US20030232403A1 - Devices and methods for the performance of miniaturized homogeneous assays - Google Patents
Devices and methods for the performance of miniaturized homogeneous assays Download PDFInfo
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- US20030232403A1 US20030232403A1 US10/602,564 US60256403A US2003232403A1 US 20030232403 A1 US20030232403 A1 US 20030232403A1 US 60256403 A US60256403 A US 60256403A US 2003232403 A1 US2003232403 A1 US 2003232403A1
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Definitions
- This invention relates to methods and apparatus for performing microanalytic and microsynthetic analyses and procedures.
- the invention relates to microminiaturization of genetic, biochemical and bioanalytic processes.
- the present invention provides devices and methods for the performance of miniaturized biochemical assays. These assays may be performed for a variety of purposes, including but not limited to screening of drug candidate compounds, life sciences research, and clinical and molecular diagnostics. Methods for performing any of a wide variety of such microanalytical or microsynthetic processes using the Microsystems apparatus of the invention are also provided.
- the function of drug candidates, targets, and the effect of the candidates on targets is assessed in the early stages of pharmaceutical development through a process of screening that typically includes: binding of a drug candidate to a portion or domain of the target molecule; immunoassays that bind to drug candidate target domains correlated with drug efficacy; enzymatic assays, in which the inhibition of an enzymatic activity of the target by the drug candidate can be used as a sign of efficacy; protein/protein binding; and protein/DNA(RNA) binding.
- Additional assays involve the use of living cells and include gene expression, in which levels of transcription in response to a drug candidate are monitored, and functional assays designed to investigate both macroscopic effects, such as cell viability, as well as biochemical effects and products produced in and by the cells as a result of treatment with the drug lead compound.
- Miniaturization can also confer performance advantages. At short length scales, diffusionally-limited mixing is rapid and can be exploited to create sensitive assays (Brody et al., 1996, Biophysical J. 71: 3430-3431). Because fluid flow in miniaturized pressure-driven systems is laminar, rather than turbulent, processes such as washing and fluid replacement are well-controlled. Microfabricated systems also enable assays that rely on a large surface area to volume ratio such as those that require binding to a surface and a variety of chromatographic approaches.
- Fluid processing in microtiter plates is also difficult.
- washing an important step in many assays can be problematic.
- Methods that reduce both the number of manipulations of fluids on the plate as well as manipulations of the plate itself can reduce cost while improving assay quality through suppression of contamination, carry-over, and fluid loss.
- This invention provides Microsystems platforms as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. Nos. 08/761,063, filed Dec. 5, 1996; 08/768,990, filed Dec. 18, 1996; 08/910,726, filed Aug. 12, 1997; 08/995,056, filed Dec. 19, 1997; and 09/315,114, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein.
- the invention provides apparatus and methods for performing microscale processes on a microplatform, whereby fluid is moved on the platform in defined channels motivated by centripetal force arising from rotation of the platform.
- the first element of the apparatus of the invention is a microplatform that is a rotatable structure, most preferably a disk, the disk comprising fluid (sample) inlet ports, fluidic microchannels, reagent reservoirs, collection chambers, detection chambers and sample outlet ports, generically termed “microfluidic structures.”
- the disk is rotated at speeds from about 1 to about 30,000 rpm for generating centripetal acceleration that enables fluid movement through the microfluidic structures of the platform.
- the disks of the invention also preferably comprise air outlet ports and air displacement channels.
- the air outlet ports and in particular the air displacement ports provide a means for fluids to displace air, thus ensuring uninhibited movement of fluids on the disk.
- Specific sites on the disk also preferably comprise elements that allow fluids to be analyzed, as well as detectors for each of these effectors.
- the discs of this invention have several advantages over those that exist in the centrifugal analyzer art. Foremost is the fact that flow is laminar due to the small dimensions of the fluid channels; this allows for better control of processes such as mixing and washing. Secondly, the small dimensions conferred by microfabrication enable the use of “passive” valving, dependent upon capillary forces, over much wider range of rotational velocities and with greater reliability than in more macroscopic systems. To this are added the already described advantages of miniaturization.
- the second element of the invention is a micromanipulation device that is a disk player/reader device that controls the function of the disk.
- This device comprises mechanisms and motors that enable the disk to be loaded and rotated.
- the device provides means for a user to operate the Microsystems in the disk and access and analyze data, preferably using a keypad and computer display.
- the micromanipulation device also advantageous provides means for actuation of on-disc elements, such active valves; the application and control of heat to the disc for purposes of chemical or biological incubation; and means for adding fluids to and removing fluids from the discs.
- the micromanipulation devices of this invention are more particularly described in co-owned U.S. Pat. No.
- the invention specifically provides Microsystems platforms comprising microfluidics components contained in one or a multiplicity of platform layers that are fluidly connected to permit transfer, mixing and assay performance on the sealed surface of the platform.
- the platforms preferably comprise reagent reservoirs containing a sufficient volume, preferably from about 1 nL to about 1 mL, of a reagent solution for a multiplicity of individual assays.
- the reagent reservoirs are fluidly connected by microchannels to one or more preferably a multiplicity of collection, and more preferably detection, chambers, and the microfluidics components arranged so that a specific volume of the reagent solution is delivered to each collection chamber.
- said reagent reservoirs are fluidly connected to mixing structures, most preferably a mixing microchannel that is also fluidly connected to a sample reservoir, so that one or a plurality of reagents are mixed with sample and the resulting mixture delivered into the detection chamber.
- the platform comprises a multiplicity of sample reservoirs and mixing structures fluidly connected with a multiplicity of detection chambers.
- the platforms of the invention permit the use of a detector, most preferably an optical detector, for detecting the products of the assay, whereby the collection chambers comprise optical cuvettes, preferably at the outer edge of the platform, most preferably wherein the platform is scanned past a fixed detector through the action of the rotary motor. Because the platforms of the invention are most preferably constructed using microfabrication techniques as described more fully below, the volumes of fluids used may be made arbitrarily small as long as the detectors used have sufficient sensitivity.
- the present invention solves problems in the current art through the use of a microfluidic disc in which centripetal acceleration is used to move fluids. It is an advantage of the microfluidics platforms of the present invention that the fluid-containing components are constructed to contain a small volume, thus reducing reagent costs, reaction times and the amount of biological material required to perform an assay. It is also an advantage that the fluid-containing components are sealed, thus eliminating experimental error due to differential evaporation of different fluids and the resulting changes in reagent concentration. Because the microfluidic devices of the invention are completely enclosed, both evaporation and optical distortion are reduced to negligible levels.
- the platforms of the invention also advantageously permit “passive” mixing and valving, i.e., mixing and valving are performed as a consequence of the structural arrangements of the components on the platforms (such as shape, length, position on the platform surface relative to the axis of rotation, and surface properties of the interior surfaces of the components, such as wettability as discussed below), and the dynamics of platform rotation (speed, acceleration, direction and change-of-direction), and permit control of assay timing and reagent delivery.
- “passive” mixing and valving i.e., mixing and valving are performed as a consequence of the structural arrangements of the components on the platforms (such as shape, length, position on the platform surface relative to the axis of rotation, and surface properties of the interior surfaces of the components, such as wettability as discussed below), and the dynamics of platform rotation (speed, acceleration, direction and change-of-direction), and permit control of assay timing and reagent delivery.
- metering structures as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000 and incorporated by reference herein, are used to distribute aliquots of reagent to each of a multiplicity of mixing structures, each mixing structure being fluidly connected to one of a multiplicity of sample reservoirs, thereby permitting parallel processing and mixing of the samples with a common reagent.
- This reduces the need for automated reagent distribution mechanisms, reduces the amount of time required for reagent dispensing (that can be performed in parallel with distribution of reagent to a multiplicity of reaction chambers), and permits delivery of small (nL-to- ⁇ L) volumes without using externally-applied electromotive means.
- the assembly of a multiplicity of collection chambers on the platforms of the invention also permits simplified detectors to be used, whereby each individual collection/detection chamber can be scanned using mechanisms well-developed in the art for use with, for example, CD-ROM technology.
- the platforms of the invention are advantageously provided with sample and reagent entry ports for filling with samples and reagents, respectively, that can be adapted to liquid delivery means known in the art (such as micropipettors).
- the platforms of the invention reduce the demands on automation in at least three ways.
- the total number of fluid “delivery” events on the microfluidic platform is reduced relative to microtiter plates.
- microfluidic structures that sub-divide and aliquot common reagents (such as reagent solutions, buffers, and enzyme substrates) used in all assays performed on the platform, the number of manual or automated pipetting steps are reduced by at least half (depending on the complexity of the assay).
- a reduction in fluid transfers to the device can reduce total assay time. Examples of these structures have been disclosed in co-owned U.S. Pat. 6,063,589, issued May 16, 2000, and incorporated by reference herein.
- the invention also provides on-platform means for mixing reagents with sample and washing the resulting reaction products, removing the need for transferring the assay collection chamber(s) to a separate “wash” station. This also reduces manipulation of the assay device as well as providing controlled and integrated fluid processing.
- FIG. 1 depicts an exploded,oblique view of a microsystems platform of the invention.
- FIG. 2 depicts a plan view of one component of the Microsystems platform shown in exploded, oblique view in FIG. 1, the reservoir layer.
- FIG. 3 is a detail of a section of the reservoir layer illustrated in FIG. 2.
- FIG. 4 shows a plan view of another component of the microsystems platform of FIG. 1, the microfluidic layer.
- FIG. 5 is a detail of one segment of the microfluidic layer of FIG. 4 comprising the microfluidic channels for two microfluidic assay structures.
- FIG. 6 is a segment of the assembled reservoir and microfluidic layers comprising the Microsystems platform of FIG. 1.
- FIGS. 7 a through 7 f illustrate the sequence of fluid motions through a single segment of the Microsystems platform comprising two microfluidic assay structures.
- FIG. 8 illustrates kinetic data for enzymatic inhibition assays performed with the devices of the invention.
- FIG. 9 is a dose-response curve illustrating enzymatic activity as a function of inhibitor concentration for enzymatic inhibition assays performed with the devices of the invention, as disclosed in Example 1.
- FIGS. 10 a and 10 b is another dose-response curve illustrating enzymatic activity as a function of inhibitor concentration for for enzymatic inhibition assays performed with the devices of the invention, as disclosed in Example 2.
- This invention provides a microplatform and a micromanipulation device as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. Nos. 08/761,063, filed Dec. 5, 1996; 08/768,990, filed Dec. 18, 1996; 08/910,726, filed Aug. 12, 1997; 08/995,056, filed Dec. 19, 1997; 09/315,114, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein, adapted for performing microanalytical and microsynthetic assays of biological samples.
- sample will be understood to encompass any fluid, solution or mixture, either isolated or detected as a constituent of a more complex mixture, or synthesized from precursor species.
- sample will be understood to encompass any biological species of interest.
- biological sample or “biological fluid sample” will be understood to mean any biologically-derived sample, including but not limited to blood, plasma, serum, lymph, saliva, tears, cerebrospinal fluid, urine, sweat, plant and vegetable extracts, semen, and ascites fluid.
- a centripetally motivated fluid micromanipulation apparatus is intended to include analytical centrifuges and rotors, microscale centrifugal separation apparatuses, and most particularly the Microsystems platforms and disk handling apparatuses as described in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. Nos. 08/761,063, filed December 5, 1996; 08/768,990, filed Dec. 18, 1996; 08/910,726, filed Aug. 12, 1997; 08/995,056, filed Dec. 19, 1997; 09/315,114, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein.
- Microsystems platform is intended to include centripetally-motivated microfluidics arrays as described in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. Nos. 08/761,063, filed Dec. 5, 1996; 08/768,990, filed Dec. 18, 1996; 08/910,726, filed Aug. 12, 1997; 08/995,056, filed Dec. 19, 1997; 09/315,114, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein.
- capillary For the purposes of this invention, the terms “capillary”, “microcapillary” and “microchannel” will be understood to be interchangeable and to be constructed of either wetting or non-wetting materials where appropriate.
- the term “reagent reservoir,” “assay chamber,” “fluid holding chamber,” “collection chamber” and “detection chamber” will be understood to mean a defined volume on a Microsystems platform of the invention comprising a fluid.
- the terms “entry port” and “fluid input port” will be understood to mean an opening on a Microsystems platform of the invention comprising a means for applying a fluid to the platform.
- exit port and “fluid outlet port” will be understood to mean a defined volume on a Microsystems platform of the invention comprising a means for removing a fluid from the platform.
- capillary junction will be understood to mean a region in a capillary or other flow path where surface or capillary forces are exploited to retard or promote fluid flow.
- a capillary junction is provided as a pocket, depression or chamber in a hydrophilic substrate that has a greater depth (vertically within the platform layer) and/ or a greater width (horizontally within the platform layer) that the fluidics component (such as a microchannel) to which it is fluidly connected.
- the fluidics component such as a microchannel
- the force hindering flow is produced by capillary pressure, that is inversely proportional to the cross sectional dimensions of the channel and directly proportional to the surface tension of the liquid, multiplied by the cosine of the contact angle of the fluid in contact with the material comprising the channel.
- capillary pressure that is inversely proportional to the cross sectional dimensions of the channel and directly proportional to the surface tension of the liquid, multiplied by the cosine of the contact angle of the fluid in contact with the material comprising the channel.
- Capillary junctions can be constructed in at least three ways.
- a capillary junction is formed at the junction of two components wherein one or both of the lateral dimensions of one component is larger than the lateral dimension(s) of the other component.
- a junction occurs at an enlargement of a capillary as described in co-owned and co-pending U.S. Ser. Nos. 08/761,063, filed Dec. 5, 1996; 08/768,990, filed Dec. 18, 1996; and 08/910,726, filed Aug. 12, 1997. Fluid flow through capillaries is inhibited at such junctions.
- capillary junctions are formed when the dimensions of the components change from a small diameter (such as a capillary) to a larger diameter (such as a chamber) in wetting systems, in contrast to non-wettable systems, where capillary junctions form when the dimensions of the components change from a larger diameter (such as a chamber) to a small diameter (such as a capillary).
- a second embodiment of a capillary junction is formed using a component having differential surface treatment of a capillary or flow-path.
- a channel that is hydrophilic that is, wettable
- may be treated to have discrete regions of hydrophobicity that is, non-wettable.
- a fluid flowing through such a channel will do so through the hydrophilic areas, while flow will be impeded as the fluid-vapor meniscus impinges upon the hydrophobic zone.
- the third embodiment of a capillary junction according to the invention is provided for components having changes in both lateral dimension and surface properties.
- An example of such a junction is a microchannel opening into a hydrophobic component (microchannel or reservoir) having a larger lateral dimension.
- capillary junctions according to the invention can be created at the juncture of components having different sizes in their lateral dimensions, different hydrophilic properties, or both.
- capillary action will be understood to mean fluid flow in the absence of rotational motion or centripetal force applied to a fluid on a rotor or platform of the invention and is due to a partially or completely wettable surface.
- capillary microvalve will be understood to mean a capillary microchannel comprising a capillary junction whereby fluid flow is impeded and can be motivated by the application of pressure on a fluid, typically by centripetal force created by rotation of the rotor or platform of the invention.
- Capillary microvalves will be understood to comprise capillary junctions that can be overcome by increasing the hydrodynamic pressure on the fluid at the junction, most preferably by increasing the rotational speed of the platform.
- the term “in fluid communication” or “fluidly connected” is intended to define components that are operably interconnected to allow fluid flow between components.
- the platform comprises a rotatable platform, more preferably a disk, whereby fluid movement on the disk is motivated by centripetal force upon rotation of the disk.
- air displacement channels will be understood to include ports in the surface of the platform that are contiguous with the components (such as microchannels, chambers and reservoirs) on the platform, and that comprise vents and microchannels that permit displacement of air from components of the platforms and rotors by fluid movement.
- microplatforms of the invention are provided to comprise one or a multiplicity of microsynthetic or microanalytic systems (termed “microfluidics structures” herein).
- microfluidics structures comprise combinations of related components as described in further detail herein that are operably interconnected to allow fluid flow between components upon rotation of the disk.
- These components can be microfabricated as described below either integral to the disk or as modules attached to, placed upon, in contact with or embedded in the disk.
- microfabricated refers to processes that allow production of these structures on the sub-millimeter scale. These processes include but are not restricted to molding, photolithography, etching, stamping and other means that are familiar to those skilled in the art.
- the invention also comprises a micromanipulation device for manipulating the disks of the invention, wherein the disk is rotated within the device to provide centripetal force to effect fluid flow on the disk. Accordingly, the device provides means for rotating the disk at a controlled rotational velocity, for stopping and starting disk rotation, and advantageously for changing the direction of rotation of the disk.
- electromechanical means and control means are provided as components of the devices of the invention.
- User interface means (such as a keypad and a display) are also provided, as further described in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. Nos. 08/761,063, filed Dec.
- the invention provides a combination of specifically-adapted microplatforms that are rotatable, analytic/synthetic microvolume assay platforms, and a micromanipulation device for manipulating the platform to achieve fluid movement on the platform arising from centripetal force on the platform as result of rotation.
- the platform of the invention is preferably and advantageously a circular disk; however, any platform capable of being rotated to impart centripetal for a fluid on the platform is intended to fall within the scope of the invention.
- the micromanipulation devices of the invention are more fully described in co-owned and co-pending U.S. Ser. Nos. 08/761,063, filed Dec. 5, 1996; 08/768,990, filed Dec. 18, 1996; 08/910,726, filed Aug. 12, 1997; 08/995,056, filed Dec. 19, 1997; and 09/315,114, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein.
- Fluid (including reagents, samples and other liquid components) movement is controlled by centripetal acceleration due to rotation of the platform.
- the magnitude of centripetal acceleration required for fluid to flow at a rate and under a pressure appropriate for a particular microfluidics structure on the microsystems platform is determined by factors including but not limited to the effective radius of the platform, the interior diameter of microchannels, the position angle of the microchannels on the platform with respect to the direction of rotation, and the speed of rotation of the platform.
- an unmetered amount of a fluid is applied to the platform and a metered amount is transferred from a fluid reservoir to a microchannel, as described in co-owned U.S. Pat.
- the metered amount of the fluid sample provided on an inventive platform is from about 1 nL to about 500 ⁇ L.
- metering manifolds comprising one or a multiplicity of metering capillaries are provided to distribute the fluid to a plurality of components of the microfluidics structure.
- microchannels comprising the surface of the platforms of the invention.
- Microchannel sizes are optimally determined by specific applications and by the amount of and delivery rates of fluids required for each particular embodiment of the platforms and methods of the invention.
- Microchannel sizes can range from 0.1 ⁇ m to a value close to the thickness of the disk (e.g., about 1 mm); in preferred embodiments, the interior dimension of the microchannel is from 0.5 ⁇ m to about 500 ⁇ m.
- Microchannel and reservoir shapes can be trapezoid, circular or other geometric shapes as required.
- Microchannels preferably are embedded in a microsystem platform having a thickness of about 0.1 to 25 mm, wherein the cross-sectional dimension of the microchannels across the thickness dimension of the platform is less than 1 mm, and can be from 1 to 90 percent of said cross-sectional dimension of the platform.
- Sample reservoirs, reagent reservoirs, reaction chambers, collection chambers, detections chambers and sample inlet and outlet ports preferably are embedded in a microsystem platform having a thickness of about 0.1 to 25 mm, wherein the cross-sectional dimension of the microchannels across the thickness dimension of the platform is from 1 to 75 percent of said cross-sectional dimension of the platform.
- delivery of fluids through such channels is achieved by the coincident rotation of the platform for a time and at a rotational velocity sufficient to motivate fluid movement between the desired components.
- the flow rate through a microchannel of the invention is inversely proportional to the length of the longitudinal extent or path of the microchannel and the viscosity of the fluid and directly proportional to the product of the square of the hydraulic diameter of the microchannel, the square of the rotational speed of the platform, the average distance of the fluid in the channels from the center of the disk and the radial extent of the fluid subject to the centripetal force. Since the hydraulic diameter of a channel is proportional to the ratio of the cross-sectional area to cross-sectional perimeter of a channel, one can judiciously vary the depth and width of a channel to affect fluid flow (see Duffy et al., 1998, Anal. Chem. 71: 4669-4678 and co-owned and co-pending patent applications U.S. Ser. Nos. 08/761,063, filed Dec. 5, 1996 and 08/768,990, filed Dec. 18, 1996, incorporated by reference).
- fluids of higher densities flow more rapidly than those of lower densities given the same geometric and rotational parameters.
- fluids of lower viscosity flow more rapidly than fluids of higher viscosity given the same geometric and rotational parameters. If a microfluidics structure is displaced along the radial direction, thereby changing the average distance of the fluid from the center of the disc but maintaining all other parameters, the flow rate is affected: greater distances from the center result in greater flow rates. An increase or a decrease in the radial extent of the fluid also leads to an increase or decrease in the flow rate. These depencies are all linear.
- Variation in the hydraulic diameter results in a quartic dependence of flow rate on hydraulic diameter (or quadratic dependence of fluid flow velocity on hydraulic diameter), with larger flow rates corresponding to larger diameters. Finally, an increase in the rotational rate results in a quadratic increase in the flow rate or fluid flow velocity.
- Input and output (entry and exit) ports are components of the microplatforms of the invention that are used for the introduction or removal of fluid components. Entry ports are provided to allow samples and reagents to be placed on or injected onto the disk; these types of ports are generally located towards the center of the disk. Exit ports are also provided to allow products to be removed from the disk. Port shape and design vary according specific applications. For example, sample input ports are designed, inter alia, to allow capillary action to efficiently draw the sample into the disk. In addition, ports can be configured to enable automated sample/reagent loading or product removal. Entry and exit ports are most advantageously provided in arrays, whereby multiple samples are applied to the disk or to effect product removal - from the microplatform.
- the inlet and outlet ports are adapted to the use of manual pipettors and other means of delivering fluids to the reservoirs of the platform.
- the platform is adapted to the use of automated fluid loading devices.
- an automated device is a single pipette head located on a robotic arm that moves in a direction radially along the surface of the platform.
- the platform could be indexed upon the spindle of the rotary motor in the azimuthal direction beneath the pipette head, which would travel in the radial direction to address the appropriate reservoir.
- Another embodiment is a pipettor head adapted to address multiple reservoirs, either a subset of or all of the reservoirs on the platform surface.
- a single head can involve three pipetting units: one for each of the fluids used in a given assay, arranged in a configuration such that all three reservoirs may be addressed without changing the spatial relationship of the pipettes to one another.
- This could be enlarged to a head with six such pipettors, three for the inner ring and three for the outer ring, as illustrated in FIG. 1. Larger numbers of heads could be used but “sparsely” distributed around the platform (for example, 24 heads over 360 degrees). Indexing of an arm containing the head or the table beneath the head could be used to effect filling of the entire disc.
- specially-designed heads that address all reservoirs are also advantageously provided.
- air displacement channels Also included in air handling systems on the disk are air displacement channels, whereby the movement of fluids displaces air through channels that connect to the fluid-containing microchannels retrograde to the direction of movement of the fluid, thereby providing a positive pressure to further motivate movement of the fluid.
- Platforms of the invention such as disks and the microfluidics components comprising such platforms are advantageously provided having a variety of composition and surface coatings appropriate for particular applications.
- Platform composition will be a function of structural requirements, manufacturing processes, and reagent compatibility/chemical resistance properties.
- platforms are provided that are made from inorganic crystalline or amorphous materials, e.g. silicon, silica, quartz, inert metals, or from organic materials such as plastics, for example, poly(methyl methacrylate) (PMMA), acetonitrile-butadiene-styrene (ABS), polycarbonate, polyethylene, polystyrene, polyolefins, polypropylene and metallocene.
- PMMA poly(methyl methacrylate)
- ABS acetonitrile-butadiene-styrene
- PCC polycarbonate
- polyethylene polystyrene
- polyolefins polypropylene and metallocene
- the platforms may also be made from thermoset materials such as polyurethane and poly(dimethyl siloxane) (PDMS).
- PDMS poly(dimethyl siloxane)
- platforms made of composites or combinations of these materials for example, platforms manufactures of a plastic material having embedded therein an optically transparent glass surface comprising the detection chamber of the platform.
- platforms composed of layers made from different materials may be made.
- the surface properties of these materials may be modified for specific applications, as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. Nos. 08/761,063, filed Dec. 5, 1996; 08/768,990, filed Dec. 18, 1996; 08/910,726, filed Aug. 12, 1997; 08/995,056, filed Dec. 19, 1997; and 09/315,114, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein.
- the disk incorporates microfabricated mechanical, optical, and fluidic control components on platforms made from, for example, plastic, silica, quartz, metal or ceramic. These structures are constructed on a sub-millimeter scale by molding, photolithography, etching, stamping or other appropriate means, as described in more detail below. It will also be recognized that platforms comprising a multiplicity of the microfluidic structures are also encompassed by the invention, wherein individual combinations of microfluidics and reservoirs, or such reservoirs shared in common, are provided fluidly connected thereto. An example of such a platform is shown in FIG. 1.
- Microfluidics structures are provided embedded in a substrate comprising the Microsystems platform of the invention.
- the platform is preferably manufactured and assembled as layers containing separate components that are bonded together.
- the exemplified embodiment of the platforms of the invention comprise two layers, a reservoir layer and a microfluidics layer. Platforms having additional layers are also within the scope of the invention.
- the reservoir layer of the platform is manufactured from a thermoplastic material such as acrylic, polystyrene, polycarbonate, or polyethylene.
- fabrication methods include machining and conventional injection molding.
- the mold inserts that are used to define the features of the platform can be created using standard methods of machining, electrical discharge machining, and other means known in the art.
- the reservoir layer of the platform can also be manufactured from a thermoset material or other material that exists in a liquid form until subjected to heat, radiation, or other energy sources.
- thermoset materials include poly(dimethyl siloxane) (PDMS), polyurethane, or epoxy.
- PDMS poly(dimethyl siloxane)
- polyurethane polyurethane
- epoxy epoxy
- these materials are obtained from the manufacturer in two parts; the two parts are mixed together in a prescribed ratio, injected into or poured over a mold and subjected to heat to initiate and complete cross-linking of the monomers present in the pre-polymer fluid.
- the process of rapidly injecting a pre-polymer fluid into a mold and then cross-linking or curing the part is often referred to as reaction injection molding (RIM).
- RIM reaction injection molding
- Mold inserts for RIM or casting can be fabricated using standard methods of machining, electrical discharge machining, and other means known in the art.
- the microfluidics layer of the platform can also be manufactured from a thermoplastic material such as acrylic, polystyrene, polycarbonate, or polyethylene. Because the dimensions of the channels and cuvettes may be much smaller than those found in the reservoir layer, typical fabrication methods with these materials may include not only machining and conventional injection molding but also compression/injection molding, and embossing or coining.
- a thermoplastic material such as acrylic, polystyrene, polycarbonate, or polyethylene.
- typical fabrication methods with these materials may include not only machining and conventional injection molding but also compression/injection molding, and embossing or coining.
- the mold inserts that are used to define the features of this layer of the platform can be created using conventional methods such as machining or electrical discharge machining. For mold inserts with features too fine to be created in conventional ways, various microfabrication techniques are used.
- Etched silicon may also be used to create a metal mold insert through electroplating using, for example, metallic nickel.
- Silicon etched using any one of a variety of techniques such as anisotropoic or isotropic wet etching or deep reactive ion etching (DRIE) may serve as a basis for a metal mold.
- a seed layer of nickel is deposited through evaporation on the silicon; once such an electrically-conductie seed layer is formed, conventional electroplating techniques may be used to build a thick nickel layer.
- the silicon is then removed (Larsson, 1997, Micro Structure Bull. 1: 3).
- the insert is then used in conventional injection molding or compression/injection molding.
- molds can alternatively be created using photolithography without etching the silicon.
- Photoresist patterns are created on silicon or other appropriate substrates. Rather than etching the silicon wafer as in silicon micromachining, the photoresist pattern and silicon are metallized through electroplating, thermal vapor deposition, or other means known in the art. The metal relief pattern then serves as a mold for coining, injection molding, or compression/injection molding as described above.
- the microfluidic layer of the platform can also be manufactured using a thermoset material as described above for production of the reservoir layer, wherein the mold pattern for thermosets of the microfluidics layer is prepared as described above. Because reaction-injection molding and casting do not require the high pressures and temperatures of injection molding, a wider variety of mold patterns may be used. In addition to the use of a silicon or metal mold insert, the photoresist pattern as described can also be used as a mold relief itself. While the photoresist would not withstand the high pressures and temperatures of injection molding, the milder conditions of casting or RIM create no significant damage.
- the assembly of the platform involves registration and attachment of the microfluidic layer to the reservoir layer.
- certain microfluidics pathways in the reservoir layer must be connected to certain microfluidics pathways in the microfluidics layer. Registration of these microfluidics pathways may be accomplished through optical alignment of fiducial marks on the microfluidic and reservoir layers or through mechanical alignment of holes or depressions on the microfluidic layer with pins or raised features on the reservoir layer. The required registration tolerances may be relaxed by designing the microfluidics pathway in the reservoir layer to be much larger than the microfluidics pathway in the microfluidics layer, or vice versa.
- Attachment may be accomplished in a number of ways, including conformal sealing, heat sealing or fusion bonding, bonding with a double-sided adhesive tape or heat-sealable film, bonding with a ultraviolet (UV) curable adhesive or a heat-curable glue, chemical bonding or bonding with a solvent.
- conformal sealing heat sealing or fusion bonding
- bonding with a double-sided adhesive tape or heat-sealable film bonding with a ultraviolet (UV) curable adhesive or a heat-curable glue
- UV ultraviolet
- a requirement for conformal sealing is that one or both of the layers are made of an elastomeric material and that the surfaces to be bonded are free of dust or debris that could limit the physical contact of the two layers.
- an elastomeric microfluidics layer is registered with respect to and then pressed onto a rigid reservoir layer.
- the elastomeric microfluidics layer may be advantageously made of silicone and the rigid reservoir layer may be advantageously made of acrylic or polycarbonate. Hand pressure allows the layers to adhere through van der Waals forces.
- both the reservoir and microfluidics layers are made of thermoplastic materials and that the sealing occurs at temperatures above the glass transition temperatures, in the case of amorphous polymers, or melting temperatures, in the case of semi-crystalline polymers, of both of the layer materials.
- the microfluidics layer is registered with respect to and pressed onto the reservoir layer, this composite disk is then placed between two flat heated blocks and pressure is applied to the composite through the heated blocks.
- the temperature versus time profile at each of the faces of the composite disk and by adjusting the pressure versus time profile that is applied to the composite system, one can determine the time-temperature-pressure profile that allows for bonding of the two layers yet minimizes variation of the features within each of the layers. For example, heating two acrylic disks from room temperature to a temperature just above the glass transition temperature of acrylic at a constant pressure of 250 psi over one hour is a recipe that allows for minimal variation of 250 ⁇ m wide fluidic channels.
- the bond surfaces of the microfluidics and reservoir layers are separately heated in a non-contact fashion with radiative lamp and when the bond surfaces have reached their glass transition temperatures the microfluidics layer is registered with respect to and pressed onto the reservoir layer.
- a double-sided adhesive tape or heat sealable film may be used to bond the microfluidics and reservoir layers. Before bonding, holes are first cut into the tape (or film) to allow for fluid communication between the two layers, the tape (or film) is registered with respect to and applied onto the reservoir layer, and the microfluidics layer is registered with respect to and applied onto the tape(or film)/reservoir layer composite.
- a heat-sealable film In order to bond a heat-sealable film to a surface, it is necessary to raise the temperature of the film to above the glass transition temperature, in the case of an amorphous polymer, or the melting temperature, in the case of a semicrystalline polymer, of the film's adherent polymer material.
- an adequate bond can typically be achieved with hand pressure.
- a photopolymerizable polymer for example, a UV-curable glue
- a heat-curable polymer may be used to adhere the microfluidics and reservoir layers.
- this glue is applied to one or both of the layers.
- Application methods include painting, spraying, dip-coating or spin coating. After the application of the glue the layers are assembled and exposed to ultraviolet radiation or heat to allow for the initiation and completion of cross-linking or setting of the glue.
- the microfluidics and reservoir layers are each fabricated with a set of fluid channels that are to be used only for the glue. These channels may, for example, encircle the fluid channels and cuvettes used for the assay.
- the microfluidics layer is registered with respect to and pressed onto the reservoir layer.
- the glue is pipetted into the various designated channels and after the glue has filled these channels, the assembled system is exposed to ultraviolet radiation or heat to allow for the cross-linking or setting of the glue.
- a requirement for solvent bonding is that the bond surfaces of both the microfluidics and reservoir layers can be solvated or plasticized with a volatile solvent.
- the bond surfaces are each painted with the appropriate solvating fluid or each exposed to the appropriate solvating vapor and then registered and pressed together. Plasticization allows the polymer molecules to become more mobile and when the surfaces are brought in contact the polymer molecules become entangled; once the solvent has evaporated the polymer molecules are no longer mobile and the molecules remain entangled, thereby allowing for a physical bond between the two surfaces.
- the microfluidics and reservoir layers are each fabricated with a set of fluid channels that are to be used only for the solvent and the layers are bonding much like they are with the UV-curable or heat-curable glue as described above.
- FIG. 1 shows an exploded view of an example of a disc appropriate for large numbers of similar or identical microfluidics structures for performing, inter alia, liquid-phase assays.
- the disc shown here performs 96 assays of the general form: mix first fluid A with second fluid B, and then mix the combined fluids (A+B) with third fluid C.
- These assays are “independent”: fluids A, B, and C are loaded through appropriate entry ports into the individual reservoirs for each assay. In this way, fluids A, B, and C need not be identical for all assays, but can be chosen by the user.
- platforms having a greater number of such microfluidics structures are contemplated by the invention.
- This disc illustrates that identical assays may be made by repeating microfluidics structures around the disc at a given radius as well as modifying the structures for placement at different radial positions. In this way, it is possible to fully cover the surface of the disc with microfluidics structures for performing assays.
- the maximum number of assays that may be performed will depend upon the volume of fluid that may be manipulated reproducibly, i.e., the minimum reproducible dimensions with which the disc may be fabricated, and the amount of hydrodynamic pressure required to drive small volumes of fluid through microchannels at convenient rotational rates. Taking these considerations into account, it is estimated that greater than 10,000 assays having volumes of 1-5 nL can be created in a circular platform having a 6 cm radius.
- platform 100 is composed of at least two component layers.
- a reservoir layer 201 is bonded to a microfluidics layer 301 .
- the bottom face of the reservoir layer when mated with the microfluidic layer described below, forms a complete network of enclosed channels and reservoirs through which fluids flow under the impetus of centripetal force created by rotation of the platform about a central axis.
- Fluid flow permits mixing of various component fluids in the assay and movement of the fluids from sample and reagent reservoirs through mixing structures and into assay collection chambers.
- fluid flow can be effectuated to include incubation and wash steps, using structures disclosed in co-owned U.S. Pat. No.
- Platform 100 is preferably provided in the shape of a disc, a circular planar platform having a diameter of from about 10 mm to about 50 mm and a thickness of from about 0.1 mm to about 25 mm.
- Each layer comprising the platform preferably has a diameter that is substantially the same as the other layers, although in some embodiments the diameters of the different layers are not required to completely match.
- Each layer has a thickness ranging from about 0.1 mm to about 25 mm, said thickness depending in part on the volumetric capacity of the microfluidics components contained therein.
- FIG. 2 depicts the “bottom” face to more clearly illustrate this embodiment of the platforms of the invention. By illustrating the surface in this way it is easier to see how the features are aligned with the microfluidic layer 301 (as shown in FIGS. 4 and 5 as described more fully below).
- Reservoir layer 201 is preferably provided in the shape of a disc, a circular planar platform having a diameter of from about 10 mm to about 50 mm and a thickness of from about 0.1 mm to about 25 mm.
- the layer preferably comprises a center hole 202 for mounting on a spindle, having a diameter of from about 1 mm to about 20 mm.
- Center hole 202 can be replaced by an extruded fitting for connection to a spindle, or may be absent entirely, in which case registry and connection to the spindle is accomplished using the attached microfluidic layer or another portion of the surface of the platform.
- Reservoir layer 201 can also include registry features such as the groove 203 that permits a clamping fixture above the platform to be brought in proximity with, but not in contact with, the top surface of the platform when the platform is loaded into the spindle.
- a pin on the clamping fixture preferably spring-loaded, slips into the groove as the disc is spun at low rpm, and captures the clamping fixture, thus determining the platform's orientation with respect thereto.
- the platform comprises “home-flag” 204 , that is a reflective or absorbing stripe that can be positioned on the surface of the platform and sensed by an emitter/photodiode pair as the disc is spun, thus permitting the orientation of the disc with respect to the instrument to be determined.
- FIG. 3 illustrates an expanded view of a section of the reservoir layer.
- this embodiment of the platforms of the invention contains three reservoirs plus one collection/detection chamber for each assay, arrayed in two concentric rings of assays.
- Each reservoir has dimensions of from about 0.05 mm to about 5 mm wide, from about 0.05 mm to about 20 mm long, and from about 0.05 mm to about 5 mm thick, and has a volumetric capacity of from about 0.1 nL to about 500 ⁇ L.
- Reservoirs 205 , 206 , and 207 are designed to contain fluids A, B, and C for the inner ring of assays, while reservoir 211 is the detection chamber for the inner set of assays.
- reservoirs 208 , 209 , and 210 contain fluids A, B, and C for the outer ring of assays, with 212 the corresponding detection chamber.
- the reservoirs of the inner rings also contain loading holes, 213 , through which the fluids are loaded prior to use.
- Loading holes 213 preferably have dimensions adapted to automated loading devices such as micropipettors, for example, a standard 200 ⁇ L plastic pipette tip having a tip diameter of 1.5 mm; micropipette tips of diameter 1 mm; piezoelectric or ceramic drop delivery systems (such as are sold by the IVEK Corp., Springfield, Vt.); and inkjet-based fluid delivery systems.
- the dimensions of the ports must be a few times greater than the size of the droplets, e.g., 0.2 mm for a 1 nL drop.
- the assay collection/detection chambers also contain air displacement holes 214 that allow air displaced by the motion of fluids to escape, having a cross-sectional dimension of from about 100 to about 500 ⁇ m. These holes may optionally be replaced by a manifold or series of channels connecting the receiving reservoirs to one or more air hole.
- the collection/detection chambers are designed to be accessible to optical interrogation, for example, by being composed of optically-transparent plastics or other materials.
- FIGS. 4 and 5 The microfluidics layer of the embodiment of the platform of the invention is shown in FIGS. 4 and 5.
- Microfluidics layer 301 is optimally of the same lateral dimensions as the microfluidics layer. There is also an optional center hole 302 for mounting on a spindle, although this is not required in all configurations.
- the microfluidics layer contains an array 303 of microfluidic structures 304 , the number of structures in the array being equal to one half of the number of parallel assays to be run on the platform. In the embodiment illustrated in the Figures, there are 48 such structures repeated with angular spacing of 7.5°.
- Microfluidics structures 304 preferably comprise microchannels having cross-sectional dimensions of from about 5 ⁇ m to about 5001 ⁇ m and a depth in the microfluidics layer of from about 10 ⁇ m to about 3 mm.
- FIG. 5 is an expanded view of a single unit of microfluidic structures.
- Each microfluidics structure comprises the microfluidics for one inner and one outer ring assay.
- the microfluidic structure consists of depressions in the surface of the microfluidic disc of a single or multiple depths ranging between 2 microns and 1000 microns, while the widths of the depressions varies from about 2 ⁇ m to about 500 ⁇ m, as further described below.
- microchannels 305 and 306 are aligned by assembly between the reservoir layer and microfluidics layer so that the microchannels protrude into reservoirs 205 and 206 , respectively.
- the microchannel 307 is an expansion space for air displaced by fluids flowing through microchannels 305 and 306 into the, microchannel 308 .
- Microchannels 305 and 306 in some embodiments narrow to form capillary junctions 309 before joining mixing microchannel 308 .
- Mixing microchannels are configured to provide mixing of different solutions as the mixture traverses the longitudinal extent of the microchannel.
- the degree of mixing is dependent on the flow rate of the fluids and the longitudinal extent of the mixing microchannel, which is proportional to the amount of time the two fluids are in contact and are mixed together.
- the degree of mixing is also dependent on the lateral extent of the mixing microchannel, and is further dependent on the diffusion constants of the fluids to be mixed.
- the mixing microchannels are provided as shown in FIG. 5. Mixing is promoted as illustrated in FIG.
- Mixing microchannel 308 has a length of from about 1 mm to about 100 mm, its length in some cases achieved through the use of bends.
- Mixing microchannel 308 is provided with a capillary junction at 310 wherein the interior diameter of the microchannel changes by between about 0 to 95%, and then joins capillary junction 311 .
- Capillary junction 311 is larger in the lateral or vertical direction or both than the capillary junction at 310 .
- the mixing time is 500 seconds, an unacceptably long time for most reactions.
- This mixing time may be reduced by mechanical stirring, for example, but stirring is difficult to obtain in fluids confined in small structures because the flow of the fluid is laminar and does not contain turbulent eddies that are known to promote mixing. If, instead of placing fluids A and then B in a 1 mm 3 container, fluids A and B were placed side-by-side in a long, thin capillary of lateral dimension d, the relevant time for mixing is much shorter. If, for example, d is 100 microns, mixing time t is 20 seconds.
- the mixing channels of the device simulate the placement of fluid in a long capillary by co-injecting fluid streams A and B into a capillary microchannel. These fluids flow side-by-side down the channel initially. As the fluid is pushed through the microchannel due to centrifugal force produced by rotation of the platform, diffusion occurs between the fluids.
- a capillary of sufficiently narrow diameter, sufficient length, and a pumping rate that is sufficiently low the portion of A and B of the total volumes of A and B present in the channel during pumping can be caused to mix.
- w is the lateral size of the channel.
- the amount of time necessary to traverse the channel is simply the length of the channel divided by the fluid velocity, the velocity being calculated as described in co-owned and co-pending U.S. Ser. No. 08/910,726, filed Aug. 12, 1997, and Duffy et al. (1999, Anal. Chem.
- the fluid properties are the density ⁇ and viscosity ⁇ , ⁇ R and ⁇ R> are the extent along the radius and average radial position of the fluid subject to centripetal acceleration, and l and d H are the length and hydraulic diameter of the channel.
- Entry 312 to microchannel 313 protrudes into reservoir 207 and preferably forms capillary junction 314 , having dimensions substantially the same as capillary junction 311 .
- Microchannel 313 passes through a change in the lateral dimension at 315 wherein the interior diameter of the microchannel changes by between 0% and 95% and then joins capillary junction 311 .
- the capillary junction leads to a further mixing microchannel 327 that terminates at end 328 and that protrudes into detection chamber 211 .
- Mixing microchannel 327 has a length of from about 1 mm to about 100 mm, and preferably comprises one or a multiplicity of bends as illustrated in the Figures.
- the structure of the outer ring microfluidics is as follows. Microchannels 316 and 317 are aligned by assembly between the reservoir layer and microfluidics layer so that the microchannels protrude into reservoirs 208 and 209 , respectively.
- the microchannel 318 is an expansion space for air displaced by fluids flowing through microchannels 316 and 317 into microchannel 319 .
- Microchannels 316 and 317 in some embodiments narrow to form capillary junctions 320 before joining mixing microchannel 319 .
- Mixing microchannel 319 passes through a change in the lateral dimension at 321 to the capillary junction 322 .
- the entry 323 to microchannel 324 protrudes into reservoir 210 and may form a capillary junction 325 .
- Microchannel 324 passes through a change in the lateral dimension at 326 to the capillary junction 322 .
- the capillary junction leads to a further mixing microchannel 329 that terminates at end 330 that protrudes into detection chamber 212 .
- Mixing microchannel 329 has a length of from about 1 mm to about 100 mm, and preferably comprises one or a multiplicity of bends as illustrated in the Figures.
- the dimensions of the microfluidic components for the outer ring structures can (as here) be the same as for the corresponding components described above for the inner ring microfluidics structures.
- the dimensions of the components are chosen so that the fluid flow rates are substantially the same in each microfluidics structure, without regard to the position of the structure on the platform.
- the important considerations for designing the platforms of the invention are microchannel diameters, lengths, and positioning on the disc, which determines the flow-rate of fluid at a given rotational rate; and the radial position of and diameters of channels leading into capillary junctions, which determines the rotational speed at which passive or capillary valving occurs.
- the assays would run identically (i.e., at the same rotational speeds) using the same component dimensions for the inner and outer ring structures. This is a consequence of the length of the mixing microchannels, which were long enough to permit substantially complete mixing to occur even through the flow-rate of fluids in the outer assay ring is higher than that in the inner ring. Additionally, the separation in rotational speed between the two capillary valving events for each assay was large enough that the sequence of fluidic motions necessary for performing the assay was obtained for both inner and outer assay rings. In alternative embodiments, the dimensions of the microfluidics components between the inner and outer rings may be different, depending on the considerations set forth herein.
- k is a constant dependent upon the fluid density, surface tension, and contact angle of the fluid on the capillary junction; ⁇ R ij > is the average radial position of the fluid radially-inward of the capillary junction; ⁇ R ij is the radial extend of the fluid radially-inward of the capillary junction; and d ij H is the hydraulic diameter of the channel used as a capillary junction (as discussed above).
- ⁇ R ij > and ⁇ R ij are somewhat constrained by placement on the disc.
- ⁇ R ij > is proportional to the radial position; ⁇ R ij is the same for all i; and therefore d ij H must be varied inversely proportional to the radial position to maintain a constant product of the three variables, and hence the valving rotational speed.
- FIG. 6 illustrates three assay sectors of the assembled platform, in which the reservoirs of the reservoir layer are mated to microchannels from the microfluidics layer.
- the platform layers were mated as described in more detail above and in Example 1.
- capillary valving is understood to depend on geometry, fluid properties and rotational rate, as disclosed more fully in U.S. Ser. Nos. 08/761,063, filed Dec. 5, 1996; 08/768,990, filed Dec. 18, 1996; and 08/910,726, filed Aug. 12, 1997, incorporated by reference herein.
- Alternative arrangements of the microfluidic layers of the platforms of the invention can be provided to contain any number of concentric rings of assays consistent with the amount of surface area available on the platform surface and the extent of the surface taken up by any one embodiment of microfluidics required to perform an assay.
- a detector that can access cuvettes at a variety of radial and azimuthal positions.
- An example of a scanning optical system would be one in which the optical signal is scanned radially, while the disc could be indexed beneath the optics azimuthally. In this way the optics can address any point on the disc surface.
- Scanning methods include a detector on a linear drive that moves radially; alternately, the optical signal may be scanned radially through the use of a galvanometrically-controlled mirror system.
- fluidic design A number of variations in fluidic design are possible, either dictated by assay requirements, fluidic requirements, ease-of-use or reduction in automation or all of these factors.
- capillary valves have been shown to retain fluids in an intermediate chamber at elevated temperatures, used for incubation (as disclosed more extensively in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, incorporated by reference).
- Assays that require intermediate incubations, for example, because of slow chemical kinetics, may be performed in such structures.
- assays for which diffusional mixing is insufficient may require agitation of the fluid to effect mixing.
- active valves can be used, which retain the fluids against the sudden pressure changes induced by agitation, as described in more fully in co-owned and co-pending U.S. Ser. No. 09/315,114, filed May 19, 1999.
- a platform as depicted in the Figures was prepared as follows.
- the reservoir layer was manufactured through machining of acrylic using computer/numerical code machining using a Light Machines VMC5000 milling machine running Light Machines “Benchman” software (Light Machines Corporation, Manchester, N.H.).
- the microfluidics layer was manufactured as follows.
- a microfluidics structure such as the structure shown in FIG. 5 was designed using in a computer aided design package such as AutoCAD (Autodesk, San Rafael Calif.) and Freehand (Macromedia Inc., San Fransisco, Calif.). This design was converted into a photomask by printing at high resolution (3386 dpi) on a transparent plastic sheet.
- a 125-mm diameter silicon wafer was coated with a layer of negative photoresist (SU-8(50)) and spun on a spin-coater (obtained from Chemat Technology, Northridge, Calif.) at a speed sufficient (200 to 8000 rpm) to give a desired thickness between 5 ⁇ m and 500 ⁇ m.
- the silicon wafer was baked to have a smooth surface and then the photoresist partially cured.
- the silicon wafer was exposed to ultraviolet (UV) light using a conventional UV source and mask aligner.
- UV ultraviolet
- the photoresist was then developed in propylene glycol methyl ether acetate and non-crosslinked photoresist removed through washing in dichloromethane.
- the resulting relief was then passivated by exposure to a vapor of tridecafluoro-1,1,2,2-tetrhydrooctyl-1-trichlorosflane and used as a mold for microfabrication (as described in Duffy et al., 1998, Anal. Chem. 70: 4974-4984).
- PDMS polydimethylsiloxane
- crosslinking agent Sylgard 184, Dow Corning
- the microplatform was assembled by forming a reversible, conforming seal between the PDMS microfluidics layer and the acrylic reservoir layer made through simple physical contact of the two components. This seal is based on physical adhesion forces alone—van der Waals attraction forces and potentially static electrical charge present on the surfaces—and was sufficient to seal the disc against leakage due to the centripetally-induced pressures used.
- FIG. 1 The platform shown in FIG. 1 and prepared as described herein was used to perform simultaneously and in parallel ninety-six enzyme inhibition assays, in order to demonstrate that the microfluidics platforms of the invention provide an alternative to enzyme assays performed in conventional 96-well microtitre plates. Fluids were deposited in the reservoirs formed in reservoir layer 201 when reservoir layer is mated or bonded with microfluidics layer 301 . Platform 100 was then rotated using a rotational profile designed to drive fluids through the microchannels within microfluidics disc 301 .
- FIGS. 1 through 6 The platform shown in FIGS. 1 through 6 was used to perform 96 simultaneous enzyme inhibition assays as model homogeneous assays.
- an enzyme inhibition assay the effect of a compound present in a first fluid (“A”) upon the capacity of an enzyme present in a second fluid (fluid “B”) to catalyze a reaction, typically of a substrate in a third fluid (“C”) is determined.
- the reaction was chosen to give a change in a readily-measured parameter of the fluid, such as its optical density, or to produce a fluorescent moiety.
- a readily-measured parameter of the fluid such as its optical density, or to produce a fluorescent moiety.
- fluid A if an inhibitor was present in fluid A, mixing fluid A with fluid-B resulted, after a sufficient time in a chemical reaction or other change induced by the inhibitor in most or all of the enzyme molecules, rendering them incapable of catalyzing the desired reaction. If this solution was mixed with the substrate solution, little or no change in the measured parameter was seen.
- the system chosen to model homogeneous assays consisted of theophylline as inhibitor, alkaline phosphatase as the enzyme, and p-nitrophenol phosphate (PNPP) as the substrate.
- PNPP p-nitrophenol phosphate
- Theophylline was used in concentrations of 0.01 mM to 100 mM to provide a standard dose-response curve in the inhibitor.
- Alkaline phosphatase was used in a 1 mg/mL solution, and PNPP was used as a 0.5 mM solution. All solutions were made in a buffer of 0.1M glycine and 0.5 mM MgCl 2 in deionized water.
- the dimensions of the platform used for these assays were as follows.
- the overall platform diameter was 12 cm.
- the reservoir layer was about 3.2 mm thick.
- the radial positions of the ends of reservoirs 205 , 206 , and 207 most proximal to the center of the disc were about 3.25 cm and will be denoted as “inner radii;” the radial positions of the ends 205 , 206 , and 207 most distant from the center of the disc were about 3.7 cm and will be denoted “outer radii.”
- Reservoirs 205 , 206 and 207 were about 1.2 mm deep. The angle subtended by each reservoir was chosen such that it could accommodate appropriate volume.
- Reservoirs 205 and 206 subtended angles of about 1.6 degrees, while reservoir 207 subtended an angle of about 2.1 degrees such that reservoirs 205 and 206 could accommodate a 3 ⁇ L volume and reservoir 207 accommodated a 6 ⁇ L volume.
- the inner radii were about 4.1 cm, the outer radii were 4.5 cm, and the platform was about 3.2 mm thick.
- Detection chambers 211 and 212 were constructed of optically-transparent material and had an outer radius of about 5.7 cm and an inner radius of about 5 cm, were 1 mm deep, and had a subtended angle of 2.7°.
- the microfabricated layer was also 12 cm in diameter and had a thickness between 1 and 5 mm (although the thickness is not important) and was composed of white PDMS.
- the depth of all microfluidic structures (that was determined by the height of the SU-8 relief) was 100 ⁇ m.
- the width of mixing microchannels 308 , 313 , 327 , 319 , 324 and 329 was 100 ⁇ m.
- the lengths of the mixing microchannels was chosen to provide sufficient time for mixing via diffusion with liquids of moderate diffusion constant (8 ⁇ 10 ⁇ 6 cm 2 /s) as fluids were pumped through them under the influence of centripetal acceleration.
- Fluid flow was controlled on the platforms by capillary valving as described in co-owned U.S. Pat. No. 6,063,589, and co-owned and co-pending U.S. Ser. Nos. 08/761,063, filed Dec. 5, 1996; 08/768,990, filed Dec. 18, 1996; and 08/910,726, filed Aug. 12, 1997 incorporated by reference.
- For each inner assay microfluidics structure there were four capillary valving points: at the junction of microchannels 305 and 306 with microchannel 307 ; at the junction of microchannel 312 with microchannel 314 ; at the junction of constriction 315 with capillary junction 311 ; and at the junction of constriction 310 with capillary junction 311 .
- the widths of the channels leading into the junctions was 100 ⁇ m for microchannels 305 and 306 ; 200 ⁇ m for microchannel 312 ; and 50 ⁇ m for capillary junctions 310 and 315 .
- the corresponding microchannels have identical widths at those junctions as for the junctions in the inner assay arrays (100 ⁇ m for microchannels 316 and 317 ; 200 ⁇ m for microchannel 323 ; and 50 ⁇ m for capillary junctions 321 and 326 .
- the assays were run as follows. 3 ⁇ L aliquots of alkaline phosphatase and theophylline solutions having the concentrations set forth above were loaded into reservoirs 205 and 208 (alkaline phosphatase) and 206 and 209 (theophylline). 6 ⁇ L aliquots of PNPP solution were loaded into reservoirs 207 and 210 . The platform was placed on the spindle of an instrument containing a diffuse reflectance optical head capable of three-color measurements.
- the platform was first rotated at 750 rpm for 10-30 seconds in order to “prime” the microchannels 305 , 306 , 312 , 316 , 317 , and 323 (i.e., to pump the fluid into the microchannels to the point where they were retained at the enlargements forming the capillary junctions).
- the rate of rotation was then increased at an acceleration rate of 45 rpm/sec to a rotational speed of 840 rpm.
- the pressure induced by centrifugation at capillary junctions 309 , 314 , 320 and 325 is sufficient to overcome the capillary pressure and fluids entered channels 308 , 313 , 319 and 324 .
- the rotational rate of 840 rpm was maintained for 30 seconds (though in practice this could be reduced as low as a few seconds), during which time fluids traversed the channels to the capillary junctions at 310 , 315 , 321 and 326 , where they were retained due to capillary pressure.
- the rotational rate was then increased at an acceleration rate of about 180 rpm/sec to a rotational speed of 1200 rpm and maintained there for 1 sec. At this rotation rate the fluids burst past the capillary junctions at 311 and 322 and entered microchannels 327 and 329 .
- the rotational velocity was then reduced to 600 rpm at a rate of 150 rpm/s and maintained for 50 seconds to effect pumping of the fluids into the detection chambers 211 and 212 .
- FIGS. 7 a through 7 f illustrate the sequence of fluid flows.
- fluid began to flow from reservoirs 205 , 206 and 207 and was halted at constriction 310 .
- the substrate solution flowed from 207 to constriction 315 . If either the fluid in 205 or 206 flowed into 307 and 308 first, the narrowness of the channel 308 provided sufficient resistance that fluid the fluid accumulated in expansion area 307 , thereby wetting the protruding liquid interface of the opposite channel. In this way both fluids are guaranteed to be brought into contact and into the microchannel together.
- the diameters of the capillary junctions x and y may be chosen to pre-dispose one or the other of the fluids to flowing at a lower rotational rate. For example, if there is variability in the surface tension of one fluid because of compositional variations (for example, if it is a biological fluid), it may be desirable to effect the motion of the other fluid first. In this case the capillary junction of the variable fluid is made narrower in a way to insure that the capillary valving rpm for all reasonable variations in fluid properties is higher than that of the other fluid.
- a A and B B are the cross-sectional area of the reservoirs 205 and 206 as a function of time, or alternately, radial position of the meniscus as fluid is removed from the reservoirs. If it was desired that the ratio of flows is constant (as was the case here), it was sufficient to maintain a constant ratio of cross-sectional areas as a function of radial position. Note that this does not imply that the cross sections are constant, just that their ratio is. The ratio expressed in the equation can be manipulated by altering the ratio of cross-sectional areas of-the reservoirs, as disclosed more fully in co-owned U.S. Pat. No.6,063,589, issued May 16, 2000 and incorporated by reference.
- This equation and analysis also accurately describes the significance of the ratio of the three fluids in mixing microchannel 327 .
- the pressure induced by rotation works against the hydraulic resistance to flow the reservoirs 205 , 206 , and 207 ; the hydraulic resistance of the channels 308 , 313 , and 319 ; and the mixing microchannel 327 .
- the lengths and diameters of 308 , 313 , and 319 are chosen to be identical, the hydraulic resistance across these channels are identical as well.
- the hydraulic resistance of mixing microchannel 327 and the reservoirs 205 , 206 , and 207 are the determining factors for the relative flow-rates of the fluids.
- microchannel 327 is much greater than that of the reservoirs due to its much smaller diameter, the pressure-equalization phenomenon described above again results in meniscuses for all three fluids that move from the inner portion of the reservoirs to the outer portion as fluid is drained.
- alkaline phosphatase and theophylline solutions enter microchannel 308 as co-flowing laminar streams. Because microchannel 308 is long and the flow rates are controlled by rotational rate, these co-flowing streams are present for a time long enough for diffusion across the interface between these streams to effect complete mixing of the solutions. Similar statements may be made about fluid flow in microchannel 327 .
- reflectance optics was used to measure the reflected radiation at an off-specular (diffuse) angle at two wavelengths, 430 nm (absorbing for the expected reaction product, PNP) and 630 nm. As there is no absorbance from reaction product PNP at 630 nm, this wavelength can be used to correct for optical imperfections in the platform, stray scattering, or unintended air bubbles in the optically-transparent chamber.
- the optical system also advantageously contained a beam-splitter that sent a fraction of the incident light to a reference photodiode.
- the assay detector which measured diffusely-reflected light
- the reference detector which measured a fraction of the incident light. Measurements at each detector were made when both the 430 nm and 630 nm light sources were active as well as when they were “dark” or off. The measured voltages were thus:
- C PNP is the concentration of yellow product, p-nitrophenol; this concentration is inversely related to the concentration of theophylline in the initial solution.
- FIG. 8 shows data for 48 assays run simultaneously on the platform, representing three-fold replicates for each of fifteen theophylline concentrations ranging from 0 to 10 mM.
- End-point analysis is also possible by choosing a time—here, 2 minutes—after the fluid is pumped into the detection chambers for the obtaining optical data.
- FIG. 9 shows the end-point measurement (dose-response curve).
- an inhibition constant of (9.7 ⁇ 0.9)mM was determined.
- the coefficients of variation are approximately 3 to 3.5%, of which approximately 1.5% was due to instrumentation and disc variations, as determined through analysis of known concentrations of pre-mixed reagents or dyes.
- microplatform systems according to the invention can be used as a substitute for conventional 96-well microtitre plates for performing enzyme assays to determine enzymatic activity thereof.
- a further type of homogeneous assays used in high-throughput drug screening are serine protease assays. Interest in these enzymes derives from their implication in a wide variety of normal and pathologic metabolic conditions. Serine proteases include:
- HTS serine protease high throughput screening
- An exemplary serine protease is trypsin. It is a vital part of intercellular metabolism but is released when cells lyse. Its proteolytic activity is so potent that animals produce a constitutive inhibitor, ⁇ 1 -antitrypsin ( ⁇ 1 -AT), that is maintained at significant blood levels, even pre-natally. A number of genetic mutations that reduce or eliminate ⁇ 1 -AT activity in the blood have been identified. An estimated 10% of the US population is heterozygous or homozygous for one of these mutations. Even the heterozygous population is prone to chronic liver and lung diseases. In addition, chronic inflammation releases trypsin levels that help to overwhelm the body's ability to make extra ⁇ 1 -AT in heterozygous normal individuals.
- Example 1 The platform described in Example 1 was used to demonstrate a model assay for a trypsin inhibitor, ovomucoid inhibitor.
- manual performance requires: mixing of trypsin with ovomucoid inhibitor; incubation at 37° C. for 30 minutes; mixing of first fluid mixture with substrate BODIPY FI (casein substrate); and fluorescence detection
- the assay was performed on the bench using the above protocol. Trypsin was used at a concentration 50 ⁇ g/mL. Ovomucoid inhibitor was used in the ranges of 0-50 ⁇ g/mL. The fluorescent substrate BODIPY Fl was used in a concentration of 33 ⁇ g/mL.
- This substrate yields a fluorescent peak at 520 nm when it has been cleaved by trypsin. Incubation was performed using a standard laboratory incubator. The fluid was then transferred into the detection cuvettes of the disc described above. The disc was placed on an epiflourescence microscope with a band-pass filter centered at 520 nm. The disc was indexed beneath the objective of the microscope and the fluorescent signal measured.
- FIGS. 10 a and 10 b show both the using the disc and instrument of the invention. These data show comparable performance on-disc for relevant concentrations of ovomucoid inhibitor, 0-50 ⁇ g/mL, even in the absence of the incubation step.
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Also Published As
Publication number | Publication date |
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EP1230544A2 (en) | 2002-08-14 |
WO2000079285A9 (en) | 2002-10-03 |
AU5494900A (en) | 2001-01-09 |
EP1230544B1 (en) | 2004-07-28 |
WO2000079285A2 (en) | 2000-12-28 |
WO2000079285A3 (en) | 2002-06-20 |
JP2004529312A (ja) | 2004-09-24 |
ATE272213T1 (de) | 2004-08-15 |
US6582662B1 (en) | 2003-06-24 |
DE60012562D1 (de) | 2004-09-02 |
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