JP5502275B2 - Thermal reaction device and method of using the thermal reaction device - Google Patents

Description

(Priority claim)
This application claims priority to each of the following applications:
US patent application Ser. No. 11 / 058,106 filed Feb. 14, 2005; U.S. Patent Application No. 11 / 043,895 filed Jan. 25, 2005; filed Jun. 23, 2004 US patent application Ser. No. 10 / 876,046; and US patent application Ser. No. 10 / 837,885 filed May 2, 2004. Each of these is incorporated by reference in its entirety for all purposes.

(Cross-reference to related applications)
This application also includes US patent application Ser. No. 10 / 818,642, filed Apr. 5, 2004; U.S. Provisional Patent Application No. 60 / 460,634, filed Apr. 3, 2003; US Patent Application No. 10 / 819,088 filed on May 5, US Patent Application No. 10 / 818,642 filed April 5, 2004; US filed November 27, 2002 Patent application No. 10/3 No. 06,798; also relates to and U.S. provisional Patent application No. 60 / 335,292; which filed US provisional Patent application No. 60 / 391,529 on June 24, 2002. Each of these is incorporated herein by reference in its entirety for all purposes.

(background)
In recent years, coordinated efforts have been made to develop and manufacture microfluidic systems that perform various chemical and biochemical analyzes and syntheses, both for preparative and analytical applications. The goal of making such devices arises from the significant benefits that can be realized by miniaturization for analysis and synthesis performed on a macro scale. Such benefits include the substantial time, cost and space reduction required for the devices utilized to perform the analysis or synthesis. In addition, the microfluidic device has the potential to be adapted for use with automated systems, thereby providing the additional benefit of further cost savings and reduced operator error due to reduced human involvement. . Microfluidic devices have been proposed for use in a variety of applications, including, for example, capillary electrophoresis, gas chromatography, and cell separation.

  However, the realization of these benefits is often frustrated due to the various complexities associated with microfluidic devices that have been manufactured so far. For example, many of the current microfluidic devices are manufactured from silica-based substrates, but these materials are difficult and complex to machine, and devices made from such materials are fragile . Furthermore, the transfer of fluid through many existing microfluidic devices requires complex electric field control to transfer the fluid in a controlled manner through the device.

  Thus, in view of the aforementioned benefits that can be achieved using microfluidic devices and the current limitations of existing devices thereto, they are designed for use in performing various chemical and biochemical analyses. There is a need for additional microfluidic devices. Because of its importance in modern biochemistry, there is a particular need for devices that can be used to perform various nucleic acid amplification reactions, while also having sufficient versatility for use in other types of analysis. To do.

  Devices with the ability to perform nucleic acid amplification have a variety of utilities. For example, such a device can be used as an analytical tool to determine whether a particular target nucleic acid of interest is present or absent in a sample. Thus, the device can be used to test for the presence of specific pathogens (eg, viruses, bacteria or fungi) and for identification purposes (eg, paternity and forensic applications). Such devices can also be used for the detection or characterization of specific nucleic acids previously associated with a particular disease or genetic disease. When used as an analytical tool, the device can also be used to perform genotyping analysis and gene expression analysis (eg, differential gene expression studies). Alternatively, the device can be used in a preparative manner to amplify sufficient nucleic acid for further analysis (eg, sequencing of amplification products, cell typing, DNA fingerprinting, etc.). The amplified product can also be used in a variety of genetic engineering applications, such as insertion into a vector that can be used to transform cells for production of the desired protein product.

(Summary)
Various devices and methods for performing microfluidic analysis are provided herein, including devices that can be used to perform thermal cycling reactions (eg, nucleic acid amplification reactions). This device differs from conventional microfluidic devices in that it comprises an elastomeric component, and in some examples, most or all of this device is composed of an elastomeric material.

  Certain devices are designed to perform a thermal cycling reaction (eg, PCR) using a device that includes one or more elastomeric valves to control the flow of solution through the device. Accordingly, a method for performing an amplification reaction using a device of this design is also provided.

  Some devices comprise a blind flow channel with a region that functions as a reaction site. Certain such devices comprise a flow channel formed in the elastomeric material and a plurality of blind flow channels in fluid communication with the flow channel, each blind flow channel region defining a reaction site . The device may also include one or more control channels that overlap and intersect each of the blind flow channels, where the elastomeric membrane is from the blind flow channel at each intersection. The channels are separated. The elastomeric membrane in such a device is arranged to bend or leave the blind flow channel in response to a driving force. The device can optionally further comprise a plurality of protective channels formed in the elastomeric material and overlaying the flow channel and / or one or more reaction sites. This protective channel is designed to allow fluid to pass therethrough to reduce evaporation from the flow channel and reaction site of the device. In addition, the device optionally includes one or more reagents disposed at each of the reaction sites.

  In certain devices, the flow channel is one of a plurality of flow channels, each of which is in fluid communication with a number of blind flow channels that diverge therefrom. In devices of this design, in some examples, multiple flow channels can be interconnected with each other so that fluid can be introduced into each reaction site via a single inlet. However, in other devices, multiple flow channels are separated from each other so that fluid introduced into one flow channel cannot flow to another flow channel and each flow channel is injected at one or both ends. An inlet is provided and fluid can be introduced into the inlet.

Other devices comprise an array of reactive sites having a density of at least 50 sites / cm ² , which are typically formed in an elastomeric material. Other devices have even higher densities (eg, at least 250 sites / cm ² , 500 sites / cm ² or 1000 sites / cm ² ).

  Still other devices comprise reaction sites formed in the elastomeric substrate, where reagents for conducting the reaction are immobilized non-covalently. The reagent can be one or more reagents for performing essentially any type of reaction. The reagent in some devices includes one reagent for performing a nucleic acid amplification reaction. Thus, in some devices, the reagent includes a primer, a polymerase, and one or more nucleotides. In other devices, the reagent is a nucleic acid template.

Various matrix-based or array-based devices are also provided. Specific these devices are the following:
(I) a first plurality of flow channels formed in the elastomer substrate;
(Ii) a second plurality of flow channels formed in the elastomeric substrate that intersect with the first plurality of flow channels to define the reaction sites of the array;
(Iii) a plurality of separation valves disposed in the first and second plurality of flow channels, wherein the valves are actuated to separate the solution in each reaction site from the solution in other reaction sites; A separation valve, and (iv) a plurality of protective channels that overlap one or more flow channels and / or one or more reaction sites to prevent evaporation of the solution from the reaction sites;
Is provided.

  The devices described above can be used to perform many different types of reactions, including reactions involving temperature control (eg, thermal cycling of nucleic acid analysis). A method performed using a particular blind channel type device includes providing a microfluidic device. The device comprises a flow channel formed in the elastomeric material; and a plurality of blind flow channels in fluid communication with the flow channel, with the end of each blind flow channel defining a reaction site. At least one reagent is introduced at each reaction site, and then the reaction is detected at one or more of this reaction site. The method optionally includes heating at least one reagent within the reaction site. Thus, for example, the method includes introducing a component for a nucleic acid amplification reaction, and then thermally cycling the component to form an amplification product.

  Another method includes providing a microfluidic device comprising one or more reaction sites, each reaction site being non-covalently disposed on an elastomeric substrate for a first analysis to be performed. Contains reagents. A second reagent is then introduced into the one or more reaction sites whereby the first reagent and the second reagent are mixed to form a reaction mixture. Reactions between the first reagent and the second reagent at one or more reaction sites are subsequently detected.

Yet another method includes providing a microfluidic device, the microfluidic device comprising an array of reaction sites formed in a substrate and having a density of at least 50 sites / cm ² . At least one reagent is introduced into each reaction site. A reaction at at least one reaction site is then detected.

  Still other methods include providing a microfluidic device, the microfluidic device including at least one reaction site formed in the elastomeric substrate, and a plurality of protections formed in the elastomeric substrate as well. With channels. At least one reagent is introduced into each reaction site and then heated within that reaction site. Fluid flows through the protective channel before or during heating to reduce evaporation from at least one reaction site. A reaction within the at least one reaction site is subsequently detected.

  Additional devices designed to reduce the evaporation of fluid from the device are also provided. In general, such devices comprise a cavity that is part of a microfluidic network formed in an elastomeric substrate; and a plurality of protective channels that overlap the cavity and are separated from the cavity by an elastomeric membrane. The protective channel in such a device is in the cavity by (i) allowing the solution to flow therethrough and (ii) bending the membrane as a result of applying a driving force to the protective channel. The size is such that there is no substantial reduction when entering, exiting or passing through. Other such devices comprise (i) one or more flow channels and / or one or more reaction sites; and (ii) a plurality of protective channels that overlap the microfluidic system and are separated therefrom by an elastomer. Where the gap between the protective channels is between 1 μm and 1 mm. In other devices, the gap is between 5 μm and 500 μm, in other devices between 10 μm and 100 μm, and in other devices between 40 μm and 75 μm.

  Compositions for performing nucleic acid analysis at the reaction site of a particular microfluidic device are also provided. Certain such compositions include one or more of the following: factors and surfactants that block protein binding sites on the elastomeric material. This blocking factor is typically selected from the group consisting of proteins (eg, albumin such as gelatin or bovine serum albumin (BSA)). The surfactant can be, for example, SDS or Triton.

(Detailed explanation)
(I. Definition)
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of a number of terms used in the present invention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MULTICULAR BIOLOGY (2nd edition, 1994); TECHNOLOGY (Walker, 1988); THE GLOSSARY OF GENETICS, 5th edition, R.A. Rieger et al. (Eds.), Springer Verlag (1991); and Hale.
& Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

  A “flow channel” generally refers to a flow path through which a solution can flow.

  The term “valve” refers to a configuration where flow and control channels intersect and are separated by an elastomeric membrane, unless otherwise indicated. The elastomeric membrane may bend into or leave the flow channel in response to a driving force.

A “blind channel” or “dead-end channel” refers to a flow channel that has an inlet but does not have another outlet. Thus, solution flow into and out of the blind channel occurs at the same location. The process of filling one or more blind channels is sometimes referred to simply as “blind injection”.

  A “separated reaction site” generally refers to a reaction site that is not in fluid communication with other reaction sites present on the device. When used with a blind channel, this isolated reaction site is a region at the end of the blind channel that can be blocked by a valve connected to the blind channel.

The "pore" refers to a channel formed in an elastomeric device to provide a flow body access between the external ports and one or more flow channels of the device. Thus, the holes can function as sample inlets and outlets, for example.

  The terms “elastomer” and “elastomeric” have the general meaning used in the art. Thus, for example, Allcock et al. (Contemporary Polymer Chemistry, 2nd edition) generally describe elastomers as polymers that exist at temperatures between their glass transition temperature and liquefaction temperature. Elastomeric materials exhibit stretch properties. Because the polymer chain responds easily to forces, in the absence of that force, the skeletal chain reacts to take its original shape, causing a torsional movement that allows the skeletal chain to return straight Because. In general, an elastomer deforms when a force is applied, but then returns to its original shape when the force is removed. This stretchability exhibited by the elastomeric material can be characterized by a Young modulus. The elastomeric materials used in the microfluidic devices disclosed herein typically represent between about 1 Pa and 1 TPa, in other examples between about 10 Pa and 100 GPa, yet other examples. Has a Young's modulus between about 20 Pa and 1 GPa, yet in other examples between about 50 Pa and 10 MPa, and in particular examples between about 100 Pa and 1 MPa. Elastomeric materials having a Young's modulus outside these ranges can also be used depending on the needs of a particular application.

Some of the microfluidic devices described herein, GE RTV 615 (formulation) is prepared from an elastomeric polymer such as vinylsilane crosslinked (type) silicon elastomer (family). However, the microfluidic system of the present invention is not limited to this one formulation, type or polymer of this family, but rather almost any elastomeric polymer is suitable. Given the wide variety of polymer chemistries, precursors, synthesis methods, reaction conditions and potential additives, there are a number of possible elastomer systems that can be used to make integral elastomeric microvalves and pumps. To do. The choice of material typically depends on the specific material properties (eg, solvent resistance, stiffness, gas permeability, and / or temperature stability) required for the application being performed. Further details regarding the type of elastomeric materials that may be used in the manufacture of the components of the infinitesimal fluid device that is disclosed herein, Unger et al. (2000) Science 288: 113-116, and PCT publication WO02 / 43,615 and WO01 / 01025. These are incorporated herein in their entirety for all purposes.

The terms “nucleic acid”, “polynucleotide”, and “oligonucleotide” as used herein encompass polymeric forms of nucleotides of any length, including but not limited to ribonucleotides or deoxyribonucleotides. Used to be.
There is no intended length difference between these terms. Furthermore, these terms refer only to the primary structure of the molecule. Thus, in certain embodiments, these terms can encompass triple-stranded DNA, double-stranded DNA and single-stranded DNA as well as triple-stranded RNA, double-stranded RNA and single-stranded RNA. These also include modified and unmodified forms of the polynucleotide, such as by methylation and / or capping. More specifically, the terms “nucleic acid”, “polynucleotide” and “oligonucleotide” include polydeoxyribonucleotides (including 2-deoxy-D-ribose), polyribonucleotides (including D-ribose), N— Any other type of polynucleotide that is a purine or pyrimidine base of a glycoside or C-glycoside, as well as other polymers containing non-nucleotide backbones (eg, polyamides (eg, peptide nucleic acids (PNA)) and polymorpholinos (Anti-Virals) , Inc., Corvallis, Oregon, Neugene) and other synthetic sequences that provide nucleobase-containing polymers in a configuration that allows base pairing and base deposition as found in DNA and RNA. Specific nucleus Including a polymer).

  A “probe” is a nucleic acid that can bind to a complementary sequence of target nucleic acid through one or more types of chemical bonds. This chemical bond is usually by complementary base pairing by the formation of hydrogen bonds, thereby forming a double-stranded structure. A probe binds or hybridizes to a “probe binding site”. The probe can be labeled with a detectable label to allow easy detection of the probe, particularly once the probe has hybridized to its complementary target. The label attached to the probe can include any of a variety of different labels known in the art that can be detected, for example, by chemical or physical means. Suitable labels that can be attached to the probe include radioactivity, fluorescence, chromophores, mass labels, electron density particles, magnetic particles, spin labels, chemiluminescent molecules, electrochemically active molecules, enzymes, cofactors And enzyme substrates, but are not limited to these. Probes can vary considerably in size. Some probes are relatively short. Generally, the probe is at least 7-15 nucleotides in length. Other probes are at least 20, 30 or 40 nucleotides in length. Still other probes are somewhat longer, at least 50, 60, 70, 80, 90 nucleotides in length. Still other probes are longer, at least 100, 150, 200 or longer nucleotides in length. The probe can be any specific length that falls within the aforementioned ranges.

  A “primer” is in an appropriate buffer under appropriate conditions (ie, in the presence of four different nucleotide triphosphates and a factor for polymerization (eg, DNA or RNA polymerase or reverse transcriptase)). A single-stranded polynucleotide that can act as a disclosure point for template-dependent DNA synthesis at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer, but is typically at least 7 nucleotides long, more typically in the range of 10 to 30 nucleotides long. Other primers can be somewhat longer (eg, 30-50 nucleotides in length). Short primer molecules generally require lower temperatures in order to form a sufficiently stable hybrid complex with the template. A primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template. The term “primer site” or “primer binding site” refers to the segment of target DNA to which a primer hybridizes. The term “primer pair” is a set of 5 ′ “upstream primer” that hybridizes to the complement of the 5 ′ end of the DNA sequence to be amplified and 3 ′ “downstream primer” that hybridizes to the 3 ′ end of the sequence to be amplified. Means.

  A “fully complementary” primer has a completely complementary sequence over its entire length and has no mismatches. The primer is typically completely complementary to a part (partial sequence) of the target sequence. A “mismatch” refers to a site where the nucleotides in the primer and the nucleotides in the aligned target nucleic acid are not complementary. The term “substantially complementary” when used in reference to a primer is that the primer is not completely complementary to its target sequence; instead, the primer is in its respective desired primer binding site. Is sufficiently complementary only to selectively hybridize with the other strand.

  The term “complementary” means that one nucleic acid is identical to or selectively hybridizes to another nucleic acid molecule. Hybridization selectivity exists when hybridization is selective over the overall lack of specificity. Typically, selective hybridization will have at least about 55%, preferably at least 65%, more preferably at least 75%, and most preferably at least 90% identity over a single at least 14-25 nucleotides. Happens when you do. Preferably, one nucleic acid specifically hybridizes to the other diffusion. M.M. Kanehisa, Nucleic Acids Res. 12: 203 (1984).

  The term “label” refers to a molecule or embodiment of a molecule that can be detected by physical, chemical, electromagnetic and other relevant analytical techniques. Examples of detectable labels that can be used include radioactivity, fluorescence, chromophores, mass labels, electron density particles, magnetic particles, spin labels, chemiluminescent molecules, electrochemically active molecules, and nucleic acid probes. Examples include, but are not limited to, linked enzymes, cofactors and enzyme substrates. The term “detectably labeled” refers to an inherent property that allows an agent to be detected without being conjugated to a label or conjugated to a separate label (eg, , Size, shape or color).

  A “polymorphic marker” or “polymorphic site” is a locus where branching occurs. Preferred markers have at least two alleles, each occurring more frequently than 1% of the selected population, and more preferably occurring more frequently than 10% or 20%. A polymorphic locus can be as small as one base pair. Polymorphic markers include restriction enzyme fragment length polymorphism, variable number of tandem repeats (VNTR), hypervariable region, minisatellite, dinucleotide repeat, trinucleotide repeat, tetranucleotide repeat, simple sequence repeat, insertion such as Alu Element. The first identified allelic form is arbitrarily designated as the reference form, and other allelic forms are designated as alternative or variant allelic forms. The most frequently occurring allelic form in a selected population is sometimes referred to as the wild-type form. A diploid organism may be homozygous or heterozygous for the allelic form. Two alleles have two forms of polymorphism. The three allelic polymorphisms have three forms.

  A “single nucleotide polymorphism” (SNP) occurs at a polymorphic site occupied by a single nucleotide and is the site of variation between allelic sequences. This site is usually present before and after highly conserved allelic sequences (eg, sequences that vary by less than 1/100 or 1/1000 of the population). Single nucleotide polymorphisms usually arise due to substitution of one nucleotide for another at the polymorphic site. A transition is a substitution of one purine for another purine or a substitution of a pyrimidine for another pyrimidine. Transversion is a purine to pyrimidine or pyrimidine to purine substitution. Single nucleotide polymorphisms can also result from nucleotide deletions or nucleotide insertions relative to a reference allele.

“Reagent” refers broadly to any factor used in a reaction. Reagents can include a single factor that can be monitored by itself (eg, a substance that is monitored when heated) or a mixture of two or more. The reagent may be alive (eg, a cell) or not alive. Exemplary reagents for nucleic acid amplification reaction, buffers, metal ions, polymerase, primers, cast type nucleic acid, nucleotides, labels, dyes, but such nucleases include, but are not limited to. Reagents for enzymatic reactions include, for example, substrates, cofactors, conjugated enzymes, buffers, metal ions, inhibitors, and activators. Reagents for cell-based reactions include, but are not limited to, cells, cell-specific dyes, and ligands that bind to cell receptors (eg, agonists and antagonists).

  A “ligand” is any molecule in which there is another molecule that binds specifically or non-specifically to that ligand (ie, an “anti-ligand”). This binding is due to recognition of a portion of the ligand by the anti-ligand.

(II. Overview)
A number of different microfluidic devices (sometimes referred to as chips) with unique flow channel structures are provided herein as well as the use of such devices to perform various high-throughput analyzes. The This device is designed for use in analyzes that require temperature control, especially for thermal cycling (eg, nucleic acid amplification reactions). This microfluidic device incorporates specific design features that provide a significantly smaller footprint than many conventional microfluidic devices, allowing the device to be easily integrated into other means And provide automated analysis.

Some of the microfluidic devices use a design typically referred to herein as a “blind channel” or “blind fill”. These are flow channels that have closed or separated ends so that the solution can only flow in and out of that blind channel from one end, as shown in the definition section (ie for blind channels). There are no separate inlets and outlets). In these devices, only one valve per blind channel is required to separate the blind channel regions to form separate reaction sites. During the manufacture of this type of device, one or more reagents for performing the analysis are placed at the reaction site, thereby resulting in a significant reduction in the number of inputs and outputs. In addition, this blind channel is connected to the channel's interconnect network so that all reaction sites can be filled with a single limited number of sample inputs. Due to the reduced complexity at the input and output and the use of only one valve that separates each reaction site, the space available for the reaction site is increased. Thus, the characteristics of these devices are that each device can be equipped with a large number (eg, up to tens of thousands) of reactive sites and achieve a high reactive site density (eg, greater than 1000-4000 reactive sites / cm ² ). It means you can. Both individually and collectively, these features have also been directly translated into a significant reduction in the size of the microfluidic device of the present invention when compared to conventional microfluidic devices.

Other microfluidic devices disclosed herein, using a matrix design. In general, this type of microfluidic device uses a plurality of intersecting horizontal and vertical flow channels to define an array of reaction sites at the intersection. Thus, the device of this design also has an array of reaction sites, but there are multiple sample inlets and corresponding outlets to accommodate multiple samples in this design. A valve system called a switchable flow array structure allows the solution to flow selectively only in the lateral direction or through the flow channels, thereby allowing a switchable separation of the various flow channels in the matrix. . Thus, blind channel devices are designed to perform a large number of analyzes under different conditions using a limited number of samples, while matrix devices analyze a large number of samples under a limited number of conditions. To be built. Still other devices are a hybrid of these two design types.

The microfluidic devices described above are further characterized in part by utilizing various components (eg, flow channels, control channels, valves and / or pumps made of elastomeric materials). In some examples, an essentially complete device is made from an elastomeric material. Consequently, such devices, the majority of conventional microfluidic devices that are formed from silicon-based materials, form and function are significantly different.

This device design allows it to be utilized in combination with a number of different heating systems. Therefore, this device is useful for performing various analyzes that require temperature control. Furthermore, microfluidic devices for use in heating applications can incorporate additional design features to minimize sample evaporation from the reaction site. This type of device generally comprises a number of protective channels formed in the elastomeric device through which water flows to increase the water vapor pressure in the elastomeric material, and the device is separated from the elastomeric material. Formed, thereby reducing evaporation of the sample from the reaction site.

In another embodiment, the thermal cycling device can be used to control the temperature of the microfluidic device. Preferably, the microfluidic device is adapted to be in thermal contact with the microfluidic device. If the microfluidic device is supported by a substrate material (eg, a glass slide) or the bottom of a carrier plate (eg, a plastic carrier), a window is formed in the area of the carrier or slide, resulting in a microfluidic device, Preferably the device having an elastomeric block can be in direct contact with the heating / cooling block of the thermal cycling device. In a preferred embodiment, the heating / cooling block has a groove therein and communicates with a microfluidic device, preferably a vacuum source for applying suction to the part where the reaction takes place. Alternatively, a hard, thermally conductive plate can be coupled to a microfluidic device that couples with a heating block and a cooling block to produce efficient heat conduction.

  The array format of a particular device means that the device can achieve high throughput. Collectively, high throughput and temperature control capabilities make the device useful for performing multiple nucleic acid amplifications (eg, polymerase chain reaction-PCR). Such reactions are discussed in detail herein as an illustration of the usefulness of the device (especially as an example of use in any reaction requiring temperature control). However, it should be understood that the device is not limited to these particular applications. This device can be utilized for a wide variety of other types of analysis or reactions. Examples include protein-ligand interactions and interactions between cells and various compounds. Further examples are provided below.

(III. General structure of microfluidic device)
(A. Pump and valve)
The microfluidic devices disclosed herein are typically composed at least in part of an elastomeric material, and include single and multi-layer soft lithography (MSL) techniques and / or sacrificial-layer encapsulation methods. (e.g. Unger et al. (2000) Science 288: 113-116 and PCT Publication WO 01/01025, both of which are hereby incorporated by reference in their entirety for all purposes). As a). Utilizing such methods, a microfluidic device is controlled by one or more control channels in which the solution flowing through the device's flow channel is at least partially separated from the flow channel by an elastomeric membrane or segment. Can be designed to The membrane or segment can be deflected into or retracted from the flow channel, to which the control channel is coupled by applying an actuation force to the control channel. By controlling the degree to which the membrane is deflected into or withdraws from the flow channel, the solution flow can be slowed or completely blocked through the flow channel. Using this type of control and flow channel combination, as described in extensive detail in Unger et al. (2000) Science 288: 113-116 and PCT Publications WO / 02/43615 and WO 01/01025, Various different types of valves and pumps can be made to regulate the solution flow.

  The devices provided herein incorporate such pumps and valves to selectively isolate reaction sites where reagents can react. The reaction site can be located at any of a number of different locations within the device. For example, in some matrix type devices, the reaction site is located at the intersection of a set of flow channels. In blind channel devices, the reaction site is located at the end of the blind channel.

  When this device is utilized in a temperature controlled reaction (eg, thermal cycling reaction), the elastomeric device is typically secured to a support (eg, a glass slide), as described in more detail below. The resulting structure can then be placed on a temperature control plate, for example, to control the temperature of the various reaction sites. In the case of a thermal cycling reaction, the device can be placed on any of a number of thermal cycling plates.

Since the device is made from a relatively optically clear elastomeric material, the reaction can be easily monitored using a variety of different detection systems at essentially any location of the microfluidic device. Most typically, however, the detection occurs at its own reaction site (eg, in a region with the intersection of flow channels or the blind ends of the flow channels). This device is also the fact that is manufactured from a substantially transparent material, particular detection system is unusable with conventional silicon-based microfluidic devices, utilized with current device It can be done. Detection can be accomplished using a detector that is incorporated into the device or remote from the device, but aligned within the area of the device to be detected.

(B. Protection channel)
To reduce sample and reagent evaporation from the elastomeric microfluidic devices provided herein, multiple protective channels can be formed in the device. This protective channel is similar to the control channel in that it is typically formed in an elastomeric layer that covers the flow channel and / or reaction site. Thus, like the control channel, the protective channel is separated from the underlying flow channel and / or reaction site by a membrane or segment of elastomeric material. However, unlike the control channel, the protection channel has a fairly small cross-sectional area. In general, a membrane with a smaller area is less deflected than a membrane with a larger area under the same applied pressure. The protective channel is designed at a pressure that allows the solution (typically water) to flow into the protective channel. Water vapor originating from the protective channel can diffuse into the pores of the elastomer adjacent to the flow channel or reaction site, thus increasing the concentration of water vapor adjacent to the flow channel or reaction site and reducing evaporation of the solution therefrom. Let

In general, the protective channel is small enough so that when pressurized, the membrane separating the protective channel from the underlying flow channel or reaction site is within, outside of or within the flow channel or reaction site covered by the protective channel. Does not substantially limit the flow of the solution through. As used in this context, the term “substantially restrictive” or other similar terms refers to solutions flowing in, out of or through a flow channel or reaction site, and flow of solution through a protective channel. Compared to the flow of solution in the flow channel or reaction site under the same conditions when the protective channel is not pressurized to achieve, the flow of solution to them, or the flow of solution through them, Means greater than%, typically less than 30%, usually less than 20%, and in some cases less than 10%. Usually, this means that the guard channels have a cross-sectional area or cross-sectional area not any integer or integer therebetween, between 100 [mu] m ² and 50,000 ^2. Thus, for example, in some instances, the cross-sectional area is less than 50,000 ^2, less than 10,000 ² In another example, in yet another embodiment, less than 1,000 .mu.m ^2, Note In other examples, it is less than 100 μm ² . Protection channel may take any of a variety of shapes, is in it found a circular, oval, square, rectangular, hexagonal, and the shape of the octagonal include, but are not limited to.

The protection channel provides less than 50% evaporation of sample and reagent from the device, and in other instances less than 45%, less than 40%, and 30 during the time required to perform the thermal cycling reaction. Less than 25%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5% or less than 1%. Thus, for example, a typical PCR reaction of 40 cycles can be performed within 120 minutes. The protection channel system is designed to reduce evaporation during approximately this time frame for the following set of limits. In order to achieve this level of evaporation reduction, the protection channels are typically present at a density of at least 10 lines / cm ^{2 to} 1000 lines / cm ² , or any integer density level therebetween. More particularly, the protection channel is typically at least 25 lines / cm ² , in other examples at least 50 lines / cm ² , in still other examples at least 100 lines / cm ² , and in still other examples at least 500 lines / cm ² . Line / cm ² . To achieve this level of evaporation reduction, the protection channel typically has a spacing between 1 mm and 1 μm, measured from the outer end of one line to the nearest outer end of an adjacent line, Or any integer density level between them. More particularly, the protection channels are generally spaced between 500 μm and 5 μm, in other examples between 100 μm and 10 μm, and in still other examples between 75 μm and 40 μm. Yes. Thus, the spacing is typically at least 1 μm, but less than 1 mm, in other examples less than 500 μm, in yet other examples, less than 400 μm, and in still other examples, less than 300 μm. In other examples, less than 200 μm, and in still other examples, less than 100 μm, less than 50 μm, or less than 25 μm.

  The protection channel may be formed as a separate network of channels or may be smaller than a channel that is a branch of the control channel. The protection channel can extend beyond the device or can extend beyond a specific area of the device. Typically, the protection channels are placed adjacent to the flow channel and the reaction site, since it is the first position where evaporation is first involved. Exemplary locations of protection channels on specific matrix devices are shown in FIG. 1C, exemplary locations on specific blind channel devices are shown in FIGS. 3B and 3C, and will be discussed in more detail below. .

The solution flowing through the protective channel includes any substance that can reduce water evaporation. This material is a material that increases the concentration of water evaporation adjacent to the flow line and / or reaction site, or the concentration of water vapor despite blocking water vapor from the flow line and / or reaction site. It may be a substance that does not increase (blocking factor). Thus, one option is to utilize essentially any aqueous solution, where appropriate solutions include water and buffer solutions (eg, TaqMan buffer solution and phosphate buffered saline). However, it is not limited to these. Suitable blocking factors include, for example, mineral oil.

  The protective channel is typically formed in the elastomer using MSL techniques and / or the sacrificial layer encapsulation method cited above.

  The following sections describe in more detail a number of specific configurations that can be utilized to perform various analyzes, including those that require temperature control (eg, nucleic acid amplification reactions). However, it should be understood that these arrangements are exemplary and modifications to these systems will be apparent to those skilled in the art.

(IV. Matrix design)
(Overview)
Devices that use a matrix design typically have multiple vertical and horizontal flow channels that intersect to form the junctions of the array. Since various samples and reagents (or sets of reagents) can be introduced into each flow channel, a large number of samples can be tested in a high throughput format for a relatively large number of reaction conditions. Thus, for example, be introduced into different samples of M different vertical flow channels, different reagents (or set of reagents) is, when introduced into each of the N horizontal flow channels, M × N number of Different reactions can occur simultaneously. Typically, matrix devices comprise valves that allow switchable separation of vertical and horizontal flow channels. In other words, the valves are arranged to allow selective flow through only vertical or horizontal flow channels. This type of device allows flexibility with respect to the type and number of samples, and the choice of number and types of reagents, so that these devices can screen a large number of samples against a relatively large number of reaction conditions. It is well suited for performing the desired analysis. The matrix device can incorporate protective channels as needed to help prevent sample and reactant evaporation.

  The present invention provides a high density matrix design that utilizes fluid communication holes between layers of a microfluidic device to assemble control and fluid lines through the device. For example, having a fluid line in each layer of a two layer elastomeric block allows for a higher density reaction cell arrangement. FIG. 21 shows an exemplary matrix design, in which a first elastomer layer 2110 (first layer) and a second elastomer layer 2120 (second layer), each having a fluid channel, are formed therein. . For example, a reagent fluid channel in the first layer 2110 is connected to a reagent fluid channel in the second layer 2120 through a hole 2130, but the second layer 2120 has a sample channel therein. The sample channel and reagent channel terminate in the sample and reagent chamber 2180, respectively. The sample chamber and reagent chamber 2180 are in fluid communication with each other through an interface channel 2150 that has an interface valve 2140 coupled to it to control fluid communication between each of the chambers 2180 of the reaction cell 2160. In use, the interface is first closed, reagent is introduced into the reagent channel from the reagent inlet, sample is introduced into the sample channel through the sample inlet, and the containment valve 2170 is then closed, each reaction cell 2160 Is separated from the other reaction cell 2160. Once the reaction cell 2160 is separated, the interface valve 2140 is opened, causing fluid communication with the sample chamber and reagent chamber with each other so that the desired reaction can occur.

  Accordingly, a preferred aspect of the present invention provides a microfluidic device for reacting M different samples with N different reagents, the following: a plurality of reaction cells (each reaction cell comprising a sample chamber and a reagent chamber). A plurality of sample chambers and reagent chambers in fluid communication with the interface channel having an interface valve coupled to the interface channel to control fluid communication between the sample chamber and the reagent chamber, each in fluid communication with the sample chamber A plurality of reagent inlets in fluid communication with each of the sample inlets and reagent chambers, one of the sample inlets or one of the reagent inlets being one of the sample chambers or one of the reagent chambers, respectively. It is in fluid communication through a hole. Certain embodiments have a reaction cell formed in an elastomeric block formed from multiple layers bonded together, and the interface valve is a deflectable membrane; in fluid communication with the sample chamber via the sample channel A sample inlet, wherein the reagent inlet is in fluid communication with the reagent chamber via a reagent channel, wherein a portion of the sample channel and a portion of the reagent channel are oriented substantially parallel to each other and include a containment valve. A containment valve coupled to each for controlling fluid communication therethrough; a valve in communication with the sample channel; the valves in communication with the reagent channel are in operative communication with each other through a common containment control channel; The containment common control channel is in a row that is approximately perpendicular to one of the sample or reagent channels. They are placed me.

Another aspect of the present invention provides a method for creating a feature in an elastomeric block comprising the steps of: providing a first elastomeric layer; on the surface of the first elastomeric layer Applying a photoresist layer; applying a light pattern to the photoresist layer to form a pattern of reactive photoresist material; leaving a pattern of reacted photoresist on the surface of the first elastomer layer; Removing unreacted photoresist material; applying an etching reagent to the first elastomer surface to etch the surface of the first elastomer layer not covered by the pattern of reacted photoresist material, thereby A process for removing regions of the first elastomer layer not covered by the reacted photoresist pattern ; And reacted photoresist material step can place a corresponding pattern of the elastomeric layer to the pattern of. Certain preferred embodiments of the method, the reacted photoresist material that has a step of removing the pattern of; caused by applying an adhesive tape to a surface of the step Gae elastomer layer to be removed, then reacted Images Separating the adhesive tape from the elastomeric layer while having some or all of the resist material pattern removed from the surface of the elastomeric layer; having a photoresist that is SU8; the etching reagent is tetrabutylammonium fluoride Including a hydrate; having a characteristic of being a pore; having an elastomer block comprising a plurality of elastomer layers bonded together, wherein two or more elastomer layers have recesses formed therein. And one recess in one elastomer layer is connected to another elastomer through the hole. It involves communicating with the recess of the over layer.

The microfluidic device of the present invention may be further incorporated into a carrier device as described in pending co-owned US patent application Ser. No. 60 / 557,715, filed March 29, 2004 by Unger, This patent application is incorporated herein for all purposes. Unger's carrier provides on-board continuous fluid pressure and maintains valve blockage away from a source of fluid pressure (eg, house pressure). Unger further provides an automated system for charging and operating the valves of the present invention as described therein. In another preferred embodiment, the accumulator is loaded, automated system for actuating the valve, using Lud devices that have a platen to which pair against one or more surfaces of the microfluidic device, the The platen comprises at least two or more ports in fluid communication with a controlled vacuum or pressurized source and includes a mechanical part (eg, including but not limited to a check valve) for the operating part of the microfluidic device. Not).

Another aspect of the present invention provides for use as an elastomeric block, a substrate for stabilizing a carrier, preferably having one or more of the following characteristics: at least one formed in or with the carrier A well or reservoir in fluid communication with the elastomer block through the channel; an accumulator in fluid communication with the elastomer block through at least one channel formed in or with the carrier; and a fluid port in fluid communication with the elastomer block, preferably vacuum Or a fluid port accessible to an automated source of pressurization (eg, the automated system described above), wherein the automated source further comprises a platen having a port mated with the fluid port, between the automated system Separate fluid communication Formed, to apply fluid pressure or vacuum to the elastomeric block. In certain embodiments, the automated source may create fluid communication with one or more accumulators that couple with the carrier to increase and decrease the pressure maintained in the accumulator. In certain embodiments, the carrier may further comprise a region located within the carrier that contacts the microfluidic device, the region being made from a different material than another portion of the carrier, the material of the region Are selected for improved heat transfer and distribution characteristics that are different from other parts of the carrier. Preferred materials for improved thermal conduction and distribution, including but silicone is not limited thereto, preferably, highly polished silicon (e.g., cut from the wafer and or wafer polished in the semiconductor field portion ( for example, chips) available types of silicon) can be mentioned as.

Another aspect of the invention provides for using a heat source (eg, but not limited to a PCR thermocycler), which can be modified from its original manufactured state. The heat source has a thermally regulated portion that can be mated with a portion of the carrier, preferably the heat conducting portion and the heat distributing portion of the carrier, thereby reducing the heat conducting portion and the heat distributing portion of the carrier. Temperature control of the elastomer block. In a preferred embodiment, the thermal contact is improved by applying a vacuum source to one or more channels formed in the thermally regulated portion of the heat source, which channel is a thermally conductive portion of the carrier. And is formed to contact the surface of the heat distribution portion and applies suction to the heat transfer portion and heat distribution portion of the carrier to maintain their position. In a preferred embodiment, the heat transfer portion and heat distribution portion of the carrier are not in physical contact with the carrier, but the heat transfer portion and heat distribution portion are attached only to the elastomeric block, By locating a gap surrounding the edge, the carrier and the elastomeric block are combined by reducing the parasitic temperature effect by the carrier. It should be understood that in many aspects of the invention described herein, the preferred elastomeric block can be replaced with any known microfluidic device in the art that is not described herein. As the device, for example, Affymetrix (R), Santa Clara, California, Gene Chip (R) of USA or Caliper of Mountain View, California, Gene Chip (R) of USA. U.S. patents issued to Soane, Parce, Fodor, Wilding, Ekstrom, Quake or Unger are other thermal advantages and improvements (e.g., the location of suction, other A microfluidic device or medium-scale fluidic device is described that can be used to replace the elastomeric block of the present invention utilizing reduced superheated heat conduction to the region.

(B. Exemplary design and use)
FIG. 1A provides an illustration of one exemplary matrix device. The device 100 includes seven vertical flow channels 102 and seven horizontal flow channels 104 that intersect to form an array of 49 different intersections or reaction sites 106. This particular device thus allows 7 samples to react with 7 different reagents or sets of reagents. A row valve 110 that regulates the flow of vertically oriented solution can be controlled by a control channel 118 that can be all actuated by a single inlet 114.

  Similarly, the row valve 108 regulates the horizontally oriented solution flow; these are controlled by a control channel 116 that is activated by a single control inlet 112. As shown in FIG. 1A, the width of the control channel 116 for adjusting the row valve 108 varies depending on its position. If the control channel 116 intersects the vertical flow channel 102, the control channel 116, when activated, is sufficient to not deflect the vertical flow channel 102 so as to substantially reduce the flow of solution through the vertical flow channel 102. It is thin. However, the width of the control channel 116 increases when the control channel 116 is above one horizontal flow channel 104, which causes the control channel membrane to block the flow of solution through the horizontal flow channel 104. To be big enough.

  In operation, reagents R1-R7 are introduced into their respective horizontal flow channels 104 and samples S1-S7 are injected into their respective vertical flow channels 102. Thus, the reagent in the horizontal flow channel 104 is mixed with the respective sample of the vertical flow channel 102 at an intersection 106, which in this particular device is in the shape of a well or chamber. Thus, in the specific case of a nucleic acid amplification reaction, for example, reagents necessary for the amplification reaction are introduced into each of the horizontal flow channels 104. Various nucleic acid templates are introduced into the vertical flow channel 102. In a particular analysis, the primers introduced as part of the reagent mixture introduced into each of the horizontal flow channels 104 can vary between the flow channels. This allows each nucleic acid template to react with a number of different primers.

  1B-E show enlarged plan views of adjacent reaction sites of the device shown in FIG. 1A to illustrate in more detail how the device operates during analysis. For purposes of clarity, the intersection 106 is not shown in the shape of the reaction well, the control channels 116, 118 are omitted, and only the row and column valves 110, 108 are shown (rectangular). As shown in FIG. 1B, the analysis closes the row valve 110 and opens the column valve 108 to allow solution flow through the horizontal flow channel 104 while blocking flow through the vertical flow channel 102. To start. Reagent R 1 is then introduced into horizontal flow channel 104 and flows completely over the entire length of horizontal flow channel 104, thus filling all reaction sites 106. Solution flow through the horizontal channel 104 can be achieved by an external pump, but more typically described in detail, for example, in Unger et al. (2000) Science 288: 113-116 and PCT Publication No. WO 01/01025. As is achieved, it is achieved by incorporating a peristaltic pump into the elastomeric device itself.

  Once R1 is introduced, the column valve 108 is closed and the row valve 102 is opened (see FIG. 1C). This allows samples S1 and S2 to be introduced into vertical flow channel 102 and flow through their respective flow channels. As the sample flows through the vertical flow channel 102, R1 is drained from the reaction site 106, thus leaving the sample at the reaction site 106. The column valve 108 is then opened, allowing S1 and S2 to diffuse and mix with R1, as shown in FIG. 1D. Thus, a mixture of samples and reactants (R1S1 and R1S2) is obtained at the area of each intersection, ie, the reaction site 106. After allowing sufficient time for S1 and S2 to diffuse with R1, all column valves 108 and row valves 110 are closed, separating S1 and S2 in the region of their respective reaction sites 106, and S1 And mixing of S2 is prevented (see FIG. 1E). The mixture can then react, and the reactant is detected by monitoring the intersection 106 or a cross-shaped area that includes the intersection 106. For analyzes that require heating (eg, thermal cycling during the amplification reaction), the device is placed on a heater and heated while the sample remains separated.

  A modified version of the device shown in FIG. 1A is shown in FIG. 1F. The general structure has many similarities to the structure shown in FIG. 1A, and elements common to both figures share the same reference numbers. The device 150 illustrated in FIG. 1F differs in that it is a pair of horizontal flow channels 104 that couple to a common inlet 124. This essentially allows two sets of reagents to be introduced into two adjacent flow channels with only a single injection into the inlet 124. The use of a common inlet is further expanded with respect to the vertical flow channel 102. In this particular example, each sample is introduced into five vertical flow channels 102 with a single injection into the sample inlet 120. Thus, in this particular device, there are essentially ten repeated reactions for each particular combination of sample and reagent. Of course, the number of repeated reactions can be varied as desired by changing the number of vertical flow channels 102 and / or horizontal flow channels 104 connected to a common inlet 120, 124.

The device shown in FIG. 1F also includes a separate control channel inlet 128 that regulates the control channel 130 that can be used to control the flow of solution to the outlet 132, and a control that regulates the flow of solution to the outlet 136. Another control channel inlet 132 for controlling the channel 134 is provided. In addition, device 150 captures protection channel 138. In this particular design, protection channel 138 is formed as part of control channel 116. As indicated above, the protection channel 138 is smaller than the column valve 108; consequently film protection channel 138, the flow of the solution is not deflected to the horizontal flow channels 104 in the lower layer so as to interrupt.

Finally, the design object shown in FIG. 1F, the reaction is not intended that arise in the intersection of the horizontal flow line and vertical flow line well, with the difference that occurs in the intersection itself.

(V. Blind channel design)
(A. Outline)
Devices that utilize blind channel designs have certain characteristics. First, the device comprises one or more flow channels from which one or more blind channels are branched. As indicated above, the terminal regions of such channels can serve as reaction sites. A valve formed by placing on the flow channel can be actuated to separate the reaction sites at the end of the blind channel. The valve provides a mechanism for switchably separating the reaction sites.

  Second, the flow channel network in communication with the blind channel is configured such that all or most reaction sites can be filled with a single or a limited number (eg, less than 5 or less than 10) of inlets. The The ability to fill blind flow channels is possible because the device is made from an elastomeric material. The elastomeric material is sufficiently porous so that air in the flow and blind channels can escape through these pores when a solution is introduced into the channel. The lack of porosity of materials utilized in other microfluidic devices makes it impossible to use blind channel designs. This is because the air in the blind channel will never leak if the solution is injected unless a vent is provided somewhere along the fluid path.

A third feature is that one or more reagents are non-covalently disposed on the elastomeric base layer in the reaction site during manufacture (see below for further details of the manufacturing process). ) The reagent is bound non-covalently. This is because the reagent is designed to dissolve when the sample is introduced into the reaction site. The number of analyzes in order to maximize the variety of reaction monomers other set of reactants is disposed in each of the different reaction sites.

  Certain blind channel devices are designed such that the reaction sites are arranged in the form of an array.

Thus, in a blind channel device designed to perform a nucleic acid amplification reaction, for example, one or more reagents necessary to perform an extension reaction are placed at each reaction site during device manufacture. Such reagents include, for example, all or some of the following: primers, polymerases, nucleotides, cofactors, metal ions, buffers, intervening dyes, and the like. In order to maximize high throughput analysis, different primers selected to amplify different regions of DNA are placed at each reaction site. Consequently, when a nucleic acid template is introduced into the reaction site via an inlet, multiple extension reactions can be performed on different segments of the template. Thermal cycling required for the amplification reaction, the device was placed on a thermal cycling plate, may be achieved by cycling the device between the various required temperatures.

  Reagents can be immobilized in various ways. For example, in some examples, one or more reagents are non-covalently disposed at a reaction site, while in other examples, one or more reagents are covalently bound to a substrate at the reaction site. When covalently bound, the reagent can be bound to the substrate via a linker. A variety of linker types can be utilized, such as photochemical / photosensitive linkers, thermolabile linkers and linkers that can be cleaved by enzymes. Some linkers are bifunctional (ie, the linker contains a functional group at each end, which is reactive with the group located on the component to which the linker is attached) These functional groups may be the same or different. Examples of suitable linkers that can be used in some assays include linear or branched carbon linkers, heterocyclic linkers, and peptide linkers. Various types of linkers are available from Pierce Chemical Company, Rockford, Illinoi, EPA 188,256, US Pat. No. 4,671,958; 4,659,839, 4,414, 148, 4,669,784, 4,680,338, 4,569,789, and 4,589,071, and Eggenweiler, H. et al. M.M. , Pharmaceutical Agent Discovery Today 1998, 3, 552. NVOC (6-nitroveratryloxycarbonyl) linkers and other NVOC-related linkers are examples of suitable photochemical linkers (see, eg, WO90 / 15070 and WO92 / 10092). Peptides having a protease cleavage site are discussed, for example, in US Pat. No. 5,382,513.

  FIG. 2 is a simplified plan view of one exemplary device that utilizes a blind channel design. Device 200 includes a flow channel 204 formed in an elastomeric substrate 202 and a set of branch flow channels 206 that branch off therefrom. Each branch flow channel 206 terminates at a reaction site 208, thereby forming an array of reaction sites. A control channel 210 separated from the branch flow channel 206 by the membrane 212 is placed over the branch flow channel 206. Actuation of the control channel 210 deflects the membrane 212 into the branch flow channel 206 (ie, to function as a valve), thus allowing each of the reaction sites 208 to be separated from other reaction sites.

  Operation of such a device involves injecting a test sample into the flow channel 204 and then flowing the solution through each of the branch channels 206. Once the sample fills each branch channel 206, the control channel 210 is activated, causing the valve / membrane 212 to be activated and deflected into the branch channel 206, thereby sealing each reaction site 208. As the sample flows into and remains in the reaction site 208, the sample dissolves the pre-spotted reagent at each of the reaction sites 208. Once dissolved, the reagent can react with the sample. The valve 212 prevents the reagent dissolved at each reaction site 208 from mixing by diffusing. The reaction between the sample and the reagent is then typically detected within the reaction site 208. The reaction can optionally be heated as described in the temperature control section below.

  FIG. 3A illustrates an example of a slightly more complex composite blind flow channel design. In this particular design 300, each of a set of horizontal flow channels 304 connects with two vertical flow channels 302 at each end. A plurality of branch flow channels 306 extend from each of the horizontal flow channels 304. The branch flow channels 304 in this particular design are interleaved, so a branch channel 306 attached to any given horizontal flow channel 304 is two branches that couple directly adjacent to the horizontal flow channel 304. Located between the channel 306 or between a branch flow channel 306 that couples directly adjacent to one of the flow channel 304 and the vertical flow channel 302. Similar to the design shown in FIG. 3A, each branch flow channel 302 terminates within a reaction site 308. Also, consistent with the design shown in FIG. 3A, control channels 310 are placed on each branch channel and separated from the underlying branch channel by membrane 312. The control channel operates at port 316. The vertical flow channel 302 and the horizontal flow channel 304 are interconnected so that injection of sample into the inlet 314 allows solution flow through the network of horizontal and vertical flow channels and eventually branches. Allows flow to each of the reaction sites 308 via the flow channel 306.

  Thus, during operation, the sample is injected into the inlet and introduces the solution into each reaction site. Once the reaction site is filled, the valve / membrane is activated and traps the solution in the reaction site by pressurizing the control channel at the port. Reagents previously placed in the reaction site are resuspended in the reaction site, thereby allowing a reaction between the reagent placed in each reaction site and the sample. The reaction within the reaction site is monitored by a detector. As before, the reaction is controllably heated, if necessary, according to the method shown in the temperature control section below.

  An even more complex version of the general design illustrated in FIG. 3A is shown in FIG. 3B. The device shown in FIG. 3B is a device in which the organizational unit of the horizontal branch flow channel 302 shown in FIG. 3A is repeated multiple times. The device shown in FIG. 3B further illustrates providing a protective channel 320 for a device utilized for applications involving heating (eg, thermal cycling). An exemplary orientation of protection channel 320 with respect to flow channel 304 and branch channel 306 is shown in the enlarged view shown in the right panel of FIG. 3B. A protection channel 320 is placed over the branch flow channel 306 and the reaction site 308. As discussed above, water flows through the protective channel 320 during heating of the device 300, increasing the local concentration of water in the device, thereby increasing the solution in the flow channel 306 and reaction site 308. Reduces water evaporation from.

The features of the blind channel device discussed at the beginning of this section minimize the device footprint, allow multiple reaction sites to be formed on the device, and achieve high density. For example, this type of device with 2500 reaction sites can be easily manufactured to fit on a standard microscope slide (25 mm × 75 mm). The features described above also allow a very high density of reactive sites to be obtained using a device that utilizes a blind channel design. For example, at least 50, 60, 70, 80, 90 or 100 reaction sites / cm ² , or any integer density value therebetween, can be easily obtained. However, certain devices have a higher density range (eg, 100-4000 reaction sites / cm ² or any integer density value therebetween). For example, some devices have a density of at least 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 sites / cm ² . Devices with very high density of at least 2000, 3000 or 4000 sites / cm ² are also available. Such high density is interpreted directly as a large number of reaction sites on the device. Devices that utilize blind channel structures typically have at least 10 to 100 reaction sites, or any integer value therebetween. More typically, the device has at least 100 to 1000, or any integer number of sites therebetween. Higher density devices may have even more reactive sites (eg, at least 1,000-100,000 reactive sites, or any integer number in between). Thus, a particular device is at least 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, depending on the overall size of the device. It has 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000 or 100,000 reaction sites.

  The large number of reaction sites and densities that can be obtained are also a result of the ability to make very small wells or holes. For example, a hole or well typically has a volume of less than 50 nL, in other examples, a volume of less than 40 nL, less than 30 nL, less than 20 nL, or less than 10 nL, and in still other examples, a volume of less than 5 nL or less than 1 nL. Have. In a particular example, a particular device has a well that is 300 microns long, 300 microns wide, and 10 microns deep.

  The blind channel devices provided herein are of the specific design discussed in PCT / US01 / 44549 (published as WO02 / 43615) and PCT / US02 / 10875 (published as WO02 / 082047). Features and methodologies may be used, such as strategies for filling dead-end channels, liquid priming, compressed gas discharge priming, and for replacing gas while filling microfluidic channels There are various methods. Both of these PCT publications are incorporated herein by reference in their entirety for all purposes.

(VI. Hybrid design)
Still other devices are matrix hybrid and blind fill designs. The design of this type of device is the blind channel shown in FIG. 3A, except that each horizontal flow channel is connected to its own sample inlet port and the horizontal flow channel is not interconnected via a vertical flow channel. Similar to device. As a result, a sample introduced into any given horizontal flow channel fills only that horizontal flow channel and the reaction sites connected to it. In contrast, in the blind flow channel device shown in FIG. 3A, samples can flow between the horizontal flow channels 304 via the vertical flow channels 302.

  An example of this general device is shown in FIG. Device 400 includes a plurality of horizontal flow channels 404, each having a plurality of branch flow channels 406 extending therefrom and its own sample inlet 414. A control 410 is placed on each of the branch flow channels 406 and a membrane (valve) 412 separates the control channel 410 from the underlying branch flow channel 406. Similar to the blind flow channel design, actuation of the control channel at the inlet 416 causes deflection of the membrane 412 into the branch flow channel 406 and separation of the reaction site 408. In this design variation, each horizontal flow channel 404 may be provided with an inlet 414 at each end, thus allowing samples to be introduced from both ends.

  In some examples, the reagent is placed at the reaction site during manufacture of the device. This allows a large number of samples to be tested under a relatively large number of reaction conditions in a short time without requiring the addition of time consuming reagents required for the matrix device. . Alternatively, the reaction mixture can be prepared prior to injection into the chip. Once the mixtures are injected, they can be analyzed or further processed (eg, heated).

  By injecting various samples into each of the horizontal flow channels, a large number of samples can be analyzed quickly. Assuming that the reagents were pre-arranged at the reaction site, the presence of the same reagent at each reaction site associated with any given horizontal flow channel is an easy way to perform multiple replicate reactions with each sample. I will provide a. Instead, if the reagents at the reaction site are different for any given flow channel, each sample is exposed to a variety of different reaction conditions at essentially the same time.

  Thus, the devices provided herein are specifically tailored for a variety of different types of research. If the study involves screening of a relatively large number of different samples under user-controlled conditions (eg, 100 samples for a user-selected 100 reagents), the matrix device provides a useful solution. . However, if the study involves the analysis of one or a limited number of samples under a wide variety of reaction conditions (eg, one sample for 10,000 reaction conditions), the blind channel design is Is useful. Finally, if you want to test a relatively large number of samples against defined reaction conditions without injecting reagents (eg 100 samples for 100 predefined reagents) The device is useful.

(VII. Temperature control)
(A. Devices and components)
A number of different options of different sophistication are available to control the temperature of a selected region of the microfluidic device or the entire device. Thus, as used herein, the term temperature controller refers to an entire microfluidic device or a portion of a microfluidic device (eg, within a particular temperature region or within a matrix of blind channel microfluidic devices). It is meant to broadly refer to a device or element that can regulate the temperature (at more than one junction).

  Generally, the device is placed on a thermal cycling plate to temperature circulate the device. A variety of such plates are readily available from commercial sources such as ThermoHybaid Px2 (Franklin, Mass.), MJ Research PTC-200 (South San Francisco, Calif.), Eppendorf Part # E5331 (Nestbury, Westy ), Techne Part # 205330 (Princeton, NJ).

  The array device is in contact with the thermal control device, so that the thermal control device is in thermal communication with the thermal control source, and the temperature of the reaction in the at least one reaction chamber is such that the temperature of the thermal control source changes. As a result changes.

In different embodiments, the heat transfer device, a semiconductor (e.g., silicon) may include, may contain a reflective material and / or metal may be contained.

The heat control device may be adapted to apply a force to the heat transfer device to propel the heat transfer device against a heat control source. The force may include magnetic force, electrostatic force or vacuum force in different embodiments. For example, in one embodiment, the force includes a vacuum force applied to the heat transfer device via a channel formed in the surface of the heat control device or heat transfer device. The level of vacuum achieved between the surface of the thermal control device and the surface of the heat transfer device can be detected. Such detection may be performed using a vacuum level detector located at a location along the channel distal from the location of the vacuum source. If the vacuum does not exceed a pre-set level, a warning can sound or a reconditioning protocol can be engaged.

  The array device can contact the thermal control device through the use of one or more mechanical or electromechanical positioning devices. The implementation of this method can be automatically controlled and monitored. For example, such automatic control and monitoring may be performed using an automatic control system in operative communication with a robotic control system for introducing and removing array devices from the thermal control device. The progress of the reaction can also be monitored.

  A unit comprising a thermal control device may be provided. A system comprising an array device and a thermal control device may be provided.

  In order to ensure the accuracy of the thermal cycling process, it is useful to incorporate sensors in a particular device that detect temperature in various regions of the device. One structure for detecting temperature is a thermocouple. Such thermocouples can be made as thin film wires patterned under the substrate material or as wires incorporated directly into the microfabricated elastomeric material.

The temperature can also be measured via a change in electrical resistance. For example, the change in resistance of the thermistor which is processed into a semiconductor board underneath using conventional techniques can be calibrated to a given temperature change. Alternatively, the thermistor can be inserted directly into the microfabricated elastomeric material. Yet another approach to detecting temperature by resistance is described in Wu et al., “MEMS Flow Sensors for Nano-fluidic Applications”, Sensors and Actuators, A89, 152-158, (2001), which is described herein. The entire document is incorporated by reference. This document describes the use of polysilicon Con structure doped to both control and measure the temperature. For polysilicon Con and other semiconductor materials, the temperature coefficient of resistance may be precisely controlled by the identity and amount of dopant, thus, to optimize the performance of the sensor for a given application.

Thermo-chromatic material is another type of structure that can be used to detect the temperature in the region of the amplification device. In particular, certain materials change color dramatically and reproducibly through different temperatures. Such materials can be added to the solution as it passes through different temperatures. Temperature chromatic materials are groups Saitama other underlying may be formed embedded within the elastomeric material. Alternatively, the temperature chromatic material can be added to the sample solution in the form of particles.

Another approach to detecting temperature is through the use of an infrared camera. Infrared cameras are used that are connected with the microscope, it can determine the temperature profile of the amplification structure overall. The permeability of the elastomeric material to irradiation facilitates this analysis.

  Yet another approach for temperature detection is through the use of pyroelectric sensors. In particular, some crystalline materials, especially those exhibiting piezoelectric behavior, also exhibit a pyroelectric effect. This effect describes the phenomenon responsible for the polarization of the material's crystal lattice, so the voltage across the material is highly temperature dependent. Such materials can be incorporated on a substrate or elastomer and used to detect temperature.

  Other electrical phenomena (eg, capacitance and inductance) can be utilized to detect temperature in accordance with embodiments of the present invention.

(B. Accurate thermal cycling evaluation)
As described in more detail in the fabrication section below, the blind channel device has a basal layer in which reagents are disposed. A structure comprising two layers comprising a flow channel and a control channel covers the surface on the basal layer so that the flow channel is aligned with the deposited reagent. The opposite side of the base layer is then placed on a substrate (eg, glass). Typically, the reaction site where the reaction takes place is about 100-150 microns above the substrate / glass interface. Calculate the time required for the temperature in the reaction site to reach the temperature that the controller seeks to maintain, using known formulas for thermal diffusivity and the appropriate values for the elastomers and glasses utilized in the above devices. Can do. The calculated values shown in Table 1 indicate that the elastomer and glass are much thicker than those utilized in devices where the reaction sites are about 100-150 microns (ie, typical distances for the devices described herein). It demonstrates that the temperature is achieved rapidly even when using this layer.

  (Table 1: Calculated thermal diffusion length through PDMS and glass layer for the indicated time period)

図５は、ブラインドチャネルデバイスを使用して所望の温度が達成される速度を示す。

FIG. 5 shows the rate at which the desired temperature is achieved using a blind channel device.

  In another embodiment, the temperature can be measured by using a double stranded oligonucleotide polymer with a known tm, where its interpolation indicates that the oligonucleotide has hybridized or denatured. An intercalation dye (eg, SYBR Green ™ or ethidium bromide) can be prepared by introducing a solution containing the above oligonucleotide together with the dye into a chamber of a microfluidic device having an array of reaction chambers, for example. It can be used to determine that the degree of chamber temperature is constant throughout the array. In this embodiment, when the temperature rises above tm, the intervening dye changes its relationship to the oligonucleotide as it denatures into a single stranded oligonucleotide. Alternatively, if the temperature is above and below tm, dye insertion into the oligonucleotide that is being annealed at that time can be monitored. In essence, the use of a dye provides an “oligonucleotide thermometer” that changes properties (eg, fluorescence) in response to temperature changes associated with the tm of the oligonucleotide. By designing or using oligonucleotides with a selected tm, the extent to which the array of reaction chambers changes temperature in a similar manner can be determined.

(VIII. Detection)
(A. Overview)
Many different detection strategies can be utilized with the microfluidic devices provided herein. The selection of an appropriate system is known in part with respect to the type of event and / or the factor to be detected. Detectors can be designed to detect many different signal types, including but not limited to: radioisotopes, fluorophores, chromophores, electron density particles, magnetism Signals from enzymes associated with particles, spin labels, chemiluminescent molecules, electrochemically active molecules, enzymes, cofactors, nucleic acid probes and enzyme substrates.

  Exemplary detection methodologies suitable for use in the microfluidic devices of the present invention include light scattering, multichannel fluorescence detection, UV and visible wavelength absorption, luminescence, differential reflection, and confocal laser scanning methods. However, it is not limited to these. Additional detection methods that can be used in specific applications include scintillation proximity assay techniques, radiochemical detection, fluorescence polarization, fluorescence correlation spectroscopy (FCS), time-resolved energy transfer (TRET), fluorescence resonance energy transfer (FRET) and variations ( For example, bioluminescence resonance energy transfer (BRET)). Additional detection options include electrical resistance, resistivity, impedance, and voltage sensing.

  Detection occurs in the “detection section” or “detection region”. These terms and other related terms refer to the portion of the microfluidic device where detection occurs. As indicated above, for devices that utilize blind channel designs, the detection section is generally a reaction site that is isolated by a valve that connects to each reaction site. The detection section for matrix-based devices is usually in the region of the flow channel adjacent to the intersection, the intersection itself, or the region that includes the intersection and the surrounding region.

  The detection section may communicate with one or more of a microscope, a diode, a photostimulation device (eg, a laser), a photomultiplier tube, a processor, and combinations of the foregoing, which work together for specific events and A signal associated with the factor is detected. In many cases, the detected signal is an optical signal detected in the detection section by an optical detector. The optical detector includes one or more photodiodes (eg, avalanche photodiodes), fiber-optic light guide reading (eg, photomultiplier tubes, microscopes, and / or video cameras (eg, CCDs). Camera)).

  The detector may be microfabricated within the microfluidic device or may be a separate element. If the detector is present as a separate element and the microfluidic device comprises multiple detection sections, detection can occur within a single detection section at any given moment. Alternatively, a scanning system may be used. For example, certain automated systems scan the light source for microfluidic devices, and other systems scan the emission on the detector or comprise a multi-channel detector. As a specific illustrative example, the microfluidic device can be coupled to a translatable stage and scanned under a microscope objective. The signal thus obtained is then routed to a processor for signal analysis and processing. An array of photomultiplier tubes can also be utilized. In addition, an optical system having the ability to collect signals from all different detection sections and simultaneously determine the signal from each section can be utilized.

External detectors are also available. This is because the devices provided are either made entirely from optically transparent materials at the wavelength being monitored or are made primarily from those materials. This feature makes it possible to use a number of optical detection systems devices described is not possible by conventional silicon-based microfluidic devices in this specification.

  A particularly preferred detector uses a CCD camera and an optical path that provides a large field of view and many apertures to maximize the amount of light collected from each reaction chamber. In this regard, the CCD is used as an array of photodetectors, where each pixel or group of pixels corresponds to a reaction chamber rather than a chamber used to produce an image of the array. Thus, the optics can be modified to increase the depth of field of an optical system that degrades image quality or is out of focus and collects more light from each reaction chamber. In a preferred embodiment, using a high aspect ratio, or a cylindrical chamber for concentrating the sample is provided by a detector along the optical axis of the optical system, and preferably the focus of the image to increase the depth of field. It is useful to be able to send a response command signal by shifting. The use of a low NA lens system, preferably a symmetrical lens system is used. It is also useful to use a detector device, such as one or more CCD devices having a size of the area of the elastomeric block to be imaged, or larger. When used with low NA optics, improved detection sensitivity can be achieved.

  The detector may comprise a light source for stimulating a reporter that produces a detectable signal. The type of light source utilized will depend, in part, on the nature of the reporter being activated. Suitable light sources include, but are not limited to, lasers, laser diodes, and high intensity lamps. If a laser is utilized, it can be used to scan a set of detection sections or an entire single detection section. The laser diode can be microfabricated into the microfluidic device itself. Alternatively, the laser diode can be fabricated on another device that is placed adjacent to the microfluidic device utilized to perform the thermal cycling reaction, whereby the laser light from the diode is directed to the detection section.

  Detection can involve many non-optical approaches as well. For example, the detector can also comprise, for example, a temperature sensor, a conductivity sensor, a potentiometric sensor (eg, a pH electrode) and / or an amperometric sensor (eg, for monitoring oxidation and reduction reactions).

  Many commercially available external detectors can be utilized. Many of these are fluorescence detectors because the preparation of fluorescent labeling reagents is easy. Specific examples of detectors that may be used include, but are not limited to, Applied Precision Array WoRx (Applied Precision, Issaqah, WA).

(B. Detection of amplified nucleic acid)
(1. Intervening dye)
Certain intervening dyes that fluoresce only upon binding to double stranded DNA can be used to detect double stranded amplified DNA. Examples of suitable dyes include SYBR ^™ and Pico Green (obtained from Molecular Probes, Inc. of Eugene, OR), ethidium bromide, propidium iodide, chromomycin, acridine orange, Hoechst 33258, Toto-1, Yoyo-1 And DAPI (4 ′, 6-diamidino-2-phenylindole hydrochloride). For further discussion on the use of intervening dyes, see Zhu et al., Anal. Chem. 66: 1941-1948 (1994), which is hereby incorporated by reference in its entirety.

(2. FRET-based detection method)
This type of detection method involves detecting a change in fluorescence from a donor (reporter) and / or acceptor (quencher) fluorophore in a donor / acceptor fluorophore pair. The donor and acceptor fluorophore pair is selected such that the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. Thus, energy transfer from the donor to the acceptor can occur if the pair of fluorophores is brought sufficiently close to each other. This energy transfer can be detected.

(FRET and template extension reaction)
These methods generally utilize a primer labeled with one member of a donor / acceptor pair and a nucleotide labeled with the other member of the donor / acceptor pair. Prior to incorporating labeled nucleotides into the primer during the template-dependent extension reaction, the donor and acceptor are placed sufficiently apart so that no energy transfer can occur. However, if the labeled nucleotide is incorporated into the primer and its location is close enough, energy transfer occurs and can be detected. These methods are particularly useful in carrying out single base pair extension reactions in detecting single nucleotide polymorphisms (supra) and are described in US Pat. No. 5,945,283 and PCT Publication WO 97/22719. Is done.

(Quantitative RT-PCR)
Various so-called “real-time amplification” or “real-time quantitative PCR” methods also determine the amount of target nucleic acid present in a sample by measuring the amount of amplification product formed during or after the amplification process. Can be used to determine. The fluorogenic nuclease assay is one particular example of a real-time quantification method that can be successfully used with the devices described herein. This method of monitoring the formation of amplification products involves continuously measuring PCR product accumulation using a dual-labeled fluorogenic oligonucleotide probe. In the reference, this approach is often referred to as the “TaqMan” method.

  The probes used in such assays are typically short (about 20-25 bases) polynucleotides that are labeled with two different fluorescent dyes. The 5 'end of the probe is typically attached to a reporter dye and the 3' end is attached to a quencher dye, although these dyes may be attached to other positions on the probe as well. . The probe is designed to have at least substantial sequence complementarity with the probe binding site on the target nucleic acid. Upstream and downstream PCR primers that bind to the region adjacent to the probe binding site are also included in the reaction mixture.

  If the probe is intact, energy transfer occurs between the two fluorophores and the quencher quenches the emission from the reporter. During the extension phase of PCR, the probe is cleaved by the 5 ′ nuclease activity of a nucleic acid polymerase, such as Taq polymerase, which releases the reporter from the polynucleotide-quencher and can be measured by an appropriate detector. This causes an increase in strength.

  For example, one detector specifically adapted for measuring fluorescence emission as generated during a fluorogenic assay is described in Applied Biosystems, Inc. ABI 7700 manufactured by In Foster City, CA. The computer software provided with this instrument can record the fluorescence intensity of the reporter and quencher during the amplification process. These recorded values can then be used to continually calculate an increase in normalized reporter luminescence intensity and finally quantitate the amount of mRNA amplified.

  Further details regarding the theory and operation of fluorescence generation methods for determining the concentration of amplification products in real time are described, for example, in US Pat. No. 5,210,015 to Gelfand et al., US patent to Livak et al. No. 5,538,848, and US Pat. No. 5,863,736 to Haaland, and Heid, C .; A. Et al., Genome Research, 6: 986-994 (1996); Gibson, U., et al. E. M et al., Genome Research 6: 995-1001 (1996); Holland, P. et al. M.M. Et al., Proc. Natl. Acad. Sci. USA 88: 7276-7280, (1991); and Livak, K .; J. et al. Et al., PCR Methods and Applications 357-362 (1995), each of which is incorporated herein by reference in its entirety.

  Therefore, as the amplification reaction proceeds, the amount of dye that binds increases and at the same time the amount of signal increases.

  For example, the intervening dyes described above can be utilized in different approaches to quantitative PCR methods. As noted above, these dyes bind preferentially to double stranded DNA (eg, SYBR GREEN) and only generate a signal once bound. Thus, as the amplification reaction proceeds, the amount of dye that binds increases and simultaneously the signal that can be detected increases.

(Molecular beacon)
With respect to molecular beacons, changes in the conformation of the probe when hybridizing to the complementary region of the amplification product form a detectable signal. The probe itself includes two sections: one is a 5 ′ end section and the other is a 3 ′ end section. These sections are adjacent to and complementary to the section of the probe that anneals to the probe binding site. One end section is typically attached to the reporter dye and the other end section is usually attached to the quencher dye.

  In solution, the two end sections can hybridize to each other to form a hairpin loop. In this conformation, the reporter dye and quencher dye are close enough so that the fluorescence from the reporter dye is efficiently quenched by the quencher dye. In contrast, hybridized probes produce a linear conformation with a reduced degree of quenching. Therefore, it is possible to indirectly monitor the formation of amplification products by monitoring the luminescence changes for the two dyes. This type of probes and methods of their use are further described, for example, in: Piatek, A. et al. S. Et al., Nat. Biotechnol. 16: 359-63 (1998); Tyagi, S .; And Kramer, F .; R. , Nature Biotechnology 14: 303-308 (1996); and Tyagi, S .; Et al., Nat. Biotechnol. 16: 49-53 (1998), each of which is incorporated herein by reference in its entirety for all purposes.

(Invader)
Invader assays (Third Wave Technologies, (Madison, Wis.)) Are used for SNP genotyping and are named oligonucleotides (which can be targeted to target nucleic acids (DNA or RNA) or polymorphic sites). Is complementary). A second oligonucleotide, termed invader oligo, contains the same 5 ′ nucleotide sequence, but the 3 ′ nucleotide sequence contains a nucleotide polymorphism. This invader oligo interferes with the binding of the signal probe to the target nucleic acid, whereby the 5 ′ end of the signal probe forms a “flap” at the nucleotide containing the polymorphism. This complex is recognized by a structure-specific endonuclease called the Cleavease enzyme. Cleavease cleaves the 5 'flap of nucleotides. The released flap binds to a third probe with a FRET label, thereby forming another duplex structure that is recognized by the Cleavease enzyme. This time, the Cleavease enzyme generates a fluorescent signal by cleaving off the fluorophore from the quencher. For SNP genotyping, the signal probe is designed to hybridize with either a reference (wild type) allele or a variant (mutant) allele. Unlike PCR, there is a linear amplification of the signal without nucleic acid amplification. Further details sufficient to guide one skilled in the art can be found in, for example, Neri, B. et al. P. Et al., Advances in Nucleic Acids and Protein Analysis 3826: 117-125, 2000).

(Nasba)
Nucleic Acid Sequence Based Amplification (NASBA) is a detection method that uses RNA as a template. The primer complementary to the RNA contains the sequence for the T7 promoter site. This primer allows binding of template RNA and added reverse transcriptase (RT) to create a complementary strand from 3 ′ to 5 ′. Subsequently, RNase H is added to degrade the RNA and the single stranded cDNA remains intact. The second copy of the primer can then bind to the single stranded cDNA and become a double stranded cDNA. To make many RNA copies from the T7 promoter site incorporated into the cDNA sequence by the first primer, T7 RNA polymerase is added. All the enzymes mentioned can function at 41 ° C. (see, eg, Compton, J. Nucleic Acid Sequence-based Amplification, Nature 350: 91-91, 1991).

(Scorpion)
This method is described, for example, by Thelwell N.W. Et al., Nucleic Acids Research, 28: 3752-3761,2000, which is incorporated by reference in its entirety for all purposes, and FIG. 20 shows the scheme. Here, the Scorpion probing mechanism is as follows. Step 1: Initial denaturation of target and Scorpion stem sequence. Step 2: Annealing the Scorpion primer to the target. Step 3: Extension of the Scorpion primer produces double stranded DNA. Step 4: Denaturation of the double-stranded DNA produced in Step 3. This results in a single stranded target molecule to which the Scorpion primer is bound. Step 5: Upon cooling, the Scorpion probe sequence binds to its target in an intramolecular manner. This is preferred for intramolecular binding of complementary target strands. Scorpion (shown in FIG. 24) consists of specific probe sequences held in the hairpin loop configuration by complementary 5 ′ and 3 ′ stem sequences of the probe. 5 'fluorophore attached to a terminal, the third the loop' part component attached to the end (usually methyl red) is quenched by. The hairpin loop is ligated to the 5 ′ end of the primer via a PCR stop sequence (stopper). After extension of the primer during PCR amplification, the specific probe sequence can bind to the complement within the same strand of DNA. This hybridization event cleaves the hairpin loop so that fluorescence is no longer quenched and an increase in signal is observed. The PCR stop sequence prevents readthrough that cleaves the hairpin loop in the absence of a specific target sequence. Such readthrough results in the detection of non-specific PCR products (eg, primer dimers or mispriming events).

(3. Capacitive DNA detection)
There is a linear relationship between the DNA concentration and the change in capacitance caused by the passage of nucleic acid through a 1-kHz electric field. This relationship has been found to be species independent (see, eg, Sonn et al., (2000) Proc. Natl. Acad. Sci. USA 97: 10687-10690). Thus, in certain devices, nucleic acids within the flow channel (eg, the substantially circular flow channel of FIG. 1 or the reaction chamber of FIG. 2) are subjected to such a field to determine the concentration of the amplified product. . Alternatively, a solution containing the amplification product is recovered and then subjected to an electric field.

(IX. Composition of the mixture for carrying out the reaction)
Reactions performed using the microfluidic devices disclosed herein are typically performed with certain additives to facilitate the reaction. So, for example, in the case of a device on which reagents are deposited, these additives can be spotted with one or more reactants, for example at the reaction site. One set of additives is a blocking reagent that blocks protein binding sites on the elastomeric substrate. A wide variety of such compounds can be utilized, including many different proteins (eg, gelatin and various albumin proteins (eg, bovine serum albumin)) and glycerol.

  Surfactant additives may also be useful. Any of a number of different surfactants can be utilized. Examples include, but are not limited to, SDS and various Triton surfactants.

  In particular cases of nucleic acid amplification reactions, many different types of additives can be included. One category is enhancers that facilitate the amplification reaction. Such additives include, but are not limited to, reagents that reduce secondary structure in nucleic acids (eg, betaine), and factors that reduce mispriming events (eg, tetramethylammonium chloride).

  Some polymerases have also been found to give enhanced results when performing certain amplification reactions. For example, good results were obtained using AmpliTaq Gold polymerase from Thermus aquaticus (Applied Biosystems, Foster City, Calif.), While in some cases using DyNAzyme polymerase obtained from Finnzyme, Espoo, Finland An improved response was obtained. This polymerase is derived from Thermus blockianus, a thermophilic bacterium. Other exemplary polymerases that may be utilized include, but are not limited to: rTH polymerase XL (which is a combination of Thermos thermophilus (Tth) and Thermococcus literalis (Tli)) Thermophilic archaeon Pyrosoccus wesei (Pwo) and Tgo DNA polymerase. It may also be useful to combine two or more polymerases in the reaction mixture to increase the fidelity or sensitivity of the PCR reaction. The addition of other reagents (eg, DMSO) (especially adding those containing intervening dyes to the PCR cocktail) may further assist in performing the reaction. Using a glycerol or PEG solution in the control line fluid can also improve the performance of the PCR reaction.

  Further details regarding additives useful in conducting reactions (including nucleic acid amplification reactions) using specific devices disclosed herein are provided in the examples below.

(X. Exemplary application)
Since the microfluidic devices provided herein can be fabricated to include many reaction sites, the devices are useful in a wide variety of screening and analysis methods. Generally, the device is for detecting a reaction between a species that reacts to form a detectable signal, or a product that interacts with another species that produces a detectable signal. Can be used. In view of the use of various types of temperature control systems, the device can also be utilized in many different types of analyzes or reactions required for temperature control.

(A. Nucleic acid amplification reaction)
The devices disclosed herein can be used to perform essentially all types of nucleic acid amplification reactions. Thus, for example, the amplification reaction may be linear amplification (amplification with a single primer) or exponential amplification (ie, amplification performed by forward and reverse primer sets). Also good.

  When blind channel type devices are utilized to perform nucleic acid amplification reactions, the reagents typically deposited within the reaction site are those necessary to perform the desired type of amplification reaction. Typically this means that some or all of the following (eg, primers, polymerase, nucleotides, metal ions, buffers, and cofactors) are deposited. In such a case, the sample introduced into the reaction site is a nucleic acid template. Alternatively, however, the template can be deposited and the amplification reagent can flow to the reaction site. As discussed above, when a matrix device is utilized to perform the amplification reaction, the sample containing the nucleic acid template flows through the vertical flow channel and the amplification reagent flows through the horizontal flow channel. Alternatively, the sample containing the nucleic acid template flows through a horizontal flow channel and the amplification reagent flows through a vertical flow channel.

  Although PCR is perhaps the best known amplification technique, the device is not limited to performing PCR amplification. Other types of amplification reactions that can be performed include, but are not limited to: (i) ligase chain reaction (LCR) (Wu and Wallace, Genomics 4: 560 (1989) and Landegren et al., Science 241: (See 1077 (1988)); (ii) transcription amplification (see Kwoh et al., Proc. Natl. Acad. Sci. USA 86: 1173 (1989)); (iii) autonomous sequence replication (Guatelli et al.). , Proc. Nat. Acad. Sci. USA, 87: 1874 (1990)); and (iv) Nucleic acid-based sequence amplification (NASBA) (Sooknanan, R. and Malek, L., BioTechnology 13: 563). -65 (19 5) see). Each of the aforementioned references is incorporated herein by reference in its entirety for all purposes.

  Detection of the resulting amplification product can be accomplished using any of the detection methods described above for detecting amplified DNA.

(B. SNP analysis and genotyping)
(1. Overview)
Many diseases associated with genomic alterations in either the host or infecting organism are the result of a few nucleotide changes and are often associated with single nucleotide changes. Such a single nucleotide change is referred to as a single nucleotide polymorphism or simply a SNP, and the site where the SNP occurs is typically referred to as the polymorphic site. The devices described herein can be utilized to determine the identity of nucleotides present at such polymorphic sites. Extending this capability, the device can be utilized in genotyping analysis. Genotyping determines that a diploid organism (ie, an organism with two copies of each gene) has two copies of the reference allele (reference homozygote), one for each of the reference allele and the variant allele. This includes determining whether to include a copy (ie, a heterozygote) or whether to include two copies of a variant allele (ie, a variant homozygote). When performing genotyping analysis, the methods of the invention can be utilized to interrogate a single variant site. However, as described further below in the section on multiplexing, this method is used to determine an individual's genotype at many different DNA loci (either the same gene, different genes, or combinations thereof). Can also be used.

  The device to be used for performing genotyping analysis utilizes an appropriately sized reaction site and, from a statistical point of view, each copy of the two alleles is valid for a diploid subject. It is designed to ensure that it is present at the reaction site at the correct DNA concentration. Otherwise, the analysis yields results suggesting that the heterozygote is a homozygote simply because no copy of the second allele is present at the reaction site. Table 2 below shows the number of genome copies present in a 1 nl reaction volume at various exemplary DNA concentrations that can be utilized by the devices described herein.

  (Table 2: number of genome copies present in 1 nL volume at the indicated DNA concentrations)

一般的な問題として、サンプルの確率論的比率に起因して、増幅反応が開始される前に存在するコピー数は、測定における誤差の可能性を決定する。特定のデバイスを使用する遺伝子型決定分析は、代表的に約０．１０μｇ／μＬのＤＮＡ濃度を有するサンプルを用いて行われるが、本発明は、反応部位１つにつき単一のゲノムが存在する濃度においてＴａｑＭａｎ反応を首尾よく行う。

As a general matter, due to the stochastic ratio of the sample, the copy number present before the amplification reaction is initiated determines the potential for error in the measurement. Although genotyping analysis using a specific device is typically performed with a sample having a DNA concentration of about 0.10 μg / μL, the present invention has a single genome per reaction site Successful TaqMan reaction at concentration.

(2. Method)
Genotyping analysis can be performed using a variety of different approaches. In these methods, it is generally sufficient to obtain a “yes” or “no” result. That is, detection only needs to be able to answer the question of whether a given allele is present. Thus, analysis can only be performed with primers or nucleotides that are potentially required to detect the presence of one allele at the polymorphic site. More typically, however, primers and nucleotides are included to detect the presence of each allele, potentially at a polymorphic site. An example of a suitable approach is:

(Single base pair extension (SBPE) reaction)
The SBPE reaction is one technique that has been specifically developed to perform genotyping analysis. Many SPBE assays have been developed, but the general approach is quite similar. Typically, these assays involve hybridizing a primer that is complementary to the target nucleic acid, whereby the 3 ′ end of the primer is directly 5 ′ of the variant site, or Adjacent to it. Extension occurs in the presence of one or more labeled non-extendable nucleotides that are complementary to the nucleotide occupying the variant site and the polymerase. This non-extendable nucleotide is a nucleotide analog that prevents further extension by the polymerase once incorporated into the primer. If the added non-extendable nucleotide is complementary to the nucleotide at the variant site, the labeled non-extendable nucleotide is incorporated at the 3 ′ end of the primer to produce a labeled extension product. . Thus, the extension primer provides an indication that the nucleotide is present at the variant site of the target nucleic acid. Such and related methods are described, for example, in US Pat. No. 5,846,710; US Pat. No. 6,004,744; US Pat. No. 5,888,819; US Pat. No. 5,856,092. And U.S. Pat. No. 5,710,028; and WO 92/16657.

  The detection of extension products can be detected utilizing the FRET detection approach described for the extension reaction in the previous detection section. Thus, for example, using the device described herein, a primer labeled with one member of a donor / acceptor fluorophore, 1 to 4 labeled non-extendable nucleotides (greater than 1 If non-extendable nucleotides are included, they are differentially labeled), and a reagent mixture containing a polymerase is introduced (or previously spotted) into the reaction site. A sample containing template DNA is then introduced into the reaction site, and template extension can occur. Any extension product formed is detected by formation of a FRET signal (see, eg, US Pat. No. 5,945,283 and PCT Publication WO 97/22719). This reaction can be thermocycled as needed to increase the signal using the temperature control methods and equipment described above.

(Quantitative PCR)
Genotyping analysis can be performed using previously described quantitative PCR methods. In this case, differentially labeled probes complementary to each of the allelic forms are included as reagents along with primers, nucleotides and polymerases. However, the reaction can be performed using only a single probe, which means whether the lack of signal is due to the absence of a particular locus or simply due to failure of the reaction In terms of ambiguity can arise. For a representative biallelic case where two alleles are likely for a polymorphic site, the two differentially labeled probes are each for one of the alleles. These probes are usually included in the reagent mixture along with amplification primers, nucleotides and polymerase. A sample containing the target DNA is introduced into the reaction site. If an allele for which the probe is complementary is present in the target DNA, then amplification occurs, thereby producing a detectable signal as described in the detection above. If a differential signal is obtained, nucleotide identity at the polymorphic site can be determined. If both signals are detected, then both alleles are present. Thermal cycling during the reaction is performed as described in the temperature control section above.

(B. Gene expression analysis)
(1. Overview)
Gene expression analysis involves determining the level at which one or more genes are expressed in a particular cell. The determination can be qualitative, but is generally quantitative. In differential gene expression analysis, the level of a gene in one cell (eg, test cell) is compared to the expression level of the same gene in another cell (control cell ) . A wide variety of such comparisons can be made. Examples include comparison between healthy and diseased cells, comparison between cells from one drug-treated individual and cells from another untreated individual, exposure to certain toxins Examples include, but are not limited to, comparison between cells and unexposed cells. Genes that differ in expression levels between test cells and control cells can serve as markers and / or targets for therapy. For example, if a particular group of genes is found to be up-regulated in diseased cells rather than healthy cells, such genes can function as markers for the disease and are utilized as a basis for diagnostic testing. There is a possibility to get. These genes can also be targets. Strategies for treating the diseases include procedures that result in decreased expression of upregulated genes.

  The device designs disclosed herein are useful to facilitate various gene expression analyses. Since the device includes many reaction sites, many genes and / or samples can be tested simultaneously. For example, using blind flow channel devices, the expression levels of hundreds to thousands of genes can be determined simultaneously. The device also facilitates differential gene expression analysis. For example, using a matrix design, it is possible to test a sample from diseased cells in the immediate adjacent channel while testing a sample obtained from healthy cells in one flow channel. This feature enhances the ease of detection and the accuracy of the results. This is because two samples are tested simultaneously and under the same conditions on the same device.

(2. Sample preparation and concentration)
In order to measure the transcription level (and thereby the expression level) of a gene, a nucleic acid sample comprising the mRNA transcript of the gene or gene fragment, or a nucleic acid derived from the mRNA transcript is obtained. A nucleic acid derived from an mRNA transcript refers to a nucleic acid whose mRNA transcript or subsequence thereof ultimately serves as a template for its synthesis. Therefore, cDNA reverse transcribed from mRNA, RNA transcribed from cDNA, DNA amplified from cDNA, and RNA transcribed from amplified DNA are all derived from the mRNA transcripts described above. , An indication of the presence and / or abundance of the original transcript in the sample. Thus, suitable samples include, but are not limited to: mRNA transcripts of genes, cDNA reverse transcribed from mRNA, cRNA transcribed from cDNA, DNA amplified from genes, amplified DNA Transcribed RNA.

  In some methods, the nucleic acid sample is total mRNA isolated from the biological sample; in other examples, the nucleic acid sample is total RNA from the biological sample. The term “biological sample” as used herein refers to a sample (eg, cell, biological tissue and fluid) obtained from an organism or from a component of an organism. In some methods, the sample is from a human patient. Such samples include sputum, blood, blood cells (eg, white blood cells), tissue or fine needle biopsy samples, urine, ascites, and pleural fluid, or cells therefrom. A biological sample can also include a section of tissue, such as a frozen section taken for histological purposes. In many cases, two samples are provided for comparison purposes. The sample may be derived from the same original sample that has been subjected to two different treatments (eg, drug treatment and control treatment), eg, from different cells or tissue types or from different individuals. It may be derived from.

  Any RNA isolation technique that is not selected for mRNA isolation may be utilized for the purification of such RNA samples. For example, methods of nucleic acid isolation and purification are described in detail below: WO 97/10365, WO 97/27317, Laboratory Techniques in Biochemistry and Molecular Biology, Chapter 3: Hybridization With Nucleic Acid Pc. Theory and Nucleic Acid Preparation, (P. Tijssen (ed.)) Elsevier, N .; Y. (1993); Chapter 3 of Laboratory Technologies in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part1. Theory and Nucleic Acid Preparation, (P. Tijssen (ed.)) Elsevier, N .; Y. (1993); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N .; Y. (1989); Current Protocols in Molecular Biology, (Ausubel, FM et al. (Ed.)) John Wiley & Sons, Inc. , New York (1987-1993). Numerous tissue samples are easily processed using techniques known in the art, including the one-step RNA isolation process of Chomczynski, P. described in US Pat. No. 4,843,155. Can be done.

  In gene expression analysis utilizing the described device, an important factor affecting the results is the nucleic acid concentration in the sample. When the copy number is small, the noise is proportional to the square root of the copy number. Thus, the level of error that is considered acceptable depends on the number of copies required. The required copy number in a particular sample volume gives the required DNA concentration. Although not necessarily optimal, quantitative reactions can be performed with an error level of up to 50% (but preferably at a lower level). Table 3 shows the DNA concentration required to achieve a specific error level assuming a 1 nanoliter volume. As can be appreciated, for example, a 1 nanoliter volume used in a particular device has a sufficient copy number of gene expression product at a concentration that is feasible using a microfluidic device.

  (Table 3. Gene expression-DNA quantification)

さらなる計算により、１ナノリットルの反応部位を利用する本明細書において提供される特定のデバイスは、正確な発現結果を達成するための十分なＤＮＡを含むことを実証する。具体的には、代表的なｍＲＮＡ調製手順は、約１０μｇのｍＲＮＡを生じる。代表的に、１つの細胞あたり各ｍＲＮＡの１〜１０，０００コピーが存在することが実証されている。任意の所定の細胞内で発現されるｍＲＮＡのうちで、大体、４種の最も一般的なメッセージが、総ｍＲＮＡレベルの約１３％を構成する。従って、このように高く発現されたメッセージは、１．３μｇのｍＲＮＡを含む（各々、４×１０^−１２モルまたは約２．４×１０^１２コピー）。前述の発現範囲を考慮して、まれなメッセージが、約２×１０^−８コピーのレベルで存在することが予測される。標準的分析においてｍＲＮＡサンプルが１０μｌ中に溶解されている場合、まれなメッセージの濃度は約２×１０^７コピー／μｌであり、この濃度は、１ｎｌウェルあたり２０，０００コピー（または４×１０^１１Ｍ）に対応する。

Further calculations demonstrate that the particular device provided herein that utilizes a 1 nanoliter reaction site contains sufficient DNA to achieve accurate expression results. Specifically, a typical mRNA preparation procedure yields about 10 μg of mRNA. Typically, it has been demonstrated that there are 1 to 10,000 copies of each mRNA per cell. Of the mRNAs expressed in any given cell, roughly the four most common messages constitute about 13% of the total mRNA level. Thus, such a highly expressed message contains 1.3 μg of mRNA (4 × 10 ⁻¹² moles or about 2.4 × 10 ¹² copies, respectively). In view of the aforementioned expression range, rare messages are expected to be present at a level of about 2 × 10 ⁻⁸ copies. If the mRNA sample is lysed in 10 μl in a standard analysis, the rare message concentration is about 2 × 10 ⁷ copies / μl, which is 20,000 copies (or 4 × 10 ¹¹ per nl well). M).

(3. Method)
Since expression analysis typically involves quantitative analysis, detection is typically accomplished using one of the quantitative real-time PCR methods described above. Thus, when the TaqMan approach is utilized, the reagents introduced (or pre-spotted) at the reaction site can include one or all of primers, labeled probes, nucleotides, and polymerases. Where an intervening dye is utilized, the reaction reagent mixture typically includes one or all of a primer, a nucleotide, a polymerase, and an intervening dye.

(D. Multiplexing)
The array-based devices described herein (see, eg, FIGS. 1A, 1F, 2, 3A and 3B, and the accompanying text) are originally designed to perform multiple amplification reactions simultaneously. The However, this feature can be more easily extended by performing multiple analyzes (eg, genotyping and expression analysis) within each reaction site.

  Multiplex amplification can be performed even during a thermal cycling process, for example, within a single reaction site by utilizing multiple primers, each specific for a particular target nucleic acid of interest. The presence of different amplification products can be detected by performing a quantitative RT-PCR reaction using a differentially labeled probe, or by using a differentially labeled molecular beacon (see above). checking). In such an approach, each differentially labeled probe is designed to hybridize only to a specific amplification label. By judicious selection of the different labels utilized, an analysis can be performed in which different labels are excited and / or detected at different wavelengths of a single reaction. Additional indicators for the selection of suitable fluorescent labels suitable for such an approach include the following: Fluorescence Spectroscopy (Pesce et al. (Ed)) Marcel Dekker, New York, (1971); White et al., Fluorescence Analysis: A Practical Approach, Marc Dekker, New York, (1970); Berman, Handbook of Fluorescence Spectra of Aromatic Physics, Second Edition, Academic Prey. lecules, Academic Press, New York, (1976); Indicators (Bishop (ed.)). Pergamon Press, Oxford, 19723; and Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene (1992).

Multiple genotyping and expression analysis can be performed at each reaction site as needed. For example, when the quantitative PCR methods such as TaqMan is utilized, a primer for amplifying a different region of the target DNA of interest are included in a single reaction site. A differentially labeled probe for each region is utilized to identify the product formed.

(E. Non-nucleic acid analysis)
While the device is useful for performing a wide variety of nucleic acid analyses, the device can also be utilized in many other applications as well. As indicated above, the device can be any signal between two or more species that produces a reaction product that can react with a detection reagent that produces a signal upon interaction with a detectable signal or reaction product. It can be used to essentially analyze the interaction.

  Thus, for example, the devices can be utilized in many screening applications to identify test agents having a particular desired activity. As a specific example, the device can be utilized to screen compounds for activity as substrates or inhibitors of one or more enzymes. In such an analysis, test compounds and other necessary enzyme assay reagents (eg, buffers, metal ions, cofactors and substrates) are introduced into the reaction site (otherwise deposited in advance). An enzyme sample is then introduced and a reaction (if the test compound is a substrate) or an inhibition of the reaction (if the test compound is an inhibitor) is detected. Such a reaction or inhibition can be achieved by standard techniques such as monitoring directly or indirectly the loss of substrate and / or the appearance of product.

  Devices with sufficiently large flow channels and reaction sites can be utilized to perform cellular assays to detect interactions between cells and one or more reagents. For example, a particular analysis involves the determination of whether a particular cell type is present in a sample. One example to accomplish this is to utilize cell specific dyes that react preferentially with specific cell types. Thus, such a dye can be introduced into the reaction site and then cells can be added. Cell staining can be detected using standard microscopic techniques. As another example, test compounds can be screened for the ability to induce or inhibit cellular responses (eg, signal transduction pathways). In such an analysis, a test compound is introduced into the site and then cells are added. The reaction site is then examined to detect the formation of a cellular response.

  Additional considerations regarding related devices and the application of such devices are described in co-pending and co-owned US Provisional Patent Application 60 / 335,292 (filed November 30, 2001), which is for all purposes. The entirety of which is incorporated herein by reference.

(XI. Production)
(General aspects)
As suggested above, the provided microfluidic devices generally have single and multi-layer soft lithography (MSL) technology and / or sacrificial-layer encapsulation method. ). The basic MSL approach involves casting a series of elastomeric layers in a small machine mold, removing the layers from the mold, and then fusing the layers together. In the sacrificial layer encapsulation approach, a pattern of photoresist is deposited where the channel is desired. The use of these techniques in these techniques and methods of manufacturing microfluidic devices is discussed in detail, for example, as follows: Unger et al. (2000) Science 288: 113-116, Chou et al. (2000) “Integrated Elastomer. Fluidic Lab-on-a-chip-Surface Patterning and DNA Diagnostics, in Proceedings of the Solid State Actuators and Sensor Works, "Hilton Head. C. As well as PCT publication WO 01/01025, each of which is incorporated herein by reference in its entirety for all purposes.

Briefly, fabrication method described above, by photolithography by photoresist (Shipley SJR 5740) on silicon wafers, the upper layer (e.g., an elastomer layer having a control channel) and the lower layer (e.g., an elastomeric layer having a flow channel A mother mold for the production of the mold. The channel height can be precisely controlled by the spin coating speed. The photoresist channel is formed by exposing the photoresist to UV light and then developing. Thermal reflow processes and protective treatments are typically A. Unger, H .; -P. Chou, T .; Throsen, A.M. Scherer and S. R. This is accomplished as described by Quake, Science (2000) 288: 113, which is hereby incorporated by reference in its entirety. Then, mixed 2 parts of silicon elastomer (GE RTV 615) is poured in drop (spun) and the upper mold in a mold under each. Here, spin coating can also be utilized to control the thickness of the underlying polymer fluid layer. The partially cured upper layer is peeled from the mold by baking in an 80 ° C. oven for 25 minutes and assembled side by side with the lower layer. A final bake at 80 ° C. for 1.5 hours bonds the two layers irreversibly. Once peeled off from the lower side of the silicon mother mold, this RTV device is typically being treated with HCL (0.1 N, 30 min at 80 ° C.). This treatment acts to break some Si—O—Si bonds, thereby exposing the hydroxy groups and making the channel more hydrophilic.

  The device can then be hermetically sealed onto the support as needed. This support may be made from essentially any material, but its surface is flat to ensure a good seal if the seal formed is primarily due to adhesive forces. Should be. Examples of suitable supports include glass, plastic and the like.

  A device formed according to the foregoing method produces a substrate (eg, a glass slide) that forms one wall of the flow channel. Alternatively, the device once removed from the mother mold is sealed with a thin elastomeric membrane, thereby enclosing the flow channel entirely with the elastomeric material. The resulting elastomeric device can then be coupled to a substrate support as needed.

(B. Device using blind channel design)
(1. Layer formation)
Microfluidic devices based on blind channel designs in which reagents are deposited at the reaction site during manufacture are typically formed from three layers. The lower layer is the layer on which the reagent is deposited. The underlying layer can be formed from a variety of elastomeric materials as described in the references listed for the MLS method above. Typically, the material is a polydimethylsiloxane (PMDS) elastomer. Based on the desired placement and location of reaction sites for a particular device, it is possible to determine the location on the lower layer where the appropriate reagent is to be spotted. Since PMDS is hydrophilic, the deposited aqueous spots become smaller and form very small spots. The deposited reagent is deposited such that no covalent bond is formed between the reagent and the surface of the elastomer. This is because, as described above, the reagent is intended to dissolve in the sample solution when the reagent is introduced into the reaction site.

  The other two layers of the device are the layer in which the flow channel is formed and the layer in which the control channel and any protection channel are formed. These two layers are prepared according to the general method described earlier in this section. The resulting two-layer construct is then placed on the first layer on which the reagent is deposited. Specific examples of these three layer compositions are as follows (ratio of component A to component B): first layer (sample layer) 30: 1 (weight); second layer (flow channel) Layer) 30: 1; and third layer (control layer) 4: 1. However, other compositions and ratios of these elastomer components can be utilized as well. Use of other methods of bonding two or more layers or substrates includes adhesive or plasma bonding (preferably air plasma bonding) to form elastomeric blocks or other parts of the microfluidic device. Can be. In some embodiments, an additional layer (preferably an elastomeric layer) interconnected with other layers through pores can be used to increase the density of reactants per unit area of the elastomeric block, or The size of the elastomer block can be reduced.

During this process, the reaction site is aligned with the deposited reagent so that the reagent is positioned within the appropriate reaction site. Figure 6 shows a diagram obtained from the four corners of the device. These figures demonstrate that the deposited reagents can be accurately aligned within the reaction site using the approach described above. These figures show the protective channel and reaction site positioned at the end of the branch flow channel. The white circle indicates the position of the reagent deposited with respect to the reaction site. As shown, each reagent spot is well within its reaction site.

(2. Spotting)
The reagents can be deposited using a variety of established spotting techniques utilizing any of a number of commercially available reagent spotters. Examples of suitable spotters that can be utilized in the preparation of the devices include pin spotters, acoustic spotters, automatic micropipetters, electrophoretic pumps, ink jet printer devices, ink drop printers, and certain osmotic pumps. Commercial spotters include Cartesian Technologies MicroSys 5100 (Irvine, Calif.), Hitach SPBIO (Alameda, CA), Genetix Q-Array (United Kingdom Dom), Affymetrix 417 ). Generally, very small spots of reagents are deposited; typically less than 10 nl spots are deposited, in other examples less than 5 nl, less than 2 nl or less than 1 nl, and in still other examples 0.5 nl Spots of less than, less than 0.25 nl, or less than 0.1 nl are deposited.

  An array of materials is also described in Föder et al., US Pat. No. 5,445,934: the title of the invention “Array of oligonucleotides on a solid substrate”, which is incorporated herein by reference. Where oligonucleotide probes (eg, SNP probes) are synthesized in situ using spatial light-directed photolithography. Such an array is used as a substrate or base for the microfluidic device of the present invention so that the area of the substrate corresponding to the reaction site (eg, a blind fill chamber) is the substrate. One or preferably more than one oligonucleotide probe placed in a known position. In the case of a split microfluidic structure (eg, as shown in FIG. 15 herein), the reaction site (shown as a square box along a curved fluid channel) includes a plurality of different SNP probes. A collection of SNP probes suitable for identifying individuals, preferably from a population of individuals, and preferably a plurality of reaction sites along this curved fluid channel (fluids containing nucleic acid sequences from a plurality of individuals) Multiple valves communicating with the bent flow channel when activated (if the sample is introduced into the bent flow channel) and dividing the bent flow channel, thereby defining the fluid sample in each reaction site. Each reaction site is separated from each other so as to include a minute. Amplification of the components of the sample can be performed to increase the number of molecules (eg, nucleic acid molecules) to bind to the array of SNP probes located within each reaction site. In some embodiments, each reaction site along the curved fluid channel is the same array, i.e., has the same SNP probe positioned, and in other embodiments, along the curved fluid channel. Two or more reaction sites have different sets of SNP probes. Other split fluid channel architectures (eg, branching systems and / or branched branching systems, etc.) may also be used. Other placement techniques (e.g., spotting as described herein) may similarly be used to form an array located within a segmented reaction site along a curved or generally branched fluid channel. Can be used.

  The following examples are provided to further illustrate certain aspects of the devices and methods disclosed herein. These examples should not be construed as limiting the invention.

Example 1
(Signal strength evaluation)
I. Introduction The purpose of this series of experiments is to demonstrate that a successful PCR reaction can be performed with a signal intensity of over 50% for the Macro TaqMan reaction using the microfluidic device of the design shown herein. there were.

II. Microfluidic device A three-layer microfluidic device assembled using the MSL process was designed and assembled to perform the experiments described in the examples below. FIG. 7A shows a cross-sectional view of this device. As shown, the device 700 includes a layer 722 in which a flow channel is formed. This fluid layer 722 is sandwiched between an overlying layer 720 and an underlying sealing layer 724, the overlying layer comprising a control layer and a protective layer . This sealing layer 724 forms one side of the flow channel. The resulting three-layer structure is secured to a substrate 726 (in this example, a slide or cover slip), which provides structural rigidity, increases thermal conductivity, and the microfluidic device 700. Helps prevent evaporation from the bottom.

  FIG. 7B shows a schematic diagram of the flow channel design in the flow layer 722 and the control and protection channel design in the control / protection layer 720. Device 700 consists of 10 independent flow channels 702, each having its own inlet 708 and branching blind channel 704, each blind channel 704 having 1 nl reaction site 706. Device 700 includes a network of control lines 712 that separate reaction sites 706 when sufficient pressure is applied, and a series of protective channels 716 also allow liquid to evaporate from reaction sites 706. Provided to prevent; fluid is introduced through inlet 718.

II. Experimental Setup A PCR reaction using β-actin primer and TaqMan probe was performed on device 700 to amplify exon 3 (Promega, Madison WI) of β-actin gene derived from male human genomic DNA. This TaqMan reaction consists of the following components: 1 × TaqMan buffer A (50 mM KCl, 10 mM Tris-HCl, 0.01 M EDTA, 60 nM Passive Reference 1 (PR1), pH 8.3); 3.5-4.0 mM MgCl; 200 nM dATP, dCTP, dGTP, 400 nM dUTP; 300 nM β-actin forward and reverse primers; 200 nM FAM-labeled β-actin probe; 0.01 U / μl AmpEraseUNG (Applied Biosystems, Foster 0.1% to 0.2 U / μl DyNAzyme (Finzyme, Espoo, Finland); 0.5% Triton-x-100 (Sigma, St. Lo) It is, MO); 0.8μg / μl gelatin (Calbiochem, San Diego, CA); 5.0% glycerol (Sigma, St.Louis, MO); genomic DNA of deionized water and men. This reaction component was added to yield a total reaction volume of 25 μl. A negative control consisting of all the TaqMan reaction components other than the target DNA was included in each set of PCR reactions.

Once the above TapMan reaction samples and controls were prepared, they were injected into the microfluidic device 700 by using a gel loading pipette tip attached to a 1 ml syringe. The pipette tip was filled with the reaction sample and then inserted into the fluid via 708. The flow channel 702 was manually filled by applying back pressure to the syringe until all blind channels 704 and reaction sites 706 were filled. After all samples were loaded into flow lines 702, 704, control line 712 was filled with deionized water and pressurized to 15-20 psi. The pressurized control line 712 was activated to close the valve and the sample was separated into 1 nl well 706. The protective channel 716 was then filled with deionized water and pressurized to 5-7 psi. Mineral oil (15 μl) (Sigma) was placed on the thermocycler flat plate, and then the microfluidic device / cover glass 700 was placed on the thermocycler. The microfluidic device 700 was then thermally cycled using an initial ramp and either a 3-stage or 2-stage thermal cycling profile:
1. Initial ramp to 95 ° C and hold for 1 minute (1.0 ° C per second up to 75 ° C, 0.1 ° C per second up to 95 ° C)
2. 2. 3 cycles of thermal cycling for 40 cycles (92 ° C. for 30 seconds, 54 ° C. for 30 seconds, and 72 ° C. for 1 minute); or 40 cycles of 2-stage thermal cycling (92 ° C. for 30 seconds and 60 ° C. for 60 seconds).

  A MicroAmp tube (Applied Biosystems, Foster City, Calif.) Containing the remaining reaction mixture (referred to as the Macro TaqMan reaction to distinguish them from reactions performed in a microfluidic device) was replaced with a GeneAmp PCR System 9700 (Applied Biosystems, Foster Systems, , CA) and thermal cycling in 9600 mode. The Macro TaqMan reaction served as a macro control over the reaction performed with the microfluidic device. A thermal cycling protocol was set up to be consistent with the microfluidic device protocol except that the initial ramp rate was not controlled for the Macro TaqMan reaction.

  Once thermal cycling was complete, the control and protection lines were depressurized and the chip was transferred to a glass slide (VWR, West Chester, PA). The chip was then placed in an Array WoRx Scanner (Applied Precision, Issquah, WA) with a modified carrier. The fluorescence intensity was measured for three different excitation / emission wavelengths: 475/510 nm (FAM), 510/560 nm (VIC), and 580/640 nm (Passive Reference 1 (PR1)). Using Array Works Software, the fluorescence in the microfluidic device was imaged and the signal intensity and background intensity of each 1 nl well was measured. The results were then analyzed using a Microsoft Excel file to calculate the FAM / PR1 ratio for the β-actin TaqMan reaction. For conventional Macro TaqMan, positive samples for target DNA were determined using calculations described in the protocol provided by the manufacturer (TaqMan PCR reagent kit protocol). The signal intensity was calculated by dividing the sample FAM / PR1 ratio by the control FAM / PR1 ratio. A successful response was defined as the sample ratio above the 99% confidence threshold level.

III. Results First, AmpliTaq Gold (Applied Biosystems, Foster City, Calif.) Was used in the TaqMan reaction, resulting in a FAM / PR1 / control ratio of 1.5-2.0 and a Macro TaqMan reaction of 5.0-14.0. Compared with the ratio. Although the result was positive, an increase in signal intensity was desired. Therefore, AmpliTaq Gold polymerase was replaced with DyNAzyme polymerase due to the thermal stability, calibration, and resistance to impurities of DyNAzyme polymerase. A standard Macro TaqMan DyNAzyme concentration of 0.025 U / μl was used in the microfluidic experiment. This polymerase change to DyNAzyme resulted in a FAM / ROX / control ratio of 3.5-5.8. Although the signal intensity improved, it was difficult to obtain consistent results. Reasons have been found that some proteins bind to PDMS, the concentration of polymerase increased, and additives that modify the surface were included. Tested for two increased concentrations of DyNAzyme (100 pg / nl genomic DNA in microfluidic devices, 8 times (0.2 U / μl) and 4 times (0 U) the standard concentration for Macro TaqMan .1 U / μl)). Gelatin, glycerol, and 0.5% Triton-x-100 were added to prevent the polymerase from binding to PDMS. The results of the reaction of the microfluidic device (chip) and Macro TaqMan control are shown in FIG.

  The microfluidic TaqMan reaction ratio is in the range of 4.9-8.3, while the Macro TaqMan reaction is in the range of 7.7-9.7. Thus, the signal intensity of the TaqMan reaction at the chip is up to 87% of the Macro TaqMan reaction. There was no significant difference between 4x and 8x DyNAzyme. This result indicates that the PCR reaction can be performed with a signal intensity higher than 50% in a microfluidic device when compared to the Macro TaqMan reaction. This result is consistent across at least 4 attempts.

(Example 2)
(Spotting reagent)
I. Introduction The purpose of this experiment was to demonstrate a successful spotted PCR reaction in a microfluidic device. The term “spotted” in this context refers to placing a small drop of reagent (spot) on a substrate, which is then assembled into a part of a microfluidic device. The spotted reagents are generally a subset of the reagent mixture required to perform PCR.

II. Procedure A. Reagent spotting Normal reagent spotting was performed by the contact printing process. Reagents were deposited by taking metal pins from a series of source wells and contacting the pins to the target substrate. This printing process is further outlined in FIG. As indicated, the reagents were taken from a source (eg, a microtiter plate) and then printed by contacting the filled pins with the substrate. After a washing step consisting of stirring in deionized water, it was vacuum dried. The system used to print this reagent spot is Cartesian Technologies MicroSys 5100 (Irvine, Calif.) Using TeleChem “ChipMaker” brand pins, but other systems can be used as described above. .

  The pin used was a Telechem ChipMaker 4 pin, which incorporates an electro-milled slot (see FIG. 9) to increase the capture volume (and hence printable) The number of spots increases). Under the operating conditions used (typically 75% relative humidity and temperature about 25 ° C.), more than 100 spots were printed per fill cycle with one pin. Under the above conditions, the volume of reagent spotted on the PDMS substrate is on the order of 0.1 nL.

  The dimension of the tip of the pin is 125 × 125 μm. The final spot of the dried reagent is substantially smaller than this dimension (as much as 7 μm in diameter), but the pin size defines a lower limit to the spot spacing that can be easily achieved. This achievable spacing determines the smallest well-to-well pitch in the final device. Using such a device and the method described above, an array with a spacing of 180 μm was achieved. Arrays built on working chips tend to have spacings between 600 microns and 1300 microns.

  Spotting was performed using only one pin at a time. However, the system used has a pin head that can fit up to 32 pins. Printing standard size chips (array dimensions on the order of 20 × 25 mm) takes less than 5 minutes.

B. Assembling Spotted Chips The flow layer and control layer of the PCR device are assembled according to the normal MSL process described above. The design of the microfluidic device is the same as that described in Example 1. In parallel, 30: 1 Ratio A: a substrate layer made of PDMS having a thickness of 150μm with B, formed through a spin coating plain silicon wafer, then cured at 80 ° C. 90 minutes .

  The cured plain substrate layer PDMS (sealing / substrate layer 724 in FIG. 7A) serves as a target for reagent spotting. The pattern of spots is printed on a substrate, which is still on a plain wafer. The reagents spotted for the PCR reaction were primers and probes specific for the particular gene to be amplified. The spotted reagents included a 1: 1: 1 volume ratio of 300 nM β-actin forward primer (FP), 300 nM β-actin reverse primer (RP), and 200 nM β-actin probe (Prb). . In some cases it is useful to further adjust the chemical reaction by concentrating the spotted mixture. It has been found that consistently good results can be obtained by adjusting the concentrations so that the primer and probe concentrations are equal to or slightly higher than the normal macroscopic recipe values. Yes. Therefore, the spotted reagent is concentrated to a concentration of 3 and 4 times the macro reaction. Reagent concentration is performed in a Centrivap that is heated and vacuum centrifuged and does not change the relative FP: RP: Prb ratio. The increased spot concentration results in the correct final concentration when the reagent is resuspended in a 1 nL reaction volume. The spotted reagents need not be limited to primers and probes: and also not all three (FP, RP and Prb) may be spotted. Applications that spot only the probe or even spots that spot one of the primers can be made. Experiments were performed where the sample primer / probe set was TaqMan β-actin and TaqMan RNAse-P.

  After the spotting process to the substrate layer, the combined flow layer and control layer (ie, layers 720 and 722 in FIG. 7A) were aligned and contacted with the spot pattern. Further heating at 80 ° C. for 60 to 90 minutes bonded the substrate to the remaining chips. After the chip is assembled, the remaining components of the PCR reaction (described in Example 1) are injected into the flow channel of the chip and the chip is thermocycled as described in Example 1.

III. Results The PCR reaction was successfully and reproducibly performed using a device spotted with primers (forward and reverse primers) and probe molecules. An example of data derived from a chip that has been successfully reacted is shown in FIG.

  The spotted reagent resulted in a successful PCR reaction as defined in Example 1. Successful reactions were performed using two- and three-step thermal cycling protocols.

(Example 3)
(Genetic typing)
I. Introduction The purpose of the following experiments was to demonstrate that genotyping experiments can be performed utilizing microfluidic devices or chips as described herein. Specifically, determine if the reaction performed on the device has sufficient sensitivity and ensure that in addition to β-actin, other primer / probe sets can be performed on the microfluidic device These experiments were designed as follows.

II. Method / Results RNase P experiments RNase P TaqMan reaction (Applied Biosystems; Foster City, CA) was performed on a microfluidic device as described in Example 1 to demonstrate that other primer / probe sets yield detectable results did. Since the RNase P primer / probe set detects single copy genes (2 copies / genome) as opposed to the β-actin primer / probe set, the RNase P reaction also requires a higher level of sensitivity. The β-actin set detects a single copy β-actin gene and several pseudogenes, for a total of approximately 17 copies per genome. An RNase P reaction was performed with the same components as described in Example 1 except that the β-actin primer / probe set was replaced with an RNase P primer / probe set. In addition, RNase P primer / probe sets were used at 4 times the manufacturer's recommended values to enhance the fluorescent signal. The VIC dye was coupled to a probe for RNase P and this analysis concentrated on the VIC / PR1 ratio. The result of one of the four experiments is shown in FIG.

  The VIC / PR1 / control ratio for the Macro TaqMan reaction is 1.23. The corresponding ratios for TaqMan reaction in microfluidic devices are 1.11 and 1.12. The ratio of genomic DNA samples in the microfluidic device is above the 99% confidence threshold level. Furthermore, the signal strength of TaqMan reaction in microfluidic devices is 50% and 93.7% of Macro TaqMan reaction. The control TaqMan response in the microfluidic device has a standard deviation of 0.006 and 0.012, indicating the consistency of the response across the microfluidic device. Therefore, it is determined that the TaqMan reaction on the chip is sensitive enough to detect 2 copies per genome.

B. DNA dilution experiments To further determine the sensitivity of the TaqMan reaction in microfluidic devices, dilutions of genomic DNA were tested using a β-actin primer / probe set. The reaction composition was generally configured as described in Example 1 with a 4-fold dilution of DyNAzyme and genomic DNA. Genomic DNA was diluted to 0.25 pg / nl (which corresponds to about 1 copy per nl). The results of one dilution series are shown in FIG.

  According to the Poisson distribution, if the average target number is 1, 37% of the total number of wells should be negative. Well numbers 5, 6 and 7 are below the calculated threshold and are therefore negative. This suggests that the β-actin TaqMan reaction in the microfluidic chip can detect an average of 1 copy per nl. Thus, the sensitivity of the reaction in the microfluidic device is sufficient to perform genotyping experiments.

C. Genotyping experiments Since TaqMan in microfluidic devices can detect a small number of targets, a preliminary test of SNP (Single Nucleotide Polymorphism) genotyping, Prededicated Allyl Discrimination Kit (Applied Biosystems; CA) And performed on the CYP2D6 P450 cytochrome gene. This kit is labeled against one primer set and two probes; FAM, CYP2D6 ^* 1, and CYP2D6 ^* 3 mutant alleles or variant genes labeled against a wild-type or reference allele It was, including the VIC. Positive controls (PCR products) for each allele along with genomic DNA were tested with the device using the same conditions as described in Example 1. Results from one experiment are shown in FIG. 13 and FIG. The experiment was repeated at least three times to confirm the results and demonstrate reliability.

As shown in FIG. 13, Al-1 (allele 1, CYP2D6 ^* 1 wild type allele) and genomic DNA (100 pg / nl) showed an average VIC / PR1 / control of 3.5 and 2.2, respectively. A ratio was generated, indicating that this genomic DNA was positive for CYP2D6 ^* 1 (wild type allele). These values exceed the threshold limit boundary for the reaction. The signal intensity of the TaqMan reaction in the microfluidic device is 59% and 40% of the Macro TaqMan control, respectively. Al-2 (allele 2, CYP2D6 ^* 3 mutant allele or variant gene), which should be negative in the VIC channel, shows some signal over control (1.5) This is likely due to the FAM fluorescence leaking into the detector's VIC channel. This leakage can be minimized by an improved detection process.

Al-2 positive control gave an average FAM / PR1 / Control ratio of 3.0, which is 37% of the Macro TaqMan signal exceeds the calculated threshold limit boundary (see Figure 14). The genomic sample is negative for the CYP2D6 ^* 3 variant allele, an expected result because the frequency of the CYP2D6 ^* 3 allele is low. Again, there appears to be some leakage of the Al-1, VIC probe into the FAM channel of the detector. Overall, the SNP detection reaction was successful with microfluidic devices.

(Example 4)
(Confirmation of PCR by gel electrophoresis)
I. Introduction Experiments were performed to detect PCR products by gel electrophoresis so that an alternative method of demonstrating DNA amplification occurs in microfluidic devices. The PCR reaction composition was as described in Example 1, except that the TaqMan probe was omitted and the β-actin forward primer was bound to FAM.

II. Procedure A. Microfluidic Device A three-layer microfluidic device manufactured using the MSL process was designed and manufactured to perform the experiments described in this example; FIG. 15 shows a schematic diagram of this design. The device 1500 generally consists of a sample area 1502 and a control area 1504. The sample region 1502 includes 341 1 nl reaction sites 1508 represented by rectangles disposed along the flow channel 1506, which includes an inlet hole 1510 and an outlet hole 1512. Control region 1504 includes three control flow channels 1514, each including ten 1 nl reaction sites 1518 (also represented by a rectangle) and inlet holes 1516. The network of control lines 1522 separates each reaction site 1508, 1518 when sufficient pressure is applied to the inlet hole 1524. A series of protective channels 1520 are provided to prevent liquid from evaporating from the reaction sites 1508, 1518. This device is a three-layer device as described in Example 1 (see FIG. 7A). This whole chip is placed on the coverslip.

B. Experimental Setup A microfluidic device 1500 was filled and thermocycled using the three temperature profiles described in Example 1. The remaining reaction samples were thermocycled with GeneAmp 9700 using the same thermal cycling profile as for microfluidic device 1500. The reaction product was collected after thermal cycling was complete. To recover the amplified DNA, 3 μl of water was injected into the sample input hole 1506 and 3-4 μl of product was removed from the outlet hole 1512. The reaction products from device 1500 and the Macro reaction were treated with 2 μl ExoSAP-IT (USB, Cleveland, OH), which consists of DNA Exonuclease I and Shrimp alkaline phosphatase to remove excess nucleotides and primers. Removed. The Macro product was diluted 1:10 to 1: 106. The product from device 1500 was dehydrated and resuspended in 4 μl formamide.

III. Results Both products were analyzed on polyacrylamide gels with negative controls. FIG. 16 shows the results of gel electrophoresis. An appropriately sized DNA band of 294 base pairs in length is observed in FIG.

  The product from the Macro reaction is shown on the left hand side of the gel, which corresponds to approximately 294 base pairs, the expected size of the β-actin PCR product. Negative controls lack PCR products. Similarly, the product from the device yielded the expected β-actin PCR product. Therefore, the target DNA was amplified with a microfluidic device.

(Example 5)
(Large division)
Polymerase chain reaction (PCR) has become an essential tool in molecular biology. The combination of sensitivity (amplification of a single molecule of DNA), specificity (discriminating single base mismatches) and dynamic range (10 ^{5 in} real-time measurements) make PCR one of the most powerful analytical tools in existence. . It has been demonstrated that the performance of PCR is improved to reduce the reaction volume: we have a single microfluidic chip with a single template molecule sensitivity at a volume of 90 pL per reaction, 21 1,000 simultaneous PCR reactions were performed.

  Figures 17a-17d show a single bank and a dual bank dividing the microfluidic device. Here, multilayer soft lithography (MSL) (1) is used to make an elastomeric microfluidic chip, which is used to divide each of several liquid samples into a large number of separate reaction volumes on a large scale. Active valves are used. After injection of the sample into the inlet 1703 (which communicates with the branched split channel system 1705 of the microfluidic device 1701 (FIG. 17b)), each sample's 2400 90pL volume 1709 is replaced with a simple microfluidic channel. Are separated by a closing valve 1707 spaced apart along (FIG. 17d). The chip device is then thermally cycled with a flat plate thermocycler and imaged with a commercially available fluorescence reader.

PCR performance was assessed on the chip by varying the concentration of template DNA and measuring the number of wells that produced a positive Taqman ^tm signal. We have found that digital amplification is observed when the average number of copies per well is low (FIGS. 18a and 18b). Even when the average number of copies per well is less than 1, a mixture of strong positive and clearly negative signals is observed; this means that good amplification can also occur with single copy targets . The number of positive wells was consistent with the number of wells calculated by the Poisson distribution to have more than one target copy (FIG. 19). This result demonstrates that this system consistently produces amplification even from a single copy target. Fluorescent signal intensity derived from the microfluidic ^Taqman tm PCR is Macro PCR reaction in the same DNA concentration (this macro reaction is contained template copy more than 10 per reaction ⁴⁾ was comparable to.

Probably the main source of observed fidelity is the effective concentration of the target: single molecules in a 90 pL volume are 55,000 times more concentrated than single molecules in a 5 μL volume. The number of target molecules (n _t ) does not change (ie, n _t = 1) and the number of molecules that can cause side reactions (n _s ) (ie, primer dimers and non-complementary DNA sequences in the sample) is the volume since linearly proportional to _{(i.e., n} s alpha] V) with respect to the ratio of the target to side reactions, inverse _{(n t / n s α1 /} V) to relative volume. Since side reactions are a major cause of PCR failure (4), the advantages to reducing reaction volume are clear.

  PCR amplification from a single copy of the template has been previously reported (5). However, current methods of obtaining reliable amplification from a single copy in a macrovolume are often modified thermal cycling protocols (eg, long extension times, high cycle numbers), mispriming and non-specific Preventive amplification (eg, “hot start” PCR (thermal activation of polymerase), “booster” PCR, additives to reduce non-specific hybridization, etc.), and in most cases 2 Complete with a round of PCR (an aliquot of the first PCR is used as a template in the second reaction). In contrast, this system achieves reliable amplification from a single copy using standard conditions (off-the-shelf primers and probes) and a single round of standard thermal cycling protocol. When completely encapsulated, it can withstand environmental pollution. The ability to perform a large number of PCR reactions simultaneously provides obvious logistic cost and time benefits compared to macrovolume (one chip with 21,000 reactions vs. 219 individual 96 wells) Plates, and associated time, equipment and tracking infrastructure).

  The principle of large scale partitioning with digital PCR readout can be used for absolute quantification of target concentration in a sample. For example, it can be used to genotype pooled genomic DNA samples by simply counting the number of wells that are positive for a particular allele or many of the alleles described above. Due to increased resistance to side reactions, it should also be useful in quantifying mutants in the background of wild-type DNA (problems related to cancer detection). The general principle of enrichment by resolution may also be useful in other reactions where detection of a single molecule, bacteria, virus or cell is a goal (eg, ELISA reactions for protein detection). Digital PCR is described in Brown et al., US Pat. No. 6,143,496, title of the invention “Method of sampling, amplification and quantifying sequen sing sir ming samp ri n s ing s i n s i n e n e n e n e n e s e n e n e n e n e n e e n e n e n e n e n e e n e n e n e n e n e n e n e s e n e n e n io Vogelstein et al., US Pat. No. 6,446,706, described by the title “Digital PCR”, both of which are hereby incorporated by reference in their entirety. The small volume that can be achieved using microfluidics allows both large scale parallelism and very high target-to-background concentration ratios. A high target-to-background ratio allows single-molecule amplification fidelity. These factors suggest that smaller ones for PCR are actually better.

  The present invention provides a method and device for performing digital PCR in a microfluidic environment, the method comprising providing a microfluidic device having a fluid channel therein, the fluid channel being in the channel. Having two or more valves coupled, which when actuated can divide the fluid channel into two or more reaction sites or chambers; a sample comprising at least one target nucleic acid polymer; Introducing a step of actuating a valve to divide the fluid sample into two or more parts, wherein at least one part contains the target nucleic acid polymer and another part does not contain the target nucleic acid polymer; Amplifying the target nucleic acid polymer; and determining the number of fluid channel moieties comprising the target molecule. In a preferred embodiment, the microfluidic device comprises an elastomeric material, more preferably at least one layer comprising an elastomeric material. In certain preferred embodiments, the microfluidic device further includes a bendable membrane that controls the flow of fluid within the fluid channel and / or separates a portion of the fluid channel. Bendable in and out of the fluid channel so as to divide from the portion, preferably the bendable membrane is integral to the layer of the microfluidic device having channels or recesses formed in the device; and Preferably, the bendable membrane is formed when the first channel in the first layer overlaps with the second channel in the second layer of the microfluidic device. In some embodiments, the sample fluid includes all of the components necessary to perform the amplification reaction, while in other embodiments, the microfluidic device has at least one of the amplification reactions prior to introduction of the sample fluid. Contains ingredients. In some embodiments, the microfluidic device further comprises a detection reagent, preferably within one or more nucleic acid polymers that are complementary to at least a portion of the target nucleic acid polymer, preferably within a reaction site or chamber of the microfluidic device. It includes a plurality of different nucleic acid polymers arranged separately.

  Amplification can be a thermal cycling reaction (eg, PCR), or an isothermal reaction (eg, US Patent Application No. 10 / 196,740 (published as US 2003/0138800 A1) (this is isothermal amplification). For the purpose of teaching the scheme, which is incorporated herein by reference in its entirety).

  The invention further provides for structural changes (eg, denaturation) of a protein, particularly when the temperature of protein denaturation changes when the protein interacts with another moiety (eg, a ligand or compound or other protein). Protein microcalorimetric assays using fluorescent dyes (eg, SYBER Green ™) to measure are provided. A further advantage of using SYBR Green (TM) is that this SYBR Green (TM) is used at shorter wavelengths than other UV range dyes, and therefore the background of the background typically associated with many plastic materials. It is to reduce the problem.

FIG. 22B comprises the components shown in FIG. 22A and is further attached or more preferably to elastomeric block location 2807 of substrate 2800 to form a complete microfluidic device 2899 (FIG. 22C). Shows an exploded view of a complete microfluidic device 2899 comprising an elastomeric block 2808, glued with an adhesive, and more preferably, preferably without glue and directly glued. Within elastomer block 2808 there is one or more channels in fluid communication with one or more holes 2814, which in turn provide fluid communication between the channels in the elastomer block and the channels in the substrate. And then leads to the wells 2805 in the row of wells 2806a-d to provide fluid communication between the wells 2805 of the substrate 2800 and the channels in the elastomeric block 2808. Accumulator well tops 2809 and 2810 are attached to accumulator wells 2801 and 2802 to form accumulator chambers 2815 and 2816. The accumulator well tops 2809 and 2810 include valves 2812 and 2811, respectively, which are preferably check valves for introducing and holding gas into the accumulator chambers 2815 and 2816 under pressure. Valve 2811 and 2812, it mounted inside the de Raiweru 2802 and 2804, liquid, when present in accumulator chambers 2815 and in 2816, keeping the state not in contact with the valve 2811 and 2812. Valves 2811 and 2812 preferably press a shave, pin, etc. into the preferred check valve to overcome the self-sealing force of the check valve and allow the pressure to be released from the accumulator chamber. It can be mechanically released by reducing the pressure of the fluid contained within the chamber.

FIG. 22B comprises the components shown in FIG. 22A and is further attached or more preferably to elastomeric block location 2807 of substrate 2800 to form a complete microfluidic device 2899 (FIG. 22C). is contact wear, and, even more preferably, preferably without the use of adhesives, bonded directly includes an elastomeric block 2808, it shows an exploded view of a complete microfluidic device 2899. Within elastomer block 2808 there is one or more channels in fluid communication with one or more holes 2814, which in turn provide fluid communication between the channels in the elastomer block and the channels in the substrate. And then leads to the wells 2805 in the row of wells 2806a-d to provide fluid communication between the wells 2805 of the substrate 2800 and the channels in the elastomeric block 2808. Accumulator well tops 2809 and 2810 are attached to accumulator wells 2801 and 2802 to form accumulator chambers 2815 and 2816. The accumulator well tops 2809 and 2810 include valves 2812 and 2811, respectively, which are preferably check valves for introducing and holding gas into the accumulator chambers 2815 and 2816 under pressure. Valve 2811 and 2812, it mounted inside the de Raiweru 2802 and 2804, liquid, when present in accumulator chambers 2815 and in 2816, keeping the state not in contact with the valve 2811 and 2812. Valves 2811 and 2812 preferably press a shave, pin, etc. into the preferred check valve to overcome the self-sealing force of the check valve and allow the pressure to be released from the accumulator chamber. It can be mechanically released by reducing the pressure of the fluid contained within the chamber.

  FIG. 22D shows a top view of the microfluidic device 2899 and well 2805, where the ports are wells in channels formed in the substrate 2800 (preferably on the side of the substrate 2800 opposite the well 2805). In order to allow fluid to pass therethrough, adjacent to the base (preferably the bottom) of the well or the side of the well 2805. In a particularly preferred embodiment, the substrate 2800 has a recess formed therein and the recess is made in the channel by a sealing layer (preferably an adhesive film or sealing layer).

Substrate 2800 and its associated components can be polymers such as polypropylene, polyethylene, polycarbonate, high density polyethylene, polytetrafluoroethylene PTFE or Teflon (R), glass, quartz, or metal (eg, aluminum), transparent material may be fabricated polysilicon con a cathodic et al. Accumulator wells upper 2809 and 2810 may further include a proximity screw 281 3, which may be removed to introduce or remove gas or liquid from accumulator chambers 2815 and 2816. Preferably, valves 2812 and 2811 can be actuated to relieve fluid pressure, otherwise they are held inside accumulator chambers 2815 and 2816. Notch 2817, the microfluidic device, other devices, for example, used to assist in accurate placement of the infinitesimal fluidic device or a device used to operate or analyze the reaction carried out in the . FIG. 22D further depicts a hydration chamber 2850 that surrounds the elastomeric block region 2807, which may be covered with a hydration cover 2851 to form a humidification chamber and facilitate control of humidity around the elastomeric block. . Humidity can be increased by adding a volatile liquid, such as water, to the humidification chamber 2851, preferably by wetting the blotting material or sponge. Preferably, polyvinyl alcohol is used. Humidity control is preferably achieved by changing the ratio of polyvinyl alcohol to water used to wet the blotting material or sponge. Hydration also may be controlled by using a humidity control device such as a Humidipak (trademark) humidification package which, for example, hold a salt solution having an appropriate salt concentration to maintain the desired humidity level A water vapor permeable but liquid impermeable envelope is used. See US Pat. No. 6,244,432 by Saari et al. This patent is incorporated herein by reference for all purposes, including the specific purpose of humidity control devices and methods. The hydration cover 2850 is preferably transparent so as not to prevent the events in the elastomer block from being visible during use. Similarly, the portion of the substrate 2800 below the elastomeric block region 2807 is preferably transparent, but may be impermeable or reflective.

  FIG. 22E depicts a cross-sectional view of a substrate 2800 with an elastomeric block 808 positioned in an elastomeric block region 2807 along a sealing layer 2881 that is attached to the side of the substrate 2800 opposite the elastomeric block 2808. FIG. Well 2805 is in fluid communication with elastomeric block 2808 through first port 2890, channel 2870, and second port 2892 and into a recess in elastomeric layer 2808 that is sealed by upper surface 2897 of substrate 2800 to form channel 2885. doing. Seal layer 2881 forms channel 2870 from a recess that is molded or machined into bottom surface 2898 of substrate 2800. The sealing layer 2881 is preferably a transparent material, such as polystyrene, polycarbonate, or polypropylene. In one embodiment, the sealing layer 2881 is flexible, as in an adhesive tape, and can be mechanically attached to the substrate 2800 by bonding, such as with an adhesive or heat seal, or by compression. Preferably, the material for the seal layer 2881 is adapted to form a fluid seal with each recess to form a fluid channel with minimal leakage. Seal layer 2881 can be further supported by an additional support layer (not shown) that is rigid. In another embodiment, the sealing layer 2881 is rigid.

  Details from a close distance of the fluid show the interface between the elastomer block 2808 and the elastomer block region 2807 of the substrate 2800. As described in co-pending Grossman et al., US patent application Ser. No. 11 / 043,395, the elastomeric block can be formed from multiple layers of elastomeric material bonded together to form an elastomeric block, The patent application is incorporated herein for all purposes described herein and in this patent application, and for the purpose of disclosing further details regarding the use of integrated carriers and self-actuating devices. Preferably, at least two layers of the elastomer block have a recess. For example, a first elastomer layer having a recess formed therein is bonded to a second elastomer layer having a recess formed therein to form an elastomer block having a recess formed therein. . The recess in the first elastomer layer is wholly or partially closed to form a channel in the first elastomer layer. The recess formed in the second elastomer layer is likewise closed in whole or in part when the elastomer layer is bonded to the substrate, forming a channel in the second elastomer layer, thereby A microfluidic device having a plurality of layers with channels formed therein is formed.

In FIG. 23, a first elastomer layer 2920 having a bottom surface with a first recess 2901 formed therein and a second surface having a top surface and a bottom surface with a second recess 2905 formed therein. The elastomeric layers 2923 are bonded together to form an elastomeric block having a channel 2907 formed from a first recess 2901 and a top surface of the second elastomeric layer 2923. The substrate 2800 is attached to the bottom surface of the second elastomer layer 2923 and forms a channel 2909 from the top surface 2897 of the substrate 2800 and the bottom surface of the second elastomer layer 2923. The port 2892 may connect the channel 2872 of the substrate 2800 with the channel 2909 of the second elastomeric layer formed in part by the top surface of the substrate 2800. Alternatively, as shown in FIG. 23, the port 2892 connects the channel 2872 of the substrate 2800 with the channel 2907 of the first elastomer layer 2920 of the elastomer block 2808 through the hole 2 950. The holes 2950 are formed substantially perpendicular to the substrate surface 2897, preferably in the second elastomer layer 2923, and more preferably the first and second elastomers, prior to their bonding with the elastomer layer 2920. It is formed after the layers are bonded together. See US Provisional Patent Application No. 60 / 557,715 by Unger, filed March 29, 2004 and assigned to the present applicant. This patent application is incorporated by reference for all purposes and the specific purpose of teaching via formation using automated laser ablation systems and methods. Exemplary methods for generating holes include microfabrication during formation of the second elastomer layer 2923, laser drilling, laser drilling using a CO ₂ laser, laser drilling using an excimer laser, mechanical drilling, and Including coring, preferably wherein the drilling is performed by a robot drilling system, preferably a system having an x, y automation stage.

  FIG. 23 depicts a cross-sectional view of another preferred use of the holes in the microfluidic devices described herein. The microfluidic block 2921 includes a first layer 2920 having a first layer recess (or channel when coupled to the second layer) 2907 formed therein, and a second layer recess therein. A second layer 2923 having a channel (or a channel when coupled to a substrate). The two second layer channels are in fluid communication through the first layer channel by two or more holes 2950. Preferably, at least one hole 2950 is further in fluid communication with well 2999 of substrate 2800 through substrate recess 2892 (or a channel if a seal layer (not shown) is coupled to substrate 2800). At least one second layer channel 2909 is overlapped by a portion of the first layer channel 2907 without fluid communication. In the embodiment shown in FIG. 23, a higher density of reaction and / or detection zones per unit area of the microfluidic device may be achieved. This is because a fluid channel in one layer can take a path above or below an intervening fluid channel in the same layer. An ablation residue chamber 2989 is present to capture the residue generated from the laser ablation hole 2950. Residue chamber 2989 can be cast into layer 2920 by a two-layer casting method, where after the first layer of photoresist is patterned and developed, the second layer of photoresist is first patterned. And a second pattern is formed over the pattern of the first photoresist layer such that regions of the photoresist pattern can be of different heights. Multiple layers can be built on top of each other to produce varying height patterns. Different photoresist materials can also be used, for example, so that they can flow again when the upper layer of the photoresist is heated, while the lower layer is made from a photoresist that does not flow substantially again at the same heating temperature.

  The flow channels of the present invention can be designed with different cross-sectional sizes and shapes as needed, providing different advantages depending on their desired application. For example, the cross-sectional shape of the lower flow channel may have a curved upper surface either along its entire length or in a region located below the upper cross channel. Such a curved upper surface facilitates sealing of the valve as follows. Membrane thickness profiles and flow channel cross sections contemplated by the present invention include rectangular, trapezoidal, circular, elliptical, parabolic, hyperbolic, and polygonal, and cross sections of the above shapes. More complex cross-sectional shapes are also contemplated by the present invention, such as embodiments with protrusions in the flow channel or embodiments with concave surfaces.

Further, although the present invention has been described primarily in combination with embodiments in which the flow channel walls and ceiling are formed from an elastomer and the channel floor is formed from an underlying substrate, the present invention is not limited to this particular The orientation is not limited. Only the ceiling of the flow channel is constructed from elastomer, and the walls and floor of the channel can also be formed in the underlying substrate. The elastomeric flow channel ceiling, projects downward into the channel in response to actuation force applied, thereby controlling the flow of material through the flow channel. In general, monolithic elastomeric structures are preferred for microfluidic applications. However, employing channels formed in the substrate can be useful in cases where such an arrangement provides advantages. For example, a substrate that includes an optical waveguide can be constructed such that the optical waveguide specifically directs light to the sides of the microfluidic channel.

  The microfluidic device of the present invention can be used as a stand-alone device or, preferably, can be used as part of a system provided by the present invention. FIG. 24 depicts a perspective view of a robot station operating a microfluidic device. Automated pneumatic control and accumulator charging station 3200 includes a receiving bay 3203 for holding a microfluidic device 3205 of the present invention, such as the type depicted in FIGS. The platen 3207 is adapted to contact the upper surface 3209 of the microfluidic device 3205. The platen 3207 has a port in it that aligns with the microdevice 3205 and provides fluid pressure, preferably gas pressure, to wells and accumulators in the microfluidic device 3205. In one embodiment, the platen 3207 is pushed against the top surface 3221 of the microfluidic device 3205 by movement of the arm 3211, which is hinged on the pivot 3213 and attached to the arm 3211 at one end. And moved by a piston 3215 attached to the platform 3217 at the other end. A sensor along the piston 3215 detects piston movement and relays information about the piston position to a controller, preferably a controller under the control of a computer (not shown) according to a software script. The plate detector 3219 can detect the presence of the microfluidic device 3205 inside the receiving bay 3203 and preferably can detect the proper orientation of the microfluidic device 3205. This can occur, for example, by optically detecting the presence and orientation of the microfluidic device 3205 by reflecting light from the side of the microfluidic device 3205. The platen 3207 can be lowered by a robot, by aerodynamic force, electrically, or the like. In some embodiments, the platen 3207 is manually lowered to engage the device 3205.

  In one embodiment, the fluid line leading to the platen 3207 is located in the arm 3211 and is connected to a fluid pressure source, preferably an automatic pneumatic pressure source under the control of the controller. This pressure source provides a controlled fluid pressure to a port in a platen surface (not shown) of the platen 3207 and provides a controlled pressurized fluid to the microfluidic device 3205. Fine positioning of the platen 3207 is achieved, at least in part, by employing a gimbal connection 3223 so that the platen 3207 can angularly move about an axis perpendicular to the top surface 3221 of the microfluidic device 3205. Attached to the arm 3211.

Figure 25 is depicts a partially cut-away side view of the platen 3207 pressed against the upper surface 3221 of the infinitesimal fluid device 3205. The platen 3207 is pushed against the upper surface 3221 of the microfluidic device 3205 to provide a fluid tight seal between the microfluidic device 3205 and the platen surface 3227 or between a portion of the device 3205 and a portion of the surface 3227. Form. The platen surface 3227, in one embodiment, includes or is made of a corresponding material such as an elastic elastomer, preferably a chemical resistant rubber. Inside the platen 3207 is a separate fluid pressure line, preferably a gas pressure line, that mates with various locations on the top surface 3221 of the microfluidic device 3205. A check valve purge actuator 3233 is also shown, which is preferably actuated by aerodynamic forces, and when activated, pushes the pin 3231 down into the check valve 2812 to release and relieve fluid pressure, Alternatively, the introduction of fluid through check valve 2812 is permitted by overcoming its open resistance. In one embodiment, the platen 3207 has first and second purge actuators 3233 that engage the check valves 2811 and 2812 (see FIG. 22B).

  In another embodiment, the chip or device 3205 is manufactured with a normally closed containment and / or interface valve. In this embodiment, an accumulator will not necessarily be needed to keep the valve closed during incubation. Pressure may be applied to the carrier or device 3205 well region when it is desired that the interface and / or closing valve be opened. During all or most other times, the valve may remain closed, separating the various chip experiments from each other and / or separating the reagent and protein wells on the chip from each other.

  FIG. 25 depicts a partial cutaway view of the platen 3207 that is pushed against the top surface 3221 of the microfluidic device 3205, where the pressure cavity 3255 contacts the platen surface 3227 against the ridge 3250 of the top surface 3221. To form on the well row 2806. Fluid pressure, preferably gas pressure, is then applied to pressure cavity 3225 by introducing fluid into pressure cavity 3255 from a pressure line running down arm 3211 of charging station 3200. The pressure is controlled by a pressure regulator combined with the charging station 3200, preferably by an electrically controlled variable pressure regulator which can change the output pressure according to a signal sent by a charging station controller under computer control. Preferably it is adjusted. The fluid pressure inside the pressure cavity 3255 then causes the liquid in the well 2805 to flow through the channel in the substrate 2800 and into the chamber of the channel and / or elastomer block 2808, filling the channel or chamber, Or actuate the deflectable portion of the elastomeric block 2808, which is preferably a deflectable membrane valve as described above.

  FIGS. 27A and 27B depict a particular embodiment of system 3500 and, more particularly, interface plate 3520. In FIG. 27A, the interface plate 3520 is coupled to the integrated chip and carrier 3400 in a manner that fluidically seals a particular area thereof. In particular, the fluid seal may include interface plate 3520 and carrier 3400 and one or more regions of the chip (eg, first protein region 3430, second protein region 3432, first well region 3420, second well region 3422). , Interface accumulator 3460, check valve 3465, confinement accumulator 3450, and / or check valve 3455). In one embodiment, interface plate 3520 provides a fluid seal to regions 3420, 3422, 3430, 3432 and accumulators 3450 and 3460. In one embodiment, interface plate 3520 provides one or more check valve actuators 3570 as best observed in FIG. 27B.

In some embodiments, interface plate 3520 provides all desired fluid seals to carrier 3400 and microfluidic device. In doing so, the interface plate 3520 may include a seal gasket 3580. Seal gasket 3580 may include a wide range of materials, but are not limited to, include silicon rubber, elastomer and the like. In some embodiments, the gasket 3580 includes a corresponding material that forms to assist the fluid seal at a desired location. In this way, the system 3500 can provide the desired pressure to the appropriate area of the chip and carrier 3400. In other embodiments, the interface plate 3520 is two or more components. For example, a region or port on the carrier 3400 and microfluidic device can each be fluidly coupled to a separate plate 3520 adapted to fit that port or region. System 3500 then includes the required number of interface plates 3520 for the various ports or areas. Further, in some embodiments, more than one region or port is coupled to a particular interface plate 3520 while other regions or ports are coupled to a separate interface plate 3520. Other combinations of interface plates and carrier / chip areas and ports are within the scope of the present invention.

  Operation of system 3500 includes, in one embodiment, loading one or more carriers 3400 into receiving station (s) 3510. In some embodiments, carrier 3400 includes a microfluidic device coupled thereto and has the desired reagents and proteins loaded into a carrier well prior to placing the carrier in receiving station 3510. In other embodiments, the carrier 3400 is placed in a receiving station 3510 and then loaded with reagents and proteins. The carrier 3400 can further be loaded with a hydrating fluid. Hydration fluid may be placed in the hydration chamber 3440. After the carrier 3400 is loaded into the system 3500, the interface plate 3520 is lowered or otherwise moved to engage the carrier 3400. Plate 3520 is lowered manually, by a robot, or otherwise, to fluidly seal part or all of chip / carrier 3400. Hydrating fluid is provided to interface accumulator 3460 and / or confinement accumulator 3450 and into the chip by applying an appropriate pressure to accumulator 3450, 3460 using a pressure source coupled to interface plate 3520. Shed. In certain embodiments, the system 3500 automatically performs this process, which in certain embodiments occurs within about 20 hours after the hydrating fluid is added to the carrier 3400. As a result, the tip is fully loaded with hydraulic fluid and, as described herein, and more particularly in the patents and applications previously incorporated herein by reference, tip containment. And / or actuate the interface valve.

  The solution containing the reagent and sample is dispensed into the chip by applying the desired pressure to the properly sealed chip area around the appropriate inlet. For example, applying a pressure between about 1 psi and about 35 psi to the first and second well regions 3420 and 3422 operates to flow the reagent through the chip. Similarly, applying a pressure between about 1 psi and about 35 psi to the first and second protein regions 3430 and 3432 operates to cause the protein to flow through the chip. In certain embodiments, this occurs within about 60 minutes after loading the tip with hydraulic fluid. Once the solution is flowed into the desired well, chamber, reservoir, etc. in the chip, the interface valve in the chip is opened by releasing the check valve 3465 in the interface accumulator 3460. In certain embodiments, when the check valve 3465 is released and the system 3500 activates the check valve actuator 3570 that engages the check valve 3465, it opens the interface valve in the chip. In some embodiments, check valve actuator 3570 includes pins, posts, etc. adapted to engage check valve 3465 to relieve pressure in interface accumulator 3460. In an alternative embodiment, check valve 3465 is manually released or opened.

The solution is allowed to mix for a desired time using convection mixing or other processes such as repeated opening and closing of interface valves to increase diffusion or fluid movement in the chamber. After this, the interface valve is closed. Pressure is applied to actuators 3450 and / or 3460 to maintain a closed interface valve and confinement valve. The carrier 3400 may be removed from the system 3500 for incubation, storage, or use in the processes described herein. Actuator 3450 and 3460, anti-tool or that containment and interface valve opens, or to prevent and support, from a few hours to a few days, to maintain this pressure for a desired period of time. In certain embodiments, actuators 3450 and 3460 maintain the pressure in the chip above a threshold pressure sufficient to confine and / or maintain the interface valve closed. In one embodiment, actuators 3450 and 3460 maintain the pressure above the threshold pressure for at least 2 days, at least 7 days, etc. For a given time, actuators 3450 and 3460 maintain the desired pressure, depending in part on the incubation temperature. Depending in part on the desired incubation period length and / or incubation conditions, the carrier 3400 can be returned to the system 3500 to recharge or repressurize the actuators 3450, 3460. In this way, the incubation period can be this way.

  In certain embodiments, the integrated carrier 3400 and the microfluidic device are adapted to perform the desired experiment according to embodiments of the present invention by using the system of the present invention. More particularly, as shown in FIG. 26A, system 3500 includes one or more receiving stations 3510, each adapted to receive a carrier 3400. In certain embodiments, the system 3500 includes four receiving stations 3510, although fewer or greater numbers of stations 3510 are provided in alternative embodiments of the invention. FIG. 26B depicts a combination of the carrier 3400 and device located in the station 3510 under the interface plate 3520. FIG. The interface plate 3520 is adapted to move downward in FIG. 26B, and the interface plate 3520 engages the top surface of the carrier 3400 and its microfluidic device. In some embodiments, station 3510 and platen 3520 are similar to station 3200 and platen 3207. Interface plate 3520 includes one or more ports 3525 for connection to a region in carrier 3400 that is adapted to receive fluid, pressure, and the like. In some embodiments, the interface plate 3520 includes two ports, three ports, four ports, five ports, six ports, seven ports, eight ports, nine ports, ten ports, etc. Including. In a preferred embodiment, interface plate 3520 is coupled to two lines to provide a mechanism for activating check valves 3455 and 3465 to provide pressure to a desired region of carrier 3400. The

As shown in FIG. 26A, the system 3500 further includes a processor, which in one embodiment is a processor associated with a laptop computer or other computing device 3530. Computing device 3530 includes a memory adapted to maintain software, scripts, etc., software, scripts, etc. for performing the desired processes of the present invention. Further, the computing device 3530 includes a screen 3540 for depicting the results of microfluidic device research and analysis, and in one embodiment, a GUI display is used with the system 3500. System 3500, pressurized fluid to the microfluidic devices that are connected by an interface plate (s) 3520 and a fluid, for delivering such gases, one or more pressure source (pressurized fluid, such as gas) and Connected.

  FIGS. 27A and 27B depict a particular embodiment of system 3500, and more particularly, interface plate 3520. FIG. In FIG. 27A, interface plate 3520 is coupled to integrated chip and carrier 3400 in a manner that fluidically seals that particular area. In particular, the fluid seal includes an interface plate 3520, a first protein region 3430, a second protein region 3432, a first well region 3420, a second well region 3422, an interface accumulator 3460, a check valve 3465, a confinement accumulator. A fluid seal is provided between the carrier 3400, such as the modulator 3450, and / or the check valve 3455, and one or more regions of the chip. In one embodiment, interface plate 3520 provides a fluid seal for regions 3420, 3422, 3430, 3432 and for accumulators 3450 and 3460. In one embodiment, interface plate 3520 provides one or more check valve actuators 3570 as best seen in FIG. 27B.

In some embodiments, interface plate 3520 provides all desired fluid seals to carrier 3400 and microfluidic device. In doing so, the interface plate 3520 may include a seal gasket 3580. Seal gasket 3580 may include a wide range of materials, but are not limited to, include silicon rubber, elastomer and the like. In some embodiments, the gasket 3580 includes a corresponding material that forms to assist the fluid seal at a desired location. In this way, the system 3500 can provide the desired pressure to the appropriate area of the chip and carrier 3400. In other embodiments, the interface plate 3520 is two or more components. For example, a region or port on the carrier 3400 and microfluidic device can each be fluidly coupled to a separate plate 3520 adapted to fit that port or region. System 3500 may then include as many interface plates 3520 as necessary for various ports or areas. Further, in some embodiments, more than one region or port is coupled to a particular interface plate 3520 while other regions or ports are coupled to a separate interface plate 3520. Other combinations of interface plates and carrier / chip areas and ports are also within the scope of the present invention.

  Operation of system 3500 includes, in one embodiment, loading one or more carriers 3400 into receiving station (s) 3510. In some embodiments, carrier 3400 includes a microfluidic device coupled thereto and has the desired reagents and proteins loaded into a carrier well prior to placing the carrier in receiving station 3510. In other embodiments, the carrier 3400 is placed in a receiving station 3510 and then loaded with reagents and proteins. The carrier 3400 can further be loaded with a hydrating fluid. Hydration fluid may be placed in the hydration chamber 3440. After the carrier 3400 is loaded into the system 3500, the interface plate 3520 is lowered or otherwise moved to engage the carrier 3400. Plate 3520 is lowered manually, by a robot, or otherwise, to fluidly seal part or all of chip / carrier 3400. Hydrating fluid is provided to interface accumulator 3460 and / or confinement accumulator 3450 and into the chip by applying an appropriate pressure to accumulator 3450, 3460 using a pressure source coupled to interface plate 3520. Washed away. In certain embodiments, the system 3500 automatically performs this process, which in certain embodiments occurs within about 20 hours after the hydrating fluid is added to the carrier 3400. As a result, the tip is fully loaded with hydraulic fluid and, as described herein, and more particularly in the patents and applications previously incorporated herein by reference, tip containment. And / or actuate the interface valve.

  The solution containing the reagent and sample is dispensed into the chip by applying the desired pressure to the properly sealed chip area around the appropriate inlet. For example, applying a pressure between about 1 psi and about 35 psi to the first and second well regions 3420 and 3422 operates to cause the reagent to flow into the chip. Similarly, applying a pressure between about 1 psi and about 35 psi to the first and second protein regions 3430 and 3432 operates to cause the protein to flow into the chip. In certain embodiments, this occurs within about 60 minutes after loading the tip with hydraulic fluid. Once the solution is flowed into the desired well, chamber, reservoir, etc. in the chip, the interface valve in the chip is opened by releasing the check valve 3465 in the interface accumulator 3460. In certain embodiments, when the check valve 3465 is released and the system 3500 activates the check valve actuator 3570 that engages the check valve 3465, it opens the interface valve in the chip. In some embodiments, check valve actuator 3570 includes pins, posts, etc. adapted to engage check valve 3465 to relieve pressure in interface accumulator 3460. In an alternative embodiment, check valve 3465 is manually released or opened.

The solution is allowed to mix for a desired time using convection mixing or other processes such as repeated opening and closing of interface valves to increase diffusion or fluid movement in the chamber. After this, the interface valve is closed. Pressure is applied to actuators 3450 and / or 3460 to maintain a closed interface valve and confinement valve. The carrier 3400 may be removed from the system 3500 for incubation, storage, or use in the processes described herein. Actuator 3450 and 3460, anti-tool or that containment and interface valve opens, or to prevent and support, from a few hours to a few days, to maintain this pressure for a desired period of time. In certain embodiments, actuators 3450 and 3460 maintain the pressure in the chip above a threshold pressure sufficient to confine and / or maintain the interface valve closed. In one embodiment, actuators 3450 and 3460 maintain the pressure above the threshold pressure for at least 2 days, at least 7 days, etc. For a given time, actuators 3450 and 3460 maintain the desired pressure, depending in part on the incubation temperature. Depending in part on the desired incubation period length and / or incubation conditions, the carrier 3400 can be returned to the system 3500 to recharge or repressurize the actuators 3450, 3460.

  In some embodiments, an integrated chip carrier (ICC) and an elastomer chip attached thereto are used to facilitate the polymerase chain reaction (PCR), as generally described herein. However, when attempting PCR with a PCR chip, the thermal conductivity of the plastic ICC may not efficiently generate a uniform thermal field between the array of reactions contained in this elastomer chip. For example, the ICC depicted in FIG. 28A is useful for protein crystallization, among other processes, but may not transfer heat uniformly enough to the chip attached to it. Further, the operation of the ICC of FIG. 28A may require that the ICC be held against the platen. As shown in FIG. 29A, the application of compressive force can adversely affect the chip. Thus, in some embodiments of the invention, an improved homogeneous heat field is provided using the ICC embodiment described in conjunction with FIG. 28B, and an improved ICC retention method is shown in FIG. 29B. .

  In some embodiments, the elastomeric chip is designed such that the fluid connection with the ICC is located on the outer surface of the elastomeric chip. In this manner, a portion of the ICC need not be relied upon for fluid transport. One such embodiment of ICC 3800 is shown in FIG. 28A. In some embodiments, the area of the elastomer chip that is not in contact with the ICC surface is where the array of reaction chambers is placed. This reaction region 3810 comprises an elastomeric chip and a conductive material that is fitted to the underside of the chip (the side of the elastomeric block that otherwise contacts the plastic portion of the ICC). The size and shape of the reaction region 3810 can vary within the scope of the present invention, and one embodiment shown in FIG. 29C shows the relationship between the ICC and the chip.

In this manner, thermal energy (eg, from a PCR device) can be transferred to the elastomeric block with minimal or reduced thermal impedance. In some embodiments, thermal conduction material includes silicon (Si). In certain embodiments, silicon (similar to or the same as that used in the semiconductor industry) derived from a polished and smoothed silicon wafer is used. Other low thermal impedance materials can also be used within the scope of the present invention, depending on the nature of the thermal profile required. In some embodiments, thermal conduction material has a low thermal mass (i.e., is a good heat conductor, resulting in rapid changes in temperature material (e.g., copper)). In some embodiments, polished silicon is used in real time or during PCR reaction endpoint analysis to enhance the mirror effect and to reduce the amount of light that can be collected by the detectors used in the system. Increase. These benefits can also improve the isothermal response.

  Those who desire to perform PCR using ICC 3800 may perform PCR by fitting the reaction region of the elastomer block with a thermal control source. This thermal control source may comprise a PCR machine, a heating platen, a separate heat source, and the like. In some embodiments, the ICC is compatible with standard PCR machines that accept flat-bottom reaction plates and / or has a flat thermal plate, where various types of tube-based PCR are used. Can be used. However, each of these configurations generally relies on pressing the plate down to obtain good uniform thermal contact as shown in FIG. 29A. In some embodiments of the present invention, the elastomeric tip is unacceptable for downward compression because the plastic valve and the elastomeric chamber are deformed causing undesirable fluid action within the elastomeric tip.

In other embodiments, the negative effect of squeezing the ICC downwards is achieved by using a vacuum chuck to fit against a thermal material that is otherwise coupled to the underside of the exposed elastomeric block. Reduced or avoided. In this manner, a tight seal is created between the vacuum chuck and the ICC thermal material when a vacuum is applied to the chuck. In one embodiment, as shown in FIG. 29B, the vacuum chuck 3950 includes or is one or more materials that are good thermal conductors coupled to a thermal control source (eg, one or more Peltier devices). Made from different materials. In one embodiment, a plurality of heat control is used to generate a thermal gradient between the reaction of the array.

In use, ICC is placed on the vacuum chuck 3950, it is moved in the case of or otherwise lowered below so as to contact the heat portion 3920 of the ICC 3930 comprising an integrated chip 3910 and the vacuum chuck. Again, in one embodiment, the thermal portion 3920 includes silicon. The thermal portion 3920 is drawn to connect to an elastomeric block or chip 3910, and such connection is accomplished using an adhesive or the like in some embodiments. As shown, block 3910 engages ICC 3930 and, in one embodiment, gap 3940 is maintained between thermal portion 3920 and ICC 3930. Gap 3940 assists in thermally isolating ICC 3930 from a thermal heat source (eg, chuck or platen 3950). Further, in one embodiment, gap 3940 allows some curvature of block 3910 and / or thermal portion 3920. In this manner, gap 3940 forms a seal when, in some embodiments, a vacuum is applied to chuck 3950 through one or more vacuum ports 3960 and thermal portion 3920 is pulled toward chuck 3950. To help. The amount of vacuum achieved can be monitored to confirm that good thermal contact has actually been created between the chuck 3950 and the thermal portion 3920. In some embodiments, one or more ports of the vacuum chuck are used to monitor the vacuum level. In certain embodiments, such as those shown in FIGS. 29D and 30, one or more ports used to monitor the level of vacuum are located distal to the vacuum port and pass through each other (preferably serpentine). Fluid communication through a narrow channel. In this manner, there is an attempt to reposition the ICC in the vacuum chuck, where the difference between one side of the vacuum chuck—the thermal part of the ICC is compared to the other side of the vacuum chuck—the thermal part of the ICC. In some cases, the problem of loss of vacuum can be solved. This is an iterative process that is performed either automatically or manually, or some combination thereof.

  FIG. 30 shows one embodiment of a chuck according to the present invention. The nature of the serpentine passages used to achieve an accurate measurement of the differential vacuum level over the vacuum chuck hot part area and to make a homogeneous or relatively homogeneous vacuum application to the hot part of the ICC. It can be changed within the range. By using this approach, the elastomeric block used to perform the PCR reaction can be made without compromising the functionality of the elastomeric (curvable) portion of the elastomeric block when normal downward compression is used. Good thermal contact with the PCR machine.

  Although the present invention has been described herein with reference to specific embodiments thereof, certain acceptable modifications, various changes and substitutions are contemplated in the above disclosure, and in some cases, It will be understood that some features of the invention may be utilized without a corresponding use of other features without departing from the scope of the invention as described above. For example, in addition to the pressure-based actuation system described above, any electrostatic and magnetic actuation system is also contemplated. It is also possible to operate the device by causing fluid flow in the control channel based on the application of thermal energy, either by thermal expansion or by generating a gas from the liquid. Furthermore, in another embodiment, centrifugal force is used to place proteins and reagents into the chip. Accordingly, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope and spirit of the invention. While the invention is not limited to the specific embodiments disclosed as the best mode contemplated for carrying out the invention, the invention includes all embodiments and equivalents falling within the scope of the appended claims Is intended to be included.

(reference)
1. Unger et al., Science 288, 113-116 (2000).

2. Sample channels and control lines are filled with “blind filling” —PDMS is fully gas permeable, liquid pressurized at a few psi exits the gas out of the channel, and the channel is completely filled with liquid Meet. See Hansen et al., PNAS 99,16531-16536 (2002).

3. The 294 bp segment of the human β-actin gene was amplified using a 5′-exonuclease assay (Taqman). The forward primer and reverse primer sequences were 5′-TCACCCACACTGTGCCCCATCTACGA3 ′ and 5′-CAGCGGAACCGCCTCATGCCAATGG3 ′, respectively. FIG. 1 b was obtained using the TAMRA-based FRET probe, sequence 5 ′-(FAM) ATGCCC-X (TAMRA) -CCCCCATGCCATCCTGGCGTp-3 ′. The data in FIG. 1c was acquired using a dark quencher-based probe. This is because many of these primer-probe sets are commercially available. The reaction was 1 × Taqman buffer A (50 mM KCl, 10 mM Tris-HCl, 0.01 M EDTA, 60 nM passive reference 1, pH 8.3), 4 mM MgCl ₂ , 200 nM dATP, dCTP, dTTP, 400 nM dUTP, 300 nM forward primer, 300 nM reverse primer, 200 nM probe, 0.01 U / μL Amperase UNG (all from Applied Biosystems, Foster City, Calif.), 0.2 U / μL Dynazyme (Finzyme, Espoo, Finland), 0.5% Triton-x-100, 0.8 μg / μl gelatin (Calbiochem, San Diego, Calif.), 5.0% glycerol, deionized water and human male genomic DNA (Pr mega) contained.

4). Quantitative PCR Tschnology, Chapter on “Gene Quantification”, LJ McBride, K Livak, M Lucero et al., Editor, Francois Ferre, Birkauser, Boston, MAp.

E. T.A. Lagally, I .; Medintz, R.M. A. Mathies, Anal Chem 73 (3), 565-570 (2001), and B.M. Vogelstein, K .; W. See Kinzler, PNAS 96, 9236-9241 (1999).

  The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes are suggested to one of ordinary skill in the art in view thereof, and the various modifications or changes are described herein. It is understood that the spirit and scope of the document should be encompassed within the scope of the appended claims and the appended claims. All publications, patents, and patent applications mentioned in this specification are intended to indicate that each individual publication, patent, or patent application is clearly and individually incorporated by reference. As such, it is incorporated by reference in its entirety for all purposes to the same extent.

FIG. 1A is a schematic diagram of an exemplary device for matrix design of fluid channels that intersect vertically or horizontally. 1B-1E show an enlarged view of a portion of the device shown in FIG. 1A and illustrate its operation. FIG. 1F is a schematic diagram of another exemplary matrix design device that uses protective channels to reduce sample evaporation. FIG. 2 is a plan view of an exemplary blind channel device. FIG. 3A is a plan view of another exemplary blind channel device. FIG. 3B is a schematic diagram of a more complex blind channel device based on the general design unit shown in FIG. 3A. FIG. 4 is a plan view of a device using a hybrid design. FIG. 5 is a graph showing rise and fall times for performing a thermal cycling reaction. FIG. 6 shows the position of the spotted reagent within the reaction site in a blind channel type device and illustrates the proper alignment of the reagent within the reaction site at the corner of the device. 7A and 7B are cross-sectional and schematic views, respectively, of another hybrid type microfluidic device, showing the type of device used to perform the experiments described in Examples 1-4. FIG. 8 shows a bar graph in which the average FAM / PR1 control ratio is plotted against six different β-actin Taqman reactions. The reaction was heat cycling definitive microfluidic device shown in FIG. 7B (chip) (solid bars) and Macro TaqMan reactions (hatched bars). Controls are first and fourth bars set without DNA. Error bars are the standard deviation of the ratio. FIG. 9 is a diagram illustrating an exemplary pin spotting process. Reagents are picked up from a source (eg, a microtiter plate) and then printed by contacting the filled pins with the substrate. The washing step consists of stirring with deionized water and then vacuum drying. FIG. 10 is a bar graph showing FAM signal intensity for the microfluidic device (chip) described in Example 1 (see FIG. 7B), based on the experiment described in Example 2. This data is in the form of (FAM signal / PR1 signal) scaled by the FAM / PR1 ratio of the reference lane. Error bars are the standard deviation of the lane. The notations “1.3 ×” and “1 ×” refer to the spotted primer and probe concentrations relative to their normal values. FIG. 11 is a bar graph showing the average VIC / PF1 / control ratio for 9-10 wells for Macro TaqMan (striped bars) and Tag Man reaction (solid bars) in microfluidic devices. Two samples containing two negative controls (control) and 100 pg / nl genomic DNA were thermocycled with the reaction components described above using a standard amount of 4 times primers / probes. Error bars indicate the standard deviation of the average ratio. FIG. 12 is a bar graph showing the FAM / PR1 / control ratio for each 10-1 nl well branching from a single flow channel of the microfluidic device (see FIG. 7B). The amount of genomic DNA is 0.25 pg / nl, which yields an average target of 1 copy per well. FIG. 13 is a bar graph showing the average VIC / PR1 / control ratio for the CYP2D6 SNP response using the microfluidic device shown in FIG. 7B. Allele 1 (Al-1) is a positive control for the VIC probe relative to the reference or wild type allele CYP2D6 ^* 1. Allele 2 (Al-2) is a positive control for the FAM probe for the variant or mutant allele CYP2D6 ^* 3. The control has no DNA template. Genomic DNA was used at either 100 pg / nl or 20 pg / nl. Error bars are the standard deviation of the ratio. FIG. 14 is a bar graph showing the average FAM / PR1 / control ratio for the CYP2D6 SNP response in the microfluidic device shown in FIG. 7B. The samples are the same as described with respect to FIG. 13 and in Example 3. FIG. 15 is a schematic view of a microfluidic device used for the experiment in Example 4. FIG. 16 is a polyacrylamide gel containing PCR products from Macro PCR and the PCR reaction in the microfluidic device shown in FIG. 7B. The results on the left show proper migration of various DNA base pair lengths. Lanes containing interspersed bands are molecular weight markers. Lanes labeled “Macro” are PCR products from macro reactions at different dilutions. The lane labeled “In Chip” is the PCR product generated in the chip. Lanes containing many bands throughout the gel are non-specific background signals. Figures 17a-17d show two preferred designs of partitioned microfluidic devices with the valve off and valve actuated. FIG. 18a shows an image of a partitioned microfluidic device after a thermal cycling reaction has been performed. FIG. 18a shows a two-color image. FIG. 18b shows an image of the partitioned microfluidic device after the thermal cycling reaction has been performed. FIG. 18b shows the signal remaining after subtraction of the control red signal. FIG. 19 shows a graph comparing the average number of copies per well against the number of positive wells. FIG. 20 shows SCORPION, an isothermal amplification scheme. FIG. 21 shows a top view of an exemplary matrix microfluidic device. FIG. 22A shows a substrate of a microfluidic device having an integrated pressure accumulating well according to an embodiment of the present invention. FIG. 22B is an exploded view of the microfluidic device shown in FIG. 8A and further comprising an elastomer block. FIG. 22C is an overall view of the microfluidic device shown in FIG. 22B. FIG. 22D is a plan view of the microfluidic device shown in FIG. 22B. FIG. 22E shows a cross-sectional view of the microfluidic device shown in FIG. 22B. FIG. 23 is a cross-sectional view of a hole for use in some embodiments of the microfluidic device of the present invention. FIG. 24 is a perspective view of a station for operating a microfluidic device according to the present invention. FIG. 25 is a cross-sectional view of a platen moved toward the top surface of a microfluidic device according to an embodiment of the present invention. FIG. 26A is a simplified overall view of a system according to an embodiment of the present invention. FIG. 26B is a perspective view of a receiving station in the system of FIG. 26A. FIG. 26C is a plan view of the back of the fluid path in a plate interface or platen according to another embodiment of the present invention. FIG. 27A is a side cross-sectional view showing an interface plate fitted to a carrier according to an embodiment of the present invention. FIG. 27B is a side cross-sectional view showing an interface plate fitted to a carrier according to an embodiment of the present invention. FIG. 28A is a perspective view of an integrated carrier according to an embodiment of the present invention. FIG. 28B is a perspective view of an integrated carrier according to another embodiment of the present invention for use with PCR. FIG. 29A is a simplified cross-sectional view of a system for holding the carrier of FIG. 28A. FIG. 29B is a simplified cross-sectional view of a system for holding the carrier of FIG. 28B. FIG. 29C is a simplified plan view of a portion of the carrier of FIG. 28B. FIG. 29D is a simplified top view of a vacuum chuck for use with the system of FIG. 29B. FIG. 30 is a simplified overall view of a vacuum chuck for use with the system of FIG. 28B.

Claims (11)

A method of reacting over time at a selected temperature or temperature range, the method comprising:
Providing an array device for housing a plurality of separate reaction chambers, the array device comprising an elastomeric block formed from a plurality of layers, wherein at least one layer is within the layers; At least one recess formed in the substrate, the recess having at least one bendable membrane integral with the layer comprising the recess, the array device further comprising a heat transfer device, The heat transfer device is a hard thermally conductive plate, coupled to the elastomer block and configured to contact a thermal control source;
Providing a thermal control device including a thermal control source;
Introducing into the array device a reagent for performing a desired reaction; and
As the heat transfer device is communicated with the thermal heat control source, the array device comprising the steps of contacting a thermal control device, as a result, the temperature of the reaction in at least one of said reaction chamber, Changing as a result of a change in temperature of the thermal control source,
Encompasses, heat control devices are added to force the heat transfer device adapted to heat-transfer device to move toward the heat control source, method. The method of claim 1, wherein the heat transfer device comprises a light reflective material. Wherein Ru rigid silicon der thermal conductivity of the plate is polished, the method according to claim 2. The method of claim 1 , 2 or 3 , wherein the force comprises a magnetic force, an electrostatic force, or a vacuum. The method of claim 4 , wherein the force comprises a vacuum applied to the heat transfer device through a channel formed in the surface of the heat control device or the heat transfer device. 6. The method of claim 5 , further comprising detecting the level of vacuum achieved between the surface of the thermal control device and the surface of the heat transfer device. If the vacuum level does not exceed a predetermined level, a warning appears or a readjustment protocol is engaged, where the readjustment protocol removes the vacuum and controls the array device to the thermal control. 7. The method of claim 6 , comprising repositioning with respect to the source . The method of claim 1, wherein contacting the array device with the thermal control device is performed by using one or more mechanical or electromechanical positioning devices. The method of claim 1, further comprising automatically controlling and monitoring execution of the method. The step of automatically controlling and monitoring the execution of the method is operatively communicated to a robotic control system for introducing the array device into the thermal control device and for removing the array device from the thermal control device. The method of claim 9 , wherein the method is implemented by an automated control system. The method of claim 1, further comprising monitoring the progress of the reaction.

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