JP2005536233A - Determination and / or control of reactor environmental conditions - Google Patents

Abstract

The present invention relates generally to chemical, biological, and / or biochemical reactor chips and other reaction systems, such as microreactor systems, and systems and methods for constructing and using such devices. In one aspect, a chip or other reaction system can be constructed to promote cell growth therein. In certain embodiments, the chip or other reaction system of the present invention includes one or more reaction sites. This reaction site is very small, for example, may have a volume of less than about 1 ml. In one aspect of the invention, environmental factors associated with one or more reaction sites, such as temperature, pressure, CO ₂ concentration, O ₂ concentration, relative humidity, pH, etc., are measured using one or more sensors, actuators. Can be detected, measured and / or controlled by the chip by using a processor and / or control system.

Description

Background (Field of the Invention)
The present invention generally relates to chemical, biological and / or biochemical reactor chips and other reaction systems such as microreactor systems.

(Description of related fields)
A wide variety of reaction systems are known for the production of products of chemical and / or biochemical reactions. Chemical plants, biochemical fermenters, production plants and other system hosts involved in the catalyst are well known. Biochemical treatment can involve the use of live microorganisms (eg, cells) to produce the material of interest.

  Cells are cultured for various reasons. Interestingly, cells are cultured for proteins or other useful substances that they produce. Many cells require specific conditions such as a controlled environment. The presence of other factors such as nutrients, metabolic gases such as oxygen and / or carbon dioxide, humidity, and temperature can affect cell growth. Cells take time to grow, and favorable conditions must be maintained during this time. In some cases, for example, successful cell culture can be performed in about 24 hours using specific bacterial cells. In other cases, when using certain mammalian cells, a successful culture can take about 30 days or more.

  Typically, cell culture is performed in a medium that is suitable for cell growth and contains the necessary nutrients. Cells are generally cultured in a place such as an incubator that can control environmental conditions. Incubators traditionally range from the size of a small incubator (eg, about 1 cubic foot) for a few cultures to the entire room (s) in which the desired environmental conditions can be carefully maintained.

  As described in International Patent Application PCT / US01 / 07679, published September 20, 2001, as WO 01/68257, entitled “Microreactors”, which is now incorporated by reference herein. The cells are also cultured on a very small scale (ie, on the order of 2 to 3 milliliters or less) so that, among other things, many cultures can be performed in parallel.

(Summary of the Invention)
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 the present invention relates in some cases to correlated products, alternative solutions to a particular problem, and / or a plurality of different applications of one or more systems and / or objects.

In one aspect, the invention is an apparatus. The apparatus, in one set of embodiments, comprises a chip with a predetermined reaction site having a volume of less than about 1 ml. In one embodiment, the device also controls environmental factors associated with the chip in response to a signal of a condition indicator associated with the chip, thereby supporting viable cells within the predetermined reaction site. Active control system. The apparatus, in another embodiment, includes a control system that can control environmental factors associated with a given reaction site, such as relative humidity, pH, dissolved O ₂ concentration, dissolved CO ₂ concentration, and media components. At least one of the concentrations.

  According to another embodiment, the apparatus causes the change in the first environmental factor associated with the predetermined reaction site to be within one second of the change in the second environmental factor associated with the predetermined reaction site and A control system may be provided that can occur in response thereto. In yet another embodiment, the device may comprise an active control system that can control the environment within a given reaction site and thereby support viable cells for at least one day. In yet another embodiment, the device is substantially transparent to incident electromagnetic radiation in the infrared to ultraviolet range, having a pore size of less than 2.0 microns in liquid communication with a given reaction site. With a membrane.

  In another embodiment, the apparatus also comprises a component that separates the predetermined reaction site from a source of a non-neutral pH composition. In yet another embodiment, the apparatus may comprise a precursor that can react to form a gaseous factor that can substantially change the pH of the substance within a given reaction site, It is arranged to allow the gaseous non-liquid permeation of this factor to a given reaction site. In yet another embodiment, the apparatus comprises a pH modifier dispersion unit integrally connected to the chip in liquid communication with a predetermined reaction site. According to another embodiment, the present invention comprises a gas supply source integrally connected to the chip. In another embodiment, the present invention comprises a laser waveguide in optical communication with a surface that defines a predetermined reaction site.

  In another embodiment, the apparatus comprises a sensor integrally connected to the chip, which can determine environmental factors associated with a given reaction site. This environmental factor is pH, dissolved gas concentration, molar concentration, osmolality, glucose concentration, glutamine concentration, pyruvate concentration, apatite concentration, color, turbidity, viscosity, amino acid concentration, vitamin concentration, hormone concentration, serum concentration , At least one of ion concentration, shear rate, and degree of agitation. In some cases, the device may also include an actuator integrally connected to the chip, which may change environmental factors.

  In another embodiment, the apparatus comprises a first sensor integrally connected to the chip and a second sensor integrally connected to the chip, the first sensor comprising a temperature and At least one of the pressures can be determined and a second sensor can determine a second environmental factor. The second environmental factor is, in certain cases, pH, dissolved gas concentration, molar concentration, osmolality, glucose concentration, glutamine concentration, pyruvate concentration, apatite concentration, color, turbidity, viscosity, amino acid concentration, vitamin Concentration, hormone concentration, serum concentration, ion concentration, shear rate, degree of agitation. In some cases, the apparatus may also include an actuator integrally connected to the chip that can change at least one of temperature, pressure, and environmental factors.

The apparatus may comprise a sensor capable of determining an environmental factor associated with at least one of the predetermined reaction sites, according to another embodiment of the invention. The environmental factor may be at least one of CO ₂ concentration, glucose concentration, glutamine concentration, pyruvate concentration, apatite concentration, serum concentration, vitamin concentration, amino acid concentration, and hormone concentration.

  In another set of embodiments, the apparatus comprises a chip with a predetermined reaction site having an inlet, an outlet, and a volume of less than about 1 ml. This predetermined reaction site is constructed and arranged to maintain at least one of the viable cells at the predetermined reaction site. In certain embodiments, the chip is constructed and arranged to stably connect to other similar chips in a predetermined aligned relationship.

  In one set of embodiments, the apparatus comprises a chip with a predetermined reaction site having an inlet, an outlet, and a volume of less than about 1 ml, such that the chip can be stably connected to the microplate. Built and arranged. The apparatus, according to another set of embodiments, comprises a chip with a predetermined reaction site having an inlet, an outlet, and a volume of less than about 1 ml, the chip constructed to handle the wells of a microplate Constructed and arranged to be in fluid communication with the device to be arranged and arranged. In yet another set of embodiments, the apparatus comprises a chip comprising a predetermined reaction site having an inlet, an outlet, and a volume of less than about 1 ml, each predetermined reaction site being at least one of the microplates. It fills one well. The apparatus, in yet another embodiment, comprises a substantially liquid-tight tip comprising a predetermined reaction site having a volume of less than about 1 ml, wherein the predetermined reaction site is at least one at the predetermined reaction site. Constructed and arranged to maintain viable cells.

  The apparatus, in one set of embodiments, is by a chip produced by a process that includes fixing two components to produce a portion of the chip that defines a predetermined reaction site having a volume of less than about 1 ml. At least in part, wherein the predetermined reaction site is constructed and arranged to maintain at least one viable cell at the predetermined reaction site. The apparatus, in another set of embodiments, comprises a chip comprising a predetermined reaction site having a volume of less than about 1 ml, wherein the predetermined reaction site maintains at least one viable cell at the predetermined reaction site. Constructed and arranged in this manner, this given reaction site has a non-zero evaporation rate of less than about 100 μl per day.

  According to another set of embodiments, the apparatus is a predetermined reaction site having a volume of less than about 1 ml that is enhanced or monitored by electromagnetic radiation within a predetermined wavelength range. Required to promote or monitor the reaction, having a predetermined reaction site constructed and arranged to perform a biological reaction, and a pore size of less than 2.0 microns in fluid communication with the predetermined reaction site And a film that is transparent to electromagnetic radiation within a predetermined wavelength range to the extent.

  According to another set of embodiments, the device is at least partially defined by a chip comprising a first predetermined reaction site and a second predetermined reaction site having a volume of less than about 1 ml. The chip defines a passage that fluidly connects the first predetermined reaction site and the second predetermined reaction site, the passage traversing the membrane.

  The apparatus, in one set of embodiments, includes a reaction site having a first portion and a second portion separated by a membrane, and at least a first and second fluid in fluid communication with the second portion of the reaction site. With channels.

  The present invention is a method in another aspect. This method, in one set of embodiments, includes the task of infiltrating the pH modifier into a predetermined reaction site having a volume of less than about 1 ml. According to another set of embodiments, the method provides a chip comprising a predetermined reaction site having a volume of less than about 1 ml, generating an acid or base proximal to the predetermined reaction site, wherein the acid or It includes at least the operation of bringing a base into contact with a substance in the predetermined reaction site to substantially change the pH of the substance. In another set of embodiments, the method includes providing a chip defining at least one compartment, the chip further comprising a predetermined reaction site having a volume of less than about 1 ml; Permeating components located between the predetermined reaction site and this compartment.

  According to one set of embodiments, the method includes generating gas in a chip with a predetermined reaction site having a volume of less than about 1 ml by directing a laser to at least a portion of the chip.

  According to one set of embodiments, the method is a method for generating a chip with a predetermined reaction site having a volume of less than 1 ml, and for assisting in generating a part of the chip defining the predetermined reaction site. Bonding the first component of the chip to the second component of the chip in the presence or absence of a mechanical adhesive.

  The method, in yet another set of embodiments, includes the act of providing a material having a surface that produces a plurality of reaction sites, the at least one reaction site having a volume of less than about 2 ml. Divided into at least a cell culture portion containing cells and a reservoir portion free of cells by a substantially cell impervious membrane, the reservoir portion being connected to at least first and second channels made on the surface of the substance Liquid connected. The method also includes 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.

  In another aspect, the invention relates to a method of making one or more embodiments described herein, eg, a chip such as a microreactor system or other reaction system. In yet another aspect, the invention relates to a method of using one or more embodiments described herein, eg, a chip or other reaction system, eg, a microreactor system. In yet another aspect, the invention relates to a method of promoting one or more embodiments described herein, eg, a chip or other reaction system, eg, a microreactor system.

  In another aspect, the invention relates to a method of making a chip and / or reactor system, eg, as described in any embodiment herein. In yet another aspect, the invention relates to a method of using a chip and / or reactor system as described, for example, in any embodiment herein, eg, the Examples. In yet another aspect, the present invention relates to a method for promoting a chip and / or reactor system, as described in any embodiment herein.

  Other advantages and novel features of the invention will become apparent from the following detailed description of various non-limiting embodiments of the invention, taken in conjunction with the accompanying drawings. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.

(Detailed explanation)
The following application is incorporated herein by reference: US Provisional Patent Application No. 60 / 282,741 filed Apr. 10, 2001, entitled “Microfermentor Device and Cell Based Screening Method” by Zarur et al. No .; US Patent Application No. 10 / 119,917 filed April 10, 2002, entitled “Microfermentor Device and Cell Based Screening Method” by Zarur et al .; International Patent Application No. PCT / US02 / 11422, filed 10 April 2002, entitled “Method”; Rodgers et al. US Provisional Patent Application No. 60 / 386,323, filed 5 June 2002, entitled “Materials and Reactors having Humidity and Gas Control”; Miller et al., “Reactor Having Lighting-Interacting Component”. US Provisional Patent Application No. 60 / 386,322 filed June 5, 2002; entitled “Fluidic Device and Cell-Based Screening Method” filed August 19, 2002 by Schreyer et al. No. 10 / 223,562, entitled “Protein Production and Screening Methods” by Zarur et al. US Provisional Patent Application No. 60 / 409,273, filed Sep. 24, 2002; Miller et al., US Patent Application No. 60 / 409,273, filed Jun. 5, 2003, entitled “Reactor Systems Responsive to Internal Conditions”. No. 10 / 457,048; US Provisional Patent Application No. 10 / 456,934, filed June 5, 2003, entitled “Systems and Methods for Control of Environment Environments” by Miller et al., By Rodgers et al., US patent application Ser. No. 10 / 456,133, filed Jun. 5, 2003, entitled “Microreactor Systems and Methods”; Rodgers et al., “Materia US Patent Application No. 10 / 457,049, filed June 5, 2003, entitled “S and Reactor Systems having Humidity and Gas Control”; Rodgers et al., “Materials and Reactor Systems Hastingham Hast. US Patent Application No. 10/457, filed June 5, 2003, entitled “Reactor Systems Having a Light-Interacting Component” by Miller et al. , 015; Miller et al., “Reactor Systems Having a Light-Interacting Co”. International Patent Application filed June 5, 2003, entitled “ponent”; US Patent Application No. 10/10, filed June 5, 2003, entitled “System and Methods for Process Automation” by Rodgers et al. US patent application Ser. No. 10 / 456,929, filed Jun. 5, 2003, entitled “Apparatus and Methods for Manufacturing Substrates” by Zarur et al.

The present invention relates generally to chemical, biological, and / or biochemical reactor chips and other reaction systems, such as microreactor systems, and systems and methods for constructing and using such devices. In one aspect, a chip or other reaction system may be constructed to promote cell growth therein. In certain embodiments, the chip or other reaction system of the present invention includes one or more reaction sites. This reaction site is very small, for example, may have a volume of less than about 1 ml. In one aspect of the invention, environmental factors associated with one or more reaction sites, such as temperature, pressure, CO ₂ concentration, O ₂ concentration, relative humidity, pH, etc., are measured using one or more sensors, actuators. , Can be detected, measured and / or controlled by the chip by using a processor and / or control system. In another aspect, the invention relates to materials and systems with humidity and / or gas control, for example, for use with a chip. Such materials may have oxygen permeability and / or low water vapor permeability. The present invention, in yet 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 to FIG. 1, a portion of a chip according to one embodiment is schematically illustrated. The part shown is a layer 2 with a series of voids therein, which is located between two layers (top and bottom layers (not shown) relative to the plane of FIG. 1). If so, a series of enclosed channels and reaction sites are defined. The overall configuration in which layer 2 can be assembled to form a chip will be more clearly understood from the following figures with respect to other figures.

  FIG. 1 corresponds to an embodiment comprising six reaction sites 4 (similar to reaction site 125 of FIG. 3A or reaction site 112 of FIG. 5A described below, for example). The reaction site 4 defines a series of normally arranged, elongated, rounded rectangular voids in a relatively thin, generally planar piece of material defining the layer 2. The reaction site 4 can be addressed by a series of channels including a channel 6 for delivering species to the reaction site 4 and a channel 8 for removal of the species from the reaction site. Of course, any combination of channels can be used to deliver species to and / or remove species from the reaction site. For example, channel 8 can be used to deliver species to the reaction site, while channel 6 can be used to remove species. As indicated by the lines in FIG. 1, it should be understood that the channels 6 and 8 define a void in the layer 2, which is encapsulated when covered above and / or below by other layers. Can be a channel. Each of channels 6 and 8 is addressed by port 9 in the embodiment illustrated in FIG. If port 9 is connected to an inlet channel, it may define an inlet port, and if it is liquid connected to an outlet channel, it may define an outlet port. In the illustrated embodiment, the port 9 is a void that is wider than the width of the channel 6 or 8. Those skilled in the art are aware of various techniques for evaluating and utilizing ports 9 to introduce species into channels and / or remove species from the channels addressed by those ports. As an example, port 9 may be a “self-sealing” port addressable by a needle (described in more detail below), with at least one side of port 9 having a layer of material ( (Not shown), when the needle is inserted and withdrawn through this material, it is usually impervious to species such as liquid introduced into or removed from the tip through this port. Form a seal.

  Also shown in FIG. 1 is a series of ports 15 that are not shown to be fluidly connected or connectable to any inlet channel, outlet channel, or reaction site of the chip. It is. Port 15 may be defined by voids in layer 2 and is used to allow a fluid connection between and within the various layers of the chip and / or the external environment to this chip. Also good. For example, if layer 2 forms part of a multi-layer chip that includes a plurality of reaction sites in various layers, another layer comprises layer 2 (an intermediate layer (single or A plurality of) may be provided on one side of) and optionally another layer may be provided on the opposite side of layer 2 that is also similarly separated by an intermediate layer and port. 15 may define a passage or route of liquid connection between the reaction sites and / or conduits of the tip layer opposite layer 2. The port 15 can also be connected to a channel that communicates with a chamber aligned with the chamber defining the reaction site 4 separated from the reaction site 4 by a membrane, eg, a semi-permeable membrane. In this method, the liquid may be independently flowed into, through and / or through the space on one side of the membrane, one or both defining the chamber and / or reaction site. It may flow independently through the space on the other side of the membrane.

  In FIG. 1, each reaction site 4 together with associated liquid communication (eg, channels 6 and 8, port 9 and port 15) defines a reactor 14 as indicated by the dotted line. In FIG. 1, layer 2 includes six such reactors, each reactor having substantially the same configuration. In other embodiments, the reactor may include more than one reaction site, channel, port, and the like. Further, the layers of the chip may have reactors that do not have substantially the same configuration.

  Also shown in FIG. 1 is a series of devices 16 that are used to secure layer 2 to other layers of the chip and / or other layers to which the chip is bonded as needed. And / or alignment of layer 2 with other systems can be ensured. Device 16 may define a screw, post, press fit (ie, conform to a corresponding protrusion of another layer or device), and the like. Those skilled in the art are aware of various suitable techniques for securing layers to other components and / or chips of the invention to other components or systems using devices such as these.

  Various provisions are presently provided to assist in understanding the present invention. Consistent with and incorporating these provisions is further disclosure, which includes a drawing description that details the invention. The components shown in the following figures can generally be used in combination with layer 2 of FIG. In FIG. 1, and in all other figures, the arrangement of channels addressing the sequence of reaction sites, the number of reaction sites, the reaction sites, ports, etc. is merely shown as examples that fall within the present invention.

  As used herein, the term “determining” generally refers to, for example, quantitatively or qualitatively measuring and / or analyzing a substance (eg, within a reaction site) or the presence or absence of this substance. Refers to detection. “Determining” also refers to the measurement and / or analysis of an interaction between two or more substances, eg, quantitatively or qualitatively or by detecting the presence or absence of this interaction. Examples of techniques suitable for use in the present invention include gravimetric analysis, calorimetry, pressure or temperature measurement, spectroscopy, eg infrared, absorption, fluorescence, UV / visible, FTIR (“Fourier Transform Infrared Spectroscopy (Fourier Transform Infrared Spectroscopy))), or Raman; gravimetric technique; ellipsometry; piezoelectric measurement; immunoassay; electrochemical measurement; optical measurement, eg optical density measurement; circular dichroism; Examples include, but are not limited to, quasi-electron scattering; ellipsometry; refractometry; or turbidimetry including nephelometry.

  A “chip” as used herein is an integral object that includes one or more reactors. “Integral article” means a monolithic material or an assembly of components integrally connected to each other. As used herein, the term “integrated connected” when referring to two or more objects has become unseparated from each other during the course of normal use, eg Means an object that cannot be manually separated; separation requires at least the use of tools and / or causes damage to at least one of the components, for example by breaking, peeling, etc. The components to be fixed together via adhesives, tools, etc.).

  The chip may be connected to or inserted into a large framework that defines the overall reaction system, eg, a high throughput system. The system is mainly by other chips, chassis, cartridges, cassettes and / or larger machines or sets of channels or conduits, reactant sources, cell types and / or nutrients, inlets, outlets, sensors , Actuators, and / or control devices. Typically, the chip may be a generally flat or planar object (ie, having one dimension that is relatively small compared to the other dimensions); however, in some cases, the chip is For example, the chip may have a cubic shape, a curved surface, a solid or block shape, and the like.

  As used herein, a “membrane” is a three-dimensional material having an arbitrary shape, so that one of the dimensions is substantially smaller than the other dimension. In some cases, the membrane may generally be flexible or non-rigid. For example, the membrane may be a rectangular or round object having a length and width greater than or equal to millimeters, centimeters, and a thickness of less than millimeters, in some cases less than 100 microns, less than 10 microns, or less than 1 micron. There may be. The membrane may define a reaction site and / or a portion of the reactor, or the membrane is used to divide the reaction site into two or more parts that may have substantially the same or different volumes or dimensions. Also good. Some membranes may be semipermeable membranes that are recognized by those skilled in the art to be membrane permeable for at least one species, but not readily permeable for at least one other species. . For example, a semi-permeable membrane may allow oxygen to pass therethrough but does not allow water vapor to pass therethrough or allows water vapor to pass therethrough, provided that at least one of Permeability can be as small as a single digit. Alternatively, the semi-permeable membrane may be selected so that water passes across it, but certain ions do not pass. For example, the membrane may be cation permeable and substantially anion impermeable, or anion permeable and cation substantially impermeable. (For example, a cation exchange membrane and an anion exchange membrane). In another example, the membrane may be substantially impermeable to molecules having a molecular weight greater than or equal to about 1 kilodalton, 10 kilodalton, or 100 kilodalton. In one embodiment, the membrane may be impermeable to cells, but may be selected to be permeable to a variety of selected substances; for example, the membrane may be a nutrient It may be permeable to proteins and other molecules produced by cells, waste, etc. In other cases, the membrane may be gas impermeable. Some films are transparent to certain light (eg, infrared, UV, or visible light; the wavelength of light with which the device utilizing the film interacts; visible light unless otherwise indicated). If the film is substantially transparent, the film absorbs no more than 50% of light, or in other embodiments, no more than 25% or 10% of light, as described in more detail herein. In some cases, the membrane may be both semi-permeable and substantially transparent. In other embodiments, a membrane may be used to divide a reaction site constructed and arranged to support a cell culture from a second portion, eg, a reservoir. For example, the reaction site may be divided into three parts, four parts, or five parts. For example, the reaction site may be divided into a first cell culture portion and a second intracellular culture portion adjacent to the first reservoir portion and the two additional reservoir portions, one of which is the first The cell culture portion is separated by a membrane and the other is separated from the second cell culture portion by a membrane. Of course, one skilled in the art can design other sequences having various numbers of cell culture portions, reservoir portions, etc., as described further below.

  As used herein, a “substantially transparent” material (eg, a film) allows electromagnetic radiation to penetrate the material without significant scattering, so that The intensity of electromagnetic radiation that passes through the substance allows it to interact with substances on other surfaces of this substance, such as chemical, biochemical or biological reactions, or with cells A substance that is sufficient to make it. In some cases, the material is substantially incident to electromagnetic radiation having a wavelength ranging between infrared and ultraviolet (including visible light), particularly between about 400-410 nm and about 1,000 nm. It is transparent. In some cases, the material may be transparent to electromagnetic radiation at a wavelength between about 400-410 nm and about 800 nm, and in certain embodiments, the material is between about 450 nm and 700 nm. It may be substantially transparent to radiation of a wavelength of. This substantially transparent material may be capable of transmitting electromagnetic radiation in some cases, so that most of the radiation incidence is unchanged with respect to the passage of this material, and in certain embodiments At least about 50% of this incident radiation, at least about 75% in other embodiments, at least about 80% in other embodiments, at least about 90% in yet other embodiments, and at least about 95 in yet other embodiments. %, In yet other embodiments at least about 97%, and in still other embodiments at least about 99% may pass through the material without modification. In certain cases, this material may be used, for example, to promote and / or monitor physical, chemical, biochemical and / or biological reactions that occur within the reaction site, as previously described. To the extent necessary, it is at least partially transparent to electromagnetic radiation within the above-mentioned wavelength range. In other embodiments, the material may be transparent to electromagnetic radiation within the wavelength range described above to the extent necessary to monitor, observe, stimulate and / or control cells within the reaction site.

  As used herein, a “reactor” is a reaction site, any chamber (including the reaction chamber and attached chamber), channel, port, inlet and / or outlet (ie, connected to or Derived from), a combination of components including sensors, actuators, processors, controllers, membranes, etc., which together, at the reaction site, biological, chemical or biochemical reaction, interaction , Operates to facilitate and / or monitor operations or experiments, and may be part of the chip. For example, a chip may include at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, or at least 1,000 or more reactors. Examples of reactors include chemical or biological reactors and cell culture devices and international patent applications published on September 20, 2001 as WO 01/68257, incorporated herein by reference. And the reactor described under the number PCT / US01 / 07679. The reactor may comprise one or more reaction sites or chambers. This reactor can be used for any chemical, biochemical and / or biological purposes such as cell growth, drug production, chemical synthesis, hazardous chemical production, drug screening, substance screening, drug development, chemical weaponry It can be used for chemical therapy and the like. For example, the reactor can be used to facilitate very small scale culture of cells or tissues. In one set of embodiments, the reactor of the present invention comprises a matrix or substrate sized from a few millimeters to a few centimeters, which comprises channels sized for example tens or hundreds of micrometers. The reagent of interest may be allowed to flow through these channels, for example, to a reaction site, or between various reaction sites, and the reagent may be mixed or reacted in some manner. Good. The product of such a reaction may be recovered, separated and processed in this system in certain cases.

  As used herein, a “reaction site” is constructed and arranged to produce a physical, chemical, biochemical and / or biological reaction during use of the reactor. Defined as a site within the reactor. More than one reaction site may be present in the reactor or 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 reactive sites, at least 7 reactive sites, at least 10 reactive sites, at least 15 reactive sites, at least 20 reactive sites, at least 30 reactive sites, at least 40 reactive sites, at least 50 reactive sites There may be sites, at least 100 reaction sites, at least 500 reaction sites, or at least 1,000 or more reaction sites in the reactor or chip. This reaction site may be defined as the region in which the reaction occurs; for example, the reactor is constructed and arranged to generate a reaction within a channel, in one or more chambers, at the intersection of two or more channels, etc. Also good. The reaction may be, for example, a mixing or separation process, a reaction between two or more chemicals, a photo-initiated or light-inhibited reaction, a biological process, and the like. In certain embodiments, the reaction may involve an interaction with light that does not cause a chemical change, for example, a photon of light is absorbed by a substance associated with the reaction site and converted to thermal energy or It can be re-emitted as fluorescence. In certain embodiments, the reaction site may include one or more cells and / or tissues. Thus, in some cases, the reaction site may be defined as a region around the location where cells are to be placed in the reactor, eg, a cytophilic region in the reactor.

  In some cases, a reaction site containing cells may include a region containing a gas (eg, a “gas head space”) if, for example, the reaction site is not completely filled with liquid. This gas headspace may in some cases be separated from the reaction site separately through the use of a gas permeable or semi-permeable membrane. In some cases, the gas headspace may include various sensors to monitor temperature and / or other reaction conditions.

  Many embodiments and configurations of the present invention have been described with respect to chips or reactors, and those skilled in the art will recognize that the present invention is applicable to either or both. For example, the configuration of the channel may be described in one situation, but it will be appreciated that this arrangement can be applied to other situations (or typically both: reactors that are part of the chip). All descriptions given herein in the context of the reactor or chip are otherwise consistent with the description of the configuration in the context of the “chip” and “reactor” definitions herein. It should be understood that this also applies.

  In certain embodiments, the reaction site may be defined by geometric considerations. For example, a reaction site may be defined as a chamber in a reactor, a channel, the intersection of two or more channels, or other locations defined in some manner (eg, a substance that may define a reactor and / or a chip) Formed or etched in). Other methods of defining the reaction site are possible. In certain embodiments, the reaction site is raised on a surface, for example, by two or more liquid intersections or junctions (eg, within one or several channels), or, for example, to constrain liquid flow Or it may be made artificially by constraining the liquid on the surface by using a ridge. In other embodiments, the reactive site may be defined through an electrical, magnetic and / or optical system. For example, the reaction site may be defined as the intersection between a beam of light and a liquid channel.

  The volume of the reaction site may be very small in certain embodiments. Specifically, the reaction site is, in various embodiments, less than 1 liter, less than about 100 ml, less 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 μl, less than about 300 μl, It may have a volume of less than about 200 μl, less than about 100 μl, less than about 50 μl, less than about 30 μl, less than about 20 μl or less than about 10 μl. The reaction site may also have a volume of less than about 5 μl, or less than about 1 μl in certain cases. The reaction site may have any convenient size and / or shape. In another set of embodiments, the reaction site may have a dimension of a depth of 500 microns or less, a depth of 200 microns or less, or a depth of 100 microns or less.

  In some cases, cells may be present at the reaction site. The sensor (s) associated with the chip or reactor may be able to determine, in certain cases, the number of cells, cell density, cell status or health, cell type, cell physiology, etc. Absent. In certain cases, the reactor may also maintain or control one or more environmental factors associated with the reaction site, such as in a manner that supports chemical reactions or viable cells. In one set of embodiments, the sensors may be connected to actuators and / or microprocessors that can make appropriate changes in environmental factors within the reaction site. The actuator may be connected to an external pump, the actuator may cause the release of material from the reservoir, or the actuator may generate acoustic or electromagnetic energy to heat the reaction site or be sensitive to that energy These types of cells may be selectively killed. The reactor may comprise two or more reaction sites and one or more sensors, actuators, processors, and / or control systems associated with the reaction site (s). It should be understood that any reaction site or sensor technology disclosed herein may be provided in combination with any combination of other reaction sites and sensors.

  As used herein, a “channel” is a conduit associated with a reactor and / or a chip (within, connected to, or connected to a reaction site), for example, As described, one or more liquids can be specifically moved from one location to another, eg, from a reactor or chip inlet to a reaction site. Substances (eg, liquids, cells, particles, etc.) may flow through the channel continuously, randomly, intermittently, etc. The channel may be a closed channel or an opening, eg, a channel open to the external environment surrounding the reactor or chip containing the reactor. The channel is in a feature that facilitates control over liquid transport, such as structural features (eg, elongated press-fit), physical / chemical features (eg, hydrophobic versus hydrophilic), and / or within the channel Other features that can exert a force (eg, including a force) on the liquid may be provided. The liquid in the channel may partially or completely fill the channel. In some cases, the liquid may be retained or constrained in some manner, for example, using surface tension, inside the channel or in a portion of the channel (i.e., the liquid is meniscus, e.g., To be retained in a channel in a concave or convex meniscus). The channel may have any suitable cross-sectional shape that allows liquid transport, e.g., a square channel, a circular channel, a rounded channel, a rectangular channel (e.g., having any aspect ratio) ), Triangular channels, irregular channels, etc. The channel can be any size in the reactor or chip. For example, the channel may be less than about 1000 μm in some cases, less than about 500 μm in other cases, less than about 400 μm in other cases, less than about 300 μm in other cases, and less than about 200 μm in other cases, In still other cases, it may have a maximum dimension normal to the direction of liquid flow in the channel, less than about 100 μm, or even less than about 50 or 25 μm. In certain embodiments, the dimensions of the channel can be selected such that, for example, if the liquid contains cells, the liquid can flow freely through the channel. The dimensions of the channel may also be selected in certain cases, for example to allow for a specific quantitative or linear flow rate of the liquid in the channel. In one embodiment, the depth of the other largest dimension perpendicular to the direction of liquid flow may be similar to the depth of the reaction site with which this channel is in liquid communication. Of course, the number of channels, the shape or geometry of the channels, and the position of the channels within the chip can be determined by those skilled in the art.

  The chip of the present invention may also be capable of receiving and / or outputting any of a variety of reactants, products and / or liquids, eg, toward one or more reactors and / or reaction sites. An inlet and / or outlet may be provided. In some cases, the inlet and / or outlet may allow aseptic transfer of the compound. At least some of the plurality of inlets and / or outlets may be in fluid communication with one or more reaction sites in the chip. In certain embodiments, the inlet and / or outlet may also include one or more sensors and / or actuators, as described further below. In essence, the chip may have any number of inlets and / or outlets from one to thousands that can be in fluid communication with one or more reactors and / or reaction sites. Less than 5 or 10 inlets and / or outlets may be provided in the reactor and / or reaction site (s) for certain reactions, eg, biological or biochemical reactions. In some cases, each reactor has about 25 inlets and / or outlets, in other cases about 50 inlets and / or outlets, and in other cases about 75 inlets and / or outlets. Or, in other cases, may have about 100 or more inlets and / or outlets.

As an example, the inlets and / or outlets of the chip, which are directed to one or more reactors and / or reaction sites, are for liquids, eg gases or liquids, eg waste streams, reactant streams, production Inlets and / or outlets for material flow, inactive flow, etc. may be provided. In some cases, the chip may be constructed and aligned such that liquid entering or exiting the reactor and / or reaction site does not substantially interfere with reactions that may occur therein. For example, the liquid may not interfere with the rate of reaction in chemical, biochemical and / or biological reactions that occur within the reaction site, may interfere with cells that may be present within the reaction site, and / or It can enter and / or exit the reaction site without destroying it. Examples of inlet and / or outlet of the _gas, CO 2, CO, oxygen, hydrogen, NO, NO _2, water vapor, nitrogen, ammonia, and acetic acid and the like without limitation. As another example, the inlet and / or outlet liquid may include liquid and / or other substances contained therein, such as water, saline, cells, cell culture media, blood or other body fluids, Antibiotics, pH buffers, solvents, hormones, carbohydrates, nutrients, growth factors, therapeutic agents (or what seems to be therapeutic factors), antifoams (eg, prevent the generation of bubbles and bubbles), proteins, antibodies, etc. May be included. Inlet and / or outlet liquids may also contain metabolites in some cases. As used herein, a “metabolite” is any molecule that can be metabolized by a cell. For example, the metabolite may be or include an energy source such as a carbohydrate or sugar such as glucose, fructose, galactose, starch, corn syrup and the like. Examples of other metabolites include hormones, enzymes, proteins, signaling peptides, amino acids and the like.

  Inlets and / or outlets may be formed by any suitable technique known to those skilled in the art, for example, by holes or openings that are punched, drilled, molded, squeezed, etc. in the chip, such as a substrate layer, or in a portion of the chip. It may be formed inside. In some cases, the inlet and / or outlet may be lined with an elastic material, for example. In certain embodiments, the inlet and / or outlet may be constructed with a self-sealing material that may be reused in some examples. For example, the inlet and / or outlet may be constructed from a material that makes the inlet and / or outlet liquid-tight (ie, the inlet and / or outlet will not allow liquid to pass through without the application of an external driving force). May allow the insertion of needles or other mechanical devices that can penetrate the substance under certain conditions). In some cases, upon removal of the needle or other mechanical device, this material may be able to reacquire its liquid tight properties (ie, a “self-sealing” material). Non-limiting examples of suitable self-sealing materials for use with the present invention include, for example, polymers such as polydimethylsiloxane ("PDMS"), natural rubber, HDPE, or silicone materials such as formulation RTV 108. , RTV 615, or RTV 118 (General Electric, New York, NY).

  In certain embodiments, the chip of the present invention may comprise very small elements, eg, submillimeter or microfluidic elements. For example, in some cases, the tip may have at least one reaction site having a cross-sectional dimension on the order of 100 mm, 80 mm, 50 mm, or 10 mm, for example. In certain embodiments, the reaction site may have a maximum cross-sectional dimension on the order of 100 mm, 80 mm, 50 mm, or 10 mm, for example. As used herein, “cross section” refers to the distance measured between two opposite boundaries of the reaction site, and “maximum cross section” is measured. It refers to the maximum distance between two opposite boundaries that can be obtained. In other embodiments, the cross-section or maximum cross-section of the reaction site may be less than 5 mm, less than 2 mm, less than 1 mm, less than 500 μm, less than 300 μm, less than 100 μm, less than 10 μm, or 1 μm or less. As used herein, a “microfluidic chip” is a chip that includes at least one liquid element that has a cross section of less than a millimeter, ie, a cross section of less than 1 mm. As one specific, non-limiting example, the reaction site may have a generally rectangular shape with a width of 80 mm, a width of 10 mm, and a depth of 5 mm.

  Although one reaction site may be able to hold and / or react with a small volume of liquid as described herein, the technology associated with the present invention also allows for scalability and parallelism. Become. Throughout, many reactors and / or arrays of reaction sites in a chip or in multiple chips may be constructed in parallel to create even greater capacity. For example, a plurality of chips (eg, at least about 10 chips, at least about 30 chips, at least about 50 chips, at least about 75 chips, at least about 100 chips, at least about 200 chips, at least about 300 chips, at least about 500 chips, at least about about 750 chips, or at least about 1,000 chips or more) may be operated in parallel, for example through the use of robotics, which may automatically monitor or control the chips. In addition, benefits can be gained by maintaining production capacity with small scale reactions typically performed in the laboratory using parallel scaling. It is a feature of the present invention that many reaction sites can be arranged in parallel within a chip reactor and / or within multiple chips. In particular, at least five reaction sites may 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 may be constructed in parallel, eg, to operate in a high throughput system. Good. In some cases, the number of reaction sites may be selected to produce a particular amount of multiple species or products, or to produce a particular amount of reactant. In certain cases, chip and / or reactor parallelization may allow many compounds to be screened simultaneously, or many different growth conditions and / or cell lines may be tested and / or screened simultaneously. Can be possible. Of course, the exact location and arrangement of the reaction site (s) within the reactor or chip is a function of the particular application.

  Furthermore, any of the embodiments described herein can be used in conjunction with a collection chamber that is ultimately connectable to the outlet of one or more reactors and / or chip reaction sites. The collection chamber may have a volume of 10 milliliters or in some cases greater than 100 milliliters. The collection chamber may in other cases have a volume greater than 100 liters or 500 liters, or greater than 1 liter, 2 liters, 5 liters or 10 liters. A large volume may be appropriate if the reactors and / or reaction sites are arranged in parallel in one or more chips, for example, multiple reactors and / or reaction sites may deliver product to the collection chamber. It may be possible to deliver.

  In certain embodiments, the reaction site (s) and / or channels in fluid communication with the reaction site (s) do not have active mixing elements. In these embodiments, the chip reactor produces a sway in the liquid provided through the inlet and / or outlet, thereby mixing and mixing the liquid mixture, preferably without active mixing, if mixing is desired. It may also be constructed in such a way that it is delivered. In particular, the reactor (s) and / or reaction site (s) are multiple in the liquid flow path that produces a flow of liquid through the flow path for mixing, as shown, for example, in the mixing unit 42 in FIG. You may have obstacles. These obstacles may be of essentially any geometric configuration, for example for a series of struts. As used herein, “active mixing elements” are movable with respect to the reaction site (s) and / or the channel itself, ie, define the reaction site or channel. It is meant to define a mixing element, such as a blade, stirrer, etc., that is movable relative to the part (s) of the reactor.

  The chips of the present invention can be stacked together in a predetermined pre-aligned relationship with other similar chips so that when stacked together, the chips are all oriented in a predetermined direction (e.g., all the same Oriented and oriented) may be constructed and configured. When the chip of the present invention is designed to be stacked with other similar chips, the chip is often at least a portion of the chip, such as one or more reactive sites in other chips, and / or other It may be constructed and configured to be in fluid communication with a reaction site in the chip. This configuration may find use in chip collimation, as discussed herein.

  In one set of embodiments, for example, as defined in the proposed standard, 2002 SPS / ANS1, the chip is constructed such that the chip can be stably connected to the microplate and Constructed (eg, a microplate having dimensions of approximately 127.76 ± 0.50 mm × 85.48 ± 0.50 mm). As used herein, “stable connected” means that two components are connected so that the two components are disconnected from each other with a specific movement or force. , Ie, a system that cannot be removed by random vibration or movement of the two components (eg, accidentally lightly bumping the components). This component can be stably connected by pegs, screws, snap-on parts, press-fit and protruding matching sets, and the like. “Microplate” is also sometimes referred to as a “microtiter” plate, a “microwell” plate, or other similar terminology in the field. The microplate may comprise a large number of wells. For example, as is typically commercially available, the microplate can be a 6-well microplate, a 24-well microplate, a 96-well microplate, It may be a microplate or a 1,536 well microplate. The well may be any suitable shape, such as a cylinder or a rectangle. The microplate may also have other numbers of wells and / or other well shapes or configurations, for example, in certain professional applications.

  3A-3C illustrate one set of embodiments of the present invention, where one or more reaction sites may be located in association with a chip, so that this chip is connected to other chips and / or Or, when stably connected to a microplate, one or more reaction sites of this chip may be one or more reaction sites of other chip (s) and / or one of microplate (s). These wells are positioned or aligned so as to be in chemical, biological or biochemical communication or in chemical, biological or biochemical communication. “Alignment” in this context means that the entire area of the side of a reaction site adjacent to another reaction site or well completely overlaps with another reaction site or well, and vice versa It can mean complete alignment such that at least a portion of the reaction sites can overlap at least some of the adjacent reaction sites or wells. “Chemically, biologically, or biochemically connectable” means that the reaction site is in fluid communication with another reaction site or well (ie, the liquid is in contact with each other). Fluidly communicated to other sites or wells (eg, two wells from each other, or other components that can be pierced or broken, or the two Separated by a valve that can be opened in the conduit connecting the two); or the reaction site and other sites or wells are at least some of the chemical, biological or biochemical species from each other, eg, half Means arranged so that it can be moved across the permeable membrane . For example, the chip is constructed and arranged to align with 6 wells of a 6-well microplate when the chip is stably connected to the microplate (eg, located above the microplate). A chip with 96 reaction sites is 96 wells out of 96 well microplates when the chip is stably connected to the microplate. And may be constructed and arranged to be aligned with the wells. Of course, in some cases, the chip is such that a single reaction site of the chip is aligned with two or more microplate wells and / or two or more other reaction sites and / or 2 It can be constructed and arranged such that one or more microplate wells and / or two or more other reaction sites are aligned with a single reaction site of the chip.

  The chip of the present invention also provides a device in which at least one reaction site and / or reactor of the chip is constructed and arranged so as to address at least one well of the microplate, such as a seed in a well of the microplate. Add and / or remove a species from its well and / or fluidly communicate with a device capable of testing the species in the well of the microplate and / or chemical, biological or biochemical May be constructed and arranged to be connectable to each other. With this arrangement, the device can add species to and / or remove species from the reaction site of the chip and / or test the species at the reaction site. In this embodiment, the reaction sites are typically arranged in alignment with the wells of the microplate.

  Referring to FIGS. 3A and 3B, an example is shown. Here, the chip 120 of the present invention can be stably connected to a commercially available microplate 123. In FIG. 3A, the chip 120 is such that when the chip 120 is stably connected to the microplate 123, at least some of the reaction sites 125 of the chip 120 are aligned with at least some of the wells 127 of the microplate 123, and / or Or it may be positioned so that it can be connected. Similarly, in FIG. 3B, when the chip 120 is stably connected to the microplate 23, at least some of the reaction sites 125 can be aligned and / or connected to at least a portion of the wells 127 on the microplate 123. May be constructed and aligned in this manner. In FIG. 3C, another embodiment of the present invention is shown, where the chips 130, 131,... 132 are constructed and arranged so that the chips can be stably connected to each other. In some cases, when the chips 130, 131,... 132 are stably connected to each other, the reaction site 135 of the chip 130 may be combined with one or more other reaction sites on another chip, for example, the chip It is constructed and arranged to be arranged with reaction sites 136 in 131 and / or reaction sites 137 in chip 132.

  The chip of the present invention may be substantially liquid-tight in one set of embodiments. As used herein, a “substantially liquid-tight chip” or “substantially liquid-tight reactor” means that the chip or reactor, respectively, When filled with a liquid such as water, this liquid simply passes through the defined inlet and / or outlet of the chip or reactor, regardless of the orientation of the chip or reactor when the chip is assembled for use. A chip or reactor constructed and arranged so that it can enter or exit the lever chip or reactor. In this set of embodiments, the chip is less than about 100 μl per day, less than about 50 μl per day, when the chip or reactor is filled with water and the inlet and / or outlet is sealed, Or constructed and arranged to have an evaporation rate of less than about 20 μl per day. In certain cases, the chip or reactor exhibits evaporation of a non-measurable and non-zero amount of water per day. A substantially liquid-tight chip or reactor may otherwise have a water evaporation rate of zero.

  The chip of the present invention can be manufactured using any suitable manufacturing technique for producing a chip having one or more reactors each having one or more reaction sites, and the chip is at least 1 It can be constructed from any substance or combination of substances that can support the liquid network necessary to supply and define one reaction site. Non-limiting examples of microfabrication processes include wet etching, chemical vapor deposition, strong reactive ion etching, anodic bonding, injection molding, hot pressing and LIGA. For example, the chip can be manufactured by etching or molding silicon or other substrates, for example, via standard lithographic techniques. The chip is also micro-assembly or micro-machining methods such as stereolithography, laser chemistry three-dimensional entry methods, modular assembly methods, replica molding techniques, injection molding techniques, milling techniques, as known to those skilled in the art. (Milling techniques) or the like. The chip can also be, for example, by patterning multiple layers on the substrate (which can be the same or different), as described further below, or by various known rapid prototyping techniques or masking. It may be manufactured by using technology. Examples of materials that can be used to form the chip include polymers, silicones, glasses, metals, ceramics, inorganic materials and / or combinations thereof. This material may be opaque, translucent or transparent, and may be gas permeable, translucent or gas impermeable. In some cases, the tip may be formed from a material that can be etched to create reactors, reaction sites and / or channels. For example, the chip may comprise an inorganic material such as a semiconductor, fused silica, quartz or metal. This semiconductor material may be, for example, but not limited to, silicon, silicon nitride, gallium arsenide, indium arsenide, gallium phosphide, indium phosphide, gallium nitride, indium nitride, other Group III / V compounds, II / VI It may be a compound having three or more elements, such as a group compound, a group III / V compound, a group IV compound and the like. The semiconductor material may also be formed from combinations of these and / or other semiconductor materials known in the art. In some cases, the semiconductor material can be etched via known processes such as lithography. In certain embodiments, the semiconductor material may be formed from a wafer, for example, as typically produced by the semiconductor industry.

  In some cases, the chip of the present invention comprises a polyacrylate, polymethacrylate, polycarbonate, polystyrene, polyethylene, polypropylene, polyvinyl chloride, polytetrafluoroethylene, fluorinated polymer, silicone, such as polydimethylsiloxane, polyvinylidene chloride, It may be formed from or may include a polymer such as, but not limited to, bis-benzocyclobutene (“BCB”), polyimide, fluorinated derivatives of polyimide, and the like. Combinations, copolymers or mixtures comprising polymers including those mentioned above are also envisaged. The chip may also be formed from a hybrid material, such as a mixture of polymer and semiconductor material.

  In some embodiments, the chip, or at least a portion thereof, is deformed so that the chip is sufficiently robust to be handled by commercially available microplate handling equipment and / or the chip is deformed after routine use. It is rigid so that it will not be. One skilled in the art can select a material or combination of materials for a chip configuration that not only conforms to the present specification but also conforms to other specifications for the use for which the particular chip is intended. However, in other embodiments, the chip may be a semiconductor or plastic.

In certain embodiments, the chip may include a sterilizable material. For example, the chip may have several in order to kill or otherwise inactivate biological cells (eg, bacteria), viruses, etc. before it is used or reused. Can be sterilized in a manner. For example, the chip may be sterilized with a chemical, irradiated (eg, using ultraviolet light and / or ionizing radiation), heat treated, or otherwise. Suitable sterilization techniques and protocols are known to those skilled in the art. For example, in one embodiment, the chip is autoclaved, i.e., the chip is used in commonly used autoclave conditions (e.g., at temperatures greater than about 100 ° C or about 120 ° C, often at elevated pressures, for example, Constructed and composed of a material that can resist exposure to at least one atmosphere of pressure, so that the chip does not substantially deform or become unusable after sterilization. Other examples of sterilization techniques include exposure to ozone, alcohol, ferroics, halogens, heavy metals (eg, silver nitrate), surfactants, quaternary ammonium components, ethylene oxide, CO ₂ , aldehydes, and the like. In another embodiment, the chip can resist ionizing radiation, such as shortwave, high intensity radiation, such as gamma rays, electron beams or X-rays. In some cases, the ionizing radiation may be generated from a nuclear reaction, for example from the decay of ⁶⁰ Co or ¹³⁷ Cs.

  In one set of embodiments, at least a portion of the chip may be manufactured without using an adhesive. For example, at least two components of the chip (eg, two layers of the chip are a multi-layer structure of the chip, a layer or substrate of the chip and membrane, two films, a chip of a microfluidic system, and a component object, etc. Case) may be secured together without the use of an adhesive. For example, the components may be connected, such as through the application of a pressure sensitive material, by using methods such as heat sealing, acoustic welding. In one embodiment, the component can be mechanically held in place. For example, the chip (or part thereof) may be mechanically held together using screws, posts, cantilevers, press fit matching, and the like. In other embodiments, the two components of the chip are not limited to, but are limited to heat sealing methods (e.g., more components of the chip are below the glass transition temperature before being joined to other components). Can be heated to a high temperature or the melting temperature of this component), or acoustic melting technology (eg, vibrational energy, eg, acoustic energy, can be applied to one or more components of the chip, thereby May be coupled together using a technique such as at least partially liquefying or softening.

  In one embodiment, the two components of the chip can be secured by a heat sealing method. For example, one or more components of the chip can be heated to a temperature above the glass transition temperature or the melting temperature of the component (ie, the temperature at which the component begins to soften or liquefy). The components can be placed in contact with each other and cooled to below the glass transition temperature or the melting temperature, thereby allowing the components to be secured together.

  In another embodiment, the two components may be secured via acoustic welding techniques. In one example, vibrational energy (eg, acoustic energy) may be applied to one or more components of the chip. The applied vibrational energy at least partially liquefies or softens the component (s) or at least a portion of the component (s). The components may then be placed together. The vibrational energy may then be stopped, which allows the components to be secured together. In some cases, this component will only have this component's energy director region so that this vibrational energy can terminate in a particular region of this component (the “energy director” region). It may be designed such that it can be liquefied under the influence of vibration energy. For example, as shown in FIG. 4A (lateral side) and FIG. 4B (upper side), the side of component 75 of this chip is illustrated, which shows energy director region 73. When vibrational energy is applied to component 75, a substantial fraction of this energy can be concentrated in energy director region 73, which allows at least a portion of energy director 73 to soften or liquefy. Become. The softened and / or liquefied region may then be connected to other components of the chip and cured, so that the two components of the chip are, for example, as shown in FIG. 4C The component 75 is fixed to the component 77 here.

  In another set of embodiments, two or more components of the chip may be joined together using an adhesive. As used herein, “adhesive material” is described in its ordinary sense as used in the art, that is, as an adhesive material capable of fixing or linking two other materials together. The Non-limiting examples of adhesives suitable for use with the present invention include silicone adhesives such as pressure sensitive silicone adhesives, neoprene based adhesives and latex based adhesives. One or more components of the chip may be used in any suitable manner, for example, by applying the adhesive to the chip components as a liquid or as a semi-solid material such as a viscoelastic solid. May be added. For example, in one embodiment, the adhesive has a transfer tape (e.g., having an adhesive attached thereto so that if the tape is applied to the component, the tape is removed from the component. When removed, the adhesive or at least a portion of the adhesive may be applied to the component (s) using tape that remains attached to the component. In one set of embodiments, the adhesive may be a pressure sensitive adhesive, i.e. the material is neither normal nor substantially adhesive, but pressure, e.g. about 6 atm or about 13 atm (about 100 psi). Or become adhesive under the influence of pressures of about 200 psi) or higher and / or increase its bond strength. Non-limiting examples of pressure sensitive adhesives include AR Clad 7876 (available from Adhesives Research, Inc., Glen Rock, PA) and Trans-Sil Silicone PSA NT-1001 (available from Dielectric Polymers, Holyoke, MA). ).

  In another embodiment, the adhesive may be applied to at least one component of the chip using a solvent bonding system. In solvent coupled systems, one or more components of the chip are placed in a solvent vapor rich environment, i.e., the environment in which the component (s) is placed is saturated or supersaturated with a solvent. So that the solvent can be concentrated on the component (s) placed in the environment under appropriate conditions (eg, when pressure and / or temperature is reduced). The components may then be contacted together in the environment and allowed to be fixed together, for example, when the environment (including solvent) is removed. As one specific example, the two polymer bonate components of the chip of the present invention may be secured together in a methylene chloride environment. For example, a thin layer of solvent, i.e. methylene chloride, may be added to the surface. The two surfaces to be joined may then be pressed together and / or clamped under pressures that ensure bonding.

  In certain embodiments of the invention, the chip is at least one by two or more components in which one or more reaction sites are secured together as previously described (ie, with or without adhesive). The parts may be constructed and arranged so that they can be defined. In some cases, the reaction site may not include any adhesive that is adjacent or otherwise in contact with one or more surfaces that define the reaction site, and for example, if the adhesive is It may be advantageous if it otherwise leaches into the liquid at the reaction site. Of course, the adhesive may be used elsewhere in the chip, for example at other reaction sites. Similarly, in certain cases, the reaction site may be constructed using an adhesive so that at least a portion of the adhesive used to construct the reaction site remains in the chip, and the As a result, the adhesive remains adjacent to or otherwise in contact with one or more surfaces defining the reaction site. Of course, other components of the chip may be constructed without the use of adhesives as previously discussed.

  Referring now to FIG. 2, an example of a microfluidic chip 40 of the present invention is shown. The chip 40 comprises four general units including a mixing unit 42, a heating / dispersing unit 44, a reaction site 46, and a separation unit 48. One or more sensors, processors, and / or actuators (not shown) may each be involved in sensing or operating communications using the chip, as needed. “Sensing communication” and “acting communication”, as used herein, refer to the sensor or actuator, respectively, as to the reaction site environment and / or reaction site contents. It means to be located somewhere in association with the chip so that it can be determined and / or controlled. The sensor or actuator may be included in the chip, for example, incorporated in the reaction site or integrally connected to the reaction site, located in or on the chip, or away from the chip. However, it may be arranged in physical, electrical and / or optical connection with the reaction site, whereby the factors in this reaction site can be detected or acted upon. For example, the sensor may not have any physical connection to the chip, but may be electromagnetic radiation, e.g., directed to, or passed through, or reflected or diffracted by this site It may be arranged to detect the result of an infrared, ultraviolet or visible light interaction. In another example, the sensor may be located on or in the chip and may detect activity at the reaction site by being optically connected to the reaction site via a waveguide. The chip can also be connected directly or indirectly to a network or control system for overall control of detection and operation. Sensing and actuating communication may also be provided, where the reactive site is in fluid, optical or visual, thermal, pneumatic, with a sensor or actuator. It is possible to detect the state of the reaction site and / or the contents of the site by being communicated electrically or the like. In one example, the sensor may be located one downstream of the outlet, or behind a membrane or transparent cover that separates the reaction site from the sensor. Further discussion of the sensing and operation arrangement is given below.

  FIG. 5 illustrates another embodiment of the present invention. FIG. 5A illustrates a top view of chip 105 and FIG. 5B illustrates a side view of chip 105. In this embodiment, the chip 105 consists of three layers of material: an upper layer 100 (which is transparent in the illustrated embodiment), an intermediate layer 115 and a lower layer 110. Of course, in other embodiments of the present invention, the chip 105 may have more or less layers of material (eg, only one layer), depending on the particular aspect. In the embodiment shown in FIG. 5, the intermediate layer 115 has one or more voids 112 that define a plurality of predetermined reaction sites as discussed below. One or more channels 116, 117 may also be defined in the intermediate layer 115 in liquid communication with the reaction site 112. In some cases, one or more ports 114, 118 may allow external access to the channel, eg, through upper layer 110.

  The top layer 100 may cover or at least partially cover the intermediate layer 115, thereby partially defining the reactive site (s) 112. In some cases, the top layer 110 may be permeable to gas or liquid, for example, where a gas or liquid factor is allowed to penetrate or penetrate through the top layer 110. For example, the top layer 100 may be formed from a polymer, such as PDMS or silicone, which may be detectable through it or thin enough to allow measurable gas transport. In some cases, gas transport through the upper layer 100 may be possible, but liquid transport through the upper layer 100 is generally not possible within a reasonable time frame. In certain cases, the top layer 100 may also be substantially transparent or translucent, for example in embodiments that use light to initiate the reaction or activate the process (eg, within the reaction site). There may be. In some cases, the top layer 100 may be formed from a polymer that allows a gaseous pH modifier to penetrate. In certain cases, the top layer 100 may be formed from a self-sealing material, i.e., the material may be infiltrated by a solid object, but generally reacquires its shape after such infiltration. For example, the top layer 100 may be infiltrated by a mechanical device such as a needle, but may be formed from an elastic material that seals and closes once the needle or other mechanical device is removed.

  The intermediate layer 115 comprises four voids in the embodiment illustrated in FIG. Of course, in other embodiments, more or fewer voids may exist in the intermediate layer 115. In the embodiment illustrated in FIG. 5, the voids in the intermediate layer 115 may define reaction sites 112 along the upper layer 110 and the lower layer 110. In the embodiment of FIG. 5, there are four reaction sites 112, which are substantially the same; however, in other embodiments of the invention, there may be more or less predetermined reaction sites present. Well, and the reactive sites may be the same or different from each other. In the embodiment shown, each void is substantially identical and has two liquid channels 116, 117 in liquid communication with the void. Of course, in other embodiments of the present invention, more or less channels may flow through the chip. In the embodiment of FIG. 5, liquid channel 116 is connected to port 118 in layer 115 and liquid channel 117 is connected to port 114 in layer 115; of course, in other embodiments, liquid channels 116 and 118 are One or more reaction sites relative to each other may be liquidally connected to one or more liquid ports and / or to one or more other components in the chip 105. Ports 114 and / or 118 may be used to introduce or aspirate liquid or other material from the reactor in some cases. In certain embodiments of the invention, the reaction site 112 and / or one or more liquid channels may be bonded to two layers, for example, in one or more layers of the chip, eg, simply within one layer. In a gap spanning three layers, etc.

  Ports 114 and 118 may be in fluid communication with one or more reaction site (s) 112. Ports 114 and 118 may be accessible by inserting needles or other mechanical devices through top layer 100 in some cases. For example, in some cases, the top layer 100 may be infiltrated, or the space in the top layer 100 may allow external access to the ports 114 and / or 118. In some cases, the top layer 110 may be composed of a flexible or elastic material that may be self-sealing in some cases. In certain cases, the top layer 110 may have a passage formed in the top layer 110 that allows direct or indirect access to the ports 114 and / or 118, or the port. 114 and / or 118 may be formed in the upper layer 110 and connected to the channels 116 and 117 through channels defined in the layer 100.

  The lower layer 110 forms the bottom of the chip 105 as illustrated in FIG. As previously described, a portion of the bottom layer 110 may partially define the reaction site 112 in certain cases. In some cases, the bottom layer 110 may be formed from a relatively rigid or rigid material, which may provide a relatively rigid structural support for the chip 105. Of course, in other embodiments, the lower layer 110 may be formed from a flexible or elastic (ie, non-rigid) material. In some cases, bottom layer 110 may include one or more channels defined therein and / or one or more ports defined therein. In some cases, the material defining the boundary of the reaction site, such as the bottom layer 110 (or the top layer 110), can be, for example, reacted to the reaction site 112 or distal to it in some manner. Salts and / or other substances may be included when producing factors that can be transported. This factor can be any material previously discussed, such as gases, liquids, acids, bases, tracer compounds, small molecules (eg, molecules having a molecular weight of less than about 1000 Da to less than 1500 Da), drugs, proteins, etc. And transport may occur by any suitable mechanism, such as diffusion (neutral or accelerated) or leaching. In one embodiment, this factor is caused by a pyrolysis reaction that can be initiated externally, for example, using current or light (eg, using a laser). In other particular cases, the material defining the boundary of the reaction site, such as bottom layer 110 or top layer 100, may include one or more reservoirs of factors that are not in liquid contact with reaction site 112, Here the factor can be transported to or distal to the reaction site, for example by making at least one liquid connection between the reservoir and the reaction site. This transport may be controlled or driven externally in some cases, for example using electric or magnetic fields for direct liquid motion. Of course, in still other cases, the bottom layer 110 and / or the top layer 100 may not include any factors or other reservoirs.

  It should be understood that the chips and reactors of the present invention may have a wide variety of different configurations. For example, the chip may be formed from a single material, or the chip may include more than one type of reactor, reservoir and / or factor. In some cases, the chip may include two or more systems that can change one or more environmental factor (s) in one or more reaction sites in the chip. For example, the chip may include a sealed reservoir and a top layer that allows non-pH neutral gas to permeate across.

  The chip of the present invention can be constructed and arranged such that, for example, a sensor can be used to detect or determine one or more environmental conditions associated with the reaction site of the reactor. In some cases, each reaction site may be determined independently. Detection of environmental conditions is performed by a sensor, for example, which may be located within the reaction site or proximal to the reaction site, i.e. the sensor may be arranged to communicate with the reaction site in some manner. May be. In some cases, such detection may occur in real time. This sensor may be, for example, a pH sensor, an optional sensor, an oxygen sensor, a sensor capable of detecting a substance concentration, or the like. Other examples of sensors are further described below. The sensor may be embedded in and integrally connected to the chip (eg, in a component that defines at least a portion of the reaction site channel in fluid communication with the reaction site), and in some cases may be separated from the chip. Good (eg, in the process of detecting contact). Also, the sensor may be integrally connected to or separated from the reaction site in certain embodiments.

As used herein, “environmental factor” or “environmental condition” refers to a detectable and / or measurable environment within and / or associated with a reaction site. Conditions (e.g. by sensor), e.g. temperature or pressure. This factor or condition may be detected and / or measured within the reaction site and / or at a location proximal to the reaction site (eg, upstream or downstream of the reaction site), so that the environment within this reaction site Conditions are known and / or controlled. For example, the environmental factor may be the concentration of a gas or dissolved gas associated with or at the reaction site (eg, upstream or downstream of the reaction site separated from the reaction site by a membrane or the like). This gas may be, for example, oxygen, nitrogen, water (ie, relative humidity), CO ₂ and the like. This environmental factor may also be the concentration of the substance in some cases. For example, environmental factors may be collective quantities such as molarity, osmolarity, salinity, total ion concentration, pH, color, optical density, and the like. This concentration may also be the concentration of one or more compounds present in the reaction site, for example ionic concentrations such as sodium, potassium, iron or chloride ions; or biologically active compounds such as proteins, lipids or carbohydrates It may be the concentration of a source (eg sugar) such as glucose, glutamine, pyruvate, apatite, amino acids or oligopeptides, vitamins, hormones, enzymes, proteins, growth factors, serum and the like. In some cases, the material within this reaction site may include one or more metabolic indicators, such as found in the culture medium or produced as cell-derived waste. In the presence of cells, this sensor is also a sensor for determining all of viability, cell density, cell motility, cell differentiation, cell products (eg, proteins, lipids, small molecules, drugs, etc.). May be.

  The environmental factor may also be the liquid properties of the liquid in the reaction site, such as pressure, viscosity, turbidity, shear rate, degree of agitation, or liquid flow rate. This liquid may be, for example, a liquid or a gas. In one set of embodiments, the environmental factor is an electrical condition within the reaction site, such as charge, current, voltage, electric field strength, or resistivity or conductivity of a liquid or another substance. In one set of embodiments, the environmental condition is temperature or pressure. In certain embodiments, the sensor is a ratiometric sensor, ie, a sensor that can determine the difference or ratio between two (or more) signals, eg, measurement and control signals, two measurements, etc. There may be.

Non-limiting examples of sensors useful in the present invention include dye-based detection systems, affinity-based detection systems, microfabricated gravimetric analyzers, CCD cameras, optical detectors, optical microscope systems, electrical systems, thermocouples And thermistors and pressure sensors. One skilled in the art can identify other sensors for use in the present invention. For example, in one set of embodiments, the chip may comprise a sensor that includes one or more detectable chemicals that are responsive to one or more environmental factors, eg, a dye (or combination of dyes), fluorescent molecules, and the like. Good. Alternatively, one or more dyes, fluorescent or chromogenic molecules that are sensitive to specific environmental condition (s) can be selected by one skilled in the art. Non-limiting examples of such dyes, or fluorescent or chromogenic molecules, include pH sensitive dyes such as phenol red, bromothymol blue, chlorophenol red, fluorescein, HPTS, 5 (6) -carboxy- 2 ′, 7′-dimethoxyfluorescein SNARF and phenothalein; calcium sensitive dyes such as Fura-2 and Indo-1; chloride sensitive dyes such as 6-methoxy-N- (3-sulfopropyl) -quinolinine and Lucigenin; a nitric oxide sensitive dye such as 4-amino-5-methylamino-2 ′, 7′-difluorofluorescein; a dissolved oxygen sensitive dye such as tris (4,4′-diphenyl-2,2 ′ -Bipyridine) ruthenium (II) chloride pentahydrate Dyes sensitive to dissolved CO _2; susceptible to fatty dye, e.g., BODIPY 530- labeled glycero-phosphoethanolamine; protein sensitive dye, such as 4-amino-4'-benzal bromide stilbene -2-2'- disulfonic acid (Sensitive to serum albumin), X-Gal or NBT / BCIP (sensitive to specific enzymes), Tb ³⁺ from TbCl ₃ (sensitive to specific calcium binding proteins), BODIPY FL faracidin (sensitive to actin), or BOCILLIN FL (Sensitive to specific penicillin binding proteins); dyes sensitive to the concentration of glucose, lactose or other components, or dyes sensitive to proteases, lactases or other metabolic by-products, dyes sensitive to proteins, antibodies or other cellular products ,example , Calcein (calcein) AM, ethidium bromide or resazurin (sensitive to viability) and the like.

  In one embodiment, the dye or fluorescent molecule may be immobilized in one or more walls in the chip, for example, in one or more walls that define a reaction site. In another embodiment, the dye or fluorescent molecule may be immobilized in a gel located within the chip, for example in liquid communication with the reaction site. In yet another embodiment, the dye or fluorescent molecule may be dissolved, for example, in a medium that is passed through the reaction site. The dye or fluorescent molecule may have a response that is generally proportional to the value of one or more environmental factors and / or other variable (s) of interest. This response can be measured, for example, as a fluorescent signal, absorbance signal, wavelength or frequency. The reactor and / or reaction site in the chip may be combined with light delivery and / or other light interacting component (s). For example, the light interacting component may comprise a detection system in which light (eg, having a predetermined wavelength) originating from a dye, fluorescent molecule, etc. may be detected and / or measured.

  The sensor may in some cases comprise a colorimetric detection system that may be outside the chip, or in certain cases may be microfabricated on the chip. In one embodiment, the colorimetric detection system may be external to the chip, but may be an optical fiber or other optical interactive component that may be embedded in the chip (eg, those described below, etc. ) May be optically coupled to the reaction site. For example, if a colorimetric detection system is used with a dye or fluorescent molecule, the colorimetric detection system can detect the frequency of the dye or fluorescent molecule and / or respond to changes or shifts in one or more environmental factors within the reaction site. Or a change or shift in intensity can be detected. In a specific example, Ocean Optics Inc. (Dunedin FO) provides a fiber optic probe and spectrometer for the measurement of pH and dissolved oxygen concentration.

  In certain aspects of the invention, any of the above chips is a chip or portion thereof, eg, one or more reaction sites, of environmental conditions within or associated with the reaction site, eg, by use of a control system. It may be constructed and arranged to be responsive to changes. In some cases, each reaction site in the chip may be controlled independently in some manner. As used herein, a “control system” can detect and / or measure one or more environmental factors within or associated with a reaction site and within this reaction site or reaction site. A system that produces a response or change in the environmental conditions associated with (e.g., maintaining the environmental conditions at a particular value). In some cases, the control system may control environmental factors in real time. The response produced by the control system may be based on environmental factors in certain cases. As used herein, an “active” control system is a system that can cause changes in environmental factors associated with a reaction site as a direct response to a measurement of environmental conditions. The active control system can provide a factor that can be delivered or released from the reaction, which is controlled in response to a sensor that can determine the conditions associated with the reaction site. A “passive” control system, as used herein, is a system that can maintain or cause changes in the environmental conditions of the reaction site without the need to measure environmental factors. This passive control system can control environmental factors within the reaction site, but does not need to respond to sensors or measurements. This passive control system may allow certain factors to enter and exit the reaction site without active control. For example, a passive control system may comprise an oxygen membrane and / or a water permeable membrane that maintains oxygen and / or water vapor content within the reaction site, eg, within certain predetermined limits. Can do. This control system can be used, for example, for one day, one week, thirty days, sixty days, ninety days, one year, or in some cases indefinitely, any one or more conditions within or associated with a reaction site. Can be controlled.

  The control system may include a number of control elements, such as sensors operably connected to an actuator, and optionally a processor. One or more components of the control system may be integrally connected to or separated from the chip with the reaction site. In some cases, the control system comprises components that are integral to the chip and other components that are separated from the chip. This component may be within the reaction site or proximal to the reaction site (eg, upstream or downstream of the reaction site, etc.). Of course, in some cases, the control system comprises two or more sensors, processors, and / or actuators, depending on the application and environmental factor (s) being detected, measured and / or controlled. May be. An example of a control system is shown in FIG. 5, where the environmental condition 50 in the chip 105 detected by the sensor 52 is converted into a signal 51 that is transmitted to the processor 54 for proper processing. . The processor 54 then produces a signal 53 that is sent to the actuator 56 where the signal is converted to a response 60. In some cases, the control system can detect on an environmental factor such as temperature or pH at a stimulus or change in stimulus (eg, less than 5 seconds, less than 1 second, less than 100 ms, less than 10 ms, or less than 1 ms). It may be possible to produce very rapid changes in environmental factors in response to

  As used herein, a “processor” or “microprocessor” receives a signal from one or more sensors, stores a signal, and / or, for example, a formula or an electrical circuit Alternatively, any component or device that can convert this signal into one or more responses for one or more actuators by using a computer circuit. In one embodiment, the processor may be a specialized system. This signal may be any suitable signal of an indicator of environmental factors determined by the sensor, such as a pneumatic signal, an electrical signal, an optical signal, a mechanical signal, and the like. The processor 54 may be any device suitable for determining a response to a signal, for example, a mechanical device such as a semiconductor chip or an electrical device. Depending on the application, the processor may be integrally connected, incorporating the reaction site or chip, or separated from the reaction site or chip. In one embodiment, the processor is programmed using a process control algorithm, which can take, for example, an input signal from a sensor and convert this signal to an appropriate output to an actuator. Any suitable algorithm (s) may be used in the processor 54, such as a PID control system, a feedback or feedforward system, a fuzzy logic control system, and the like. The processor may be programmed or otherwise designed to control environmental conditions within the reaction site, for example, by operation of an actuator.

  For example, in one embodiment, the processor 54 maintains one or more environmental conditions (eg, temperature or pressure) at a certain predetermined level within a predetermined reaction site of the chip, eg, the chemistry therein. Reaction can be promoted. In another embodiment, the processor 54 can change one or more environmental conditions within one or more predetermined reaction sites of the chip according to a predetermined pattern or in response to a specific condition; The processor allows the actuator to raise the pH within a given reaction site at a particular rate, or the processor may allow the actuator to move to a given reaction site once a particular temperature or other environmental condition is reached. Or the processor allows the actuator to allow, prevent, increase or decrease the flow of a substance or factor to a given reaction site. To do. In certain embodiments, the processor 54 may rely on several environmental conditions within a given reaction site, such as at least 2, 3, 4, 5, 6, 7 or more conditions depending on the degree of application and control desired. Thus, it can be controlled simultaneously or almost simultaneously. For example, the processor 54 may be in communication with one or more sensors and / or one or more actuators.

  In certain embodiments, the processor 54 may be programmed or designed to maintain one or more environmental conditions within one or more reaction sites. For example, the processor 54 may be programmed or designed to maintain one or more environmental conditions such as within 3 reaction sites, within 7 reaction sites, within 10 reaction sites, etc. . For example, if there are multiple reaction sites, one subset of reaction sites may be held at one temperature, but different subsets of reaction sites may be held at various temperatures. In another example, one subset of reactive sites may have a first compound added thereto, while a second subset of reactive sites may have a different compound added thereto. . Combinations of subsets may also use different subsets having different compounds, temperatures, etc., for example. Thus, many different environmental conditions can be controlled simultaneously with different values within one chip. In some cases, the reaction site control and monitoring pattern may be changed in time, ie, during the experiment. Thus, for example, two reaction sites that are monitored and / or controlled simultaneously at a first point in time may be monitored and / or controlled separately at a second point in time. Control and monitoring may be preset, determined automatically or manually.

In one set of embodiments, the processor 54 may be programmed or designed to maintain conditions appropriate to support the metabolism or growth of cells (eg, bacterial or mammalian cells). For example, the processor 54 can control one or more of temperature, relative humidity, pressure, oxygen concentration, CO ₂ concentration, serum concentration, nutrient concentration, shear rate or pH within the reaction site of the chip. May be possible. Other environmental factors suitable for supporting cell growth are further described below.

As used herein, an “actuator” is a device that can affect the environment in or near one or more reaction sites, or in an inlet or outlet in fluid communication with one or more reaction sites. (For example, as in channels 116 and 117 in FIG. 5A). This actuator may be remote from the reaction site or chip, or may be integrally connected. For example, in certain embodiments, the actuator may include a substance or factor into or out of the reaction site, such as a chemical solution, a buffer (eg, pH buffer), a gas such as CO ₂ or O ₂ , a nutrient solution, Valves or pumps (microvalves and pumps) that can control, change and / or prevent the flow of solutions containing saline, acids, bases, carbon sources, nitrogen sources, inhibitors, promoters, hormones, growth factors, inducers, etc. Including a micropump). The material to be transported depends on the specific application. In some cases, the pump may be outside the tip. In one example, the actuator may selectively open a valve that allows CO ₂ or O ₂ to enter the reaction site. In other cases, the pump may be inside the tip. For example, the pump may be a piezoelectric pump or a mechanical activation pump (eg, activated by pressure, electrical stimulation, etc.). In one embodiment, the pump is activated by creating a gas in the chip that can cause a liquid to flow in the chip; for example, the gas is light such as laser light to a reactant that initiates a gas generating reaction. Or the gas may be generated by applying an electric current to the reactant (eg, an electric current may be passed through the water to produce a gas). In another example, the actuator may include a pumping system that may cause liquid communication with the reactants as needed. In one particular example, a gas permeable chip may be placed in an incubator or other encapsulated environment and the atmosphere in the incubator or other environment may be controlled thereby reacting You may control the environmental condition in a site | part.

  In yet another example, the actuator may comprise a heating or cooling element, such as a heat exchanger (eg, shown in FIG. 2), a resistive heater or a Peltier cooler. In other embodiments, the actuator may be an electrical system, eg, an electrical system that maintains a constant current or a constant electric field gradient within the reaction site. In another embodiment, at least two liquid streams enter and exit the reaction site, but the actuator may include a valve or pump that can control the ratio of flow rates between the two liquid streams. For example, the actuator can operate in response to a signal to increase the inlet flow rate and decrease the outlet flow rate within the reaction site.

  In one set of embodiments, the actuator may comprise an energy source, such as an electromagnetic energy source, a heat source, a mechanical energy source, or an ultrasonic source. In certain embodiments, the electromagnetic radiation is in the optical or visual range of wavelengths or frequencies (eg, having a wavelength of about 400 nm to 700 nm), infrared wavelengths (eg, having a wavelength of about 300 nm to 700 nm), ultraviolet Wavelength (eg, having a wavelength of about 400 nm to about 10 nm), and the like. In some cases, the light is, for example, in the range of about 350 nm to about 1000 nm, about 300 nm to about 500 nm, about 500 nm to about 1 nm, about 400 nm to about 700 nm, about 600 nm to about 1000 nm, or about 500 nm to about 50 nm. Can cover the frequency. In other cases, the light may be monochromatic (ie, having a single frequency or a narrow frequency distribution), for example, the frequency commonly produced by commercially available lasers, or the frequency at which the fluorescent agent is excited. For example, this frequency may be a frequency centered around 366 nm, 405 nm, 436 nm, 546 nm, 578 nm, 457 nm, 488 nm, 514 nm, 532 nm, 543 nm, 594 nm, 633 nm, 568 nm or 647 nm. The monochromatic beam of light may have a narrow distribution of frequencies. For example, 90% or 95% of the frequency may be within ± 5 nm or ± 3 nm of the average frequency. In certain cases, the light may be polarized (eg, linearly or circularly), or two or more wavelengths of light may be used, eg, sequentially or simultaneously. In certain embodiments, the light interacting component may change the wavelength of light in the device.

  In another embodiment, the actuator may be constructed and arranged to selectively kill or inactivate specific cells or cell types, preferably without affecting nearby or adjacent cells. For example, the actuator may comprise an energy source substantially directed to a reaction site or an inlet or outlet in fluid communication with the reaction site; upon detection of a particular cell or cell type by a sensor By, for example, directing energy to the cell, killing the cell, or otherwise inactivating the cell in the same manner (eg, by damaging its DNA sufficient to prevent replication) Can be targeted. The energy targeted to the cell may be any energy that can inactivate the cell, such as electromagnetic or ionizing radiation, ultrasound or thermal energy.

  In one set of embodiments, the chip controls environmental factors associated with a reaction site by transporting factors that may affect environmental factors, or precursors of factors that may affect these factors, to or near the reaction site. (Ie, to affect environmental factors within the reaction site). Control of the delivery of this factor (or precursor) to the reaction site can be used in certain cases to control environmental factors.

  In another set of embodiments, environmental factors within or associated with the reaction site directly contact the reaction site with an agent, e.g., an exogenous or unsterile factor such as a liquid or gas. Can be changed and / or controlled without any changes. For example, the reaction site may include a biological specimen or material for use in a biological setting where sterilization and / or isolation is required; or the reaction site is, for example, subject to a change in liquid or pH It may include reactions that are sensitive to water, for example, water-sensitive reactions that only take place in a moisture-free environment, where direct contact between this factor and the reaction site is difficult.

  In one set of embodiments, the chip may be constructed and arranged to allow certain factors to penetrate or diffuse into the reaction site. For example, the reaction site can be defined, at least in part, by a component, such as a chip wall or layer, through which an agent can permeate. This factor may be capable of changing and / or controlling one or more environmental factors within or associated with the reaction site. For example, the component may comprise a membrane that is permeable across the factor, such as a permeable membrane or a semi-permeable membrane (eg, with respect to the factor). In some cases, this component may be chemically or physically inert with respect to this factor. In certain cases, factor flow may occur on one side of the component. In certain embodiments, the flow of the factor on one side of the component is performed, for example, through a serpentine or non-linear path so that the point of contact between the factor and the component is extended. Also good. For example, in FIG. 2, if compartment 20 is separated from compartment 42 by a membrane (not shown) through which the factor can permeate, the flow of the factor may occur along tortuous passageway 281.

  In one embodiment, the chemical agent that occurs anywhere in the chip may be made to interact with the reaction site (s) that control the environmental factor (s) therein, or One or more liquid pathways are created (eg, opened) in the chip, thereby causing factors stored in or off the chip to contact the reaction site or otherwise affect the reaction site May be possible. This factor may be any factor that can alter and / or control one or more environmental factors within the reaction site. For example, the factor may be a composition that is not pH neutral or a pH modifier as previously described. For example, in FIG. 5A, the chip 105 can be constructed to allow factors to penetrate and / or diffuse into the reaction site. For example, the reaction site can be a wall of a chip (eg, the wall of a given reaction site 112) or one or more layers of a chip (eg, the top layer 100) through which factors can permeate and affect the reaction site. A component may be provided. In another example, a component that can be penetrated by the agent in a manner may comprise a membrane that can be penetrated across the agent, such as a membrane that is permeable or semi-permeable (eg, semi-permeable with respect to the factor). And may be defined by the membrane. In some cases, the component may be chemically or physically inert with respect to the agent; for example, the component may be substantially damaged by an acidic or alkaline compound. It may be possible to permeate across this reaction site without doing or changing. In certain cases, the flow of the factor can occur on one side of the component. In some cases, the flow of the factor on one side of the component may occur along a serpentine or non-linear path, for example, to extend the contact time between the factor and the component.

  For example, in the embodiment of the invention shown in FIG. 7A, a chip 205 having a predetermined reaction site 207 and a permeable upper layer 220 is illustrated. In this embodiment, the dispersion unit 228 is located proximal to the reaction site, so that the dispersion unit allows the dispersion unit to be within a desired time frame, for example 2-3 seconds or tens of seconds, depending on the application. Factors can be generated that can penetrate into and interact with the reaction site 207 in sufficient or hours. Dispersion unit 228 is also connected to one or more chemical sources, eg, one or more sources of gas and / or pH modifiers, eg, sources 222 and 224 as shown in the illustrative figures. May be. For example, source 222 may be an acidic source, source 224 may be an alkaline source, and source 222 and source 224 may each be an acidic source or an alkaline source. The source 222 may be a source of cellular media and the source 224 may be a source such as glucose or saline. FIG. 7B illustrates an enlarged view of a droplet 225 that includes factors dispersed on the surface of the chip 205 on the upper layer 220 (eg, factors dispersed by the dispersion unit 228). In this view, the portion 226 of the droplet 225 partially penetrates the reaction site 207 through the layer 220. Over time, the infiltration region 226 can be expanded as the factor penetrates the upper layer 220 until the factor contacts the reaction site 207 and affects environmental factors within the reaction site.

In certain embodiments, as shown in Equation 1, the permeability (P) of a substrate (eg, a component or layer of the chip) for a factor is the capacitive transfer rate (v) × thickness (T) of the factor. ) / Area (a) × time (t) × partial pressure difference (p):
P = vT / atp (1)
The thickness (T) of the substance is, for example, cm or mm, the time (t) is seconds, the pressure (p) is Pa, atm, cmHg or mmHg, the area (a) is cm ² or mm ² , and The capacitive transfer rate (v) of this factor may be measured in cm ³ , which refers to a temperature of 273.15 K (0 ° C.) and a pressure of 101 325 Pa (1 atm) or other standard conditions, Measured at temperature STP ("standard temperature and pressure"). Therefore, the permeability is a unit of, for example, cm ³ _STP mm / scm ² cmHg. Thus, for example, in part, the substrate is at least about 400 × 10 ⁻⁹ (cm ³ _STP mm / scm ² cmHg), at least about 500 × 10 ^{−9 for} ammonia, acetic acid and / or CO ₂ . (Cm ³ _STP mm / scm ² cmHg), at least about 590 × 10 ⁻⁹ (cm ³ _STP mm / scm ² cmHg), at least about 700 × 10 ⁻⁹ (cm ³ _STP mm / scm ² cmHg), or at least about It may have a permeability of 800 × 10 ⁻⁹ (cm ³ _STP mm / scm ² cmHg). In one specific example, where the substrate is a membrane having a thickness of approximately 100 μm, the substrate is permeable to about 172 mol / day m ² atm for ammonia and about 150 mol / day m ² for acetic acid. It may have atm permeability and / or permeability of about 150 mol / day m ² atm for CO ₂ .

  In one example, if the environmental factor within or associated with the reaction site is pH, the factor can be delivered or transported to or near this reaction site to control the pH therein. It may be. As used herein, a “pH-altering agent” is any factor that can change the pH of the environment within or associated with the reaction site, such as an acid, base or reaction site. It may be a factor capable of reacting internally or proximally to form an acid or base. In certain embodiments, the pH modifier is inert to the reaction site and / or other component (s) of the chip. This pH modifier is dependent on the required sensitivity and the specific application, for example at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.8, 1, 2 or 3 or more. It may be possible to change the pH of the environment within or associated with the reaction site to a significant or measurable degree by the pH unit. The required pH sensitivity can be readily determined by one skilled in the art. For example, a chemical process that requires a change in pH to initiate the reaction may require a large pH change, but a process that adjusts the pH of the reaction site near the optimum value may require a slight change in pH. Sensitivity to may be required.

As used herein, “acid” is shown in its usual definition as used in chemistry. In some cases, the acid may have a pH of less than about 7, less than 5, less than 4, less than 3, less than 2 pH units, depending on the strength of the acid. Similarly, "base" or "alkaline" is described in the usual definition used in the chemical domain. In some cases, the base or alkali may have a pH of at least about 7, at least about 8, at least about 9, at least about 11, or at least about 12 pH units. “Non-neutral” or “non-pH-neutral” is either an acidic or basic composition (ie, the composition is preferably a significant amount). Each having a pH of either greater than or less than 7, eg, at least 1 or 2 pH units). A composition that is not pH neutral may in some cases be a solid, liquid or gas. As used herein, a “gaseous” acid or base is a composition that is in the gas phase or is generally volatile (ie, has a high vapor pressure) and is easily gasified. Enter the phase. For example, the gaseous acid or base may have a vapor pressure of at least about 300 mmHg, at least about 400 mmHg, at least about 500 mmHg, at least about 600 mmHg, or at least about 700 mmHg. Non-limiting examples of gaseous acids include acetic acid, formic acid, propionic acid, pyruvic acid, lactic acid, SO ₂ , CO ₂ , CO, NO ₂ or butyric acid; non-limiting examples of gaseous bases Examples include ammonia, phosphine or arsine.

  In some embodiments, the chip components (eg, layers or membranes) include polymers that are permeable to molecules (eg, small molecules), such as nylon, polyethylene, polypropylene, polycarbonate, polydimethylsiloxane or copolymers or It may be a mixture or may contain them. In another set of embodiments, the component may comprise a polymer that is substantially impermeable to the factor being transported, but the component may be, for example, porous if the factor is porous. Or it may be constructed or designed to allow transport through a region with multiple channels. In yet other embodiments, the component may be impermeable to the factor being transported, but the component is converted to an permeable form upon addition of the osmotic factor. May be. As used herein, “permeation” and “permeate” refer to any suitable non-bulk transport process. Non-bulk transport generally refers to a transport process in which no substantial conversion or bulk flow occurs within the substrate. For example, the permeability of the factor may occur through passive diffusion, for example, through the bulk material of the component, or through pores or other gaps that may be present in the component; Then, it may be facilitated or enhanced, for example, by osmolarity, electrodiffusion, electroosmosis, leaching, or by the use of permeation enhancing compounds within this component. In certain embodiments, the transport of this factor may be facilitated using an externally applied field, such as an electric field, magnetic field or centripetal field.

  In some cases, this component may be designed such that the factor is transported through it within a predetermined time or under certain conditions. In these cases, the exact thickness, density, porosity, twist, composition, or other characteristics of the component may be determinable by one skilled in the art. For example, in some cases, the diffusion of a factor across this component may generally be Fick diffusion, and the time it takes for this factor to diffuse across this component can be determined using Fick's law. Can be determined. In some cases, transport of factors across this component may be relatively fast, for example when relatively thin components are used. For example, depending on the application, the component may be constructed such that the agent is transported in less than about 10 minutes, less than about 5 minutes, less than about 3 minutes, or less than about 1 minute.

For example, in another set of embodiments, as shown in FIG. 8A, the laser 230 directs the laser beam 232 to a compartment 235 of the chip 205, for example, environmental factors within a given reaction site 207, such as pH. Alternatively, it activates a reaction that produces a factor that can change the concentration. In other embodiments, it will be appreciated that other forms of energy, such as heat or electrical energy, may be applied to compartment 234 (or generally to chip 205) to activate this factor. An enlarged view of FIG. 8A is shown in FIG. 8B. Laser beam 232 may be directed directly into section 235 in virtually any direction or angle (as shown in FIG. 8) or indirectly, for example through a waveguide (not shown). May be. As shown in FIG. 8, the laser beam 232 may optionally pass through one or more other layers and / or components of the chip 205 before reaching the compartment 235 (eg, these layers and And / or if the component is substantially transparent). Upon absorption of energy from the laser beam 232 by the factor generating precursor (s) 237 in the compartment 235, the factor generating precursor (s) 237 can yield factor 238 in this example. Factor 238 may be a gas such as a pH changing gas in this example, for example, ammonium, acetic acid, CO, CO ₂ , O ₂ , N ₂ , HCl, and the like. The factor 238 can then permeate through at least a portion of the chip 205 (eg, in a channel or through a component and / or layer of the chip) to interact with a predetermined reaction site 207. In this way, controlled application of light or other energy to compartment 235 can result in alteration and / or control of environmental factors within a given reaction site 207.

  In certain embodiments, environmental factors within the reaction site may be altered by generating one or more factors in the chip from one or more precursors, such as, for example, precursor 237 of FIG. 8B. The factor (s) may interact with or alter environmental factors within the reaction site in some way. In one embodiment, the factor can be generated within the reaction site. In another embodiment, the factor may be generated anywhere in the chip and transported in some manner, eg, liquid, to the reaction site. For example, the chemical agent may be generated and / or stored (eg, in a reservoir) in various compartments associated with or outside the chip, and then, for example, a channel or other liquid It may be transported to the reaction site by connection or by allowing it to permeate or diffuse across the membrane or another component. In one embodiment, the agent may be generated at a location proximal to the reaction site, for example, the agent may be generated at a location where it can be easily moved or transported to the reaction site, eg, a few seconds or tens of seconds. May be generated within. In another embodiment, the factor may, for example, prevent liquid communication between the compartment and the reaction site, while preventing non-gaseous products from entering the reaction site, or through other membranes or other barriers. It may be a gas that is transported over the reaction site to the reaction site. In certain embodiments of the invention, the reaction (s) of the precursor (s) that yield this factor can be initiated on the outside. For example, a light source such as a laser may be added to the precursor (s) or other energy source such as current or heat to initiate the reaction of the precursor (s) May be used. In yet another embodiment, a liquid connection can be created reversibly between this compartment and the reaction site, for example. For example, the liquid connection can be made by opening a valve, such as a mechanical valve or a micromechanical valve that separates the compartment from the reaction site.

  In certain embodiments, additional compounds may be combined with the precursor (s) to prevent, for example, degradation or modification of the precursor (s), the ability of the precursor (s) to react. Enhancing (eg, catalyst or enzyme) or enhancing absorption of incident energy into this precursor (s), eg, increasing the reaction rate at which this precursor (s) forms factors Can be made. In some cases, a material that allows absorption of incident electromagnetic radiation, eg, a darkened or “black” material, may be added to the precursor (s) to, for example, absorb energy. Can be improved. Non-limiting examples of such materials include quartz, black glass, silicon (silicon), black sand, carbon black and the like. The additional compound may be substantially non-reactive and unable to form a transportable factor (ie, moveable through the chip phase or component) or the additional compound is associated with the reactive site. The generation of factors or the control of environmental factors may not be substantially disturbed.

  This factor, in certain embodiments, can be produced by a reaction that is activated at a particular temperature, for example, a thermal decomposition or a thermal denaturation reaction. In some cases, the reaction to the generated factor is at least a specific temperature at which the precursor (s) can activate the reaction, eg, at least about 200 ° C., 300 ° C., 400 ° C. or It can be initiated when exposed to a temperature of 500 ° C. The temperature required to activate this reaction can be determined by any suitable technique, for example, the precursor (s) to light energy, heat, electrical energy (eg, resistive heating), exothermic chemical reaction. , Etc., can be produced within this precursor (s) upon exposure.

In certain embodiments, the factor thus generated may be a gas, such as O ₂ , CO, CO ₂ , NO, NO ₂ , HCl, and the like. In some cases, the factor producing reaction may produce one or more gases and / or one or more non-gas products. In some cases, the gaseous factor (s) can then be transported into or near the reaction site (eg, through the membrane or across the barrier), but non-gaseous products (eg, Liquid or solid) may be prevented from entering this reaction site in the same manner.

This factor may be a pH modifying factor in certain cases. In some cases, the pH modifier may be a base, such as ammonia. The base can be generated by any suitable reaction that can generate an alkaline factor, such as through a thermal decomposition reaction of an alkali precursor salt. For example, ammonia can be generated by thermal decomposition of ammonium precursor salts such as ammonium nitrate, ammonium carboxylate, ammonium bicarbonate, ammonium chloride, ammonium bromide, ammonium fluoride, and the like. In other cases, the pH modifier may be an acid, such as acetic acid or formic acid. The acid can be generated using any suitable reaction that can generate an acidic factor, such as thermal denaturation of an acid precursor salt. For example, acetic acid can be produced by thermal denaturation such as sodium acetate, potassium acetate, calcium acetate, lithium acetate, magnesium acetate. Similarly, formic acid can be generated by thermal denaturation of sodium formate, potassium formate, calcium formate, lithium formate, magnesium formate, and the like. In some cases, the pH modifier may not be an acid or a base, but may be in a form that can be converted to an acid or base within the chip or reaction site. For example, the pH modifier may react with water to form an acid or base within the chip or reaction site. In a non-limiting example, a gas such as CO ₂ can react with water to produce carbonic acid, for example:
CO ₂ + H ₂ O <—> H ₂ CO ₃ <—> H ⁺ + HCO ₃ ⁻
In yet another set of embodiments of the present invention, this factor may be present in a compartment that is not in fluid communication with the reaction site; within the reaction site, to the reaction site to change or control environmental factors. If exposure of this factor is desired, a fluid passageway can be created to allow this factor to enter or otherwise interact with the reaction site. For example, the created liquid passage may be a new passage, i.e. a non-existing passage, or a passage made in an area that does not previously contain a passage of liquid; It may be made in an area previously provided with a fluid passageway that has been modified to prevent fluid communication. In some cases, a new passage is created within the chip by removing or damaging the chip components, such as the layers, membranes, walls or channels in fluid communication with the reaction site that define the reaction site. May be. In another example, the liquid passage may be a closed existing liquid passage, such as a valve or switch, that can be opened and / or modified under certain conditions. In one embodiment, the compartment is a sealed compartment, eg, a compartment without access to the external environment and / or reaction site. In another embodiment, the compartment is accessible from the outside (ie, through the inlet or outlet) but is not in fluid communication with the reaction site.

  The chip of the present invention may comprise one or more liquid passages for species delivery or removal of species from the reaction site. In some cases, the liquid passageway separates the compartment from the reaction site (eg, as a wall or membrane), or is in fluid communication with the reaction site and permeates components that separate the compartment from the liquid passageway, or It can be made in situ by damage (after chip construction, during chip deployment and / or during chip use). For example, in certain embodiments of the invention, a liquid passageway or other means for liquid communication includes compartments containing this factor (and / or precursor (s) of the factor) from liquid communication with the reaction site. Permeates or damages (reversibly or irreversibly) components that separate the compartment from channels or other liquid passages that are in fluid communication with the reaction site, thereby reacting with the compartment. It can be made by creating a fluid communication between the sites. For example, the component can be infiltrated by heating the component to increase the permeability of the chemical agent or by melting or vaporizing the component. In some cases, the permeability of this component can be increased to a magnitude of 1, 2, or 3 or more. In some cases, the permeability of this component may be reversible or at least partially reversible, for example, by reducing the temperature or by introducing an impermeable factor.

  This component is in some cases damaged through a reaction, such as a chemical or electrochemical reaction, or otherwise altered or permeable to create a liquid connection with the reaction site. Also good. For example, the component may include a metal, such as gold, silver, or copper, that can be electrolyzed when an appropriate current is applied. As yet another example, the component may be chemically etched using, for example, reactive species.

  In still other embodiments, components such as those discussed above may be mechanically modified and created, for example, by creating a liquid passage between the compartment and the reaction site through the component having microneedles. And / or may be damaged. Microneedles or other mechanical devices may come from inside or outside the chip. In one embodiment, the component may be reversibly changed, for example, the component may be self-sealing and / or include an elastomeric material that may be resealed.

  This component can also be damaged in some cases without mechanical force or use of chemicals. For example, energy may be applied to the surface to damage it. In certain embodiments, this component may be cut using, for example, heat or light. If light is used, this light may in some cases be directed through a waveguide to the surface, or the light may be applied directly to this surface.

  This component may include a material that may enhance the creation of the liquid passage in certain embodiments of the invention. For example, the enhancer can facilitate the absorption of light or other forms of energy, or increase the chemical reaction or transport rate. For example, in one embodiment, this component is a material that can absorb incident electromagnetic radiation, ie, a darkened or “black” material, such as quartz, black glass, silicon, black sand, carbon black. Etc. may be included. In other examples, the component may include a catalyst, enzyme, or permeation enhancer.

  In one aspect, the invention relates to a chip in which the gas or humidity can be controlled. The present invention allows, in some cases, humidity control to be passively incorporated into a chip that can be used, for example, to perform chemical or biochemical reactions or cultured cells. In one embodiment, humidity control or maintenance may be provided to the chip in the form of a humidity controller and / or film that has a low water permeability relative to the oxygen permeability, if desired. As used herein, a “humidity controller” allows certain gases, such as oxygen, carbon dioxide or nitrogen, to enter the chip but inhibits the passage of water vapor to the chip. It is a device. This humidity control device may allow a small amount of water vapor to pass through the chip, but does not allow at least one other gas, such as the above gas, to enter the chip. Examples include, but are not limited to, membranes and thin layers (eg, films having a thickness of less than 2 mm). In certain embodiments, the humidity control device may be located as or on the wall of the chip, such as in the wall of the reactor unit or reaction site. In other embodiments, the humidity control device may be positioned in liquid communication with one or more reaction sites. In certain embodiments, each reaction site in the chip may be adjacent to the humidity controller and / or in fluid communication. In some cases, the humidity control device may substantially seal at least a portion of the chip.

  The humidity control device of the present invention may comprise a humidity control material designed to maximize gas passage therein and / or minimize water vapor passage. The humidity control material of the present invention may allow the passage of certain desired gases, such as oxygen and / or carbon dioxide, but inhibits the passage of other gases, such as water vapor. The material of the present invention is suitable for use as a humidity control device in a chip, but is not limited to such use; rather, this material holds water vapor or other specific gases in or out. Can be used anywhere that allows the passage of oxygen and / or other gases. For example, the humidity control materials of the present invention may be useful in greenhouses or wound dressings.

  In one set of embodiments, the humidity control material may comprise a membrane or thin film selected to control the passage of gas and / or water vapor therethrough. In one embodiment, the humidity control device is a membrane or thin film that has the desired permeability to one or more gases. This membrane or film may be placed on any of the chips that can affect one or more reaction sites in a manner. For example, the membrane or thin film may be arranged such that it defines the surface of one or more reaction sites.

  In one set of embodiments, the membrane or thin film is greater than about 10 μm, in some cases greater than about 25 μm, in some cases greater than about 50 μm, in some cases, as further described herein. A thickness that is greater than about 75 μm, in some cases greater than about 100 μm, or in some cases greater than about 150 μm, but still allows sufficient oxygen permeation therethrough, for example to allow cell culture. Have In some cases, a film or thin film having a thickness greater than about 50 μm may be particularly useful, for example, during chip manufacture. This film may in some cases have a thickness of less than 1 or 2 μm.

  In some cases, it may be desirable to incorporate a humidity control material into the structural shape of the chip or to incorporate the structural shape of the chip into the humidity control material. If the humidity control material is intended to provide or complement the support, or if itself is not fully supported, the humidity control material may also comprise a support layer. The support layer may include any material or materials that provide the desired support. For example, the support layer may include one of the layers that may be included in the humidity control material for permeability, such as polydimethylsiloxane or polyfluoro organic material, or the support layer may be a different material. , For example, glass (eg, PYREX® glass by Corning Glass of Corning, NY; or indium / tin coated glass), latex, silicon, and the like. This support layer may be located anywhere within the humidity control material, for example, as an outer layer or an intermediate layer, and may be arranged to help protect one or more delicate layers. In some cases of the present invention, the use of a support layer may allow most or nearly all of the reaction sites, reactors or chips to be constructed as humidity control materials. Preferably, this support layer does not significantly affect the permeability of the humidity control material, or the change in permeability can be accounted for in the design of the humidity control material.

  If the chip of the present invention is intended for use with a substance such as a reactant that can damage, reduce function, otherwise react with or degrade the humidity control substance, The membrane may comprise a protective layer. This protective layer may be located as an optional component of the humidity control substance, for example, as a surface layer, or intervened between the sensitive area of the humidity control substance and the substance or environment that can adversely affect it. Also good. For example, the protective layer may be disposed on the inside surface of the humidity control material, particularly if the hazardous material is inside the chip, or on the outside surface of the humidity control material, especially if the harmful material is outside the chip. May be arranged. This protective layer may also be placed between other layers as long as it can perform a protective function. Preferably, this protective layer does not significantly affect the permeability of the humidity control material, and the change in permeability can be accounted for in the design of the humidity control material.

  For example, a chip 140 comprising a humidity control device according to one embodiment of the present invention is illustrated in FIG. 9A. The chip includes a reaction site 142, an inlet 144, an outlet 146 and an inner wall 148. The inner wall 148 is defined on one side of the humidity control device 150. In this embodiment, the humidity control device 150 includes a film having a first layer 152 and a second layer 154.

  Another embodiment of a chip 140 with a humidity control device is illustrated in FIG. 9A. In this embodiment, the humidity controller 150 includes a multilayer film that defines the walls of the reaction chamber 142 and also defines the walls of the inlet and outlet. In addition to the first and second layers 152 and 154 provided primarily for the purpose of providing the desired permeability, the membrane also supports a support located between the first and second layers 152 and 154. Layer 156 is provided. Other arrangements for the permeability control layer (s) and support layer (s) are possible. Also provided in the chip 140 in this particular example is a cell adhesion layer 158 located on the inner wall 148 of the reaction site 142, which promotes cell growth there, but not the inlet 144 and outlet 146. . In other embodiments, the cell adhesion layer may extend further beyond or throughout the surface of the humidity control device 150. It is also understood that the geometry of the tip 140 as shown in FIGS. 9A and 9B is shown for illustrative purposes only, and that many other arrangements and tip shapes may be useful in certain embodiments. It should be.

  In one set of embodiments, the humidity control material is selected to have a specific permeability and / or a specific transmission rate. As used herein, the “permeability” of a substance is described as its inherent meaning as used in the art, ie, an inherent property that generally represents the ability of a gas to pass through the substance. The In contrast, as used herein, the “permeance” of a material is the actual rate of gas transport through the sample of material, ie, an external property. The permeation rate of a material sample is affected by factors such as the area or thickness of the material, differential pressure across the material, and the like. For example, FIG. 11 shows that the oxygen transmission rate of the two membranes depends on the thickness of the membrane.

The chip of the invention, in one set of embodiments, is greater than about 3.9 × 10 ⁻⁸ cm ³ / s, and in some cases, greater than about 4.3 × 10 ⁻⁸ cm ³ / s against oxygen. Humidity control material having permeability and / or permeability to water vapor less than about 1.7 × 10 ⁻⁷ cm ³ / s and in some cases less than about 1.0 × 10 ⁻⁷ cm ³ / s ( For example, a film or a thin film) may be provided. Oxygen control is used herein as an example, but other gases, such as nitrogen or carbon dioxide, may be controlled instead, as described above, or a combination of gases may be controlled. It should be understood that it is good. In the cell examples described further below, the lower limit of oxygen transfer and the upper limit of water vapor transfer may typically be desired to be controlled, but in other applications, such as chemical synthesis operations, other parameters It should also be understood that it may be desirable to control, for example, the upper limit of oxygen transfer and the lower limit of water vapor transfer, or the lower and upper limits of other gases such as nitrogen or carbon dioxide.

  The humidity control materials of the present invention can be used in a wide variety of reactions and interactions. An example of a reaction is, for example, a cell culture to maintain a cell culture, to increase the number of effective cells or cell types, or to produce a desired cell product. In some cases, the humidity control material may allow sufficient oxygen to enter by diffusion through it to support cell growth. In some cases, the humidity control material may also be highly impermeable to microorganisms and to other cells, for example to prevent contamination. Preferably this substance is less toxic.

  In embodiments where the present invention is used with cultured cells, cell culture may be performed over various lengths of time, depending on the cells being cultured and other factors known to those skilled in the art. Thus, the chip design and the nature of the humidity control material can be adapted to the incubation time. For example, the chip or humidity control material may be designed such that it can resist the time required for incubation and preferably it may be reused many times. In various embodiments, the cell culture is 24 hours, 48 hours, 1 week, 2 weeks, 4 weeks, 6 weeks, 3 months, 1 year, continuous, or any other required for a particular cell culture. It may be done for hours.

  In some cases, the humidity control material is selected to have a permeability and / or permeation rate for one or more gases that correspond to a range suitable for culturing particular cells. For example, the humidity control substance has a sufficiently high permeability and / or permeation rate for oxygen and / or a sufficiently low permeability and / or permeation rate for water vapor to allow cell culture. Also good. Examples of such permeability include the above-described permeability. Those skilled in the art will be able to permeate specific cells and cell lines, and specific ranges of specific materials suitable for the successful cultivation of larger cell populations, such as microorganisms and mammalian cells, tissues, tissue engineering constructs, etc. Sex can be identified.

  Thus, in one embodiment, the present invention is a method for identifying oxygen and humidity requirements of a particular cell, high enough oxygen permeability to meet the oxygen requirement of the cell and low enough to meet the humidity requirement of the cell It includes a method for selecting a substance having water vapor permeability, and a method for culturing cells in a chip including a reaction site. This reaction site is formed at least partially from a selected material.

An example of the permeability range of a humidity control material for use in the present invention, eg, for use in culturing a broad range of cells, is about 100 (cm ³ _STP mm / m ² atm day) for oxygen. Greater permeability and less than about 6 × 10 ⁻⁶ (cm ³ _STP mm / m ² atm day) for water vapor. As used herein, “STP” refers to “standard temperature and pressure”, which is a temperature of 273.15 K (0 ° C.) and about 10 ⁵ Pa (1 atm). Refers to pressure. In another embodiment, the humidity control material is less than about 100 (cm ³ _STP mm / m ² atm day), and in other embodiments about 30 (cm ³ _STP mm / m ² atm day) or about 10 Permeability to water of less than (cm ³ _STP mm / m ² atm day), and at least less than about 6 × 10 ⁶ (cm ³ _STP mm / m ² atm day), and in some embodiments 1 × 10 ⁷ (Cm ³ _STP mm / m ² atm day) and in other embodiments about 3 × 10 ⁷ (cm ³ _STP mm / m ² atm day) or 1 × 10 ⁸ (cm ³ _STP mm / m ² atm day) It can have oxygen permeability. Any combination of oxygen permeability and water vapor permeability listed herein may be used. For microbial cells, examples of suitable ranges of oxygen permeability are provided by membranes having oxygen permeability greater than about 1 × 10 ³ (cm ³ _STP mm / m ² atm days) and / or water vapor permeability. Is about 6 × 10 ⁶ (cm ³ _STP mm / m ² atm day). For mammalian cells, examples of suitable ranges are oxygen permeability greater than about 100 (cm ³ _STP mm / m ² atm days) and less than about 1 × 10 ⁵ (cm ³ _STP mm / m ² atm days) Provided by the membrane of the present invention having a water vapor permeability of

For humidity control materials that are permeable to oxygen and water vapor, in certain cases this material is very high oxygen permeability and very low for water vapor, as shown for example in FIG. 12 as “goal” region 80. It may be desirable to be permeable. For example, the material is greater than about 1000 (cm ³ _STP μm / m ² atm day), in some cases greater than about 10,000 (cm ³ _STP μm / m ² atm day), and in some cases about Oxygen permeability greater than 100,000 (cm ³ _STP μm / m ² atm day) and / or less than about 1000 (g μm / m ² day), in some cases less than about 100 (g μm / m ² day), and In some cases, it may have a water vapor permeability of less than about 10 (g μm / m ² days). For example, as illustrated in FIG. 5, high density polyethylene (“HDPE”), polyethylene terephthalate (“PET”), polypropylene (“PP”) or poly (4-methylpentene-1) (“PMP”). Results of such materials are shown, and these may be suitable for use with the present invention, as further described below. Other materials and combinations of materials are also contemplated, for example, as described further below.

  In certain embodiments, the humidity control material does not promote cell adhesion, but a cell adhesion layer (or cell) that can be any of a wide variety of hydrophilic, cytophilic and / or biocompatible materials. An adhesive layer may be provided on this material). Examples of materials that may be suitable for cell adhesion layers on humidity control materials include, but are not limited to, polyfluoroorganic materials, polyester, PDMS, polycarbonate, polystyrene, and aluminum oxide. As another example, the humidity control material may include a layer coated with a material that promotes cell adhesion, eg, using an RGD peptide sequence. In certain embodiments, it may be desirable to modify the surface of the cell adhesion layer, for example, by attachment, bonding, dipping or other treatment. Exemplary molecules that promote cell adhesion include, but are not limited to, fibronectin, laminin, albumin or collagen. When the substance includes a cell adhesion layer, the cell adhesion layer may be positioned as an inner layer or a surface layer of the membrane, or may be in contact with the inside of the chip. Preferably, the cell adhesion layer does not significantly affect the permeability or permeation rate of the humidity control material, or the change in permeability or permeation rate can 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 its layers may be selected based on the gas permeability of the material, for example, as described below. The person skilled in the art knows how to determine the gas permeability of a substance. One particular exemplary method includes a sample of material (eg, a membrane) having a known exposed area and thickness between two chambers and a gas (or liquid) in one chamber. Also good. The experimental time taken for the gas (or liquid) to diffuse across this material to other chambers and then detected in an appropriate manner can then be related to the gas (or liquid) permeability of the material. .

  In one set of embodiments, the humidity control material may comprise a polymer (eg, a single polymer type, a copolymer, a polymer mixture, a polymer derivative, etc.). Examples of polymers that can be used in humidity control materials include polyfluoro organic materials such as polytetrafluoroethylene (eg, commercially available as TEFLON® from DuPont, Wilmington, DE, for example, TEFLON® ) AF) or certain amorphous fluoropolymers; polystyrene; PP; silicon, eg, polydimethylsiloxane; polysulfone; polycarbonate; acrylic, eg, polymethyl acrylate and polymethyl methacrylate; polyethylene, eg, high density polyethylene ( “HDPE”), low density polyethylene (“LDPE”), linear low density polyethylene (“LLDPE”), ultra-low density polyethylene (“ULDPE”), etc .; PET; polyvinyl chloride (“PVC”) Substance, for example, Midland, Dow Chemical Co. of MI Such as those commercially available under the name of SARAN® by NON; nylon as commercially available as DARTEK® from Dupont; thermoplastic elastomers and the like. Another example of a suitable material is the BIOFIL® polymer membrane made by VivaScience (Hanover, Germany). In one embodiment, the polymer is poly (4-methylpentene-1) (“PMP”):

であってもよく、これはある場合には、約３１７．２（ｍ^３ _ＳＴＰｍ／ｓ ｍ Ｐａ）という酸素についての透過効率を有し得る。ＰＭＰの例としては、Ｍｉｔｓｕｉ Ｐｌａｓｔｉｃｓ（Ｗｈｉｔｅ Ｐｌａｉｎｓ，ＮＹ）からＴＰＸ（商標）の名称で市販されるものが挙げられる。他の実施形態では、ポリマーは、ポリ（４−メチルヘキセン−１）、ポリ（４−メチルヘプテン−１）ポリ（４−メチルオクテン−１）などであってもよい。別の実施形態では、このポリマーは、ポリ（１−トリメチルシリル−１−プロピエン）（「ＰＴＭＳＰ」）：

Which in some cases may have a transmission efficiency for oxygen of about 317.2 (m ³ _STP m / s m Pa). Examples of PMP include those commercially available under the name TPX ™ from Mitsui Plastics (White Plains, NY). In other embodiments, the polymer may be poly (4-methylhexene-1), poly (4-methylheptene-1) poly (4-methyloctene-1), and the like. In another embodiment, the polymer is poly (1-trimethylsilyl-1-propene) (“PTMSP”):

であってもよく、これはある場合には、約５．７８×１０^５（ｃｍ^３ _ＳＴＰｍｍ／ｍ^２日ａｔｍ）という酸素についての透過効率を有し得る。ある場合には、これらのコポリマーおよび／または他のポリマーが、湿度制御物質において用いられてもよい。

Which in some cases may have a transmission efficiency for oxygen of about 5.78 × 10 ⁵ (cm ³ _STP mm / m ² days atm). In some cases, these copolymers and / or other polymers may be used in humidity control materials.

  Of course, the first and second layers may also each comprise a mixture of materials in certain embodiments. For example, a layer may include at least 50% by weight of a material using an equilibrium that includes one or more other materials. In another embodiment, each layer consists essentially of a single material.

  In certain embodiments, the area and thickness of the humidity control material, or layers or portions thereof, can be used to select the desired degree of transmission rate and / or permeability. In one example, the additional water vapor permeable material may be made thicker or smaller to reduce the amount of water vapor that reaches or exits the area or region where humidity control is desired. Good. In some cases, this material may be designed to be about 10 μm to 2 mm thick. Within this range, the relative thickness of the layers in the multilayer or a portion of this material may vary. For example, a relatively thin layer of polyfluoro organic material and a relatively thin layer of vinylidene chloride may be useful in certain embodiments. As a further example, 2-3 μm polytetrafluoroethylene may be deposited on or coated on a layer of polydimethylsiloxane, or 2-3 μm HDPE may be co-molded using PDMS. May be.

In some cases, the polymer (or mixture of polymers) used in the humidity control material may be sufficiently hydrophobic so that the polymer can retain water (ie, water vapor readily passes through the polymer). Can not). For example, the permeability of water through the hydrophobic polymer, as described before, about 1000 (gμm / ^{m 2} day), about 900 (gμm / ^{m 2} day), about 800 (gμm / ^{m 2} day), It may be about 600 (gμm / m ² days) or less.

In certain embodiments, the polymer (s) used in the humidity control material may have a molecular structure that is sufficiently open to allow oxygen to pass therethrough easily. For example, the molecular structure may allow for the movement of oxygen across the polymer, as described previously, of about 1000 (cm ³ _STP μm / m ² days atm) or greater. In one embodiment, the polymer has an oxygen transfer through the polymer under environmental conditions of, for example, about 1000 (cm ³ _STP μm / m ² days atm) or 500 (cm ³ _STP μm / m ² The structure is substantially branched so that a structure (for example, a dense crystal structure) limited to less than (day atm) cannot be formed.

In another embodiment, the polymer may include bulky groups that prevent easy formation of structures that limit oxygen movement therethrough under environmental conditions. As used herein, a “bulky group” on a polymer refers to the movement of oxygen through it under environmental conditions of about 1000 (cm ³ _STP μm / m ² days) or 500 (cm ³ _STP μm / m ² days) is a sufficiently large part that the polymer cannot form a crystal structure that is limited to less than This bulky group may be part of the backbone of the polymer or side chain, for example. Non-limiting examples of bulky side groups include groups containing a cyclopentyl moiety, isopropyl moiety, cyclohexyl moiety, phenyl moiety, isobutyl moiety, tert-butyl moiety, cycloheptyl moiety, trimethylsilyl or other trialkylsilyl moieties, etc. Is mentioned. For example, in one set of embodiments, the polymer has the structure:

を有してもよく、この各々のＲは独立して少なくとも１つの原子を含み、そしてＢｋはかさ高い基である。ある場合にはＲは、水素基であってもまたはアルキル基であってもよい。

Wherein each R independently contains at least one atom and Bk is a bulky group. In some cases, R may be a hydrogen group or an alkyl group.

  Of course, it should be understood that the polymer may have some or all of the above characteristics. For example, the polymer may be a polymer mixture or copolymer that is sufficiently hydrophobic so that it can retain water while having a sufficiently open molecular structure that allows sufficient oxygen permeability therethrough. .

  In one embodiment, the present invention achieves the goal of permeability 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 can also be used in other embodiments. By combining various materials and adjusting their relative thicknesses, the desired oxygen and water vapor permeability can be achieved. In one embodiment where the humidity control material comprises two layers or portions, they may be formed from polymers of the same material or different materials. For example, the humidity control material may comprise a first layer comprising at least about 55% by weight of the first polymer or copolymer and a second layer comprising no more than about 45% by weight of the first polymer or copolymer. Good. In another example, the humidity control material comprises a first layer comprising at least about 60%, about 70%, or about 80% by weight of the first polymer or copolymer and about a first polymer or copolymer. And a second layer comprising no more than 40%, about 30% or about 20% by weight. In certain embodiments, the first polymer may comprise about 100% of the first layer and essentially no second layer. In some cases, at least a portion of this first layer may be copolymerized using a second layer.

  Where the humidity control material of the present invention is constructed as a membrane comprising two or more layers, the two or more layers can be joined in any manner that provides sufficient strength to the membrane. In some cases, the two or more layers may be sufficiently self-supporting and may not need to be bonded to the layer, which means that space is desired if desired. It means that it may be left in the meantime. In other embodiments, additional layers can be used to support the membrane. In embodiments where it is desired to connect two or more layers to provide a mutual support or another, an example of an acceptable means of connecting the layers is to layer the layers together. Including mixing, at least partially mixing the layers, and copolymerizing the layers together. When the layers are mixed, the resin forming each layer can be mixed partially or wholly before the film is formed. For example, the liquid prepolymer may be mixed and then the culture agent added, or the two partially cured layers may be connected between them with a curing agent that cures the layers together. Good.

  In another set of embodiments, the humidity control material of the present invention allows light to pass through it. This makes it possible to use this material when light is important, for example to promote reactions such as photocatalysis, to promote cell or plant growth, to cause biochemical changes, etc. Can be. This material may also allow observation of areas such as reactors or reaction sites that are protected by humidity control substances or placed behind humidity controlled areas. In one embodiment, the humidity control material is translucent, and in some cases is at least substantially transparent. One skilled in the art will recognize that there are varying degrees of translucency or transparency and can select the desired properties based on the particular application.

  The chip may comprise various other components. For example, the chip may be, for example, a light source, a flow meter (eg, for measuring gas or liquid flow), a circuit such as a self-contained circuit, a reservoir (eg, for a solution), as described further below. , Components such as micromachines or MEMS (“microelectromechanical system”) components, microvalves, micropumps, and the like. This component can be fabricated on the chip using the same techniques used in standard microfabrication, similar to those used to fabricate semiconductors (Madou Fundamentals of Microfabrication, CRC Press, Boca Raton, FL 1997; and Maluf, An Introduction of Micromechanical Systems Engineering, Artech House Boston, MA 2000). In some embodiments, at least one, two, three or more components are integrally connected to the chip. In certain embodiments, all of this component is integrally connected to the chip.

  Other examples of components suitable for use with the present invention include a pylon-like blockage placed in the flow path of the chip, reactor and / or stream to enhance mixing within the reaction site, or a chip, reactor And / or heating, separation and / or dispersion units within the reaction site. For example, if a heating unit is present, this heating unit may be a miniaturized traditional heat exchanger.

  For example, in one set of embodiments, the present invention comprises a membrane, such as a membrane that can control humidity (eg, as previously described) and / or may be substantially transparent. Also good. If a membrane is present, it may be located in any of the reactors in the chip. In one embodiment, the membrane is positioned to define the surface of one or more reaction sites and / or to divide the reaction site into two or more parts that may have the same or different dimensions. . For example, in FIG. 10A, a membrane 410 that can be a humidity controller and / or can be substantially transparent defines the surface of the reaction site 411. In FIG. 10B, membrane 410 defines the surface of reaction site 411 and the surface of reaction site 412. In another example, the membrane may be placed in liquid communication with one or more reaction sites of the chip. In some cases, the membrane may be arranged such that a passage connecting the first reaction site and the second reaction site across the membrane crosses the membrane. In another embodiment, the membrane may be placed in liquid communication with one or more reaction sites of the chip. In some cases, the membrane may be arranged such that a passage connecting the first reaction site and the second reaction site across the membrane crosses the membrane. For example, in FIGS. 10C and 10D, membrane 410 does not define the surface of reaction site 411 or 412 but is arranged such that at least one passage that fluidly connects reaction site 411 and reaction site 412 traverses membrane 410. The

  In one example, in one embodiment, the membrane may be a porous membrane having a number of average pore sizes, for example, greater than about 0.03 μm and less than about 5 μm. In other embodiments, the pore size of the membrane is less than about 4 μm, less than about 3 μm, less than about 2 μm, less than about 1.5 μm, less than about 1.0 μm, less than about 0.75 μm, less than about 0.6 μm, about It may be less than 0.5 μm, less than about 0.4 μm, less than about 0.3 μm, less than about 0.1 μm, less than about 0.07 μm, and in other embodiments less than about 0.05 μm. In certain cases, the pores are also greater than 0.03 μm or greater than 0.08 μm. In some cases, the membrane may be selected to prevent passage of specific cells therethrough (eg, bacterial cells, yeast cells, mammalian cells, etc.). For example, a membrane with a pore size of about 0.2 μm can prevent the passage of bacterial cells, and a membrane with a pore size of about 1 μm can prevent passage of mammalian cells. In certain embodiments, the membrane may be selected to prevent or allow passage of certain molecules, such as macromolecules having a certain size and / or charge, ie, a charge and / or size selective membrane. .

  The membrane may be a polymer or other material such as polyethylene terephthalate (PET), polysulfone, polycarbonate, acrylic, such as polymethyl methacrylate, polyethylene, polypropylene, regenerated cellulose, nitrocellulose, aluminum oxide, glass, glass fiber, etc. , May include them. In certain embodiments, the membrane may also be substantially transparent, for example, as previously described. In one embodiment, for example, the membrane is a substantially transparent polyethylene terephthalate membrane having a pore size of 2 μm or less, such as Oxyphen US S. A. , Inc. It is a ROTRAC (registered trademark) capillary membrane manufactured by (New York, NY).

  In one set of embodiments, the chip of the invention may comprise a structure adapted to promote a reaction or interaction intended to occur therein (eg, within a reaction site). For example, if the chip is intended to function as one or more bioreactors for cell culture, the chip comprises a structure or structures that can improve or promote cell growth. Also good. For example, in certain embodiments, the surface of the reaction site may be a surface that can promote cell binding or adhesion, or the reactor and / or reaction site in the chip may be any of a wide variety of hydrophilic, cellular, A structure including a cell adhesion layer that may include an affinity and / or biocompatible substance may be provided. For example, the surface can be ionized, coated with any of a wide variety of hydrophilic, cytophilic and / or biocompatible materials, such as materials having exposed carboxylic acid, alcohol and / or amino groups. It may be micropatterned (eg in a support material). Examples of materials that may be suitable for the cell adhesion layer include, but are not limited to, polyfluoro organic materials, polyesters, PDMS, polycarbonate, polystyrene, and aluminum oxide. In another example, the structure may include a layer that is coated with a substance that promotes cell adhesion, such as an RGD peptide sequence, or the structure may be treated in a manner that can promote cell adhesion, For example, the surface may be treated such that the surface is relatively more hydrophilic, cytophilic and / or biocompatible. In certain embodiments, it may be desirable to modify the surface of the cell adhesion layer, for example, with an agent that promotes cell adhesion, for example, by attachment, bonding, dipping, or other treatment. Exemplary substances that promote cell adhesion include, but are not limited to, fibronectin, laminin, albumin or collagen. For example, in other embodiments where certain types of bacteria or anchorage-independent cells are used, the surface may be formed from a hydrophilic, cytophilic and / or biocompatible material, or the surface May be treated in some way, making it hydrophilic, cytophilic and / or biocompatible, for example by using aliphatic hydrocarbons and / or fluorocarbons.

  In some embodiments, the chip is a “light-interacting component”, eg, producing light, reacting to light, producing a change in the characteristics of light, directing light. Doing, changing the light, etc. may include components that interact with the light. As used herein, a “light interacting component” affects a sample in a chip or reactor and / or determines something about the sample (existence of sample, sample characteristics, etc.) In some manner relating to the chip and / or reactor function, for example by generating light, reacting to light, causing changes in the properties of light, directing light, changing light, etc. Is a component that interacts with. In one embodiment, this component produces light, such as in light emitting diodes (“LEDs”) or lasers. In another embodiment, the light interacting component may be a light sensitive or light responsive component, such as a photodetector or a photovoltaic cell. In yet another embodiment, the light-interacting component is in some way, for example, to focus or collimate the light, or to split the light, as in a lens, or to a diffraction grating Alternatively, the light can be manipulated or modified by spectrally dispersing the light, as in a prism. In another embodiment, the light-interacting component changes the direction of the light in some way, for example along a bent path or along a corner, for example in a waveguide or mirror. Or it may be possible to redirect. In yet another embodiment, the light interacting component may change the characteristics of light incidence on the component, such as the degree of polarization or frequency, as in a polarizer or interferometer, for example. Other devices or combinations of devices are possible. In general, the term “light interacting component” refers to a component or device that passively transmits light without significant modification, alteration or redirection, eg, air, or flat glass or plastic (eg, , “Window”). The term “light-interacting component” also generally does not include components that passively absorb substantially all incident light without response, as found in opaque materials.

  In embodiments where the light interacting component is provided in combination with a reactor, it may be placed either on or in the reactor. For example, the light interacting component may be placed in or proximal to the reaction site. In some cases, the light interacting component is integrally connected with the reaction site, for example, as a wall or surface of the reaction site.

  In another example, the light-interacting component is either in the chip or with the chip so that at least a portion of the light entering the light-interacting component is in optical communication with the reaction site. They can be connected together. As used herein, the term “optical communication” generally refers to any path that provides electromagnetic radiation, eg, transmission of visible light. Optical communication includes direct “line-of-sight” communication. Optical communication can also be facilitated by the use of optical devices such as lenses, filters, optical fibers, waveguides, diffraction gratings, mirrors, beam splitters, prisms and the like. In certain embodiments, the light interacting component may direct light to or from two or more reaction sites, or the light interacting component may react from two or more light sources to the reaction site. You may direct the light. In certain embodiments, there may be more than one light interacting component.

  This optically interacting component may comprise a waveguide in some cases. As used herein, the term “waveguide” is given its ordinary meaning in the art and may include optical fibers. A waveguide generally receives light and directs a portion of that light to a target site that is not within “view direction” communication, eg, along bends, corners, and similar obstacles without substantial loss. Can guide or transmit to the light (however, the waveguide can transmit light to the viewing direction region).

  In certain embodiments, the waveguide is a “core” of material that is at least partially embedded or surrounded by a second “cladding” material that may have a lower refractive index. An area may be provided. The core may have any shape, for example, a slab (slab), strip (strip), or cylindrical material.

  The waveguide, or at least a portion of the waveguide, can be constructed from any material capable of transmitting or illuminating light to or from the reaction site. The waveguide may be substantially transparent or translucent in some cases. In some embodiments, the waveguide is a silicon-based material, such as glass, ion-implanted glass, quartz, silicon, silicon oxide, silicon nitride, silicon carbide, polysilicon, coated glass, conductive glass, indium tin oxide. You may form from glass etc. In other embodiments, the waveguide may include other transparent or opaque organic or inorganic materials. For example, in certain embodiments, waveguides include, but are not limited to, polyacrylates, polymethacrylates, polycarbonates, polystyrene, polypropylene, polyethylene, polyimides, polyvinylidene fluoride, ion exchange polymers, and fluorinated derivatives as described above. It may contain polymers that are not. Combinations, mixtures or copolymers are also possible.

  In one embodiment, the waveguide or portion thereof may be surrounded or coated with a highly reflective material, such as silver or aluminum. In another embodiment, the waveguide is formed to include a central material (eg, a core) having a first refractive index and a peripheral material (eg, a coating material) having a second refractive index. Also good. The coating material may have a refractive index that is less than the refractive index of the central substance. In yet another embodiment, the refractive index of the core or coating material can vary across the cross section. In one example, the core may be a graded optical fiber whose refractive index is typically highest near the center of the core.

  Under these conditions, a substantial portion of the movement of light through the central material can be internally reflected as a result of this refractive index difference (“total internal reflection”). The electromagnetic radiation entering one end of the waveguide can be confined to the central region due to the phenomenon of total internal reflection at the core-coating material boundary. Light can be transmitted through the core without significant absorption by the coating material or other ambient material until the end of the waveguide or a predetermined region of the waveguide from which the light exits. The movement of light through the central region can be detected along corners and other obstacles without significant loss of intensity, for example with an attenuation coefficient of less than about 10 db / cm or 20 db / cm. In another embodiment, the waveguide may have more than one central material or more than one peripheral material.

  As an example of a waveguide, both the central and surrounding materials that form the waveguide may each be glass. In another example, the waveguide may be formed from a polymer and silicon-based material such that a material with a low refractive index surrounds a material with a higher refractive index. In yet another example, the waveguide may be constructed from, for example, a single material surrounded by air or a portion of a chip having a higher refractive index than the waveguide, resulting in waveguide / air. Or a condition occurs where total internal reflection can occur at the waveguide / chip interface.

  The waveguide may be formed by any suitable technique known to those skilled in the art, for example, by rolling, polishing or machining (eg, cutting or etching the channel into the chip substrate, and then using a sealant as necessary. , By depositing a substance in the channel). Waveguides can also be formed, for example, by depositing a layer of material during the chip manufacturing process. In some cases, this deposited material may have a higher refractive index than the surrounding reactor substrate, thus forming a common core-coating material structure, as previously described. The waveguide can also be constructed by laser etching of the material forming the chip, such as glass or plastic, in such a way as to manipulate / change the refractive index relative to the surrounding material. In some cases, the refractive index of this etched / unetched portion may be controlled to form a core-coating material structure.

  In some embodiments, the light interacting component may be a light source or a light source. The light source may be any light source that is in optical communication with the reaction site. For example, the light source may be external or ambient light, a coherent or monochromatic beam of light as produced in an LED, or a laser such as a semiconductor laser or quantum well laser. The light source may be connected integrally with a part of the chip, for example in a laser diode manufactured as part of the chip, or the light source may be separated from the chip or connected integrally therewith. However, it may be arranged to allow optical communication with the reaction site. The light source may produce a single wavelength or a substantially monochromatic wavelength, or a wide range of wavelengths, as previously described. In certain embodiments, the light source is also involved in a chemical reaction or biological process, such as a chemical reaction that produces a photon, such as GFP (“green fluorescence protein”) or luciferase, or It can be produced in a reaction through fluorescence or phosphorescence. For example, incident electrons, currents, friction, heat, chemical reactions or biological reactions can be performed, for example, in a sample located at the reaction site or in a chip in optical communication with the reaction site. From which can be added to produce light.

  In certain cases, the light interacting component may comprise a filter, such as a low pass filter, a high pass filter, a notch filter, a spatial filter, a wavelength selective filter, and the like. The filter can, for example, substantially reduce or eliminate some of the incident light. For example, the filter can eliminate or substantially reduce light having a wavelength less than about 350 nm or greater than about 1000 nm. In another embodiment, the filter may be able to reduce noise in the incident light or increase the signal to noise ratio of the incident light. In yet another embodiment, the filter may be able to polarize incident light, for example, linearly or annularly.

  In certain embodiments, the light interacting component may comprise an optical element in optical communication with the reaction site. As used herein, an “optical element” is a path of light that enters or exits this optical element, for example, by focusing or collimating light, or by splitting light. Refers to any element or device that can be modified. For example, the optical element may focus the incident light at a single point or small area, or the optical element may collimate or redirect the branched beam of light to collimate or redirect the light. Can be formed. The term “focus” generally refers to the ability to focus a line of light onto a point or small area. The term “collimate” generally refers to the ability to improve the focusing of a line of light, but not necessarily to a point or small area, for example, so that the beam is focused at an infinite distance. In one example, the diverging light beam may be collimated to a parallel beam of light. In certain embodiments, the optical element may disperse or diverge light, such as in a diverging lens. In other embodiments, the optical element may be, for example, a beam splitter, optical coating (eg, dichroic, antireflective or reflective coating), optical diffraction grating, diffraction grating, and the like.

  In one set of embodiments, the optical element may be a lens. The lens may be any lens such as a condensing lens or a diverging lens. The lens may be, for example, a meniscus (concave / convex lens), plano-convex lens, plano-concave lens, biconvex lens, biconcave lens, Fresnel lens, spherical lens, aspheric lens, binary lens, and the like. The optical element may also be a mirror, such as a plane mirror, a phase mirror, a parabolic mirror, and the like. In other embodiments, the optical element may disperse light, such as in a diffraction grating or prism.

  In some cases, materials having various refractive indices may be used. For example, in embodiments where light reaches the optical element through a waveguide, the optical element can be a material having a different refractive index than the waveguide. In some cases, the refractive index of the optical element is approximately the same as or greater than the refractive index of the waveguide.

  In some cases, a material having a graded refractive index (“GRIN” material) may be used as the optical element. This GRIN material can minimize the amount of diffusion inherent in the light reaching the GRIN material. For example, a uniform thickness material may be made to function as a lens by changing its refractive index along the cross section of the element. In one embodiment, the GRIN material may redirect the diffused lines of light to a parallel arrangement. In another embodiment, the GRIN material need not have a uniform thickness, and a combination of the graded refractive index of the material and the shape of the material can be used to focus or collimate the light.

  This light-interacting component is, in one embodiment, a component capable of converting light into electricity, such as a photosensitive element or photodetector, a photomultiplier tube, a photocell, a photodiode, such as an avalanche photodiode, photo A diode array, a CCD chip (“charge-coupled device”), and the like may be provided. This component may in some cases be related to the state of the substance in the reaction site, for example by radiation (including fluorescence or phosphorescence), absorbance, scattering, optical density, polarimetry or other measurements, including those by the naked eye. Can be used to determine conditions.

  In other cases, the light interacting component may be used for imaging purposes, e.g., to image a reaction site or a portion of a cell or other substance located proximally thereof, or a cell It can be used to determine whether it is adhered to a surface.

  In some cases, light interacting components can be used to generate electricity. In one embodiment, the photovoltaic cell may be fabricated integrally in a chip using one or more layers that include a semiconductor material.

  In certain embodiments, light can be directed to a reaction site, for example, to activate or inhibit a chemical reaction. For example, the reaction can require the use of light for activation, or the photosensitive enzyme can be inhibited by applying light to the enzyme. In certain embodiments, light directed at the reaction site may be used as a probe or signal source. The light can be delivered in a controlled manner to the reaction site in certain embodiments, for example, so that light reaching the reaction site has a particular wavelength, polarization, or intensity.

  In certain embodiments, a portion of the light originating from the reaction site can be detected and analyzed. The light originating from the reaction site may be reflected or refracted light, for example light directed to the reaction as described previously, or this light may be transmitted through physical means, for example, It may be generated through fluorescence or phosphorescence. In some cases, light may be generated within the reaction site as previously described. The light from the reaction site can be any suitable analysis technique, such as infrared spectroscopy, FTIR (“Fourier Transform Infrared Spectroscopy”), Raman spectroscopy, absorptiometry, fluorescence spectroscopy. , Optical density, circular dichroism, light scattering, ellipsometry, refractometry, turbidity measurement, pseudo-electron scattering or any other suitable technique. In another embodiment, imaging of the reaction site can be performed using, for example, optical imaging or infrared imaging.

  In certain embodiments of the invention, the reactors and / or reaction sites in the chip may be constructed and arranged to maintain an environment that promotes the growth of one or more types of living cells, for example, at the same time. In some cases, the reaction site may be provided with conditions similar to those found in living tissue, eg, tissue from which cells are derived, such as fluid flow, oxygen, nutrient distribution. Thus, the chip may be able to provide conditions that are closer in vivo than those provided by a batch culture system. In embodiments where one or more cells are used at the reaction site, the cells may be any cell or cell type, eg, prokaryotic cells or eukaryotic cells. For example, the cells may be bacteria or other unicellular organisms, plant cells, insect cells, fungal cells or animal cells. If the cell is a unicellular organism, the cell may be, for example, a protozoan, trypanosoma, amoeba, yeast cell, algae, and the like. Where the cell is an animal cell, the cell can be, for example, an invertebrate cell (eg, a cell from Drosophila), a fish cell (eg, a zebrafish cell), an amphibian cell (eg, a frog cell), a reptile cell, Avian or mammalian cells such as primate cells, bovine cells, horse cells, pig cells, goat cells, dog cells, cat cells, or rodents such as rats or mice It may be a cell. If the cell is derived from a multicellular organism, the cell may be derived from any part of the organism. For example, if the cells are of animal origin, the cells are cardiac cells, fibroblasts, keratinocytes, hepatocytes, chondrocytes, neurons, bone cells, muscle cells, blood cells, endothelial cells, immune cells (eg, T cells, B cells, macrophages, neutrophils, basophils, mast cells, eosinophils), stem cells and the like may be used. In some cases, the cell may be a genetically engineered cell. In certain examples, the cells may be Chinese hamster ovary (“CHO”) cells or 3T3 cells. In certain embodiments, more than one cell type may be used simultaneously, for example, fibroblasts and hepatocytes. In certain embodiments, cell monolayers, tissue cultures or cell constructs (eg, cells located in non-viable scaffolds) and the like may also be used at the reaction site. The exact environmental conditions required at the reaction site for a particular cell type (s) can be determined by one skilled in the art.

  In certain embodiments, the cells may produce chemical or biological compounds for therapeutic and / or diagnostic purposes, for example, in nanogram, microgram, milligram, or gram quantities. For example, cells can be monoclonal antibodies, proteins such as recombinant proteins, amino acids, hormones, vitamins, drugs or pharmaceuticals, other therapeutic molecules, artificial compounds, polymers, tracers such as GFP ("green fluorescent protein" ) ") Or products such as luciferase can be produced. In one set of embodiments, the cells can be used for drug development and / or drug development purposes. For example, the cell may be exposed to a factor suspected of interacting with the cell. Non-limiting examples of such factors include carcinogenic or mutagenic compounds, synthetic compounds, hormones or hormone analogs, vitamins, tracers, drugs or pharmaceuticals, viruses, prions, bacteria, and the like. For example, in one embodiment, the present invention can be used in automated cell culture to allow high throughput processing of monoclonal antibodies and / or other compounds of interest. In another embodiment, the invention can be used to screen cells, cell types, cell growth conditions, etc. to determine, for example, self-viability, self-generation rate, and the like. In some cases, the present invention can be used in high throughput screening techniques. For example, using the present invention, the effect of one or more selected compounds on cell proliferation, normal or abnormal biological function of the cell or cell type, expression of proteins or other factors produced by the cell, etc. Can be evaluated. The present invention can also be used to study the effects of various environmental factors on cell growth, cell biological function, cell product generation, and the like.

  In some cases, the reactor and / or reaction site in the chip is used to prevent, promote and / or determine a chemical or biochemical reaction using live cells in the reaction site (eg, for live cells). May be constructed and sequenced (to determine the effects, if any, of factors such as drugs, hormones, vitamins, antibiotics, enzymes, antibodies, proteins, carbohydrates, etc.). For example, one or more factors suspected of being able to interact with the cell may be added to the reactor and / or reaction site containing the cell, and the response of the cell to this factor (s) It may be determined using systems and methods.

  In some cases, the cell may be sensitive to light. For example, the cells may be plant cells that respond to photostimulation or are photosynthetic. In another embodiment, light can be used to grow cells such as mammalian cells or plant cells that are sensitive to light. In yet another embodiment, the cells may be bacteria that are attracted or repelled by light. In another embodiment, the cell may be an animal cell having a photoreceptor or other light signaling response, such as a rod cell or a cone cell. In yet another embodiment, the cell degrades or forms a reactive entity upon exposure to a photoreceptor or another photosensitive molecule, such as light, or stimulates a biological process to occur. It may be a genetically engineered cell that has something. In other cases, the cells may be insensitive to light; the light applied to the chip may be used for analysis of the cells, eg, detection, imaging, counting, morphological analysis or spectroscopic analysis. Can be used for In still other cases, the cells may be killed using light, for example, directly or by inducing an apoptotic response.

  In certain embodiments, the chip can be constructed and arranged, for example, such that the cells can grow and divide so that the cells in the chip can be maintained in a metabolically active state. For example, the chip may be constructed such that one or more additional surfaces can be added to the reaction site, eg, as a series of plates, or the chip is constructed to allow cells to divide while attached to the substrate. May be. In some cases, the chip collects cells as needed, for example, through the outlet of the chip or by removing the surface from the reaction site without substantially interfering with other cells present in the chip. And may be constructed so that it can be recovered from the chip. The chip can maintain cells in a metabolically active state at any suitable time, eg, 1 day, 1 week, 30 days, 60 days, 90 days, 1 year or in some cases indefinitely. Can be possible.

  In one aspect, the present invention provides any of the above chips packaged in a kit, optionally equipped with an apparatus for use of the chip. That is, the kit may comprise, for example, instructions for use of the chip, for use with a microplate, or a device adapted to a handle microplate. As used herein, “instructions” may define the contents of instructions and / or promotions and typically include written instructions for or related to the packaging of the present invention. . The instructions may also include any oral or electrical instructions provided in any manner so that the user of the chip clearly recognizes that the instructions are related to the chip. In addition, the kit may include other components depending on the particular application, eg, container, adapter, syringe, needle, replacement part, and the like. As used herein, “promoted” refers to education, hospitals and other clinical facilities, chemical research, drug discovery or development, academic exploration, drug sales. Includes all methods of conducting business, including pharmaceutical industry activities, and any advertising or other promotional activity methods including any form of written, verbal and telecommunications related to the present invention.

  The functionality and advantages of these and other embodiments of the invention will be more fully understood from the following examples. The following examples are merely intended to illustrate the advantages of the present invention and do not exemplify the overall scope of the invention.

(Example 1)
In this example, a chip was prepared according to an embodiment of the present invention, as generally illustrated in FIG. 5A.

  The first chip layer, with liquid channels, ports, chambers, other reaction sites, etc. therein, was injection molded or machined from an acrylic or polycarbonate stock sheet. This first layer was bonded to a machined or injection molded flat bottom plate (also acrylic or polycarbonate) by pressure sensitive silicone adhesives (Dielectric Polymers). A 0.2 μm pore size membrane (Osmonics, Minnetonka, Minn.) Was also adhered to the upper side of the first layer with a pressure sensitive silicone adhesive.

  A second chip layer (including the chamber top) with associated liquid channels, ports, chambers, other reaction sites, etc. therein was cast into a mold using PDMS. The second layer is formed in alignment with the first chip layer. This second layer is aligned with the chamber of the first chip layer and bonded with a pressure sensitive silicone adhesive to form a complete chip. The PDMS top functions as a septum or a self-sealing membrane by itself, or in some cases, additional partial layers of PDMS can be bonded across the inlet or outlet of the chip using a pressure sensitive adhesive .

(Example 2)
In this example, pH detection was demonstrated using the embodiment of this experiment.

  Several chips similar to those described in Example 1 were prepared. Each chip contained a predetermined reaction site as defined by the chamber within the chip. The chamber depth of the bottom chamber (ie, the distance of the chamber from the surface of the chip) was about 3 mm.

Fourteen solutions of 0.1 M phosphate buffer (K ₂ HPO ₄ / KH ₂ PO ₄ , both Sigma-Aldrich, Milwaukee, WI) with various pH were prepared using a 5 μM solution of CDMF. CDMF (5 (6) -carboxy-2 ′, 7′-dimethoxyfluorescein; Helix Research, Springfield, OR) is a fluorescent pH dye. A series of reaction sites on three different chips were each filled with CDMF solution.

  The fluorescence intensity (“I”) of the CDMF solution in each chamber within each chip was measured upon excitation at two wavelengths, 510 nm and 450 nm. The light source used for excitation was high intensity light-emitting diodes (LEDs, LXHL-BE01 and -BR02; Lumileds, San Jose, CA). 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) and then directed to the chip. This emitted light is collected by a 25.4 mm f-1 lens (Thorlabs, Newton, NJ) and optically communicated to another 600 micron fiber which is then 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 intensity ratio is plotted against the pH of the solution. Upon excitation with ₄₅₀ nm light (I _{450 nm} ) and 510 nm light (I _{510 nm} ), the intensity was measured at 560 nm and the ratio of these values was plotted as (I _{450 nm} / I _{510 nm} ). It has been found that the response of the fluorescent signal correlates well with pH over a range of at least about 6 to at least about 8.

  Thus, this experiment demonstrates the ability to optically address one embodiment of the present invention to measure and control pH using ratiometric fluorescence techniques.

(Example 3)
This example illustrates the preparation of a chip according to an embodiment of the present invention.

  A chip layer with liquid channels, ports, chambers, etc. therein was cast into polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning, Midland, MI) using machined aluminum molding. The PDMS layer was cured at 90 ° C. for 20 minutes. The PDMS layer was adhered to the bottom plate with a pressure sensitive silicone adhesive (Dielectric Polymers, Holyoke, Mass.). The bottom plate was made from acrylic or polycarbonate and either machined from sheet stock or injection molded. The layers were combined by pressing the layers in a hydraulic press (Carver, Wabash, IN) to form a complete chip. The top of the PDMS can function as the septum itself, or in some cases, an additional partial layer of PDMS could be bonded across the inlet or outlet of the chip using a pressure sensitive silicone adhesive.

(Example 4)
This example illustrates the control of the pH within the reaction site of the chip according to another embodiment of the present invention.

  Multiple chips similar to the chip described in Example 3 having a shape similar to the embodiment illustrated in FIG. 5 were prepared using PDMS. Each chip had three predetermined reaction sites defined by chambers within the chip. The chamber depth (distance from the surface of the chip) was 500 microns.

  Each of the three chambers of one chip was filled with 50 μM chlorophenol red dye (Sigma-Aldrich, Milwaukee, Wis.). Chlorophenol red is known to undergo a color change from yellow to purple when the solution becomes basic (ie, when 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 site in the chip was optically determined. A light source (tungsten halogen; LH-1; Ocean Optics) was connected to an optical fiber (P100-2; Ocean Optics) terminated with a collimator lens (74-UV; Ocean Optics) (these components are shown in FIG. 15). Absent). The fiber optic assembly transmitted light 310 to the reaction site 320. Transmitted light 315, now attenuated at least partially by the turbidity of sample 325 within reaction site 320, is collected using a separate collimator lens / fiber assembly (not shown) and is computer controlled spectrophotometric. A total of 330 (USB-2000; Ocean Optics) was transmitted. The optical density (“OD”) was calculated as OD = log (I / I ₀ ).

To control the pH within a given reaction site 320, a small amount (about 20 μl) of ammonia solution (Sigma-Aldrich, Milwaukee, Wis.) Was placed on the chip, generally proximal to the reaction site 320. The light absorbance at 574 nm of the reaction site was monitored over the course of 2 hours. As shown in FIG. 14, three concentrations of ammonia were used: 4.0M NH ₄ OH (black circles), 1.5M NH ₄ OH (black squares) and control water (black triangles). In FIG. 14, the optical density at 574 nm was plotted against time using 50 μM chlorophenol red as the pH indicator for the three solutions. Initial and final pH values were estimated from the observed changes in OD.

  Volatile ammonia can permeate PDMS and enter the reaction site, whereby gaseous non-liquid transport through PDMS, that is, direct liquid contact to the liquid in a given reaction site. Rather, it was found that the pH of the solution within the reaction site substantially increased. It has been demonstrated that both the overall change in pH and the rate of change within a given reaction site can be independently controlled by controlling the concentration of ammonia. The rate of pH change within the reaction site was also controlled by adjusting the thickness of the cover and the permeability of the cover material.

  Similar results were also demonstrated using a method similar to that described above, where the pH was controllably lowered instead of being raised. In those experiments, acetic acid was used as a pH modifier.

(Example 5)
This example illustrates an embodiment of the invention used to adjust the pH within a given reaction site without any liquid contact therein.

  A microreactor was constructed from polydimethylsiloxane (PDMS). This unique device had a 127.77 mm x 85.48 mm footprint, generally the same size as a 96 microwell plate. This unique device was assembled by combining various layers of materials, membranes and barrier / interface layers to form a stacked composite structure with 200 μl chambers as described in Example 1.

  The pH of this chamber was monitored using chlorophenol red, a pH modifier, in the cell culture chamber. The emission spectrum of the chamber was recorded every 10 seconds for about 90 minutes. At the first time, a drop of ammonia (20 μl, 4.0M) was placed on a thin layer of PDMS covering the chamber. As the gas enters the chamber across the PDMS, it diffuses the ammonia gas, thereby illustrating the gaseous non-liquid transport of the factor to a given reaction site.

  For the time of this experiment, a plot of the optical density of the chamber is shown in FIG. 16 for wavelengths of 480 nm, 574 nm and 700 nm. The wavelength of 480 nm is an index of chlorophenol red which is a factor having a high optical density value indicating a further alkaline condition. These data show a rapid increase in optical density at 574 nm over about 3 minutes, starting at about 5 minutes, which represents a rapid change in pH to more alkaline conditions during this experiment. Show. In this experiment, it was observed that the pH in the chamber increased rapidly from the initial value of 4.35 to the final value of 10.5.

  Thus, this example illustrates a controlled change in the pH of the chamber where there is no direct contact between the chamber and the liquid.

(Example 6)
This example illustrates a ratiometric determination of the pH within the reaction site of a chip according to an embodiment of the present invention.

  A chip was prepared using a method similar to that of Example 1. The pH sensor of the chip was constructed by immobilizing a fluorescent pH sensitive dye in the gel. The gel was prepared as follows. A stock solution of 15 ml tetraethoxysilane (TEOS) and 20 ml ethanol (both Sigma-Aldrich, Milwaukee, Wis.) Was prepared and kept sealed until use. To make the sol / gel, 1 ml TEOS solution was mixed with 1 ml solution containing 500 μM carboxyfluorescein (Sigma-Aldrich) in 1: 1 ethanol and water solution. To this mixture, 0.1 ml of 0.5 M hydrochloric acid (SIgma-Aldrich) was added to catalyze the formation of sol / gel. A 20 μl aliquot of the catalyzed mixture was pipetted into a small well (500 micron depth) in the bottom plate. The plate with this sol / gel mixture was then allowed to gel for 48 hours in a humid environment. After the gel was completely solidified, carboxyfluorescein dye was immobilized on the bottom plate.

  The gel was placed in liquid contact with the reaction site. A solution having a known pH value was added to the reaction site. The fluorescence of the gel in contact with the reaction site, which is an indicator of the pH within the reaction site, was monitored using a ratiometric fluorescence procedure. In this procedure, the fluorescence response of pH sensitive dyes at two different wavelengths (510 and 480 nm) in response to pH was determined using a commercial UV-visible spectrometer. By using solutions with various known pH's within the reaction site, the ratio of the response at 510 nm and the response at 480 nm is shown to be proportional to the pH of the solution, thus A ratiometric decision was demonstrated.

(Example 7)
In this example, control of the pH within the reaction site of the chip was demonstrated according to one embodiment of the present invention.

  A chip similar to that described in Example 1 was bonded to the control system. A computer was used to record the pH value determined using the ratiometric procedure described above and using a control algorithm, but this computer does not require a control action to adjust the pH within the reaction site. Could be determined. When the computer determined that control action was required, a fluid connection was established between the tip and the external pumping system by opening a valve that connected the tip to the external pumping system. An amount of acid (eg, ammonium hydroxide) or base (eg, acetic acid) was then added to the external pumping system to adjust the pH of the liquid in the reaction site to the required predetermined point. The amount of acid or base to be added was determined by computer using a control algorithm.

(Example 8)
In this example, control of the pH within the reaction site was demonstrated according to another embodiment of the present invention.

  A chip similar to that described in Example 1 was bonded to the control system. A fluorescent pH sensitive dye was immobilized on the gel according to Example 6 and a computer was connected to a chip similar to the method described in Example 7. If the computer determines that a control action is required to adjust the pH in the reaction site, the computer causes the liquid system to be placed on the permeable membrane in liquid communication with the reaction site in a determined amount. Ammonium hydroxide (base) or acetic acid (acid) was given. Control of the pH was then achieved by the action of acid or base diffusing through the membrane entering the reaction site.

Example 9
This example illustrates various chips of the present invention formed from multiple layers of different materials. Various adhesives are used to fix the boundary layer to a strong cell culture or sealing layer, depending on the material involved. One of the adhesives used for PDMS bonding to polycarbonate was 2 parts urethane epoxy mixed with uncured PDMS. The bonding process used to bond the strong polycarbonate layers to each other was either acoustic welding or a hot press. The reaction site was designed to be about 200 microns thick and had a volume of about 20 μm.

  In this example, a chip 280 having a reaction site 240 was manufactured. A polycarbonate layer 244 was bonded to the PDMS layer 242 as shown in FIG. 17A. As shown in FIG. 17A, the gap in the PDMS layer 242 defined the reaction site 240 when the chip was assembled. The PDMS layer 242 was bonded to the polycarbonate layer 244 using 2 parts of urethane epoxy mixed with the above uncured PDMS.

  A similar chip is illustrated in FIG. 17B. In this figure, the reaction site 240 was defined by a layer 245, which is a thin, strong layer of polycarbonate. Between layers 242 and 245 was a gas permeable film 246 (BIOFOIL® made by VivaScience). Using the bonding process described above, layers 244, 245, 246 and 242 of chip 80 were bonded.

(Example 10)
This example illustrates various chips of the present invention formed from multiple layers of different materials. Various adhesives were used to fix the boundary layer to a strong cell culture or sealing layer, depending on the material involved. One of the adhesives used for PDMS bonding to polycarbonate was 2 parts urethane epoxy mixed with uncured PDMS. The bonding process used to bond the strong polycarbonate layers to each other was either acoustic welding or a hot press. The reaction site was designed to be about 200 microns thick and had a volume of about 20 μm.

  The manufacture of the chip illustrated in FIGS. 18A and 18B was similar to that described in Example 9, including the bonding method. In FIG. 18A, reservoir layer 248 was formed from polycarbonate and was placed between gas permeable film 246 (BIOFOIL®) and polycarbonate layer 244. The reservoir layer 248 has a gap (ie, a hole or a partially hollow exterior space) that defines the reaction site 50 that was the reservoir in this example. In FIG. 18A, the reaction site 240 is defined by the gap interface layer 242.

  In FIG. 18B, the reaction site 250 was defined using a polycarbonate layer 248. In addition, a second gas permeable membrane 249 (BIOFOIL®) was used between the polycarbonate layer 245 (which defines the reaction site 240) and the polycarbonate layer 248.

(Example 11)
This example illustrates the manufacture of an embodiment of the present invention without the use of an adhesive. The reaction site was designed to be about 200 microns thick and had a volume of about 20 μm.

  The arrangement of the present invention illustrated in FIG. 19 is similar to the arrangement illustrated in FIG. 18B of Example 10 except that an additional compression layer 252 is used to mechanically hold the other layers in place. In this example, no adhesive was used. Instead, a chip 280 was manufactured with the screw 253 extending from the polycarbonate layer 252 through another layer of chips fixed to the layer 244.

(Example 12)
In this example, embodiments of the present invention are illustrated as used in a chip sealed by a membrane having a sufficiently high oxygen permeability to allow for culturing of viable cells. The amount of oxygen required in this example is a function of the number of cells present and the oxygen demand required for cell metabolism. This is illustrated in equations 2-4 below.

これらの式では、Ｐは、透過性（代表的には、ｃｍ^３ _ＳＴＰｍｍ／ｍ^２ａｔｍ日の単位で測定された）であり、Ａは面積（代表的には、ｍ^２で測定した）であり、ｐ_ｉｎは、チップの酸素分圧（代表的にはａｔｍで測定した）であり、ｐ_ｏｕｔは、チップの外側の酸素分圧（代表的にはａｔｍで測定した）であり、ｌは、膜厚（代表的にはμｍで測定した）であり、Ｖは、チップの容積（代表的にはμｌで測定した）であり、ｄは細胞培養チャンバ厚（代表的にはμｍで測定した）であり、ｎは細胞密度（代表的には１ｍｌあたりの細胞で測定した）であり、そしてｒは細胞１個あたりの比酸素消費量（代表的には１時間細胞１個あたりのＯ_２で測定した）である。

In these equations, P is permeability (typically measured in units of cm ³ _STP mm / m ² atm days) and A is area (typically measured in m ² ). in and, p _in is the oxygen partial pressure of the chip (typically measured in atm), p _out is the oxygen partial pressure outside the chip (typically measured in atm), l Is the film thickness (typically measured in μm), V is the chip volume (typically measured in μl), and d is the cell culture chamber thickness (typically measured in μm). N is the cell density (typically measured in cells per ml) and r is the specific oxygen consumption per cell (typically O / cell per hour). ₂ ).

  Equation 4 shows a mass balance that regards the oxygen consumed by the growing culture as equivalent to the oxygen available through diffusion through the film. Equation 2 sets the volume of the culture chamber equal to the cross-sectional area of the membrane in contact with the outer chamber positive product on both sides. Rearrangement yields Equation 3, which represents the minimum oxygen permeability required to maintain a given population density and metabolic rate of cells as a function of film thickness and chamber depth.

The value of P is generally dependent on the polymer and the permanent system, and in this example 39,000 (cm ³ _STP mm / m ² atm day) for silicon to 0.01 (cm ³ for EVA). oxygen was changed for that between the _STP mm ^/ m 2 atm _{day); p in} is varied _{0.05atm~0.2atm, p out} was assumed to 0.2 atm. The film thickness l varied between 1 μm and 2 mm. V was held to be less than 1 ml and cell culture depth d ranged between 30 μm and 2 mm. The cell density n was assumed in this example to be 10 ⁵ cells / ml to 10 ⁷ cells / ml for mammalian cells and 10 ⁹ cells / ml to 10 ¹¹ cells / ml for bacteria. The specific oxygen required per cell ranged from 0.5 to 5 × 10 ⁻¹² mol O ₂ / cell · hour.

20 and 21 were then generated using equations 2-4. FIG. 20 is a graph of oxygen permeability requirements for bacterial cell culture as a function of film thickness and device shape. FIG. 21 is a graph of oxygen permeability requirements for bacterial cell culture as a function of film thickness and device shape. In both figures, a flat horizontal line probably corresponds to the permeability of the membrane or thin layer construct material, while the diagonal line corresponds to the highest and lowest oxygen demands expected. In these figures, the cell density n and the specific reaction rate r is set to high and low values, and partial pressure difference of _{(p in -p out)} was set to 0.05 atm. The required permeability was then linear in the product of d, chip depth and cover film thickness l.

(Example 13)
This example illustrates the use of an embodiment of the invention to determine the turbidity of a solution. This example generally corresponds to the usual operation of measuring the cell density of bacterial cells by the turbidimetric method (light scattering measured at 90 ° to the primary beam). See, generally, Methods for General Bacteriology, P.M. Edited by Gerhardt, 1981 Washington D.C. C. p. See 197.

  A chip having an integrated waveguide was constructed as follows. The top layer of the chip was prepared and cast using polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning, Midland, MI) using a machined aluminum mold.

  A short section of polymer waveguide (500 micron square, acrylic; South Coast Fiber, Alachua, FL), with one end adjacent to the mold end and the other end extending to the mold end. Placed in a machined aluminum mold. Liquid PDMS was poured into a mold and allowed to cure. PDMS was cured at 90 ° C. for 20 minutes, and the waveguide was fixed in the chip to create a light path from the end of the chip to a predetermined reaction site and chamber. The cured PDMS layer was fixed to a flat polystyrene bottom layer to form a complete chip (PDMS layer was naturally fixed to the polystyrene layer). The depth of the chamber from the surface of the chip was about 1 mm.

  Light scattering was measured from a series of turbid solutions contained in the chip. Referring to FIG. 22, the output of a helium neon laser (05-LHP-991, wavelength = 632.8 nm; Melles Griot Lasers, Carlsbad, Calif.) Is focused on the end of waveguide 540, which transmits the light to reaction site 520. It was. Detector 530 includes a collimator lens (74-UV f / 2 lens; Ocean Optics, Dunedin, FL), an optical fiber (P600-2, 600 micron diameter; Ocean Optics), and a mounted spectrophotometer (USB-2000F). ; Ocean Optics). The detection angle was about 90 ° from the waveguide axis.

  The reaction site was filled with a series of turbid solutions of non-dairy creamers (Sugar Foods, New York, NY) with absorbance values ranging from 0.05 to 1.85 at 632 nm. A plot of scattered light intensity (632 nm) versus relative concentration is shown in FIG. A linear correlation was observed for the solution with optical density values ranging from 0.05 to 0.5. At high concentrations, the response of scattered light was non-linear.

(Example 14)
This example demonstrates an optically addressable reaction site according to an embodiment of the present invention.

  A chip was prepared using a method similar to that in Example 13. The chips used in this experiment were generally prepared. The distance from the surface of the chip to the reaction site was about 200 microns. As discussed below, the chip was optically addressed to measure optical density using an arrangement similar to that shown in FIG.

A light source (tungsten halogen, LH-1; Ocean Optics) was connected to an optical fiber (P100-2; Ocean Optics) ending with a collimator lens (74-UV; Ocean Optics) (not shown in FIG. 15). The fiber optic assembly transmitted light 310 to the reaction site 320. The transmitted light 315, now at least partially attenuated by the turbidity of sample 325, is collected using another collimator lens / fiber assembly (not shown), which is then detected by detector 330, computer Transmitted to a controlled spectrophotometer 330 (USB-2000; Ocean Optics). The optical density was calculated as OD = log (I / I ₀ ).

  The optical density (“OD”) of the bacterial culture (E. coli BL21 in chemically defined medium w / glucose) was monitored over a 13 hour growth period at the reaction site. The results from this experiment are shown in FIG. 24, which shows E. coli at 30 ° C. and 37 ° C. at the reaction site of the chip monitored by a fiber optic spectrometer. The growth of E. coli BL21 is illustrated. Thus, these data demonstrate the validity of cell proliferation measurements by optically addressing the reactions of the present invention.

(Example 15)
FIG. 25 is a perspective view of a planar solid substrate having a single reaction site (eg, chamber) and various channels. The planar substrate comprises two separately molded silicone sheets 605 and 615. In this embodiment, reaction site 610 and channels 640 and 650 are formed by juxtaposing elements molded in silicone sheets 605 and 615.

  The chamber 610 of FIG. 25 includes a bottom cell culture portion 620 and an upper reservoir portion 630. The bottom cell culture portion 620 is in fluid communication with two bottom channels 640 located at opposite corners of the bottom cell culture portion 620. The upper reservoir portion 630 is in fluid communication with two upper channels 650 located at opposite corners of this bottom cell culture portion 630. The upper reservoir portion 630 and its associated upper channel 650 are molded into the silicone sheet 605 while the lower cell culture portion 620 and its associated lower channel 640 are molded into the lower silicone sheet 615. The upper reservoir portion 630 and the lower cell culture portion 620 are separated by a membrane 655 that extends beyond the chamber 610 between the upper silicone sheet 605 and the lower silicone sheet 615. In this example, the membrane is substantially impermeable to mammalian cells, but is permeable to proteins, small molecules, and the like. Of course, in other embodiments, other impermeable or semi-permeable membranes such as humidity control membranes may be used.

  In FIG. 25, each of the upper channels 650 terminates in an upper port 665 that passes completely through the upper silicone sheet 615. This arrangement allows the upper port to be connected to additional channels, supply chambers, waste chambers, production chambers, etc. connected to the upper surface of the upper silicone sheet. Of course, access to the upper channel can be obtained in other ways.

  In FIG. 25, each of the bottom channels 640 terminates in a lower port 660 that passes up the lower silicone sheet 605. Each lower port 660 is aligned with an opening 670 in the upper silicone sheet 615. This arrangement allows access to each lower port through the upper silicone sheet 615, and each lower port is connected to an upper surface of the upper silicone sheet, a further channel, a supply chamber, a waste chamber, It becomes possible to connect to a production chamber or the like. Of course, access to the lower channel can be obtained in other ways.

  As described above, the chamber and the accompanying upper reservoir portion of the channel are molded directly into the top silicone sheet, while the bottom cell culture portion of the chamber and its associated channel are molded separately into the bottom silicone sheet. Thus, prior to assembly of the device as shown in FIG. 25, each silicone sheet comprises an open top or bottom of the chamber and several open channels. A fully closed two-part chamber and a closed channel are formed by sandwiching a selectively permeable membrane between opposite top and bottom silicone sheets. In the embodiment of FIG. 25, the bottom silicone sheet functions to close the open top channel and the top silicone sheet functions to close the open bottom channel. As shown in FIG. 25, the membrane may extend beyond the walls of the chamber so that it is between the upper and lower silicone sheets. The two silicone sheets are held together using any convenient fixation.

  The silicone that forms the chamber and part of the channel is sufficiently gas permeable and provides sufficient gas exchange for the growth of aerobic cells in the chamber of the device.

  FIG. 26A is a plan view of the bottom silicone sheet 605 showing the bottom cell culture portion 620 of the chamber and the channel 640 and bottom port 660 leading to it. The wall 680 of the bottom cell culture portion 620 lacks abrupt transitions and corners. This facilitates complete mixing and dispersion of the substance introduced into the bottom cell culture part.

  26B is a cross section of the bottom silicone sheet 605 along A-A ′ in FIG. 2. The base 690 of the bottom cell culture portion 620 is substantially planar and perpendicular to the wall 680 of the bottom cell culture portion 620. In this embodiment, the base 690 bends gently upward to match the wall 680. In this example, the absence of sharp corners facilitates complete mixing and dispersion of materials in the bottom cell culture portion 620.

  FIG. 26C is a plan view of the upper silicone sheet 615, showing the upper reservoir portion 630 of the chamber and the channel 650 leading to it, both of which end at the upper port 665, thereby removing the upper silicone sheet 615 from the top. Access to the channel is gained. The wall 695 of the upper reservoir portion 630 lacks abrupt transitions and corners in this example. This facilitates complete mixing and dispersion of the substance introduced into the upper reservoir portion 630. In this assembled device, the passage 670 in the top silicone sheet 615 is aligned with the bottom port bottom silicone sheet, thereby allowing access to the bottom channel through the top silicone sheet.

  FIG. 26D is a cross section of the upper silicone sheet 615 along B-B ′ in FIG. 26C. As can be seen in this figure, the passage 670 provides an opening through the top silicone sheet 615. This opening is aligned with one of the bottom ports when the top and bottom silicone sheets are joined to form a complete chamber. An upper port 665 is formed in the upper silicone sheet 615 to gain access to the upper channel.

  FIG. 26E is a perspective view of the upper reservoir portion of the chamber with the channel. Upper reservoir portion 620 and associated channel 650 are molded into upper silicone 615. The base 628 of the upper reservoir 620 is planar in this embodiment. In this embodiment, the wall of the upper reservoir portion 618 is perpendicular to the upper base 628. Base 628 bends gently upward to conform to wall 618 to facilitate mixing and dispersion of material at the top. An upper port 665 located at the end of the channel 650 allows for the introduction of material into the channel. The top silicone sheet 615 includes two passages 670 that allow access to the bottom port when the top and bottom silicone sheets are combined to form a complete chamber.

(Example 16)
In this example, a device was manufactured using three layers. In this embodiment, the bottom layer is a solid slab. The intermediate layer has a membrane molded therein that separates the upper reservoir portion from the bottom cell culture portion both molded into the intermediate layer. The upper reservoir portion and the upper microchannel are molded into the upper surface of the intermediate layer, and the bottom cell culture portion and the lower microchannel are molded into the lower surface of the intermediate layer. An opening through the intermediate layer allows access to the bottom microchannel. The top layer has four openings therethrough to serve as ports for the four microchannels. The top layer functions to seal the upper reservoir portion and its associated microchannels while still allowing access to all ports. The bottom layer functions to seal the bottom cell culture portion and the accompanying microchannels.

(Example 17)
In this prophetic example, the fluid pathway device of the present invention is used to test the effect of chemical factor A on bacterial fermentation. Each has a single chamber, which juxtaposes 12 fluid paths with cell culture and reservoir portions in parallel. This fluid pathway is sterile and pumps sterile growth medium through the liquid delivery system to each cell culture portion. The reservoir portion of the six liquid channels receives a measured aliquot of chemical factor A and growth medium through the liquid delivery system, and the remaining six receive only the growth medium. By having six fluid paths for each case, a redundancy measure for statistical purposes is obtained. A cell culture portion of each of the 12 fluid pathways is inoculated with a volume of concentrated cells, which is about 1/20 to 1/10 volume of the cell culture portion. Through the use of appropriate sensors in this fluid path, microbial growth is monitored in each of the 12 fluid paths by measuring pH, dissolved oxygen concentration, and cell density. Fluid path heat exchanger, chemical addition and air flow rate, this fluid path may control temperature, pH and dissolved oxygen concentration, respectively. When the cells reach the stationary phase, the average cell growth rate and average final cell concentration are calculated for the six fluid pathways with chemical factor A and for the six fluid pathways without this factor. By comparing these averages, it can be said that chemical factor A enhances cell growth, has no significant effect, or interferes with cell growth.

(Example 18)
In this prophetic example, the fluid pathway device of the present invention is used to provide an environment for growing cells or tissues that mimics the environment found in humans or mammals. For drug screening, the fluid pathway device can monitor the response of cells to drug candidates. These responses can include an increase or decrease in cell growth rate, cell metabolic changes, cell physiological changes, or changes in the uptake or release of biological molecules. By manipulating many fluid pathways in parallel, different cell lines may be tested, along with screening multiple drug candidates or combinations of different drugs. By incorporating the necessary electronics and software to monitor and control the array of fluid passages, the screening process can be automated.

  20 fluid passages each containing a single chamber divided into a cell culture portion and a reservoir portion are sterilized. Sterile animal cell culture medium is pumped through the liquid delivery system into each cell culture portion of the chamber. Each fluid passage is then inoculated with mammalian cells that have been genetically engineered to produce a therapeutic protein. The cells are allowed to grow to the production stage, with their growth and environment monitored by sensors in the fluid pathway. Through control of temperature, pH, and airflow rate, the fluid pathway can maintain an optimal environment for cell growth. Once in the production stage, the fluid passages are divided into four groups out of five. Three of the four groups receive various cocktails of therapeutic protein inducers, while the fourth group serves as a control and therefore does not accept inducers. This inducer and control sample are introduced into the reservoir portion of the chamber through a liquid delivery system. A marker compound that binds to the therapeutic protein is introduced along with the inducer. When the culture is irradiated with light of a wavelength that excites the bound marker compound, the compound, and thus fluorescence, and the intensity of fluorescence are proportional to the concentration of the therapeutic protein in the culture. Both the illumination light and the fluorescent signal pass through a detection window that covers the liquid chamber. The fluorescent signal is picked up by a photodetector outside the fluid passage. The production of therapeutic protein can be monitored for each of the four groups, and at the end of production, the average production rate and average total production can be calculated for each group. A comparison of the production between the four groups can then determine the effectiveness of the various inducers on protein production.

(Example 19)
In this prophetic example, a fluid pathway device is used in an adsorption assay to model, for example, the adsorption of drugs and other factors in the intestine. For example, the fluid path device may comprise a chamber divided into two parts by a polycarbonate membrane having a pore size of 3.0, 2.0 or 1.0 microns. Caco-2 (colorectal cancer cells) is grown on one surface of the membrane in the first part of the chamber until it is differentiated. Drugs or other factors are introduced into the portion of the chamber that contains the cells. Monitor the passage of drugs or other factors through the cell layer to the second part of the chamber.

  Similar arrangements may be used for cell migration assays. Such an assay uses a membrane having a pore size of 5.0-12.0 microns.

(Example 20)
Useful amounts of multiple target proteins are generated according to this prophetic example.

  A microfabricated bioreactor containing one or more cell growth chambers is sterilized and a sterile growth medium is pumped into each growth chamber through a liquid delivery system. For convenience, the bioreactor may comprise, for example, 96 cell growth chambers arranged in the same manner as the wells of a 96 well plate. Each chamber receives an aliquot of mammalian cells and an aliquot of DNA encoding the protein of interest, and optionally one or more selectable markers. Cells are transfected with the added DNA by calcium phosphate transfection or other techniques.

  After completion of transfection, each chamber contains cells that express various proteins of interest. Cells may be cultured to produce a useful amount of the protein of interest, and then this protein may be recovered and analyzed, or analyzed through the microchannel and using the microreactor system described above. May be.

  Alternatively, the cells may be transfected with the DNA molecule of interest prior to introduction into the growth chamber.

(Example 21)
Useful amounts of a large number of target proteins are produced according to this prophetic example.

  A microfabricated bioreactor containing one or more cell growth chambers is sterilized and a sterile growth medium is pumped into each growth chamber through a liquid delivery system. Each chamber receives an aliquot of mammalian cells and an aliquot of a mixture of DNA molecules encoding the protein of interest, and optionally one or more selectable markers. Cells are transfected with the added DNA by calcium phosphate transfection or some other technique.

  After completion of transfection, each chamber contains cells that express one or more of the various proteins of interest. Cells may be cultured to produce a useful amount of the protein of interest, and then this protein may be recovered and analyzed, or analyzed through the microchannel and using the microreactor system described above. May be.

(Example 22)
In this prophetic example, useful quantities of a large number of target proteins are produced as follows.

  The microfabricated bioreactor is sterilized and a sterile growth medium is pumped through the liquid delivery system into each growth chamber. Each chamber receives an aliquot of mammalian cells. Different factors are added to each chamber or each chamber is incubated under different conditions. As a result of different treatments, the cells in each chamber potentially produce different groups of proteins.

  Cells may be cultured to produce a useful amount of the protein of interest, and then this protein may be recovered and analyzed, or analyzed through the microchannel and using the microreactor system described above. May be.

(Example 23)
In this prophetic example, useful quantities of a large number of target proteins are produced as follows.

  A microfabricated bioreactor containing one or more cell growth chambers is sterilized and a sterile growth medium is pumped into each growth chamber through a liquid delivery system. Each chamber receives an aliquot of mammalian cells and an aliquot of DNA or a mixture of DNA encoding the protein of interest, and optionally one or more selectable markers. Cells are transfected with the added DNA by calcium phosphate transfection or some other technique.

  After completion of transfection, the cells in each chamber are genetically mutated by the action of ionizing radiation, ultraviolet light, or other physical, chemical or biological mutagens. After gene mutation, each chamber contains cells that express one or more of the various proteins of interest, potentially at various rates and under various gene expression profiles. The cells can be cultured to produce a useful amount of the protein of interest, and then the protein can be collected and analyzed, or analyzed through the microchannel and through the microreactor system described above. May be.

(Example 24)
Useful amounts of multiple target proteins may also be produced according to this prophetic example.

  A microfabricated bioreactor containing one or more cell growth chambers is sterilized and a sterile growth medium is pumped into each growth chamber through a liquid delivery system. Each chamber receives an aliquot of bacterial or fungal cells and an aliquot of a mixture of DNA molecules encoding the protein of interest within a genetic vector.

  After the cellular genetic modification is complete, each chamber contains cells that express one or more of the various proteins of interest. Cells may be cultured to produce a useful amount of the protein of interest, and then this protein may be recovered and analyzed, or analyzed through the microchannel and using the microreactor system described above. May be.

(Example 25)
In this prophetic example, a useful amount of multiple target proteins may be produced as follows.

  A microfabricated bioreactor containing one or more cell growth chambers is sterilized and a sterile growth medium is pumped into each growth chamber through a liquid delivery system. Each chamber is implanted with a tissue sample exhibiting the desired phenotype.

  The tissue sample can be incubated to produce a useful amount of the protein of interest, and then this protein can be collected and analyzed, or analyzed through the microchannel and using the microreactor system described above. May be.

(Example 26)
This example illustrates the construction of a specific embodiment of the present invention.

  FIG. 27A shows a cross-sectional view of a cell growth chamber of a microfabricated bioreactor device useful in the methods of the present invention. The cell growth chamber 710 is a cylinder having a total volume of 3.85 μl with a diameter of about 7 mm and a height of about 0.1 mm. This chamber is liquid connected to three microchannels. The first microchannel 720 is 0.44 mm wide × 0.1 mm deep and functions as a liquid inlet. The second microchannel 730 has similar dimensions and functions as a liquid outlet. The third microchannel 740 is 0.2 mm wide × 0.1 mm deep. This microchannel can be used to introduce cells or any desired substance into the chamber. Three microchannels and a cell growth chamber are etched into the solid support material.

  FIG. 27B shows a cross-sectional view of the headspace portion of gas coupled to the cell growth chamber. This allows a continuous supply of air through the microfabricated bioreactor. A cylindrical chamber 750 having a diameter of about 7 mm and a height of about 0.05 mm is glassed along a gas inlet microchannel 760 and a gas outlet microchannel 770 that are both about 0.05 mm wide by about 0.05 mm deep. Etch into. A cylindrical chamber in the gas headspace portion is matched across the cell growth chamber. The halves may then be joined together, thereby creating a tight seal.

  In this example, a membrane may be positioned to separate the gas headspace from the liquid-filled bioreactor to prevent gas flow through the gas headspace from removing liquid in the cell growth chamber. This membrane delays the passage of water and allows the passage of air. Various microchannels are connected to the supply unit or the waste unit. These units and mixing devices, control valves, pumps, sensors and monitoring devices may be incorporated into the substrate, where the cell growth chamber can be built or provided externally. The entire assembly may be controlled above or below the heat exchanger (or sandwiched between two heat exchangers) to control the temperature of this unit.

  In this particular embodiment, the silicone from which the chamber and part of the microchannel are molded is sufficiently gas permeable to provide a sufficient gas exchanger for the growth of aerobic cells in the device chamber. .

  Although some embodiments of the present invention are described or illustrated herein, one of ordinary skill in the art will appreciate that the functions described and / or results or advantages described herein may be implemented. Various other means and constructs are readily envisioned to obtain and each such variation or modification is deemed to be within the scope of the invention. More generally, one skilled in the art will mean that all parameters, dimensions, materials and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials and configurations are It will be readily understood that the teachings herein depend on the particular application in which they are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, it is to be understood that the foregoing embodiments are merely examples, and that the invention can be practiced otherwise than as specifically described within the scope of the appended claims and the equivalents thereto. Should be understood. The present invention is directed to each individual feature, system, material and / or method described herein. Further, any combination of two or more such features, systems, materials and / or methods is within the scope of the invention if such features, systems, materials and / or methods are consistent with each other. Is included.

  In the claims (and herein above), “comprising,” “including,” “carrying,” “having”. All provisional phrases such as “including”, “containing”, “related, involving”, etc. are unconstrained, ie, including but not limited to. It should be understood to mean. Only the provisional phrases “consisting of” and “consisting essentially of” are the only terms in the United States Patent and Trademark Office Patent Examination Guidelines, Section 2111.03 (United States Patent Office). Manual of Patent Exchanging Procedure, Section 2111.03), respectively, are closed or semi-closed transitional phrases.

Non-limiting embodiments of the present invention will now be described with reference to the accompanying drawings, which, by way of example, are schematic and are not intended to describe scale. In the drawings, each identical or nearly identical illustrated component is typically indicated by a single numeral. For purposes of simplicity, not all components are shown in every drawing, and are not shown in every embodiment of each embodiment of the invention, which is illustrated to allow those skilled in the art to understand the invention. Not necessary. In this drawing:
FIG. 1 illustrates one embodiment of the present invention. FIG. 2 illustrates in an enlarged view an example of a microfluidic chip for use with the present invention comprising a mixing, heating / dispersing, reaction and separation unit. 3A-3C illustrate various stockable configurations of the chip of the present invention. 4A-4C illustrate various energy detectors for use with the present invention in certain embodiments. 5A 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 present invention. 7A and 7B illustrate a device according to another embodiment of the present invention having a distribution unit. 8A and 8B illustrate a device according to another embodiment of the present invention that uses a laser to generate a response. 9A and 9B are cross-sectional views of certain embodiments of the invention. 10A-10D illustrate certain membranes of the present invention in fluid communication with various reaction sites. FIG. 11 is an illustration of the dependence of oxygen penetration on a thin layer in one embodiment of the present invention. FIG. 12 is a plot of oxygen permeability versus water vapor passage for various membranes, including specific membranes used in the present invention. FIG. 13 is a graph of pH versus relative intensity, according to one embodiment of the present invention. FIG. 14 is a graph of optical density vs. time illustrating control of environmental factors according to an embodiment of the present invention. FIG. 15 illustrates one embodiment of the present invention showing optical interaction with the reaction site. FIG. 16 illustrates the change in pH index over time in one embodiment of the invention. 17A and 17B (enlarged) illustrate some of the various chips according to one embodiment of the present invention. 18A and 18B illustrate enlarged views of portions of various chips according to another embodiment of the present invention. FIG. 19 illustrates an enlarged view of a portion of a chip according to yet another embodiment of the present invention. FIG. 20 is a graph illustrating oxygen permeability of an embodiment of the present invention when used in bacterial culture. FIG. 21 is a graph illustrating oxygen permeability for an embodiment of the invention used in mammalian cell culture. FIG. 22 illustrates another embodiment of the invention having a wavelength. FIG. 23 is a graph of intensity (relative unit) versus relative concentration in an embodiment of the present invention. FIG. 24 is a graph of optical density at 480 nm versus time for experiments using embodiments of the present invention. FIG. 25 illustrates a solid substrate having reaction sites and channels, according to one embodiment of the present invention. 26A-26E illustrate various views of the embodiment illustrated in FIG. And FIGS. 27A and 27B illustrate bioreactors made according to various embodiments of the present invention.

Claims (76)

A chip comprising a predetermined reaction site having a volume of less than about 1 ml, the predetermined reaction site having an inlet and an outlet;
An active control system capable of controlling environmental factors associated with the chip in response to a signal that is indicative of a condition associated with the chip to support viable cells within the predetermined reaction site;
A device comprising: The apparatus of claim 1, wherein the chip comprises a plurality of reactors, one of the plurality of reactors comprising the predetermined reaction site. The apparatus of claim 1, wherein the active control system is integrally connected to the chip. The apparatus of claim 1, wherein the predetermined reaction site has a volume of less than about 500 μl. The apparatus of claim 1, wherein the predetermined reaction site has a volume of less than about 100 μl. The apparatus of claim 1, wherein the predetermined reaction site has a volume of less than about 10 μl. The apparatus of claim 1, wherein the predetermined reaction site has a volume of less than about 1 μl. The apparatus of claim 1, wherein the predetermined reaction site has a maximum dimension of less than about 1 cm. The apparatus of claim 1, wherein the predetermined reaction site has a maximum dimension of less than about 1 mm. The apparatus of claim 1, wherein the predetermined reaction site has a maximum dimension of less than about 100 μm. The apparatus of claim 1, wherein the predetermined reaction site has a maximum dimension of less than about 10 μm. The apparatus of claim 1, wherein at least one surface of the predetermined reaction site comprises an inorganic substance. The apparatus of claim 12, wherein the inorganic material comprises a semiconductor. The apparatus of claim 12, wherein the inorganic material comprises a metal. The device of claim 1, wherein the viable cell is a mammalian cell. The device of claim 1, wherein the viable cell is a bacterium. The apparatus of claim 1, wherein the cell is a plant cell. The device of claim 1, wherein the viable cells are part of a tissue culture. The apparatus of claim 1, wherein at least one surface of the predetermined reaction site comprises a polymer. The apparatus of claim 19, wherein the at least one surface consists essentially of a polymer. 20. The polymer is selected from the group consisting of silicone, polycarbonate, polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene chloride, bis-benzocyclobutene, polystyrene, polyacrylate, polymethacrylate, polyimide, and combinations thereof. The device described in 1. The control system is:
Relative humidity, pH,
Molar concentration, dissolved gas concentration,
Osmolality, glucose concentration,
Glutamine concentration, pyruvic acid concentration,
Apatite concentration, color,
Turbidity, viscosity,
Amino acid concentration, vitamin concentration,
Hormone concentration, serum concentration,
Ion concentration, shear rate,
The degree of stirring, temperature,
Pressure, O ₂ concentration,
CO ₂ concentration,
The apparatus of claim 1, wherein at least one environmental factor within a predetermined reaction site selected from the group consisting of and a concentration of oligopeptides is controllable. The apparatus of claim 1, wherein the control system is capable of controlling a temperature within the predetermined reaction site. The apparatus of claim 1, wherein the control system is capable of controlling pressure within a predetermined reaction site. A chip comprising a predetermined reaction site having a volume of less than about 1 ml, the predetermined reaction site having an inlet and an outlet;
A control system capable of controlling an environmental factor associated with the predetermined reaction site, wherein the environmental factor is at least one of relative humidity, pH, dissolved O ₂ concentration, dissolved CO ₂ concentration and medium component concentration. A control system;
An apparatus comprising: 26. The apparatus of claim 25, wherein the control system is integrally connected to the chip. 26. The apparatus of claim 25, wherein the chip is constructed and arranged to maintain at least one viable cell at a predetermined reaction site. 26. The apparatus of claim 25, wherein the medium component is selected from the group consisting of a carbohydrate source, serum, growth factor, enzyme, hormone, amino acid, or oligopeptide. 26. The apparatus of claim 25, wherein the carbohydrate source is glucose. 26. The apparatus of claim 25, wherein the chip comprises a plurality of reactors, and one of the plurality of reactors comprises the predetermined reaction site. 26. The apparatus of claim 25, wherein the predetermined reaction site has a volume of less than about 500 [mu] l. 26. The apparatus of claim 25, wherein at least one surface of the predetermined reaction site comprises an inorganic material. 26. The apparatus of claim 25, wherein at least one surface of the predetermined reaction site comprises a polymer. 26. The device of claim 25, wherein the viable cell is a mammalian cell. A chip comprising a predetermined reaction site having a volume of less than about 1 ml, the predetermined reaction site having an inlet and an outlet;
A sensor integrally connected to the chip, wherein the sensor can determine an environmental factor associated with the predetermined reaction site, the environmental factor being:
pH, dissolved gas concentration,
Molarity, osmolarity,
Glucose concentration, glutamine concentration,
Pyruvate concentration, apatite concentration,
Color, turbidity,
Viscosity, amino acid concentration,
Vitamin concentration, hormone concentration,
Serum concentration, ion concentration,
Shear rate and degree of agitation;
A sensor that is at least one of:
An actuator integrally connected to a chip capable of changing the environmental factor;
An apparatus comprising: 35. The apparatus of claim 34, wherein the chip is constructed and arranged to maintain at least one viable cell at a predetermined reaction site. 35. The apparatus of claim 34, wherein the actuator is capable of transferring energy to a predetermined reaction site. 40. The apparatus of claim 36, wherein the energy comprises thermal energy. 40. The apparatus of claim 36, wherein the energy comprises acoustic energy. 40. The apparatus of claim 36, wherein the energy comprises mechanical energy. 35. The apparatus of claim 34, further comprising a processor capable of determining a response for the actuator based on measurements from a sensor. 41. The apparatus of claim 40, wherein the processor is integrally connected to the object. 41. The apparatus of claim 40, wherein the processor comprises an electrical circuit. 35. The apparatus of claim 34, wherein the chip comprises a plurality of reactors, and one of the plurality of reactors comprises the predetermined reaction site. 35. The apparatus of claim 34, wherein the predetermined reaction site has a volume of less than about 500 [mu] l. 35. The apparatus of claim 34, wherein at least one surface of the predetermined reaction site comprises an inorganic material. 35. The apparatus of claim 34, wherein at least one surface of the predetermined reaction site comprises a polymer. 35. The device of claim 34, wherein the viable cell is a mammalian cell. 35. The apparatus of claim 34, further comprising a processor that can receive a signal from the sensor and generate a signal to the actuator. A chip comprising a predetermined reaction site having a volume of less than about 1 ml, the predetermined reaction site having an inlet and an outlet;
A first sensor integrally connected to the chip capable of determining at least one of temperature and pressure;
A second sensor integrally connected to the chip that can determine a second environmental factor, wherein the second environmental factor is:
pH, dissolved gas concentration,
Molarity, osmolarity,
Glucose concentration, glutamine concentration,
Pyruvate concentration, apatite concentration,
Color, turbidity,
Viscosity, amino acid concentration,
Vitamin concentration, hormone concentration,
Serum concentration, ion concentration,
Shear rate and degree of agitation;
A second sensor that is at least one of the following;
An actuator integrally connected to the chip capable of changing at least one of temperature, pressure and environmental factors;
An apparatus comprising: 50. The apparatus of claim 49, wherein the chip is constructed and arranged to maintain at least one viable cell at a predetermined reaction site. A chip comprising a plurality of predetermined reaction sites having a volume of less than about 1 ml, the predetermined reaction sites having inlets and outlets;
A sensor capable of determining an environmental factor associated with at least one of the predetermined reactive sites, wherein the environmental factor is:
CO ₂ concentration, glucose concentration,
Glutamine concentration, pyruvic acid concentration,
Apatite concentration, serum concentration,
Vitamin concentration, amino acid concentration,
Hormone concentration,
A sensor that is at least one of:
An apparatus comprising: 52. The apparatus of claim 51, wherein the sensor is integrally connected to the chip. The apparatus of claim 51, further comprising a temperature sensor. 52. The apparatus of claim 51, further comprising a pressure sensor. 52. The apparatus of claim 51, wherein the chip comprises a plurality of reactors, and one of the plurality of reactors comprises the predetermined reaction site. 52. The apparatus of claim 51, wherein the at least one predetermined reaction site has a volume of less than about 500 [mu] l. 52. The apparatus of claim 51, wherein at least one surface of the at least one predetermined reaction site comprises an inorganic material. 52. The apparatus of claim 51, wherein at least one surface of the at least one predetermined reaction site comprises a polymer. 52. The device of claim 51, wherein the viable cell is a mammalian cell. 52. The apparatus of claim 51, further comprising an actuator that can change an environmental factor associated with at least one of the predetermined reaction sites. 61. The apparatus of claim 60, further comprising a processor that can receive a signal from a sensor and generate a signal to the actuator. A chip with a predetermined reaction site having a volume of less than about 1 ml;
A control system capable of generating a change in a first environmental factor associated with the predetermined reaction site within one second and responsive to a change in a second environmental factor associated with the predetermined reaction site;
A device comprising: 64. The apparatus of claim 62, wherein the control system is integrally connected to the chip. 64. The apparatus of claim 62, wherein the chip is constructed and arranged to maintain at least one viable cell at the predetermined reaction site. 64. The apparatus of claim 62, wherein the control system can produce a change in the first environmental factor within 100ms of a change in the second environmental factor. 64. The apparatus of claim 62, wherein the control system can produce a change in the first environmental factor within 10 ms of a change in the second environmental factor. 64. The apparatus of claim 62, wherein the control system can produce a change in the first environmental factor within 1 ms of the change in the second environmental factor. 64. The apparatus of claim 62, wherein the chip comprises a plurality of reactors, one of the plurality of reactors comprising the predetermined reaction site. 64. The apparatus of claim 62, wherein the predetermined reaction site has a volume of less than about 500 [mu] l. 64. The apparatus of claim 62, wherein at least one surface of the predetermined reaction site comprises an inorganic material. 64. The apparatus of claim 62, wherein at least one surface of the predetermined reaction site comprises a polymer. 64. The device of claim 62, wherein the viable cell is a mammalian cell. A chip comprising a predetermined reaction site having a volume of less than about 1 ml, the predetermined reaction site having an inlet and an outlet;
An active control system capable of controlling the environment within the predetermined reaction site to support viable cells for at least one day;
A device comprising: 74. The apparatus of claim 73, wherein the control system is integrally connected to the chip. 74. The apparatus of claim 73, wherein the chip is constructed and arranged to maintain at least one viable cell at the predetermined reaction site.

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