Patent Grant
7815868
Microtiter plates have become a standard tool in chemistry, biology and medical laboratories. The plates are typically flat glass or plastic trays in which an array of circular reagent wells are formed. Each well can typically hold between from a few microliters to hundreds of microliters of fluid reagents and samples, which may be loaded into the wells with automated delivery equipment. Plate readers are used to detect biological, chemical and/or physical events in the fluids placed in each well.
As the fields of combinatorial chemistry and high throughput screening have grown, so has equipment and laboratory instrumentation that has been designed to fill, manipulate and read microtiter plates. Unfortunately, the equipment makers made little effort develop systems that were cross-compatible with the systems of other manufacturers. By the mid-1990s, the Society for Biomolecular Screening (SBS) formed a standards group to address these cross-compatibility problems. A final set of standards was published by SBS and the American National Standards Institute 2003.
These standards define the overall dimensions of a compliant microtiter plate, as well as the diameter, depth and spacing of the reagent wells in the plate. The plates may include 96, 384, 1536, etc., wells arranged in a 2:3 rectangular matrix. While some manufacturers have made plates packing even larger numbers of reagent wells into the dimensions of an SBS-formatted plate, the small-sizes of the wells can make filling and reading the plates more difficult. Thus, there is a need for devices, systems and methods that can rapidly and accurately deliver small volumes of samples and reagents to reaction sites in high throughput microtiter plates. There is also a need for devices, systems and methods that provide monitoring, detecting and reading of reactions performed at the reaction sites of such microtiter plates. There is also a need to design such microtiter plates to SBS compatible standards, so they can take advantage of the large amount of SBS-formatted equipment and instrumentation that is currently in use. These and other needs are addressed by the present invention.
Embodiments of the invention include SBS-formatted microfluidic devices that include a plate having an upper surface and a lower surface, where the geometry of the plate defines an array of interrogation areas. The perimeter of each interrogation area defines a region on the plate where SBS-compatible equipment can measure and/or influence reactions taking place on the plate. Typically, the interrogation areas have the same size and position as the reaction wells in a conventional SBS-formatted microtiter plate. Each interrogation area may encompass one or more reaction sites. These reaction sites may be sealable chambers in which reactants, samples, etc., may be brought into contact and reacted. The reaction sites may be isolated from each other, allowing multiple different reactions to take place at the same time in the same interrogation area. The reaction sites may lie beside each other within an interrogation area and be defined by the presence of one or more reactants or analytes that have been deposited upon a specific area on the substrate surface of the plate.
The devices may also include one or more elastomeric layers, generally positioned upon a rigid substrate surface, wherein a plurality of channels is disposed within the elastomeric layers or is defined at the interface between the elastomeric layers or at the interface of an elastomeric layer and the rigid substrate surface. In certain embodiments, a first elastomeric layer in contact with the upper surface of the plate, where the first elastomeric layer comprises a network of microfluidic flow channels formed therein in fluid communication with an array of reaction sites. The devices may further include a second elastomeric layer in contact with the first elastomeric layer, where the second elastomeric layer includes a network of control channels formed therein, configured to control the flow of fluids within the flow channels.
It should be noted that all reference to relative positions of the plate and elastomeric layers, such as “upper surface” and “lower surface” are provided solely for the purpose of conceptual convenience, and are not meant to restrict the invention to such arrangements. The devices of the invention may be made with the plate either above or below the elastomeric layers or in any other arrangement such that the elements provide a functioning microfluidic system.
Embodiments of the invention may also include systems of SBS-formatted microfluidic devices that can process at least one flowable reagent. The reagent may include any liquid, and may include a dye or other reagent detectable by optical or other sensors. The systems may also be compatible with machines used to read the device or to monitor a reaction performed upon the device. The machines may be SBS compatible and able to accept, use, manipulate, and read an SBS-formatted plate. In addition, the systems may include machines for dispensing and/or pumping fluids into the microfluidic devices. These dispensing and pumping machines may also be SBS compatible and able to accept an SBS-formatted plate.
Embodiments may still further include systems to manipulate, read and/or monitor one or more reactions performed upon or within a SBS-formatted microfluidic device. The systems may include a SBS-formatted microfluidic device and a machine for reading, monitoring and/or manipulating a reaction performed upon or within the SBS-formatted microfluidic device.
Embodiments of the invention may still also include processes for conducting microfluidic high throughput sample reaction measurements. The processes may include the step of providing a microfluidic testing device comprising a reaction plate that has an array of reaction sites, such as within one or more wells. In certain embodiments, each well defines a single reaction site for the performance of a single reaction. In additional embodiments each well has at least two reaction sites, allowing two or more reactions to be performed in a single well. The reaction sites may be physically defined by a reaction chamber. The reaction chambers may be coupled to reagent flow channels and sample flow channels that are formed in a first elastomeric layer adjacent to the reaction plate. The processes may also include loading a set of reagents into an array of reagent reservoirs, and loading a set of samples into an array of sample reservoirs of the microfluidic testing device. Each of the reagent reservoirs is fluidly coupled to one of the reagent flow channels, and each of the sample reservoirs is fluidly coupled to one of the sample flow channels. The processes may further include transporting at least one of the reagents through at least one of the reagent flow channels to a designated reaction chamber and at least one of the samples through at least one of the sample flow channels to the designated reaction chamber. The control channels control the flow of the reagent and the sample through the flow channels to the designated reaction chamber, and are formed in a second elastomeric layer adjacent to the first elastomeric layer. The processes may still further include the step of measuring a reaction response from the reagent and the sample in the designated reaction chamber.
In certain embodiments, two or more reaction sites may be present within a single interrogation site. The interrogation site may be defined, for example, as an imaging area upon an SBS-formatted chip, which is imaged by a single lens at a single set position. This interrogation area may have defined within it any number of reaction sites. Any or all of these reaction sites may be imaged simultaneously by a lens positioned above the reaction site. A single image of a single interrogation area captured by a single lens may contain information pertaining to any number of reaction sites within the imaged interrogation area. The information associated with each reaction site may be parsed and differentiated from the information associated with any other reaction site in a number of ways.
For example, a device having four reaction sites within a single reaction area (e.g.,
Embodiments of the invention still also include systems having lens to focus light on the reaction sites in interrogation areas.
Additional embodiments and features are set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the specification or may be learned by the practice of the invention.
Devices, systems and processes are described that integrate microfluidic sample delivery and reaction technology with existing laboratory devices and methods that may be used for performing high throughput testing of chemical and biological samples, and for carrying out chemical and biological reactions, especially those employing small liquid volumes. These devices and systems may include fluorescent plate readers, real-time PCR machines, robotic plate handlers, and pipetting robots, and devices designed to load, manipulate and read elastomeric microfluidic devices, among other types of equipment. The devices, systems and methods of the invention are compatible with one or more standards that allow them to easily interface with existing laboratory equipment. For example, the components may conform to the widely adopted SBS format for automated laboratory instrumentation.
The devices and systems of the invention may include arrays of reaction reservoirs that are formatted for compatibility with automated reactant loading equipment (e.g., pipetting robots) that already exist and are in common usage in laboratories and manufacturing facilities. The devices and systems may also include arrays of reaction product reservoirs that receive reaction products from reactions chambers where selected combinations of the reactants were combined. The reaction product reservoirs may also be formatted for compatibility with pre-existing automated sample extraction equipment to transport reaction product samples to analysis equipment, storage containers, etc.
Integrating microfluidic sample delivery technology with high throughput testing equipment combines advantages from both fields. Microfluidic systems have fewer moving parts and simpler operational logistics than robotic fluid delivery systems. In general, the microfluidic systems cost less to manufacture and require less maintenance and repair. In addition, microfluidic systems can be manufactured with smaller sized conduits and chambers, allowing them to deliver smaller volumes of samples, reagents, etc., than practicable with, for example, pipetting robots. This can reduce the costs and waste products generated for large screening studies involving thousands or more combinations of reagents and samples. The small volumes can also make screening and combinatorial studies practical when only a small amount of a sample is available.
Smaller component dimensions also permit more densely packed arrangements of the reaction sites. For example, two, four, eight, or more microfluidic reaction chambers (each defining a reaction site) may be packed into the interrogation area of a single site for a standardized high throughput screening device. This can allow the microfluidic device to achieve a twofold, fourfold, eightfold, or more, increase in the throughput rate using an existing screening device.
Microfluidic Device Overview
As
The reservoir arrays 110a-b may be filled with reagents, reactants, samples, reaction products, etc., in any configuration. For example, one of the reservoir arrays (e.g., array 110a) may be filed with reagents, and the second array 110b may be filled with samples. Individual combinations of the reagents and samples may be combined at the reaction sites in array 102 and monitored by SBS-formatted measuring equipment that defines an interrogation area 108 each of the reaction sites. In another example, one of the reservoir arrays (e.g., array 110a) may hold reactants, and the second array 110b may receive reaction products from the reactions that take place in the reaction sites of array 102. The reservoir arrays 110a-b may also be subdivided into sub-arrays and/or at the level of individual reservoirs into different groupings of fluids.
The space around the perimeter of plate 100 occupied by the reservoir arrays 110a-b, channels that supply fluid to the array 102, pumping equipment, etc., may take up space that would otherwise be occupied by wells in a conventional microtiter plate. This may result in fewer interrogation areas 108 in plate 100 than wells in a conventional plate (which can define the size and position of the interrogation areas 108). For example, the reaction site array 102 of plate 100 shown in
For example, if just 2 reaction sites 106 were packed into each interrogation area 108 (i.e., a 2:1 ratio of reaction sites to interrogation areas) the total number of reaction sites would be back up to 384 (i.e., equal to the number of wells in a conventional 384 well SBS microtiter plate). Using microfluidics production technology, arrays of reaction sites can be constructed with 2, 3, 4, 6, 8, 10, 11, 12, 20, or more reaction sites overlapping each SBS-formatted interrogation area. This results in throughputs that are double, triple, quadruple, etc., those of a conventional SBS-formatted microtiter plate.
For example,
The 2×2 unit cells of reaction sites 206a-d encompassed by the interrogation areas 208 can boost the efficiency of SBS-formatted equipment a number of ways. For example, SBS-formatted screening equipment can monitor sequential reactions that would be run at each of the reaction sites 206a-d and then read. This may start with a sample and reagent being introduced to the first reaction site 206a and reading the results (e.g., a change in absorbance or fluorescence caused by a product species). The first site 206a may be bleached or washed after the run. Then, a second sample and reagent may be introduced to the second reaction site 206b while reading the results of this combination. The process may then be repeated for the third site 206c and, finally, the fourth reaction site 206d.
The plate has 192 interrogation areas that each have 4 reaction sites, allowing a total of 768 reactions to be run on the plate, either simultaneously or sequentially. This is twice the number of reactions possible using a conventional 384 well microtiter plate. Still more reactions are possible by packing 5, 6, 7, 8, 9, 10, etc., reaction sites 206 into each of the interrogation areas 208.
Additional methods include running reactions in all four reaction sites 206a-d simultaneously instead of sequentially. For example, reagents may be labeled with different colored dyes in each of the reaction sites 206a-d that absorb and/or fluoresce light of different colored wavelengths. Spectrally discriminating detection equipment that monitors each wavelength independently can detect changes in all four reaction sites at the same time. These multiple color reactions can significantly increase the throughput rate of for an SBS-formatted reaction plate by running two, three, four, five, etc., times the number of reactions in the same period of time as a conventional 384 well microtiter plate.
There are also methods and processes for using reaction sites 206 to make reaction product samples that can be transported to product sample reservoirs in the reservoir arrays 110 on plate 100. SBS-formatted equipment (e.g., pipetting robots) can be programmed to automatically extract the product samples from the reservoirs and place them in storage containers or instrumentation for additional chemical and/or biological analysis.
Embodiments of these methods may also include multi-stage reactions and syntheses where reaction products from one reaction site are actually intermediates in a more complex reaction scheme. In these methods, a first reaction product formed from reactants in a first reaction site 206 are combined with additional reagents and/or reaction products from a second reaction site. The products made by reacting the first reaction product with the additional reactants and/or reaction products may be the final reaction products that are transported to the product sample reservoir. The methods may include even more complexity by forming a plurality of reaction product intermediates in multiple reaction sites 206 that are combined at various stages leading to a final product or group of products.
At each reaction site, SBS-formatted equipment may be used to monitor and/or influence the reactions taking place in the reaction sites 206. For example, spectral analysis equipment may be used to monitor the consumption or production of chemical species in a reaction site 206. Equipment may also be used to apply localized heating and/or light exposure (e.g., UV light exposure) to influence the types of reaction products that are generated.
Further methods include using the reaction sites 206 in high-throughput molecular genetics analyses. For example, SBS-formatted plate 100 may be used with a standard real-time PCR instrument to perform an automated DNA analysis of single nucleotide polymorphisms (SNPs) from one or more samples of genetic material. Multiple SNP reactions (e.g., SNP restriction fragment length polymorphism) may be performed in reaction sites 206 and the reaction products amplified by polymerase chain reaction (PCR). The PCR product samples may then be transported to product reservoirs 110 where automated DNA analysis equipment can extract the samples and run them through electrophoresis equipment (e.g., gel or capillary electrophoresis equipment).
Fluids may be supplied via supply channels (not shown) to the reaction sites 106 in the reaction site array 102 from the reservoir arrays 110a-b. The reservoir arrays 110a-b may be filed with samples and reagents that are delivered in various combinations to the reaction sites 106. As shown in
By selectively actuating the control channels 414, specific combinations of fluid samples from the fluid reservoir array (not shown) may be added to individual reaction sites 406. For example, a first reservoir array may hold M samples and a second reservoir array may hold N reagents. Selective actuation of the control channels 414 may be done to deliver combinations of each M sample and N reagent to one of the N×M discrete reaction sites 406. Measurements may then be taken to determine how each combination of sample and reagent react.
Exemplary Device Optics
Optical lenses may be used to position and concentrate light on the reactions sites in the microfluidic plates. This can increase the signal sensitivity and/or reduce the cross-talk in spectroscopic detection equipment measuring spectral events (e.g., absorption spectroscopy, fluorescence spectroscopy, etc.). The lenses can also concentrate light that may be used to enhance photochemical reactions in a selected reaction site.
The lenses may be integral to the microfluidic plate, for example, present within the elastomeric structure of the device. An integrated lens may be formed from any material that has an index of refraction that is different from the surrounding material. In certain embodiments, the lens is provided by molding a generally bi-convex-shaped void into the structure of the elastomeric material. Air or another fluid is pumped into the void such that it inflates to provide a pre-determined bi-convex shape within the elastomeric material that acts as a lens. Alternatively, the space may be evacuated to cause the surface layer to recess into the plate and form a concave lens. Adjustments in the pressure may be made to adjust the curvature and optical properties of the lenses, such as the focal point. The integral lenses may also be used with external lenses to form compound lenses. In certain embodiments, two or more integral lenses are provided to provide a multi-lens system within the device. Integral lenses are generally positioned within the device above a reaction site so as to provide optical magnification of the reaction site. In certain embodiments, the integrated lens is inflated with a gas, such as air, and in additional embodiments, the lens may be inflated using a liquid such as water, saline, an oil, an alcohol, a polyol, an isobutyrate, etc. The inflating fluid may be selected to provide the desired optical (e.g., light focusing) properties.
As shown in
Additional types of lenses (not shown) may also be used to control the position and concentration of light impinging on the microfluidics plate. For example, plano-covex lenses, convex-concave lenses, meniscus lenses, plano-concave lenses, and fresnel lenses may also be used with the invention. The lenses may be used alone (e.g., a simple lens) or in combination (e.g., a compound lens) to control the focus of the light impinging on, or emitted from, the microfluidics plate.
The SBS Dimensional Standards for Microplates
The Society for Biomolecular Screening (“SBS”) has developed formatting standards for microplates used in high throughput screening processes for biological and chemical compounds. These automated processes included the use of robot pipetteing to transfer fluid samples to an array of reaction wells formed in the microplate. Detection equipment was aligned with the wells to observe and measure events (e.g., chemical reactions, enzymatic catalysis, crystallizations, etc.). As the number of vendors and systems proliferated, standards were clearly needed to address compatibility problems. SBS developed dimensional standards for microplates that are followed by a significant number of microplate manufacturers and instrument makers that utilize microplates.
SBS has defined dimensional standards for 96, 384, and 1536 well microplates. In each case, the microplate has a rectangular shape that measures 127.76 mm±0.5 mm in length by 85.48 mm±0.5 mm in width. The four corners of the plate are rounded with a corner radius to the outside of 3.18±1.6 mm. The complete definitions for these standards were published by the American National Standards Institute on Mar. 28, 2005, in publications ANSI/SBS 1-2004, ANSI/SBS 2-2004; ANSI/SBS 3-2004; and ANSI/SBS 4-2004, the entire contents of which are herein incorporated by reference for all purposes. A summary of the definitions for 96, 384 and 1536 well plates are provided here:
The 96 Well Format
The 384 Well Format
The 1536 Well Format
Carry-On Mixing and Metering in the SBS Formatted Devices
This invention further relates to microfluidic devices and methods of using the devices that provide precise metering of fluid volumes and efficient mixing of the metered volumes. The devices are useful for analytical assays for research or diagnostic purposes where high density, high throughput, sample parsimony, and lower cost are desired. The devices and methods are also useful as tools for the synthesis, sorting, and storage of high value chemical and biological entities. The application of microfluidic devices with high sensitivity and reproducibility may require the carry-on mixing and metering approach of the present invention in order to achieve practical and enhanced sensitivity and dynamic range to be of practical value in a research setting.
The microfluidic devices of the present invention may also utilize a configuration in which at least one solution is metered into a segment of a flow channel, typically through a junction disposed between valves along the flow channel. The junction typically has an on/off valve or a one way check valve at the inlet portion of the junction. The flow channel valves that bracket the junction are closed and the junction inlet valve is opened. A solution is instilled into the segment of the flow channel. The filling of the segment is preferably performed by “blind filling” the segment. Blind filling takes advantage of the permeability of the material defining at least one side of the flow channel to gases and not to liquid. The first solution is filled into the flow channel segment by placing the solution under pressure at the junction and allowing the first solution to fill the segment as the gases that are present in the flow channel diffuses out through the gas permeable material. Once the segment of the flow channel defined by the valves is filled, the inlet junction is closed or allowed to close and a precisely defined volume is contained within the flow channel segment. The exact volume is determined by the flow channel dimensions and the spacing of the valves along the segment that are closed to define the blind filled portion of the flow channel. With the valves remaining closed, a second solution is introduced into an empty portion of the flow channel by blind filling against one of the closed valves. By maintaining the second solution under pressure and then opening the valves on the flow channel segment, the second solution pushes the first solution through the flow channel. In a preferred embodiment, the flow channel segment valve opposite the valve against which the second solution is blind filled, is adjacent to a reaction well of a defined volume. When the flow channel segment valves are opened, the second solution pushes the first solution into the reaction well. By fabricating the reaction well such that the reaction well volume is greater that the volume of the first solution in the flow channel, a precisely defined amount of the second solution is pushed into the reaction well along with the known volume of the first solution. The volume of the second solution that fills the reaction well is defined by the volume of the reaction well minus the volume of the first solution. As both solutions fill the reaction well, mixing of the solutions occurs. The mixing of the solutions is typically efficient and is greater that 25% efficient, preferably greater than 35% efficient, more preferably greater than 50% efficient, more preferably greater that 65% efficient, more preferably greater than 75% efficient, more preferably greater than 85% efficient, more preferably greater than 90% efficient, more preferably greater than 95% efficient, more preferably greater that 99% efficient, and more preferably about 100% efficient. In one embodiment, the reaction well has only the flow channel entering the chamber defined by its volume. In another embodiment, the reaction well has a separate opening for an additional inlet or outlet channel. In an additional embodiment, the reaction well has a plurality of openings for other channels in addition to the described flow channel.
Various terms such as “pulse-chase,” “carry-on,” slug mixing,” pig mixing,” and “bolus mixing” may be used to describe the described mixing technique.
Microfluidic Chip Construction
Preferred Layer, Channel, and Reaction Well Dimensions:
Microfabricated refers to the size of features of an elastomeric structure fabricated in accordance with an embodiment of the present invention. In general, variation in at least one dimension of microfabricated structures is controlled to the micron level, with at least one dimension being microscopic (i.e. below 1000 μm). Microfabrication typically involves semiconductor or MEMS fabrication techniques such as photolithography and spincoating that are designed for to produce feature dimensions on the microscopic level, with at least some of the dimension of the microfabricated structure requiring a microscope to reasonably resolve/image the structure.
In preferred aspects, flow channels preferably have width-to-depth ratios of about 10:1. A non-exclusive list of other ranges of width-to-depth ratios in accordance with embodiments of the present invention is 0.1:1 to 100:1, more preferably 1:1 to 50:1, more preferably 2:1 to 20:1, and most preferably 3:1 to 15:1. In an exemplary aspect, flow channels have widths of about 1 to 1000 microns. A non-exclusive list of other ranges of widths of flow channels in accordance with embodiments of the present invention is 0.01 to 1000 microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to 500 microns, more preferably 1 to 250 microns, and most preferably 10 to 200 microns. Exemplary channel widths include 0.1 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, and 250 μm.
Flow channels have depths of about 1 to 100 microns. A non-exclusive list of other ranges of depths of flow channels in accordance with embodiments of the present invention is 0.01 to 1000 microns, more preferably 0.05 to 500 microns, more preferably 0.2 to 250 microns, and more preferably 1 to 100 microns, more preferably 2 to 20 microns, and most preferably 5 to 10 microns. Exemplary channel depths include including 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, and 250 μm.
Elastomeric layers may be cast thick for mechanical stability. In an exemplary embodiment, a layer is 50 microns to over a centimeter thick, and more preferably approximately 4 mm thick. A non-exclusive list of ranges of thickness of the elastomer layer in accordance with other embodiments of the present invention is between about 0.1 micron to 1 cm, 1 micron to 1 cm, 10 microns to 0.5 cm, 100 microns to 10 mm.
Accordingly, membranes separating flow channels have a typical thickness of between about 0.01 and 1000 microns, more preferably 0.05 to 500 microns, more preferably 0.2 to 250, more preferably 1 to 100 microns, more preferably 2 to 50 microns, and most preferably 5 to 40 microns. Exemplary membrane thicknesses include 0.01 μm, 0.02 μm, 0.03 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 750 μm, and 1000 μm.
Furthermore, embodiments of the present invention provide reduced reaction volumes. In embodiments of the present invention, reaction volumes ranging from 10 picoliters to 100 nanoliters are utilized. In some embodiments, reaction volumes greater than 100 nanoliters are utilized. Reaction wells may also be in the microliter, nanoliter, picoliter, femtoliter or lower range of volume. In one embodiment, the reaction well volume is between 1-1000 femtoliters. Merely by way of example, in an embodiment, the methods and systems of the present invention are utilized with reaction volumes of 10 picoliters, 50 picoliters, 100 picoliters, 250 picoliters, 500 picoliters, and 1 nanoliter. In alternative embodiments, reaction volumes of 2 nanoliters, 5 nanoliters, 10 nanoliters, 20 nanoliters, 30 nanoliters, 40 nanoliters, 50 nanoliters, 75 nanoliters, and 100 nanoliters are utilized. In another embodiment, the reaction well volume is between 1-1000 picoliters. In another embodiment, the reaction well volume is between 0.01-100 nanoliters, preferably between 1-75 nanoliters. In one embodiment the reaction well volume is about 50 nanoliters. In one embodiment the reaction well volume is about 7.6 nanoliters. In another embodiment, the reaction well volume is 6 nL. The volume defined for the first solution in the flow channel (the slug volume or carry-on volume) is a fraction of the reaction well volume. In various embodiments, the fraction may be ⅞, ¾, ⅝, ½, ⅜, ¼, ⅕, ⅛, 1/10, 1/12, 1/20, 1/25, 1/50, 1/100, or less of the total reaction well volume.
Multilayer Soft Lithography Construction Techniques and Materials:
Soft Lithographic Bonding:
Preferably, elastomeric layers are bonded together chemically, using chemistry that is intrinsic to the polymers comprising the patterned elastomer layers. Most preferably, the bonding comprises two component “addition cure” bonding.
In one aspect, the various layers of elastomer are bound together in a heterogenous bonding in which the layers have a different chemistry. Alternatively, a homogenous bonding may be used in which all layers would be of the same chemistry. Thirdly, the respective elastomer layers may optionally be glued together by an adhesive instead. In a fourth aspect, the elastomeric layers may be thermoset elastomers bonded together by heating.
In one aspect of homogeneous bonding, the elastomeric layers are composed of the same elastomer material, with the same chemical entity in one layer reacting with the same chemical entity in the other layer to bond the layers together. In one embodiment, bonding between polymer chains of like elastomer layers may result from activation of a crosslinking agent due to light, heat, or chemical reaction with a separate chemical species.
Alternatively in a heterogeneous aspect, the elastomeric layers are composed of different elastomeric materials, with a first chemical entity in one layer reacting with a second chemical entity in another layer. In one exemplary heterogenous aspect, the bonding process used to bind respective elastomeric layers together may comprise bonding together two layers of RTV 615 silicone. RTV 615 silicone is a two-part addition-cure silicone rubber. Part A contains vinyl groups and catalyst; part B contains silicon hydride (Si—H) groups. The conventional ratio for RTV 615 is 10A:1B. For bonding, one layer may be made with 30A:1B (i.e. excess vinyl groups) and the other with 3A:1B (i.e. excess Si—H groups). Each layer is cured separately. When the two layers are brought into contact and heated at elevated temperature, they bond irreversibly forming a monolithic elastomeric substrate.
Alternatively, other bonding methods may be used, including activating the elastomer surface, for example by plasma exposure, so that the elastomer layers/substrate will bond when placed in contact. For example, one possible approach to bonding together elastomer layers composed of the same material is set forth by Duffy et al, “Rapid Prototyping of Microfluidic Systems in Poly (dimethylsiloxane)”, Analytical Chemistry (1998), 70, 4974-4984, incorporated herein by reference. This paper discusses that exposing polydimethylsiloxane (PDMS) layers to oxygen plasma causes oxidation of the surface, with irreversible bonding occurring when the two oxidized layers are placed into contact.
Yet another approach to bonding together successive layers of elastomer is to utilize the adhesive properties of uncured elastomer. Specifically, a thin layer of uncured elastomer such as RTV 615 is applied on top of a first cured elastomeric layer. Next, a second cured elastomeric layer is placed on top of the uncured elastomeric layer. The thin middle layer of uncured elastomer is then cured to produce a monolithic elastomeric structure. Alternatively, uncured elastomer can be applied to the bottom of a first cured elastomer layer, with the first cured elastomer layer placed on top of a second cured elastomer layer. Curing the middle thin elastomer layer again results in formation of a monolithic elastomeric structure.
Elastomeric layers may be created by spin-coating an RTV mixture on microfabricated mold at 2000 rpm for 30 seconds yielding a thickness of approximately 40 microns. Additional elastomeric layers may be created by spin-coating an RTV mixture on microfabricated mold. Both layers may be separately baked or cured at about 80° C. for 1.5 hours. The additional elastomeric layer may be bonded onto first elastomeric layer at about 80° C. for about 1.5 hours.
Suitable Elastomeric Materials:
Allcock et al, Contemporary Polymer Chemistry, 2nd Ed. describes elastomers in general as polymers existing at a temperature between their glass transition temperature and liquefaction temperature. Elastomeric materials exhibit elastic properties because the polymer chains readily undergo torsional motion to permit uncoiling of the backbone chains in response to a force, with the backbone chains recoiling to assume the prior shape in the absence of the force. In general, elastomers deform when force is applied, but then return to their original shape when the force is removed. The elasticity exhibited by elastomeric materials may be characterized by a Young's modulus.
The systems of the present invention may be fabricated from a wide variety of elastomers. In an exemplary aspect, elastomeric layers may preferably be fabricated from silicone rubber. However, other suitable elastomers may also be used.
In an exemplary aspect of the present invention, the present systems are fabricated from an elastomeric polymer such as GE RTV 615 (formulation), a vinyl-silane crosslinked (type) silicone elastomer (family). However, the present systems are not limited to this one formulation, type or even this family of polymer; rather, nearly any elastomeric polymer is suitable. An important requirement for the preferred method of fabrication of the present microvalves is the ability to bond multiple layers of elastomers together. In the case of multilayer soft lithography, layers of elastomer are cured separately and then bonded together. This scheme requires that cured layers possess sufficient reactivity to bond together. Either the layers may be of the same type, and are capable of bonding to themselves, or they may be of two different types, and are capable of bonding to each other. Other possibilities include the use an adhesive between layers and the use of thermoset elastomers.
Given the tremendous diversity of polymer chemistries, precursors, synthetic methods, reaction conditions, and potential additives, there are a huge number of possible elastomer systems that could be used to make monolithic elastomeric microvalves and pumps. Variations in the materials used will most likely be driven by the need for particular material properties, i.e. solvent resistance, stiffness, gas permeability, or temperature stability.
There are many, many types of elastomeric polymers. A brief description of the most common classes of elastomers is presented here, with the intent of showing that even with relatively “standard” polymers, many possibilities for bonding exist. Common elastomeric polymers include polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), the polyurethanes, and silicones.
Polyisoprene, Polybutadiene, Polychloroprene:
Polyisoprene, polybutadiene, and polychloroprene are all polymerized from diene monomers, and therefore have one double bond per monomer when polymerized. This double bond allows the polymers to be converted to elastomers by vulcanization (essentially, sulfur is used to form crosslinks between the double bonds by heating). This would easily allow homogeneous multilayer soft lithography by incomplete vulcanization of the layers to be bonded; photoresist encapsulation would be possible by a similar mechanism.
Polyisobutylene:
Pure polyisobutylene has no double bonds, but is crosslinked to use as an elastomer by including a small amount (≈1%) of isoprene in the polymerization. The isoprene monomers give pendant double bonds on the polyisobutylene backbone, which may then be vulcanized as above.
Poly(styrene-butadiene-styrene):
Poly(styrene-butadiene-styrene) is produced by living anionic polymerization (that is, there is no natural chain-terminating step in the reaction), so “live” polymer ends can exist in the cured polymer. This makes it a natural candidate for the present photoresist encapsulation system (where there will be plenty of unreacted monomer in the liquid layer poured on top of the cured layer). Incomplete curing would allow homogeneous multilayer soft lithography (A to A bonding). The chemistry also facilitates making one layer with extra butadiene (“A”) and coupling agent and the other layer (“B”) with a butadiene deficit (for heterogeneous multilayer soft lithography). SBS is a “thermoset elastomer”, meaning that above a certain temperature it melts and becomes plastic (as opposed to elastic); reducing the temperature yields the elastomer again. Thus, layers can be bonded together by heating.
Polyurethanes:
Polyurethanes are produced from di-isocyanates (A—A) and di-alcohols or di-amines (B—B); since there are a large variety of di-isocyanates and di-alcohols/amines, the number of different types of polyurethanes is huge. The A vs. B nature of the polymers, however, would make them useful for heterogeneous multilayer soft lithography just as RTV 615 is: by using excess A—A in one layer and excess B—B in the other layer.
Silicones:
Silicone polymers probably have the greatest structural variety, and almost certainly have the greatest number of commercially available formulations. The vinyl-to-(Si—H) crosslinking of RTV 615 (which allows both heterogeneous multilayer soft lithography and photoresist encapsulation) has already been discussed, but this is only one of several crosslinking methods used in silicone polymer chemistry.
Cross Linking Agents:
In addition to the use of the simple “pure” polymers discussed above, crosslinking agents may be added. Some agents (like the monomers bearing pendant double bonds for vulcanization) are suitable for allowing homogeneous (A to A) multilayer soft lithography or photoresist encapsulation; in such an approach the same agent is incorporated into both elastomer layers. Complementary agents (i.e. one monomer bearing a pendant double bond, and another bearing a pendant Si—H group) are suitable for heterogeneous (A to B) multilayer soft lithography. In this approach complementary agents are added to adjacent layers.
Other Materials:
In addition, polymers incorporating materials such as chlorosilanes or methyl-, ethyl-, and phenylsilanes, and polydimethylsiloxane (PDMS) such as Dow Chemical Corp. Sylgard 182, 184 or 186, or aliphatic urethane diacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCB Chemical may also be used.
The following is a non-exclusive list of elastomeric materials which may be utilized in connection with the present invention: polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), the polyurethanes, and silicone polymers; or poly(bis(fluoroaLkoxy)phosphazene) (PNF, Eypel-F), poly(carborane-siloxanes) (Dexsil), poly(acrylonitrile-butadiene) (nitrile rubber), poly(1-butene), poly(chlorotrifluoroethylene-vinylidene fluoride) copolymers (Kel-F), poly(ethyl vinyl ether), poly(vinylidene fluoride), poly(vinylidene fluoride-hexafluoropropylene) copolymer (Viton), elastomeric compositions of polyvinylchloride (PVC), polysulfone, polycarbonate, polymethylmethacrylate (PMMA), and polytertrafluoroethylene (Teflon).
Doping and Dilution:
Elastomers may also be “doped” with uncrosslinkable polymer chains of the same class. For instance RTV 615 may be diluted with GE SF96-50 Silicone Fluid. This serves to reduce the viscosity of the uncured elastomer and reduces the Young's modulus of the cured elastomer. Essentially, the crosslink-capable polymer chains are spread further apart by the addition of “inert” polymer chains, so this is called “dilution”. RTV 615 cures at up to 90% dilution, with a dramatic reduction in Young's modulus.
Other examples of doping of elastomer material may include the introduction of electrically conducting or magnetic species, as described in detail below in conjunction with alternative methods of actuating the membrane of the device. Should it be desired, doping with fine particles of material having an index of refraction different than the elastomeric material (i.e. silica, diamond, sapphire) is also contemplated as a system for altering the refractive index of the material. Strongly absorbing or opaque particles may be added to render the elastomer colored or opaque to incident radiation. This may conceivably be beneficial in an optically addressable system.
Finally, by doping the elastomer with specific chemical species, these doped chemical species may be presented at the elastomer surface, thus serving as anchors or starting points for further chemical derivitization.
In the present application, references are made to certain types of “reaction” chambers or wells in a microfluidic device. In general and in addition to those characteristics described above, these include processing sites, processing chambers, and/or reaction sites, any combination of these, and the like. These chambers may be closed, partially closed, open, partially open, sealed, or combinations thereof, including any temporary or transient conditions involving any of these states, and the like. In some embodiments, the chambers are sealed, capable of being sealed, closeable, isolated, capable of being isolated, and combinations thereof, and any combination or single condition of any temporary or transient conditions involving any of these states, and the like. Therefore, use of the term reaction chamber is not intended to limit the present invention, but to include these other structures. Additionally, the term chamber is not intended to limit the present invention, but should be used in its ordinary meaning, unless specific features associated with the chamber have been recited. Of course, there can be other variations, modifications, and alternatives.
The microfluidic devices that are described herein are further characterized in part in some embodiments by utilizing various components such as flow channels, control channels, valves and/or pumps fabricated from elastomeric materials. In some instances, essentially the entire device is made of elastomeric materials. Consequently, such devices differ significantly in form and function from the majority of conventional microfluidic devices that are formed from plastics or silicon-based materials. The number of reaction chambers provided varies according to embodiments of the present invention. Other microfluidic devices fabricated by non elastomeric materials such as glass, silicon, rigid plastics, and metal are possible, however, elastomeric microfluidic devices are a preferred embodiment of the invention.
The design of the devices enables them to be utilized in combination with a number of different heating systems. Thus, the devices are useful in conducting diverse analyses that require temperature control. Additionally, those microfluidic devices adapted for use in heating applications can incorporate a further design feature to minimize evaporation of sample from the reaction sites. Devices of this type in general include a number of guard channels and/or reservoirs or chambers formed within the elastomeric device through which water can be flowed to increase the water vapor pressure within the elastomeric material from which the device is formed, thereby reducing evaporation of sample material from the reaction sites.
In another embodiment, a temperature cycling device may be used to control the temperature of the microfluidic devices. Preferably, the microfluidic device would be adapted to make thermal contact with the microfluidic device. Where the microfluidic device is supported by a substrate material, such as a glass slide or the bottom of a carrier plate, such as a plastic carrier, a window may be formed in a region of the carrier or slide such that the microfluidic device, preferably a device having an elastomeric block, may directly contact the heating/cooling block of the temperature cycling device. In a preferred embodiment, the heating/cooling block has grooves therein in communication with a vacuum source for applying a suction force to the microfluidic device, preferably a portion adjacent to where the reactions are taking place. Alternatively, a rigid thermally conductive plate may be bonded to the microfluidic device that then mates with the heating and cooling block for efficient thermal conduction resulting.
The array format of certain of the devices means the devices can achieve high throughput. Collectively, the high throughput and temperature control capabilities make the devices useful for performing large numbers of nucleic acid amplifications (e.g., polymerase chain reaction (PCR)). Such reactions will be discussed at length herein as illustrative of the utility of the devices, especially of their use in any reaction requiring temperature control. However, it should be understood that the devices are not limited to these particular applications. The devices can be utilized in a wide variety of other types of analyses or reactions. Examples include analyses of protein-ligand interactions and interactions between cells and various compounds. Further examples are provided throughout the present specification.
The microfluidic devices disclosed herein are typically constructed at least in part from elastomeric materials and constructed by single and multilayer soft lithography (MSL) techniques and/or sacrificial-layer encapsulation methods (see, e.g., Unger et al. (2000) Science 288:113-116, and PCT Publication WO 01/01025, both of which are incorporated by reference herein in their entirety for all purposes). Utilizing such methods, microfluidic devices can be designed in which solution flow through flow channels of the device is controlled, at least in part, with one or more control channels that are separated from the flow channel by an elastomeric membrane or segment. This membrane or segment can be deflected into or retracted from the flow channel with which a control channel is associated by applying an actuation force to the control channels. By controlling the degree to which the membrane is deflected into or retracted out from the flow channel, solution flow can be slowed or entirely blocked through the flow channel. Using combinations of control and flow channels of this type, one can prepare a variety of different types of valves and pumps for regulating solution flow as described in extensive detail in Unger et al. (2000) Science 288:113-116, and PCT Publication WO/02/43615 and WO 01/01025.
If the device is to be utilized in temperature control reactions (e.g., thermocycling reactions), then, as described in greater detail infra, the elastomeric device is typically fixed to a support (e.g., a glass slide). The resulting structure can then be placed on a temperature control plate, for example, to control the temperature at the various reaction sites. In the case of thermocycling reactions, the device can be placed on any of a number of thermocycling plates.
Because the devices are made of elastomeric materials that are relatively optically transparent, reactions can be readily monitored using a variety of different detection systems at essentially any location on the microfluidic device. Most typically, however, detection occurs at the reaction site itself (e.g., within a region that includes an intersection of flow channels or at the blind end of a flow channel). The fact that the device is manufactured from substantially transparent materials also means that certain detection systems can be utilized with the current devices that are not usable with traditional silicon-based microfluidic devices. Detection can be achieved using detectors that are incorporated into the device or that are separate from the device but aligned with the region of the device to be detected.
Some high-density matrix designs utilize fluid communication vias between layers of the microfluidic device to weave control lines and fluid lines through the device. For example, by having a fluid line in each layer of a two layer elastomeric block, higher density reaction cell arrangements are possible. As will be evident to one of skill in the art, multi-layer devices allow fluid lines to cross over or under each other without being in fluid communication. The microfluidic devices utilized in embodiments of the present invention may be further integrated into the carrier devices described in co-pending and commonly owned U.S. patent application Ser. No. 11/058,106 by Unger filed on Feb. 14, 2005, which is incorporated herein for all purposes. The carrier of Unger provides on-board continuous fluid pressure to maintain valve closure away from a source of fluid pressure, e.g., house air pressure. Unger further provides for an automated system for charging and actuating the valves of the present invention as described therein. An another preferred embodiment, the automated system for charging accumulators and actuating valves employs a device having a platen that mates against one or more surfaces of the microfluidic device, wherein the platen has at least two or more ports in fluid communication with a controlled vacuum or pressure source, and may include mechanical portions for manipulating portions of the microfluidic device, for example, but not limited to, check valves. Check valves may be incorporated into the chip design to provide for a one-way flow of fluid (either reagent, sample, first reactant, second reactant, third reactant, etc.). Check valves and their fabrication are described in commonly owned U.S. Provisional Application Ser. No. 60/849,223 by Wang filed on Oct. 4, 2006 which is incorporated herein for all purposes.
Another device utilized in embodiments of the present invention provides a carrier used as a substrate for stabilizing an elastomeric block. Preferably the carrier has one or more of the following features; a well or reservoir in fluid communication with the elastomeric block through at least one channel formed in or with the carrier; an accumulator in fluid communication with the elastomeric block through at least one channel formed in or with the carrier; and, a fluid port in fluid communication with the elastomeric block, wherein the fluid port is preferably accessible to an automated source of vacuum or pressure, such as the automated system described above, wherein the automated source further comprises a platen having a port that mates with the fluid port to form an isolated fluid connection between the automated system for applying fluid pressure or vacuum to the elastomeric block. In devices utilized in certain embodiments, the automated source can also make fluid communication with one or more accumulators associated with the carrier for charging and discharging pressure maintained in an accumulator. In certain embodiments, the carrier may further comprise a region located in an area of the carrier that contacts the microfluidic device, wherein the region is made from a material different from another portion of the carrier, the material of the region being selected for improved thermal conduction and distribution properties that are different from the other portion of the carrier. Preferred materials for improved thermal conduction and distribution include, but are not limited to silicon, preferably silicon that is highly polished, such as the type of silicon available in the semiconductor field as a polished wafer or a portion cut from the wafer, e.g., chip.
As described more fully below, embodiments of the present invention utilize a thermal source, for example, but not limited to a PCR thermocycler, which may have been modified from its original manufactured state. Generally the thermal source has a thermally regulated portion that can mate with a portion of the carrier, preferably the thermal conduction and distribution portion of the carrier, for providing thermal control to the elastomeric block through the thermal conduction and distribution portion of the carrier. In a preferred embodiment, thermal contact is improved by applying a source of vacuum to a one or more channels formed within the thermally regulated portion of the thermal source, wherein the channels are formed to contact a surface of the thermal conduction and distribution portion of the carrier to apply suction to and maintain the position of the thermal conduction and distribution portion of the carrier. In a preferred embodiment, the thermal conduction and distribution portion of the carrier is not in physical contact with the remainder of the carrier, but is associated with the remainder of the carrier and the elastomeric block by affixing the thermal conduction and distribution portion to the elastomeric block only and leaving a gap surrounding the edges of the thermal conduction and distribution portion to reduce parasitic thermal effects caused by the carrier. It should be understood that in many aspects of the invention described herein, the preferred elastomeric block could be replaced with any of the known microfluidic devices in the art not described herein, for example devices produced such as the GeneChip (R) by Affymetrix(R) of Santa Clara, Calif., USA, or by Caliper of Mountain View, Calif., USA. U.S. patents issued to Soane, Parce, Fodor, Wilding, Ekstrom, Quake, or Unger, describe microfluidic or mesoscale fluidic devices that can be substituted for the elastomeric block of the present invention to take advantage of the thermal advantages and improvements, e.g., suction positioning, reducing parasitic thermal transfer to other regions of the fluidic device, which are described above in the context of using an elastomeric block.
Utilizing systems and methods provided according to embodiments of the present invention, throughput increases are provided over 384 well systems. As an example, throughput increases of a factor of 4, 6, 12, and 24 and greater are provided in some embodiments. These throughput increases are provided while reducing the logistical friction of operations. Moreover the systems and methods of embodiments of the present invention enable multiple assays for multiple samples. For example, in a specific embodiment 24 samples and 24 assays are utilized to provide a total of 576 data points. In another embodiment, 32 samples and 32 assays are utilized to provide a total of 1024 data points. In another embodiment, 48 samples and 48 assays are utilized to provide 2304 data points. In another embodiment, 96 samples and 48 assays are utilized to provide 4608 data points. In another embodiment, 96 samples and 96 assays are utilized to provide a total of 9,216 data points. In a particular example, the 96 assays are components of a TaqMan 5′ Nuclease Assay.
Depending on the geometry of the particular microfluidic device and the size of the microfluidic device and the arrangement of the fluid communication paths and processing site, embodiments of the present invention provide for a range of processing site (or reaction chamber) densities. In some embodiments, the methods and systems of the present invention are utilized with chamber densities ranging from about 100 chambers per cm2 to about 1 million chambers per cm2. Merely by way of example, microfluidic devices with chamber densities of 250, 1,000, 2,500, 10,000, 25,000, 100,000, and 250,000 chambers per cm2 are utilized according to embodiments of the present invention. In some embodiments, chamber densities in excess of 1,000,000 chambers per cm2 are utilized, although this is not required by the present invention.
Operating microfluidic devices with such small reaction volumes reduces reagent usage as well as sample usage. Moreover, some embodiments of the present invention provide methods and systems adapted to perform real-time detection, when used in combination with real-time quantitative PCR. Utilizing these systems and methods, three, four, five, or six orders of linear dynamic range are provided for some applications as well as quantitative resolution high enough to allow for the detection of sub-nanoMolar fluorophore concentrations in 10 nanoliter volumes.
Smart Chip Assays and Reactions
Through the present application, references are made to fluorescent indications from a microfluidic device. Included within the scope of the present invention are not only fluorescent indications, but luminescent indications, including chemiluminescent, electroluminescent, electrochemiluminescent, and phospholuminescent, bioluminescent, and other luminescent processes, or any other processing involving any other type of indications that may be detected using a detection device. As will be evident to one of skill in the art, methods and systems operable in the detection and analysis of these fluorescent and luminescent indications are transferable from one indication to another. Additionally, although some embodiments of the present invention utilize spectral filters as optical elements, this is not required by the present invention. Some fluorescent and luminescent applications do not utilize spectral filters in the optical excitation path, the optical emission path, or both. As described herein, other embodiments utilize spectral filters. One of skill in the art will appreciate the differences associated with particular applications.
In some embodiments, a variety of devices and methods for conducting microfluidic analyses are utilized herein, including devices that can be utilized to conduct thermal cycling reactions such as nucleic acid amplification reactions. The devices differ from conventional microfluidic devices in that they include elastomeric components; in some instances, much or all of the device is composed of elastomeric material. For example, amplification reactions can be linear amplifications, (amplifications with a single primer), as well as exponential amplifications (i.e., amplifications conducted with a forward and reverse primer set).
The methods and systems provided by some embodiments of the present invention utilize blind channel type devices in performing nucleic acid amplification reactions. In these devices, the reagents that are typically deposited within the reaction sites are those reagents necessary to perform the desired type of amplification reaction. Usually this means that some or all of the following are deposited: primers, polymerase, nucleotides, metal ions, buffer, and cofactors, for example. The sample introduced into the reaction site in such cases is the nucleic acid template. Alternatively, however, the template can be deposited and the amplification reagents flowed into the reaction sites. As discussed in more detail throughout the present specification, when a matrix device is utilized to conduct an amplification reaction, samples containing nucleic acid template are flowed through the vertical flow channels and the amplification reagents through the horizontal flow channels or vice versa.
PCR is perhaps the best known amplification technique. The devices utilized in embodiments of the present invention are not limited to conducting PCR amplifications. Other types of amplification reactions that can be conducted include, but are not limited to, (i) ligase chain reaction (LCR) (see Wu and Wallace, Genomics 4:560 (1989) and Landegren et al., Science 241:1077 (1988)); (ii) transcription amplification (see Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173 (1989)); (iii) self-sustained sequence replication (see Guatelli et al., Proc. Nat. Acad. Sci. USA, 87:1874 (1990)); and (iv) nucleic acid based sequence amplification (NASBA) (see, Sooknanan, R. and Malek, L., BioTechnology 13: 563-65 (1995)). Each of the foregoing references is incorporated herein by reference in their entirety for all purposes.
Moreover, certain devices are designed to conduct thermal cycling reactions (e.g., PCR) with devices that include one or more elastomeric valves to regulate solution flow through the device. Thus, methods for conducting amplification reactions with devices of this design are also provided.
Amplification products (amplicons) can be detected and distinguished (whether isolated in a reaction chamber or at any subsequent time) using routine methods for detecting nucleic acids. Many different signal moieties may be used in various embodiments of the present invention. For example, signal moieties include, but are not limited to, fluorophores, radioisotopes, chromogens, enzymes, antigens, heavy metals, dyes, phosphorescence groups, chemiluminescent groups, and electrochemical detection moieties. Exemplary fluorophores that may be used as signal moieties include, but are not limited to, rhodamine, cyanine 3 (Cy 3), cyanine 5 (Cy 5), fluorescein, VIC™, LIZ™, Tamra™, 5-FAM™, 6-FAM™, and Texas Red (Molecular Probes). (VIC™, LIZ™, Tamra™, 5-FAM™, and 6-FAM™ (all available from Applied Biosystems, Foster City, Calif.) Exemplary radioisotopes include, but are not limited to, 32P, 33P, and 35S. Signal moieties also include elements of multi-element indirect reporter systems, e.g., biotin/avidin, antibody/antigen, ligand/receptor, enzyme/substrate, and the like, in which the element interacts with other elements of the system in order to effect a detectable signal. Certain exemplary multi-element systems include a biotin reporter group attached to a probe and an avidin conjugated with a fluorescent label. Detailed protocols for methods of attaching signal moieties to oligonucleotides can be found in, among other places, G. T. Hermanson, Bioconjugate Techniques, Academic Press, San Diego, Calif. (1996) and S. L. Beaucage et al., Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, New York, N.Y. (2000).
Amplicons comprising double-stranded DNA can be detected using intercalation dyes such as SYBR™, Pico Green (Molecular Probes, Inc., Eugene, Oreg.), ethidium bromide and the like (see Zhu et al., 1994, Anal. Chem. 66:1941-48) and/or gel electrophoresis. More often, sequence-specific detection methods are used (i.e., amplicons are detected based on their nucleotide sequence). Examples of detection methods include hybridization to arrays of immobilized oligo or polynucleotides, and use of differentially labeled molecular beacons or other “fluorescence resonance energy transfer” (FRET)-based detection systems. FRET—based detection is a preferred method for detection according to some embodiments of the present invention. In FRET-based assays a change in fluorescence from a donor (reporter) and/or acceptor (quencher) fluorophore in a donor/acceptor fluorophore pair is detected. The donor and acceptor fluorophore pair are selected such that the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. Thus, when the pair of fluorophores are brought within sufficiently close proximity to one another, energy transfer from the donor to the acceptor can occur and can be detected. A variety of assays are known including, for example and not limitation, template extension reactions, quantitative RT-PCR, Molecular Beacons, and Invader assays, these are described briefly below.
FRET and template extension reactions 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 incorporation of the labeled nucleotide into the primer during an template-dependent extension reaction, the donor and acceptor are spaced far enough apart that energy transfer cannot occur. However, if the labeled nucleotide is incorporated into the primer and the spacing is sufficiently close, then energy transfer occurs and can be detected. These methods are particularly useful in conducting single base pair extension reactions in the detection of single nucleotide polymorphisms and are described in U.S. Pat. No. 5,945,283 and PCT Publication WO 97/22719. The reactions can optionally be thermocycled to increase signal using the temperature control methods and apparatus described throughout the present specification.
A variety of so-called “real time amplification” methods or “real time quantitative PCR” methods can also be used to determine the quantity of a target nucleic acid present in a sample by measuring the amount of amplification product formed during or after the amplification process itself. Fluorogenic nuclease assays are one specific example of a real time quantitation method which can be used successfully with the devices described herein. This method of monitoring the formation of amplification product involves the continuous measurement of PCR product accumulation using a dual-labeled fluorogenic oligonucleotide probe—an approach frequently referred to in the literature as the “TaqMan” method. See, for example, U.S. Pat. No. 5,723,591.
With molecular beacons, a change in conformation of the probe as it hybridizes to a complementary region of the amplified product results in the formation of a detectable signal. The probe itself includes two sections: one section at the 5′ end and the other section at the 3′ end. These sections flank the section of the probe that anneals to the probe binding site and are complementary to one another. One end section is typically attached to a reporter dye and the other end section is usually attached to a quencher dye. In solution, the two end sections can hybridize with each other to form a hairpin loop. In this conformation, the reporter and quencher dye are in sufficiently close proximity that fluorescence from the reporter dye is effectively quenched by the quencher dye. Hybridized probe, in contrast, results in a linearized conformation in which the extent of quenching is decreased. Thus, by monitoring emission changes for the two dyes, it is possible to indirectly monitor the formation of amplification product. Probes of this type and methods of their use are described further, for example, by Piatek et al., 1998, Nat. Biotechnol. 16:359-63; Tyagi, and Kramer, 1996, Nat. Biotechnology 14:303-308; and Tyagi, et al., 1998, Nat. Biotechnol. 16:49-53 (1998).
The Scorpion detection method is described, for example, by Thelwell et al. 2000, Nucleic Acids Research, 28:3752-3761 and Solinas et al., 2001, “Duplex Scorpion primers in SNP analysis and FRET applications” Nucleic Acids Research 29:20. Scorpion primers are fluorogenic PCR primers with a probe element attached at the 5′-end via a PCR stopper. They are used in real-time amplicon-specific detection of PCR products in homogeneous solution. Two different formats are possible, the ‘stem-loop’ format and the ‘duplex’ format. In both cases the probing mechanism is intramolecular. The basic elements of Scorpions in all formats are: (i) a PCR primer; (ii) a PCR stopper to prevent PCR read-through of the probe element; (iii) a specific probe sequence; and (iv) a fluorescence detection system containing at least one fluorophore and quencher. After PCR extension of the Scorpion primer, the resultant amplicon contains a sequence that is complementary to the probe, which is rendered single-stranded during the denaturation stage of each PCR cycle. On cooling, the probe is free to bind to this complementary sequence, producing an increase in fluorescence, as the quencher is no longer in the vicinity of the fluorophore. The PCR stopper prevents undesirable read-through of the probe by Taq DNA polymerase.
Invader assays (Third Wave Technologies, Madison, Wis.) are used particularly for SNP genotyping and utilize an oligonucleotide, designated the signal probe that is complementary to the target nucleic acid (DNA or RNA) or polymorphism site. A second oligonucleotide, designated the Invader Oligo, contains the same 5′ nucleotide sequence, but the 3′ nucleotide sequence contains a nucleotide polymorphism. The Invader Oligo interferes with the binding of the signal probe to the target nucleic acid such that 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 Cleavase enzyme. Cleavase cleaves the 5′ flap of the nucleotides. The released flap binds with a third probe bearing FRET labels, thereby forming another duplex structure recognized by the Cleavase enzyme. This time the Cleavase enzyme cleaves a fluorophore away from a quencher and produces a fluorescent signal. For SNP genotyping, the signal probe will be designed to hybridize with either the reference (wild type) allele or the variant (mutant) allele. Unlike PCR, there is a linear amplification of signal with no amplification of the nucleic acid. Further details sufficient to guide one of ordinary skill in the art are provided by, for example, Neri, B. P., et al., Advances in Nucleic Acid and Protein Analysis 3826:117-125, 2000) and U.S. Pat. No. 6,706,471.
A variety of multiplex amplification systems can be used in conjunction with the present invention. In one type, several different targets can be detected simultaneously by using multiple differently labeled probes each of which is designed to hybridize only to a particular target. Since each probe has a different label, binding to each target to be detected based on the fluorescence signals. By judicious choice of the different labels that are utilized, analyses can be conducted in which the different labels are excited and/or detected at different wavelengths in a single reaction. See, e.g., Fluorescence Spectroscopy (Pesce et al., Eds.) Marcel Dekker, New York, (1971); White et al., Fluorescence Analysis: A Practical Approach, Marcel Dekker, New York, (1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd ed., Academic Press, New York, (1971); Griffiths, Colour and Constitution of Organic Molecules, 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).
Many diseases linked to genome modifications, either of the host organism or of infectious organisms, are the consequence of a change in a small number of nucleotides, frequently involving a change in a single nucleotide. Such single nucleotide changes are referred to as single nucleotide polymorphisms or simply SNPs, and the site at which the SNP occurs is typically referred to as a polymorphic site. The devices described herein can be utilized to determine the identify of a nucleotide present at such polymorphic sites. As an extension of this capability, the devices can be utilized in genotyping analyses. Genotyping involves the determination of whether a diploid organism (i.e., an organism with two copies of each gene) contains two copies of a reference allele (a reference-type homozygote), one copy each of the reference and a variant allele (i.e., a heterozygote), or contains two copies of the variant allele (i.e., a variant-type homozygote). When conducting a 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, the methods can also be used to determine the genotype of an individual in many different DNA loci, either on the same gene, different genes or combinations thereof.
Genotyping analyses can be conducted using a variety of different approaches. In these methods, it is generally sufficient to obtain a “yes” or “no” result, i.e., detection need only be able to answer the question whether a given allele is present. Thus, analyses can be conducted only with the primers or nucleotides necessary to detect the presence of one allele potentially at a polymorphic site. However, more typically, primers and nucleotides to detect the presence of each allele potentially at the polymorphic site are included.
Single Base Pair Extension (SBPE) reactions are one technique specifically developed for conducting genotyping analyses. Although a number of SPBE assays have been developed, the general approach is quite similar. Typically, these assays involve hybridizing a primer that is complementary to a target nucleic acid such that the 3′ end of the primer is immediately 5′ of the variant site or is adjacent thereto. Extension is conducted in the presence of one or more labeled non-extendible nucleotides that are complementary to the nucleotide(s) that occupy the variant site and a polymerase. The non-extendible nucleotide is a nucleotide analog that prevents further extension by the polymerase once incorporated into the primer. If the added non-extendible nucleotide(s) is(are) complementary to the nucleotide at the variant site, then a labeled non-extendible nucleotide is incorporated onto the 3′ end of the primer to generate a labeled extension product. Hence, extended primers provide an indication of which nucleotide is present at the variant site of a target nucleic acid. Such methods and related methods are discussed, for example, in U.S. Pat. Nos. 5,846,710; 6,004,744; 5,888,819; 5,856,092; and 5,710,028; and in WO 92/16657.
Genotyping analyses can also be conducted using quantitative PCR methods. In this case, differentially labeled probes complementary to each of the allelic forms are included as reagents, together with primers, nucleotides and polymerase. However, reactions can be conducted with only a single probe, although this can create ambiguity as to whether lack of signal is due to absence of a particular allele or simply a failed reaction. For the typical biallelic case in which two alleles are possible for a polymorphic site, two differentially labeled probes, each perfectly complementary to one of the alleles are usually included in the reagent mixture, together with amplification primers, nucleotides and polymerase. Sample containing the target DNA is introduced into the reaction site. If the allele to which a probe is complementary is present in the target DNA, then amplification occurs, thereby resulting in a detectable signal as described in the detection above. Based upon which of the differential signal is obtained, the identity of the nucleotide at the polymorphic site can be determined. If both signals are detected, then both alleles are present. Thermocycling during the reaction is performed as described in the temperature control section supra.
Gene expression analysis involves determining the level at which one or more genes is expressed in a particular cell. The determination can be qualitative, but generally is quantitative. In a differential gene expression analysis, the levels of the gene(s) in one cell (e.g., a test cell) are compared to the expression levels of the same genes in another cell (control cell). A wide variety of such comparisons can be made. Examples include, but are not limited to, a comparison between healthy and diseased cells, between cells from an individual treated with one drug and cells from another untreated individual, between cells exposed to a particular toxicant and cells not exposed, and so on. Genes whose expression levels vary between the test and control cells can serve as markers and/or targets for therapy. For example, if a certain group of genes is found to be up-regulated in diseased cells rather than healthy cells, such genes can serve as markers of the disease and can potentially be utilized as the basis for diagnostic tests. These genes could also be targets. A strategy for treating the disease might include procedures that result in a reduction of expression of the up-regulated genes.
The devices of the present application are not limited to biological assays such as PCR reactions. The efficient mixing of the carry-slug design is an enhancement to any chemical or biochemical assay and can be utilized in chemical synthesis (in particular where it is desirable to monitor reaction progress or degree of completion of the reaction in an SBS formatted reader). Other assays that benefit from the present invention include immunological and enzymatic assays.
Exemplary Chip Holder
Microfluidic device 11008 is positioned within recessed area 11006 of bottom portion 11002 of chip holder 11000. Microfluidic device 11008 comprises an active region 11011 that is in fluidic communication with peripheral wells 11012 configured in first and second rows 11012a and 11012b, respectively. Wells 11012 hold sufficient volumes of material to allow device 11008 to function. Wells 11012 may contain, for example, solutions of crystallizing agents, solutions of target materials, or other chemical reagents such as stains. Bottom portion 11002 contains a window 11003 that enables active region 11011 of chip 11008 to be observed.
Top portion 11014 of chip holder 11000 fits over bottom chip holder portion 11002 and microfluidic chip 11008 positioned therein. For ease of illustration, in
Lower surface 11014a of top holder portion 11014 includes raised annular rings 11020 and 11022 surrounding recesses 11024 and 11026, respectively. When top portion 11014 of chip holder 11000 is pressed into contact with chip 11008 utilizing screws 11016, rings 11020 and 11022 press into the soft elastomeric material on the upper surface of chip 11008, such that recess 11024 defines a first chamber over top row 11012a of wells 11012, and recess 11026 defines a second chamber over bottom row 11012b of wells 11012. Holes 11030 and 11032 in the side of top holder portion 11014 are in communication with recesses 11024 and 11026 respectively, to enable a positive pressure to be applied to the chambers through pins 11034 inserted into holes 11030 and 11032, respectively. A positive pressure can thus simultaneously be applied to all wells within a row, obviating the need to utilize separate connecting devices to each well.
In operation, solutions are pipetted into the wells 11012, and then chip 11008 is placed into bottom portion 11002 of holder 11000. The top holder portion 11014 is placed over chip 11008, and is pressed down by screws. Raised annular rings 11020 and 11022 on the lower surface of top holder portion 11014 make a seal with the upper surface of the chip where the wells are located. Solutions within the wells are exposed to positive pressures within the chamber, and are thereby pushed into the active area of microfluidic device.
The downward pressure exerted by the chip holder may also pose the advantage of preventing delamination of the chip from the substrate during loading. This prevention of delamination may enable the use of higher priming pressures.
The chip holder shown in
Exemplary Systems for Delivery Fluids to Reaction Sites
The positive-displacement cross-injection metering scheme allows for sequential injection of precise sample aliquots from a single microfluidic channel into an array of reaction chambers through a positive displacement cross-injection (PCI) junction.
Reaction cells are fabricated in 100 nL (
To operate the carry-slug reaction cell, control channel 1 is pressurized to deflect the elastomeric membrane valve and close the slug reagent flow channel at its interface with the sample to slug via and the slug to reaction chamber via. The reagent input flow channel is pressurized and the entire slug reagent flow channel is blind filled with the desired reagent. Simultaneously, the sample input flow channel is also pressurized and the entire sample flow channel is blind filled up to the valve delineated by control channel 1. Control channel 2 is then pressurized to deflect the elastomeric membrane valve that closes the slug reagent flow channel near its interface with the reagent input flow channel. The sample input flow channel is then re-pressurized and control channel 1 is depressurized to open the slug reagent flow channel at its via connections. The contents of the slug reagent flow channel are then transferred into the reaction chamber through blind filling and under pressure from the sample input flow channel. The volume of the reaction chamber is in excess of the volume of the slug reagent flow channel which allows for sample to continue flowing from the sample input flow channel and the slug reagent flow channel and to fill up the reaction chamber in the amount that is the difference of the reaction chamber volume and the slug reagent flow channel volume. Control channel 1 is repressurized to close off the reaction chamber. The reagent and sample are retained in a mixed solution in the reaction chamber and the reaction is allowed to proceed.
A 32×32 elastomeric microfluidic matrix reaction array with slug mixing was constructed and mixing efficiency was compared to a conventional 32×32 elastomeric microfluidic matrix reaction array constructed with a sample/reaction chamber adjacent to a reagent chamber and separated by an interface valve.
The matrix reaction array was constructed with each reaction cell comprising a central 50 nL reaction chamber (400) and a 5 nL slug channel (250) as shown in
Operation of the matrix reaction array was performed by the following steps. Control channel 1 (260) was pressurized to close the valves that fluidically isolate the ends of the slug channel (
Having described several embodiments, it will be recognized by those of skill in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the invention. Additionally, a number of well known processes and elements have not been described in order to avoid unnecessarily obscuring the present invention. Accordingly, the above description should not be taken as limiting the scope of the invention.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.
As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a process” includes a plurality of such processes and reference to “the electrode” includes reference to one or more electrodes and equivalents thereof known to those skilled in the art, and so forth.
Also, the words “comprise,” “comprising,” “include,” “including,” and “includes” when used in this specification and in the following claims are intended to specify the presence of stated features, integers, components, or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps, acts, or groups.
This application claims priority to U.S. Provisional Application Ser. No. 60/777,972 filed Feb. 28, 2006, U.S. Provisional Application Ser. No. 60/849,223 filed Oct. 4, 2006, and U.S. Provisional Application Ser. No. 60/881,627 filed Jan. 19, 2007, of which the entire contents of all the applications are herein incorporated by reference for all purposes. In addition, embodiments of microfluidic structures and materials used in the devices of the present invention are described in U.S. patent application Ser. No. 11/006,522, filed Dec. 6, 2004, and published Sep. 22, 2005 as U.S. Pat. Pub. No. 20050205005, with the title “MICROFLUIDIC PROTEIN CRYSTALLOGRAPHY”, the entire contents of which are hereby incorporated by reference for all purposes. Applications of the methods and devices of the present invention are also related to the inventions described in PCT application PCT/US01/44869, filed Nov. 16, 2001, and entitled “CELL ASSAYS AND HIGH THROUGHPUT SCREENING”; U.S. patent application Ser. No. 10/116,761, filed Apr. 3, 2002, and published Jan. 9, 2003 as U.S. Pat. Pub. No. 20030008411, with the title “COMBINATORIAL SYNTHESIS SYSTEM”; U.S. patent application Ser. No. 10/416,418, filed Nov. 16, 2001, and published Jun. 17, 2004 as U.S. Pat. Pub. No. 20040115838, with the title “APPARATUS AND METHODS FOR CONDUCTING ASSAYS AND HIGH THROUGHPUT SCREENING”; and U.S. patent application Ser. No. 10/118,466, filed Apr. 5, 2002, and issued Nov. 1, 2005 as U.S. Pat. No. 6,960,437, with the title “NUCLEIC ACID AMPLIFICATION UTILIZING MICROFLUIDIC DEVICES”, which are also hereby incorporated by reference for all purposes. Additional information relating to the formation of microfabricated fluidic devices utilizing elastomer materials are described generally in U.S. Pat. No. 6,408,878, filed Feb. 28, 2001, entitled “MICROFLUIDIC ELASTOMERIC VALVE AND PUMP SYSTEMS”; U.S. Pat. No. 6,899,137, filed Apr. 6, 2001, entitled “MICROFABRICATED ELASTOMERIC VALVE AND PUMP SYSTEMS”; U.S. patent application Ser. No. 09/724,784 filed Nov. 28, 2000, entitled “MICROFABRICATED ELASTOMERIC VALVE AND PUMP SYSTEMS”; and Ser. No. 09/605,520, filed Jun. 27, 2000, entitled “MICROFABRICATED ELASTOMERIC VALVE AND PUMP SYSTEMS.” These patents and patent applications are also hereby incorporated by reference for all purposes.
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