • Fig. 26E is a perspective view of the upper reservoir portion of the chamber along with associated channels.
  • the upper reservoir portion 620 and associated channels 650 are molded into an upper silicone sheet 615.
  • the base 628 of the upper reservoir portion 620 is planar in this example. In this embodiment, the wall of the upper reservoir portion 618 is perpendicular to the base 628 of the upper portion.
  • the base 628 can curve gently upward to meet the wall 618 in order to facilitate mixing and dispersion of material in the upper portion.
  • the upper portion ports 665 located at the ends of the channels 650 allow the introduction of material into the channels.
  • the upper silicone sheet 615 includes two passages 670 that permit access to the lower portion ports when the upper silicone sheet and lower silicone sheet are joined to form a complete chamber.
  • Example 16 In this example, a device was fabricated using three layers. In this embodiment, the bottom layer is a solid slab.
  • the middle layer has a membrane molded into it that separates an upper reservoir portion from a lower cell culture portion, both of which are molded into the middle layer.
  • the upper reservoir portion and the upper portion microchannels are molded into the upper surface of the middle layer and the lower cell culture portion and the lower portion microchannels are molded into the lower surface of the middle layer. Openings passing through the middle layer permit access to the lower portion microchannels.
  • the top layer has four openings passing through it to serve as ports for the four microchannels. The top layer serves to seal the upper reservoir portion and its associated microchannels, while allowing access to all ports.
  • the bottom layer serves to seal the lower cell culture portion and its associated microchannels.
  • Example 17 In this prophetic example, a fluidic device of the invention is used to examine the effect of chemical agent A on fermentation of a bacterium. Twelve fluidics, each bearing a single chamber having a cell culture portion and reservoir portion are aligned in parallel.
  • the fluidic heat exchangers, addition of chemicals, and airflow rate, the fluidic can control temperature, pH, and dissolved oxygen concentration, respectively.
  • the average cell growth rate and average final cell concentration are computed for the six fluidics with chemical agent A and for the six fluidics without. By comparing these averages, chemical agent A can be said to enhance cell growth, have no significant effect, or hinder cell growth.
  • Example 18 In this prophetic example, a fluidic device of the invention is used to provide an environment in which to grow cells or tissue that closely resembles that found in humans or mammals. With respect to drug screening, the fluidic device can monitor responses of cells to a drug candidate.
  • These responses can includes increase or decrease in cell growth rate, cell metabolic changes, cell physiological changes, or changes in uptake or release of biological molecules.
  • different cell lines can be tested along with screening multiple drug candidates or various drug combinations.
  • the screening process can be automated. Twenty fluidics each containing a single chamber divided into a cell culture portion and a reservoir portion are sterilized. Sterile animal cell culture media is pumped into the cell culture portion of each of the chambers through the fluid delivery system. Each fluidic is then inoculated with mammalian cells that are genetically engineered to produce a therapeutic protein. The cells are allowed to grow to production stage all the while their growth and environment is monitored by sensors in the fluidic.
  • the fluidic through control of temperature, pH, and air flow rate, is able to maintain an optimal environment for growth of the cells.
  • the fluidics are separated into four groups of five. Three of the four groups receive various cocktails of inducers for the therapeutic protein while the fourth group serves as a control and thus receives no inducers.
  • the inducers and control sample are introduced into the reservoir portions of chambers through the fluid delivery system.
  • a marker chemical that binds with the therapeutic protein is introduced along with inducers. When the culture is irradiated with light at a wavelength that excites the bound marker chemical, the chemical then fluoresces, and the intensity of fluorescence is proportional to the concentration of therapeutic protein in the culture.
  • a fluidic device is used in an adsorption assay, for example, to model the adsorption of drugs and others agents in the gut.
  • the fluidic device can be provided with a chamber divided into two portions by a polycarbonate membrane having a 3.0, 2.0, or 1.0 micron pore size.
  • Caco-2 colon carcinoma cells
  • Caco-2 colon carcinoma cells
  • a drug or other agent is introduced into the portion of the chamber containing the cells. Passage of the drug or other agents through the cell layer into a second portion of the chamber is monitored.
  • a similar arrangement can be used for a cell migration assay. In such an assay, a membrane with a 5.0-12.0 micron pore size is used.
  • Example 20 Useful quantities of a large number of target proteins are produced as follows in this prophetic example. A micro fabricated bioreactor containing one or more cell growth chambers is sterilized and sterile growth media is pumped into each growth chamber through a fluid delivery system.
  • Example 25 In this prophetic example, useful quantities of a large number of target proteins can also be produced as follows. A microfabricated bioreactor containing one or more cell growth chambers is sterilized and sterile growth media is pumped into each growth chamber through a fluid delivery system. Each chamber is implanted with a tissue sample displaying a phenotype of interest. The tissue samples are incubated so as to produce useful quantities of the proteins of interest which can then be harvested and analyzed or passed through microchannels to be analyzed using the microreactor system described above.
  • Example 26 This example illustrates the construction of certain embodiments of the invention.
  • FIG. 27B depicts a cross-sectional view of a gas headspace portion associated with a cell growth chamber. This allows a continuous supply of air to pass through the microfabricated bioreactor.
  • a cylindrical chamber 750 that is about 7 mm in diameter and about 0.05 mm in height is etched in glass along with a gas inlet microchannel 760 and gas outlet microchannel 770, both of which are about 0.05 mm wide by about 0.05 mm deep.
  • the cylindrical chamber of the gas headspace portion is matched over the cell growth chamber. The two halves can then be bonded together so as to form a tight seal.
  • a membrane can be placed in so as to separate the gas headspace from the liquid filled bioreactor.
  • the membrane retards passage of water and allows for the passage of air.
  • the various microchannels are connected to supply units or waste units. These units as well as mixing devices, control valves, pumps, sensors, and monitoring devices can be integrated into the substrate in which the cell growth chamber is built or can be externally provided. The entire assembly can be placed above or below a heat exchanger (or sandwiched between two heat exchangers) to control the temperature of the unit.
  • the silicone into which the portions of the chamber and microchannels are molded, in this particular example, is sufficiently gas permeable to provide adequate gas exchange for the growth of aerobic cells in the chamber of the device.

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Abstract

L'invention concerne, dans son ensemble, des puces de réaction chimique, biologique et/ou biochimique et d'autres systèmes de réaction tels que des systèmes de microréacteur, ainsi que des systèmes et des procédés pour réaliser et utiliser de tels dispositifs. Dans un mode de réalisation, une puce ou un autre système de réaction sont conçus pour favoriser la croissance cellulaire à l'intérieur de ceux-ci. Dans certains modes de réalisation, les puces ou les autres systèmes de réaction comportent un ou plusieurs sites de réaction, lesquels peuvent être très petits, par exemple, avoir un volume inférieur à environ 1 ml. Dans un autre mode de réalisation, la puce peut détecter, mesurer et/ou contrôler un facteur environnemental, tel que température, pression, concentration de CO2, concentration de O2, humidité relative, pH, etc., associé à un ou plusieurs sites de réaction, au moyen d'un ou de plusieurs capteurs, actionneurs processeurs et/ou systèmes de contrôle. La présente invention porte également sur des matériaux et des systèmes dotés de contrôle d'humidité et/ou de gaz pour, par exemple, être utilisés avec une puce, ces matériaux pouvant présenter une grande perméabilité à l'oxygène et/ou une faible perméabilité à la vapeur d'eau. Cette invention porte aussi, dans son ensemble, à des composants interagissant avec la lumière et pouvant être utilisés dans des puces et d'autres systèmes de réaction. Ces composants peuvent comprendre des guides d'ondes, des fibres optiques, des sources de lumière, des photodétecteurs, des éléments optiques, etc.
PCT/US2003/025943 2001-04-10 2003-08-19 Architecture de microréacteur et procédés associés WO2004069983A2 (fr)

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CA002496017A CA2496017A1 (fr) 2002-08-19 2003-08-19 Architecture de microreacteur et procedes associes
AU2003303303A AU2003303303A1 (en) 2002-08-19 2003-08-19 Microreactor architecture and methods
EP03815293A EP1567637A2 (fr) 2002-08-19 2003-08-19 Architecture de micror acteur et procedes associes
JP2005515706A JP2006521786A (ja) 2002-08-19 2003-08-19 マイクロリアクター構造および方法
US10/664,067 US20050032204A1 (en) 2001-04-10 2003-09-16 Microreactor architecture and methods

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US22356202A 2002-08-19 2002-08-19
US10/223,562 2002-08-19
US40927302P 2002-09-09 2002-09-09
US60/409,273 2002-09-09
US45704803A 2003-06-05 2003-06-05
US45613303A 2003-06-05 2003-06-05
US45693403A 2003-06-05 2003-06-05
US10/457,015 US20040058407A1 (en) 2001-04-10 2003-06-05 Reactor systems having a light-interacting component
US10/457,049 US20040058437A1 (en) 2001-04-10 2003-06-05 Materials and reactor systems having humidity and gas control
US10/456,934 2003-06-05
US10/456,133 2003-06-05
US10/457,015 2003-06-05
US10/457,049 2003-06-05
US10/457,048 2003-06-05

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