Note: Descriptions are shown in the official language in which they were submitted.
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SEALING FOR MICROFLUIDIC DEVICES
INTRODUCTION
Field of the Invention
The field of this invention is microfluidic devices.
Background
Microfluidic devices offer great promise for the accurate manipulation of very
small
volumes, the rapid execution of a wide variety of operations, the minimal use
of reagents, as
well as many other benefits. As with all situations, the benefits come with
challenges. For
many purposes, one wishes to have a number of independent electrokinetic units
in a single
substrate. Since each unit will frequently comprise a plurality of reservoirs
and channels, it is
important that the individual units do not communicate except as required by
the design of the
device. Also importantly, is the problem of the very small volumes, which are
frequently
involved with the operations, due to evaporation. Where there is an interest
in doing a
quantitative analysis of an operation involving kinetics, it is important that
the solvent volume
remain substantially constant, so that the concentrations of the reactants are
not changing due to
decreasing volume. To this end, methods are required to minimize evaporation.
Additionally,
particularly where long incubation times and/or long reaction times are
involved, there is an
interest in preventing contamination. A further concern is adventitious
pressurization, during
closing of a microstructure vessel, which could prematurely move the liquid
from a reservoir
into a channel.
In response to these concerns, lids have been used to seal the ports of
microfluidic
devices. Lids may provide sealing through the pressure of their weight, by
providing adhesion,
using various forms of latches or clasps, or by fitting snugly around a part
and held by friction.
There is an interest in developing devices and methods to substantially
diminish the
injurious events that may occur due to open ports of a microfluidic device,
where the devices are
effective and can be readily fabricated and methods readily performed.
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Relevant Art
U. S. Patent no. 5,4443, 890 and references cited therein describe leakage-
proof sealing of
microfluidic devices. W099/43432 describes microfluidic devices and systems
incorporating
cover layers. U.S. Patent no. 5,545,280 describes applying adhesive to
protrusions on a
substrate.
SUMMARY OF THE INVENTION
Improved microfluidic devices are provided for use in performing operations
involving
the manipulation of small volumes. Substrates are formed comprising channels
and reservoirs,
where the channels communicate with the reservoirs and the channels are
otherwise enclosed,
and the reservoirs have an aligned collar in relief, extending beyond the
planar surface of the
substrate and outwardly from the border of the reservoir. The reservoirs are
more efficiently
sealed with an appropriate cover which contacts the crown of the collar. The
substrates may be
formed by plastic molding or other means. Of particular interest are devices
employing
electrokinesis for movement of solutions from one site to another in the
device, where the
substrate comprises a plurality of individual electrokinetic units and the
volumes involved
generally are below about 5~1.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a plan view of a device with collars around microstructures; Fig. 1
a is a cross-
sectional view of the device of Fig. 1 along lines 1 a-1 a;
Fig. 2 is a plan view of the device of Fig. 1, with the collars removed to
provide details
of the individual units; Fig. 2a is a diagrammatic exploded view of the units
of Fig. 2;
Fig. 3 is a side elevation cross-sectional view of a reservoir microstructure
of a unit with
a cover.
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DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The following examples are offered by way of illustration of the present
invention, not
limitation. Microfluidic devices are provided for manipulation of small
volumes, where the
devices comprise a substrate, usually an organic substrate in which are
channels and reservoirs,
where the reservoirs have a raised collar above the planar surface of the
substrate. A bottom
film, including a rigid substrate, is adhered to and encloses the channels and
the bottoms of the
reservoirs. The reservoirs can be sealed on the top sideusing a film, which
seals to the upper
surface of the collar.
The microfluidic devices will be characterized by having one or more
operational units
present in the substrate, where the number of units may vary from 16 to 1536
units, more usually
not more than about 384 units, the number of units frequently being related to
the number of
wells in a microtiter well plate. Each unit will have at least one channel and
at least two
reservoirs, usually having at least two channels and at least four reservoirs.
The total number of
reservoirs for a device will generally be in the range of about 4 to 1600,
more usually in the
range of about 64 to 1500.
The sealing cover or lid will be a film, which forms a seal about the collar,
so as to at
least substantially inhibit fluid flow from the reservoir. The cover will
provide for sealing
interaction with the collar upper surface, as a result of a compliant surface
contacting the collar
or an adhesive surface adhering to the upper surface of the collar,
particularly an adhesive
surface, which is removable. Contact will usually be minimal or not at all
between the sealing
cover or lid and the planar surface. The forces providing the sealing may be
gravity, adhesive
forces, or mechanical forces. For compliant surfaces, such as elastomeric
films, skin-surface
(closed-cell) foams, soft films, pressure would be applied, as a result of a
weighted backing,
latching or gripping devices for holding the film against the collars, a
vacuum chuck which
holds the film in position and can release the film, as appropriate, etc. The
film may be stretched
across the collars, held in position by clasps at the periphery of the
substrate, a sealing pliable
band around the periphery, a vacuum chuck, etc. A continuous sealing film may
be used, which
may be unrolled from a reel as the devices are moved in a continuous manner,
for example, on a
wheel or moving belt. The films may be natural rubber, polyisoprene, ethylene-
propylene
elastomers, polyurethane foams, polydimethylsiloxane, etc. The films may be
thin or thick, so
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long as they have a minimum dimension, which provides for their sealing of the
collars.
Generally, the films will be at least about SOp in thickness. Alternatively,
films may be used,
which have a thin adherent layer, which will adhere to the surface of the
collar and after the film
has fulfilled its function, the adhesive may be removed. Useful adhesives
include pressure
sensitive adhesives, such as ethylene-containing polymers, urethane polymers,
butyl rubber,
butadiene-acrylonitrile polymers, butadiene-acrylonitrile-isoprene polymers,
and the like. See,
for example, U.S. Patent no. 5,908,695 and references cited therein.
The substrate will generally have a thickness of at least about 20~m, more
usually at
least about 40~m, and not more than about O.Scm, usually not more than about
0.25cm. The
width of the substrate will be determined by the number of units to be
accommodated and may
be as small as about 2mm and up to about 6cm or more. The dimension in the
other direction
will generally be at least about O.Scm and not more than about 20cm, usually
not more than
about l Ocm. The substrate may be a flexible film or relatively inflexible
solid, where the
microstructures, such as reservoirs and channels, may be provided by
embossing, molding,
machining, etc. The collars may be formed at the same time using the same
process, although
more expensive processes maybe used, such as photolithography or laser
ablation. In this case,
the collar regions would be protected while the substrate was eroded. The
channel dimensions
will generally be in the range of about 0.1 ~m to lmm deep and about O.S~m to
SOO~m wide,
where the cross-section will generally be 0.1 ~.mZ to about 0.25mm'. The
channel lengths will
vary widely depending on the operation for which the channel is to be used,
generally being in
the range of about O.OSmm to lOcm, more usually in the range of about O.Smm to
lcm. The
reservoirs will generally have volumes in the range of about lOnl to 101, more
usually have
volumes in the range of about 20n1 to 1 ~1. The reservoirs may be
cylindrically shaped or
conically shaped, particularly inverted cones, where the diameter of the port
will be from about
1.5 to 25 times, usually 1.5 to 15 times, the diameter of the bottom of the
reservoir, where the
reservoir connects to the channel.
Depending upon whether a film is embossed to produce the device or the device
is
molded, whether the microfeatures are left open will depend upon whether a
supporting film
and/or an enclosing film is provided. The supporting film will generally be at
least about 40~m
and not more than about Smm thick. The film used to enclose the channels and
the bottom of
the reservoirs will generally have a thickness in the range of about 1 Oym to
2mm, more usually
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in the range of about 20pm to 1 mm. The selected thickness is primarily one of
convenience and
assurance of good sealing and the manner in which the devices will be used to
accommodate
instrumentation. Therefore, the ranges are not critical.
The collars surrounding the reservoir ports will generally have a height from
the planar
surface of the substrate in the range of about 0.1 to 1 mm, more usually about
0.2 to 1 mm, and
preferably about 0.25 to 0.75mm. The crown will be thick enough to provide a
good seal
between the sealing film or lid and the crown, so that usually it will be
about 0.05 to 1 mm thick,
more usually about 0.1 to about O.Smm thick. The collars may be considered
extensions of the
inner walls of the microstructures, having an inner wall aligned with the
inner wall of the
microstructure, where the collars then extend outwardly from the inner wall,
much like the
structure of a volcano. Alternatively, the collar inner wall may be displaced
from the reservoir
inner wall, generally displaced less than about lmm, usually less than about
O.Smm and may be
less than about O.lmm. In this way the inner wall is offset from the edge of
the reservoir,
serving as a fence around the reservoir.
The area occupied by a single unit will vary widely, depending on the number
of units of
the device, the function of the units, and the like. As illustrative, for the
most part, where the
devices are designed to be compatible with 96 to 384 microtiter well plates,
the units will have
from about 4.5 to 9mm spacings.
As indicated, the substrate may be a flexible film or inflexible solid, so the
method of
fabrication will vary with the nature of the substrate. For embossing, at
least two films will be
used, where the films may be drawn from rolls, one film embossed and the other
film adhered to
the embossed film to provide a physical support. The individual units may be
scored, so as to be
capable of being used separately, or the roll of devices retained intact. See,
for example,
application serial no. PCT/98/21869. Where the devices are fabricated
individually, they will
usually be molded, using conventional molding techniques. The substrates and
accompanying
film will generally be plastic, particularly organic polymers, where the
polymers include
addition polymers, such as acrylates, methacrylates, polyolefins, polystyrene,
etc. or
condensation polymers, such as polyethers, polyesters, polyamides, polyimides,
etc. Desirably,
the polymers will have low fluorescence inherently or can be made so by
additives or bleaching.
The underlying enclosing film will then be adhered to a substrate by any
convenient means, such
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as thermal bonding, adhesives, etc. The literature has many examples of
adhering such films,
see, for example, U.S. Patent nos. 4,558,333; and 5,500,071.
Liquids may be moved through the units by any convenient means, including
electrokinesis, pneumatics, sonics, thermal, etc. Electrokinetic devices will
usually have two or
more reservoirs connected by microchannels, where the microchannels may cross,
providing for
injection of a plug from one microchannel into another microchannel. The
devices may find use
in sequencing of nucleic acids, detection of binding between two entities,
e.g. proteins with
proteins, small molecules or cells, or various assays for the determination of
drugs, single
nucleotide polymorphisms, etc.
The methods employing the subject devices may be associated with the transfer
to the
microstructures of the devices of volumes ranging from about l Onl to 5001,
with reaction
volumes ranging from about 20n1 to O.SmI, usually SOnI to O.lml. The volumes
may be
transferred by any efficient means, including pins, ink jet dispensers, other
piezoelectric devices,
pipettes, etc. After the liquid is dispensed, the applicable seal may be
applied. Instead of
dispensing liquid into the microstructure, the process may involve withdrawing
liquid from the
microstructure. Where the seal is in place, the seal would be removed, the
liquid withdrawn
from the microstructure and the seal replaced. In this way the integrity of
the concentration of
the solution in the microstructure may be maintained. Alternatively, one may
have a self sealing
film, where the seal would be pierced for the transfer of liquid.
For further understanding of the invention, the drawings will now be
considered. In Fig.
1 is depicted a plan view of a microfluidic device having 96 units with the
spacing appropriate to
a 96 well microtiter plate, where the spacing of the collars is shown. The
device 100 has a
substrate 102 with collars 104 associated with the reservoirs 106. In Fig. 1
a, a cross-sectional
view is shown of the device 100. The device 100 is shown with reservoirs 106,
only two
reservoirs being shown for clarity. Above the reservoirs 106 on the upper
planar surface 108 of
the device are collars 104. An enclosing film 110 provides the bottom of the
device, serving to
enclose the reservoirs 106 and channels 112.
In Fig. 2, the device 150 is shown with the sealing film removed to provide
the detail of
the individual microfluidic units 152. An expanded version of the microfluidic
units 152 is
depicted in a diagrammatic plan view in Fig. 2a. This unit is for illustration
purposes only and
demonstrates a unit for performing a reaction with incubation, followed by
separating the
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components of the reaction mixture using electrophoresis and identifying the
product with a
detector, not shown. The unit 152 has a separation channel 154, beginning with
electrophoresis
buffer reservoir 156 and terminating in waste reservoir 158. The assay channel
160 begins with
a reagent incubation reservoir 162 and ends with a second waste reservoir 164.
The reagent
incubation reservoir 162 receives the various components of the reaction where
the reagents may
react. Some of the liquid in the reagent incubation reservoir 162 may wick by
capillary action
through the separation channel to a stop junction reservoir 168 provided in
assay channel 160 to
stop the movement of the reaction solution. After sufficient time for
incubation liquid remaining
in the reagent incubation reservoir 162 is moved by pressure to the stop
junction reservoir 168
and the electrophoretic process begun by introducing electrodes into the waste
reservoirs 158
and 164, the stop junction reservoir 168 and the buffer reservoir 156. By
activating the
electrodes in the stop junction reservoir 168 and the waste reservoir 164, the
reaction
components may be moved to cross-section 166 for injection into separation
channel 154. The
electrodes in the reservoirs 164 and 168 may then be allowed to float, while
the electrodes in
buffer reservoir 156 and waste reservoir 158 are activated for electrophoretic
transport and
separation in separation channel 154 for detection of product.
In Fig. 3 is shown a cross-section of a reservoir with the collar on a
substrate. The
substrate 200 has an upper planar surface 202. Extending upward from upper
planar surface 202
is the external wall of collar 204. As shown in the Figure, the collar 204
meets with the upper
opening 206 of reservoir 208. The wall 210 of reservoir 208 is shown as
conical having a
linearly even surface, but could be vertical or irregular, as needed. The
reservoir wall 210 is
aligned with the collar wall 204, having a smooth transition, being a single
feature when
molding the substrate. As already indicated, the collar inner wall may be
offset from the port
edge. The reservoir 208 terminates at the bottom into channel 212. The channel
212 is
enclosed by film 214, which adheres to the bottom surface 216 of the
substrate. The reservoir
208 is enclosed by a conformable cover film 218, backed by a weighted backing
220, to hold the
film 218 in sealing relationship with the top surface 222 of collar 204.
The subject invention provides many advantages in enclosing, usually
reversibly, small
reservoirs or other microstructure. The subject collar structure has a small
contact area, which
serves to concentrate the force produced by whatever means of application of
the lid onto a
much smaller area, as compared to a cover which bonds to the entire surface of
the device. A
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reduction in differential pressures created during application of the lid is
achieved. Where the
upper surface is flat, without.areas in relief, a conformal lid comes down in
such a way that it
will usually first make contact with a large ring around the area to be
sealed. The air trapped
within this ring is pressurized into the volume to be sealed. By contrast with
the subject
structure, the lid makes contact before it reaches the device main surface,
avoiding trapping large
volumes of air. In addition, for lid attachment mechanisms like gravity,
friction and mechanical
clips, where the force is not directly related to the contact area, the
subject method increases the
local pressure with which the lid is attached to the part. This increased
pressure generally
improves the seal and improves the proximity of conformal lids. This improved
seal can enable
the use of a weighted lid to produce an airtight seal without requiring a
large mass or an
extremely conformable lid material. Where the seal is substantially air tight,
the lid will act to
resist or prevent fluid flow. Capillary stop junctions may be prone to failure
by condensation or
other mechanism, in which case the sealed lid provides a backup mechanism.
Also, the sealed
lid can easily counteract relatively strong fluidic forces, such as surface
tension. Yet another
advantage of the collar is that the effective leakage path (surface path)
between two adjacent
wells is increased. This increases the resistance to arcing due to high
differential voltages in two
adjacent wells. In fact, multiple concentric collars would be beneficial in
further reducing this
problem. Finally, during application and removal of a lid, there is a
potential for liquids to wick
between the lid and the surface of the device and microstructures. The short
distance between
the device and the lid can result in very strong capillary pressures. In the
subject invention the
lid is only in close proximity to the collar surface, so that fluid can only
wick along that surface.
By avoiding continuous ridges between different microstructures, movement of
liquid between
the microstructures can be obviated.
All publications and patent applications mentioned in this specification are
indicative of
the level of skill of those skilled in the art to which this invention
pertains. All publications and
patent applications are herein incorporated by reference to the same extent as
if each individual
publication or patent application was specifically and individually indicated
to be incorporate by
reference.
The invention now having been fully described, it will be apparent to one of
ordinary
skill in the art that many changes and modifications can be made thereto
without departing from
the spirit or scope of the appended claims.
