U.S. patent application number 10/378795 was filed with the patent office on 2003-08-14 for bidirectional flow centrifugal microfluidic devices.
This patent application is currently assigned to Tecan Trading AG.. Invention is credited to Carvalho, Bruce L., Kellogg, Gregory J..
Application Number | 20030152491 10/378795 |
Document ID | / |
Family ID | 22757246 |
Filed Date | 2003-08-14 |
United States Patent
Application |
20030152491 |
Kind Code |
A1 |
Kellogg, Gregory J. ; et
al. |
August 14, 2003 |
Bidirectional flow centrifugal microfluidic devices
Abstract
This invention relates to methods and apparatus for performing
microanalytic and microsynthetic analyses and procedures. The
invention particularly provides microsystem platforms for achieving
efficient mixing of one or a plurality of fluids on the surface of
the platform when fluid flow is motivated by centripetal force
produce by rotation.
Inventors: |
Kellogg, Gregory J.;
(Cambridge, MA) ; Carvalho, Bruce L.; (Watertown,
MA) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Assignee: |
Tecan Trading AG.
|
Family ID: |
22757246 |
Appl. No.: |
10/378795 |
Filed: |
March 4, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10378795 |
Mar 4, 2003 |
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09858581 |
May 15, 2001 |
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6527432 |
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60204264 |
May 15, 2000 |
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Current U.S.
Class: |
422/400 ;
366/182.1; 366/220; 422/72; 436/45 |
Current CPC
Class: |
Y10T 436/111666
20150115; G01N 2035/00524 20130101; B01L 2300/0806 20130101; B01L
2400/0677 20130101; B01L 2300/10 20130101; B01F 35/71 20220101;
B01L 2200/0605 20130101; B01L 3/50273 20130101; B01L 3/5025
20130101; B01L 3/502715 20130101; B01L 3/502738 20130101; B01L
2300/0874 20130101; G01N 35/00069 20130101; B01L 2400/088 20130101;
G01N 21/07 20130101; B01L 7/00 20130101; B01L 7/525 20130101; B01F
33/30 20220101; B01L 2300/0864 20130101; B01L 2300/1805 20130101;
G01N 2035/00514 20130101; B01L 2300/0867 20130101; G01N 2035/00504
20130101; B01F 25/4331 20220101; B01L 2400/0409 20130101; A63B
63/083 20130101; B01L 2400/0688 20130101; B01L 3/5027 20130101;
B01L 3/502723 20130101; G01N 2035/00495 20130101; B01L 2400/0406
20130101; B01F 35/71725 20220101 |
Class at
Publication: |
422/99 ; 422/72;
436/45; 366/182.1; 366/220 |
International
Class: |
G01N 001/00; B01F
001/00; B01L 003/02 |
Claims
We claim:
1. A centripetally-motivated microsystems platform comprising: a) a
rotatable platform comprising a substrate having an axis of
rotation and a surface comprising one or a multiplicity of
microfluidics structures embedded in the surface of the platform,
wherein each microfluidics structure comprises i) one or a
plurality of fluid reservoirs, ii) one or a plurality of detection
chambers, iii) one or a plurality of mixing microchannels wherein
the interior surface of each of the microchannels comprises a
graded hydrophobic surface, wherein the hydrophobicity of the
surface increases with distance from the axis of rotation, and
wherein each of said fluid reservoir is fluidly connected to a
mixing microchannel that is fluidly connected to a detection
chamber, and wherein fluid within the microchannels of the platform
is moved through said microchannels by centripetal force arising
from rotational motion of the platform for a time and a rotational
velocity sufficient to move the fluid through the
microchannels.
2. A centripetally-motivated microsystems platform comprising: a) a
rotatable platform comprising a substrate having an axis of
rotation and a surface comprising one or a multiplicity of
microfluidics structures embedded in the surface of the platform,
wherein each microfluidics structure comprises i) one or a
plurality of fluid reservoirs, ii) one or a plurality of detection
chambers, iii) one or a plurality of mixing microchannels wherein
the interior surface of each of the microchannels comprises a
graded surface to volume ratio, wherein the surface to volume ratio
increases with distance from the axis of rotation, and wherein each
of said fluid reservoir is fluidly connected to a mixing
microchannel that is fluidly connected to a detection chamber, and
wherein fluid within the microchannels of the platform is moved
through said microchannels by centripetal force arising from
rotational motion of the platform for a time and a rotational
velocity sufficient to move the fluid through the
microchannels.
3. A microsystems platform of claim 1, wherein the interior surface
of each of the microchannels comprises a graded surface to volume
ratio, wherein the surface to volume ratio increases with distance
from the axis of rotation
4. A method for mixing two or a plurality of different fluids,
comprising the steps of: a) applying a volume of a first fluid to
one or a plurality of fluid reservoirs of a microsystem platform of
claim 1 when the platform is stationary; b) applying a volume of a
second fluid to one or a plurality of fluid reservoirs of a
microsystem platform of claim 1, wherein the fluid reservoir
containing the first fluid is the same fluid reservoir containing
the second fluid, or the fluid reservoirs containing the first and
second fluids are fluidly connected to the same mixing
microchannel; c) rotating the platform at an increasing rotational
speed sufficient to motivate fluid flow from the fluid reservoir to
the most distal extend of the mixing microchannel without
motivating fluid flow into the detection chamber; d) rotating the
platform at a decreasing rotational speed until all fluid in the
mixing microchannel returns to the fluid reservoir e) repeating
steps (c) and (d) for a number of repetitions sufficient to
homogeneously mix the first and second fluids into a homogeneous
mixture; f) rotating the platform at a rotational speed greater
than the maximum speed of rotation in step (c) at a speed
sufficient to motivate the homogeneously mixed fluid into the
detection chamber; and g) detecting the homogenous mixture.
5. A method for mixing two or a plurality of different fluids,
comprising the steps of: a) applying a volume of a first fluid to
one or a plurality of fluid reservoirs of a microsystem platform of
claim 2 when the platform is stationary; b) applying a volume of a
second fluid to one or a plurality of fluid reservoirs of a
microsystem platform of claim 2, wherein the fluid reservoir
containing the first fluid is the same fluid reservoir containing
the second fluid, or the fluid reservoirs containing the first and
second fluids are fluidly connected to the same mixing
microchannel; c) rotating the platform at an increasing rotational
speed sufficient to motivate fluid flow from the fluid reservoir to
the most distal extend of the mixing microchannel without
motivating fluid flow into the detection chamber; d) rotating the
platform at a decreasing rotational speed until all fluid in the
mixing microchannel returns to the fluid reservoir e) repeating
steps (c) and (d) for a number of repetitions sufficient to
homogeneously mix the first and second fluids into a homogeneous
mixture; f) rotating the platform at a rotational speed greater
than the maximum speed of rotation in step (c) at a speed
sufficient to motivate the homogeneously mixed fluid into the
detection chamber; and g) detecting the homogenous mixture.
Description
[0001] This application claims priority to U.S. Provisional
Application, Serial No. 60/204,264, filed May 15, 2000., the
disclosure of which is explicitly incorporated by reference
herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to chemical and biological assay
technology carried out in disposable plastic assemblies, and in
particular the devices referred to as microfluidic systems as
disclosed in U.S. Pat. No. 6,063,589, issued May 16, 2000, and
co-owned and co-pending patent applications U.S. Ser. No.
08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18,
1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No.
08/995,056, filed Dec. 19, 1997; and Ser. No. 09/315,114, filed May
19, 1999, the disclosures of each of which are explicitly
incorporated by reference herein.
[0004] 2. Background of the Related Art
[0005] Microfluidic systems are closed interconnected
networks/systems of channels and reservoirs with characteristic
dimensions ranging from microns to millimeters. By introducing
fluids, reagents and samples into the devices, chemical and
biological assays can be carried out in an integrated and automated
way. In a conventional assay, two or more fluids are mixed and
incubated within a microfluidic device and during, or after, this
incubation period, a reaction product may be detected. It is
typically the case that this microfluidic device, specifically the
depths, cross-sectional dimensions and connectivity and layout of
the microfluidic systems, defines the relative volumes of these
fluids.
[0006] A problem in the art is that microfluidic devices, once
fabricated, do not allow the user to redefine the relative volumes
of the fluids to be mixed. An additional problem in the art
concerns the degree and efficiency of mixing. Because the flow
within a microfluidic device is laminar, mixing is brought about
through mass diffusion. A typical mixing device consists of a long
capillary. Two or more fluids may enter this capillary as separate
fluids and leave as a single fluid. The degree of mixing can be
enhanced and the time to mix these fluids can be decreased by
decreasing the cross-sectional dimension of the capillary and by
increasing the length of the capillary channel, but such a device
can occupy a fair amount of space within a microfluidic system.
SUMMARY OF THE INVENTION
[0007] This invention describes the use of air-ballasts and
microchannels with graded surface properties to allow for
bidirectional fluid flow. Bidirectional flow within microchannels
allows different fluids to laminate and mix within a microchannel.
The combination of air-ballasts, microchannels with graded surface
properties, (passive) capillary valves and (active) wax valves
allow for mixing and aliquotting of arbitrary volumes within a
defined microfluidic system.
DESCRIPTION OF THE FIGURES
[0008] FIG. 1 describes bidirectional flow centrifugal micro
fluidic devices, azimuthally arrayed on a disc.
[0009] FIG. 2 describes an individual bidirectional flow
device.
[0010] FIG. 3 describes a sequence of flow events at different disc
rotation rates.
[0011] FIG. 4 describes mixing in a microchannel.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0012] This invention provides a microplatformn 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. No. 08/761,063, filed Dec. 5, 1996; Ser. No.
08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug.
12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No.
09/315,114, filed May 19, 1999, the disclosures of each of which
are explicitly incorporated by reference herein, adapted for
performing efficient mixing of a plurality of different fluids and
solutions.
[0013] For the purposes of this invention, the term "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.
[0014] For the purposes of this invention, the term "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. No.
08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18,
1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No.
08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19,
1999, the disclosures of each of which are explicitly incorporated
by reference herein.
[0015] For the purposes of this invention, the term "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. No. 08/761,063, filed Dec. 5, 1996; Ser. No.
08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug.
12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No.
09/315,114, filed May 19, 1999, the disclosures of each of which
are explicitly incorporated by reference herein.
[0016] 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.
[0017] For the purposes of this invention, the term "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. For liquids having a contact angle less than 90.degree.
(such as aqueous solutions on platforms made with most plastics,
glass and silica), flow is impeded as the channel cross-section
increases at the interface of the capillary junction. 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. The factors
relating to capillarity in microchannels according to this
invention have been discussed in co-owned U.S. Pat. No. 6,063,589,
issued May 12, 2000 and in co-owned and co-pending U.S. patent
application, Ser. No. 08/910,726, filed Aug. 12, 1997, incorporated
by reference in its entirety herein.
[0018] Capillary junctions can be constructed in at least three
ways. In one embodiment, 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. As an example, in microfluidics components
made from "wetting" or "wettable" materials, such a junction occurs
at an enlargement of a capillary as described in co-owned and
co-pending U.S. Serial Nos. U.S. Ser. No. 08/761,063, filed Dec. 5,
1996; Ser. No. 08/768,990, filed Dec. 18, 1996; and Ser. No.
08/910,726, filed Aug. 12, 1997. Fluid flow through capillaries is
inhibited at such junctions. At junctions of components made from
non-wetting or non-wettable materials, on the other hand, a
constriction in the fluid path, such as the exit from a chamber or
reservoir into a capillary, produces a capillary junction that
inhibits flow. In general, it will be understood that 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).
[0019] A second embodiment of a capillary junction is formed using
a component having differential surface treatment of a capillary or
flow-path. For example, 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.
[0020] 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.
Those of ordinary skill will appreciate how 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.
[0021] For the purposes of this invention, the term "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.
[0022] For the purposes of this invention, the term "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.
[0023] For the purposes of this invention, the term "in fluid
communication" or "fluidly connected" is intended to define
components that are operably interconnected to allow fluid flow
between components.
[0024] For the purposes of this invention, the term "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.
[0025] For the purposes of this invention, 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.
[0026] For the purposes of this invention, the term "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.
[0027] The microplatforms of the invention (preferably and
hereinafter collectively referred to as "disks"; for the purposes
of this invention, the terms "microplatform", "microsystems
platform" and "disk" are considered to be interchangeable) are
provided to comprise one or a multiplicity of microsynthetic or
microanalytic systems (termed "microfluidics structures" herein).
Such microfluidics structures in turn 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. For the
purposes of this invention, the term "microfabrcated" 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.
[0028] 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. Both electromechanical means and
control means, as further described herein, 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. No.
08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18,
1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No.
08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19,
1999, the disclosures of each of which are explicitly incorporated
by reference herein.
[0029] 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. Serial Nos. U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser.
No. 08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed
Aug. 12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; and Ser.
No. 09/315,114, filed May 19, 1999, the disclosures of each of
which are explicitly incorporated by reference herein.
[0030] The components of the platforms of the invention are in
fluidic contract with one another. In preferred embodiments,
fluidic contact is provided by 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 .mu.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 .mu.m to about 500 .mu.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,
detecflons 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. In preferred embodiments, 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.
[0031] 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. Specifically, 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. These may
be used with unmodified or modified surfaces as described below.
The platforms may also be made from thermoset materials such as
polyurethane and poly(dimethyl siloxane) (PDMS). Also provided by
the invention are 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.
Alternately, 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. No. 08/761,063, filed Dec.
5, 1996; Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No.
08/910,726, filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec.
19, 1997; and Ser. No. 09/315,114, filed May 19, 1999, the
disclosures of each of which are explicitly incorporated by
reference herein.
[0032] Preferably, 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.
[0033] PLATFORM MANUFACTURE AND ASSEMBLY
[0034] Referring now to the Figures for a more thorough description
of the invention, FIG. 1 show a plan view of a disc of the
microsystem platform. In this embodiment, platform 100 is composed
of at least two layers, a fluidics layer 101 and a sealing layer
199 (not shown). At the center of this disc is a hole 102 for
affixing the disc to a rotary spindle; other means such as extruded
features for connection to a spindle, or features not on the axis
of the disc, are also possible.
[0035] The construction of this disc is made to illustrate the
concept of centrifugally driven, bidirectional flow. It is
understood that the elements shown here may be complete for the
purposes of performing certain assays or fluid processing, or may
be part of a larger system of reservoirs and channels. This disc
illustrates that identical assays may be made by repeating assay
structures around the disc at a given radius. Here, structure 103
is repeated azimuthally around the platform layer 101.
[0036] 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. A variety of materials may be used to fabricate 101 but
preferred materials are polymer materials, including
thermoplastics, thermosets and elastomeric materials. Examples of
thermoplastic materials include acrylics, polycarbonates, cyclic
olefin copolymers and polyolefins such as polypropylene.
Polyurethane thermosets and silicone are examples of thermoset and
elastomeric materials, respectively. A variety of standard
fabricating methods may be used to define features within 101,
including high-speed machining, injection molding, compression
injection molding and embossing. Reaction injection molding may be
used to fabricate discs made with thermoset materials. The sealing
layer (199) may consist of thermoplastic lids that are diffusion
bonded to the 101 with temperature and pressure or adhesive films
that are applied with hand pressure. Depending on the choice of
materials of 101 and 199, it is possible to functionalize the
opposing surfaces of 101 and 199 to achieve chemically bond 101 to
199, when the surfaces are brought into contact.
[0037] Referring to FIG. 2, a single structure for the performance
of a mixing assay is illustrated. Among the components of the
structure are a fluid entry port 201 through which fluids may be
added to the reservoir 202. Reservoir 202 is preferably sized to
contain the maximum amount of fluid that might be processed in a
series of assays, being in the range of 1 nL to 100 .mu.L.
Extending from reservoir 202 is microchannel 203. Microchannel 203
is preferably sized such that its volume is between 1 and 2 times
the maximum volume of reservoir 202 and with a cross-sectional size
in the range of 5-500 .mu.m. At the radially-distal end of 203 is a
detection cuvette 204 with a volume between 0.5 and 2 times the
maximum volume of the reservoir 202. Connected to 204 by channel
205 is an air-ballast 206, an enclosed reservoir containing air or
another gas. The size chosen for 206 is a function of the desired
operating parameters of the device and is generally in the range of
0.1-10 times the maximum volume of the reservoir 202.
[0038] In use, the disc would function in the following fashion. A
first liquid sample is added via port 201 to reservoir 202. The
device may be rotated to drive the fluid to the radially-distal end
of the reservoir. A second fluid is added to port 201. The
situation at rest is illustrated in FIG. 3 a. The disc is now spun
at a rotational speed sufficient to drive the fluids into channel
203. Opposing flow into 203 is a restoring force due to the
compression of he trapped air in the remainder of 203, the cuvette
204, channel 205, and ballast 206. For any mode of mixing, the
fluid must be driven such that the entire combined volume has
entered the channel; however, the rotational rate must not be so
high as to result in the fluid leaving channel 203 and entering
reservoir 204.
[0039] The rotational rates necessary may be determined from the
following considerations. It can be shown that the hydrostatic
pressure generated by a column, reservoir, or channel of liquid due
to rotation is
P.sub.R=.rho..omega..sup.2.DELTA.r{overscore (r)} (1)
[0040] where .rho. is the density of the fluid (average density in
the case of multiple fluids); .omega. is the angular velocity with
which the device rotates; .DELTA. r is the radial extent of the
liquid, that is, the difference in radial position between the
liquid interface in contact with the compressed air and the radial
position of the trailing interface of the liquid with the air in
202; and <r> is the average radial position of the liquid as
defined by the radial position of those two interfaces.
[0041] Let the various volumes be defined as follows: V.sub.T=total
combined fluid volumes added; V.sub.C=volume of channel 203;
V.sub.B=volume of 204 plus that of 205 and 206. As the liquid
enters 203, it displaces some of the volume of 203; if the device
is rotated with sufficient speed, all fluid leaves 202, and the
displaced volume can be greater than V.sub.T. Let the volume of 203
that is displaced volume for a specific radial position of the
liquid of interest be defined as V.sub.D. If the fluid has not
entered chamber 204, the restoring pressure due to compressed gas
is then 1 P C = P ATM V D V c + V B ( 2 )
[0042] Where P.sub.ATM is the ambient pressure when the fluid is
loaded into the device.
[0043] Fluid motion will halt when 2 2 r r _ = P ATM V D V c + V B
= [ P ATM r r _ ( V D V c + V B ) ] 1 / 2 ( 3 )
[0044] This relationship allows one to determine the appropriate
rotational velocity for desired displacement volume.
[0045] FIG. 3 illustrates the motion of the fluid. In FIG. 3a, the
first and second fluids are seen to be layered, due to being added
sequentially. In FIG. 3b, the fluids are shown as they begin to
move into 203 at a non-zero rotational rate. In FIG. 3c, has
completely entered 203 and is stationary at a rotational rate given
in Eq. 3. As the rotational rate is decreased, as in FIG. 3d, the
fluid is expelled from the channel 203, until it is completely
expelled into 202 at zero rotational rate.
[0046] The device illustrated in the figures is only one possible
construction for affecting bidirectional flow. Alternative
constructions include a reservoir 202 that is also a detection
cuvette. For such a device, a single ballast chamber may be at the
end of channel 203, with no additional detection cuvette.
[0047] Another alternative embodiment uses surface forces, rather
than forces due to compression of gas, to drive flow in the reverse
direction. If, for example, the surfaces of the disc are coated or
functionalized to have a contact angle of greater than 90 degrees
surface energy considerations show that the preferred state of
fluids is one with minimum contact with the hydrophobic surfaces.
Aqueous liquids in hydrophobic channels are naturally expelled into
chambers with smaller surface area to volume ratios. It should be
noted that fluid in a channel with constant cross-section is
subjected to no force driving in either direction along the
channel. Only if the cross-section decreases along the outward
direction will a restoring force exist. In such an application,
either gradual or abrupt narrowing of channels can provide this
force.
[0048] Another alternative embodiment can use surfaces with a
gradient in contact angle along the flow path in the channel. A
channel of fixed cross-section will provide a restoring force if
the contact angle at the leading edge of the fluid is larger than
that at the trailing edge of the liquid, that is, the surface grows
progressively more hydrophobic along the flow path. Such a device
may be fabricated through surface functionalization and patterning
of hydrophobic patches on the surface of a channel with fixed
cross-section.
[0049] Combinations of surface treatment and trapped air may also
be used. For example, the all surfaces in the disc of FIGS. 1-3 may
be treated to be hydrophobic. This has the advantage of preventing
condensation of liquid onto surfaces if the liquid is heated.
Heated liquids preferentially recondense onto the air-liquid
interface rather than onto hydrophobic surfaces in such cases.
[0050] The use of the device for mixing is now demonstrated. In
FIG. 4a, the first and second fluids are seen to be layered, due to
being added sequentially. In FIG. 4b, the fluids are shown as they
begin to move into 203 at a non-zero rotational rate. In FIG. 4c,
has completely entered 203 and is stationary at a rotational rate
given in Eq. 3. . FIG. 4d is a magnified view of the fluid in the
channel. It is not to scale, and the lateral dimension is
exaggerated for clarity. The shape of the interface between the two
fluids is seen to be a broadening along the direction of the
channel. This is due to the laminar flow that occurs in small
channels for low flow velocities and is a familiar feature from
flow injection analysis. Note also that this is a cross-sectional
representation, and that the broadening exists three-dimensionally
and is a function of the inverse second power of the lateral
channel dimensions. The important feature in FIG. 4d is that there
is a large amount of interface between fluids A and B relative to
the interface seen in FIG. 4a, and that the average distance of
elements of fluid A from elements of fluid B is much smaller than
in FIG. 4a.
[0051] More interface may be created between the fluids by driving
them further into 203 by approximately 1/2 of the length occupied
in FIG. 4b. FIG. 4e shows this situation. Here the interface
between the two fluids occupies most of the length of the fluid
column within 203.
[0052] Diffusion now acts to mix the fluids. Diffusional timescales
are of the order of 3 t x 2 D ( 4 )
[0053] where x is distance over which diffusion must take place and
D is the diffusion constant of chemical species, molecules, etc.,
which must be mixed. For example, if x=100 .mu.m and
D=5.times.10.sup.-6cm.sup.2s.su- p.-1, t=20 seconds. In this case,
the relevant dimension is the lateral size of the channel.
[0054] In order to ensure complete mixing, the device is brought to
near 0 rotational speed, and the fluid is expelled by air pressure
into 202, as shown in FIG. 4f. The device is then accelerated once
again to drive fluids into 203 the appropriate distance. Multiple
iterations of acceleration, holding, and deceleration allow
additional fluid motion within reservoir 202 to provide additional
mixing.
[0055] Additional fluids may be added via 201 and the mixing
process repeated.
[0056] The device is then rotated at a second, higher rotational
velocity, at which point it is expelled into cuvette 204. Air from
channel 205 and ballast 206 then enters the end of 203; a pathway
for air from 205 into 203 relieves the restoring force on the fluid
already present In 204, and the device may be slowed while fluid is
retained in 204.
[0057] The theoretical effectiveness of the mixing device may be
compared to the diffusional mixing which would occur in reservoir
202 without the use of bidirectional motion. Assume 202 is a cube
designed to contain 8 .mu.L and as such is 2 mm.times.2 mm.times.2
mm in size. If two fluids are added sequentially with volumes of 4
.mu.L and allowed to diffusionally mix, the expected time for this
mixing is over 2 hours. This is in contrast to perhaps 2 minutes to
effect 5 acceleration/hold/deceleration cycles using a 100 .mu.m
channel. Even volumes of 100 nL may require up to 10 minutes to
diffusionally mix without the use of bidirectional flow.
[0058] It can be seen that the device as illustrated is capable of
performing homogeneous assays in which active mixing must take
place. The turnover of p-nitrophenol phosphate by alkaline
phosphatase in the presence of theophylline is a model system for
examining enzyme inhibition and requires efficient mixing of the
enzyme and inhibitor before the addition of substrate. This
enzymatic reaction can be monitored colorimetrically through the
conversion of p-nitrophenol phosphate to p-nitrophenol, which is
yellow and absorbs at wavelength of 410 nm. The absorbance at, or
near, 410 nm decreases as the inhibitor (theophylline)
concentration is increased. To perform such an assay in a
bidirectional flow centrifugal microfluidic device, an aliquot of
enzyme solution would be pipetted into the device, the disc would
be rotated at a rotation rate sufficient to drive the fluid into
the reservoir (202) and away from the entry port (201), an aliquot
of inhibitor solution would then be pipetted into the device and
the disc would undergo an acceleration/hold/deceleration cycle to
allow for dispersional and diffusional mixing of the enzyme and
inhibitor solutions within the channel (203) and reservoir (202),
The calculation above shows that hold times of approximately 20
seconds are required to allow for diffusional mixing across 100
.mu.m (assuming a diffusion coefficient of
5.times.10.sup.-6cm.sup.2s.sup.-1). After several
acceleration/hold/decel- eration cycles, disc rotation would be
stopped and an aliquot of substrate solution would be pipetted into
the device. A similar sequence of acceleration/hold/deceleration
cycles would be performed to allow for mixing of enzyme/inhibitor
with substrate.
[0059] Bidirectional flow can also be used to perform polymerase
chain reaction (PCR) on a disc. In this application, the disc of
FIG. 1 forms a mechanical and thermal contact with a co-rotatable
platen. Electrical signals are distributed from a stationary power
and control unit to the rotating platen through the use of an
electrical commutator. In a simplified version of this idea, the
surface of the platen has three bands of annular resistive heaters,
each maintained at temperatures appropriate for the denaturation,
annealing and extension of nucleic acids in the PCR process. More
specifically, the heaters are arranged on the platen so that
meandering capillary (203) has three distinct temperature when the
disc is mated to the platen. Bidirectional flow can be used to
drive fluid across these three different temperature zones. And
because the ratio of surface to volume can be quite high within a
microchannel, it is expected that fluid that traverses a defined
temperature zone quickly comes to the temperature of this zone,
thereby allowing the reaction to take place. A recent report by
Chiou et al. demonstrates the use of gas to drive plugs of fluid
through capillaries that are in thermal contact with a set of
heaters; when used to perform PCR, it was found that a 500 base
pair product could be amplified in 23 minutes with 30 complete
temperature cycles and 78% amplification efficiency (J. Chiou, P.
Matsudaira, A. Sonin and D. Erlich, "A Closed-Cycle Capillary
Polymerase Chain Reaction Machine, Analytical Chemistry, 2001, 73,
2018-2021).
[0060] Bidirectional flow may also be used to perform inhomogeneous
assays in the following fashion. Referring to FIG. 4, the device
may be manufactured such that immunochemicals or other ligands are
immobilized into the channel 203, for either the entire length of
the channel or for a portion. A fluid sample is added to 202. The
sample is driven into channel 203 cyclically as described above,
allowing molecules within the fluid to bind to the ligands on the
surface of the channel. The disc is then rotated at its third
rotational rate, sufficient to drive all fluid into chamber 204;
the emptying of 203 removes the restoring force. A second fluid may
be added now, for example, a complementary molecule which may can
bind to the molecule of interest whose presence is being assayed in
the first fluid. This in turn may be bound to gold colloid
particles for visual detection, or linked to enzymes for exposure
to substrate. The liquid is now driven bidirectionally into the
channel, and the complementary molecule allowed to bind to the
first, immobilized molecule of the analyte. Direct visual detection
in reflection mode using blue light will reveal the presence of
gold colloid particles. For enzyme-linked complements, the process
may be repeated with a third fluid consist of substrate for the
enzyme; action of the enzyme may cause a colored or fluorescent
product to be formed.
[0061] An important element of bidirectional flow in such an
inhomogeneous assay is that the multiple passes of fluid across the
surface covered with immobilized ligand allows trace amounts of
analyte to be concentrated into a small area of the surface. It is
possible to bind all of the analyte in a large volume of liquid
onto a small area much more quickly than could be achieved by bulk
diffusion in an unmoving fluid.
[0062] Another application of such a method is nucleic acid
hybridization. Complementary strands of DNA or RNA of interest may
be immobilized into channels or intermediate reservoirs between 202
and 204. If necessary, the nucleic acids in the fluid sample may be
denatured by application of heat, and then driven across the
immobilized nucleic acids. Detection may be performed using common
methods such as molecular beacons or intercalating dyes for
double-stranded DNA.
[0063] This invention is additionally taught through the
non-limiting example described below.
EXAMPLE 1
[0064] An experimental demonstration of bidirectional flow in a
centrifugal microfluidic device was performed. Discs were
fabricated from cast acrylic sheet (PMMA, ICI Acrylics, St. Louis,
Mo.) using a computer controlled milling machine (Benchman
VMC-4000, Light Machines Corp., Manchester, N.H.) and a selection
of end-mills that ranged in diameter from 250 .mu.m to 1.6 mm. The
machined acrylic surfaces were polished with methylene chloride
vapor and then sealed with a layer of doubled-sided tape (467MP Hi
Performance Adhesive, 3M, Minneapolis, Minn.) and subsequently
backed with a white polyester sheet. Liquids were pumped through
the channels by rotating the discs on a spindle driven by a dc
servomotor with an integral optical encoder (DC MicroMotor
3042/HEDS-55401, MicroMo, Clearwater, Fla.). The servomotor was
operated via a motor controller card (PIC-Servo, HdB Electronics,
Redwood City, Calif.) and a host PC using a program written in
Visual Basic (Microscoft, Redmond, Wash.). The speed of the motor
could be programmed to give rates of rotation between 0 and 4600
rpm. The encoder triggered external devices such as a tachometer, a
stroboscope and frame buffer. Liquid flow was monitored using
stroboscopic video microscopy. A fast response stroboscope
(NovaStrobe DA116, Monarch Instruments, Amherst, N.H.) was
triggered by the encoder and illuminated the spinning disc for 30
.mu.s at each revolution. An image of the spinning disc was
continually recorded by a 1/3 inch CCD color video camera
(GP-KR222, Panasonic, Tokyo, Japan) with macrofocus zoom lens. The
rate of rotation was recorded by the tachometer (08212,
Cole-Parmer, Veron Hills, Ill.) and displayed simultaneously using
a digital video mixer. To give a continuous illuminated image of
the disc, dark frames were filtered out using a frame buffer (Ultra
II, Coreco, Saint-Laurent, Quebec, Canada). The experiments
described in this example were performed with food colored aqueous
solutions.
[0065] As described in FIG. 2, a bidirectional flow centrifugal
microfluidic device may consist of an entry port, an entry
reservoir, a meandering capillary, a detection cuvette, and an
air-ballast, all in fluid communication. As described in FIG. 1,
bidirectional flow devices may be arrayed around the circumference
of a circular disc and aligned so that fluid can flow back and
forth between positions close to the inner and outer diameters of
the disc. For this particular set of experiments, a bidirectional
flow device was fabricated using the designs of FIGS. 1 and 2. The
device was located on discs such that the center of the detection
cuvette was at a distance of 54.4 mm from the center of rotation.
The entry ports were sized to easily accommodate a plastic pipette
tip and to allow air to escape as fluid was dispensed into the
device. The entry reservoir (202) acommodated 15 .mu.L with a
length, width and depth of 15 mm, 2 mm and 0.5 mm, respectively.
The meandering capillary (203) had a depth and cross-section of
approximately 250 .mu.m and had a volume of 15 .mu.L so that all of
the fluid could be driven from the entry reservoir into the channel
at high rotation rates; the diameter and depth of the detection
cuvette (204) were 3 mm and 0.5 mm, respectively, yielding a volume
of approximately 3.5 .mu.L; the volume of the air-ballast (206) was
fixed close to 16 .mu.L so that it would be possible to drive fluid
from the meandering capillary to the detection cuvette. It was
found that with a smaller air-ballast, the restoring forces were
too high for the instrumentation at hand and fluid could not be
driven into the detection cuvette at achievable rotation rates;
significantly larger air-ballasts did not provide enough restoring
force to rapidly drive the fluid from the meandering capillary back
into the entry reservoir to achieve the required degree of mixing.
It was experimentally determined that spinning this device with 12
.mu.L of aqueous solution at 4600 rpm for 30 seconds was sufficient
to fill the detection cuvette and that after stopping the disc
rotation, an amount of fluid remained within the detection cuvette
defined by the volume of the detection cuvette that is radially
outboard of the junction between 205 and 204.
[0066] The table below reports the extent of the fluid front for a
number of sequential rotation rates.
1 Radial Elapsed Time Rotation Position of Fluid (seconds) Rate
(rpm) Front 0 500 30.0 mm 20 1500 35.1 mm 40 500 30.0 mm 60 1500
35.1 mm 80 500 30.0 mm 100 1500 35.1 mm 120 500 30.0 mm 140 2300
41.7 mm 160 500 29.5 mm 180 2300 41.7 mm 200 500 29.5 mm 260 3800
50.9 mm 290 4000 54.4 mm 320 4200 54.4 mm 350 4400 54.4 mm 360 4600
54.4 mm 390 0 54.4 mm
[0067] It is worth noting that at 4000 rpm fluid begins to flow
into the detection cuvette (204) but it takes an increased rotation
rate of 4600 rpm to fill the cuvette up to the junction of 205 and
204.
[0068] This example shows that bidirectional flow can be achieved
in a microfluidic device with the combination of a centrifugal
drive and air-ballasts.
[0069] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
all modifications or alternatives equivalent thereto are within the
spirit and scope of the invention.
* * * * *